Enantiomeric 2-acetamido-1,4-dideoxy-1,4-iminoribitols as potential pyrrolidine hexosaminidase inhibitors

Article abstract of DOI:10.1016/j.crci.2010.03.020

2-Acetamido-1,4-imino-1,2,4-trideoxy-D-ribitol (DRBNAc) and the enantiomeric LRBNAc were prepared as potential hexosaminidase inhibitors from L- and D-lyxonolactone, respectively. Neither N-benzyl-DRBNAc nor N-benzyl-LRBNAc showed any inhibition of α- or

Full text of DOI:10.1016/j.crci.2010.03.020

C. R. Chimie 14 (2011) 126–133
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´
Full paper/Memoire
Enantiomeric 2-acetamido-1,4-dideoxy-1,4-iminoribitols as potential
pyrrolidine hexosaminidase inhibitors
J.S. Shane Rountree a,b, Terry D. Butters b, Raymond A. Dwek b, George W.J. Fleet a,
*
a Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Mansfield Road, Oxford OX1 3TA, United Kingdom
b Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom
A R T I C L E I N F O
A B S T R A C T
Article history:
2-Acetamido-1,4-imino-1,2,4-trideoxy-D-ribitol (DRBNAc) and the enantiomeric LRBNAc
were prepared as potential hexosaminidase inhibitors from L- and D-lyxonolactone,
Received 7 January 2010
Accepted after revision 22 March 2010
Available online 7 May 2010
respectively. Neither N-benzyl-DRBNAc nor N-benzyl-LRBNAc showed any inhibition of
or -hexosaminidases or any other glycosidase.
a-
b
ß 2010 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
Keywords:
Iminosugars
Carbohydrates
Hexosaminidase
Inhibitors
Pyrrolidines
Lyxonolactone
1. Introduction
other synthetic piperidine pyranose mimics [10] – are
potent inhibitors of
b-hexosaminidases (Fig. 1). The
Deficiencies of
sible for a number of lysosomal storage diseases; lack of
enzymes to cleave a terminal N-acetyl- -D-glucosamine
unit gives rise to Sanfilippo [1], Tay-Sachs and Sandhoff [2]
diseases whereas lack of an enzyme to hydrolyze a
a
- and
b
-hexosaminidases are respon-
natural product nagstatin 2 [11], with a galacto-config-
uration in the piperidine ring, is also a potent inhibitor of
b
b
-hexosaminidases [12]. The analogue of N-acetyl-
galactosamine 3 is the only powerful inhibitor of N-acetyl-
-D-galactosaminidases (GalNAcases) yet reported [13].
a-D-
a
terminal N-acetyl-
a
-D-galactosamine component is asso-
Hydroxyazepane [14] mimics of N-acetyl-hexosamines,
such as the azepanes 4 and 5, are specific sub-micromolar
ciated with Schindler-Kanzaki disease [3]. Inhibition of
hexosaminidases may also provide strategies for the
treatment of osteoarthritis [4], cancer [5], and type II
diabetes [6]; thus the design of specific and potent
hexosaminidase inhibitors provides an attractive synthetic
target.
inhibitors of
b-hexosaminidases [15]. Pochonicine 6 [or its
enantiomer], a nanomolar pyrrolizidine
b
-hexosaminidase
inhibitor, has been isolated from Pochonia suchlasporia var.
suchlasporia TAMA 87 [16].
Pyrrolidine analogues of pentoses provide examples of
powerful glycosidase inhibitors. The natural product DAB
7D, isolated from Arachniodes standishii and Angylocalyx
Iminosugars
– in which the ring oxygen of the
carbohydrate is replaced by nitrogen – constitute a family
of both natural and synthetic inhibitors of glycosidases [7].
boutiqueanus [17], shows strong inhibition of
a-glucosi-
DNJNAc 1 [8], the corresponding analogue of N-acetyl-
a
-
dases and weaker inhibition of several other glycosidases;
D-glucosamine, and its N-alkyl analogues [9] – as well as
the unnatural enantiomer LAB 7L is also a potent – but
highly specific – inhibitor of
a-glucosidases [18] (Scheme
1). A significant number of L-enantiomers of imino-D-
sugar mimics show inhibition of the same glycosidases
[19]. Both DABNAc 8D and LABNAc 8L, the NAc analogues
of DAB 7D and LAB 7L were synthesised from the
* Corresponding author.
E-mail addresses: terry.butters@biochem.ox.ac.uk (T.D. Butters),
george.fleet@chem.ox.ac.uk (George W.J. Fleet).
1631-0748/$ – see front matter ß 2010 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.crci.2010.03.020

J.S.S. Rountree et al. / C. R. Chimie 14 (2011) 126–133
127
Fig. 1. Potent inhibitors of hexosaminidases.
enantiomers of lyxonolactone [20]. LABNAc 8L and its N-
alkyl analogues [such as the N-benzyl derivative] were
micromolar – whereas DABNAc 8D analogues were weak –
involves two successive SN2 reactions; initial inversion by
trifluoroacetate displacement of a triflate to afford the
epimeric alcohol [26], with a subsequent SN2 displacement
of the epimeric triflate by azide to give overall retention of
configuration [27]. An alternative approach depends on the
observation that, in general, azides which are cis to all the
other substituents on a lactone ring are thermodynami-
cally more stable than epimers in which the azide is the
sole trans substituent [28]; there are even examples where
base catalysed epimerization of the less stable kinetic
triflate is so rapid that it is not possible to isolate the kinetic
azide but only the thermodynamic azide with retention of
configuration [29]. Esterification of 14D with triflic
(trifluoromethanesulfonic) anhydride in pyridine gave
the corresponding triflate 15D in quantitative yield [extra
care must be taken in the handling of this triflate, as a co-
worker experienced a strong allergic response to this
compound or its decomposition products]. Treatment of
the lyxono-triflate 15D with sodium azide in DMF
produced 83% yield of the xylono-azide 16D under kinetic
control using a short reaction time (50–60 min) and just
under stoichiometric amounts of sodium azide (0.97
equivalents) during the reaction [21,28]. However, when
the triflate 15D was left for 20 h at room temperature in the
presence of excess sodium azide and pyridinium p-
toluenesulfonate (PPTS) the thermodynamic lyxono-azide
11D was formed in 86% yield with a trace of the easily
separable xylono-azide 16D (4%).
specific inhibitors of
b-hexosaminidases; neither showed
any inhibition of -GalNAcases [21].
a
The imino ribitol 9, isolated from the mulberry tree
Morus alba [22] and synthesized from carbohydrate [23]
and non-carbohydrate [24] starting materials, was a weak
inhibitor of several glycosidases (IC50 = 230–610
but was shown to mimic a galactose motif by its moderate
inhibition of a -D-galactosidase (rat intestinal lactase,
IC50 17 M) [25]. LRBNAc 12L and DRBNAc 12D are related
mM) [13],
b
m
to 9 in the same way that LABNAc 8L and DABNAc 8D are
related to LAB 7L and DAB 7D. Accordingly, in the search for
GalNAcase inhibitors, the syntheses of LRBNAc 12L from D-
lyxonolactone 10D and of DRBNAc 12D from L-lyxono-
lactone 10L (Scheme 1) – which depend on the efficient
introduction of an azide at C2 with retention of configura-
tion in suitably protected intermediates 11L and 11D – are
herein described. However, this paper reports that neither
DRBNAc 12D nor LRBNAc 12L showed significant inhibi-
tion of any glycosidase.
The key steps in the synthesis of LABNAc 8L from D-
lyxonolactone 10D were introduction of nitrogen by
nucleophilic substitution with inversion at C2, and
formation of the pyrrolidine ring by nucleophilic substitu-
tions by benzylamine between C5 and C2 [with inversion
of configuration] of the D-lyxo sugar to give the required L-
ribo configuration. Reaction of D-lyxonolactone 10D with
benzaldehyde and hydrochloric acid gave the highly
crystalline benzylidene derivative 14D (90% yield) in
which only the C2-OH group of the lactone is unprotected
(Scheme 2).
The sensitivity of
a-azidolactones to base frequently
results in low yields of direct conversion of the lactone to
the corresponding diol by hydrides [27]. Attempted
reductions of the azidolactone 11D with lithium borohy-
dride, sodium borohydride or Super-Hydride1 gave low
yields of the lyxo-diol 17D. However, L-Selectride1 in THF
at ꢀ30 8C gave acceptable yields for the formation of lyxo-
There are two strategies for the introduction of azide at
C2 of 10D with retention of configuration. One route
Scheme 1. Relationships between pyrrolidine glycosidase inhibitors.

128
J.S.S. Rountree et al. / C. R. Chimie 14 (2011) 126–133
Scheme 2. Reagents and conditions: (i) PhCHO, HCl, 90% (ii) Tf2O, pyridine, ꢀ50 8C, 2.5 h, 100%; (iii) NaN3, PPTS, DMF, RT, 20 h, 86% 11D, 4% 16D; (iv) L-
SelectrideØ, THF, ꢀ78 8C, 2 h, 57%; (v) MsCl, Et3N, DCM, 0 8C, 2 h, 92%; (vi) BnNH2, 95 8C, 20 h, 80%; (vii) AmberlystØ 15 beads, 1,4-dioxane, H2O, 95 8C, 14 h;
(viii) PPh3, H2O, THF, RT, 14 h, 89%; (ix) Ac2O, pyridine, RT, 14 h, 85%, (x) NaOMe, MeOH, RT, 2 h, 91%; (xi) H2/Pd black, 1,4-dioxane, H2O, r.t., 18 h, 88%.
diol 17D (57%); after the reduction was complete, it was
necessary to employ an oxidative workup procedure using
sodium hydroxide and aqueous hydrogen peroxide to
remove the borane adducts in order to obtain a reasonable
yield of pure material after flash chromatography.
Esterification of the lyxo-diol 17D with mesyl (methane-
sulfonyl) chloride in dichloromethane in the presence of
triethylamine at 0 8C afforded the dimesylate 18D in 90%
yield. Reaction of the dimesylate 18D with benzylamine at
95 8C for 20 h gave the aminobenzylmesylate 19D in 80%
yield; there was no subsequent intramolecular cyclization
since the resulting pyrrolidine would have a benzylidene
acetal trans to the pyrrolidine ring. Deprotection of the
benzylidene acetal of 19D which would allow subsequent
cyclization to the pyrrolidine 20L, was achieved by heating
19D, in aqueous dioxane in the presence of acid ion-
exchange resin at 95 8C for 14 h. After filtering off the resin,
washing with 2 M ammonium hydroxide solution, solvent
removal and flash chromatography, the L-ribo-pyrrolidine
20L was isolated in 89% yield. Staudinger reduction [30] of
the azide 20L by triphenylphosphine and water in THF
gave, after purification by ion-exchange chromatography,
the L-ribo-amine 21L in 89% yield. Reaction of the
aminodiol 21L with acetic anhydride in pyridine afforded
the triacetyl derivative 22L (85% yield). The O-acetyl
groups in 22L were selectively removed by treatment with
catalytic sodium methoxide in methanol to give N-benzyl-
LRBNAc 13L, in 91% yield; the structure of 13L was
unequivocally established by X-ray crystallographic anal-
ysis [31]. Removal of the N-benzyl group in 13L by
hydrogenolysis in the presence of palladium black in
aqueous 1,4-dioxane gave LRBNAc 12L in 88% yield. The
preparation of LRBNAc 12 from D-lyxonolactone was
achieved in 18% overall yield over the 12 steps.
2. Inhibition studies
Neither N-benzyl-DRBNAc 13D nor N-benzyl-LRBNAc
13L showed significant inhibition of the hydrolysis of the
corresponding p-nitrophenyl glycosides by any of the
following range of enzymes:
glucosidase (almond), -galactosidase (coffee bean),
galactosidase (jack bean), -mannosidase (jack bean),
mannosidase (snail), -fucosidase (bovine),
(Aspergillus niger), -N-acetylhexosaminidase (jack bean),
and -N-acetylgalactosaminidase (Charonia lampas). In
a
-glucosidase (yeast),
b
b
b
-
-
-
a
a
a
b-xylosidase
b
a
particular, there was no inhibition of any
hexosaminidases.
a- or b-
3. Summary
The synthesis of LRBNAc 12L from D-lyxonolactone 10D
over 12 steps proceeded in a good overall yield of 18%. The
enantiomer DRBNAc 12D was also prepared from L-
lyxonolactone. Assays of N-benzyl-LRBNAc 13L and N-
benzyl-DRBNAc 13L showed there was no significant
inhibition of either
b-N-acetylhexosaminidase (jack bean)
or -N-acetylgalactosaminidase (Charonia lampas).
a
4. Experimental
THF was distilled from sodium benzophenone ketyl or
purchased dry from Aldrich in Sure/SealTM bottles and
recapped with Oxford Sure/SealTM valve-caps. N,N-
Dimethylformamide (DMF) and pyridine were purchased
dry from Aldrich in Sure/SealTM bottles and were recapped
with Oxford Sure/SealTM valve caps. Water was distilled.
All other solvents were used as supplied (analytical or
HPLC grade) without prior purification. Reactions per-
formed under an atmosphere of nitrogen, argon, or
hydrogen gas were maintained by an inflated balloon.
Imidazole was recrystallised from dichloromethane. All
other reagents were used as supplied without prior
purification. Thin-layer chromatography (TLC) was per-
DRBNAc 12D was prepared from 3,5-benzylidene-L-
lyxonolactone 10L by an identical procedure; the benzy-
lidene lactone 10L was formed in 95% yield [21] from 2,3-
isopropylidene-L-lyxonolactone, readily available on a
large scale from D-ribose [32].

J.S.S. Rountree et al. / C. R. Chimie 14 (2011) 126–133
129
formed on aluminium-backed sheets coated with 60 F254
silica. Sheets were developed by dipping in a solution of
cerium(IV) sulfate (0.2% w/v) and ammonium molybdate
inhibitor. The concentrations of the enzyme were adjusted
so that the reading for the final absorbance was in the
range of 0.5–1.5 units over a reaction time of 20 min. For
(5%) in 2 M sulfuric acid and heating with a heat gun. Flash
column chromatography was performed using Sorbosil1
C60 40/60 silica. Ion-exchange chromatography was
performed using either Amberlite1 CG-150 or Dowex1
50WX8–200, as stated. NMR spectra were recorded on a
Bruker DQX 400 (1H: 400 MHz, 13C: 100 MHz) spectro-
meter at room temperature in the deuterated solvent
example, b-N-acetyl-D-hexosaminidase was diluted from
6 U mLꢀ1 to 0.4 U mLꢀ1 by experiment. Linearity over the
time course of the reaction was checked using a series of
incubation times. The following were combined in the well
of a flat-bottomed 96-well (300 mL) microtitre plate: 5
m
L
L
enzyme solution, inhibitor solution, and 40 m
5
m
L
substrate solution. The reaction mix was incubated at
stated. All chemical shifts (
d
) are quoted in ppm. Coupling
37 8C for 20 min (Denley Wellwarm 1) and was quenched
constants (3J) are quoted in Hz. Residual signals from the
solvents were used as an internal reference. The 13C
resonances were assigned using either DEPT or APT
sequences or from 1H–13C HMQC and HMBC spectra.
Melting points were recorded on a Kofler hot block and are
uncorrected. IR spectra were recorded at room tempera-
ture on a PerkinElmer 1750 IR Fourier transform spectro-
photometer or a Bruker Tensor 27 FTIR spectrometer, using
thin films on NaCl or Ge plates, as stated, and were
by the addition of 200 mL of 0.5 M Na2CO3. Absorbance at
405 nm was measured immediately using a microtitre
plate reader (Molecular Devices UVmax kinetic microplate
reader and SOFTmax 2.35 software).
6. 2-Azido-3,5-O-benzylidene-2-deoxy-D-lyxono-1,4-
lactone 11D
Triflic anhydride (2.44 mL, 14.5 mmol, 1.6 equivalents)
was added dropwise to a stirred solution of the benzyli-
dene lactone 14D21 (2.14 g, 9.06 mmol) in pyridine (20 mL)
at ꢀ50 8C under an atmosphere of nitrogen. The tempera-
ture was allowed to rise to room temperature during the
course of the reaction. After 2.5 h, analysis by TLC (ethyl
acetate) showed no residual starting material (Rf 0.07) and
a major spot due to the product (Rf 0.50). The solvents were
removed in vacuo, by coevaporation with toluene (high
vacuum). The residue was partitioned between ethyl
acetate (80 mL) and 0.1 M HCl (50 mL). The resulting
biphasic solution was separated and the organic layer
washed with brine (50 mL). The aqueous phase was
extracted with ethyl acetate (3 ꢁ 15 mL) and the combined
organic phase was dried (magnesium sulfate) and the
solvents removed in vacuo. Sodium azide (1.46 g,
22.6 mmol, 2.5 equivalents) was added to a stirred solution
of crude triflate 15D (ca. 3.34 g, 9.06 mmol) in DMF (35 mL)
at room temperature under an atmosphere of nitrogen.
After 1 h, PPTS (2.73 g, 10.9 mmol) was added. After a
further 18 h, analysis by TLC (1:1, ethyl acetate/cyclohex-
ane) showed a major product spot (Rf 0.17) and a minor
spot (Rf 0.41). The solvents were removed in vacuo, by
coevaporation with toluene (high vacuum); the residue
was purified by flash chromatography (3:1 ! 1:1, cyclo-
hexane/ethyl acetate) to give the azido-lyxono-lactone 11D
(2.04 g, 7.81 mmol, 86%) as a white solid, m.p. 122-123 8C,
128-130 8C (recrystallised from ethyl acetate/cyclohexane)
recorded over 16 or 32 scans at a resolution of 4 cmꢀ1
.
Only the characteristic peaks are quoted. Mass spectro-
metry: high-resolution mass spectra (HRMS) were
recorded on a micromass Autospec 500 OAT spectrometer
using the technique of chemical ionisation (CI), or on a
Waters 2790-Micromass LCT mass spectrometer using the
technique of electrospray ionisation (ESI). Optical rota-
tions were recorded on a PerkinElmer 241 polarimeter
with a path length of 1 dm. Concentrations are quoted in
g(100 mL)ꢀ1. The wavelength at which the rotations were
measured corresponds to the sodium D line (ca. 589 nm).
Elemental analyses were performed by the microanalysis
service of the Inorganic Chemistry Laboratory, University
of Oxford.
5. Enzyme inhibition assays
All enzymes and corresponding PNP substrates were
obtained from Sigma, apart from coffee bean
galactosidase, jack bean -D-galactosidase, -D-manno-
sidase and -N-acetyl-D-hexosaminidase, and Charonia
lampas -N-acetyl-D-galactosaminidase, which were pur-
a-D-
b
a
b
a
ified from the natural sources in the Oxford Glycobiology
Institute. Enzyme solutions: For the purposes of this
investigation, all enzyme solutions were made up using
50 mM citrate–phosphate buffer (anhydrous disodium
hydrogen phosphate dissolved in citric acid) at pH 5
containing 1 mg mLꢀ1 bovine serum albumin (BSA) and
[Lit. [28] 121-122 8C (acetone)];
½
a 2D3
+37.5 (c 0.99,
ꢂ
0.02% NaN3, except
a
-D-glucosidase, for which Dulbecco’s
acetone) [Lit. [28] ½a D24
ꢂ
+37.4 (c 1.00, acetone)]; nmax
phosphate buffered saline (PBS) buffer at pH 7.4 was used.
These solutions were stored on ice whilst in use and at
ꢀ20 8C when not in use. Inhibitor solutions: NBn-LRBNAc
13L and NBn-DRBNAc 13D were dissolved in distilled
water at a concentration of 10 mg mLꢀ1. These solutions
were stored on ice whilst in use and at ꢀ20 8C when not in
use. Enzyme substrate solutions: Substrate solutions were
made from a stock solution (3 mM) in 10 mL batches, as
required, by dissolving the appropriate mass of substrate
in the appropriate buffer solution for the enzyme. These
were kept at 4 8C when not in use. Method: Assays were
carried out in triplicate, using H2O as a blank in place of the
(NaCl) 2119 (N3), 1796 (C = O) cmꢀ1
; dH(400 MHz;
0
(CD3)2CO) 4.40 (1H, dd, J5,5 13.8 Hz, J5,4 2.0 Hz, H-5),
4.49 (1H, dd, J5 ,5 13.8 Hz, J5 ,4 1.3 Hz, H-50), 4.60 (1H, d, J2,3
0
0
0
4.1 Hz, H-2), 4.65 (1H, dt, J4,5 1.3 Hz, J4,5 2.0 Hz, J4,3 2.0 Hz,
H-4), 5.13 (1H, dd, J3,4 2.0 Hz, J3,2 4.1 Hz, H-3), 5.80 (1H, s,
CHPh), 7.36–7.52 (5H, m, CH(Ar)). dC(100 MHz, (CD3)2CO)
62.4 (C-2), 66.5 (C-5), 71.9 (C-4), 75.3 (C-3), 99.1 (CHPh),
126.5, 128.5, 129.4 (CH(Ar)), 138.1 (C(Ar)), 171.7 (C = O)
ppm; Found: C, 55.13; H, 4.33; N, 16.08; C12H11N3O4
requires C, 55.17; H, 4.24; N, 16.09%; a small amount of
azido-xylono-lactone 16D21 (97.2 mg, 0.372 mmol, 4%) was
also isolated.

130
J.S.S. Rountree et al. / C. R. Chimie 14 (2011) 126–133
The enantiomeric azidolactone 11L, m.p. 128-130 8C;
cyclohexane/ethyl acetate) to afford the dimesylate 18D
(421 mg, 92%) as a white crystalline solid, m.p. 139-140 8C;
½
a 2D5
ꢂ
ꢀ41.3 (c 0.99, CHCl3), was prepared by a similar
method. All other physical data was identical to that of
½
a 2D0
ꢂ
ꢀ75.5 (c 0.97, CHCl3);
nmax (NaCl) 2108 (N3), 1346 and
H(400 MHz, CDCl3) 3.08, 3.20 (6H,
11D.
1180 (S = O) cmꢀ1
; d
2 ꢁ s, 2 ꢁ SO2CH3), 3.94 (1H, dd, J3,2 9.7 Hz, J3,4 1.6 Hz, H-3),
0
7. 2-Azido-3,5-O-benzylidene-2-deoxy-D-lyxitol 17D
4.06 (1H, ddd, J2,3 9.7 Hz, J2,1 5.9 Hz, J2,1 2.6 Hz, H-2), 4.14
0
0
(1H, dd, J5,5 13.4 Hz, J5,4 1.2 Hz, H-5), 4.50 (1H, dd, J1,1
A 1 M solution of L-Selectride1 in THF (2.87 mL, 2.5
equivalents) was added dropwise to a stirred solution of
the lyxono-lactone 11D (300 mg, 1.15 mmol) in THF
(3.0 mL) at ꢀ78 8C under an atmosphere of argon in the
10.9 Hz, J1,2 5.9 Hz, H-1), 4.58 (1H, dd, J5 ,5 13.4 Hz, J5 ,4
0
0
1.8 Hz, H-50), 4.61 (1H, dd, J1 ,1 10.9 Hz, J1 ,2 2.6 Hz, H-10),
4.87-4.90 (1H, m, H-4), 5.57 (1H, s, CHPh), 7.37-7.50 (5H,
m, CH(Ar)); dC (100 MHz, CDCl3): 37.7, 39.2 (2 ꢁ SO2CH3),
58.7 (C-2), 67.8 (C-1), 69.4 (C-5), 70.0 (C-4), 75.4 (C-3),
101.3 (CHPh), 126.0, 128.4, 129.5 (CH(Ar)), 136.5 (C(Ar));
m/z HRMS (ESI+): Found 444.0511, C14H19N3O8Na
[M + Na]+ requires 444.0511. Found: C, 39.81; H, 4.78; N,
9.95; C14H19N3O8S2 requires C, 39.90; H, 4.54; N, 9.97%.
The enantiomeric dimesylate 18L, m.p. 138-140 8C;
0
0
˚
presence of molecular sieve (4 A). After 2 h, analysis by TLC
(ethyl acetate) showed a major spot due to the product (Rf
0.50) and no residual starting material (Rf 0.59). Methanol
(0.5 mL) was added slowly to destroy the excess hydride
and after 15 min a further portion (2.5 mL) was added.
After a further 2 h, the mixture was filtered over Celite1
diethyl ether (10 mL) and a 50% solution of 4 M NaOH/
;
v
/v
½
a 2D3
+78.5 (c 0.96, CHCl3), was prepared by a similar
method. All other physical data was identical to that of
ꢂ
35% aqueous H2O2 (5 mL) were added to the filtrate and the
reaction mixture allowed to stir for 1 h. The resulting
biphasic solution was separated and the aqueous phase
was extracted using ethyl acetate (3 ꢁ 2 mL). The com-
bined organic phase was dried (magnesium sulfate) and
the solvents were removed in vacuo. The residue was
passed through a silica plug (9:1 ethyl acetate/methanol)
to yield the diol 17D (175 mg, 57%) as a white amorphous
18D.
9. 1-Aminobenzyl-2-azido-3,5-O-benzylidene-1,2-
dideoxy-4-O-methanesulfonyl-D-lyxitol 19D
The dimesylate 18D (669 mg, 1.59 mmol) was dis-
solved in benzylamine (14.0 mL) and the resulting
solution heated at 95 8C. After 20 h, analysis by TLC
(1:1, cyclohexane/ethyl acetate) showed one major spot
(Rf 0.32) and a UV active spot (Rf 0.59) due to residual
benzylamine. The solvents were removed in vacuo by co-
evaporation with toluene. The resulting orange residue
was partitioned between ethyl acetate (30 mL) and brine
(40 mL). The layers were separated and the aqueous phase
was extracted using ethyl acetate (3 ꢁ 15 mL). The
combined organic phase was dried (magnesium sulfate),
the solvents were removed in vacuo and the resulting
residue was purified by flash chromatography (9:1 ! 1:1,
cyclohexane/ethyl acetate) to yield the aminomesylate
19D (551 mg, 1.27 mmol, 80%) as a white amorphous
solid, m.p. 102–103 8C; ½a D23
ꢂ
ꢀ42.6 (c 0.345, CHCl3); nmax
H(400 MHz, CDCl3)
(NaCl) 3417 (OH), 2103 (N3) cmꢀ1
; d
2.14 (1H, br s, OH), 2.77 (1H, br s, OH), 3.77 (1H, apparent q,
0
J 1.5 Hz, H-4), 3.81 (1H, dd, J1,1 11.4 Hz, J1,2 4.8 Hz, H-1),
3.84–3.89 (1H, m, H-2), 3.91 (1H, dd, J3,2 9.2 Hz, J3,4 1.2 Hz,
H-3), 3.98 (1H, dd, J1 ,1 11.4 Hz, J1 ,2 3.0 Hz, H-10), 4.10 (1H,
0
0
0
0
dd, J5,5 12.1 Hz, J5,4 1.4 Hz, H-5), 4.29 (1H, dd, J5 ,5 12.1 Hz,
0
J5 ,4 2.0 Hz, H-50), 5.57 (1H, s, CHPh), 7.35–7.48 (5H, m,
CH(Ar)); dC(100 MHz, CDCl3) 62.0 (C-1), 62.1 (C-2), 63.3 (C-
4), 72.4 (C-5), 78.0 (C-3), 101.4 (CHPh), 125.8, 128.4, 129.3
(CH(Ar)), 137.2 (C(Ar)); m/z HRMS (ESI-): Found 264.0985,
C
12H14N3O4 [M-H]+ requires 264.0984.
The enantiomeric diol 17L, m.p. 101-102 8C; ½a D23
ꢂ
+43.8
(c 0.85, CHCl3), was prepared by a similar method. All other
physical data was identical to that of 17D.
solid, m.p. 89-91 8C; ½a D20
ꢀ45.6 (c 0.97, CHCl3); nmax
ꢂ
(NaCl) 3355 (N-H), 2106 (N3), 1354 and 1175 (S = O)
cmꢀ1
;
d
H(400 MHz, CDCl3) 2.00 (1H, br s, NHBn), 2.95 (1H,
0
0
8. 2-Azido-3,5-O-benzylidene-2-deoxy-1,4-di-O-
methanesulfonyl-D-lyxitol 18D
dd, J1,1 13.0 Hz, J1,2 6.1 Hz, H-1), 3.16 (1H, dd, J1 ,1 13.0 Hz,
0
J1 ,23.3 Hz, H-10), 3.17 (3H, s, SO2CH3), 3.82 (1H, d, J
13.3 Hz, NCHH’Ph), 3.87 (1H, ddd, J2,3 9.7 Hz, J2,1 6.1 Hz,
0
Triethylamine (0.60 mL, 4.32 mmol,
4
equiv.) was
J2,1 3.3 Hz, H-2), 3.93 (1H, d, J 13.3 Hz, NCHH’Ph), 4.02
0
added to a stirred solution of the diol 17D (287 mg,
1.08 mmol) in dichloromethane (6.0 mL) at 0 8C under an
atmosphere of nitrogen. After 20 min, methanesulfonyl
chloride (0.34 ml, 4.32 mmol, 4 equiv.) was added drop-
wise. After 2 h, analysis by TLC (ethyl acetate) showed no
residual starting material (Rf 0.56) and a major spot due to
the product (Rf 0.67) [Note: change in the colour of the spot
made it easier to see the progress of the reaction]. The
solvents were removed in vacuo and the residue was
partitioned between ethyl acetate (20 mL) and brine
(30 mL). The layers were separated and the aqueous phase
was extracted using ethyl acetate (3 ꢁ 10 mL). The
combined organic phase was dried (magnesium sulfate),
the solvents were removed in vacuo, and the remaining
residue was purified by flash chromatography (1:1,
(1H, dd, J3,2 9.7 Hz, J3,4 1.1 Hz, H-3), 4.12 (1H, dd, J5,5
0
0
13.4 Hz, J5,4 1.0 Hz, H-5), 4.61 (1H, dd, J5 ,5 13.4 Hz, J5 ,4
1.6 Hz, H-50), 4.87 (1H, apparent d, J 1.3 Hz, H-4), 5.57
(1H, s, CHPh), 7.22–7.45 (5H, m, CH(Ar));
dC(100 MHz,
CDCl3) 39.42 (SO2CH3), 48.7 (C-1), 53.8 (NCH2Ph), 60.3
(C-2), 69.6 (C-5), 71.0 (C-4), 76.7 (C-3), 101.2 (CHPh),
126.0, 127.2, 128.1, 128.3, 128.5, 129.3 (CH(Ar)), 136.9,
139.5 (C(Ar)); m/z HRMS (ESI+): found 433.1547,
C
20H25N4O5S [M + H]+ requires 433.1546. Found: C,
55.57; H, 5.69; N, 12.56;
55.54; H, 5.59; N, 12.95%.
C20H24N4O5S requires C,
The enantiomeric aminomesylate 19L, m.p. 91-92 8C;
+40.5 (c 1.0, CHCl3), was prepared by a similar
method. All other physical data was identical to that of
½ ꢂ
a 2D2
19D.

J.S.S. Rountree et al. / C. R. Chimie 14 (2011) 126–133
131
10. N-Benzyl-2-azido-1,4-imino-1,2,4-trideoxy-L-ribitol
20L
The enantiomeric diamine 21D, oil; ½a D23
ꢂ
ꢀ64.2 (c 0.965,
H2O), was prepared by a similar method. All other physical
data was identical to that of 21L.
Amberlyst1 15 (wet) beads were added to a stirred
solution of the aminomesylate 19D (540 mg, 1.25 mmol) in
1,4-dioxane (20 mL) and water (20 mL) and the resulting
mixture was heated at 95 8C under an atmosphere of
nitrogen. After 14 h, analysis by TLC (ethyl acetate) showed
one spot (Rf 0.23) due to the product. After cooling to room
temperature, 2 M NH4OH (aq) solution (10 mL) was added
and the mixture was stirred for 2 h. The beads were filtered
off, washing thoroughly with 2 M NH4OH (3 ꢁ 5 mL) and
1,4-dioxane (10 mL). The solvents were removed in vacuo
and the remaining residue was purified by flash chroma-
tography (ethyl acetate) to give the imino-L-ribitol 20L
12. 2-Acetamido-N-benzyl-3,5-di-O-acetyl-1,4-imino-
1,2,4-trideoxy-L-ribitol 22L
Acetic anhydride (0.15 mL, 1.620 mmol) was added
dropwise to a stirred solution of the amine 21L (60.0 mg,
0.270 mmol) in pyridine (2.0 mL) at room temperature
under an atmosphere of argon. After 14 h, analysis by TLC
(ethyl acetate) showed no residual starting material
(baseline) and a spot due to the product (Rf 0.21). The
solvents were removed in vacuo using toluene to aid by
coevaporation; the remaining residue was purified by flash
chromatography (98:2, ethyl acetate/triethylamine) to
form the triacetate 22L as a colourless oil (79.8 mg,
(275 mg, 89%) as a pale brown oil; ½a D20
ꢂ
+23.9 (c 0.92,
CHCl3); nmax (NaCl) 3384 (OH), 2106 (N3) cmꢀ1
;
0
d
H(400 MHz, CDCl3) 2.69 (1H, dd, J1,1 9.9 Hz, J1,2 7.3 Hz,
0.229 mmol, 85%); ½a D22
ꢂ
+79.2 (c 0.98, CHCl3); nmax (NaCl)
3286 (N-H), 1742 (C = O ester), 1654 (C = O amide band I),
0
H-1), 2.77 (1H, br s, OH), 2.85 (1H, m, H-4), 3.26 (1H, dd, J1 ,1
9.9 Hz, J1 ,2 6.1 Hz, H-10), 3.57-3.67 (3H, m, NCHH’Ph, H-5,
1542 (N-H amide band II) cmꢀ1
; dH(400 MHz, C6D6) 1.65
0
H-50), 3.91 (1H, dt, J2,1 7.3 Hz, J 5.7 Hz, H-2), 3.97 (1H, d, J
(3H, s, amide CH3), 1.72 (3H, s, C(O)CH3), 1.89 (3H, s,
0
13.0 Hz, NCHH’Ph), 4.24 (1H, apparent t, J 4.9 Hz, H-3)
C(O)CH3), 2.27 (1H, dd, J1,1 8.1 Hz, J1,2 10.7 Hz, H-1), 2.94
0
7.26–7.38 (5H, m, CH(Ar));
d
C(100 MHz, CDCl3) 55.0 (C-1),
(1H, ddd, J4,5 5.9 Hz, J4,5 4.3 Hz, J4,3 1.5 Hz H-4), 3.15 (1H, dd,
0
0
58.9 (NCH2Ph), 60.0 (C-5), 61.9 (C-2), 70.8 (C-4), 74.1 (C-3),
127.7, 128.6, 128.8 (CH(Ar)), 137.6 (C(Ar)); m/z HRMS
(ESI+): Found 249.1344, C12H17N4O2 [M + H]+ requires
249.1352.
J1 ,1 8.1 Hz, J1 ,2 6.6 Hz, H-10), 3.37 (1H, d, J 13.2 Hz, NCH2Ph),
0
3.92 (1H, d, J 13.2 Hz, NCH2Ph), 4.23 (1H, dd, J5,5 11.5 Hz, J5,4
4.3 Hz, H-5), 4.27 (1H, dd, J5 ,5 11.5 Hz, J5 ,4 5.9 Hz, H-50), 4.82
(1H, dddd, J2,1 10.7 Hz, J2,1 6.6 Hz, J2,3 5.5 Hz, J2,NH 8.6 Hz, H-
2), 5.26 (1H, dd, J3,2 5.5 Hz, J3,4 1.5 Hz, H-3), 5.42 (1H, d, JNH,2
0
0
0
The enantiomeric benzylamine 20D, oil; ½a D23
ꢂ
ꢀ31.5 (c
0.955, CHCl3), was prepared by a similar method. All other
8.6 Hz, NH), 7.25-7.35 (5H, m, CH(Ar)); dC(100 MHz, C6D6)
physical data was identical to that of 20L.
20.7, 20.7 (2 ꢁ OC(O)CH3), 22.9 (NHC(O)CH3), 49.3 (C-2),
55.6 (C-1), 59.1 (NCH2Ph), 63.5 (C-5), 68.8 (C-4), 76.4 (C-3),
127.7, 128.8, 129.3 (CH(Ar)), 138.9 (C(Ar)), 168.9, 169.4,
170.6 (3 ꢁ C = O); m/z HRMS (ESI+): Found 349.1756,
11. 2-Amino-N-benzyl-1,4-imino-1,2,4-trideoxy-L-
ribitol 21L
C
18H25N2O5 [M+H]+ requires 349.1763.
Triphenyl phosphine (169 mg, 0.644 mmol) was added
The enantiomeric triacetate 22D, oil; ½a D23
ꢀ77.7 (c 0.65,
ꢂ
to
a
stirred solution of the azide 20L (80.0 mg,
L, 0.322 mmol) in THF
CHCl3), was prepared by a similar method. All other
0.322 mmol) and water (12
m
physical data was identical to that of 22L.
(1.0 mL) at room temperature under an atmosphere of
nitrogen. After 14 h, analysis by TLC (ethyl acetate)
showed a major product spot (baseline) and no residual
starting material (Rf 0.23). The solvents were removed in
vacuo and the remaining residue was partitioned between
chloroform (3 mL) and water (2 mL). The aqueous layer
was extracted with chloroform (3 ꢁ 1 mL) to remove
excess triphenylphosphine oxide. The organic layer was
extracted with water (3 ꢁ 1 mL); the combined aqueous
phase was purified by ion exchange chromatography
(Dowex1 50WX8-200) to afford the amine 21L (64.0 mg,
13. 2-Acetamido-N-benzyl-1,4-imino-1,2,4-trideoxy-L-
ribitol (N-benzyl-LRBNAc) 13L
Sodium methoxide (3.2 mg, 0.059 mmol) was added to
a
stirred solution of the triacetate 22L (103 mg,
0.296 mmol) in methanol (1.0 mL) at room temperature
under an atmosphere of nitrogen. After 2 h, analysis by TLC
(acetone) showed no residual starting material (Rf 0.55)
and a spot due to the product (Rf 0.29). The solvents were
removed in vacuo and the resulting residue was purified by
flash chromatography (ethyl acetate, then acetone) to give
the N-benzyl-acetamide 13L (71.1 mg, 91%) as a pale
yellow oil that was recrystallised from acetonitrile, m.p.
0.288 mmol, 89%) as a colourless oil; ½a D22
ꢂ
+63.9 (c 0.98,
H2O); nmax (Ge) 3356 (OH, NH2), 1585 (NH2) cmꢀ1
;
d
H(400 MHz, D2O) 2.38 (1H, apparent t, J 10.1 Hz, H-1),
2.72 (1H, dt, J4,3 2.6 Hz, J4,5 5.9 Hz, J4,5 5.9 Hz, H-4), 2.96
(acetonitrile) 81–82 8C; ½a D22
ꢂ
+76.8 (c 0.72, MeOH); nmax
(NaCl) 3318 (O-H and N-H), 1643 (C = O amide band I),
0
(1H, dd, J1 ,1 9.3 Hz, J1 ,2 6.5 Hz, H-10), 3.19 (1H, ddd, J2,1
0
0
10.9 Hz, J2,1 6.5 Hz, J2,3 5.6 Hz, H-2), 3.40 (2H, d, J5/5 ,4
5.9 Hz, H-5, H-50), 3.59 (1H, d, J 12.3 Hz, NCHHPh), 3.85
(1H, d, J 12.3 Hz, NCHHPh), 3.94 (1H, dd, J3,2 5.6 Hz, J3,4
1551 (N-H amide band II) cmꢀ1
;
d
H(400 MHz, CD3CN): 1.86
0
0
0
(3H, s, amide CH3), 2.29 (1H, dd, J1,1 8.3 Hz, J1,2 10.3 Hz, H-
1), 2.34 (1H, br s, OH), 2.61 (1H, dt, J4,3 2.3 Hz, J 4.0 Hz, H-4),
2.6 Hz, H-3), 7.30-7.41 (5H, m, CH(Ar)); dC(100 MHz, D2O):
2.92 (1H, dd, J1 ,1 8.3 Hz, J1 ,2 6.5 Hz, H-10), 3.44-3.49 (3H, m,
H-5, H-50, NCH2Ph), 3.94 (1H, d, J 13.1 Hz, NCH2Ph), 3.99
(1H, dd, J3,2 5.7 Hz, J3,4 2.3 Hz, H-3), 4.01-4.09 (1H, m, H-2),
6.49 (1H, d, JNH,2 5.7 Hz, amide NH), 7.21-7.36 (5H, m,
0
0
51.3 (C-2), 57.0 (C-1), 59.5 (NCH2Ph), 61.7 (C-5), 72.6 (C-
4), 73.4 (C-3), 128.2, 128.9, 130.4 (CH(Ar)), 137.1 (C(Ar));
m/z HRMS (ESI+): Found 223.1448, C12H19N2O2 [M + H]+
requires 223.1447.
CH(Ar)); dC(100 MHz, CD3CN) 22.5 (NHC(O)CH3), 51.2 (C-

132
J.S.S. Rountree et al. / C. R. Chimie 14 (2011) 126–133
(d) S. Bin Mohamad, H. Nagasawa, Y. Uto, H. Hori, Anticancer Res. 22
(2002) 4297;
(e) B. Woynarowska, H. Wikiel, M. Sharma, G.W.J. Fleet, R.J. Bernacki,
Proc. Amer. Assoc. Cancer Res. 30 (1989) 91;
(f) B. Woynarowska, H. Wikiel, M. Sharma, N. Carpenter, G.W.J. Fleet,
R.J. Bernacki, Anticancer Res. 12 (1992) 161.
2), 55.9 (C-1), 59.0 (NCH2Ph), 61.8 (C-5), 73.0 (C-3), 73.5 (C-
4), 127.4, 128.6, 129.2 (CH(Ar)), 139.7 (C(Ar)), 170.5
(C = O); m/z HRMS (ESI+): found 265.1556, C14H21N2O3
[M + H]+ requires 265.1552. Found: C, 63.74; H, 7.64; N,
10.62; C14H20N2O3 requires C, 63.62; H, 7.63; N, 10.60%.
[6] K. Voseller, L. Wells, M.D. Lane, G.W. Hart, Proc. Natl. Acad. Sci. 99
(2002) 5315.
The enantiomeric acetamide 13D, oil; ½a D23
ꢀ74.3 (c
ꢂ
[7] (a) N. Asano, Cell. Mol. Life Sci. 66 (2009) 1479;
(b) P. Compain, O.R. Martin, Iminosugars: from synthesis to therapeu-
tic application, ISBN-0-470-03391-3, John Wiley & Son, 2007.
(c) N. Asano, R.J. Nash, R.J. Molyneux, G.W.J. Fleet, Tetrahedron: asym-
metry 11 (2000) 1645;
0.79, MeOH), was prepared by a similar method. All other
physical data was identical to that of 13L.
14. 2-Acetamido-1,4-imino-1,2,4-trideoxy-L-ribitol
(LRBNAc) 12L
(d) A.A. Watson, G.W.J. Fleet, N. Asano, R.J. Molyneux, R.J. Nash, Phy-
tochemistry 56 (2001) 265.
[8] (a) G.W.J. Fleet, L.E. Fellows, P.W. Smith, Tetrahedron 43 (1987)
979;
Palladium black (10.0 mg) was added to a stirred
solution of N-benzyl derivative 13L (40.0 mg, 0.151 mmol)
in 1,4-dioxane (0.5 mL) and water (0.5 mL) at room
temperature under an atmosphere of H2. After 18 h,
analysis by TLC (acetone) showed no residual starting
material (Rf 0.15) and a spot due to the product on the
(b) G.W.J. Fleet, P.W. Smith, R.J. Nash, L.E. Fellows, R.B. Parekh, T.W.
Rademacher, Chem. Lett. (1986) 1051;
(c) H. Boshagen, F.R. Heiker, A.M. Schueller, Carbohydr. Res. 164 (1987)
141.
[9] A.J.Steiner,G.Schitter,A.E.Stutz, T.M.Wrodnigg,C.A.Tarling,S.G.Withers,
D.J. Mahuran, M.B. Tropak, Tetrahedron: asymmetry 20 (2009) 832.
[10] (a) H.C. Dorfmueller, V.S. Borodkin, M. Schimpl, D.M.F. van Aalten,
Biochem. J. 420 (2009) 221;
baseline. The palladium black was filtered off over Celite1
,
(b) B. Shanmugasundaram, A.W. Debowski, R.J. Dennis, G.J. Davies, D.J.
Vocadlo, A. Vasella, Chem. Commun. (2006) 4372;
(c) S. Knapp, D. Fash, M. Abdo, T.J. Emge, P.R. Rablen, Bioorg. Med.
Chem. 17 (2009) 1831.
washing with methanol. The solvents were removed in
vacuo and the remaining residue was purified by ion-
exchange chromatography (Dowex1 50WX8-200) to
[11] T. Aoyama, H. Naganawa, H. Suda, K. Uotani, T. Aoyagi, T. Takeuchi, J.
Antibiot. 45 (1992) 1557.
[12] (a) K. Tatsuta, S. Miura, H. Gunji, Bull. Chem. Soc. Jpn. 70 (1997) 427;
(b) S. Takahashi, H. Terayama, H. Kuzuhara, Tetrahedron 52 (1996)
13315;
(c) K. Tatsuta, S. Miura, Tetrahedron Lett. 36 (1995) 6721;
(d) K. Tatsuta, S. Miura, S. Ohta, H. Gunji, J. Antibiot. 48 (1995) 286.
[13] D. Best, P. Chairatana, A.F.G. Glawar, E. Crabtree, T.D. Butters,
F.X. Wilson, C.Y. Yu, W.B. Wang, Y.M. Jia, I. Adachi, A. Kato,
G.W.J. Fleet, Tetrahedron Lett., 51 (2010) doi 10.1016/j.tet-
let.2010.02.063.
afford LRBNAc 12L (23.3 mg, 88%) as an orange oil; ½a D22
ꢂ
+31.4 (c 0.36, H2O); nmax (Ge) 3289 (O-H and N-H), 1651
(C = O amide band I), 1554 (N-H amide band II) cmꢀ1
;
0
dH(400 MHz, D2O) 1.91 (3H, s, COCH3), 2.75 (1H, dd, J1,1
11.4 Hz, J1,2 8.6 Hz, H-1), 3.07 (1H, a-dt, J4,3 5.0 Hz, J 5.4 Hz,
H-4), 3.19 (1H, dd, J1 ,1 11.4 Hz, J1 ,2 7.0 Hz, H-10), 3.55 (1H,
0
0
0
0
dd, J5,5 11.8 Hz, J5,4 5.8 Hz, H-5), 3.61 (1H, dd, J5 ,5 11.8 Hz,
0
J5 ,4 5.0 Hz, H-50), 3.97 (1H, dd, J3,2 6.0 Hz, J3,4 5.0 Hz, H-3),
[14] R. Andreana, T. Sanders, A. Janczuk, J.I. Warrick, P.G. Wang, Tetrahedron
Lett. 43 (2002) 6525.
[15] H.Q. Li, F. Marcelo, C. Bello, P. Vogel, T.D. Butters, A.P. Rauter, Y.M.
Zhang, M. Sollogoub, Y. Bleriot, Bioorg. Med. Chem. 17 (2009) 5598.
[16] H. Usuki, M. Toyo-oka, H. Kanzaki, T. Okuda, T. Nitoda, Bioorg. Med.
Chem. 17 (2009) 7248.
4.09 (1H, a-dt, J2,1 8.6 Hz, J 6.5 Hz, H-2); dC(100 MHz, D2O)
22.1 (NHCOCH3), 48.0 (C-1), 52.3 (C-2), 61.5 (C-5), 65.8 (C-
4), 71.5 (C-3), 174.7 (C = O); m/z HRMS (ESI+): found
175.1078, C7H15N2O3 [M + H]+ requires 175.1083.
The enantiomeric DRBNAc 12D, oil; ½a D22
ꢀ27.2 (c 0.34,
ꢂ
[17] (a) J. Furukawa, S. Okuda, K. Saito, S.I. Hatanaka, Phytochemistry, 24
(1985) 593;
H2O), was prepared by a similar method. All other physical
(b) R.J. Nash, E.A. Bell, J.M. Williams, Phytochemistry, 24 (1985) 1620;
(c) D.W.C. Jones, R.J. Nash, E.A. Bell, J.M. Williams, Tetrahedron Lett., 26
(1985) 3125.
data was identical to that of DRBNAc 12L.
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