N-Alkyl-, 1-C-Alkyl-, and 5-C-Alkyl-1,5-dideoxy-1,5-imino-(l)-ribitols as Galactosidase Inhibitors

Article abstract of DOI:10.1002/cmdc.201500485

A series of 1,5-dideoxy-1,5-imino-(l)-ribitol (DIR) derivatives carrying alkyl or functionalized alkyl groups were prepared and investigated as glycosidase inhibitors. These compounds were designed as simplified 4-epi-isofagomine (4-epi-IFG) mimics and were expected to behave as selective inhibitors of β-galactosidases. All compounds were indeed found to be highly selective for β-galactosidases versus α-glycosidases, as they generally did not inhibit coffee bean α-galactosidase or other α-glycosidases. Some compounds were also found to be inhibitors of almond β-glucosidase. The N-alkyl DIR derivatives were only modest inhibitors of bovine β-galactosidase, with IC₅₀ values in the 30-700 μm range. Likewise, imino-l-ribitol substituted at the C1 position was found to be a weak inhibitor of this enzyme. In contrast, alkyl substitution at C5 resulted in enhanced β-galactosidase inhibitory activity by a factor of up to 1000, with at least six carbon atoms in the alkyl substituent. Remarkably, the 'pseudo-anomeric' configuration in this series does not appear to play a role. Human lysosomal β-galactosidase from leukocyte lysate was, however, poorly inhibited by all iminoribitol derivatives tested (IC₅₀ values in the 100 μm range), while 4-epi-IFG was a good inhibitor of this enzyme. Two compounds were evaluated as pharmacological chaperones for a GM1-gangliosidosis cell line (R301Q mutation) and were found to enhance the mutant enzyme activity by factors up to 2.7-fold.

Full text of DOI:10.1002/cmdc.201500485

DOI: 10.1002/cmdc.201500485
Full Papers
N-Alkyl-, 1-C-Alkyl-, and 5-C-Alkyl-1,5-dideoxy-1,5-imino-
(l)-ribitols as Galactosidase Inhibitors
Sophie Front,[a] Estelle Gallienne,[a] Julie Charollais-Thoenig,[b] StØphane Demotz,[b] and
Olivier R. Martin*[a]
A series of 1,5-dideoxy-1,5-imino-(l)-ribitol (DIR) derivatives car-
rying alkyl or functionalized alkyl groups were prepared and in-
vestigated as glycosidase inhibitors. These compounds were
designed as simplified 4-epi-isofagomine (4-epi-IFG) mimics
and were expected to behave as selective inhibitors of b-galac-
tosidases. All compounds were indeed found to be highly se-
lective for b-galactosidases versus a-glycosidases, as they gen-
erally did not inhibit coffee bean a-galactosidase or other a-
glycosidases. Some compounds were also found to be inhibi-
tors of almond b-glucosidase. The N-alkyl DIR derivatives were
only modest inhibitors of bovine b-galactosidase, with IC50
values in the 30–700 mm range. Likewise, imino-l-ribitol substi-
tuted at the C1 position was found to be a weak inhibitor of
this enzyme. In contrast, alkyl substitution at C5 resulted in en-
hanced b-galactosidase inhibitory activity by a factor of up to
1000, with at least six carbon atoms in the alkyl substituent.
Remarkably, the ‘pseudo-anomeric’ configuration in this series
does not appear to play a role. Human lysosomal b-galactosi-
dase from leukocyte lysate was, however, poorly inhibited by
all iminoribitol derivatives tested (IC50 values in the 100 mm
range), while 4-epi-IFG was a good inhibitor of this enzyme.
Two compounds were evaluated as pharmacological chaper-
ones for a GM1-gangliosidosis cell line (R301Q mutation) and
were found to enhance the mutant enzyme activity by factors
up to 2.7-fold.
Introduction
b-Galactosidase-linked lysosomal storage diseases (LSD) such
as GM1-gangliosidosis, Morquio disease type B, and Krabbe
disease remain major medical challenges, as there is no treat-
ment available.[1] Significant advances have been achieved in
the handling of certain LSDs using replacement enzymes
(enzyme replacement therapy, ERT);[2] however, this approach
is currently limited to non-neuronopathic forms of these dis-
eases. The use of small organic molecules as a mode of treat-
ment has shown some success in the form of substrate reduc-
tion therapy (SRT):[3] Miglustat (N-butyl-1-deoxynojirimycin, N-
Bu-DNJ, an inhibitor of glucosylceramide synthase) was found
to significantly improve patient conditions and to decrease
symptoms when administered to type 1 Gaucher disease pa-
tients.[4] The use of organic compounds that act as molecular
chaperones to stabilize mutant enzymes during their biosyn-
thesis and transport to the lysosome (i.e., pharmacological
chaperone therapy, PCT) is an approach in the treatment of
LSD and mucopolysaccharidoses (MPS) which is gaining in-
creasing importance and recognition.[5] Such chaperones,
which are usually potent enzyme inhibitors, were shown to
significantly increase the activity of certain mutant enzymes,
provided the mutation does not completely abolish enzymatic
activity. A significant advantage of small molecules over ERT or
treatment with other biomolecular drugs is their potential ca-
pacity to cross the blood–brain barrier, as has been shown for
certain iminosugar derivatives.[6] 1-Deoxygalactonojirimycin
(DGJ), an inhibitor of lysosomal a-galactosidase, has successful-
ly completed phase III clinical evaluations for Fabry disease,[7]
establishing the adequacy of this approach for the therapeutic
management of LSD. This situation has prompted various ini-
tiatives, and several other iminosugars are currently in preclini-
cal development.
Following our initial work on pharmacological chaperones
for Gaucher disease,[8] we became interested in the application
of PCT toward b-galactosidase-linked LSDs.[9] As regards GM1-
gangliosidosis, Suzuki et al. developed the carbasugar N-octyl-
4-epi-b-valienamine (NOEV) as a potential treatment of this dis-
ease.[10] This compound was shown to enhance the activity of
a number of mutated forms of lysosomal b-galactosidase.[11]
Preclinical development of this compound was hampered by
difficulties in its synthesis. More readily available, bicyclic DGJ
derivatives were reported by Garcia-Fernandez and co-work-
ers,[12] with interesting activities on a large number of GM1-
gangliosidosis-linked mutations. DGJ derivatives exhibiting
potent b-galactosidase chaperone activity were also reported
by Paschke, Wrodnigg and colleagues, but these compounds
have not been developed past the discovery phase.[13]
[a] S. Front, Dr. E. Gallienne, Prof. O. R. Martin
Institut de Chimie Organique et Analytique (ICOA), UMR 7311
UniversitØ d’OrlØans, Centre National de la Recherche Scientifique (CNRS)
Rue de Chartres, 45067 OrlØans (France)
E-mail: Olivier.Martin@univ-orleans.fr
[b] Dr. J. Charollais-Thoenig, Dr. S. Demotz
Dorphan SA, EPFL Innovation Park, 1015 Lausanne (Switzerland)
We have recently shown that 1-C-alkyl DGJ derivatives
(Figure 1) are quite weak inhibitors of b-galactosidases, al-
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500485.
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Figure 1. Iminosugars as galactoside mimics.
though they are potent inhibitors of a-galactosidases, includ-
ing human lysosomal a-galactosidase.[9] In agreement with
Heightman and Vasella’s analysis of glycosidase inhibition by
iminosugars,[14] we considered that selective inhibition of b-
galactosidase might arise more effectively from structures in
which the basic nitrogen atom occupies the anomeric position
in the corresponding galactoside (isofagomine series): 4-epi-
isofagomine (4-epi-IFG, Figure 1) is indeed a very potent gal-
actosidase inhibitor.[15] We have shown that this compound is
also a potent inhibitor of galactocerebrosidase (GALC), as sub-
sequently confirmed by Deane and co-workers,[16] and of lyso-
somal b-galactosidase.[9] To reach a diversity of galactoside
mimics more readily, we decided to investigate iminoribitol
derivatives as simplified 4-epi-IFG mimics, and to prepare
a series of N-alkyl as well as 1-C-alkyl and 5-C-alkyl imino-l-ri-
bitols (=5-C-alkyl and 1-C-alkyl imino-d-ribitols). These com-
pounds have the same configuration as a galactoside at the
C3, C4, and C5 positions, but contain an OH group at C5 in-
stead of CH2OH. The new iminoribitol derivatives were evalu-
ated for their efficiency and selectivity as b-galactosidase in-
hibitors, and compared with 4-epi-IFG and its N-nonyl deriva-
tive, NN-4-epi-IFG.
Scheme 1. Synthesis of N-alkyl imino-ribitol and -arabinitol derivatives. Re-
agents and conditions: a) For details, see Ref. [21a], 33%; b) Dess–Martin peri-
odinane, CH2Cl2, quant.; c) R-NH2 (R=Bn, C6H13, C9H19, (CH2)6NHAc), AcOH,
10% Pd/C, MeOH, 25–91%; d) Dowex 50WX8 (H+), 1,4-dioxane/H2O (9:1),
59–91%.
(8), N-nonyl (10), and N-(6-acetamidohexyl) (14) derivatives of
DIR were prepared using the corresponding alkylamines. A
very small amount of N-methyl DIR 6 was formed during the
synthesis of 4 which involved hydrogenation in methanol.[24]
We also isolated a small amount of the C4 epimer (l-arabino
configuration) of 9, compound 11, probably resulting from the
epimerization of the aldehyde d-2 or the corresponding imini-
um salt during the reductive amination reaction. This inter-
mediate was converted into N-nonyl imino-l-arabinitol 12
(Scheme 1).
Very few 1/5-C-alkylated iminoribitols have been reported so
far. Since the pioneering work of Nicotra and co-workers,[25]
a few iminoribitol derivatives carrying a C-linked functionalized
alkyl group have been described.[26] A significant investigation
with medicinal chemistry implications was published recently
by Siriwardena et al.[27] A number of imino-d-ribitol C-glyco-
sides were prepared as galactoside mimics by way of cycload-
ditions between various alkenes and a d-ribose-derived cyclic
nitrone. The resulting compounds were found to be modest
glycosidase inhibitors with little selectivity between a- and b-
galactosidases, as well as between different hexose configura-
tions.
Results and Discussion
Synthesis of iminoribitol derivatives
Iminoribitol 4 is a known compound: it has been isolated from
a plant used in folk medicine[17] and was subsequently pre-
pared by several research groups semisynthetically, by starting
from sugars (mostly d-ribose derivatives),[18] or by total synthe-
sis.[19] We selected an approach based on a double reductive
amination,[20] starting from aldehydo-d-ribodialdose derivative
2, a known aldehyde.[21] The protocol we used is similar to that
recently reported by Cardona and colleagues[22] from the d-
lyxo epimer of d-2. The reaction of d-2 in the presence of an
amine under hydrogenation conditions followed by hydrolysis
of the isopropylidene group gave 1,5-dideoxy-1,5-imino-ribitol
(4, DIR, using benzylamine) and N-alkylated derivatives in good
yields. The N-propyl[23] and N-butyl derivatives[18a] are the only
N-alkylated DIRs that have been reported so far. The N-hexyl
Our approach toward 1- and 5-C-alkylated iminoribitols fol-
lowed the methodology we developed to reach similar com-
pounds in the iminoxylitol and imino-l-arabinitol series.[28] Key
intermediates were the tert-butanesulfinyl imines derived from
ribo-pentodialdofuranoses d-15 and l-15, and chain extension
was performed by the addition of organomagnesium reagents
(Schemes 2 and 3). The reactions were performed on the imine
prepared from racemic tert-butanesulfinamide, thereby allowing
the simultaneous preparation of both stereoisomers at C5 and
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Scheme 2. Synthesis of 1-C-hexyl imino-l-ribitols. Reagents and conditions:
a) (rac)-tBuS(O)NH2, CuSO4, CH2Cl2, 69%; b) C6H13MgBr, PhMe, À788C!RT,
83%; c) HCl, MeOH, RT, 17: 61%, 20: quant.; d) H2, 10% Pd/C, 1,4-dioxane/
H2O/AcOH (8:1:1), (1S): 75%, (1R): 82%; e) Dowex 50WX8 (H+), 1,4-dioxane/
H2O (2:1), 18: 79%, 21: 45%.
hence both pseudo-anomers of the glycoside mimics.1 The con-
figuration of the newly created stereocenters was determined
after cyclization to piperidines 18, 21, 24, and 27 through de-
tailed NMR analysis. Final steps involved cleavage of the tert-bu-
tanesulfinyl group by methanolysis, intramolecular reductive
amination upon hydrogenolysis of the benzyl glycoside, and hy-
drolysis of the protective group. A single example of 1-C-alkyl
imino-l-ribitol was prepared from d-15, namely the C-hexyl de-
rivatives in pseudo-b (18) and pseudo-a (21) configurations
(Scheme 2). In the enantiomeric series, 5-C-alkylated imino-l-ribi-
tols carrying a C4-, C6-, or C9-alkyl group as well as a 2-phenyl-
ethyl or 2-(4-trifluoromethylphenyl)ethyl group were prepared
in most cases in both pseudo-anomeric configurations at posi-
tion C5 (Scheme 3). The 5-C-hexyl derivatives 24a and 27a are
thus enantiomers of 21 and 18, respectively.
Scheme 3. Synthesis of 5-C-alkyl imino-l-ribitols. Reagents and conditions:
a) For details, see Ref. [21a], 40%; b) Dess–Martin periodinane, CH2Cl2,
quant.; c) (rac)-tBuS(O)NH2, CuSO4, CH2Cl2, 53%; d) RMgBr, PhMe, À788C!
RT, 45–84%; e) HCl, MeOH, RT, 40–57%; f) H2, 10% Pd/C, 1,4-dioxane/H2O/
AcOH (10:6:1); g) Dowex 50WX8 (H+), 1,4-dioxane/H2O (3:1), 30–84% (two
steps).
The following observations were made: 1) All compounds
are highly selective for b-galactosidases over a-glycosidases, as
they generally do not inhibit the coffee bean a-galactosidase
(with the exception of NN-4-epi-IFG), although iminosugars in
the DGJ series are very powerful inhibitors of this enzyme, and
have little effect on a-glucosidase. 2) Some compounds are
also inhibitors of almond b-glucosidase (down to the micromo-
lar range for the most active ones), an enzyme that is known
to have b-galactosidase activity as well. 3) The N-alkyl DIR de-
rivatives are only modest inhibitors of bovine liver b-galactosi-
dase, with IC50 values in the 30–700 mm range. Likewise, imino-
l-ribitols substituted at C1 were found to be weak inhibitors of
bovine liver b-galactosidase. In contrast, alkyl substitution at
C5 results in enhancement of b-galactosidase inhibitory activity
by a factor of up to 1000, when the chain contains at least six
carbon atoms. Remarkably, the pseudo-anomeric configuration
in this series does not seem to play a role, as very similar data
were observed for both configurations at C5. Altogether, these
results, summarized in Figure 2, indicate that the b-galactosi-
dase inhibitory activity of hexyl-substituted iminoribitol deriva-
tives is greatest when the chain is at C5, much weaker if the
chain is at N or at C1. 4) Lysosomal b-galactosidase from
human leukocyte lysate was poorly inhibited by all iminoribitol
derivatives tested (IC50 values in the 100 mm range). In contrast,
Biological investigations
All new compounds were evaluated as inhibitors of bovine b-
galactosidase, as well as inhibitors of other b-galactosidases in-
cluding the human lysosomal enzyme. Assays were also per-
formed on an a-galactosidase (coffee bean), a b-glucosidase
(almond), and an a-glucosidase (S. cerevisiae) to determine se-
lectivity. All results are listed in Table 1. 4-Epi-IFG and its N-
nonyl derivative (NN-4-epi-IFG) were also included in the
assays for comparison.
1
In this series, stereocontrol appeared to be largely due to the chiral auxiliary.
It follows that the reaction with one or the other of the stereoisomeric tert-
butanesulfinylimines gives predominantly one or the other configuration of
the addition product at C5.
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Table 1. IC50 values (mm) for 1,5-dideoxy-1,5-imino-l-ribitol (DIR) derivatives as inhibitors of glycosidases.[a]
Compd Type
b-gal’ase
b-gal’ase b-gal’ase
b-gal’ase
b-gal’ase
a-gal’ase
b-glu’ase a-glu’ase
bovine liver
E. coli A. oryzae bovine testis human leukocyte lysate green coffee beans almonds S. cerevisiae
4-epi-IFG
NN-4-epi-IFG
4
8
10
14
18
21
24a
27a
24b
27b
24d
24c
27c
27e
8
20
300
700
40
400
30
400
0.8
1
0.3
0.3
80
0.3
0.5
2
0.3
40
10
600
2
0.1
–
–
–
–
–
–
70
–
–
–
–
–
–
–
0.8
50
–
0.4
10
300
–
–
–
500
20
>1000
>1000
–
2
200
100
>1000
–
500
–
–
–
DIR
N-C6-DIR
N-C9-DIR
–
>1000
>1000
1000
–
>800
–
–
–
–
20
6
N-6AcNHC6-DIR
b-1C6-DIR
a-1C6-DIR
a-5C6-DIR
b-5C6-DIR
a-5PhEt-DIR
b-5PhEt-DIR
a-5C4-DIR
a-5C9-DIR
b-5C9-DIR
b-5-CF3PhEt-DIR
–
–
–
–
>800
–
–
–
–
–
–
350
–
–
–
–
–
–
–
60
60
–
–
–
–
–
–
150
70
90
100
500
40
70
–
>1000
>1000
–
>1000
>1000
>1000
–
>1000
>1000
–
>1000
>1000
>1000
–
–
20
500
100
–
>1000
2
>1000
[a] Gal’ase=galactosidase; glu’ase=glucosidase; “–” denotes not determined; each value is the mean of at least two experiments that were run in dupli-
cate. The variation of the absorbance and fluorescence values exploited for the determination of the IC50 is less than 20%.
Table 2. Enhancement of residual b-galactosidase activity by 24a in
GM1-gangliosidosis.
4-epi-IFG was found to be
a potent inhibitor of human b-gal-
actosidase.
Compd
Conc. [mm]
EE[a]
We next evaluated whether the
5-C-hexyl iminoribitol derivative
24a, despite its modest inhibitory
activity toward human lysosomal
b-galactosidase, is endowed with
pharmacological chaperone activi-
ty in cells from patients of GM1-
gangliosidosis. Remarkably, it was
GM03251
GM02439
4-epi-IFG
4-epi-IFG
4-epi-IFG
0
1
10
1.0
1.1
1.1
1.0
1.5
2.7
Figure 2. Structure–activity re-
lationships of iminoribitol: the
effect of the position of
a hexyl chain on the IC50
value for inhibition of bovine
liver b-galactosidase.
24a
24a
24a
0
1
10
1.0
1.0
1.1
1.0
1.1
2.1
[a] EE=Enzyme activity fold enhancement.
observed that residual b-galactosidase activity in GM02439 fi-
broblasts cultured for five days in the presence of 24a (10 mm)
was increased by twofold. Recovery of b-galactosidase activity
was most readily achieved with 4-epi-IFG, likely reflecting that
this compound has greater affinity than 24a for the human
enzyme. In contrast, no recovery of b-galactosidase activity
was observed with GM03251 cells (W273L/W509C), establishing
the likely b-galactosidase mutant specificity of the chaperone
effect (Table 2). It was recently reported that GM02439 cells
bear the R201C mutation, which is known for its chaperone
sensitivity.[29] For comparison, a fourfold enhancement in the
enzymatic activity of R201C b-galactosidase was obtained with
NOEV at a concentration of 0.2 mm.
increases the activity of the resulting iminoglycolipid on
bovine liver b-galactosidase, provided that the chain is located
at the C5 position, which indicates a highly organized recogni-
tion of the molecule by the enzyme. Such an effect is not ob-
served if the alkyl group is at C1 or on the nitrogen atom, nor
is it observed for the human lysosomal enzyme, for which the
most active compound remains 4-epi-IFG. The 5-C-hexyl DIR
derivative was found to have some pharmacological chaper-
one activity on cells from a GM1-gangliosidosis patient, an un-
expected observation considering the modest inhibitory activi-
ty of this compound toward human lysosomal b-galactosidase.
Substituents at the C5 position might therefore play a critical
role in controlling the activity of b-galactosidase chaperones in
the 1-N-iminosugar series, and further investigations are in
progress in our laboratories to access such compounds.
Conclusions
A series of new N-alkyl as well as 1- and 5-C-alkyl derivatives of
1,5-dideoxy-1,5-imino-l-ribitol (DIR) were prepared as simpli-
fied galactoside mimics in the 1N iminosugar series and evalu-
ated as inhibitors of different glycosidases including five b-gal-
actosidases, in comparison with 4-epi-IFG and its N-nonyl de-
rivative. The results of these investigations show that all com-
pounds are highly selective as b-glycosidase inhibitors, with
little or no action on a-galactosidases and a-glucosidases. The
addition of a lipophilic group in the DIR scaffold significantly
Experimental Section
Chemical syntheses
Materials and methods: All reactions requiring anhydrous condi-
tions were carried out with oven-dried glassware under an atmos-
phere of dry Ar. CH2Cl2 and Et2O were distilled from P2O5 and CaH2,
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respectively. All reagent-grade chemicals, anhydrous MeOH, and
toluene were obtained from commercial suppliers and were used
as received. High-resolution mass spectra were recorded on
3.67 (ddd, 2H, H2, H4, J=3.1, 4.1, 9.8 Hz), 3.85 ppm (brs, 1H, H3);
13C NMR (101 MHz, CD3OD): d=14.36 (CH3), 23.67 (CH2), 27.59
(CH2b), 28.32, 32.90 (CH2), 54.60 (C1, C5), 59.22 (CH2a), 69.41 (C2,
C4), 71.99 ppm (C3); HRMS (ESI): m/z [M+H]+ calcd for C11H24NO3:
218.175070, found: 218.175317.
1
a Bruker Q-TOF MaXis spectrometer. H and 13C NMR spectra were
recorded on Bruker Avance II (250 MHz) or Bruker Avance III HD
nanobay (400 MHz) spectrometers. Chemical shifts are given in
ppm and are referenced to the residual solvent signal or to TMS as
internal standard. Carbon multiplicities were assigned by distor-
tionless enhancement by polarization transfer (DEPT) experiments.
1H and 13C NMR signals were attributed on the basis of H–H and
C–H correlations. Spectral splitting patterns are designated as fol-
lows: s=singlet, d=doublet, t=triplet, q=quadruplet, m=mul-
tiplet, br=broad. Specific rotations were measured in a 1-dm cell
with a PerkinElmer 341 polarimeter. Melting points were deter-
mined in capillary tubes with a Büchi apparatus and are uncorrect-
ed. Analytical thin-layer chromatography was performed using
silica gel 60 F254 pre-coated plates (Merck) with visualization by UV
light and ceric sulfate/ammonium
Synthesis of 1-C-alkyl imino-l-ribitols
N-tert-Butanesulfinylimine of benzyl 2,3-O-isopropylidene-alde-
hydo-b-d-ribo-pentodialdo-1,4-furanoside (d-15): To a solution of
aldehyde d-2 (347 mg, 1.25 mmol) in dry CH2Cl2 (13 mL) under Ar
were added anhydrous CuSO4 (995 mg, 6.23 mmol, 5 equiv) and
racemic tert-butanesulfinamide (176 mg, 1.45 mmol, 1.2 equiv). The
mixture was stirred for 18 h at room temperature, and then the
copper salt was removed by filtration. The filtrate was concentrat-
ed under vacuum, and the residue was purified by silica gel
column chromatography (PE/EtOAc 4:1) to give d-15 (328 mg,
69%) as a 1:1 mixture of two diastereomers and as a white solid;
1H NMR (250 MHz, CDCl3): d=1.14 (s, 4.5H, CH3 tBu), 1.21 (s, 4.5H,
CH3 tBu), 1.33 (s, 3H, CH3 iPr), 1.49 (s, 1.5H, CH3 iPr), 1.50 (s, 1.5H,
CH3 iPr), 4.48 (d, 0.5H, CH2Ph, J=8.4 Hz), 4.53 (d, 0.5H, CH2Ph, J=
8.6 Hz), 4.62 (dd, 0.5H, H3, J=0.5, 5.8 Hz), 4.66 (dd, 0.5H, H3, J=
0.8, 6.0 Hz), 4.71 (d, 0.5H, CH2Ph, J=8.6 Hz), 4.76 (d, 0.5H, CH2Ph,
J=8.4 Hz), 4.91–4.95 (m, 1H, H4), 5.16 (d, 0.5H, H2, J=5.8 Hz),
5.21–5.24 (m, 0.5H, H2), 5.22 (s, 0.5H, H1), 5.25 (s, 0.5H, H1), 7.27–
7.36 (m, 5H, Har), 8.10 (d, 0.5H, H5, J=2.3 Hz), 8.12 ppm (d, 0.5H,
H5, J=2.5 Hz); 13C NMR (101 MHz, CDCl3): d=22.48, 22.53, 25.06,
25.17, 26.40, 26.47 (CH3), 57.22, 57.49 (C, tBu), 69.61, 69.79 (CH2Ph),
82.76, 82.78 (C2), 84.93, 84.97 (C3), 86.99, 87.01 (C4), 106.92, 107.20
(C1), 112.74, 112.85 (C, iPr), 128.17–128.66 (CHar), 136.45, 136.54
(Car), 168.21, 168.26 ppm (C5); HRMS (ESI): m/z [M+H]+ calcd for
C19H28NO5S: 382.168270, found: 382.168682.
molybdate solution (0.5 g/0.24 g in
500 mL 1m aqueous H2SO4). Flash
chromatography was performed with
silica gel 60 (40–63 mm).
Numbering scheme for NMR data:
Alkyl chain series labeled a, b, c…
Synthesis of N-alkyl iminoribitols
General procedure for reductive amination: To a solution of alde-
hyde d-2[21] in MeOH (0.1m) was added the alkyl amine (1.2 equiv),
AcOH (1 equiv) and 10% Pd/C (60 mgmmolÀ1). The mixture was
stirred for 48–72 h under an H2 atmosphere at room temperature.
The catalyst was removed by filtration through a membrane and
washed with MeOH. The filtrate was concentrated under vacuum,
and the residue was purified by silica gel column chromatography.
Compounds d-15-SS and d-15-SR were prepared according to the
same conditions with each epimer of the chiral tert-butanesulfin-
amide.
N-Hexyl-2,3-O-isopropylidene-1,5-dideoxy-1,5-imino-d-ribitol (7):
Compound 7 (90 mg, 25%) was obtained from d-2 (395 mg,
1.42 mmol) and hexylamine (225 mL, 1.70 mmol, 1.2 equiv). Purifica-
tion was performed using petroleum ether (PE)/EtOAc 3:2 to give
7 as a yellow syrup: 1H NMR (400 MHz, CDCl3): d=0.85–0.91 (m,
3H, CH3), 1.24–1.35 (m, 6H, 3CH2), 1.38 (s, 3H, CH3 iPr), 1.41–1.52
(m, 2H, 2Hb), 1.56 (s, 3H, CH3 iPr), 2.33–2.46 (m, 4H, H1A, H5A,
2Ha), 2.60 (ddd, 1H, H1B, J=0.8, 4.0, 11.2 Hz), 2.71 (ddd, 1H, H5B,
J=0.8, 5.4, 12.0 Hz), 3.88–3.92 (m, 1H, H2), 4.21 (t, 1H, H3, J=
4.6 Hz), 4.23–4.28 ppm (m, 1H, H4); 13C NMR (101 MHz, CDCl3): d=
14.15 (CH3), 22.70 (CH2), 26.39 (CH3 iPr), 26.85 (CH2b), 27.16 (CH2),
27.50 (CH3 iPr), 31.82 (CH2), 54.93 (C5), 55.47 (C1), 57.47 (CH2a),
66.63 (C2), 73.02 (C4), 74.75 (C3), 109.45 ppm (Cq); HRMS (ESI): m/z
[M+H]+ calcd for C14H28NO3: 258.206370, found: 258.206624.
Benzyl (5S,SS)-5-N-tert-butanesulfinylamido-5-deoxy-5-C-hexyl-
2,3-O-isopropylidene-b-d-ribofuranoside
(16)
and
benzyl
(5R,SRS)-5-N-tert-butanesulfinylamido-5-deoxy-5-C-hexyl-2,3-O-
isopropylidene-b-d-ribofuranoside (19): To a solution of imine d-
15 (193 mg, 0.506 mmol) in toluene (5 mL) at À788C under Ar was
added a commercial solution (2m in Et2O) of hexylmagnesium bro-
mide (1.26 mL, 2.52 mmol, 5 equiv). The mixture was warmed to
room temperature and stirred for 18 h. The reaction was quenched
at 08C by the addition of a saturated NH4Cl solution (5 mL), and
the aqueous phase was extracted with Et2O (3”15 mL). The com-
bined organic phases were dried over MgSO4 and concentrated
under vacuum. The residue was purified by silica gel column chro-
matography (PE/EtOAc 4:1) to give 16 (36 mg, 15%) as a single S*-
stereoisomer and as a yellow syrup, and 19 (162 mg, 68%) as an
inseparable 3:2 mixture of two S*-stereoisomers [19 (5R,SS)/19
(5R,SR)] and as a white solid.
General procedure for isopropylidene deprotection: To a solution
of protected iminoribitol in 1,4-dioxane/H2O (9:1, 0.03m) was
added Dowex 50W X8 ion-exchange resin (H+ form, 6 mLmmolÀ1),
previously washed with 1,4-dioxane/H2O (9:1). The mixture was
stirred for 4–5 h at room temperature. The resin was then poured
onto a column, washed with 1,4-dioxane/H2O (9:1), then with H2O.
The product was eluted from the resin using aqueous 0.5n
NH4OH. Fractions containing the iminoribitol derivatives were com-
bined and concentrated under vacuum.
Compound 16: 1H NMR (400 MHz, CDCl3): d=0.85–0.94 (m, 3H,
CH3 hex), 0.97 (s, 9H, CH3 tBu), 1.24–1.45 (m, 8H, 4CH2), 1.32 (s, 3H,
CH3 iPr), 1.48 (s, 3H, CH3 iPr), 1.48–1.63 (m, 2H, 2Ha), 3.18–3.28 (m,
1H, H5), 4.46 (d, 1H, H4, J=2.8 Hz), 4.56 (d, 1H, CH2Ph, J=11.2 Hz),
4.71 (d, 1H, CH2Ph, J=11.2 Hz), 4.74 (d, 1H, H3, J=6.2 Hz), 4.90 (d,
1H, NH, J=8.8 Hz), 5.12 (d, 1H, H2, J=6.2 Hz), 5.18 (s, 1H, H1),
7.29–7.38 ppm (m, 5H, Har); 13C NMR (101 MHz, CDCl3): d=14.22
(CH3 hex), 22.72 (CH2), 22.96 (CH3 tBu), 24.87 (CH3 iPr), 25.99 (CH2),
26.50 (CH3 iPr), 29.22, 31.88 (CH2), 35.00 (CH2a), 56.16 (C, tBu), 59.09
(C5), 70.86 (CH2Ph), 83.36 (C2), 86.01 (C3), 90.14 (C4), 109.32 (C1),
112.10 (C, iPr), 128.45, 128.71, 129.01 (CHar), 136.38 ppm (Car);
N-Hexyl-1,5-dideoxy-1,5-iminoribitol (8): Compound 8 (66 mg,
85%) was obtained from 7 (92 mg, 0.357 mmol) as a yellow solid:
1
mp: 89–918C; H NMR (250 MHz, CD3OD): d=0.91 (t, 3H, CH3, J=
6.5 Hz), 1.25–1.41 (m, 6H, 3CH2), 1.45–1.61 (m, 2H, 2Hb), 2.25–2.47
(m, 4H, H1A, H5A, 2Ha), 2.58 (dd, 2H, H1B, H5B, J=4.1, 10.5 Hz),
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32.92 (CH2), 34.45 (CH2a), 54.72 (C5), 71.19 (CH2Ph), 83.26 (C3),
86.98 (C2), 92.68 (C4), 109.24 (C1), 113.59 (C, iPr), 128.92, 129.03,
129.51 (CHar), 138.80 ppm (Car); HRMS (ESI): m/z [M+H]+ calcd for
C21H34NO4: 364.248235, found: 364.248379.
Benzyl
(5S)-5-amino-5-deoxy-5-C-hexyl-b-d-ribofuranoside:
1H NMR (250 MHz, CD3OD): d=0.84–0.98 (m, 3H, CH3), 1.25–1.44
(m, 8H, 4CH2), 1.44–1.57 (m, 1H, HaA), 1.57–1.74 (m, 1H, HaB), 2.75
(dt, 1H, H5, J=4.2, 7.0, 7.0 Hz), 3.74 (t, 1H, H4, J=7.0 Hz), 3.95 (d,
1H, H2, J=4.8 Hz), 4.14 (dd, 1H, H3, J=4.8, 7.0 Hz), 4.53 (d, 1H,
CH2Ph, J=11.9 Hz), 4.73 (d, 1H, CH2Ph, J=11.9 Hz), 4.96 (s, 1H, H1),
7.23–7.39 ppm (m, 5H, Har); HRMS (ESI): m/z [M+H]+ calcd for
C18H30NO4: 324.216935, found: 324.217175.
HRMS (ESI): m/z [M+H]+ calcd for C25H42NO5S: 468.277821, found:
468.277456.
1
Compound 19 (5R,SS): H NMR (400 MHz, CDCl3): d=0.79–0.92 (m,
(1S)-1-C-Hexyl-1,5-dideoxy-1,5-imino-l-ribitol (18): Compound 17
(17 mg, 0.047 mmol) was dissolved in 1,4-dioxane/H2O/AcOH
(4:0.5:0.5 mL) and 10% Pd/C was added (40 mg). The mixture was
stirred for 12 h under an H2 atmosphere at room temperature. The
catalyst was removed by filtration through a membrane and
washed with 1,4-dioxane/H2O/AcOH. The filtrate was concentrated
under vacuum, and the residue was purified by silica gel column
chromatography (CH2Cl2/acetone 2:1) to afford crude (1S)-1-C-
3H, CH3 hex), 1.10–1.41 (m, 20H, CH3 tBu, CH3 iPr, 4CH2), 1.47 (s,
3H, CH3 iPr), 1.48–1.68 (m, 1H, HaA), 1.92–2.02 (m, 1H, HaB), 3.25–
3.35 (m, 2H, H5, NH), 3.94 (d, 1H, H4, J=8.8 Hz), 4.50 (d, 1H,
CH2Ph, J=11.6 Hz), 4.67 (d, 1H, H2, J=6.0 Hz), 4.72 (d, 1H, CH2Ph,
J=11.6 Hz), 4.82 (dd, 1H, H3, J=0.8, 6.0 Hz), 5.16 (s, 1H, H1), 7.27–
7.37 ppm (m, 5H, Har); 13C NMR (101 MHz, CDCl3): d=14.18 (CH3
hex), 22.72 (CH2), 22.89 (CH3 tBu), 24.70 (CH2), 24.86 (CH3 iPr), 26.50
(CH3 iPr), 29.48 (CH2), 31.81 (CH2a), 56.55 (C, tBu), 59.44 (C5), 70.14
(CH2Ph), 82.08 (C3), 85.24 (C2), 89.94 (C4), 107.92 (C1), 112.31 (C,
iPr), 127.98–128.62 (CHar), 136.96 ppm (Car); HRMS (ESI): m/z [M+
H]+ calcd for C25H42NO5S: 468.277821, found: 468.277870.
hexyl-3,4-O-isopropylidene-1,5-dideoxy-1,5-imino-l-ribitol
(9 mg,
75%). To a solution of this compound in 1,4-dioxane/H2O (2:1;
3 mL) was added Dowex 50W X8 ion-exchange resin (H+ form,
1 mL), previously washed with 1,4-dioxane/H2O (2:1). The mixture
was stirred for 18 h at room temperature. The resin was then
poured onto a column, washed with 1,4-dioxane/H2O (2:1, 20 mL)
then with H2O (20 mL). The product was eluted from the resin
using aqueous 0.5n NH4OH (50 mL). Fractions containing the de-
sired compound were combined and concentrated under vacuum
1
Compound 19 (5R,SR): H NMR (400 MHz, CDCl3): d=0.79–0.92 (m,
3H, CH3 hex), 1.10–1.41 (m, 20H, CH3 tBu, CH3 iPr, 4CH2), 1.47 (s,
3H, CH3 iPr), 1.48–1.68 (m, 1H, HaA), 1.68–1.83 (m, 1H, HaB), 3.42–
3.50 (m, 1H, H5), 4.07 (d, 1H, NH, J=3.6 Hz), 4.30 (dd, 1H, H4, J=
1.2, 5.6 Hz), 4.55 (d, 1H, CH2Ph, J=12.2 Hz), 4.63 (d, 1H, H2, J=
6.0 Hz), 4.76 (d, 1H, CH2Ph, J=12.2 Hz), 4.97 (dd, 1H, H3, J=1.2,
6.0 Hz), 5.16 (s, 1H, H1), 7.27–7.37 ppm (m, 5H, Har); 13C NMR
(101 MHz, CDCl3): d=14.15 (CH3 hex), 22.66 (CH2), 22.84 (CH3 tBu),
25.11 (CH3 iPr), 25.50 (CH2), 26.66 (CH3 iPr), 31.79 (CH2), 32.86
(CH2a), 55.97 (C, tBu), 56.64 (C5), 69.77 (CH2Ph), 80.31 (C3), 85.87
(C2), 89.94 (C4), 107.57 (C1), 112.58 (C, iPr), 127.98–128.62 (CHar),
136.70 ppm (Car).
to give 18 (6 mg, 79%) as a yellow syrup: ½aŠ20 = +37.5 (c=1.00,
D
MeOH); 1H NMR (400 MHz, CD3OD): d=0.86–0.94 (m, 3H, CH3),
1.26–1.41 (m, 8H, 4CH2), 1.41–1.62 (m, 2H, 2Ha), 2.45 (ddd, 1H, H1,
J=1.7, 6.1, 7.9 Hz), 2.69 (dd, 1H, H5A, J=1.6, 14.0 Hz), 3.02 (dd,
1H, H5B, J=2.6, 14.0 Hz), 3.46 (t, 1H, H3, J=3.1 Hz), 3.71–3.74 (m,
1H, H2), 3.78–3.81 ppm (m, 1H, H4); 13C NMR (63 MHz, CD3OD+
1 drop of D2O): d=14.43 (CH3), 23.68, 26.96, 30.58 (CH2), 32.67
(CH2a), 32.96 (CH2), 51.60 (C5), 59.99 (C1), 71.02 (C4), 71.34 (C3),
72.57 ppm (C2); HRMS (ESI): m/z [M+H]+ calcd for C11H24NO3:
218.175070, found: 218.175636.
Note: The configuration assignments were established according
to the reactions with d-15-SR and d-15-SS, respectively. The diaste-
reoselectivities are 100% from d-15-SR (5R configuration) and 70%
from d-15-SS (5S configuration).
Benzyl (5R)-5-amino-5-deoxy-5-C-hexyl-2,3-O-isopropylidene-b-
d-ribofuranoside (20): Acetyl chloride (70 mL, 0.984 mmol,
2.9 equiv) was added to anhydrous MeOH (23 mL) under Ar. After
20 min of stirring, this solution was added to compound 19
(160 mg, 0.342 mmol) under Ar, and the reaction was stirred for
2 h 30 min at room temperature. The mixture was neutralized
using a basic ion-exchange resin (IR400, OHÀ form). The resin was
removed by filtration, and the filtrate was concentrated under
vacuum to give crude 20 (125 mg, quant.), which was used in the
next step without purification. A sample of 20 was purified by
silica gel column chromatography (CH2Cl2/MeOH 14:1) for analyti-
Benzyl (5S)-5-amino-5-deoxy-5-C-hexyl-2,3-O-isopropylidene-b-
d-ribofuranoside (17): Acetyl chloride (16 mL, 0.225 mmol, 3 equiv)
was added to anhydrous MeOH (5 mL) under Ar. After 20 min of
stirring, this solution was added to compound 16 (36 mg,
0.077 mmol) under Ar, and the reaction was stirred for 3 h 40 min
at room temperature. The mixture was neutralized using a basic
ion-exchange resin (IR400, OHÀ form). The resin was removed by
filtration, and the filtrate was concentrated under vacuum. The res-
idue was purified by silica gel column chromatography (CH2Cl2/
MeOH 12:1) to give 17 (17 mg, 61%) as a yellow syrup and a signifi-
cant amount of benzyl (5S)-5-amino-5-deoxy-5-C-hexyl-b-d-ribofur-
anoside (10 mg, 40%).
cal purposes to give a white amorphous solid: ½aŠ20 =À57 (c=
D
1
1.00, MeOH); H NMR (400 MHz, CDCl3): d=0.81–0.90 (m, 3H, CH3
hex), 1.23–1.35 (m, 8H, 4CH2), 1.32 (s, 3H, CH3 iPr), 1.42–1.56 (m,
1H, HaA), 1.47 (s, 3H, CH3 iPr), 1.67–1.82 (m, 1H, HaB), 3.00 (dt, 1H,
H5, J=4.5, 7.1, 7.1 Hz), 4.15 (dd, 1H, H4, J=1.1, 7.1 Hz), 4.54 (d,
1H, CH2Ph, J=11.6 Hz), 4.68 (d, 1H, H2, J=6.1 Hz), 4.74 (d, 1H,
CH2Ph, J=11.6 Hz), 5.04 (dd, 1H, H3, J=1.1, 6.1 Hz), 5.16 (s, 1H,
H1), 7.26–7.38 ppm (m, 5H, Har); 13C NMR (101 MHz, CDCl3): d=
14.21 (CH3 hex), 22.73 (CH2), 25.04 (CH3 iPr), 25.75 (CH2), 26.63 (CH3
iPr), 29.41, 31.84 (CH2), 32.66 (CH2a), 53.81 (C5), 70.18 (CH2Ph),
80.99 (C3), 85.78 (C2), 90.07 (C4), 107.90 (C1), 112.69 (C, iPr), 128.08,
17: ½aŠ20 =À66 (c=1.00, MeOH); 1H NMR (400 MHz, CD3OD): d=
D
0.88–0.95 (m, 3H, CH3 hex), 1.22–1.39 (m, 8H, 4CH2), 1.31 (s, 3H,
CH3 iPr), 1.44 (s, 3H, CH3 iPr), 1.43–1.60 (m, 2H, 2Ha), 2.71 (dt, 1H,
H5, J=3.6, 8.6, 8.6 Hz), 3.91 (dd, H, H4, J=1.2, 8.6 Hz), 4.58 (d, 1H,
CH2Ph, J=11.8 Hz), 4.65 (d, 1H, H2, J=6.0 Hz), 4.70 (dd, 1H, H3,
J=1.2, 6.0 Hz), 4.75 (d, 1H, CH2Ph, J=11.8 Hz), 5.15 (s, 1H, H1),
7.26–7.38 ppm (m, 5H, Har); 13C NMR (101 MHz, CD3OD): d=14.43
(CH3 hex), 23.69 (CH2), 25.16 (CH3 iPr), 26.88 (CH3 iPr), 26.90, 30.57,
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128.15, 128.70 (CHar), 136.91 ppm (Car); HRMS (ESI): m/z [M+H]+
calcd for C21H34NO4: 364.248235, found: 364.248401.
ed under vacuum, and the residue was purified by silica gel
column chromatography (PE/EtOAc 6:1) to give pure l-15 (363 mg,
53%) as a 1:1 mixture of two diastereomers and as a colorless
(1R)-1-C-Hexyl-1,5-dideoxy-1,5-imino-l-ribitol (21): Compound 20
(125 mg, 0.344 mmol) was dissolved in 1,4-dioxane/H2O/AcOH
(20:3:3 mL) and 10% Pd/C was added (80 mg). The mixture was
stirred for 12 h under an H2 atmosphere at room temperature. The
catalyst was removed by filtration through a membrane and
washed with 1,4-dioxane/H2O/AcOH. The filtrate was concentrated
under vacuum, and the procedure was repeated once under the
same conditions to afford crude (1R)-1-C-hexyl-3,4-O-isopropyli-
dene-1,5-dideoxy-1,5-imino-l-ribitol (73 mg, 82%). To a solution of
this compound in 1,4-dioxane/H2O (1:1; 6 mL) was added
Dowex 50W X8 ion-exchange resin (H+ form, 1.5 mL), previously
washed with 1,4-dioxane/H2O (1:1). The mixture was stirred for
36 h at room temperature. The resin was then poured onto
a column and washed with H2O (20 mL). The product was eluted
from the resin using aqueous 0.5n NH4OH (70 mL). Fractions con-
taining the desired compound were combined and concentrated
under vacuum to give 21 (28 mg, 45%) as a white solid; mp: 123–
1
syrup: H NMR (250 MHz, CDCl3): d=1.14 (s, 4.5H, CH3 tBu), 1.21 (s,
4.5H, CH3 tBu), 1.33 (s, 3H, CH3 iPr), 1.49 (s, 1.5H, CH3 iPr), 1.50 (s,
1.5H, CH3 iPr), 4.48 (d, 0.5H, CH2Ph, J=8.4 Hz), 4.53 (d, 0.5H,
CH2Ph, J=8.8 Hz), 4.62 (dd, 0.5H, H3, J=0.6, 5.9 Hz), 4.66 (dd,
0.5H, H3, J=0.6, 5.9 Hz), 4.71 (d, 0.5H, CH2Ph, J=8.8 Hz), 4.76 (d,
0.5H, CH2Ph, J=8.4 Hz), 4.91–4.95 (m, 1H, H4), 5.16 (d, 0.5H, H2,
J=5.8 Hz), 5.22–5.25 (m, 0.5H, H2), 5.22 (s, 0.5H, H1), 5.25 (s, 0.5H,
H1), 7.27–7.39 (m, 5H, Har), 8.10 (d, 0.5H, H5, J=2.3 Hz), 8.11 ppm
(d, 0.5H, H5, J=2.8 Hz); HRMS (ESI): m/z [M+H]+ calcd for
C19H28NO5S: 382.168270, found: 382.168463.
General procedure for the addition of Grignard reagents: To a so-
lution of imine l-15 in anhydrous toluene (0.1m, v mL) at À788C
under Ar was added a commercial solution of Grignard reagent
(5 equiv). The mixture was warmed to room temperature and
stirred for 18 h. The reaction was quenched at 08C by the addition
of a saturated NH4Cl solution, and the aqueous phase was extract-
ed with Et2O (3”2v mL). The combined organic phases were dried
over MgSO4 and concentrated under vacuum. The residue was pu-
rified by silica gel column chromatography (PE/EtOAc).
1318C (degradation); ½aŠ20 = +30.3 (c=1.05, MeOH); 1H NMR
D
(400 MHz, CD3OD): d=0.83–0.96 (m, 3H, CH3), 1.18–1.41 (m, 8H,
4CH2), 1.41–1.56 (m, 1H, HaA), 1.73–1.85 (m, 1H, HaB), 2.66 (ddd,
1H, H1, J=3.0, 8.4, 9.6 Hz), 2.70–2.78 (m, 2H, H5), 3.13 (dd, 1H, H2,
J=2.8, 9.6 Hz), 3.54–3.59 (m, 1H, H4), 3.95 ppm (t, 1H, H3, J=
2.8 Hz); 13C NMR (101 MHz, CD3OD): d=14.43 (CH3), 23.71, 26.70,
30.79, 32.80 (CH2), 32.96 (CH2a), 46.43 (C5), 55.49 (C1), 70.24 (C4),
73.20 (C3), 74.09 ppm (C2); HRMS (ESI): m/z [M+H]+ calcd for
C11H24NO3: 218.175070, found: 218.175514.
Benzyl (5S,SRS)-5-tert-butanesulfinamido-5-deoxy-5-C-hexyl-2,3-
O-isopropylidene-b-l-ribofuranoside (22a) and benzyl (5R,SR)-5-
tert-butanesulfinamido-5-deoxy-5-C-hexyl-2,3-O-isopropylidene-
b-l-ribofuranoside (25a): Compound 22a (202 mg, 47%) as an in-
separable 7:3 mixture of two S*-stereoisomers and as a white solid,
and compound 25a (102 mg, 23%) as a single S*-stereoisomer and
as a colorless syrup were obtained from l-15 (350 mg, 0.917 mmol)
using hexylmagnesium bromide (2m in Et2O; 2.3 mL, 4.6 mmol,
5 equiv). Purification was performed with PE/EtOAc 4:1.
Synthesis of 5-C-alkyl imino-l-ribitols
Benzyl 2,3-O-isopropylidene-aldehydo-b-l-ribo-pentodialdo-1,4-
furanoside (l-2): Sulfuric acid (0.2 mL) was added to a suspension
of l-ribose (5 g, 0.033 mol) in acetone (50 mL). The mixture was
stirred for 2 h, then benzyl alcohol (10.4 mL) and toluene (17 mL)
were added, and acetone was removed under vacuum. The residu-
al mixture was stirred for 3 h and then neutralized with triethyl-
amine. The mixture was diluted with CH2Cl2 (50 mL) and washed
successively with aqueous 0.1n HCl (20 mL), H2O (20 mL), a saturat-
ed NaHCO3 solution (20 mL) and H2O (20 mL). The organic phase
was dried over MgSO4 and concentrated under high vacuum at
508C to remove most of the benzyl alcohol. The residue crystal-
lized at room temperature. The crystals were washed with cold
Et2O and dried under vacuum to give an initial fraction of benzyl
Compound 22a: Rf =0.15 (PE/EtOAc 4:1); 1H NMR (250 MHz,
CDCl3): d=0.83–0.93 (m, 3H, CH3 hex), 1.16–1.40 (m, 20H, CH3 tBu,
CH3 iPr, 4CH2), 1.47 (s, 3H, CH3 iPr), 1.53–1.67 (m, 1H, HaA), 1.68–
1.82 (m, 0.7H, HaB), 1.91–2.02 (m, 0.3H, HaB), 3.21–3.32 (m, 0.6H,
H5, NH), 3.41–3.50 (m, 0.7H, H5), 3.94 (d, 0.3H, H4, J=9.3 Hz), 4.07
(d, 0.7H, NH, J=4.0 Hz), 4.29 (dd, 0.7H, H4, J=1.6, 5.6 Hz), 4.48–
4.79 (m, 3H, H2, CH2Ph), 4.82 (d, 0.3H, H3, J=6.5 Hz), 4.97 (dd,
0.7H, H3, J=1.6, 6.1 Hz), 5.16 (s, 1H, H1), 7.28–7.38 ppm (m, 5H,
Har); HRMS (ESI): m/z [M+H]+ calcd for C25H42NO5S: 468.277821,
found: 468.277954. The 1H NMR spectrum of 22a is identical to
that of compound 19 (enantiomers).
2,3-O-isopropylidene-b-l-ribofuranoside l-1 (1.54 g, 17%).
A
Compound 25a: Rf =0.28 (PE/EtOAc 4:1); 1H NMR (250 MHz,
CDCl3): d=0.85–0.95 (m, 3H, CH3 hex), 0.98 (s, 9H, CH3 tBu), 1.25–
1.41 (m, 8H, 4CH2), 1.32 (s, 3H, CH3 iPr), 1.48 (s, 3H, CH3 iPr), 1.50–
1.63 (m, 2H, 2Ha), 3.17–3.30 (m, 1H, H5), 4.46 (d, 1H, H4, J=
3.3 Hz), 4.56 (d, 1H, CH2Ph, J=11.1 Hz), 4.72 (d, 1H, CH2Ph, J=
11.1 Hz), 4.74 (d, 1H, H3, J=6.1 Hz), 4.89 (d, 1H, NH, J=8.8 Hz),
5.12 (d, 1H, H2, J=6.1 Hz), 5.18 (s, 1H, H1), 7.29–7.39 ppm (m, 5H,
Har); HRMS (ESI): m/z [M+H]+ calcd for C25H42NO5S: 468.277821,
found: 468.277971. The 1H NMR spectrum of 25a is identical to
that of compound 16 (enantiomers).
second crystallization gave another fraction of l-1 (2.15 g, 23%)
containing a small quantity of benzyl alcohol. Alcohol l-1 (500 mg,
1.78 mmol) was then dissolved in anhydrous CH2Cl2 (18 mL) under
Ar, and Dess–Martin periodinane (918 mg, 2.16 mmol, 1.2 equiv)
was added. The reaction was stirred for 5 h at room temperature,
and then concentrated under vacuum. Cold Et2O was added at
À208C and the precipitate was removed by filtration over Celite.
The filtrate was concentrated under vacuum to give crude alde-
hyde l-2 (512 mg, quant.), which was used in the next step with-
out purification. 1H NMR data are in accordance with published
data for the enantiomer d-2.[21b]
General procedure for sulfinyl cleavage: Acetyl chloride (3 equiv)
was added to anhydrous MeOH (0.03m) under Ar. After 20 min,
this solution was added to the sulfinylamine under Ar, and the mix-
ture was stirred for the indicated time at room temperature. The
solution was neutralized with Amberlite IR400 ion-exchange resin
(OHÀ form). The resin was removed by filtration, and the filtrate
was concentrated under vacuum. The residue was purified by silica
gel column chromatography.
N-tert-Butanesulfinylimine of benzyl 2,3-O-isopropylidene-alde-
hydo-b-l-ribo-pentodialdo-1,4-furanoside (l-15): To a solution of
aldehyde l-2 (496 mg, 1.78 mmol) in dry CH2Cl2 (18 mL) under Ar
were added anhydrous CuSO4 (1.42 g, 8.90 mmol, 5 equiv) and rac-
emic tert-butanesulfinamide (238 mg, 1.96 mmol, 1.1 equiv). The
mixture was stirred for 48 h at room temperature and then the
copper salt was removed by filtration. The filtrate was concentrat-
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Benzyl (5S)-5-amino-5-deoxy-5-C-hexyl-2,3-O-isopropylidene-b-l-
ribofuranoside (23a): Compound 23a (66 mg, 42%) as an amor-
phous white solid was obtained from 22a (202 mg, 0.432 mmol)
after 3.5 h of reaction. Purification was performed using CH2Cl2/
1H, H3, J=3.1 Hz), 3.71–3.74 (m, 1H, H4), 3.77–3.82 ppm (m, 1H,
H2); HRMS (ESI): m/z [M+H]+ calcd for C11H24NO3: 218.175070,
found: 218.175390.
MeOH 30:1. Rf =0.59 (CH2Cl2/MeOH 12:1); ½aŠ20 = +63.5 (c=1.15,
D
1
CHCl3); H NMR (250 MHz, CD3OD): d=0.83–0.93 (m, 3H, CH3 hex),
Biological assays
1.19–1.38 (m, 11 H, 4CH2, CH3 iPr), 1.44 (s, 3H, CH3 iPr), 1.44–1.55
(m, 1H, HaA), 1.62–1.77 (m, 1H, HaB), 2.77 (dt, 1H, H5, J=3.6, 7.8,
7.8 Hz), 3.94 (dd, 1H, H4, J=1.4, 7.8 Hz), 4.53 (d, 1H, CH2Ph, J=
11.8 Hz), 4.64 (d, 1H, H2, J=6.1 Hz), 4.73 (d, 1H, CH2Ph, J=
11.8 Hz), 4.88 (dd, 1H, H3, J=1.4, 6.1 Hz), 5.12 (s, 1H, H1), 7.26–
7.38 ppm (m, 5H, Har); HRMS (ESI): m/z [M+H]+ calcd for
C21H34NO4: 364.248235, found: 364.248541.
Enzymatic assays: Unless otherwise indicated, all enzymes and re-
agents were obtained from Sigma (St. Louis, MO, USA). The enzy-
matic assays were run as follows, using one of the following sub-
strates: b-oNPGal (2-nitrophenyl b-d-galactopyranoside), b-4MUGal
(4-methylumbelliferyl b-d-galactopyranoside), a-pNPGal (4-nitro-
phenyl a-d-galactopyranoside), b- or a-pNPGlc (4-nitrophenyl b- or
a-d-glucopyranoside). The b-galactosidase from bovine liver was
used at a final concentration of 13 mUmLÀ1 in sodium phosphate
(100 mm, pH 7.3) with b-oNPGal as substrate (2.25 mm). After 2 h
incubation at 378C, the reaction was stopped by the addition of
an equal volume of 0.2m NaOH and absorbance was determined
at l 405 nm.
Benzyl (5R)-5-amino-5-deoxy-5-C-hexyl-2,3-O-isopropylidene-b-l-
ribofuranoside (26a): Compound 26a (40 mg, 56%) as a colorless
syrup was obtained from 25a (91 mg, 0.195 mmol) after 4.5 h of
reaction. Purification was performed using CH2Cl2/MeOH 14:1. Rf =
0.44 (CH2Cl2/MeOH 14:1); ½aŠ20 = +68.0 (c=1.18, CHCl3); 1H NMR
D
(400 MHz, CD3OD): d=0.87–0.95 (m, 3H, CH3 hex), 1.21–1.40 (m,
11 H, HaA, 3.5CH2, CH3 iPr), 1.44 (s, 3H, CH3 iPr), 1.45–1.60 (m, 2H,
HaB, 0.5CH2), 2.71 (dt, 1H, H5, J=3.6, 8.5, 8.5 Hz), 3.91 (dd, H, H4,
J=1.2, 8.5 Hz), 4.58 (d, 1H, CH2Ph, J=11.8 Hz), 4.65 (d, 1H, H2, J=
6.0 Hz), 4.70 (dd, 1H, H3, J=1.2, 6.0 Hz), 4.74 (d, 1H, CH2Ph, J=
11.8 Hz), 5.15 (s, 1H, H1), 7.26–7.38 ppm (m, 5H, Har); 13C NMR
(101 MHz, CD3OD): d=14.42 (CH3 hex), 23.69 (CH2), 25.17 (CH3 iPr),
26.89 (CH3 iPr), 26.91, 30.58, 32.92 (CH2), 34.52 (CH2a), 54.71 (C5),
71.19 (CH2Ph), 83.28 (C3), 87.0 (C2), 92.74 (C4), 109.25 (C1), 113.59
(C, iPr), 128.91, 129.03, 129.50 (CHar), 138.81 ppm (Car); HRMS (ESI):
m/z [M+H]+ calcd for C21H34NO4: 364.248235, found: 364.248494.
The b-galactosidase from E. coli was used at a final concentration
of 1 UmLÀ1 in sodium phosphate (100 mm, pH 7.3), supplemented
with MgCl2 (1 mm) and 2-mercaptoethanol (112 mm), with b-
oNPGal as substrate (2 mm). After 2 h incubation at 378C, the reac-
tion was stopped by the addition of an equal volume of 0.2m
NaOH and absorbance was determined at l 405 nm.
The b-galactosidase from Aspergillus oryzae was used at a final con-
centration of 0.2 UmLÀ1 in sodium phosphate (100 mm, pH 7.3)
with b-oNPGal as substrate (2.25 mm). After 2 h incubation at
378C, the reaction was stopped by the addition of an equal
volume of 0.2m NaOH and absorbance was determined at
l 405 nm.
General procedure for reductive amination and deprotection:
The amine was dissolved in 1,4-dioxane/H2O/AcOH (10:6:1, 0.01m)
and 10% Pd/C was added (300 mgmmolÀ1). The mixture was
stirred for 48 h under an H2 atmosphere at room temperature. The
catalyst was removed by filtration through a membrane and
washed with 1,4-dioxane/H2O/AcOH. The filtrate was concentrated
under vacuum to give the protected iminoribitol. To a solution of
this compound (0.02m) in 1,4-dioxane/H2O (3:1) was added
Dowex 50W X8 ion-exchange resin (H+ form, 8 mLmmolÀ1), previ-
ously washed with 1,4-dioxane/H2O (3:1). The mixture was stirred
for 18 h at room temperature. The resin was then poured onto
a column, washed with 1,4-dioxane/H2O (1:1), then with H2O. The
product was eluted with aqueous 0.5n NH4OH. Fractions contain-
ing the iminoribitol derivatives were combined and concentrated
under vacuum to afford the deprotected compound.
The b-galactosidase from bovine testis was used at a final concen-
tration of 5 mUmLÀ1 in sodium phosphate (100 mm, pH 7.3) with
b-4MUGal as substrate (0.8 mm). After 2 h incubation at 378C, the
reaction was stopped by the addition of an equal volume of 0.15m
glycine (pH 10.2) and fluorescence emission was determined at
l 445 nm with excitation at l 365 nm.
Human peripheral blood mononuclear cell lysate was used as
a source of human b-galactosidase activity. The assay was conduct-
ed using lysate from 2”106 cells per reaction in sodium phosphate
(50 mm, pH 7.3), supplemented with protease inhibitors (Complete
Protease Inhibitor Cocktail Tablets, Roche) and 1% Triton X-100,
with b-4MUGal as substrate (0.8 mm). After 1 h incubation at 378C,
the reaction was stopped by the addition of an equal volume of
0.15m glycine (pH 10.2) and fluorescence emission was determined
at l 445 nm with excitation at l 365 nm.
(5S)-5-C-Hexyl-1,5-dideoxy-1,5-imino-l-ribitol (24a): Compound
24a (26 mg, 65%) was obtained as a white amorphous solid from
23a (67 mg, 0.184 mmol): ½aŠ20 =À34.5 (c=1.01, MeOH); 1H NMR
D
The a-galactosidase from green coffee beans was used at a final
concentration of 12.5 mUmLÀ1 in sodium citrate (100 mm, pH 6.5)
with a-pNPGal as substrate (2 mm). After 1 h incubation at room
temperature, the reaction was stopped by the addition of an equal
volume of 0.2m NaOH and absorbance was determined at
l 405 nm.
(400 MHz, CD3OD): d=0.87–0.94 (m, 3H, CH3), 1.18–1.40 (m, 8H,
4CH2), 1.43–1.54 (m, 1H, HaA), 1.75–1.85 (m, 1H, HaB), 2.61–2.68
(m, 1H, H5), 2.74 (d, 2H, H1, J=8.0 Hz), 3.13 (dd, 1H, H4, J=2.7,
9.6 Hz), 3.56 (dt, H2, J=2.7, 8.0, 8.0 Hz), 3.94 ppm (t, H3, J=2.7 Hz);
13C NMR (101 MHz, CD3OD): d=14.42 (CH3), 23.70, 26.72, 30.80
(CH2), 32.88 (CH2a), 32.96 (CH2), 46.52 (C1), 55.49 (C5), 70.37 (C2),
73.25 (C3), 74.20 ppm (C4); HRMS (ESI): m/z [M+H]+ calcd for
C11H24NO3: 218.175070, found: 218.175402.
The b-glucosidase from almonds was used at a final concentration
of 1.25 mgmLÀ1 in sodium acetate (100 mm, pH 5.0) with b-pNPGlc
as substrate (2 mm). After 1 h incubation at room temperature, the
reaction was stopped by the addition of an equal volume of 0.2m
NaOH and absorbance was determined at l 405 nm.
(5R)-5-C-Hexyl-1,5-dideoxy-1,5-imino-l-ribitol (27a): Compound
27a (17 mg, 71%) was obtained as a colorless syrup from 26a
(40 mg, 0.110 mmol): ½aŠ20 =À38.3 (c=1.00, MeOH); 1H NMR
D
(250 MHz, CD3OD): d=0.85–0.96 (m, 3H, CH3), 1.26–1.45 (m, 8H,
4CH2), 1.45–1.66 (m, 2H, 2Ha), 2.42–2.48 (m, 1H, H5), 2.70 (dd, 1H,
H1A, J=1.8, 14.0 Hz), 3.03 (dd, 1H, H1B, J=2.5, 14.0 Hz), 3.46 (t,
The a-glucosidase from Saccharomyces cerevisiae was used at
a
final concentration of 12.5 mUmLÀ1 in sodium phosphate
(100 mm, pH 6.8) with a-pNPGlc as substrate (2 mm). After 1 h in-
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equal volume of 0.2m NaOH and absorbance was determined at
l 405 nm.
´
Chaperone assays: The fibroblast cell lines derived from GM1-gan-
gliosidosis patients GM02439 and GM03251 were obtained from
Coriell Institute (Camden, NJ, USA). The chaperone assays were
conducted by following the method described by Rigat et al.[30] Fi-
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Keywords: carbohydrates
· drug design · galactosidase
inhibitors · GM1-gangliosidosis · iminosugars · pharmacological
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Received: November 16, 2015
Published online on December 8, 2015
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