n-Propyl 6-amino-2,6-dideoxy-2,2-difluoro-β-d-glucopyranoside is a good inhibitor for the β-galactosidase from E. coli

Article abstract of DOI:10.1007/s00044-021-02715-8

A convenient route has been developed for the synthesis of novel 6-amino-2,2-(or 3,3-difluoro)-2-(or 3),6-dideoxy-hexopyranoses. Biological screening showed these compounds as good inhibitors for several glycosidases. Especially n-propyl 6-amino-2,6-dideoxy-2,2-difluoro-β-d-glucopyranoside (8) was an excellent competitive inhibitor for the β-galactosidase from E. coli holding a K_i of 0.50 μM. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-021-02715-8

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1099–1107
https://doi.org/10.1007/s00044-021-02715-8
ORIGINAL RESEARCH
n-Propyl 6-amino-2,6-dideoxy-2,2-difluoro-β-D-glucopyranoside is a
good inhibitor for the β-galactosidase from E. coli
1
Immo Serbian1 Erik Prell2 Claudia Fischer1 Hans-Peter Deigner3 René Csuk
●
●
●
●
Received: 15 November 2020 / Accepted: 17 February 2021 / Published online: 5 March 2021
© The Author(s) 2021
Abstract
A convenient route has been developed for the synthesis of novel 6-amino-2,2-(or 3,3-difluoro)-2-(or 3),6-dideoxy-
hexopyranoses. Biological screening showed these compounds as good inhibitors for several glycosidases. Especially n-
propyl 6-amino-2,6-dideoxy-2,2-difluoro-β-D-glucopyranoside (8) was an excellent competitive inhibitor for the
β-galactosidase from E. coli holding a Ki of 0.50 μM.
Graphical Abstract
●
●
●
Keywords Fluorinated monosaccharides Glucosidase Galactosidase Inhibitor
Introduction
containing [8] glycomimetics such as aza- or iminosugars
are known to behave as pharmacological pharmacophores
for lysosomal storage disorders [9]. Miglitol (Fig. 1) is an α-
glucosidase inhibitor; this enzyme catalyzes the hydrolysis
of oligo-, tri-, and disaccharides to glucose and other
monosaccharides in the intestine. Thereby, an increased
blood sugar level (as usually observed after meals) is
reduced. Hence, miglitol is in principle suitable for the
therapy of diabetes mellitus type 2 [10–13]. In Germany, it
was in use until 2015 but is nowadays often replaced by
acarbose, a pseudotetrasaccharide. An analog of miglitol,
miglustat, is a drug used to treat type I Gaucher disease [14–
19] and was the first treatment to be approved for treating
progressive neurological complications in people with
Niemann–Pick disease [20–28]. Recently, miglustat has
been suggested for the therapy of COVID-19, too [29, 30].
An extended search in literature as of 2020, October,
revealed there are some 30,600 structures containing the
substructure of a 6-amino-6-deoxy-hexopyranose (or -pyr-
anoside). Monofluorinated analogs, however, have scarcely
been prepared (some glycosyl fluorides and a couple of
Glycosidases are involved in many important biological
processes, and inhibitors of glycosidases [1–3] are dis-
cussed as therapeutics for diabetes mellitus, lysosomal
storage diseases, and obesity [4–7]. Furthermore, nitrogen-
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02715-8.
* René Csuk
rene.csuk@chemie.uni-halle.de
1
Martin-Luther-University Halle-Wittenberg, Organic Chemistry,
Kurt-Mothes_Str. 2, D-06120 Halle (Saale), Germany
2
Department of Radiation Medicine, Section of Nuclear Medicine,
University Hospital Halle (Saale), Ernst-Grube Str. 40, D-06120
Halle (Saale), Germany
3
Medical and Life Sciences Faculty, Furtwangen University, Jakob-
Kienzle Str. 17, D-78054 Villingen-Schwenningen, Germany

1100
Medicinal Chemistry Research (2021) 30:1099–1107
Fig. 1 Structure of glucosidase
inhibitors miglitol and miglustat
and acarbose
Scheme 1 Synthesis of 6-amino-2,6-dideoxy-2,2-difluoro-hexopyr-
anoside 8: (a) DMSO, TFAA, DCM, −78 → 25 °C, 12 h, 89%; (b)
DAST, DCM, 25 °C, 5 days, 76%; (c) LiAlH4/AlCl3, Δ, 48 h; 4 (15%),
5 (50%); (d) PPh3, imidazole, I2, 90 °C, 2 h, 52%; (e) LiN3, DMF,
25 °C, 4 days, 89%; (f) Pd/C (10%), H2 (30 °C, 2.43 atm), 48 h, 88%
2-fluoro and 3-fluoro analogs; the latter—by and large—being
part of modified kanamycin derivatives) [31–37]—but to
the best of our knowledge, there are no 2,2- or 3,3-difluoro-
6-amino-2 (or 3), 6-dideoxy-hexopyranoses (respectively
-hexopyranosides). Some molecules containing the struc-
tural element of a 6-amino-hexose have been proposed
in-silico as possible lead structures for the therapy of
COVID-19 diseases [38–40]. Therefore, we set out for the
first synthesis of these targets and to test their ability to act
as glycosidase inhibitors. It is reserved for later studies to
have a look at their properties regarding lysosomal storage
diseases or antiviral activity.
Partial deprotection [44] of 11 gave a mixture of 12 and
13. Main component 12 was iodinated as described above to
afford 14 whose subsequent nucleophilic displacement
reaction (→ 15) and hydrogenation gave glycoside 16.
Low or absent cytotoxicity is a mandatory requirement
for subsequent therapeutic use of the compounds as gly-
cosidase inhibitors. Therefore, their cytotoxicity was
determined. Compounds 8 and 16 were tested for antitumor
activity in a panel of 15 human cancer cell lines (518A2, A-
431, A-253, FaDu, A-549, A-2780, DLD-1, HCT-8, HCT-
116, HAT-29, SW480, 8505-C, SW1736, MCF-7, and
Lipo) and for cytotoxicity on nonmalignant NIH 3T3 mouse
fibroblasts (using a sulforhodamine B (SRB) assay) [49] but
no significant cytotoxicity was observed (IC50 > 50 μM).
An in vitro evaluation of these compounds to act as an
enzyme inhibitor using a panel of commercially available
glycosidases was performed (Table 1) using p-nitrophenolate
[50] assays.
Compounds 8 and 16 were competitive inhibitors for all
enzymes. Thereby, compound 8 was a nonselective inhi-
bitor, and its highest activity was found for the ß-galacto-
sidase from E. coli. Glycoside 16 was a weaker inhibitor
than 8.
For a better interpretation of these results additional
molecular modeling calculations were carried out.
Thereby, the protein crystal structures were selected on
their genetic similarity to the enzymes employed in the
in vitro experiments. An additional focus was set to a
good resolution of the enzyme structure. This was easily
achieved for several of the enzymes, while for the
Results and discussion
Swern oxidation [41] (Scheme 1) of well-known allyl gly-
coside 1 [42] gave 89% of the ulose 2 whose difluorination
with DAST [43] in dichloromethane for 5 days gave 76% of
difluorinated 3. Compound 3 was treated with LiAlH4/AlCl3
[44] to open the benzylidene acetal, and a mixture of 4 and
5 was obtained. Product 5 was iodinated using in situ pre-
pared triiodoimidazole [45–47] resulting in the formation of
52% of 6-iodo 6. Nucleophilic displacement with lithium
azide [45] for 4 days provided 89% of 6-azido 7 whose
hydrogenation gave 88% of n-propyl-6-amino derivative 8.
For the synthesis of the 3, 3-difluoro compounds, 9 [48]
(Scheme 2) served as a starting material whose Swern
oxidation [41] (→ 10) followed by difluorination [43] with
DAST gave 82% of difluoro 11.

Medicinal Chemistry Research (2021) 30:1099–1107
1101
Scheme 2 Synthesis of 6-amino-3,6-dideoxy-3,3-difluoro-hexopyr-
anoside 16: (a) DMSO, TFAA, DCM, −78 → 25 °C, 12 h, 75%; (b)
DAST, DCM, 25 °C, 5 days, 55%; (c) LiAlH4/AlCl3, Δ, 48 h; 12
(43%), 13 (12%); (d) PPh3, imidazole, I2, 90 °C, 2 h, 78%; (e) LiN3,
DMF, 25 °C, 4 days, 77%; (f) Pd/C (10%), H2 (30 °C, 2.43 atm),
48 h, 93%
Table 1 Glycosidase inhibition
Enzyme
From
Miglitol
8
16
(Ki in μM from p-nitrophenolate
assays (mean of experiments
performed in triplicate with three
technical triplicates, each; n.a.
no activity); [50])
α-glucosidase
α-glucosidase
β-glucosidase
α-galactosidase
β-galactosidase
B. stearothermophilus
Baker’s yeast
Almonds
0.15 0.03
9.92 0.14
0.21 0.05
13.90 0.15
0.16 0.06
2.21 0.11
1.83 0.16
1.76 0.13
3.81 0.57
0.50 0.09
n.a.
4.13 0.26
n.a.
Green coffee beans
E. coli
4.30 0.24
2.16 0.93
Table 2 Results from the
Enzyme
Miglitol
8
16
molecular docking; mean ΔG in
kcal/mol of the top 5 docking
poses (standard deviation,
median) for miglitol (standard)
and compounds 8 and 16
Geobacillus sp. (α-glucosidase)
Baker’s yeast (α-glucosidase)
Rice (β-glucosidase)
White clover (α-galactosidase)
E. coli (β-galactosidase)
−3.81 (0.11, −3.82)
−4.22 (0.05, −4.22)
−4.21 (0.02, −4.20)
−5.53 (0.11, −5.52)
−5.34 (0.10, −5.32)
−5.61 (0.15, −5.71)
−4.66 (0.31, −4.64)
−4.50 (0.10, −4.49)
−5.32 (0.22, −5.39)
−5.56 (0.07, −5.57)
−5.68 (0.14, −5.62)
−3.62 (0.48, −3.30)
−5.12 (0.27, −5.01)
−4.81 (0.01, −4.82)
−5.19 (0.17, −5.10)
β-glucosidase from almonds and the α-galactosidase
(from green coffee beans) an additional search for an
enzyme from a different organism was called for. This
was performed utilizing UniProt.org eventually finding
enzymes of high similarity. Thus, miglitol and compounds
8 and 16 were evaluated in molecular docking studies
against the enzymes α-glucosidase (Geobacillus sp.; PDB:
2ZE0, Baker’s yeast; PDB: 3AXI), β-glucosidase (from
rice due to its similarity with the β-glucosidase from
almonds; PDB: 1UAS), α-galactosidase (from white clo-
ver due to its similarity to the enzyme from green coffee
beans; PDB: 1CBG), and β-galactosidase (E. coli; PDB:
1JYW). From the docking studies the binding affinity was
estimated.
A rough blind docking of 50 individual runs showed that
for compounds 8 and 16 their main binding site at the
enzymes is similar to the binding site of miglitol in each
case. Therefore, the search space was limited to the active
site of the respective enzyme. As a result, five top poses
were obtained being very closely located to each other
holding H-bond interactions with closely related
amino acids.
Thus, the 2,2- and 3,3-difluorinated miglitol analogs are
efficient competitive inhibitors of the α- and β-glucosidase
as well as of the galactosidases. Miglitol, however, showed
lowered inhibition for yeast’s and green coffee bean’s α-
glucosidase and galactosidase, while for compound 8 low Ki
values for all tested enzymes were established. This is most
likely due to favored interaction of the difluorinated com-
pounds in the active sites leading eventually to better
binding affinities for compounds 8 and 16 with yeast α-
glucosidase and green coffee beans α-galactosidase (cf.
Tables 2 and 3). Table 3 shows the main interactions of the
compounds with the amino acids of the respective enzyme.
The good binding affinities of compounds 8 and 16 as
compared to miglitol allow no explanation for the low Ki
value of miglitol for the α-glucosidase from Geobacillus sp.
(almond’s β-glucosidase) and E. coli’s β-galactosidase. We
assume that these findings are caused by different residence
times of the compounds. As a consequence of this dynamic
effect, the hydroxyl groups of miglitol may help to hold this
compound in the periphery of the binding site thereby
increasing the residence time of miglitol as compared to the
residence time of compounds 8 and 16. Comparing the

1102
Medicinal Chemistry Research (2021) 30:1099–1107
docking results from Table 2, compound 8 seems to be a
slightly better inhibitor than miglitol (−0.47 kcal/mol on
average), and compound 16 seems to be also a slightly
better inhibitor than compound 8 (−0.16 kcal/mol on
average).
Table 3 summarizes the main binding residues thus
allowing some insight into the different binding motifs of
compounds 8, 16 and miglitol. For the α-glucosidase from
Baker’s yeast, Trp468 seems to be a common amino acid
residue all evaluated compounds share H-bonding with,
whereas other amino acid residues may vary. Furthermore
compound 16 and miglitol share similar H-bonding motif;
for example, docking of miglitol, 8, and 16 with the
β-glucosidase from rice showed miglitol and 16 to share
Lys58, His256, and Trp454 as amino acid residues with H-
bonding interactions. The results for the β-galactosidase
from E. coli revealed that miglitol and compound 8 only
share Asn102, whereas 16 formed H-bonds to Asn102,
Gly489, and Lys517 as also observed for miglitol.
Figure 2 depicts some of the results from the modeling,
the interaction of miglitol and of compound 16 in the active
site of the β-galactosidase from E. coli.
Conclusion
n-Propyl 6-amino-2,6-dideoxy-2,2-difluoro-β-D-glucopyr-
anoside (8) as well as its 3,3-difluor-analog (16) were easily
accessible from allyl glycosides 1 and 9 by a sequence of
oxidation, difluorination, selective deprotection, nucleo-
philic displacement, and catalytic hydrogenation. While no
significant cytotoxicity was observed in the SRB assays
(EC50 > 50 μM), screening of these compounds in p-nitro-
phenolate assays showed especially compound 8 as a good
inhibitor for the β-galactosidase from E. coli. It also seems
to be obvious for this novel class of compounds that the
presence of a hydrophobic moiety in β-position has a
stronger influence on the inhibitory activity with respect to
β-galactosidase of E. coli than a corresponding hydrophobic
moiety in γ-position.
Experimental
Melting points are uncorrected (Leica hot stage micro-
scope), optical rotations were obtained using a Perkin-Elmer
341 polarimeter (1 cm micro cell), NMR spectra were
recorded using the Varian spectrometers Gemini 2000 or
Unity 500 (δ given in ppm, J in Hz, internal Me4Si or
internal CCl3F), IR spectra (film or KBr pellet) on a Perkin-
Elmer FT-IR spectrometer Spectrum 1000, MS spectra were
taken on a Intectra GmbH AMD 402 (electron impact,
70 eV) or on
a Thermo Electron Finnigan LCQ

Medicinal Chemistry Research (2021) 30:1099–1107
1103
Fig. 2 Interaction of miglitol (A) and of compound 16 (B) in the active site of β-galactosidase (E. coli) holding a Ki of 0.16 μM
(electrospray, voltage 4.5 kV, sheath gas nitrogen) instru-
ment; for elemental analysis a Foss-Heraeus Vario EL
instrument was used; TLC was performed on silica gel
(Merck 5554, detection by UV absorption or by treatment
with a solution of 10% sulfuric acid, ammonium molybdate,
and cerium(IV) sulfate) followed by gentle heating. The
solvents were dried according to usual procedures.
1:10, and 1:5. The ice-cooled solution of the enzymes was
contained between 4.0 and 4.5 U/mL. The kinetic assays
were performed in 96-well microtiter plates. The following
solutions were pipetted per well: p-nitrophenyl glycoside
(100 μL), enzyme (50 μL), inhibitor (100 μL), and standard
(p-nitrophenol, 5 μL). Immediately after addition, UV/Vis
absorption was measured at 42 °C and λ = 415 nm for
20 min with a single measurement interval of 50 s.
Molecular docking experimental
Cytotoxicity assay (SRB assay)
The enzyme structure was accessed through http://rcsb.org
(α-glucosidase (Geobacillus sp.; PDB: 2ZE0, Baker’s yeast;
PDB: 3AXI), β-glucosidase (rice due to its similarity with
almond β-glucosidase; PDB: 1UAS), α-galactosidase (white
clover due to its similarity to green coffee beans; PDB:
1CBG), and β-galactosidase (E. Coli; PDB: 1JYW)). The
structures were prepared with autodock tools 1.5.6 follow-
ing usual procedures. The grid and docking files of the
inhibitors and enzymes were also prepared with autodock
tools 1.5.6. The grid spacing was set to 0.25 A. The genetic
algorithm settings were 150 m populations size, 4 m eva-
luations, and 27k generations. Docking settings were 50
dockings of each individual inhibitor/enzyme pair with 4 m
evaluations each. The corresponding file formats were
prepared with open babel 2.4.1. Drawing and interactions
were analyzed with PyMOL 2.0.6 from Schroedinger LLC.
The cell lines were obtained from Department of oncology
(Martin-Luther-University Halle-Wittenberg). Cultures
were maintained as monolayers in RPMI 1640 medium with
L-glutamine (Capricorn Scientific GmbH, Ebsdorfergrund,
Germany) supplemented with 10% heat inactivated fetal
bovine serum (Sigma-Aldrich Chemie GmbH, Steinheim,
Germany) and penicillin/streptomycin (1%, Capricorn Sci-
entific GmbH, Ebsdorfergrund, Germany) at 37 °C in a
humidified atmosphere with 5% CO2.
The cytotoxicity of the compounds was evaluated using
the SRB (Kiton-Red S, ABCR) micro culture colorimetric
assay using confluent cells in 96-well plates with the
seeding of the cells on day 0 applying appropriate cell
densities to prevent confluence of the cells during the period
of the experiment. On day 1, the cells were treated with six
different concentrations (1, 3, 7, 12, 20, and 30 μM);
thereby, the final concentration of DMSO was always <
0.5%, generally regarded as nontoxic to the cells. On day 4,
the supernatant medium was discarded; the cells were fixed
with 10% trichloroacetic acid. After another day at 4 °C, the
cells were washed in a strip washer and dyed with the SRB
solution (100 μL, 0.4% in 1% acetic acid) for about 20 min
to be followed by washing of the plates (four times, 1%
Glycosidase assays
The corresponding p-nitrophenyl glycoside and the inhibi-
tors were dissolved in NaOAc buffer (0.05 M, pH = 6.0)
reaching final concentrations of 1.0, 1.11, 1.25, 1.43, 1.67,
2.0, 2.5, 3.33, 5.0, and 10 mM. Further dilutions (if neces-
sary) were made in the ratios 1:60, 1:50, 1:40, 1:30, 1:20,

1104
Medicinal Chemistry Research (2021) 30:1099–1107
acetic acid) and air-drying overnight. Furthermore, tris base
solution (200 μL, 10 mM) was added to each well and
absorbance was measured at λ = 570 nm employing a
reader (96-wells, Tecan Spectra, Crailsheim, Germany). The
IC50 values were averaged from three independent experi-
ments performed each in triplicate calculated from semi
logarithmic dose response curves applying a nonlinear four-
parameter Hills-slope equation (GraphPad Prism5; variables
top and bottom were set to 100 and 0, respectively).
Allyl 3-O-benzyl-(R)-4,6-O-benzylidene-2-deoxy-2,2-
difluoro-β-D-arabino-hexopyranoside (3)
To a solution of 2 (300 mg, 0.72 mmol) in dry DCM
(6.0 mL), DAST (417 μL 3.03 mmol) was added dropwise
under argon, and the mixture was stirred at 25 °C for 5 days.
Methanol (1.0 mL) was carefully added, and the solvents
were removed under diminished pressure. The oily residue
was dissolved in DCM (90 mL) and washed with water
(50 mL). The aqueous phase was re-extracted with DCM
(3 × 100 L); the organic layers were combined, and the
solvent was evaporated under reduced pressure. The
remaining residue was subjected to chromatography (silica
gel, n-hexane/ethyl acetate, 85:15) to afford 3 (230 mg,
76%) as a white solid; MP 79–81 °C; [α]D = −26.10° (c
0.71, CHCl3) Rƒ (n-hexane/ethyl acetate, 5:3) = 0.75; ana-
lysis calcd for C23H24O5F2 (418.43): C 66.02, H 5.78;
found: C 65.85, H 5.92.
Allyl 3-O-benzyl-(R)-4,6-O-benzylidene-β-D-glucopyranoside
(1) and allyl 2-O-benzyl-(R)-4,6-O-benzylidene-β-D-
glucopyranoside (9)
To a solution of allyl (R) 4,6-O-benzylidene-β-D-glucopyr-
anoside [42] (8.44 g, 27.27 mmol) and tetrabutylammonium
hydrogen sulfate (1.63 g, 4.80 mmol) in dry DCM
(315.0 mL) an aq. solution of sodium hydroxide (1.3 M,
31.6 mL) was added. At reflux temperature, benzyl bromide
(4.91 mL, 41.06 mmol) was added dropwise and stirring
was continued for 96 h. Usual aqueous workup followed by
chromatography (silica gel, n-hexane/ethyl acetate, 5:15)
gave 1 (2.71 g, 25%) and 11 (5.22 g, 48%).
Allyl 3,6-di-O-benzyl-2-deoxy-2,2-difluoro-β-D-arabino-
hexopyranoside (4) and allyl 3,4-di-O-benzyl-2-deoxy-2,2-
difluoro-β-D-arabino-hexopyranoside (5)
Data for 1: white solid; MP 139–140 °C (lit.: [51]
140–141 °C); [α]D = −18.96° (c 0.42, CHCl3); [lit.: [51] [α]D
= +39.9° (c 1, CHCl3)]; Rƒ (n-hexane/ethyl acetate, 5:3) =
0.53; analysis calcd for C23H26O6 (398.45): C 69.33, H 6.58;
found: C 69.09, H 6.63.
Data for 9: white solid; MP 124–125 °C (lit.: [48]
124 °C); [α]D = −17.70° (c 0.93 g, CHCl3); Rƒ (n-hexane/
ethyl acetate, 5:3) = 0.63; analysis calcd for C23H26O6
(398.45): C 69.33, H 6.58; found: C 69.17, H 6.71.
To an ice-cold solution of 3 (3.00 g, 7.17 mmol) in dry ether
(30 mL) and THF (30 mL), lithium aluminum hydride
(490 mg, 12.91 mmol) was added in several portions, and
the suspension was stirred for 15 min at 25 °C. Then, the
suspension was heated to reflux, and a solution of dry
aluminum chloride (1.63 g, 12.19 mmol) in dry ether
(30 mL) was added dropwise followed by stirring under
reflux for another 48 h. The suspension was cooled to 25 °C,
methanol (30 mL) was carefully added, and stirring was
continued for another 30 min. After usual aqueous workup
followed by chromatography (silica gel, n-hexane/ethyl
acetate, 5:3) 4 (450 mg, 15%) and 5 (1.50 g, 50%) were
obtained each as a colorless oil.
Allyl 3-O-benzyl-(R)-4,6-O-benzylidene-β-D-arabino-
hexopyranoside-2-ulose (2)
To a solution of dry DMSO (4.67 g, 59.80 mmol) in dry
DCM (40.0 mL) at −78 °C, a solution of trifluoroacetic
anhydride (8.92 g, 42.45 mmol) in dry DCM (10.0 mL) was
slowly added dropwise, and the mixture was stirred at this
temperature for 45 min followed by adding a solution of 1
(8.14 g, 20.43 mmol) in dry DCM (40.0 mL) while main-
taining the temperature at −78 °C. The mixture was stirred
for 2 h at −78 °C, then a solution of Et3N (9.0 mL,
64.57 mmol) in dry DCM (40.0 mL) was added dropwise.
The mixture was stirred at −78 °C for another 30 min and at
25 °C for 12 h. Usual aqueous workup followed by chro-
matography (silica gel, n-hexane/ethyl acetate, 5:3) gave 2
(7.18 g, 89%) as a white solid; MP 130–132 °C; [α]D =
−47.95° (c 0.73, CHCl3); Rƒ (n-hexane/ethyl acetate, 5:3)
= 0.40 (ketone); Rƒ (hexane/ethyl acetate, 5:3) = 0.19
(hydrate); analysis calcd for C23H24O6 (396.43): C 69.68, H
6.10; found: C 69.43, H 6.27.
Data for 4: [α]D = −48.23° (c 0.44, CHCl3); Rƒ (n-hex-
ane/ethyl acetate, 5:3) = 0.51; analysis calcd for C23H26O5F2
(420.45): C 65.70, H 6.23; found: C 65.55, H 6.40.
Data for 5: [α]D = −18.27° (c 0.52, CHCl3); Rƒ (n-hex-
ane/ethyl acetate, 5:3) = 0.41; analysis calcd for
C23H26O5F2 (420.45): C 65.70, H 6.23; found: C 65.47,
H 6.39.
Allyl 3,4-di-O-benzyl-2,6-dideoxy-2,2-difluoro-6-iodo-β-D-
arabino-hexopyranoside (6)
To a solution of 5 (1.50 g, 3.57 mmol) in toluene (30 mL)
containing triphenylphosphane (2.06 g, 7.85 mmol) and
imidazole (1.09 g, 16.06 mmol), iodine (1.81 g, 7.14 mmol)
was added in several portions. After stirring at 90 °C for 2 h,
the reaction mixture was decanted, and the remaining oil
was washed with ether (3 × 100 mL). The combined organic

Medicinal Chemistry Research (2021) 30:1099–1107
1105
layers were evaporated, and the remaining residue was
subjected to chromatography (silica gel, n-hexane/ethyl
acetate, 85:15) to afford 6 (900 mg, 52%) as a colorless oil;
[α]D = −25.32° (c 0.44, CHCl3); Rƒ (n-hexane/ethyl acetate,
85:15) = 0.56; analysis calcd for C23H25O4F2I (530.34): C
52.09, H 4.75; found: C 51.84, H 4.92.
then a solution of Et3N (9.0 mL, 64.57 mmol) in dry DCM
(40.0 ml) was added. The mixture was stirred at −78 °C for
another 30 min and at 25 °C for 12 h. Usual aqueous
workup followed by chromatography (silica gel, n-hexane/
ethyl acetate, 85:15) gave 10 (10.90 g, 75%) as a white
solid; MP 125–127 °C; [α]D = −64.84° (c 0.49, CHCl3); Rƒ
(n-hexane/ethyl acetate, 5:3) = 0.73; analysis calcd for
C23H24O6 (396.43): C 69.68, H 6.10; found: C 69.50,
H 6.37.
Allyl 6-azido-3,4-di-O-benzyl-2,6-dideoxy-2,2-difluoro-β-D-
arabino-hexopyranoside (7)
To a solution of 6 (940 mg, 1.77 mmol) in dry DMF
(19 mL) a solution of lithium azide (20% in water, 2.17 mL,
8.86 mmol) was added at 25 °C, and the solution was stirred
at this temperature for 4 days. The solvents were removed
under diminished pressure, and the remaining residue was
dissolved in DCM (100 mL) and water (50 mL). The aq.
phase was extracted with DCM (3 × 100 mL), and the
combined organic layers were dried (Na2SO4). The solvent
was removed under reduced pressure, and the remaining
residue was subjected to chromatography (silica gel, hex-
ane/ethyl acetate, 85:15) to afford 7 (700 mg, 89%) as a
colorless oil; [α]D = −33.01° (c 0.35, CHCl3); Rƒ (n-hex-
ane/ethyl acetate, 85:15) = 0.39; analysis calcd for
C23H25O4F2N3 (445.44): C 62.01, H 5.66, N 9.43; found: C
61.80, H 5.81, N 9.25.
Allyl 2-O-benzyl-(R)-4,6-O-benzylidene-3-deoxy-3,3-
difluoro-β-D-ribo-hexopyranoside (11)
To a solution of 10 (300 mg, 0.72 mmol) in dry DCM
(6.0 mL) DAST (417 μL, 3.03 mmol) was added dropwise,
and the mixture was stirred at 25 °C for 5 days. Methanol
(2.0 mL) was carefully added, and the solvents were
removed under diminished pressure. Usual aqueous workup
followed by chromatography (silica gel, n-hexane/ethyl
acetate, 5:3) gave 11 (167 mg, 55%) as a white amorphous
solid; [α]D = −27.89° (c 0.39, CHCl3); Rƒ (n-hexane/ethyl
acetate, 5:3) = 0.67; analysis calcd for C23H24O5F2
(418.43): C 66.02, H 5.78; H 65.79, H 5.93.
Allyl 2,6-di-O-benzyl-3-deoxy-3,3-difluoro-β-D-ribo-
hexopyranoside (12) and allyl 2,4-di-O-benzyl-3-deoxy-3,3-
difluoro-β-D-ribo-hexopyranoside (13)
n-Propyl 6-amino-2,6-dideoxy-2,2-difluoro-β-D-
glucopyranoside (8)
To an ice-cold solution of 11 (4.21 g, 10.06 mmol) in dry
ether (40 mL) and THF (40 mL) lithium aluminum hydride
(687 mg, 18.111 mmol) was added in several portions, and
the suspension was stirred for 15 min at 25 °C. Then, the
suspension was heated under reflux, and a solution of dry
aluminum chloride (2.28 g, 17.11 mmol) in dry ether
(30 mL) was added followed by stirring under reflux for
72 h. The suspension was cooled to 25 °C, methanol
(30 mL) was carefully added, and stirring was continued for
another 45 min. Usual aqueous workup followed by chro-
matography (silica gel, n-hexane/ethyl acetate, 5:3) gave 12
(490 mg, 12%) and 13 (1.82 g, 43%) each as a viscous oil;
Data for 12: [α]D = +14.57° (c 0.36, CHCl3); Rƒ (n-
hexane/ethyl acetate, 5:3) = 0.47; analysis calcd for
C23H26O5F2 (420.45): C 65.70, H 6.23; found: C 65.59,
H 6.46.
A solution of 7 (280 mg, 0.69 mmol) in dry MeOH
(20.0 mL) containing palladium on charcoal (10%, 270 mg)
was hydrogenated (30 °C, 2.43 atm, 48 h). The solution was
filtered through a pad of Celite; the pad was rinsed with
methanol (4 × 50 mL). The combined organic layers were
dried (MgSO4), the solvent was removed under reduced
pressure, and the residue was subjected to chromatography
(silica gel, methanol/ethyl acetate, 20:80) to afford 8
(80 mg, 48%) as a white foam; [α]D = −20.26° (c 1.19,
MeOH); Rƒ (methanol/ethyl acetate, 10:90) = 0.14; analysis
calcd for C9H17O4F2N (241.23): C 44.81, H 7.10, N 5.81;
found: C 44.69, H 7.32, N 5.69.
Allyl 2-O-benzyl-(R)-4,6-O-benzylidene-β-D-ribo-
hexopyranoside-3-ulose (10)
Data for 13: [α]D = −17.26° (c 0.42, CHCl3); Rƒ (n-hex-
ane/ethyl acetate, 5:3) = 0.61; analysis calcd for C23H26O5F2
(420.45): C 65.70, H 6.23; found: C 65.56, H 6.41.
To a mixture of dry DMSO (8.41 g, 107.64 mmol) and dry
DCM (72.0 ml) at −78 °C, a solution of trifluoroacetic
anhydride (16.05 g, 76.41 mmol) in dry DCM (18.0 mL)
was slowly added, and the mixture was stirred at this tem-
perature for 45 min. Then a solution of 9 (14.67 g,
36.82 mmol) in dry dichloromethane (40.0 mL) was added
dropwise, maintaining the temperature at −78 °C during
this addition. The mixture was stirred for 2 h at −78 °C,
Allyl 2,4-di-O-benzyl-3, 6-dideoxy-3,3-difluoro-6-iodo-β-D-
ribo-hexopyranoside (14)
To a solution of 12 (1.82 g, 4.33 mmol) in toluene (40 mL)
containing triphenylphosphane (2.50 g, 9.52 mmol) and

1106
Medicinal Chemistry Research (2021) 30:1099–1107
imidazole (1.33 g, 19.48 mmol) iodine (2.20 g, 3.66 mmol)
was added in several portions. After stirring at 90 °C for 1 h,
the reaction mixture was decanted and the remaining oil
was washed with ether (4 × 100 mL). The combined organic
layers were evaporated, and the remaining residue was
subjected to chromatography (silica gel, n-hexane/ethyl
acetate, 85:15) to afford 14 (1.80 g, 78%) as a colorless oil;
[α]D = +23.79° (c 0.36, CHCl3); Rƒ (n-hexane/ethyl acetate,
85:15) = 0.51; analysis calcd for C23H25O4F2I (530.34): C
52.09, H 4.75; found: C 51.84, H 4.93.
Author contributions RC and H-PD conceived the work, IS, EP, and CF
generated the data, all authors analyzed the data and wrote the paper.
Funding Open Access funding enabled and organized by
Projekt DEAL.
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Consent to publish All authors have agreed on the final version of
this paper.
Allyl 6-azido-2,4-di-O-benzyl-3,6-dideoxy-3,3-difluoro-β-D-
ribo-hexopyranoside (15)
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
To a solution of 14 (1.45 g, 2.73 mmol) in dry DMF
(29 mL) a solution of lithium azide (20% in water; 3.35 mL,
13.67 mmol) was added at 25 °C, and the solution was
stirred at this temperature for 4 days. The solvents were
removed under diminished pressure, and the remaining
residue was dissolved in DCM (100 mL) and water
(50 mL). The aq. phase was extracted with DCM (3 ×
100 ml), and the combined organic layers were dried
(Na2SO4). The solvent was removed under reduced pres-
sure, and the remaining residue was subjected to chroma-
tography (silica gel, n-hexane/ethyl acetate, 85:15) to afford
15 (1.16 mg, 77%) as a colorless oil; [α]D = +33.95° (c
0.40, CHCl3); Rƒ (n-hexane/ethyl acetate, 5:3) = 0.48;
analysis calcd for C23H25O4F2N3 (445.44): C 62.01, H 5.66,
N 9.43; found: C 61.86, H 5.71, N 9.21.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing,
adaptation, distribution and reproduction in any medium or format, as
long as you give appropriate credit to the original author(s) and the
source, provide a link to the Creative Commons license, and indicate if
changes were made. The images or other third party material in this
article are included in the article’s Creative Commons license, unless
indicated otherwise in a credit line to the material. If material is not
included in the article’s Creative Commons license and your intended
use is not permitted by statutory regulation or exceeds the permitted
use, you will need to obtain permission directly from the copyright
holder. To view a copy of this license, visit http://creativecommons.
org/licenses/by/4.0/.
References
1. Asano N. Glycosidase inhibitors: update and perspectives on
practical use. Glycobiology. 2003;13:93r–104r.
2. Karpas A, Fleet GWJ, Dwek RA, Petursson S, Namgoong SK,
Ramsden NG, et al. Aminosugar derivatives as potential anti-
human immunodeficiency virus agents. Proc Natl Acad Sci USA.
1988;85:9229–33.
n-Propyl 6-amino-3,6-dideoxy-3,3-difluoro-β-D-ribo-
hexopyranoside (16)
3. Butters TD, Dwek RA, Platt FM. Therapeutic applications of
imino sugars in lysosomal storage disorders. Curr Top Med Chem.
2003;3:561–74.
4. Dominguez LJ, Sowers JR. Metabolic syndrome therapy: pre-
vention of vascular injury by antidiabetic agents. Curr Hypertens
Rep. 2005;7:110–6.
5. Ducobu J. New developments in the treatment of diabetes and
hyperlipidemias. Rev Med Brux. 1998;19:A379–85.
6. Takada M. Molecular design of the carbohydrate active enzyme
inhibitors revealed by enzymatic methods. J Appl Glycosci.
2007;54:55–61.
A solution of 15 (200 mg, 0.45 mmol) in dry methanol
(20.0 mL) containing palladium on charcoal (10%, 200 mg)
was hydrogenated (25 °C, 2.43 atm, 24 h). Workup as
described above followed by chromatography (silica gel,
methanol/ethyl acetate, 20:80) gave 16 (100 mg, 93%) as a
white foam; [α]D = −27.95° (c 0.33, MeOH); Rƒ (methanol/
ethyl acetate, 20:80) = 0.13; analysis calcd for C9H17O4F2N
(241.23): C 44.81, H 7.10, N 5.81; found: C 44.65, H 7.34,
N 5.68.
7. Winchester B. Natural and synthetic inhibitors of glycosidases.
Biochem Soc Trans. 1992;20:699–705.
8. Parmeggiani C, Catarzi S, Matassini C, D’Adamio G, Morrone A,
Goti A, et al. Human acid beta-glucosidase inhibition by carbo-
hydrate derived iminosugars: towards new pharmacological cha-
perones for Gaucher disease. Chembiochem. 2015;16:2054–64.
9. Clemente F, Matassini C, Goti A, Morrone A, Paoli P, Cardona F.
Stereoselective synthesis of C-2 alkylated trihydroxypiperidines:
novel pharmacological chaperones for Gaucher disease. ACS Med
Chem Lett. 2019;10:621–6.
10. Dash RP, Babu RJ, Srinivas NR. Reappraisal and perspectives of
clinical drug-drug interaction potential of α-glucosidase inhibitors
such as acarbose, voglibose and miglitol in the treatment of type 2
diabetes mellitus. Xenobiotica. 2018;48:89–108.
Data availability
The data sets generated during and/or analyzed during the
current study are available from the corresponding author
on reasonable request.
Acknowledgements Many thanks are due to Dr. B. Siewert and Dr. S.
Schwarz for the cytotoxicity assays, Dr. D. Ströhl and Mrs. Y. Schiller
for measuring the NMR spectra, and to the late Dr. R. Kluge for the
MS spectra. The microanalyses as well as optical rotations and IR
spectra were taken by U. Lammel, M. Schneider, and S. Ludwig.

Medicinal Chemistry Research (2021) 30:1099–1107
1107
11. Ghani U. Re-exploring promising α-glucosidase inhibitors for
potential development into oral anti-diabetic drugs: finding needle
in the haystack. Eur J Med Chem. 2015;103:133–62.
12. Harit VK, Ramesh NG. Amino-functionalized iminocyclitols:
synthetic glycomimetics of medicinal interest. RSC Adv.
2016;6:109528–607.
13. Sugimoto S, Nakajima H, Kosaka K, Hosoi H. Review: miglitol
has potential as a therapeutic drug against obesity. Nutr Metab.
2015;12:51.
14. Dulsat C, Mealy N. Gaucher’s disease. Drugs Future. 2009;34:147–9.
15. Ficicioglu C. Review of miglustat for clinical management in
Gaucher disease type I. Ther Clin Risk Manag. 2008;4:425–31.
16. Pastores GM, Giraldo P, Cherin P, Mehta A. Goal-oriented ther-
apy with miglustat in gaucher disease. Curr Med Res Opin.
2009;25:23–37.
alpha-(1 -> 6)-mannosyltransferase. Tetrahedron Asymmetry.
2005;16:553–67.
34. Takahashi Y, Tsuneda S, Tsuchiya T, Koyama Y, Umezawa S.
Synthesis of 4’-deoxy-4’-fluorokanamycin-a and 4’-deoxy-4’-
fluorokanamycin-B. Carbohydr Res. 1992;232:89–105.
35. Brumshtein B, Aguilar-Moncayo M, Garcia-Moreno MI, Mellet
CO, Fernandez JMG, Silman I, et al. 6-Amino-6-deoxy-5,6-di-N-
(N‘-octyliminomethylidene)nojirimycin: synthesis, biological
evaluation, and crystal structure in complex with acid beta-
glucosidase. Chembiochem. 2009;10:1480–5.
36. Card PJ. Fluorinated carbohydrates—use of (diethylamino)sulfur
trifluoride in the synthesis of fluorinated sugars. J Org Chem.
1983;48:393–5.
37. Card PJ, Reddy GS. Fluorinated carbohydrates. 2. Selective
fluorination of glucopyranosides and mannopyranosides—use of
17. Belmatoug N, Di Rocco M, Fraga C, Giraldo P, Hughes D,
Lukina E, et al. Management and monitoring recommendations
for the use of eliglustat in adults with type 1 Gaucher disease in
Europe. Eur J Intern Med. 2017;37:25–32.
18. Smid BE, Hollak CEM. A systematic review on effectiveness and
safety of for type 1 Gaucher disease. Expert Opin Orphan Drugs.
2014;2:523–9.
19. Benito JM, Garciia Fernaandez JM, Mellet CO. Pharmacological
chaperone therapy for Gaucher disease: a patent review. Expert
Opin Ther Pat. 2011;21:885–903.
20. Alonzi DS, Scott KA, Dwek RA, Zitzmann N. Iminosugar antivirals:
the therapeutic sweet spot. Biochem Soc Trans. 2017;45:571–82.
21. Helquist P, Wiest O. Current status of drug therapy development
for Niemann-Pick type C disease. Drugs Future. 2009;34:315–31.
22. Lyseng-Williamson KA. Miglustat: a review of its use in
Niemann-Pick disease type C. Drugs. 2014;74:61–74.
23. Nash RJ, Kato A, Yu C-Y, Fleet GWJ. Iminosugars as therapeutic
agents: recent advances and promising trends. Future Med Chem.
2011;3:1513–21.
24. Perez-Poyato MS, Pineda M. New agents and approaches to
treatment in Niemann-Pick type C disease. Curr Pharm Bio-
technol. 2011;12:897–901.
25. Pineda M, Perez-Poyato MDS. Current and future therapies for
Niemann-Pick C disease. Expert Opin Orphan Drugs. 2013;1:915–23.
26. Stirnemann J, Belmatoug N, Camou F, Serratrice C, Froissart R,
Caillaud C, et al. A review of gaucher disease pathophysiology,
clinical presentation and treatments. Int J Mol Sci. 2017;18:441.
27. Venier RE, Igdoura SA. Miglustat as a therapeutic agent: pro-
spects and caveats. J Med Genet. 2012;49:591–7.
2-D Nmr for structural assignments.
1983;48:4734–43.
J
Org Chem.
38. Prajapat M, Shekhar N, Sarma P, Avti P, Singh S, Kaur H, et al.
Virtual screening and molecular dynamics study of approved
drugs as inhibitors of spike protein S1 domain and ACE2 inter-
action in SARS-CoV-2. J Mol Graph Model. 2020;101:107716.
39. Sencanski M, Perovic V, Pajovic SB, Adzic M, Paessler S, Glisic
S. Drug repurposing for candidate SARS-CoV-2 main protease
inhibitors by a novel in silico method. Molecules. 2020;25:3830.
40. Tariq A, Mateen RM, Afzal MS, Saleem M. Paromomycin: a
potential dual targeted drug effectively inhibits both spike (S1)
and main protease of COVID-19. Int
2020;98:166–75.
J
Infect Dis.
41. Hanessian S, Butterworth RF, Nakagawa T. Aminoglycoside
antibiotics—chemical transformation of paromamine into 3’-epi-
paromamine. Carbohydr Res. 1973;26:261–3.
42. Collins PM, Munasinghe VRN, Oparaeche NN. The photo-
chemistry of ketones derived from carbohydrates. Part 5. Photo-
chemical cross-pinacolization of acetalized pyranos-3-ulose
derivatives with methanol; a route to branched-chain sugar deri-
vatives. J Chem Soc Perkin Trans. 1977;1:2423–8.
43. Middleton WJ. New fluorinating reagents—dialkylaminosulfur
fluorides. J Org Chem. 1975;40:574–8.
44. Liptak A, Jodal I, Nanasi P. Stereoselective ring-cleavage of 3-O-
benzyl-4,6-O-benzylidenehexopyranoside and 2,3-Di-O-benzyl-
4,6-O-benzylidenehexopyranoside derivatives with Lialh4-Alcl3
reagent. Carbohydr Res. 1975;44:1–11.
45. Garegg PJ, Johansson R, Ortega C, Samuelsson B. Novel reagent
system for converting a hydroxy-group into an iodo-group in
carbohydrates with inversion of configuration. Part 3. J Chem Soc
Perkin Trans. 1982;1:681–3.
46. Ashby EC, Pham TN, Amrollahmadjdabadi A. Concerning the
mechanism of the reaction of Lialh4 with Alkyl-Halides. J Org
Chem. 1991;56:1596–603.
47. Saeeng R, Isobe M. Partial synthesis of ciguatoxin (5R)-ABC
segment. Tetrahedron Lett. 1999;40:1911–4.
48. Sanders WJ, Manning DD, Koeller KM, Kiessling LL. Synthesis
of sulfated trisaccharide ligands for the selectins. Tetrahedron.
1997;53:16391–422.
28. Wraith JE, Imrie J. New therapies in the management of Niemann-
Pick type C disease: clinical utility of miglustat. Ther Clin Risk
Manag. 2009;5:877–87.
29. Rajasekharan S, Bonotto RM, Kazungu Y, Alves LN, Poggianella M,
Orellana PM, et al., 2020. Repurposing of miglustat to inhibit the
coronavirus severe acquired respiratory syndrome SARS-CoV-2.
bioRxiv. 1–17. https://doi.org/10.1101/2020.05.18.101691.
30. Williams SJ, Goddard-Borger ED. α-glucosidase inhibitors as
host-directed antiviral agents with potential for the treatment of
COVID-19. Biochem Soc Trans. 2020;48:1287–95.
31. Kim C, Haddad J, Vakulenko SB, Meroueh SO, Wu Y, Yan H,
et al. Fluorinated aminoglycosides and their mechanistic impli-
cation for aminoglycoside 3 ‘-phosphotransferases from gram-
negative bacteria. Biochemistry. 2004;43:2373–83.
32. Pacak J, Hrinak J, Cerny M. Synthesis with anhydro sugars. 23.
Synthesis of 6-amino-2,6-dideoxy-2-fluoro-D-glucose. Collect
Czech Chem Commun. 1974;39:3332–7.
49. Skehan P, Storeng R, Scudiero D, Monks A, Mcmahon J, Vistica
D, et al. New colorimetric cytotoxicity assay for anticancer-drug
screening. J Natl Cancer I. 1990;82:1107–12.
50. Conchie J, Gelman AL, Levvy GA. Inhibition of glycosidases by
aldonolactones of corresponding configuration. The C-4- and C-6-
specificity of beta-glucosidase and beta-galactosidase. Biochem J.
1967;103:609–15.
33. Subramaniam V, Gurcha SS, Besra GS, Lowary TL. Synthetic
disaccharide analogs as potential substrates and inhibitors of a
mycobacterial polyprenol monophosphomannose-dependent
51. Gent PA, Gigg R. Allyl ether as a protecting group in
carbohydrate-chemistry. 8. Synthesis of Benzyl and Allyl Ethers
of D-glucopyranose. Carbohydr Res. 1976;49:325–33.