Synthetic deoxynojirimycin derivatives bearing a thiolated, fluorinated or unsaturated N-alkyl chain: Identification of potent α-glucosidase and trehalase inhibitors as well as F508del-CFTR correctors

Article abstract of DOI:10.1039/c5ob01526j

The synthesis of eleven 1-deoxynojirimycin (DNJ) derivatives presenting either a monofluoro, difluoro, thiolated or unsaturated N-alkyl chain of various length is described. Exploiting the unsaturated moiety on the nitrogen, fluorine has been introduced through a HF/SbF₅ superacid catalysed hydrofluorination and thiol-ene click chemistry allowed introduction of sulfur. The synthetic derivatives have been tested for their ability to inhibit glycosidases and correct F508del-CFTR. Two of the unsaturated iminosugars exhibited potency similar to Miglustat as F508del-CFTR correctors. The thioalkyl iminosugars as well as the corresponding alkyl iminosugars demonstrated low micromolar α-glucosidases and trehalases inhibition. Introduction of fluorine abolished F508del-CFTR correction and trehalase inhibition.

Full text of DOI:10.1039/c5ob01526j

Organic &
Biomolecular Chemistry
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Synthetic deoxynojirimycin derivatives bearing a
thiolated, fluorinated or unsaturated N-alkyl chain:
identification of potent α-glucosidase and
trehalase inhibitors as well as F508del-CFTR
correctors†
Cite this: DOI: 10.1039/c5ob01526j
V. Cendret,a T. Legigan,a A. Mingot,a S. Thibaudeau,a I. Adachi,b M. Forcella,c
P. Parenti,d J. Bertrand,e F. Becq,e C. Norez,e J. Désiré,*a A. Kato*b and Y. Blériot*a
The synthesis of eleven 1-deoxynojirimycin (DNJ) derivatives presenting either a monofluoro, difluoro,
thiolated or unsaturated N-alkyl chain of various length is described. Exploiting the unsaturated moiety on
the nitrogen, fluorine has been introduced through a HF/SbF5 superacid catalysed hydrofluorination and
thiol–ene click chemistry allowed introduction of sulfur. The synthetic derivatives have been tested for
their ability to inhibit glycosidases and correct F508del-CFTR. Two of the unsaturated iminosugars exhibi-
ted potency similar to Miglustat as F508del-CFTR correctors. The thioalkyl iminosugars as well as the
corresponding alkyl iminosugars demonstrated low micromolar α-glucosidases and trehalases inhibition.
Introduction of fluorine abolished F508del-CFTR correction and trehalase inhibition.
Received 23rd July 2015,
Accepted 3rd September 2015
DOI: 10.1039/c5ob01526j
www.rsc.org/obc
Introduction
The single replacement of the endocyclic oxygen by a nitrogen
atom in monosaccharides leads to iminosugars, a class of
sugar mimics with high therapeutic potential.1 Its most
famous representative, 1-deoxynojirimycin (DNJ), is a natural
product2 that exhibits potent α- and β-glucosidases inhibition.
This molecule has been since converted into two approved
medicines, Zavesca® and Miglitol® targeting Gaucher’s
disease3 and type II diabetes4 respectively. A rather simple
structural modification, the alkylation of the endocyclic nitro-
gen with either a butyl or a hydroxyethyl chain, has been
necessary to convert DNJ into these two therapeutics (Fig. 1).
Such a dramatic effect has prompted many research groups to
aUniversité de Poitiers, IC2MP, UMR CNRS 7285 Equipe“Synthèse Organique”4 rue
Michel Brunet, 86073 Poitiers cedex 09, France.
E-mail: Jerome.desire@univ-poitiers.fr, yves.bleriot@univ-poitiers.fr
bDepartment of Hospital Pharmacy, University of Toyama, 2630 Sugitani, Toyama
930-0194, Japan. E-mail: kato@med.u-toyama.ac.jp
cDepartment of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza
del Scienza 2, 20126 Milano, Italy
dDepartment of Earth and Environmental Sciences, University of Milano-Bicocca,
Fig. 1 Structure of DNJ and representative derivatives.
Piazza del Scienza 1, 20126 Milano, Italy
eLaboratoire STIM Signalisation et Transports Ioniques Membranaires, ERL7368
(0864)- CNRS/Université de Poitiers, TSA 51106 – 1, rue Georges Bonnet,
86073 Poitiers Cedex 9, France
introduce a vast array of functional groups at the endocyclic
nitrogen of DNJ including alkyl chains,5 elaborated substitu-
ents6 and fluorescent moieties7 to inactivate specific glycosyl
processing enzymes or act as probes to label these latter
†Electronic supplementary information (ESI) available: Copies of 1H, 19F and 13
C
spectra of all new compounds. See DOI: 10.1039/c5ob01526j
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(Fig. 1). Functionalized alkyl chains have been also introduced ditions that are not compatible so far with most of the sugar
to allow further chemical derivatisation including the popular protecting groups except the acetate.19 Satisfyingly, treatment
copper-catalyzed azide–alkyne cycloaddition (CuAAC).8 As a of iminosugars 4a and 4b in neat HF/SbF5 (7 : 1 ratio v : v) at
part of our continuous efforts in the area of iminosugars,9 we −20 °C for 10 min furnished the corresponding monofluori-
were interested in evaluating the impact of the introduction of nated iminosugars 5a and 5b as inseparable diastereomeric
an unsaturated N-alkyl chain on DNJ on its glycosidase inhibi- mixtures in satisfactory yield (52–62%) in which the fluorine
tory profile. Such modification might positively modify the atom is attached at the last but one carbon of the alkyl chain.
inhibition profile of this α-glucosidase inhibitor. This insatura- Subsequent peracetylation of the crude product was necessary
tion was further used as a handle to incorporate a sulfur atom to obtain the target sugar analogues in decent yield as some
or fluorine atoms in the alkyl chain.
deacetylation was observed during the process. The regio-
selectivity of the fluorination can be tentatively explained by
the formation of a transient superelectrophilic dication20 A,
the ammonium cation activating the nearby electrophilic
carbon cation allowing its fluorination, and thus despite the
very low nucleophilic character of the fluoride ions in their
antimony complexed forms.21 Introduction of a gem-difluoro-
methylene group starting from the alkynes 4c and 4d was next
examined and requires stronger fluorinating conditions that can
be achieved by increasing the SbF5/HF ratio and the temperature.
Treatment of alkynes 4c and 4d in neat HF/SbF5 (3 : 1 ratio) at
0 °C for 10 min furnished the corresponding gem-difluorinated
iminosugars 5c and 5d in satisfactory yield (59–93%). In this
case, two successive regioselective superacid-catalyzed hydro-
fluorinations, involving the superelectrophilic dications B and
C, account for the formation of the desired difluorinated
product.22 Final deacetylation of the fluorinated derivatives
5a–d required some tuning of the conditions as these com-
pounds are rather sensitive to base. While use of Zemplen con-
ditions (Na, MeOH) provided the starting alkenes and alkynes
through HF elimination, milder conditions (triethylamine,
methanol) furnished the desired fluorinated iminosugars 6a–d
in excellent yield (87–99%) (Scheme 2).
Results and discussion
Synthesis
Synthesis of unsaturated derivatives. The synthesis of the
DNJ derivatives displaying an unsaturated N-alkyl chain
started from the easily available tetra-O-benzyl DNJ 1.10
N-Alkylation of 1 with the allyl, homoallyl, propargyl and
homopropargyl bromides in acetonitrile or ethyl acetate/water
in the presence of K2CO3 under reflux yielded the corres-
ponding N-alkyl derivatives 2a–2d in good yields (64–86%). In
order to preserve the insaturation, removal of the benzyl
groups was achieved with BCl3 at −78 °C in dry CH2Cl2 to yield
the target new 3a and 3b and known 3c 11a and 3d 11b imino-
sugars in high yield. These iminosugars were also peracety-
lated to provide the tetraacetylated iminosugars 4a–d (67–93%
yield) available for further chemical transformation performed
on the N-alkyl chain (Scheme 1).
Synthesis of fluorinated derivatives. The role of fluorine in
medicinal chemistry is well recognized.12 For nitrogen contain-
ing compounds, the modification of the basicity of the nitro-
gen containing proximal functions, by using fluorine strong
electron withdrawing effect,13 has been largely used in medic-
inal chemistry SAR studies.14 It is also accepted that through a
fluorine gauche effect and through electrostatic interactions,
fluorine atoms can strongly modify the preferred conformation
of nitrogen containing biomolecules.15 Regarding imino-
sugars, introduction of fluorinated alkyl substituents on the
endocyclic nitrogen is scarce but yielded compounds with
promising biological activities.16 We reasoned that the un-
saturation on the alkyl chain could be used to perform a regio-
selective hydrofluorination17 using an in house expertise in
superacid chemistry.18 Such methodology generates harsh con-
Synthesis of thiolated derivatives. Terminal alkenes are
useful appendages to introduce additional functional groups
exploiting the thiol–ene click chemistry.23 This strategy has
Scheme 2 Synthesis of fluorinated N-alkyl DNJ 6a–d. Reagents and
conditions: (a) HF/SbF5 (7 : 1 v : v), −20 °C, 10 min then Ac2O, pyridine, rt,
overnight; (b) Et3N (4 eq.), MeOH, rt, 1–3 days; (c) HF/SbF5 (3 : 1 v : v),
0 °C, 10 min then Ac2O, pyridine, rt, overnight.
Scheme 1 Synthesis of unsaturated N-alkyl iminosugars 3a–d and per-
acetylated derivatives 4a–d. Reagents and conditions: (a) RBr, K2CO3,
CH3CN or EtOAc/H2O; (b) BCl3, CH2Cl2, −78 °C; (c) Ac2O, pyridine.
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been extensively used in carbohydrate chemistry24 but there is lysis, a reaction fundamental for insect flight31 and spore ger-
only one example involving iminosugars to the best of our mination of fungi. Due to the biological relevance of trehalase
knowledge.25 We thought this methodology could be helpful inhibitors as fungicides, insecticides or antibiotics, several tre-
to study the influence of the introduction of a sulfur atom in halose mimics have been synthesized.32 In this context, and to
the alkyl chain. Previous studies emphasized the positive role confirm the potency and evaluate the selectivity of compounds
played by a single oxygen atom introduced in the chain regard- 8a–c and 9b–d toward α,α-trehalases, these molecules were
ing glucosylceramide metabolism inhibition.26 To this end, tested for their inhibitory activity against insect trehalase of
homoallyl derivative 4b was treated with 3 equiv. of alkyl thiol midge larvae of C. riparius,33 a good model for biochemical
in the presence of catalytic 2,2-dimethoxy-2-phenylacetophe- studies and porcine kidney trehalase as the mammalian
none (DPAP) under UV irradiation to generate the expected counterpart. As shown from the IC50 values, compounds 8a–c
thioalkyl DNJ derivatives 7a–c in 57–81% yield. Final de- and 9b–d potently inhibit insect and mammalian trehalases
protection (Et3N, MeOH) furnished the desired iminosugars with inhibition in the low micromolar range. Unfortunately,
8a–c in 70–98% yield. For comparison purposes, the known no selectivity toward insect trehalases could be observed
N-butyl 9a (Zavesca®)3 N-hexyl 9b,5b N-nonyl 9c 27 and N- (Table 1).
dodecyl 9d 5b DNJ derivatives (Scheme 3) were also synthesized
Correction of F508del-CFTR function. Iminosugars have
been identified as pharmacological chaperones that can stabil-
ize or correct the structure of misfolded proteins. The Cystic
Fibrosis Transmembrane conductance Regulator (CFTR)
protein is glycosylated, even though it does not involve any
sugar metabolism; CFTR is an ABC transporter-class protein
and ion channel that transports chloride ions across the apical
membrane of epithelial cells. Mutations of the CFTR gene
affect folding and/or functioning of the CFTR chloride chan-
nels in these cell membranes, causing Cystic Fibrosis (CF).
The most common CF mutation F508del causes misfolding of
the protein and intracellular retention by the endoplasmic reti-
culum quality control and premature degradation; iminosu-
gars may help in the trafficking of the misfolded protein.
Miglustat, its multivalent derivatives34 and branched pyrroli-
dine isoLAB35 have been found to show significant rescue of
the defective F508del-CFTR function as assessed by single-cell
fluorescence imaging and/or iodide effluxes.36 N-Alkyl DNJ
derivatives 3a–d, 6a–d and 9b–d 37 were compared to miglustat
for their corrector effect on CFTR function in CF-KM4 cells38
using iodide effluxes (Fig. 2).39 Results obtained with 9b–d
indicate that a four carbon butyl chain is optimal to provide
good correctors, higher chains being detrimental to correction.
Introduction of fluorine on the chain as in 6a–d also abolishes
F508del-CFTR correction. Interestingly, the unsaturated deriva-
tives 3a–d proved the most promising derivatives amongst
which the N-homoallyl DNJ 3b and the N-propargyl DNJ 3c
showed similar activity to Miglustat.
according to literature procedures.28
Biological activity
Inhibition of glycosidases. Polyhydroxylated piperidines
3a–d, 6a–d, 8a–c, 9a–d were assayed as inhibitors of a collec-
tion of sixteen glycosidases, including glucosidases, galactosi-
dases, mannosidases, fucosidases, glucuronidase, trehalase,
amyloglucosidase and rhamnosidase. All compounds proved
to retain the α-glucosidase inhibition of DNJ, being submicro-
molar inhibitors of rice α-glucosidase and low micromolar
inhibitors of rat intestinal maltase. Most of them also demon-
strated low micromolar inhibition of HL60 glucosyl transferase
(Table S1, see ESI†). Interestingly, while unsaturated and
fluorinated derivatives 3a–c and 6a–d poorly inhibited α,α-
trehalase, Aspergillus niger and Rhizopus amyloglucosidases,
almond β-glucosidase and α-L-rhamnosidase, the N-thioalkyl
derivatives 8a–c showed some inhibition toward these
enzymes and were especially potent toward porcine kidney
α,α-trehalase. Similar potency was observed for the corres-
ponding C-alkyl derivatives 9b–d (Table S2, see ESI†). This
result forced us to explore this trehalase inactivation further.
Trehalase inhibition. Trehalase is an inverting glycosidase29
belonging to the GH37 family of the carbohydrate-active
enzyme (CAZy) classification30 that catalyses trehalose hydro-
Table 1 Effect of compounds 8a–c and 9b–d on insect and mamma-
lian trehalases
IC50 C. riparius trehalase
(μM)
IC50 porcine trehalase
(μM)
Compound
8a
8b
8c
2.19 0.32
1.46 0.04
3.75 0.28
5.87 0.31
6.94 0.36
1.99 0.15
1.21 0.30
0.46 0.05
1.66 0.25
2.22 0.04
0.94 0.06
0.27 0.06
Scheme 3 Synthesis of thiolated iminosugars 8a–c and structure of N- 9b
9c
9d
alkyl DNJ 9a–d. Reagents and conditions: (a) alkyl thiol, DPAP, MeOH,
hν, rt, 30–60 min; (b) Et3N (7 eq.), MeOH, rt, 1–3 days.
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was then evaporated under reduced pressure, co-evaporated
with toluene, then freeze dried to provide the deprotected
compound.
2,3,4,6-Tetra-O-benzyl-N-(3-prop-1-enyl)-1-deoxynojirimycin
2a. K2CO3 (5 eq., 17.67 mmol, 2.44 g) was added to a stirred
solution of 1 (3.53 mmol, 1.85 g) and allylbromide (2.5 eq.,
883 mmol, 769 µL) in a mixture EtOAc : H2O (88/11 mL). The
reaction mixture was refluxed for 24 h and the aqueous layer
was extracted then back-extracted with EtOAc (2 × 20 mL). The
combined organic layers were dried over MgSO4, filtered and
concentrated. The resulting residue was purified by silica gel
column chromatography (9 : 1 to 8 : 2, PE : EtOAc) to provide 2a
(1.60 g, 80%). Rf = 0.48 (85 : 15, PE : EtOAc); [α]2D0 = +1.0 (c =
0.30, CHCl3); 1H NMR (400 MHz, CDCl3), δ: 7.39–7.28 (m, 18H,
HAr Bn), 7.17–7.15 (m, 2H, HAr Bn), 5.95–5.85 (m, 1H, H-2′),
5.16 (dd, 2H, J = 10.1 Hz, J = 17.2 Hz, H–3′), 5.00 (d, 1H, J =
11.1 Hz, CHHPh), 4.91 (d, 1H, J = 10.8 Hz, CHHPh), 4.85 (d,
1H, J = 11.1 Hz, CHHPh), 4.70 (dd, 2H, J = 11.6 Hz, CH2Ph),
4.52 (bs, 2H, CH2Ph), 4.44 (d, 1H, J = 10.8 Hz, CHHPh),
3.76–3.61 (m, 4H, H-2, H-4, H-6a, H-6b), 3.50 (t, 1H, J3–2 = J3–4
= 9.1 Hz, H–3), 3.43 (dd, 1H, J1′a–2 = 5.4 Hz, J1′a–1′b = 14.3 Hz,
H-1′a), 3.25–3.20 (m, 1H, H-1′b), 3.16 (dd, 1H, J1a–2 = 4.9 Hz,
J1a–1b = 11.4 Hz, H-1a), 2.38–2.31 (m, 1H, H-5), 2.23 (t, 1H,
J1b–1a = J1b–2 = 10.9 Hz, H–1b); 13C NMR (100 MHz, CDCl3), δ:
139.1, 138.6, 137.9 (4 × Cq Bn), 133.4 (C-2′), 128.53, 128.50,
128.45, 128.41, 128.40, 127.98, 127.96, 127.90, 127.71, 127.62,
127.53 (CHAr·Bn), 118.7 (C-3′), 87.4 (C-3), 78.6, 78.5 (C-2, C-4),
75.4, 75.3, 73.6, 72.8 (4 × CH2Ph), 65.3 (C-6), 64.0 (C-5), 55.7
(C-1′), 54.6 (C-1); HRMS (ESI+): m/z [M + H]+ calculated for
C37H42NO4 564.3108, found 564.3131.
Fig. 2 F508del-CFTR correction by DNJ derivatives 3a–d, 6a–d and
9b–d.
Experimental
General methods
All commercial reagents were used as supplied. TLC plates
were visualized under 254 nm UV light and/or by dipping the
TLC plate into a solution of phosphomolybdic acid in ethanol
(3 g per 100 mL) followed by heating with a heat gun. Flash
columns chromatographies were performed using silica gel 60
(15–40 μm). NMR experiments were recorded with a 400
1
Bruker spectrometer at 400 MHz for H, 376 MHz for 19F and
100 MHz for 13C nuclei. The chemical shifts are expressed in
part per million (ppm) relative to TMS (δ = 0 ppm) and the
coupling constant J in hertz (Hz). NMR multiplicities are
reported using the following abbreviations: b = broad, s =
singlet, d = doublet, t = triplet, q = quadruplet, m = multiplet.
HRMS were obtained with a Q-TOF spectrometer. The melting
points were recorded with a SMP3 Stuart Scientific melting
point apparatus. Optical rotations were measured using a
Perkin-Elmer 341 polarimeter.
N-(3-Prop-1-enyl)-1-deoxynojirimycin 3a. A solution of BCl3
(1M in DCM) (20 eq., 43.17 mmol, 43.17 mL) was added slowly
to a stirred solution of compound 2a (2.70 mmol, 1.52 g) in
dry DCM (47 mL) at −78 °C. The reaction mixture was stirred
overnight at −78 °C then quenched by addition of MeOH. The
solvent was then evaporated. The residue was taken up in
water (15 mL) and extracted with DCM (2 × 30 mL). The
aqueous phase was then evaporated to afford 3a (550 mg) in
quantitative yield as a colourless oil. [α]2D0 = +8.7 (c = 0.54,
MeOH); 1H NMR (400 MHz, MeOD), δ: 6.08–5.97 (m, 1H, H-2′),
5.69–5.63 (m, 2H, H-3′a, H-3′b), 4.13 (d, 1H, J6a–6b = 12.3 Hz,
H-6a), 4.07 (dd, 1H, J1′a–2′ = 5.3 Hz, J1′a–1′b = 12.6 Hz, H-1′a),
3.96 (d, 1H, J6b–6a = 12.3 Hz, H-6b), 3.85 (dd, 1H, J1′b–2′ = 7.7
Hz, J1′b–1′a = 11.5 Hz, H-1′b), 3.72–3.66 (m, 1H, H-2), 3.61 (t,
1H, J4–3 = J4–5 = 9.5 Hz, H-4), 3.41 (dd, 1H, J1a–2 = 4.5 Hz, J1a–1b
= 11.8 Hz, H-1a), 3.38–3.33 (m, 1H, H-3), 3.06–3.01 (m, 1H,
H-5), 2.93 (t, 1H, J = 11.7 Hz, H–1b); 13C NMR (100 MHz,
MeOD), δ: 127.5 (C-3′), 127.0 (C-2′), 78.1 (C-3), 68.8 (C-4), 67.7
(C-2), 67.1 (C-5), 56.6 (C-1′), 54.8 (C-6), 54.7 (C-1); HRMS (ESI+):
m/z [M + H]+ calculated for C9H18NO4 204.1230, found
204.1242.
The authors draw the reader’s attention to the dangerous
features of superacidic chemistry. Handling of hydrogen fluor-
ide and antimony pentafluoride must be done by experienced
chemists with all the necessary safety arrangements in place.
Experiments performed in superacid were carried out in a
sealed Teflon® flask with a magnetic stirrer. No further pre-
cautions have to be taken to prevent mixture from moisture
(test reaction worked out in anhydrous conditions leads to the
same results as expected).
General procedure A for TEC reaction
To a solution of N-butenyl-2,3,4,6-tetra-O-acetyl-1-deoxynojiri-
mycin in degassed (10 min under Ar atmosphere) MeOH (C =
6 × 10−2 mol L−1) were added alkylthiol (3.0 eq.) and DPAP (0.5
eq.). The reaction mixture was then irradiated for 30 min with
a UV facial tanner (12 lamps × 15 W) under Ar atmosphere.
After concentration, the crude was purified by silica gel
column chromatography.
General procedure B for deprotection of acetylated compounds
obtained after TEC reactions
2,3,4,6-Tetra-O-acetyl-N-(3-prop-1-enyl)-1-deoxynojirimycin
4a. Acetic anhydride (10 eq., 10.8 mmol, 1.01 mL) was added
Et3N (7.2 eq.) was added to a suspension of the acetylated com- dropwise to a solution of compound 3a (1.07 mmol, 219 mg)
pound in MeOH (C = 10 × 10−2 mol L−1) and the mixture was in pyridine (15 mL) at 0 °C. The mixture was warmed up to
stirred for three to four days at room temperature. The solvent room temperature and after 15 h the reaction was quenched by
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the addition of MeOH at 0 °C and evaporated. The residue was (15 mL) and extracted with DCM (2 × 15 mL). The aqueous
dissolved in EtOAc (30 mL) then successively washed with satu- phase was then evaporated to afford 3b (285 mg) in quantitat-
rated aq. NH4Cl (20 mL), NaHCO3 (20 mL) and water (20 mL), ive yield as a white solid. [α]2D0 = −1.2 (c = 0.81, MeOH); 1H
dried over sodium sulfate, filtered and concentrated. The NMR (400 MHz, D2O), δ: 5.77–5.67 (m, 1H, H-3′), 5.14 (dd, 2H,
resulting residue was purified by silica gel column chromato- J4′a–4′b = 1.5 Hz, J4′b–3′ = 17.2 Hz, J4′a–3′ = 10.3 Hz, H-4′a, H-4′b),
graphy (combiflash 100%PE to 100% EtOAc) to give 4a 4.01 (d, 1H, J6a–6b = 13.7 Hz, H-6a), 3.91 (dd, 1H, J6b–6a = 13.4
(368 mg, 92%). Rf = 0.68 (6 : 4, PE : EtOAc); [α]2D0 = +19.2 (c = Hz, J6b–5 = 2.5 Hz, H-6b), 3.75–3.69 (m, 1H, H-2), 3.61–3.51 (m,
1
0.38, CHCl3); H NMR (400 MHz, CDCl3), δ: 5.86–5.76 (m, 1H, 2H, H-4, H-1a), 3.47–3.38 (m, 2H, H-1′a, H-3), 3.23–3.17 (m,
H-2′), 5.23–5.19 (m, 2H, H-3′a, H-3′b), 5.09–5.05 (m, 1H, H-4), 1H, H-1′b), 3.15–3.12 (m, 1H, H-5), 3.03 (t, 1H, J1b–1a = J1b–2
=
5.05–4.93 (m, 2H, H-3, H-2), 4.21 (dd, 1H, J6a–5 = 2.2 Hz, H-6a), 11.9 Hz, H-1b), 2.54–2.39 (m, 2H, H-2′a, H-2′b); 13C NMR
4.12 (dd, 1H, J6b–6a = 22.9 Hz, J6b–5 = 3.1 Hz, H-6b), 3.40 (ddt, (100 MHz, D2O), δ: 133.4 (C-3′), 119.1 (C-4′), 77.6 (C-3), 68.5
1H, J1′a–1′b = 14.6 Hz, J1′a–2′ = 5.8 Hz, J1′a–3′ = 1.3 Hz, H-1′a), (C-4), 67.5, 67.3 (C-2, C-5), 55.0 (C-6), 54.6 (C-1), 53.3 (C-1′),
3.21–3.15 (m, 2H, H-1′b, H-1a), 2.64–2.60 (m, 1H, H-5), 2.29 28.4 (C-2′); HRMS (ESI+): m/z [M
(dd, 1H, J1b–1a = 10.1 Hz, J1b–2 = 9.8 Hz, H-1b), 2.07, 2.01, 2.0, C10H20NO4 218.1386, found 218.1390.
+
H]+ calculated for
1.99 (CH3COO); 13C NMR (100 MHz, CDCl3), δ: 171.0, 170.5,
2,3,4,6-Tetra-O-acetyl-N-(4-but-1-enyl)-1-deoxynojirimycin 4b.
170.1, 169.8 (4 × CH3COO), 132.7 (C-2′), 119.4 (C-3′), 74.8 (C-3), Acetic anhydride (10 eq., 14.04 mmol, 1.32 mL) was added
69.5 (C-4), 69.4 (C-2), 61.5 (C-5), 59.4 (C-6), 55.3 (C-1′), 53.1 dropwise to a solution of N-butenyl-1-deoxynojorimycin 3b
(C-1), 20.9, 20.9, 20.8, 20.7 (4 × CH3COO); HRMS (ESI+): (1.4 mmol, 305 mg) in pyridine (25 mL) at 0 °C. The mixture
m/z [M + H]+ calculated for C17H26NO8 372.1653, found was warmed up to room temperature and after 15 h the reac-
372.1655.
tion was quenched by the addition of MeOH at 0 °C and then
2,3,4,6-Tetra-O-benzyl-N-(4-but-1-enyl)-1-deoxynojirimycin 2b. evaporated. The residue was dissolved in EtOAc (30 mL) then
To a solution of 2,3,4,6-tetra-O-benzyl-1-deoxynojirimyin 1 successfully washed with saturated aq. NH4Cl (20 mL),
(2.73 mmol, 1.43 g) and 4-bromobutene (3 eq., 3.32 mmol, NaHCO3 (20 mL) and water (20 mL), dried over sodium sulfate,
832 µL) in dry acetonitrile (13.5 mL) was added potassium car- filtered and concentrated. The resulting residue was purified
bonate (2.1 eq., 5.74 mmol, 793 mg). The reaction mixture was by silica gel column chromatography (7 : 3, PE : EtOAc) to
stirred at 82 °C for 12 h under argon then cooled. Most of the provide 4b (507 mg, 93%). Rf = 0.34 (7 : 3, PE : EtOAc); [α]D20
=
acetonitrile was evaporated under reduced pressure. Water and +10.9 (c = 0.34, CHCl3); 1H NMR (400 MHz, CDCl3), δ:
dichloromethane were added to the residue and the whole 5.76–5.66 (m, 1H, H-3′), 5.11–4.94 (m, 4H, H-4′a, H-4′b, H-3,
mixture was stirred for 10 min and then portioned. The H-4), 4.89–4.82 (m, 1H, H-2), 4.21 (dd, 1H, J6a–6b = 13.1 Hz,
aqueous layer was extracted with dichloromethane (3 times). J6a–5 = 2.1 Hz, H-6a), 4.13 (dd, 1H, J6b–6a = 13.1 Hz, J6b–5 = 3.5
The combined organic extracts were dried over sodium sulfate, Hz, H-6b), 3.17 (dd, 1H, J1a–1b = 11.3 Hz, J1a–2 = 5.3 Hz, H-1a),
filtered and concentrated under reduced pressure. The result- 2.90–2.77 (m, 2H, H-1′a, H-1′b), 2.75–2.70 (m, 1H, H-5), 2.48
ing residue was purified by silica gel column chromatography (dd, 1H, J1b–1a = 10.3 Hz, J1b–2 = 1.1 Hz, H-1b), 2.28–2.22 (m,
(100% PE to 8 : 2, PE : EtOAc) to provide 2b (1.35 g, 86%). Rf = 2H, H-2′a, H-2′b), 2.00, 1.98, 1.97, 1.94 (4s, 4 × 3H, CH3COO);
0.85 (8 : 2, PE : EtOAc); [α]2D0 −2.3 (c = 0.30, CHCl3); 1H NMR 13C NMR (100 MHz, CDCl3), δ: 171, 170.4, 170.1, 169.8 (4 ×
(400 MHz, CDCl3), δ: 7.37–7.28 (m, 18H, HAr Bn), 7.16 (dd, 2H, CH3COO), 135.6 (C-3′), 116.6 (C-4′), 74.7 (C-3 or C-4), 69.5, 69.4
J = 2.1 HZ, J = 7.8 Hz, HAr Bn), 5.76–5.66 (m, 1H, H-3′), (C-2, C-3 or C-4), 61.2 (C-5), 59.7 (C-6), 53.0 (C-1′), 51.3 (C-1),
5.06–4.98 (m, 3H, H-4′a, H-4′b, CHHPh), 4.88 (dd, 2H, J = 10.8 29.3 (C-2′), 21.0, 20.9, 20.8, 20.8 (4×CH3COO); [α]2D0 = +10.8° (c
Hz, J = 11.1 Hz, CH2Ph), 4.70 (dd, 2H, J = 11.3, CH2Ph), 4.51 = 0.34, CHCl3); HRMS (ESI+): m/z [M + H]+ calculated for
(bs, 2H, CH2Ph), 4.44 (d, 1H, J = 10.6 Hz, CHHPh), 3.71–3.47 C18H28NO8 386.1809, found 386.1811.
(m, 4H, H-2, H-4, H-6a, H-6b), 3.49 (t, 1H, J3–2 = J3–4 = 8.9 Hz,
2,3,4,6-Tetra-O-benzyl-N-(3-prop-1-ynyl)-1-deoxynojirimycin
H-3), 3.13 (dd, 1H, J1a–1b = 11.3 Hz, J1a–2 = 4.5 Hz, H-1a), 2c. To a solution of 2,3,4,6-tetra-O-benzyl-1-deoxynojirimyin 1
2.84–2.71 (m, 2H, H-1′a, H-1′b), 2.39–2.35 (m, 1H, H-1b), (3.43 mmol, 1.80 g) and propargyl bromide (5.16 mmol,
2.26–2.10 (m, 2H, H-2′a, H-2′b); 13C NMR (100 MHz, CDCl3), δ: 575 µL) in dry acetonitrile (17.2 mL) was added potassium car-
139.1, 138.6, 137.5 (4 × Cq Bn), 136.3 (C-3′), 128.5, 128.5, 128.4, bonate (7.2 mmol, 998 mg). The reaction mixture was stirred at
128.4, 128.0, 127.9, 127.9, 127.7, 127.6, 127.5 (CHAr·Bn), 116.0 82 °C for 24 h under argon then cooled. Most of the aceto-
(C-4′), 87.4 (C-3), 78.7, 78.6 (C-2, C-4), 75.4, 75.3, 73.6, 75.3 (4 × nitrile was evaporated under reduced pressure. Water (50 mL)
CH2Ph), 65.5 (C-6), 63.5 (C-5), 54.5 (C-1), 51.9 (C-1′), 28.3 (C-2′); and dichloromethane (50 mL) were added to the residue and
HRMS (ESI+): m/z [M + H]+ calculated for C38H44NO4 578.3264, the whole mixture was stirred for 10 min and then portioned.
found 578.3266.
The aqueous layer was extracted with dichloromethane (3
N-(4-But-1-enyl)-1-deoxynojirimycin 3b. A solution of BCl3 times). The combined organic extracts were dried over sodium
(1M in DCM) (20 eq., 26.11 mmol, 26.11 mL) was added slowly sulfate, filtered and concentrated under reduced pressure. The
to a stirred solution of 2b (1.3 mmol, 754 mg) in dry DCM resulting residue was purified by silica gel column chromato-
(20 mL) at −78 °C. The reaction mixture was stirred overnight graphy (Combiflash 100%PE to 100% EtOAC) to provide 2c
at −78 °C then quenched by addition of MeOH. The solvent (1.23 g, 64%). Rf = 0.24 (85 : 15, PE : EtOAc); [α]2D0 = −13.0 (c =
was then evaporated. The residue was taken up in water 0.33, CHCl3); 1H NMR (400 MHz, CDCl3), δ: 7.36–7.26 (m, 18H,
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HAr Bn), 7.12–7.10 (m, 2H, HAr Bn), 4.97 (d, 1H, J = 10.7 Hz, 75.1 (C-2′), 74.4 (C-3 or C-4), 69.4 (C-2), 69.1 (C-3 or C-4), 60.0
CHHPh), 4.88 (d, 1H, J = 10.6 Hz, CHHPh), 4.83 (d, 1H, J = 10.9 (C-5), 58.8 (C-6), 53.9 (C-1), 42.5 (C-1′), 20.9, 20.9, 20.8, 20.8
Hz, CHHPh), 4.68 (d, 1H, J = 11.6 Hz, CHHPh), 4.64 (d, 1H, J = (4 × CH3COO); HRMS (ESI+): m/z [M + H]+ calculated for
11.5 Hz, CHHPh), 4.55 (d, 1H, J = 12.1 Hz, CHHPh), 4.43 (d, C17H24NO8 370.1496, found 370.1501.
1H, J = 12.2 Hz, CHHPh), 4.35 (d, 1H, J = 10.8 Hz, CHHPh),
2,3,4,6-Tetra-O-benzyl-N-(4-but-1-ynyl)-1-deoxynojirimycin 2d.
3.77–3.70 (m, 3H, H-2, H-1′a, H-6a), 3.65 (t, 1H, J4–3 = J4–5 = 9.2 To a solution of 2,3,4,6-tetra-O-benzyl-1-deoxynojirimycin 1
Hz, H-4), 3.58 (dd, 1H, J6b–6a = 10.6 Hz, J6b–5 = 2.6 Hz, H-6b), (400 mg, 0.764 mmol) and 3-butynyl p-toluenesulfonate
3.49 (t, 1H, J3–2 = J3–4 = 9.2 Hz, H-3), 3.40 (dd, 1H, J1′a–1′b = 17.5 (514 mg, 2.29 mmol) in acetonitrile (4 mL) was added potass-
Hz, J1′b–3′ = 1.9 Hz, H-1′b), 2.98 (dd, 1H, J1ax–1eq = 11 Hz, J1ax–2
=
ium carbonate (317 mg, 2.29 mmol). The reaction mixture was
5 Hz, H-1ax), 2.55 (dd, 1H, J1eq–ax = 11 Hz, J1eq–2 = 10.6 Hz, H-1 stirred at 85 °C for 48 h and then cooled. Acetonitrile was evap-
eq.), 2.43 (ddd, 1H, J5–4 = 9.2 Hz, J5–6a = 2.2 Hz, J5–6b = 1.8 Hz, orated under reduced pressure. The residue was dissolved in
H-5), 2.22 (t, 1H, J3′–1′ = 2.2 Hz, H-3′); 13C NMR (100 MHz, CH2Cl2 (50 mL) and water (50 mL). The aqueous layer was
CDCl3), δ: 139.0, 138.6, 138.5, 137.7 (4 × Cq Bn), 128.6, 128.6, extracted three times with CH2Cl2. The combined organic
128.5, 128.5, 128.4, 128.1, 128.1, 128.0, 127.9, 127.8, 127.7, layers were dried over MgSO4, filtrated and concentrated under
127.6 (CHArBn), 87.2 (C-3), 78.2 (C-2), 75.6, 75.3 (2xCH2Ph), reduced pressure. Purification by flash chromatography (PE/
74.3 (C-2′), 73.8, 72.9 (2xCH2Ph), 64.8 (C-6), 62.2 (C-5), 55.1 EtOAc 95 : 5 to 90 : 10) afforded compound 2d (330 mg, 75%)
1
(C-1), 42.4 (C-1′); HRMS (ESI+): m/z [M + H]+ calculated for as a brown solid. Mp: 69 °C; [α]D = +9.1 (c = 1.16, CHCl3); H
C37H40NO4 562.2952, found 562.2969.
NMR (400 MHz, CDCl3), δ: 7.37–7.24 (m, 18H, ArH), 7.13–7.11
N-(3-Prop-1-ynyl)-1-deoxynojirimycin 3c. A solution of BCl3 (m, 2H, ArH), 4.96 (d, 1H, J = 11.1 Hz, CH2Ph), 4.87 (d, 1H, J =
(1 M in DCM) (20 eq., 31.6 mmol, 31.6 mL) was added slowly 10.8 Hz, CH2Ph), 4.82 (d, 1H, J = 11.0 Hz, CH2Ph), 4.71–4.64
to a stirred solution of compound 2c (1.97 mmol, 1.11 g) in (m, 2H, CH2Ph), 4.50 (dd, 2H, J = 11.9 Hz, J = 16.4 Hz, CH2Ph),
dry DCM (34 mL) at −78 °C. The reaction mixture was stirred 4.38 (d, 1H, J = 10.8 Hz, CH2Ph), 3.72–3.61 (m, 3H, H-2, H-6),
overnight at −78 °C then quenched by addition of MeOH. The 3.55 (t, 1H, J = 9.0 Hz, H-4), 3.46 (t, 1H, J = 9.0 Hz, H-3),
solvent was then evaporated. The residue was taken up in 3.11–2.92 (m, 3H, 2H-a, 1H-1), 2.43–2.30 (m, 4H, 2H-b, 1H-1,
water (15 mL) and extracted with DCM (2 × 30 mL). The 1H-5), 1.97 (t, 1H, J = 2.6 Hz, H-d); 13C NMR (100 MHz, CDCl3),
aqueous phase was then evaporated to give 3c (400 mg) as a δ: 139.0, 138.5 (2C), 137.8 (4ArC), 128.6–127.6 (20ArCH), 87.3
brown solid in quantitative yield. [α]2D0 = +3.0 (c = 0.54, MeOH); (C-3), 82.7 (C-3′), 78.5, 78.4 (C-2, C-4), 75.5, 75.3, 73.6, 72.9
1H NMR (400 MHz, MeOD), δ: 4.31 (d, 1H, J1′a–3′ = 2.8 Hz, (4CH2Ph), 69.6 (C-4′), 65.8 (C-6), 63.1 (C-5), 54.5 (C-1), 51.2
J1′a–1′b = 17 Hz, H-1′a), 4.25 (d, 1H, J1′b–3′ = 2.5 Hz, J1′b–1′a
=
(C-1′), 14.1 (C-2′); HRMS (ESI+): m/z [M + Na]+ calculated for
17 Hz, H-1′b), 4.14 (bd, 1H, J6a–6b = 12.5 Hz, H-6a), 3.88 (dd, C38H41NNaO4 598.2928, found 598.2930.
1H, J6b–6a = 12.5 Hz, J6b–5 = 2.8 Hz, H-6b), 3.73–3.67 (m, 1H,
N-(4-But-1-ynyl)-1-deoxynojirimycin 3d. A solution of BCl3
H-2 or H-4), 3.64–3.57 (m, 2H, H-1a, H-2 or H-4), 3.45 (t, 1H, (1 M in CH2Cl2) (5.28 mL, 5.28 mmol) was added slowly to a
J3′–1′ = 2.5 Hz, H-3′), 3.38–3.34 (m, 1H, H-3), 3.18–3.13 (m, 2H, stirred solution of compound 2d (152 mg, 0.264 mmol) in dry
H-1b, H-5); 13C NMR (100 MHz, MeOD), δ: 82.2 (C-2′), 78.2 CH2Cl2 (3.9 mL) at −78 °C. The reaction mixture was stirred
(C-3), 72.1 (C-3′), 68.5 (C-2 or C-4), 67.7 (C-2 or C-4), 66.8 (C-5), for 40 hours at −78 °C then quenched by addition of MeOH.
55.4 (C-1), 54.7 (C-6), 43.9 (C-1′); HRMS (ESI+): m/z [M + H]+ cal- The solvent was evaporated, the residue was taken up in water
culated for C9H16NO4 202.1074, found 202.1082.
(15 mL) and extracted extensively with EtOAc (10 × 15 mL). The
2,3,4,6-Tetra-O-acetyl-N-(3-prop-1-ynyl)-1-deoxynojirimycin 4c. aqueous phase was then evaporated under reduced pressure to
Acetic anhydride (10 eq., 7.45 mmol, 705 μL) was added drop- afford compound 3d (48 mg, 85%) as a brown oil. [α]D = +6.2
1
wise to a solution of 3c (0.745 mmol, 150 mg) in pyridine (c = 0.96, MeOH); H NMR (400 MHz, MeOD), δ: 4.11 (d, 1H,
(10 mL) at 0 °C. The mixture was warmed up to room tempera- J = 12.5 Hz, 1H-6), 3.96 (dd, 1H, J = 2.4 Hz, J = 12.5 Hz, 1H-6),
ture and after 15 h the reaction was quenched by the addition 3.75 (m, 1H, H-2), 3.64–3.51 (m, 3H, H-1′, H-1, H-4), 3.48–3.38
of MeOH at 0 °C and evaporated. The residue was dissolved in (m, 2H, H-1′, H-3), 3.19 (m, 1H, H-5), 3.10 (t, 1H, J = 11.6 Hz,
EtOAc (30 mL) then successively washed with saturated aq. H-1), 2.86–2.73 (m, 2H, H-2′), 2.59 (t, 1H, J = 2.6 Hz, H-4′); 13C
NH4Cl (20 mL), NaHCO3 (20 mL) and water (20 mL), dried over NMR (100 MHz, MeOD), δ: 79.7 (C-3′), 77.8 (C-3), 73.2 (C-4′),
sodium sulfate, filtered and concentrated. The resulting 68.8 (C-4), 67.7 (2C, C-2, C-5), 55.4 (C-6), 55.0 (C-1), 52.5 (C-1′),
residue was purified by silica gel column chromatography 14.7 (C-2′); HRMS (ESI+): m/z [M
(combiflash 100%PE to 100% EtOAC) to provide 4c (228 mg, C10H18NO4 216.1230, found 216.1233.
+
H]+ calculated for
83%). Rf = 0.66 (6 : 4, PE : EtOAc); [α]2D0 = +5.7 (c = 0.3, CHCl3);
2,3,4,6-Tetra-O-acetyl-N-(4-but-1-ynyl)-1-deoxynojirimycin 4d.
1H NMR (400 MHz, CDCl3), δ: 5.11–4.95 (m, 3H, H-4, H-3, Acetic anhydride (542 μL, 5.73 mmol) was added dropwise to a
H-2), 4.21–4.11 (m, 2H, H-6a, H-6b), 3.74 (bd, 1H, J1′a–1′b = 17.8 solution of compound 3d (123 mg, 0.571 mmol) in pyridine
Hz, H-1′a), 3.40 (dd, 1H, J1′b–1′a = 18.1 Hz, J1′b–3′ = 2.3 Hz, H-1′ (10.2 mL) at 0 °C. The mixture was stirred at room temperature
b), 3.02 (dd, 1H, J1a–1b = 11.2 Hz, J1a–2 = 5 Hz, H-1a), 2.73–2.71 for 18 h, quenched by the addition of MeOH at 0 °C and evap-
(m, 1H, H-5), 2.63–2.58 (m, 1H, H-1b), 2.29 (t, 1H, J3′–1′ = 2.3 orated. The residue was dissolved in EtOAc and water, the
Hz, H-3′), 2.07, 2.02, 2.01, 2.00 (CH3COO); 13C NMR (100 MHz, aqueous layer was extracted with EtOAc. The organic layer was
CDCl3), δ: 171.0, 170.4, 170.1, 169.8 (4 × CH3COO), 76.1 (C-3′), dried over MgSO4, filtered and concentrated. Purification by
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flash chromatography (PE/EtOAc 95 : 5 to 90 : 10) afforded com- MgSO4, filtered and evaporated. The crude was acetylated with
pound 4d (148 mg, 67%) as a white solid. Mp: 82 °C; [α]D Ac20 (8 eq., 3.73 mmol, 353 µL) and pyridine (600 µL). The
=
+21.1 (c = 1.42, MeOH);. 1H NMR (400 MHz, CDCl3), δ: reaction mixture was stirred overnight then evaporated. The
5.00–4.93 (m, 2H, H-3, H-4), 4.92–4.86 (m, 1H, H-2), 4.18–4.10 resulting residue was purified by silica gel column chromato-
(m, 2H, H-6), 3.13 (dd, J = 5.1 Hz, J = 11.5 Hz, H-1), 2.97–2.86 graphy (combiflash 100%PE to 100% EtOAc) to provide 5b
(m, 2H, H-1′), 2.77–2.73 (m, 1H, H-5), 2.43 (dd, 1H, J = 10.1 Hz, (mixture of diastereosiomers (1 : 0.8*), 117.5 mg, 62%). Rf =
1
J = 11.5 Hz, H-1), 2.29–2.24 (m, 2H, H-2′), 2.02 (s, 3H, H-OAc), 0.64 (6 : 4, PE : EtOAc); [α]2D0 = +9.6° (c = 1.0, CHCl3); H NMR
1.96 (s, 6H, H-OAc), 1.95 (s, 4H, H-OAc, H-4′); 13C NMR (400 MHz, CDCl3), δ: 5.05–4.95 (m, 4H, H-3, H-3*, H-4, H-4*),
(100 MHz, CDCl3), δ: 170.8, 170.3, 170.0, 169.7 (4C-OAc), 81.9 4.91–4.63 (m, 4H, H-2, H-2*, H-3′, H-3′*), 4.28–4.08 (m, 4H,
(C-3′), 74.4 (C-3 or C-4), 70.1 (C-4′), 69.3, 69.2 (C-3 or C-4, C-2), H-6a, H-6b, H-6a*, H-6b*), 3.19–3.14 (m, 2H, H-1a, H-1a*),
60.7 (C-5), 59.9 (C-6), 52.9 (C-1), 50.4 (C-1′), 20.8, 20.7 (4 CH3), 3.00–2.91 (m, 2H, H-1′a, H-1′a*), 2.79–2.67 (m, 4H, H-1′b, H-1′
15.2 (C-2′); HRMS (ESI+): m/z [M + Na]+ calculated for b*, H-5, H-5*), 2.49–2.38 (m, 2H, H-1b, H-1b*), 2.00–1.94 (8s,
C18H25NNaO8 406.1472, found 406.1489.
24H, CH3COO), 1.85–1.67 (m, 4H, H-2′a, H-2′b, H-2′a*, H-2′b*),
2,3,4,6-Tetra-O-acetyl-1,5-dideoxy-N-(2-fluoropropyl)-1,5- 1.35 (dd, 3H, J4′*–3′* = 6.2 Hz, J4′*–F = 23.8 Hz, H-4′*), 1.29 (dd,
imino-D-glucitol 5a. A mixture of HF/SbF5 (7/1, v : v, 5 mL) was 3H, J4′–3′ = 6.1 Hz, J4′–F = 23.8 Hz, H-4′); 13C NMR (100 MHz,
added to compound 4a (0.259 mmol, 96.5 mg) at −20 °C. The CDCl3), δ: 170.8, 170.8, 170.3, 170.1, 170.1, 170.1 (CH3COO),
reaction mixture was stirred at −20 °C for 10 min then neutral- 89.8 (d, JC-3′–F = 164.1 Hz, C-3′), 89.8 (d, JC-3′*–F = 163.5 Hz,
ized with aqueous Na2CO3 and ice until pH reached 7. The C-3′*), 75.2 (C-3), 75. 1 (C-3*), 70.4 (C-2* or C-4*), 70.3 (C-2 or
aqueous layer was then extracted with CH2Cl2 (3 × 20 mL) and C-4), 70.0 (C-2* or C-4*), 70.0 (C–2 or C-4), 62.2 (C-5*), 62.1
the organic layers were combined, dried over MgSO4, filtered (C-5), 60.4 (C-6*), 60.3 (C-6), 53.5 (C-1), 53.4 (C-1*), 48.4
and evaporated. The crude was acetylated with Ac20 (8 eq., (d, JC-1′–F = 11.6 Hz, C–1′), 48.2 (d, JC-1′*–F = 10.5 Hz, C-1′*), 33.3
2.07 mmol, 196 µL) and pyridine (400 µL). The reaction (d, JC-2′*–F = 20.9 Hz, C-2′*), 33.2 (d, JC-2′–F = 20.6 Hz, C-2′), 21.5
mixture was stirred overnight at RT then evaporated. The (d, JC-4′*–F = 22.5 Hz, C-4′*), 21.2 (d, JC-4′–F = 22.7 Hz, C-4′), 20.7,
resulting residue was purified by silica gel column chromato- 20.6, 20.6 (CH3COO); 19F NMR {1H} (376 MHz, CDCl3), δ:
graphy (Combiflash 100%PE to 100% EtOAc) to provide 5a −174.4, −174.9; HRMS (ESI+): m/z [M + Na]+ calculated for
(55.1 mg, 54%) as a mixture of diastereosiomers. Rf = 0.62 C18H28FNNaO8 428.1691, found 428.1696.
(6 : 4, PE : EtOAc); [α]D20 = +18.8 (c = 0.91, CHCl3); 1H NMR
2,3,4,6-Tetra-O-acetyl-1,5-dideoxy-N-(2,2-difluoropropyl)-1,5-
(400 MHz, CDCl3), δ: 5.09–4.72 (m, 8H, H-3, H-3*, H-4, H-4*, imino-D-glucitol 5c. A mixture of HF/SbF5 (3/1, v : v, 1 mL) was
H-2, H-2*, H-2′, H-2′*), 4.28 (dd, 1H, J6a–6b = 13.1 Hz, J6a–5 = 5.2 added to compound 4c (0.503 mmol, 185.7 mg) at 0 °C. The
Hz, H–6a), 4.20 (dd, 1H, J6b–6a = 13.1 Hz, J6b–5 = 2.9 Hz, H–6b), reaction mixture was stirred at 0 °C during 10 min then neu-
4.14 (d, 2H, J6*–5* = 3.1 Hz, H-6a*, H-6b*), 3.30 (dd, 1H, J1a–1b
=
tralized with aqueous Na2CO3 and ice until pH reached 7. The
12.1 Hz, J1a–2 = 5.1 Hz, H-1a), 3.23 (dd, 1H, J1a*–1b* = 11.5 Hz, aqueous layer was then extracted with CH2Cl2 (3 × 20 mL) and
J1a*–2* = 5.1 Hz, H-1a*), 3.02–2.72 (m, 6H, H-1′a, H-1′b, H-1′a*, the combined organic layers were dried over MgSO4, filtered
H-1′b*, H-5, H-5*), 2.62 (ddd, 1H, J1b–1a = 12.1 Hz, J1b–2 = 2 Hz, and evaporated. The crude was acetylated with Ac20 (8 eq.,
H-1b), 2.57 (dd, 1H, J1b*–1a* = 11.3 Hz, J1b–2 = 1.9 Hz, H-1b*), 4 mmol, 378 µL) and pyridine (800 µL). The reaction mixture
2.10, 2.08, 2.07, 2.03, 2.02, 2.01 (s, 24H, CH3COO, CH3COO*), was stirred overnight then evaporated. The resulting residue
1.32 (dd, 3H, J3′–F = 23.5 Hz, J3′–2′ = 6.3 Hz, H-3′), 1.26 (dd, 3H, was then purified by silica gel column chromatography (com-
J3*′–F = 23.5 Hz, J3′–2′ = 6.3 Hz, H-3′*); 13C NMR (100 MHz, biflash 100%PE to 100% EtOAc) to provide 5c (122.8 mg, 59%).
CDCl3), δ: 171.1, 170.8, 170.5, 170.5, 170.2, 170.1, 169.9, 169.9 Rf = 0.64 (6 : 4, PE : EtOAc); [α]2D0 = +15° (c = 0.24, CHCl3); 1H
(CH3COO, CH3COO*), 88.8 (d, JC2′–F = 168 Hz, C-2′), 88.7 (d, NMR (400 MHz, CDCl3), δ: 5.09–4.99 (m, 2H, H-3, H-4),
JC2′*–F = 167 Hz, C-2′*), 74.6 (C-3 or C-3*), 74.5 (C-3 or C-3*), 4.96–4.90 (m, 1H, H-2), 4.25 (dd, 1H, J6a–5 = 3.7 Hz, J6a–6b
=
69.4, 69.3, 69.3, 69.1 (C-2, C-2*, C-4, C-4*), 61.7 (C-5*), 61.2 13.1 Hz, H-6a), 4.18 (dd, 1H, J6b–5 = 2.2 Hz, J6b–6a = 13.1 Hz,
(C-5), 60.2 (C-6), 59.3 (C-6*), 56.8 (d, JC-1′–F = 20.8 Hz, C-1′), 56.4 H-6b), 3.33 (dd, 1H, J1a–2 = 5.1 Hz, J1a–1b = 12.5 Hz, H-1a),
(d, JC1′*–F = 20.8 Hz, C-1′*), 54.5 (C-1*), 53.3 (C-1), 21.0, 21.0, 3.09–2.98 (m, 3H, H-1′a, H-1′b, H-5), 2.74–2.68 (m, 1H, H-1b),
20.9, 20.9, 20.9, 20.8, 20.8 (CH3COO, CH3COO*), 19.2 (d, JC3′–F 2.07, 2.03, 2.01 (3s, 12H, 4CH3COO), 1.59 (t, 3H, J3′-F = 18.3 Hz,
= 22.3 Hz, C-3′ or C-3′*), 18.9 (d, JC3′–F = 22.4 Hz, C-3′ or C-3′*); H-3′); 13C NMR (100 MHz, CDCl3), δ: 170.8; 170.3, 170.1, 169.9
19F NMR {1H} (376 MHz, CDCl3), δ: −174.1, −174.2; HRMS (CH3COO), 124.9 (t, JC2′–F = 240 Hz, C-2′), 74.2 (C-3), 69.1 (C-4),
(ESI+): m/z [M + H]+ calculated for C17H27FNO8 392.1715, 68.9 (C-2), 61.5 (C-5), 59.9 (C-6), 55.6 (t, JC1′–F = 26.3 Hz, C-1′),
found 392.1726.
53.8 (C-1), 22.1 (t, JC3′–F = 26.7 Hz, C-3′), 21.0, 20.9, 20.9, 20.8
2,3,4,6-Tetra-O-acetyl-1,5-dideoxy-N-(3-fluorobutyl)-1,5- (CH3COO); 19F NMR {1H} (376 MHz, CDCl3), δ: −91.8 (d, J =
imino-D-glucitol 5b. A mixture of HF/SbF5 (7/1, v : v, 5 mL) was 244 Hz), −93.8 (d, J = 241 Hz); HRMS (ESI+): m/z [M + H]+ calcu-
added to compound 4b (0.469 mmol, 180.7 mg) at −20 °C. The lated for C17H26F2NO8 410.1621, found 410.1628.
reaction mixture was stirred at −20 °C during 10 min. then
2,3,4,6-Tetra-O-acetyl-1,5-dideoxy-N-(3,3-difluorobutyl)-1,5-
neutralized with aqueous Na2CO3 and ice until pH reached imino-D-glucitol 5d. A mixture of HF/SbF5 (7/1, v : v, 2 mL) was
7. The aqueous layer was then extracted with CH2Cl2 (3 × added to compound 4d (50 mg, 0.130 mmol) at 0 °C. The reac-
20 mL) and the combined organic layers were dried over tion mixture was stirred at 0 °C during 10 minutes and then
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neutralized with solid Na2CO3 and ice until pH reached 7. The 1.84–1.69 (m, 4H, H-2′a, H-2′b, H-2′a*, H-2′b*), 1.33 (d, 3H,
aqueous layer was then extracted with EtOAc (3 × 20 mL) and J4′*-F = 23.7 Hz, H-4′*), 1.32 (d, J4′-F = 23.8 Hz, H-4′); 13C NMR
the organic layers were dried over MgSO4, filtered and evapor- (100 MHz, MeOD), δ: 90.6 (d, JC3′–F = 164.8 Hz, C-3′), 90.6 (d,
ated. The crude was acetylated with Ac2O (1.04 mmol, 98 μL) JC3′*–F = 164.1 Hz, C-3′*), 80.5 (C-3*), 80.5 (C-3), 72.0 (C-4,
and pyridine (200 μL). The reaction mixture was stirred over- C-4*), 70.7 (C-2*), 70.7 (C-2), 67.2 (C-5, C-5*), 59.5 (C-6), 59.3
night then evaporated. The residue was then purified by flash (C-6*), 57.8 (C-1), 57.7 (C-1*), 32.7 (d, JC2′*–F = 20.5 Hz, C-2′*),
chromatography (PE/EtOAc 85 : 15 to 70 : 30) to afford com- 32.7 (d, JC2′–F = 20.7 Hz, C-2′), 21.5 (d, JC4′*–F = 22.7 Hz, C-4′*),
pound 5d (51 mg, 93%) as colorless oil. [α]D = +12.5 (c = 1.0, 21.4 (d, JC4′–F = 22.6 Hz, C-4′); 19F NMR {1H} (376 MHz, MeOD),
1
CHCl3); H NMR (400 MHz, CDCl3), δ: 5.04–5.00 (m, 2H, H-3, δ: −174.7, −175.4; HRMS (ESI+): m/z [M + H]+ calculated for
H-4), 4.98–4.90 (m, 1H, H-2), 4.16 (d, 2H, J = 2.7 Hz, H-6), 3.16 C10H21FNO4 238.1449, found 238.1442.
(dd, J = 5.0 Hz, J = 11.3 Hz, H-1), 3.04–2.96 (m, 1H, H-1′),
1,5-Dideoxy-N-(2,2-difluoropropyl)-1,5-imino-D-glucitol
6c.
2.84–2.77 (m, 1H, 1H-1′), 2.66–2.62 (m, 1H, H-5), 2.32 (dd, 1H, Et3N (2 eq., 0.148 mmol, 20 µL) was added to a solution of
J = 10.4 Hz, J = 11.3 Hz, H-1), 2.06 (s, 3H, H-OAc), 2.03–1.94 compound 5c (30.3 mg, 0.074 mmol) in MeOH (3.1 mL) and
(m, 11H, 2H-2′, 9H-OAc), 1.61 (t, 3H, J = 18.6 Hz, H-4′); 13C the reaction mixture was stirred for 3 days at room temperature
NMR (100 MHz, CDCl3), δ: 170.9, 170.4, 170.1, 169.8 (4C-OAc), then evaporated under reduce pressure to provide 6c (17.6 mg,
123.5 (t, J = 239.1 Hz, C-3′), 74.4 (C-3 or C-4), 69.4, 69.3 (C-2, 98%) after lyophilisation. [α]2D0 = −7.2 (c = 0.18, MeOH); 1H
C-3 or C-4), 61.5 (C-5), 59.6 (C-6), 53.0 (C-1), 45.5 (t, J = 5.3 Hz, NMR (400 MHz, MeOD), δ: 3.97 (d, 1H, J6a–6b = 12.1 Hz, J6a–5
=
C-1′), 33.5 (t, J = 25.1 Hz, C-2′), 23.8 (t, J = 27.6 Hz, C-4′), 20.9, 2.6 Hz, H-6a), 3.76 (d, 1H, J6b–6a = 11.6 Hz, J6b–5 = 4.1 Hz,
20.8 (2C), 20.7 (4 CH3); 19F NMR {1H} (376 MHz, CDCl3), δ: H-6b), 3.48–3.38 (m, 1H, H-2), 3.36–3.34 (m, 1H, H-1′a), 3.20 (t,
−89.5 (d, J = 241 Hz), −91.4 (d, J = 241 Hz); HRMS (ESI): m/z 1H, J4–3 = J4–5 = 9.1 Hz, H-4), 3.16–3.11 (m, 2H, H-1a, H-3),
[M + Na]+ calculated for C18H27F2NNaO8 446.1587, found 2.87–2.77 (m,1H, H–1′b), 2.39–2.31 (m, 2H, H-1b, H-5), 1.61 (t,
446.1611.
3H, J3′-F = 18.7 Hz, H-3′); 13C NMR (100 MHz, MeOD), δ = 126.3
1,5-Dideoxy-N-(2-fluoropropyl)-1,5-imino-D-glucitol 6a. Et3N (t, JC2′–F = 239.6 Hz, C-2′), 80.4 (C-3), 72.3 (C-4), 70.6 (C-2), 68.1
(4 eq., 0.512 mmol, 69 µL) was added to a solution of 5a (C-5), 61.2 (C-6), 59.6 (C-1), 57.2 (t, JC1′–F = 26.6 Hz, C-1′), 22.4
(0.128 mmol, 50.2 mg) in MeOH (5.5 mL) and the reaction (t, JC3′–F = 25.6 Hz, C-3′); 19F NMR {1H} (376 MHz, MeOD), δ:
mixture was stirred for 3 days at room temperature then evap- −92.6 (d, J = 241 Hz), −94.2 (d, J = 244 Hz; HRMS (ESI+): m/z
orated under reduce pressure to provide 6a (28.4 mg, 99%) [M
after lyophilisation. [α]2D0 = +4.8° (c = 0.25, MeOH); 1H NMR 242.1197.
(400 MHz, MeOD), δ: 5.04–4.98 (m, 2H, H-2′, H-2′*), 3.95–3.74 1,5-Dideoxy-N-(3,3-difluorobutyl)-1,5-imino-D-glucitol
+
Na]+ calculated for C9H18F2NO4 242.1198, found
6d.
(m, 4H, H-6a, H-6b, H-6a*, H-6b*), 3.51–3.41 (m, 2H, H-2, Et3N (4 eq., 0.424 mmol, 58 µL) was added to a solution of 5d
H-2*), 3.35–3.30 (m, 1H, H-4*), 3.27 (1H, t, J4–3 = J4–5 = 9.2 Hz, (45 mg, 0.106 mmol) in MeOH (4.6 mL) and the reaction
H-4), 3.18–3.12 (m, 2H, H-3, H-3*), 3.10–3.04 (m, 2H, H-1a, H– mixture was stirred for 3 days at room temperature then evap-
1a*), 3.02–2.98 (m, 2H, H-1′a, H-1′a*), 2.90–2.82 (m, 1H, H-1′ orated under reduced pressure and freeze dried to provide 6d
b*), 2.79–2.70 (m, 1H, H-1′b), 2.43–2.28 (m, 2H, H-1b, H-1b*), (25 mg, 92%) as a white foam. [α]2D0 = −14.0 (c = 0.14, MeOH);
2.30–2.21 (m, 2H, H-5, H-5*), 1.28 (dd, 3H, J = 23.6 Hz, 1H NMR (400 MHz, MeOD), δ: 3.90 (dd, 1H, J6a–6b = 11.9 Hz,
J = 5.4 Hz, H-3), 1.27 (dd, 3H, J = 23.4 Hz, J = 5.2 Hz, H-3*); J6a–5 = 2.5 Hz, H-6a), 3.85 (dd, 1H, J6b–6a = 11.9, J6b–5 = 2.9 Hz,
13C NMR (100 MHz, MeOD), δ: 90.8 (d, JC2′–F = 130.7 Hz, C-2′), H-6b), 3.48 (m, 1H, H-2), 3.34 (m, 1H, H-4), 3.14 (t, 1H, J3,4
=
89.1 (d, JC2′*–F = 133.8 Hz, C-2′*), 80.5 (C-3, C-3*), 72.1, J3,2 = 9.1 Hz, H-3), 3.06–2.85 (m, 3H, H-1a, 2×H-1′), 2.23 (t, 1H,
72.0 (C-4, C-4*), 70.7 (C-2, C-2*), 68.1, 67.3 (C-5, C-5*), J1b,2 = J1b,1a = 10.8 Hz, H-1b), 2.17–2.05 (m, 3H, H-5, 2 × H-2′),
60.5 (C-6, C-6*), 59.5, 59.2, 59.2, 58.7 (C-1, C-1*, C-1′, C-1′*), 1.62 (t, 3H, J4′-F = 18.6 Hz, 3 × H-4′); 19F NMR (376 MHz,
19.7 (d, JC3′–F = 22.5 Hz, C-3′), 19.3 (d, JC3′*–F = 22.0 Hz, C-3′*); CDCl3) δ: −91.5 (s); 13C NMR (100 MHz, MeOD), δ: 125.3
19F NMR {1H} (376 MHz, MeOD), δ: −174.3, −175.3; HRMS (t, JC2′–F = 237.3 Hz, C-3′), 80.5 (C-3), 71.9 (C-4), 70.7 (C-2), 66.8
(ESI+): m/z [M + H]+ calculated for C9H19FNO4 224.1293, found (C-5), 59.3 (C-6), 57.7 (C-1), 47.1 (t, JC1′–F = 5.3 Hz, C-1′), 33.2
224.1292.
(t, JC2′–F = 24.7 Hz, C-2′), 23.7 (t, JC4′–F = 27.8 Hz, C-4′); 19F NMR
1,5-Dideoxy-N-(3-fluorobutyl)-1,5-imino-D-glucitol 6b. Et3N {1H} (376 MHz, MeOD), δ: −91.5; HRMS (ESI+): m/z [M + H]+
(4 eq., 0.624 mmol, 84.4 µL) was added to a solution of 5b calculated for C10H20F2NO4 256.1355, found 256.1362.
(0.156 mmol, 63.3 mg) in MeOH (6.5 mL) and the reaction
N-Methylsulfanyl-butyl-2,3,4,6-tetra-O-acetyl-1-deoxynojiri-
mixture was stirred for 3 days at room temperature then evap- mycin 7a. Procedure A was applied at 0 °C to 4b (0.305 mmol,
orated under reduce pressure to provide 6b (32.7 mg, 87%) 117.9 mg). The solvent was then evaporated and the resulting
1
after lyophilisation. [α]2D0 = −15.9° (c = 0.32, MeOH); H NMR residue was purified by silica gel column chromatography
(400 MHz, MeOD), δ: 4.70 (dm, 2H, J = 47.6 Hz, H-3′, H-3′*), (7 : 3, PE : EtOAc) to provide 7a (107 mg, 81%). Rf = 0.37 (7 : 3,
1
4.64–4.56 (m, 1H, H-3′), 3.90–3.81 (m, 4H, H-6a, H-6b, H-6a*, PE : EtOAc); [α]2D0 = +5.7 (c = 0.14, CHCl3); H NMR (400 MHz,
H-6b*), 3.49–3.43 (m, 2H, H-2, H-2*), 3.36–3.30 (m, 2H, H-4, CDCl3), δ: 4.76–4.67 (m, 2H, H-3, H-4), 4.60–4.54 (m, 1H, H-2),
H-4*), 3.15–3.09 (m, 2H, H-3, H-3*), 3.02–2.89 (m, 4H, H-1′a, 3.93 (dd, 1H, J6a–6b = 12.9 Hz, J6a–5 = 2.5 Hz, H-6a), 3.83 (dd,
H-1′a*, H-1a, H-1a*), 2.78–2.66 (m, 2H, H-1′b, H-1′b*), 1H, J6b–6a = 12.9 Hz, J6b–5 = 3.4 Hz, H-6b), 2.89 (dd, 1H, J1a–1b
=
2.22–2.16 (m, 2H, H-1b, H-1b*), 2.13–2.08 (m, 2H, H-5, H-5*), 11.3 Hz, J1a–2 = 5.2 Hz, H-1a), 2.60–2.49 (m, 1H, H-1′a),
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2.48–2.39 (m, 1H, H-5), 2.36–2.26 (m, 1H, H-1′b), 2.24–2.20 (C-1′), 33.8 (C-4′), 26.9, 22.7 (C-2′, C-3′), 14.8 (C-5′). HRMS
(m, 2H, H-4′a, H-4′b), 2.09 (d, 1H, J1b–1a = 11.3 Hz, H-1b), 1.76, (ESI+): m/z [M + H]+ calculated for C11H24NO4S 266.1420,
1.71, 1.68, 1.65 (4s, 15H, H-5′, 4 × CH3COO); 13C NMR found 266.1423.
(100 MHz, CDCl3), δ: 170.8, 170.3, 170.2, 170.1 (4 × CH3COO),
N-Butylsulfanyl-butyl-1-deoxynojirimycin 8b. Procedure B
75.2, 70.3 (C-3, C-4), 70.1 (C-2), 62.5 (C-5), 60.2 (C-6), 53.3 was applied to compound 7b (0.146 mmol, 69.7 mg) to provide
(C-1), 51.7 (C-1′), 34.3 (C-4), 27.2, 24.8 (C-2′, C-3′), 20.7, 20.7, 8b (42.9 mg, 95%) after lyophylisation. [α]2D0 = −15.2 (c = 0.60,
1
20.6, 20.6 (4 × CH3COO), 15.2 (C-5′); HRMS (ESI+): m/z [M + H]+ MeOH); H NMR (400 MHz, D2O), δ: 3.90–3.78 (m, 2H, H-6a,
calculated for C19H32NO8S 434.1843, found 434.1845.
H-6b), 3.54–3.48 (m, 1H, H-2), 3.37–3.31 (m, 1H, H-4),
N-Butylsulfanyl-butyl-2,3,4,6-tetra-O-acetyl-1-deoxynojirimy- 3.24–3.19 (m, 1H, H-3), 3.03–2.96 (m, 1H, H-1a), 2.83–2.60 (m,
cin 7b. Procedure A was applied to 4b (0.298 mmol, 114.9 mg). 2H, H-1′a, H-1′b), 2.57–2.52 (m, 4H), 2.34–2.21 (m, 2H, H-1b,
The solvent was then evaporated and the resulting residue was H-5), 1.57–1.50 (m, 6H, H-6′a, H-6′b, 4H), 1.40–1.30 (m, 2H,
purified by silica gel column chromatography (9 : 1 to 7 : 3, PE : H-7′a, H-7′b), 0.87 (t, 2H, J = 7.5 Hz, H-8′a, H-8′b); 13C NMR
EtOAc) to provide 7b (80.6 mg, 57%). Rf = 0.81 (7 : 3, PE : (100 MHz, D2O), δ: 79.3 (C-3), 70.9 (C-4), 69.8 (C-2), 65.9 (C-5),
EtOAc); [α]2D0 = +15.6 (c = 0.16, CHCl3); 1H NMR (400 MHz, 58.4 (C-6), 56.4 (C-1), 52.6 (C-1′), 32.1, 32.1, 32.0, 27.6, 23.1
CDCl3), δ: 5.07–4.99 (m, 2H, H-3, H-4), 4.97–4.91 (m, 1H, H-2), (C-2′, C-3′, C-4′, C-5′, C-6′), 22.5 (C-7′), 14.1 (C-8′); HRMS (ESI+):
4.17–4.11 (m, 2H, H-6a, H-6b), 3.18 (dd, 1H, J1a–1b = 11.6 Hz, m/z [M + H]+ calculated for C14H30NO4S 308.1890, found
J1a–2 = 5.1 Hz, H-1a), 2.82–2.55 (m, 3H, H-1′a, H-5, H-1′b), 308.1893.
2.52–2.41 (m, 4H, H-5′a, H-5′b, 2H′), 2.34–2.28 (m, 1H, H-1b),
N-Heptylsulfanyl-butyl-1-deoxynojirimycin 8c. Procedure B
2.06, 2.00, 1.98 (3s, 12H, CH3COO), 1.57–1.49 (m, 6H, H-6′a, was applied to compound 7c (0.167 mmol, 86.5 mg) to provide
H-6′b, 4H′), 1.42–1.33 (m, 2H, H-7′a, H-7′b,), 0.89 (t, 2H, 8c (41 mg, 70%) after lyophylisation. [α]2D0 = −14 (c = 0.50,
J = 7.5 Hz, H-8′a, H-8′b); 13C NMR (100 MHz, CDCl3), δ: 171.0, MeOH); 1H NMR (400 MHz, D2O), δ: 3.78 (2dd, 2H, J6a–6b
=
170.4, 170.1, 169.8 (4 × CH3COO), 74.7 (C-3), 69.5, 69.4 12.1 Hz, J6a–5 = 2.81 Hz, J6b–5 = 3.0 Hz, H-6a, H-6b), 3.42–3.36
(C-2, C-4), 61.6 (C-5), 59.5 (C-6), 52.9 (C-1), 51.3 (C-1′), 31.9, (m, 1H, H-2), 3.26 (t, 1H, J4–3 = J4–5 = 9.1 Hz, H-4), 3.05 (t, 1H,
31.9, 27.1, 24.0, 22.1 (C-2′, C-3′, C-4′, C-5′, C-6′), 22.1 (C-7′), J3–4 = J3–2 = 9.1 Hz, H-3), 2.92 (dd, 1H, J1a–1b = 11.1 Hz, J1a–2
=
20.9, 20.9, 20.8, 20.8 (4 × CH3COO), 13.8 (C-8′); HRMS (ESI+): 4.8 Hz, H-1a), 2.78–2.71 (m, 1H, H-1′a), 2.56–2.49 (m, 1H, H-1′
m/z [M + H]+ calculated for C22H38NO8S 476.2321, found b), 2.48–2.40 (m, 4H′), 2.11 (t, 1H, J = 10.7 Hz, H-1b), 2.07–2.03
476.2314.
(m, 1H, H-5), 1.52–1.45 (m, 4H, H-2′a, H-2′b, 2H′), 1.33–1.17
N-Heptylsulfanyl-butyl-2,3,4,6-tetra-O-acetyl-1-deoxynojirimy- (m, 8H, H-10′a, H-10′b, 6H′), 0.84–0.81 (m, 2H, H-11′a, H-11′b);
cin 7c. Procedure A was applied to 4b (0.263 mmol, 101.3 mg). 13C NMR (100 MHz, D2O), δ: 80.5 (C-3), 72.0 (C-4), 70.7 (C-2),
The solvent was then evaporated and the resulting residue was 67.4 (C-5), 59.4 (C-6), 57.6 (C-1), 53.3 (C-1′), 32.9, 32.8, 32.7,
purified by silica gel column chromatography (8 : 2, PE : EtOAc) 30.8, 30.0, 29.9, 28.6, 24.4, 23.7 (C-2′, C-3′, C-4′, C-5′, C-6′, C-7′,
to provide 7c (97.1 mg, 71%). Rf = 0.26 (8 : 2, PE : EtOAc); [α]D20
=
C-8′, C-9′, C-10′), 14.4 (C-11′); HRMS (ESI+): m/z [M + H]+ calcu-
+11.2 (c = 0.17, CHCl3); 1H NMR (400 MHz, CDCl3), δ: lated for C17H36NO4S 350.2359, found 350.2362.
5.07–4.98 (m, 2H, H-3, H-4), 4.96–4.90 (m, 1H, H-2), 4.17–4.14
(m, 2H, H-6a, H-6b), 3.17 (dd, 1H, J1a–1b = 11.4 Hz, J1a–2 = 5.5
Hz, H-1a), 2.77–2.52 (m, 3H, H-1′a, H-5, H-1′b), 2.30 (t, 1H,
Biological assays
J1b–1a = J1b–2 = 11.4 Hz, H-1b), 2.05, 1.99, 1.98 (3s, 12H,
Glycosidase inhibition profiling. The glycosidase activities
CH3COO), 1.57–1.50 (m, 6H, H-2′a, H-2′b, 4H′), 1.39–1.24 were determined using appropriate p-nitrophenyl glycosides as
(m, 6H, H-10′a, H-10′b, 4H′), 0.87–0.83 (m, 2H, H-11′a, H-11′b); substrates at the optimum pH of each enzyme. The reaction
13C NMR (100 MHz, CDCl3), δ: 170.9, 170.4, 170.1, 169.8 was stopped by adding 400 mM Na2CO3. The released p-nitro-
(CH3COO), 74.7 (C-3), 69.5, 69.4 (C-2, C-4), 61.6 (C-5), 59.5 phenol was measured spectrometrically at 400 nm.
(C-6), 52.9 (C-1), 51.3 (C-1′), 32.9, 31.9, 31.8, 29.8, 29.0, 29.0,
Trehalase inhibition. Compounds were tested for their
27.1, 24.0, 22.7 (C-2′, C-3′, C-4′, C-5′, C-6′, C-7′, C-8′, C-9′, C-10′), inhibitory activity against insect trehalase of midge larvae of C.
14.2 (C-11′); HRMS (ESI+): m/z [M + H]+ calculated for riparius, a good model for biochemical studies, and porcine
C25H44NO8S 518.2782, found 518.2781.
kidney trehalase (purchased from Sigma-Aldrich) as the mam-
N-Methylsulfanyl-butyl-1-deoxynojirimycin 8a. Procedure B malian counterpart. Proteins were measured according to
was applied to compound 7a (0.233 mmol, 97 mg) to provide Bradford using bovine serum albumin as standard.40 Tre-
after freeze drying 8a (52.3 mg, 98%). [α]2D0 = −16.7 (c = 0.73, halase activity was measured through a coupled assay with
1
MeOH); H NMR (400 MHz, D2O), δ: 3.89–3.78 (m, 2H, H-6a, glucose-6-phosphate dehydrogenase and hexokinase according
H-6b), 3.54–3.48 (m, 1H, H-2), 3.36–3.31 (m, 1H, H-4), to Wegener et al.41 To examine the potential of each com-
3.24–3.19 (m, 1H, H-3), 2.99 (dd, 1H, J1a–1b = 11.4 Hz, J1a–2
=
pound as a trehalase inhibitor, dose–response curves were
4.9 Hz, H-1a), 2.75–2.70 (m, 1H, H-1′a), 2.66–2.61 (m, 1H, H-1′ established to determine the IC50 values. Experiments were
b), 2.55–2.51 (m, 2H, H-4′a, H-4′b), 2.32–2.29 (m, 1H, H-1b), performed at fixed substrate concentration close to the Km
2.26–2.22 (m, 1H, H-5), 2.06 (s, 3H, H-5′), 1.54 (bs, 4H, H-2′a, value (0.5 mM for C. riparius and 2.5 mM for porcine treha-
H–2′b, H-3′a, H-3′b); 13C NMR (100 MHz, D2O), δ: 79.0 (C-3), lase), in the presence of increasing inhibitor concentrations.
70.7 (C-4), 69.6 (C-2), 65.7 (C-5), 58.2 (C-6), 56.0 (C-1), 52.3 Initial rates as a function of inhibitor concentration were fitted
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to the following equation:
U. K. Pandit and J. M. F. G. Aerts, J. Biol. Chem., 1998, 273,
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Med. Chem., 2013, 21, 5021–5028.
vi
1
½Iꢀ
IC50
ꢀ
ꢁ
¼
n
v
1 þ
7 M. van Scherpenzeel, R. J. B. H. N. van den Berg,
W. E. Donker-Koopman, R. M. J. Liskamp, J. M. F. G. Aerts,
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T. M. Barragµn, C. Ortiz Mellet and J.-F. Nierengarten,
Angew. Chem., Int. Ed., 2010, 49, 5753; J. Diot, M. Isabel
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where vi and v are the initial rate in the presence and in the
absence of inhibitor, respectively, [I] is the inhibitor concen-
tration, IC50 is the inhibitor concentration producing half-
maximal inhibition, and n is the Hill coefficient. All enzyme
assays were performed in triplicates at 30 °C by using sample
volumes varying from 5 to 20 μL in 1 mL test and using a
Cary3 UV/Vis Spectrophotometer. Enzyme activities were ana-
lyzed by Cary Win UV application software for Windows XP.
F508del-CFTR restoration assay. CFTR activity was assayed
by iodide (125I) efflux as previously described.39 Briefly, iodide
efflux curves were constructed by plotting rate of 125I, noted
k and expressed in min−1. All comparisons were based on
maximal values for the time-dependent rates kpeak excluding
the points used to establish the baseline kbasal and were
expressed as kpeak − kbasal (min −1).
9 Y. Blériot, N. Auberger, A. T. Tran, C. Gauthier, J. Yerri,
G. Principe, J. Désiré, J. Marrot and M. Sollogoub, Org.
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2, 1492.
Conclusions
We have synthesized a small library of DNJ derivatives bearing
a thiolated, a fluorinated or an unsaturated N-alkyl chain. Flu-
orine and sulfur atoms were introduced using hydrofluorina-
tion and thiol ene click reactions respectively starting from a
common unsaturated iminosugar precursor. The thiolated
derivatives exhibit low micromolar trehalases inhibition while
the N-propargyl DNJ shows potency similar to Zavesca as
F508del-CFTR corrector.
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Acknowledgements
V. Cendret and J. Bertrand thank the “Fondation pour la
Recherche Médicale” for post-doctoral fellowships. We thank
the CNRS and Université de Poitiers for financial support.
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