Iminosugars: Effects of stereochemistry, ring size, and n-substituents on glucosidase activities

Article abstract of DOI:10.3390/ph12030108

N-substituted iminosugar analogues are potent inhibitors of glucosidases and glycosyltransferases with broad therapeutic applications, such as treatment of diabetes and Gaucher disease, immunosuppressive activities, and antibacterial and antiviral effects against HIV, HPV, hepatitis C, bovine diarrhea (BVDV), Ebola (EBOV) and Marburg viruses (MARV), influenza, Zika, and dengue virus. Based on our previous work on functionalized isomeric 1,5-dideoxy-1,5-imino-D-gulitol (L-gulo-piperidines, with inverted configuration at C-2 and C-5 in respect to glucose or deoxynojirimycin (DNJ)) and 1,6-dideoxy-1,6-imino-D-mannitol (D-manno-azepane derivatives) cores N-linked to different sites of glucopyranose units, we continue our studies on these alternative iminosugars bearing simple N-alkyl chains instead of glucose to understand if these easily accessed scaffolds could preserve the inhibition profile of the corresponding glucose-based N-alkyl derivatives as DNJ cores found in miglustat and miglitol drugs. Thus, a small library of iminosugars (14 compounds) displaying different stereochemistry, ring size, and N-substitutions was successfully synthesized from a common precursor, D-mannitol, by utilizing an S_N2 aminocyclization reaction via two isomeric bis-epoxides. The evaluation of the prospective inhibitors on glucosidases revealed that merely D-gluco-piperidine (miglitol, 41a) and L-ido-azepane (41b) DNJ-derivatives bearing the N-hydroxylethyl group showed inhibition towards α-glucosidase with IC₅₀ 41 μM and 138 μM, respectively, using DNJ as reference (IC₅₀ 134 μM). On the other hand, β-glucosidase inhibition was achieved for glucose-inverted configuration (C-2 and C-5) derivatives, as novel L-gulo-piperidine (27a) and D-manno-azepane (27b), preserving the N-butyl chain, with IC₅₀ 109 and 184 μM, respectively, comparable to miglustat with the same N-butyl substituent (40a, IC₅₀ 172 μM). Interestingly, the seven-membered ring L-ido-azepane (40b) displayed near twice the activity (IC₅₀ 80 μM) of the corresponding D-gluco-piperidine miglustat drug (40a). Furthermore, besides α-glucosidase inhibition, both miglitol (41a) and L-ido-azepane (41b) proved to be the strongest β-glucosidase inhibitors of the series with IC₅₀ of 4 μM.

Full text of DOI:10.3390/ph12030108

pharmaceuticals
Article
Iminosugars: Effects of Stereochemistry, Ring Size,
and N-Substituents on Glucosidase Activities
Luís O. B. Zamoner, Valquiria Aragão-Leoneti and Ivone Carvalho *
School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café s/n, Monte Alegre,
CEP14040-903 Ribeirão Preto, Brazil
* Correspondence: carronal@usp.br; Tel.: +55-16-33154709
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 15 June 2019; Accepted: 10 July 2019; Published: 12 July 2019
Abstract: N-substituted iminosugar analogues are potent inhibitors of glucosidases and
glycosyltransferases with broad therapeutic applications, such as treatment of diabetes and
Gaucher disease, immunosuppressive activities, and antibacterial and antiviral effects against
HIV, HPV, hepatitis C, bovine diarrhea (BVDV), Ebola (EBOV) and Marburg viruses (MARV),
influenza, Zika, and dengue virus. Based on our previous work on functionalized isomeric
1,5-dideoxy-1,5-imino-D-gulitol (L-gulo-piperidines, with inverted configuration at C-2 and
C-5 in respect to glucose or deoxynojirimycin (DNJ)) and 1,6-dideoxy-1,6-imino-D-mannitol
(D-manno-azepane derivatives) cores N-linked to different sites of glucopyranose units, we continue
our studies on these alternative iminosugars bearing simple N-alkyl chains instead of glucose to
understand if these easily accessed scaffolds could preserve the inhibition profile of the corresponding
glucose-based N-alkyl derivatives as DNJ cores found in miglustat and miglitol drugs. Thus,
a small library of iminosugars (14 compounds) displaying different stereochemistry, ring size, and
N-substitutions was successfully synthesized from a common precursor, D-mannitol, by utilizing
an SN2 aminocyclization reaction via two isomeric bis-epoxides. The evaluation of the prospective
inhibitors on glucosidases revealed that merely D-gluco-piperidine (miglitol, 41a) and L-ido-azepane
(
41b) DNJ-derivatives bearing the N-hydroxylethyl group showed inhibition towards
with IC50 41 M and 138 M, respectively, using DNJ as reference (IC50 134 M). On the other hand,
-glucosidase inhibition was achieved for glucose-inverted configuration (C-2 and C-5) derivatives,
as novel L-gulo-piperidine (27a) and D-manno-azepane (27b), preserving the N-butyl chain, with IC50
109 and 184 M, respectively, comparable to miglustat with the same N-butyl substituent (40a, IC50
172 M). Interestingly, the seven-membered ring L-ido-azepane (40b) displayed near twice the activity
(IC50 80 M) of the corresponding D-gluco-piperidine miglustat drug (40a). Furthermore, besides
-glucosidase inhibition, both miglitol (41a) and L-ido-azepane (41b) proved to be the strongest
α-glucosidase
µ
µ
µ
β
µ
µ
µ
α
β-glucosidase inhibitors of the series with IC50 of 4 µM.
Keywords: iminosugars; polyhydroxypiperidines; polyhydroxyazepanes; glucosidase inhibition;
miglustat; miglitol
1. Introduction
The major groups of glucosidase inhibitors that have been discovered are polyhydroxylated
alkaloids containing piperidines, pyrrolidines, nor-tropanes, pyrrolizidines, and indolizidines as
mono and bicyclic systems [
isolated from natural sources, such as plants (Morus alba, Commelina communis), bacteria (Bacillus,
Streptomyces), and fungi (Zygosaccharomyces rouxii for mulberry leaf fermentation) [ ], and produced
by synthetic strategies with potential inhibition properties not only over - and -glucosidases
], nucleoside phosphorylases [ ], and
1]. A great variety of these compounds, named iminosugars, have been
2
α
β
but also glycosyltransferases, glycogen phosphorylase [
3–
5
6
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Pharmaceuticals 2019, 12, 108
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sugar-nucleotide mutases (UDP-Galp mutase) [
7
]. High activity and specificity of iminosugars
are associated with the ability of the nitrogen ring to mimic the transition state of pyranosidic or
furanosidic units of natural glucosidase substrates that positively influence their shape and charge for
enzyme binding.
Access to iminosugar analogues with N-substituted side chains has led to a variety of potent
glucosidase and glycosyltransferases inhibitors with broad therapeutic applications, such as treatment of
diabetes [
and antiviral effects [12,13] against HIV [14], HPV [15], hepatitis C [16], bovine diarrhea (BVDV) [17],
Ebola (EBOV) [18] and Marburg viruses (MARV) [19], influenza [20], Zika [21], and dengue virus [22 23].
Despite the - and -glucosidase inhibition promoted by nojirimycin itself ( ), it was achieved
a better profile for the corresponding 1-deoxynojirimycin (DNJ, ) due to better stability and potency.
8] and Gaucher disease [9], and even immunosuppressive activities [10] and antibacterial [11]
,
α
β
1
2
Furthermore, a combination of these structural features can promote the cellular uptake of N-alkylated
DNJ analogues, as shown by those containing long and linear alkyl chains, which displayed better
activity in whole cells (human hepatoblastoma cells, HepG2) than purified pork glucosidase I [24].
Conversely, N-alkyl-less lipophilic N-alkyl groups, or even containing an oxygen atom, displayed
lower cytotoxicity and significant activity against
N-butyl- (N-Bu-DNJ, miglustat, ) [26], N-hydroxyethyl- (N-EtOH-DNJ, miglitol,
(N-7-oxadecyl-DNJ, ) [27 28], and N-glycyl-deoxynojirimycin ( ) [29]. In fact, miglustat is particularly
useful in the control of type I Gaucher disease [ ] and Niemann–Pick type C (NPC) lysosomal storage
α
-glucosidase, as described for N-methyl- (
3) [25],
4
5
), N-7-oxadecyl-
6
,
7
9
diseases, via “substrate reduction therapy”, as well as miglitol in the treatment of non-insulin-dependent
diabetes (type II) (Figure 1) to impair carbohydrate processing in the gut [30]. In addition, inhibition of
the target human acid β-glucosidase (glucocerebrosidase, GCase) has been achieved by a set of derived
iminosugars as new pharmacological chaperones for the treatment of Gaucher disease [31,32].
R
R2
H
N
H
N
HOH2C
HO
HOH2C
HO
R
N
HOH2C
HO
HOH2C
HO
R1
NH
OH
OH
R
OH
OH
OH
OH
OH
8 R = H
9 R = OH
10 R1= CH2OH; R2= H
11 R1= H; R2= CH2OH
1 R= OH, NJ
2 R= H , DNJ
6 R= Oxadecyl
7 R= C(O)CH2NH2
3 R= Me
4 R= Bu
5 R= EtOH
R
N
MeO
R
N
OH
N
H
N
O
HOH2C
N
N
N
OH
OH
N
R3O
OR1
HOH2C
N
HOH2C
N
OR2
OH
HO
HO
12 R1= -D-glucopyranose; R2; R3 = H
HO
OH
HO
OH
OH
13 R2
14 R3
15 R1
16 R3
17 R2
=
=
=
=
=
-D-glucopyranose; R1; R3 = H
-D-glucopyranose; R1; R2 = H
-D-glucopyranose; R2 , R3 = H
-D-glucopyranose; R1; R2 = H
-D-glucopyranose; R1; R3 = H
OH
OH
19 R= -D-glucopyranosyl
20 R= 3-deoxy-D-glucopyranosid-3-yl
21 R= 6-deoxy- -D-glucopyranosid-6-yl
22
23
R= -D-glucopyranosyl
R= 3-deoxy-D-glucopyranosid-3-yl
24 R= 6-deoxy- -D-glucopyranosid-6-yl
18
Figure 1. Examples of polyhydroxylated piperidine and azepane iminosugars reported, some of them
displaying glucosidase inhibition.
Besides the N-alkyl variations, several studies have pointed to the impact of the modification of
DNJ hydroxyl groups involving C-2 to C-6 positions, assessing the influence of both stereochemistry
and substituent variations on glycosidase activities. In general, the loss of
α-glucosidase (I and
II) and ceramide glycosyltransferase activities was evident by modifying C-2, C-3, and C-4, with
the exception of N-butyl-1-deoxy-galactonojirimycin (migalastat, used for the treatment of Fabry
disease) [30]. On the other hand, changes at C-1 and the ring nitrogen were allowed, based on the
high inhibition revealed by DNJ and 1-azasugars (isofagomine (
replacing the anomeric carbon and the ring oxygen of glucose by nitrogen and carbon, respectively,
with significant activity only against -glucosidase. Interestingly, introduction of a hydroxyl group at
the carbon (C-2) neighboring the nitrogen afforded a potent -glucosidase inhibitor (noeuromycin,
8), for instance), the latter obtained by
β
α
9)

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that preserved the original
β-glucosidase activity. Additionally, the extra hydroxymethylene group
at the anomeric position of DNJ gave rise to
with the ability to inhibit -glucosidase, which was even higher whilst bearing N-methyl or N-butyl
]. Furthermore, seven-membered ring iminosugar have shown potential
35]. Comprehensive studies on iminosugar derivatives can be found in
α-homonojirimycin (10) and β-homonojirimycin (11)
α
substituents (Figure 1) [
glucosidase inhibition [33
literature reviews [36–39].
1
–
Inspired by a series of reported deoxynojirimycin disaccharides that were decorated with equal
α-
or
β
-glucopyranose units at C-2, C-3, and C-4 DNJ positions (12 17) [40 41], along with N-glycosylated
-
,
deoxynojirimycin, MDL 73,945 (18) [42], we had reported an alternative approach, using functionalized
isomeric 1,5-dideoxy-1,5-imino-D-gulitol (L-gulo-piperidines, with inverted configurations at C-2 and
C-5 with respect to glucose or DNJ, 19
-21) and 1,6-dideoxy-1,6-imino-D-mannitol (D-manno-azepane
derivatives, 22 24) cores N-linked to different sites of glucopyranose units, such as C-1, C-3, and C-6
-
positions [43]. To reach this goal, we used a CuAAC reaction (copper azide alkyne cycloaddition
reaction), as a click chemistry strategy, to connect six- and seven-membered iminosugars to glucose in
different arrangements through triazole bridges to produce the most active
21) of the pseudo-disaccharide series, L-gulopiperidine attached to glucose C-6 position, with IC50
approximately three-fold lower than that of DNJ (Figure 1) [43].
Despite the reported loss of -glucosidase activity under modification at the C-5 position of
α-glucosidase inhibitor
(
α
the iminosugar, such as displayed by 1-deoxy-L-ido-nojirimycin (with an inverted configuration at
C-5 with respect to DNJ) [24] and for 1,5-dideoxy-1,5-iminoxylitol (lacking the C-5 hydroxymethyl
group of DNJ) [27
based on the remarkable
,
28], we have been encouraged to continue our studies on L-gulo-piperidines
-glucosidase inhibition previously obtained for pseudo-disaccharides with
α
simultaneous inverted configurations at C-2 and C-5 positions in relation to glucose stereochemistry [43].
Thus, to understand the relative contribution of the ring size, stereochemistry, and N-alkyl
substitution on glycosidase inhibition, we proceeded with the synthesis of a small library of
N-substituted 1-deoxy-L-gulo-nojirimycin and D-manno-azepane derivatives and compared them
with the corresponding classical N-substituted of 1-deoxy-D-gluco-piperidine (DNJ) and L-ido-azepane
counterparts as glucose-type carbohydrate mimetics. To reach this goal, N-hydroxyethyl and
N-butyl groups of miglitol and miglustat drugs, respectively, were investigated as highly important
N-alkyl substitutions for glucosidase inhibition, besides N-phenethyl [44], and N-propynyl [43] as
a less-active counterpart.
2. Results and Discussion
2.1. Chemistry
Initially, target 1-deoxy-L-gulo-nojirimycin (26-29a) and D-manno-azepane derivatives (26-29b)
were synthesized by a regiospecific C2-symmetric unprotected bis-epoxide opening strategy in the
presence of primary amines, followed by an SN2 aminocyclization reaction to give a mixture of
both six- and seven-membered iminosugar isomers [45–47]. As previously reported, the synthesis
of unprotected bis-epoxide 25 was promptly achieved from the simple and commercially available
starting material, D-mannitol, in two steps, by tosylation of both D-mannitol primary alcohols (71%),
followed by a base-promoted intramolecular SN2 reaction to give 1,2:5,6-dianhydro-D-mannitol 25
(29%), Scheme 1A [43]. Opening of the homochiral C2-symmetric bis-epoxide 25 by alkylamines
(at the less-hindered position of one epoxy function) led to the formation of secondary amines,
which promoted an SN2 aminocyclization reaction to give a mixture of polyhydroxy-piperidine and
-azepane by 6-exo-tet or 7-endo-tet processes, respectively. In this series, azepane was isolated in
a slightly higher proportion than DNJ, indicating the free 3,4 diol in bis-epoxide 25 did not affect
the regioselectivity. Conversely, higher yields of DNJ than azepane derivatives were previously
reported using benzyl protecting groups at C-3 and C-4 of bis-epoxide in the presence of several
amines (benzylamine, for instance) [46]. Interestingly, this ratio can be reversed in the presence of a

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Lewis acid (perchloric acid), which catalyzes epoxide opening to mainly give azepane derivatives.
Furthermore, the exclusive formation of seven-membered azasugars using a more rigid trans-acetonide
protecting group, as observed in 1,2:5,6-dianhydro-3,4-O-isopropylidene-D-mannitol or L-iditol, led
to the conclusion that formation of both polyhydroxy-piperidine and -azepane regioisomers can be
achieved by an aminocyclization of a flexible bis-epoxide bearing a free or acyclic hydroxyl protecting
groups at C-3 and C-4, and the ratio varies according to the experimental conditions [46].
Therefore, the microwave-assisted aminocyclization reaction of bis-epoxide 25 was carried out
with four different primary amines (propargylamine, butylamine, ethanolamine, or phenethylamine),
which resulted in a mixture of N-alkyl substituted polyhydroxypiperidine 26
regioisomers, being iminosugars 27a,b and 28a,b novel compounds. To prevent laborious separation
of and regioisomers, some mixtures were treated with acetic anhydride and pyridine for prompt
-29a and azepane 26b-29b
a
b
separation of the per-O-acetylated 1-deoxy-L-gulo-nojirimycin and D-manno-azepane by column
chromatography, isolated in different ratio and yields over two steps, as depicted in Table 1. However,
the separation of protected regioisomers 31a,b was more demanding and required HPLC purification,
mainly because the deprotected products 28a,b were inseparable by chromatographic column or
even HPLC under test conditions. Lastly, deprotection of the regioisomers in the presence of sodium
methoxide gave products 26-29a and 26-29b in quantitative yields. Eventually, derivatives 27a,b and
29a,b bearing a more lipophilic side chain (butyl and phenethyl, respectively) were separated without
the need of previous protection by using chromatography column eluted with DCM/MeOH (4:1).
However, the yields of pure polyhydroxy-piperidines and -azepanes were much lower (approximately
10%, 0.8:1 ratio, respectively) than using protection/deprotection strategies (Table 1).
Scheme 1.
(A) Synthesis of iminosugars 26-29a and 26-29b from D-mannitol, via bis-epoxide 25.
Reagents and conditions: (i) TsCl, py, 71%; (ii) NaOH, CH3CN:H2O, 40 ◦C, 29%; (iii) Primary amine:
propargylamine, butylamine, ethanolamine, or phenethylamine, MeOH, MW, 90 ◦C; (iv) Ac2O, Py; for
yields over two steps see Table 1; (v) NaOMe, MeOH (quant). (B) Synthesis of iminosugars 39-41a and
39 41b from D-mannitol, via bis-epoxide 35; (vi) 2,2-dimethoxypropane, TsOH, 96%; (vii) NaH, BnBr,
-
n-Bu4NI, THF, 93%; (viii) HCl, MeOH, 0 ◦C, (quant); (ix) TBDMS chloride, imidazole, DMF, 0 ◦C, 88%;
(x) MsCl, NEt3, DCM, 92%; (xi) HCl, MeOH, then NaOH, H2O, 70%; and (xii) TMSI, DCM, rt, then
MeOH, 45–100%.
Table 1. Yields obtained from microwave-assisted aminocyclization reaction of bis-epoxides 25 and 35
.
Yield (%)
Primary Amine for
Aminocyclization
Reaction
Polyhydroxy-Piperidine
Polyhydroxy-Azepane
1-deoxy-L-gulo-nojirimycin
1-deoxy-D-gluco-nojirimycin (DNJ)
D-manno-azepane
L-ido-azepane
26-29a
39-41a
26-29b
39-41b
Propargylamine
Butylamine
Ethanolamine
Phenethylamine*
32
20
17
4
40
33
22
-
35
24
21
5
37
38
28
-
*
Low yields obtained when the reaction mixture was purified directly by chromatographic column, without
previous acetylation.

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In order to keep the stereo-control during the reaction and obtain the iminosugars with the
same stereochemistry of glucose, we pursued the classical procedure based on the protection of
1,2- and 5,6- positions of D-mannitol to produce the diisopropylidene intermediate 32 [48], which
was benzylated at 3,4- positions and then deprotected under acid catalysis to give compound 33
(Scheme 1B) [47]. Briefly, selective protection of primary hydroxyl functions with bulk groups,
followed by activation of the O-2 and O-5 with mesyl chloride and treatment of 34 in MeOH
with concentrated HCl allowed the preparation of bis-epoxide 35 because of the intramolecular
attack of the released primary hydroxyl functions that displaces the leaving mesyl groups. Then,
bis-epoxide 35, comprising inverted configurations at C-2 and C-5 comparatively to 25, was
converted to the corresponding mixture of N-substituted 1-deoxy-D-gluco-nojirimycin (36
-38a) and
1,6-dideoxy-1,6-imino-L-ido-azepane derivatives (36 38b) in approximately 1:1 ratio under treatment
-
with propargylamine, butylamine, ethanolamine, or phenethylamine for the aminocyclization reaction,
as described for bis-epoxide 25. Attempts to generate the N-phenethyl derivative of this series were
unsuccessful since D-glucitol was isolated as a major product, possibly because phenethylamine
promoted a regioselective opening of partially protected 1,2-epoxide (35) and then an O-cyclization
leading to glucitol, as reported using ammonium formate [49].
After chromatographic separation of regioisomers, removal of the benzyl groups was better
achieved under treatment with trimethylsilyl iodine [50] rather than hydrogenation conditions [47] to
give final products 39-41a-b in moderate to quantitative yields (45–100%).
2.2. Biological Assays
Initially, the small library of iminosugar derivatives (26-29a,b and 39-41a,b) was screened
for
-D-glucopyranoside as substrate and prospective inhibitors at 1.0 mM concentration. To broaden the
scope of the analysis, -glucosidase (almond) activity of the same set of compounds was conducted
α-glucosidase inhibition (from Saccharomyces cerevisiae) activities using p-nitrophenyl
α
β
using the corresponding p-nitrophenyl β-D-glucopyranoside.
2.2.1. Yeast α-glucosidase Activities
Based on the IC50 values using
piperidine and azepane DNJ derivatives bearing the N-hydroxylethyl group, D-gluco-piperidine
(miglitol, 41a, IC50 41 M) and L-ido-azepane (41b, IC50 138 M), using the DNJ as the reference (IC50
134 M) (Table 2). The -glucosidase inhibition promoted by L-ido-azepane 41b was significant and
α-glucosidase, the greatest inhibition was verified for both
µ
µ
µ
α
related to DNJ, although with a three-fold lower activity than D-gluco-piperidine (41a). In spite of
finding a patent for azepane 38b, the data were inaccessible [51], and mixed results were found for
nonsubstituted L-ido-azepane with inhibition properties (Ki 4.8
D-gluco-piperidine (DNJ, Ki 0.44 M) assayed on Bacillus stearothermophilus
high (Ki 29.4 M) [52] to weak activity (IC50 772 M [33] or 35% inhibition at 1 mM [53]) using yeast
-glucosidase. In addition, weak or no -glucosidase inhibition was observed for N-propynyl (39a,b)
µM) lower than the corresponding
µ
α
-glucosidase [46] and
µ
µ
α
α
and N-butyl (40a,b) DNJ and azepane derivatives in these assays, confirming reported data for 39a
and miglustat (40a) [54] and 40b (14% inhibition at 1 mM) [33] both using yeast α-glucosidase.
In respect to L-gulo-piperidine and D-manno-azepane series, with inverted configurations at C-2
and C-5, derivatives (26-29a,b) bearing N-hydroxyethyl, N-butyl, N-propynyl [43], or N-phenethyl
chains on the endocyclic nitrogen proved to be inactive against yeast
concentration (15–2000 M), leading to loss of activity even for the N-hydroxyethyl derivatives (28a,b
when compared to 41a,b. Reported -glucosidase inhibition data for nonsubstituted L-gulo-piperidine
and D-manno-azepane were found as weak as 30% and 55%, respectively, tested at 1 mM in
Bacillus stearothermophilus [46] or 21% in yeast -glucosidase at 240 M [52]. Thus, it was evident
that -glucosidase activities were considerably affected by iminosugar stereochemistry, ring size,
and N-substitutions, and inversion of configuration was detrimental for activity regardless of the
α-glucosidase at the tested
µ
)
α
α
µ
α

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N-substituents here described, suggesting the wrong orientation of at least two hydroxyl groups
attached at C-2 and C-5, which led to reduced binding affinity at yeast α-glucosidase active sites.
2.2.2. Almond β-glucosidase Activities:
Conversely, the assessment of the series on
27a) and D-manno-azepane (27b) derivatives (inverted configurations at C-2 and C-5 related to glucose)
preserving the N-butyl chain showed significant activity, IC50 109 and 184 M, respectively, comparable
to miglustat (40a, IC50 172 M), although three- to five-fold lower than DNJ (IC50 33 M) (Table 2).
In this particular case, the N-butyl chain seems to play an important role in -glucosidase inhibition
since the reported data for nonsubstituted was low for both L-gulo-piperidine (13% at 1 mM) and
D-manno-azepane (1% at 1 mM) or no inhibition at 240 M on the same enzyme [46 52]. Interestingly, for
the same set that preserve the N-butyl chain but display glucose stereochemistry, the seven-membered
ring derivative L-ido-azepane 40b displayed nearly twice the activity (IC50 80 M) of the corresponding
D-gluco-piperidine drug (40a), which resembled the stronger -glucosidase inhibition achieved for
nonsubstituted L-ido-azepane (Ki 17 M [46], 12.8 M [52], or IC50 38 M [53]) than nonsubstituted
D-gluco-piperidine (Ki 1700 M) [46]. Furthermore, both derivatives bearing the N-hydroxyethyl chain,
as occurs in miglitol (41a) and L-ido-azepane 41b, proved to be the strongest -glucosidase inhibitors
with IC50 of 4 M. Based on all these findings, it was possible to infer that almond -glucosidase active
sites can accept and interact with a wider range of iminosugars than yeast α-glucosidase.
β-glucosidase revealed that novel L-gulo-piperidine
(
µ
µ
µ
β
µ
,
µ
β
µ
µ
µ
µ
β
µ
β
See dose-response curves obtained from Yeast
in Supplementary Materials.
α-Glucosidase and Almond β-Glucosidase assays
Table 2. α- and β-Glucosidase activities of synthesized iminosugars having alternative stereochemistry,
ring size, and N-alkyl and N-arylalkyl chains on the endocyclic nitrogen.
Iminosugars with Inverted Configuration at C-2 and C-5
Iminosugars Preserving Glucose Stereochemistry
with Respect to Glucose
Inhibition (µM)
Inhibition (µM)
α-Glucosidase
α-Glucosidase β-Glucosidase
β-Glucosidase
33.1 ± 3.1
H
N
HO
HO
-
-
-
-
DNJ
39a
134.4 ± 2.1
2527 ± 82.2
OH
OH
N
HO
HO
26a
NI
1716 ± 12.8
635.7 ± 8.5
3437 ± 70.6
OH
OH
N
N
26b
27a
NI
NI
NI
39b
40a
NI
NI
OH
HO
OH
HO
HO OH
OH
HO
OH
OH
N
N
109.7 ± 9.3
172.8 ± 1.7
80.0 ± 4.9
HO
OH
HO
OH
OH
OH
N
N
27b
2031 ± 17.1
184.6 ± 2.6
40b
NI
OH
HO
OH
HO
OH
HO
OH
HO
OH
OH
OH
N
N
HO
HO
HO
28a
28b
NI
NI
NI
NI
41a
41b
41.3 ± 10.1
138.8 ± 1.2
4.0 ± 1.5
4.0 ± 1.4
HO
OH
OH
OH
OH
OH
N
N
OH
OH
HO
HO
OH
OH
HO
HO

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Table 2. Cont.
Iminosugars with Inverted Configuration at C-2 and C-5
with Respect to Glucose
Iminosugars Preserving Glucose Stereochemistry
Inhibition (µM)
Inhibition (µM)
α-Glucosidase β-Glucosidase
α-Glucosidase
β-Glucosidase
29a
29b
NI
NI
NI
NI
-
-
-
-
-
-
-
N
HO
HO
OH
OH
-
N
HO
OH
HO
OH
Enzyme inhibition: IC50 in µM, α-Glucosidase from Saccharomyces cerevisiae and β-Glucosidase from almonds. NI:
no inhibition. DNJ; deoxynojirimycin
3. Conclusions
In summary, a series of iminosugars were successfully synthesized from a common precursor,
D-mannitol, to produce two alternative bis-epoxides, further modified by an SN2 aminocyclization
reaction to give a mixture of both N-substituted six- and seven-membered iminosugar isomers. Besides
the ring size, two additional structural variations were also pursued to broaden the scope of reported
strategies, as stereochemistry (maintenance of glucose stereochemistry or inversion of configuration at
C-2 and C-5 positions) and N-chain of the endocyclic nitrogen (N-propynyl, -butyl, -hydroxyethyl, and
-phenethyl). Classical polyhydroxypiperidines, miglustat and miglitol drugs that maintain glucose
configuration (D-gluco-nojirimycin, DNJ) and bear N-butyl and N-hydroxyethyl chains, respectively,
were synthesized and used as reference for evaluation of the series towards
Assessment of -glucosidase activity of iminosugars revealed solely miglitol as the most active of the
series, followed by the corresponding L-ido-azepane isomer. All other iminosugars proved to not be
inhibitors of yeast -glucosidase. On the other hand, all N-butyl iminosugars having either glucose
α- and β-glucosidases.
α
α
stereochemistry, D-gluco-piperidine miglustat drug and L-ido-azepane, or inverted configurations
at C-2 and C-5 related to glucose, L-gulo-piperidine and D-manno-azepane derivatives, displayed
significant inhibition of almond β-glucosidase. In spite of that, the strongest inhibition was achieved
for D-gluco-piperidine miglitol drug and the corresponding L-ido-azepane iminosugars containing
N-hydroxyethyl chains, but, in these tests, no activity was accomplished for inverted configuration
counterparts. Thus, we observed that glucosidase inhibition promoted by some polyhydroxypiperidines
was accompanied by proportional inhibition of the corresponding polyhydroxyazepane isomers bearing
the same N-chain regardless of the ring stereochemistry. In addition, the findings of this study on
β-glucosidase inhibition by L-gulo-piperidine and D-manno-azepane series are relevant considering
their straightforward synthesis compared to DNJ series.
4. Material and Methods
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a Bruker® Ultrashield 300
NMR spectrometer. 1H (400 MHz) and 13C (100 MHz) NMR spectra were recorded on a Bruker®
1
Avance 400 MHz NMR spectrometer. H (500 MHz) and 13C (125 MHz) NMR spectra were recorded
on a Bruker® Avance 500 MHz NMR spectrometer. All spectra were recorded at room temperature
◦
(~20 C) in Sigma Aldrich® deuterated solvents. Chemical shifts (
δ) were expressed in parts per
million (ppm) relative to the reference peak. Coupling constants (J) were expressed in Hertz (Hz).
Splitting patterns in 1H NMR spectra were designated as s (singlet), br s (broad singlet), d (doublet),
br d (broad doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), ddd
(doublet of doublet of doublets), and m (multiplet). Optical rotations were measured on a Jasco P-2000

Pharmaceuticals 2019, 12, 108
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polarimeter at 22.5 nd using a sodium lamp and wavelength of 589 nm at 22.5 ◦C. HPLC purifications
were designed in a Shimadzu® SCL-10A HPLC system, Diode Array Detector Shimadzu® SPD-M10A,
and processed on Class-VP software. Purification of the compounds 31a and 31b was performed in
HPLC using a Macherey–Nagel CLC-ODS semiprep column, Methanol 40%, flowrate 4.0 mL/min, and
200 nm. High-resolution mass spectra (HRMS) were obtained on a Bruker Daltonics MicrOTOF-Q
II ESI-TOF mass spectrometer, and an Exactive Plus Orbitrap mass spectrometer (Thermo Scientific,
Germany) was equipped with an electrospray (H-ESI-II) probe and operated in negative ionization
mode. The system was controlled by Xcalibur and Tune (Thermo Scientific). For biological assays,
absorbance at 405 nm was measured using SpectraMax M2 Molecular Devices®.
1,2:5,6-di-anhydro-D-mannitol (25) [43]: D-Mannitol (5.05 g, 27.4 mmol) was solubilized in
pyridine (25.0 mL) and heated to 120 ◦C for 15 min. After that, the solution was refrigerated to 0 ◦C,
treated dropwise with tosyl chloride solution (13.10 g, 68.7 mmol/10 mL pyridine) for 1 h, and stirred at
0 ◦C for 3 h then at r.t. for 1 h. The mixture was coevaporated with toluene, and the solid was diluted
in dichloromethane and washed with HCl 1 mol L
−1 and saturated NaHCO3. The organic layer was
·
dried over MgSO4 and concentrated. The crude mixture of 1,6-di-O-tosyl-D-mannitol was solubilized
in a mixture of acetonitrile and water (38.0 mL, 2:1, v/v), and a small portion of phenolphthalein was
added to it. The mixture was stirred at 35–40 ◦C and titrated with NaOH 5 mol −1 until the solution
L
·
remained pink. After that, a mixture of Na2CO3 (87.4 g) in ethyl acetate (324 mL) was added and stirred
vigorously. The solid was filtrated and washed with ethyl acetate. The organic solution was dried
over MgSO4, filtered, and concentrated. The resulting mixture was purified by flash chromatography
1
(hexane and ethyl acetate, 7:3, v/v) to yield
1
(1.12 g, 7.70 mmol, 28%). H NMR (400 MHz, CD3OD):
δ
2.77 (2H, dd, J 2.7 Hz, 5.3 Hz, H-1a, H-6a); 2.82 (2H, dd, J 4.0 Hz, 5.3 Hz, H-1b, H-6b); 3.11–3.17 (2H, m,
H-2, H-5); 3.46–3.52 (2H, m, H-3, H-4). 13C NMR (100 MHz, CDCl3):
73.3 (C-3, C-4).
δ 46.1 (C-1, C-6); 52.8 (C-2, C-5);
1,2:5,6-Di-O-isopropilidene D-mannitol (32) [48]
1,2:5,6-Dianhydro-3,4-di-O-benzyl-L-iditol (35): prepared as described by Wilkinson et al. [47]
General procedure for the synthesis of L-gulo-piperidine and D-manno-azepane derivatives
(26a,b-31a,b): 1,2:5,6-dianhydro-D-glucitol was solubilized in methanol and 2.5 eq of primary
amine (propargylamine, ethanolamine, butylamine, or phenethylamine) was added to the solution.
The mixture was heated to 90 ◦C in a microwave for 5 min (150 W) in a sealed vessel. The solvents were
evaporated in vacuum. Compounds 27a,b and 29a,b were separated in a flash column (dichloromethane
and methanol, 8:2 v/v). Compounds 26a,b and 28a,b were acetylated by the addition of pyridine
(8.0 mL) and acetic anhydride (22.8 mL) and stirred at r.t. After 3 h, ice was added to the mixture,
the product was extracted with ethyl ether, and the organic layer was evaporated and dried with
MgSO4. Compounds 30a,b were separated by column chromatography (toluene and ethyl acetate, 3:7,
v/v). Compounds 31a,b were purified in a HPLC C-18 semiprep column in methanol/water 40% and
flow rate 4.0 mL/min.
See 1H, 13C and bidimensional NMR and ESI HRMS spectra of compounds 26-31a,b and 36-41a,b
in Supplementary Materials.
General procedure for removal of Acetyl groups: Compounds 30a,b and 31a,b were dissolved
in methanol (1.0 mL), and sodium methoxide 1 M was added dropwise to the solution until pH 9.0 and
checked with Tornassol. After half an hour, TLC showed total consumption of starting material, then it
was neutralized with ion exchange resin DOWEX® 50WX4-50. After that, the mixture was filtered
through a Celite® pad and concentrated in vacuo.
General procedure for removal o Benzyl groups [50]: The corresponding product was solubilized
in dichloromethane, and Me3SI (4 eq) was added slowly. The reaction was allowed to stir at r.t. for
15 min. TLC showed total consumption of starting material, then the reaction was quenched with
methanol. The product was purified in a SPE-C18 silica pad with methanol/water 1:1 v/v.
1
N-Propynyl-1,5-dideoxy-1,5-imino-L-gulitol (26a) [43]: 80% (0.0082 g, 0.041 mmol): H NMR
(500 MHz, D2O):
δ
2.71–2.77 (2H, m, H-1a, CH); 2.85 (1H, t, J 10.9 Hz, H-1b); 2.88–2.93 (1H, m, H-5);
≡

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3.46 (1H, d, J 17.6 Hz, H-7a); 3.68 (1H, d, J 17.6 Hz, H-7b); 3.83 (1H, dd, J 5.4 Hz, 11.7 Hz, H-6a); 3.89
(1H, dd, J 3.9 Hz, 11.7 Hz, H-6b); 3.91–3.94 (1H, m, H-3); 4.05–4.14 (2H, m, H-2, H-4). ESI HRMS:
[M+H]+ calculated for C9H15NO4 202.1074; found 202.1076.
1
N-Propynyl-1,6-dideoxy-1,6-imino-D-mannitol (26b)
(500 MHz, D2O): 2.66 (1H, t, J 2.2 Hz,
[
43]: 86% (0.0088 g, 0.044 mmol): H NMR
δ
≡
CH); 2.85 (4H, m, H-1a; H-1b; H-6a; H-6b); 3.37 (1H, d,
J 17.0 Hz, H-7a); 3.41 (1H, d, J 17.0 Hz, H-7b); 3.89–3.91 (2H, m, H-3, H-4); 4.12 (2H, t, J 4.8 Hz, H-2,
H-5). ESI HRMS: [M+H]+ calculated for C9H15NO4 202.1074; found 202.1073.
N-Butyl-1,5-dideoxy-1,5-imino-L-gulitol (27a): [α]2D2.5 29.5 (c 1.8, MeOH), 1H NMR (500 MHz,
D2O):
δ 0.83 (3H, t, J 7.4 Hz, H-10); 1.17–1.28 (2H, m, H-9); 1.37–1.50 (2H, m, H-8); 2.50–2.92 (5H, m,
2xH-1, H-5, 2xH-7); 3.75 (1H, dd, J 5.6. 11.8, H-6a); 3.78–3.85 (2H, m, H-3, H-6b); 3.94–4.00 (1H, m, H-2);
4.05–4.11 (1H, m, H-4). 13C NMR (125 MHz, D2O):
δ 13.3 (C-10); 20.0 (C-9); 27.4 (C-8); 53.1 (C-1 or C-7);
55.1 (C-1 or C-7); 58.5 (C-6); 68.2 (C-4); 70.1 (C-3); 70.4 (C-3); 72.8 (C-2). ESI HRMS: [M+H]+ calculated
for C10H22NO4 220.1544; found 220.1538.
N-Butyl-1,6-dideoxy-1,6-imino-D-mannitol (27b): [α]2D2.5 −62.0 (c 2.2, MeOH), 1H NMR (500 MHz,
D2O): δ 0.76 (3H, t, J 7.3 Hz, 3xH-10); 1.07–1.25 (2H, m, 2xH-9); 1.30–1.44 (2H, m, 2xH-8); 2.47–2.57 (2H,
m, 2xH-7); 2.65–2.85 (4H, m, 2xH-1, 2xH-6); 3.73–3.79 (2H, m, H-3, H-4); 3.94–4.04 (2H, m, H-2, H-5).
13C NMR (125 MHz, D2O):
13.2 (C-10); 20.0 (C-9); 27.5 (C-8); 55.2 (C-1, C-6); 58.4 (C-7); 68.3 (C-2; C-5);
72.8 (C-4; C-3). ESI HRMS: [M+H]+ calculated for C10H22NO4 220.1544; found 220.1543.
N-Hydroxyethyl-1,5-dideoxy-1,5-imino-L-gulitol (28a): [α]2D2.5 16.1 (c 0.6, MeOH), H NMR
(400 MHz, CD3OD): 2.53–2.71 (2H, m, H-1a, H-7a); 2.78–2.89 (2H, m, H-5, H-7b); 2.97 (1H, ddd, J 4.9;
7.0; 13.5 Hz, H-1b); 3.55–3.98 (7H, m, H-2, H-3, H-4, H-6a, H-6b, H-8a, H-8b). 13C NMR (100 MHz,
CD3OD): 51.9 (C-7); 55.1 (C-1); 58.6 (C-8); 60.0 (C-6); 61.0 (C-5); 66.5, 70.9, 71.3 (C-2, C-3, C-4).
ESI HRMS: [M+H]+ calculated for C8H18NO5 208.1180; found 208.1177
N-Hydroxyethyl-1,6-imino-D-mannitol (28b): [α]2D2.5 15.3 (c 0.5, MeOH), 1H NMR (400 MHz,
CD3OD): 2.58–2.80 (4H, m, H-1a; H-6 ; 2xH-7); 2.89 (2H, dd, J 4.3; 13.2 Hz, H-1b, H-6b); 3.59 (2H, dd,
J 6.0; 12.5 Hz, 2xH-8); 3.86–3.92 (2H, m, H-3, H-4); 4.00–4.08 (2H, m, H-2, H-5). 13C NMR (100 MHz,
CD3OD):
58.4 (C-1; C-6); 60.4 (C-8); 61.8 (C-7); 70.9 (C-2; C-5); 74.0 (C-3; C-4). ESI HRMS: [M+H]+
δ
1
δ
δ
−
δ
ª
δ
calculated for C8H18NO5 208.1180; found 208.1182.
N-Phenethyl-1,5-dideoxy-1,5-imino-L-gulitol (29a) [46]
N-Phenethyl-1,6-dideoxy-1,6-imino-D-mannitol (29b) [46]
1
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol (30a) [43]: H NMR (400 MHz,
CDCl3): 2.03, 2.09, 2.13, 2.14 (12H, 4s, 4 CH3); 2.33 (1H, t, J 2.3 Hz, CH); 2.80 (1H, dd, J 4.7 Hz, 11.2 Hz,
δ
×
≡
H-1a); 2.97 (1H, dd, J 9.6 Hz, 11.2 Hz, H-1b); 3.28 (1H, ddd, J 3.3 Hz, 5.8 Hz, 10.0 Hz, H-5); 3.44 (1H, dd,
J 2.3 Hz, 17.8 Hz, H-7a); 3.67 (1H, dd, J 2.3 Hz, 17.8 Hz, H-7b); 4.20 (1H, dd, J 5.8 Hz, 11.6 Hz, H-6b); 4.24
(1H, dd, J 3.3 Hz, 11.6 Hz, H-6a); 5.20–5.27 (3H, m, H-2, H-3, H-4). 13C NMR (100 MHz, CDCl3):
δ
20.8;
20.9 (CH3); 43.9 (C-1); 49.6 (C-7); 55.5 (C-5); 61.4 (C-6); 66.5 (C-3, C-4); 68.7 (C-2); 77.2 (
≡
CH); 169.3; 169.7;
170.5 (C=O). ESI HRMS: [M+H]+ calculated for C17H24NO8 370.1496; found 370.1496.
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol (30b)
[
43]: 1H NMR
(400 MHz, CDCl3):
δ
2.05, 2.12 (12H, 2s, 4
×
CH3); 2.25 (t, J 2.3 Hz,
≡
CH); 2.91 (2H, dd, J 5.6 Hz,
13.8 Hz, H-1a, H-6a); 2.99 (2H, dd, J 4.4 Hz, 13.8 Hz, H-1b, H-6b); 3.39 (1H, dd, J 2.3 Hz, 17.3 Hz, H-7a);
3.45 (1H, dd, J 2.3 Hz, 17.3 Hz, H-7b); 5.40 (2H, dt, J 1.2 Hz, 4.6 Hz, H-2, H-5); 5.48 (2H, t, J 1.2 Hz, H-3,
H-4). 13C NMR (100 MHz, CDCl3):
δ
20.8; 21.0 (CH3); 47.9 (C-7); 54.1 (C-1, C-6); 69.9 (C-2, C-5); 70.9
(C-3 e C-4); 77.2 (
found 370.1535.
≡
CH); 169.9; 170.1 (C=O). ESI HRMS: [M+H]+ calculated for C17H24NO8 370.1496;
N-Acetoxyethyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-L-gulitol (31a): 1H NMR (400 MHz,
CDCl3):
δ 2.06; 2.08; 2.10 (18H, 3s, 5xCH3); 2.89 (1H, dd, J 5.0; 13.7 Hz, H-1a); 2.96 (2H, t, H-7, J 5.9 Hz,
H-7); 3.04 (1H, dd, J 2.6; 13.7 Hz, H-1b); 3.45 (1H, dd, J 4.8; 11.4 Hz, H-5); 4.05 (1H, dd, J 5.6; 11.3 Hz,
H-6a); 4.10–4.21 (2H, m, 2xH-8); 4.37 (1H, dd, J 7.0; 11.8 Hz, H-6b); 5.15 (1H, dd, J 3.3; 9.0 Hz, H-3);
5.20–5.25 (1H, m, H-2); 5.20–5.25 (1H, m, H-2); 5.28 (1H, dd, J 4.9; 9.0 Hz, H-4). 13C NMR (100 MHz,

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CDCl3):
(C-2, C-4).
N-Acetoxyethyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-D-mannitol (31b): 1H NMR
(400 MHz, CDCl3):
2.03; 2.07; 2.10 (18H, 3s, 5xCH3); 2.85 (2H, t, J 5.8 Hz, H-4, H-40 ); 2.91 (2H, dd, J 5.6;
δ 20.8, 20.9, 21.0 (5xCH3); 48.9 (C-1); 52.7 (C-7); 58.2 (C-5); 59.9 (C-6); 68.0 (C-3); 68.1, 68.3
δ
14.0 Hz, H-1a, H-10 a); 3.04 (2H, dd, J 4.3; 14.0 Hz, H-1b, H-10 b); 4.11 (2H, dd, J 5.7; 9.1 Hz, H-5, H-50 );
5.29-5.37 (2H, m, H-2, H-20 ); 5.43-5.47 (2H, m, H-3, H-30 ). 13C NMR (100 MHz, CDCl3):
(5xCH3); 54.8 (C-1, C-10 ); 56.4 (C-4); 62.2 (C-5); 70.2 (C-2, C-20 ); 70.6 (C-3, C-30 ).
δ 20.8; 20.9; 21.0
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol (36a): 1H NMR (400 MHz,
CDCl3):
δ
2.27 (1H, t, J 2.2 Hz, CH); 2.50 (1H, d, J 9.5 Hz, H-5); 2.60 (1H, t, J 10.7 Hz, H-1a); 2.92 (1H,
≡
dd, J 4.9; 10.9 Hz, H-1b); 3.31–3.44 (2H, m, 2xH-7); 3.62–3.91 (5H, m, H-2, H-3, H-4, 2xH-6); 4.77 (2H,
dd, J 3.8; 11.2 Hz, CH2Ph); 4.98 (2H, dd, J 11.2; 13.4 Hz, CH2Ph); 7.29–7.45 (10H, m, H-Ph). 13C RMN
(100 MHz, CDCl3):
δ
42.1 (C-1); 56.1 (C-7); 57.3 (C-6); 63.1 (C-2 or C-5); 69.4 (C-2 or C-5); 74.6 (C CH);
≡
75.2 (CH2Ph); 87.3 (C-3; C-4); 127.9, 128.6, 128.7 (Ar); 138.1 (Cq). ESI HRMS: [M+H]+ calculated for
C23H28NO4 382.2013; found 382.2002.
N-Propynyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-L-iditol (36b): 1H NMR (400 MHz,
CDCl3):
δ
2.31 (1H, t, J 2.3 Hz,
≡
CH); 2.77 (2H, dd, J 8.2; 12.3 Hz, H-1a, H-6a); 2.98 (2H, dd,
J 1.1; 12.7, H-1b, H6b); 3.40–3.56 (2H, m, 2xH-7); 3.63–3.70 (2H, m, H-3, H-4); 3.80–3.93 (2H, m, H-2,
H-5); 4.67 (2H, d, J 11.2 Hz, CH2Ph); 4.81 (2H, d, J 11.2 Hz, CH2Ph); 7.29–7.42 (10H, m, H-Ph). 13C
RMN (100 MHz, CDCl3):
δ
48.8 (C-7); 56.9 (C-1, C-6); 68.0 (C-2, C-5); 73.8 (CH2Ph); 78.2 (C CH); 86.5
≡
(C-3, C-4); 127.9, 128.0, 128.6 (Ar); 137.9 (Cq). ESI HRMS: [M+H]+ calculated for C23H28NO4 382.2013;
found 382.2001.
N-Butyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol (37a): 1H NMR (400 MHz,
CDCl3):
δ 0.94 (3H, t, J 6.9 Hz, 3xH-10); 1.21–1.38 (2H, m, 2xH-9); 1.39–1.56 (2H, m, 2xH-8); 2.26 (1H,
t, J 10.6Hz, H-5); 2.32–2.54 (3H, m, H-1a, H-1b, H-7a); 2.70–2.82 (1H, m, H-7b); 3.13 (1H, dd, J 4.6;
11.2 Hz, H-6a); 3.38 (1H, t, J 8.8 Hz, H-3); 3.60–3.73 (2H, m, H-4, H-6b); 3.77–3.93 (2H, m, H-2, H-XX);
4.75 (2H, dd, J 5.6; 11.2 Hz, CH2Ph); 4.96 (2H, t ap., J 11.0 Hz, CH2Ph); 7.29–7.48 (10H, m, Ar). 13C
RMN (100 MHz, CDCl3):
δ 14.0 (C-10); 20.6 (C-9); 27.3 (C-8); 52.1 (C-1 or C-7); 55.1 (C-1 or C-7); 57.5
(C-6); 64.9 (C-2 or C-5); 69.3(C-2 or C-5); 74.9 (CH2Ph); 75.1 (CH2Ph); 78.1 (C-3 or C-4); 86.8 (C-3 or
C-4); 127.8, 127.9, 128.0, 128.5, 128.7 (C-Ar); 138.1 (Cq); 138.5 (Cq). ESI HRMS: [M+H]+ calculated for
C24H34NO4 400.2483; found 400.2477.
N-Butyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-L-iditol (37b): 1H NMR (400 MHz, CDCl3):
δ
0.93 (3H, t, J 7.2 Hz, 3xH-10); 1.25–1.40 (2H, m, 2xH-9); 1.42–1.58 (2H, m, 2xH-8); 2.52–2.69 (4H, m,
H-1a, H-6a, 2xH-7); 2.92 (2H, d, J 12.5 Hz, H-1b, H-6b); 3.60–3.70 (2H, m, H-3, H-4); 3.78–3.89 (2H, m,
H-2, H-5); 4.66 (2H, d, J 11.2 Hz, CH2Ph); 4.79 (2H, d, J 11.2 Hz, CH2Ph); 7.29–7.43 (10H, m, H-Ph).
13C RMN (100 MHz, CDCl3):
δ 13.9 (C-10); 20.4 (C-9); 29.3 (C-8); 57.6 (C-7); 59.0 (C-1; C-6); 67.7 (C-2,
C-5); 73.6 (CH2Ph); 87.0 (C-3, C-4); 127.9, 128.6 (Ar); 137.0 (Cq). ESI HRMS: [M+H]+ calculated for
C24H34NO4 400.2483; found 400.2475.
N-Hydroxyethyl-2,3,4,6-tetra-O-acetyl-1,5-dideoxy-1,5-imino-D-glucitol (38a): 1H NMR (400 MHz,
CDCl3):
δ 2.29 (1H, dd, J 9.7, 11.3 Hz, H-1a); 2.37–2.51 (2H, m, H-5, H-7a); 2.94–3.08 (1H, m, H-7b); 3.16
(1H, dd, J 4.4; 11.5 Hz, H-1b); 3.43 (1H, t, J 8.3 Hz, H-3); 3.54–3.77 (4H, m, H-2, H-4, H-8a, H-8b); 3.88
(2H, qd, J 2.8; 12.3 Hz, H-6a, H-6b); 4.67–4.81 (2H, m, CH2Ph); 4.84–4.97 (2H, m, CH2Ph); 7.29–7.45 (10H,
m, Ph-H). 13C RMN (100 MHz, CDCl3):
δ 53.2 (C-7); 55.3 (C-1); 57.8 (C-6); 59.6 (C-8); 65.7 (C-5); 69.0
(C-2 or C-4); 74.8 (CH2Ph); 78.0 (C-2 or C-4); 85.9 (C-3); 128.0, 128.6 (Ar); 138.4 (Cq). ESI HRMS: [M+H]+
calculated for C22H30NO5 388.2119; found 388.2120.
N-Hydroxyethyl-2,3,4,6-tetra-O-acetyl-1,6-dideoxy-1,6-imino-L-iditol (38b): 1H NMR (400 MHz,
CDCl3):
δ 2.66–2.80 (4H, m, 2xH-1, 2xH-2); 2.99 (2H, dd, J 2.8; 13.1 Hz, 2xH-7); 3.62–3.72 (4H, m, H-2,
H-5, 2xH-8); 3.80–3.90 (2H, m, H-3, H-4); 4.65 (2H, d, J 11.3 Hz, CH2Ph); 4.84 (2H, d, J 11.3 Hz, CH2Ph);
7.28–7.42 (10H, m, H-Ph). 13C RMN (100 MHz, CDCl3):
δ 59.0 (C-7); 59.7 (C-8); 60.7 (C-1; C-6); 70.0 (C-3;
C-4); 74.2 (CH2Ph); 85.4 (C-2; C-5); 127.9, 128.0, 128.6, 137.9 (Ar). ESI HRMS: [M+H]+ calculated for
C22H30NO5 388.2119; found 388.2102.

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N-Propynyl-1,5-dideoxy-1,5-imino-D-glucitol (39a) [47]
N-Propynyl-1,6-dideoxy-1,6-imino-L-iditol (39b) [47]
N-Butyl-1,5-dideoxy-1,5-imino-D-glucitol (40a) [55]
N-Butyl-1,6-dideoxy-1,6-imino-L-iditol (40b): [α]2D2.5 1.3 (c 1.1, MeOH), H NMR (400 MHz,
1
D2O):
δ 0.86 (3H, t, J 8.0 Hz, 3xH-10); 1.24–1.36 (2H, m, 2xH-9); 1.56–1.74 (2H, m, 2xH-8); 3.14–3.23
(2H, m, H-7); 3.26–3.37 (4H, m, 2xH-1, 2xH-6); 3.58–3.67 (2H, m, H-2, H-5); 4.01–4.08 (2H, m, H-3, H-4).
13C RMN (100 MHz, D2O):
δ 12.9 (C-10); 19.3 (C-9); 25.6 (C-8); 58.6 (C-1; C-6; C-7) 67.1 (C-3 or C-4); 67.7
(C-3 or C-4); (C-2; C-5). ESI HRMS: [M+H]+ calculated for C10H22NO4 220.1544; found 220.1545.
N-Hydroxyethyl-1,5-dideoxy-1,5-imino-D-glucitol (41a) [55]
N-Hydroxyethyl-1,6-dideoxy-1,6-imino-L-iditol (41b): [α]2D2.5 9.1 (c 0.6, MeOH), 1H NMR (400
−
MHz, D2O):
δ 2.62–2.77 (4H, m, H-1a; H-1b; H-6a; H-6b); 2.91 (2H, dd, J 3.8;13.7 Hz, H-7a, H-7b);
3.40–3.48 (2H, m, H-2, H-3); 3.69 (4H, m, H-4, H-5, H-8a, H-8b,). 13C RMN (100 MHz, D2O):
(C-7), 59.3 (C-1, C-6, C-8), 70.8 (C-4, C-5), 75.6 (C-2, C-3). ESI HRMS: [M+H]+ calculated for C8H18NO5
δ 58.4
208.1185; found 208.1176.
Biological assays [43]: Yeast
activity was assessed using a 96-well plate assay. Assays contained 20 mM NaOAc at pH 6.8
-glucosidase) and pH 6.2 (
-glucosidase), 10 mM PIPES (piperazine-N,N0-bis(2-ethanesulfonic acid),
0.1 mM EDTA, -glucosidase (5
α-glucosidase (EC 3.2.1.20) and almond β-glucosidase (EC 3.2.1.21)
(α
β
α
µ
g/mL◦),
β-glucosidase (6 µg/mL), and inhibitor (0.1–2 mM). Enzyme
and inhibitor were equilibrated at 37 C for 30 min. The reaction was initiated by the addition of
p-nitrophenyl
then it was quenched with 100
α
-D-glucopyranoside (200
µM) or p-nitrophenyl β-D-glucopyranoside (200 µM), and
µ
L of sodium carbonate 3.0 M after 25 min incubation at 37 ◦C. Assays
were repeated in duplicate and data averaged.
Supplementary Materials: The following are available online at http://www.mdpi.com/1424-8247/12/3/108/s1,
1H, 13C and bidimensional NMR and ESI HRMS spectra of compounds 26-31a,b and 36-41a,b; Table: IC50 of final
compounds and Dose-response curves obtained from Yeast α-Glucosidase and Almond β-Glucosidase assays.
Author Contributions: Conceptualization, I.C.; methodology, L.O.B.Z., V.A.-L.; software, L.O.B.Z.; validation,
L.O.B.Z. and I.C.; formal analysis, L.O.B.Z. and I.C.; investigation, L.O.B.Z., Valquiria Aragão-Leoneti and I.C.;
resources, L.O.B.Z., Valquiria Aragão-Leoneti and I.C.; data curation, L.O.B.Z. and I.C.; writing—original draft
preparation, I.C.; writing—review and editing, L.O.B.Z. and I.C.; visualization, L.O.B.Z. and I.C.; supervision, I.C.;
project administration, I.C.; funding acquisition, I.C.
Funding: This research was funded by Fundaç
number 2007/00910-6, Conselho Nacional de Desenvolvimento Cient
503709/2011-5, and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
ão de Amparo
à
Pesquisa do Estado de S
ão Paulo (FAPESP), grant
í
fico e Tecnol gico (CNPq), ), grant number
ó
Acknowledgments: We acknowledge financial support from Fundaç o de Amparo Pesquisa do Estado de São
ã
à
Paulo (FAPESP, Proc. n. 2007/00910-6), Conselho Nacional de Desenvolvimento Cient fico e Tecnol gico (CNPq,
í
ó
Proc. n. 503709/2011-5), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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