γ-Hydroxyethyl piperidine iminosugar and N-alkylated derivatives: A study of their activity as glycosidase inhibitors and as immunosuppressive agents

Article abstract of DOI:10.1016/j.bmc.2014.09.034

An efficient and practical strategy for the synthesis of (3R,4s,5S)-4-(2-hydroxyethyl) piperidine-3,4,5-triol and its N-alkyl derivatives 8a-f, starting from the d-glucose, is reported. The chiral pool methodology involves preparation of the C-3-allyl-α-d-ribofuranodialdose 10, which was converted to the C-5-amino derivative 11 by reductive amination. The presence of C-3-allyl group gives an easy access to the requisite hydroxyethyl substituted compound 13. Intramolecular reductive aminocyclization of C-5 amino group with C-1 aldehyde provided the γ-hydroxyethyl substituted piperidine iminosugar 8a that was N-alkylated to get N-alkyl derivatives 8b-f. Iminosugars 8a-f were screened against glycosidase enzymes. Amongst synthetic N-alkylated iminosugars, 8b and 8c were found to be α-galactosidase inhibitors while 8d and 8e were selective and moderate α-mannosidase inhibitors. In addition, immunomodulatory activity of compounds 8a-f was examined. These results were substantiated by molecular docking studies using AUTODOCK 4.2 programme.

Full text of DOI:10.1016/j.bmc.2014.09.034

Bioorganic & Medicinal Chemistry 22 (2014) 5776–5782
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
c
-Hydroxyethyl piperidine iminosugar and N-alkylated derivatives: A
study of their activity as glycosidase inhibitors and as
immunosuppressive agents
Pramod R. Markad a, Dhiraj P. Sonawane a, Sougata Ghosh b, Balu A. Chopade b, Navnath Kumbhar b,
Thierry Louat c, Jean Herman c, Mark Waer c, Piet Herdewijn c, Dilip D. Dhavale a,
⇑
a Department of Chemistry, Garware Research Centre, University of Pune, Pune 411007, India
b Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India
c KU Leuven, Interface Valorisation Platform (IVAP), Kapucijnenvoer 33, 3000 Leuven, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2014
Revised 5 September 2014
Accepted 15 September 2014
Available online 7 October 2014
An efficient and practical strategy for the synthesis of (3R,4s,5S)-4-(2-hydroxyethyl) piperidine-3,4,5-triol
and its N-alkyl derivatives 8a–f, starting from the -glucose, is reported. The chiral pool methodology
involves preparation of the C-3-allyl- -ribofuranodialdose 10, which was converted to the C-5-amino
derivative 11 by reductive amination. The presence of C-3-allyl group gives an easy access to the requisite
hydroxyethyl substituted compound 13. Intramolecular reductive aminocyclization of C-5 amino group
D
a
-D
with C-1 aldehyde provided the
ated to get N-alkyl derivatives 8b–f. Iminosugars 8a–f were screened against glycosidase enzymes.
Amongst synthetic N-alkylated iminosugars, 8b and 8c were found to be -galactosidase inhibitors while
8d and 8e were selective and moderate -mannosidase inhibitors. In addition, immunomodulatory activ-
ity of compounds 8a–f was examined. These results were substantiated by molecular docking studies
using AUTODOCK 4.2 programme.
c-hydroxyethyl substituted piperidine iminosugar 8a that was N-alkyl-
Keywords:
Iminosugars
Glycosidase inhibitors
Immunosuppressive activity
Chiron approach
a
a
Reductive amination
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
by the use of competitive/non-competitive inhibitors. In this
aspect, different analogues of iminosugars7 were synthesized as
Hydrolysis of the glycosidic bond, commonly called as glycosi-
dase process, assists wide range of metabolic processes such as
digestion, lysosomal glycoconjugate catabolism and glycoprotein
biosynthesis degradation within the endoplasmic reticulum.1
Abnormalities caused in these processes result in several patholog-
selective and potent inhibitors of mannosidase, glucosidase and
galactosidase enzymes. Among piperidine iminosugars, the N-
alkylated iminosugars demonstrate higher and selective inhibitory
activity compared to their parent iminosugars.8 Asano et al.
showed that the N-alkylation of deoxynojirimycin (DNJ) induces
ical complications and disorders. For example, the
a
-mannosidase
a shift in specific inhibition of glucosidases from
to -glucosidase I and in cell culture, the N-alkylated derivatives
of DNJ were found to be more effective against -glucosidase I than
a-glucosidase II
is important for regulating the biosynthesis of several glycopro-
teins. as, it controls the N-glycosylation pathway of oligosaccha-
rides, that are partially trimmed from Glc3Man9GlcNAc2 moieties
involved in N-linked glycoprotein biosynthesis,2 which plays a piv-
otal role in cancer metastasis, carbohydrate metabolic disorders,
a
a
the DNJ.8a This fact led to the discovery of N-hydroxyethyl DNJ (1a;
Fig. 1) and N-butyl DNJ 1b as promising clinical candidates for the
treatment of type II diabetes and Gaucher disease, respectively.
Apart from N-alkylation, other type of modification that has been
sought is the variation of position and stereochemistry for the
hydroxyalkyl substituent in the six-membered iminosugar.9 This
modification resulted into isofagomine 2 and related hydroxyalkyl
substituted iminosugars.10 The bioactivity data of these com-
pounds indicated that the position and orientation of the hydroxy-
alkyl group in the piperidine iminosugar affects the efficiency and
selectivity of glycosidases.6 For example, isofagomine 2a is a better
inhibitor of b-glucosidases9k however, it’s (5S)-hydroxy substituted
viral infection and immune response.3,4 Whereas, the
a
-glucosi-
dase reduces postprandial hyperglycemia in the treatment of type
II diabetic patients4 and
-galactosidase regulates lysosomal glyco-
a
conjugate catabolism and any disorder in this process or mutation
in enzyme leads to lysosomal storage disorder (LSD)5 and Fabry’s
disease.6 The malfunctioning in all these processes is controlled
⇑
Corresponding author.
E-mail address: ddd@chem.unipune.ac.in (D.D. Dhavale).
http://dx.doi.org/10.1016/j.bmc.2014.09.034
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

P.R. Markad et al. / Bioorg. Med. Chem. 22 (2014) 5776–5782
5777
O
R
N
Bn
Cbz
H
N
N
O
Ref. 15a
a
H
HO
HO
O
D-Glucose
R1
O
OH
O
OH
R2
OH
O
OH
OH
OH
O
1a R = CH2CH2OH
1b R = nBu
2a
10
11
R
1 = CH2OH; R2 = H
2b R1 = CH2OH; R2 = OH
2c R1 = OH; R2 = CH2OH
Bn
Cbz
Bn
Cbz
H
N
HO
HO
H
N
H
N
N
N
b
c
O
O
OH
O
O
HO
OH
HO
OH
R1
R2
OH
RO
O
OH
OH
O
O
HO
HO
H
5a R1 = H; R2 = OH
13
R = H
OH
5b
R1 = OH; R2 =H
3
12
d
4
14
R = Ac
R
N
(CH2)7CH3
N
(CH2)9CH3
N
R
N
α
β
γ
e
8a R = H
R1
R1
HO
OH
8b
8c
R = (CH2)3-CH3
R = (CH2)5-CH3
HO
OH
R2
R2
HO
OH
OH
OH
OH
8d R = (CH2)7-CH3
HO
8a
8e
8f
R = (CH2)9-CH3
R = CH2CH2OH
6a R1 = H; R2 = OH
7a R1 = H; R2 = OH
R = H
6b
R1 = OH; R2 =H
HO
7b
8b R = (CH2)3-CH3
8c R = (CH2)5-CH3
R
1 = OH; R2 =H
8d
R = (CH2)7-CH3
(a) (i) BnNH2, MeOH, NaCNBH4, -20 oC to rt (ii) CBz-Cl, NaHCO3
MeOH:H2O, rt, 85 % two steps (b) (i) K2OsO4 .2H2O, NMO,
Acetone:H2O, 0 oC to rt (ii) NaIO4, Acetone:H2O,0 oC to rt, 79 %
(c) NaBH4,THF:H2O,0 oC to rt, 87 % (d) Ac2O, Pyridine,0 oC to rt,
94 % (e) (i)TFA:H2O, 0 oC to rt, (ii) Pd / C, H2, 200 psi, rt, 81 %
(iii) R-Br, K2CO3, DMF, 80 oC.
8e R = (CH2)9-CH3
8f R = CH2CH2OH
Figure 1. Piperidine iminosugars and related compounds.
analogue 2b is a better inhibitor towards both
a-and b-glucosi-
Scheme 1. Synthesis of iminosugars 8a–f.
dases while; (5R)-hydroxy isofagomine 2c is a mild b-mannosidase
inhibitor.7c,15d We have reported earlier the synthesis of piperidine
4 with the hydroxyethyl group at the
which was found to be a selective and potent inhibitor of b-gluco-
sidase.11 Other analogues such as 5a and 5b, with
-gem-dihy-
droxymethyl substitution, were found to be selective inhibitors
of
-glucosidases in the nanomolar range12 while; introduction
of the hydroxyethyl group at the b-position of the piperidine imi-
nosugar 3 resulted in the loss of activity with very weak b-galacto-
sidase inhibition. In addition to the glycosidase inhibition study,
iminosugars have been evaluated as immunomodulatory agents.
Wang et al. reported hydroxyalkyl- and N-alkyl iminosugars such
as 6, 7 as potent immunosuppressive agents.13 A report from Our
group demonstrated that the indolizidine iminosugars are also act-
ing as immunomodulating agents.14
c-position of piperidine,
NMO gave triol that was directly subjected to oxidative cleavage
using sodium metaperiodate to give aldehyde 12. Reduction of
12 using sodium borohydride in THF–water afforded diol 13 that
on selective protection of primary hydroxy group, using acetic
anhydride in pyridine, gave acetyl derivative 14. In the next steps,
removal of the 1,2 acetonide group using TFA–water afforded
hemiacetal (as evident from the 1H NMR of the crude product) that
on reductive aminocyclization using 10% Pd/C in methanol under
hydrogen pressure afforded 3,4,5 trihydroxy-4-hydroxyethyl
piperidine 8a as a white solid. This one pot four step process
involves hydrogenolysis of N-benzyl and N-Cbz groups to give
primary amine that concomitantly underwent reductive aminocy-
clization with C-1 aldehyde to give six membered cyclic iminium
ion which was in situ reduced to afford piperidine iminosugar. In
the same process, we noticed that the de-acetylation of primary
acetate group took place to give iminosugar 8a in overall 44% yield
from 10.16 The high yields at each step allowed us to provide 8a in
good amount.
a
a
Encouraged by these results and as a part of our continuing
efforts in this area,15 we report herein the synthesis of (3R,4s,5S)-
4-(2-hydroxyethyl) piperidine-3,4,5-triol 8a and of a series of N-
alkyl derivatives 8b–f and the evaluation of their glycosidase
inhibition as well as immunosuppressive activity. Amongst these
iminosugars, compounds 8b and 8c showed
bition while 8d and 8e showed selective and moderate inhibition
of -mannosidase. Compound 8e was found to be a weak inhibitor
a-galactosidase inhi-
Having iminosugar 8a in hand, N-alkylation was performed
using appropriate alkyl bromides in DMF-K2CO3 as reported earlier
in the literature.17 Thus, reaction of 8a, separately with n-butyl, n-
hexyl, n-octyl, n-decyl, and hydroxyethyl bromide using K2CO3 in
DMF at 80 °C afforded corresponding N-alkylated iminosugars
8b–f in good yields (60–70%).
a
of the IgG secretion by CpG oligonucleotide stimulated B-cells.
2. Result and discussion
As shown in Scheme 1, the synthesis of target molecules started
2.1. Conformational assignments of 8a–f
from aldehyde 10 which was derived from D-glucose in five steps
as reported by us.15a In the subsequent steps, the reductive amina-
tion of C-5 aldehyde group in compound 10 with benzylamine and
sodium cyanoborohydride in methanol followed by reaction with
benzyloxycarbonyl chloride and sodium bicarbonate in metha-
nol–water afforded the N-Cbz protected compound 11. Dihydroxy-
lation of 11 with catalytic amount of K2OsO4ꢀ2H2O (5 mol %) and
An axial/equatorial orientation of the hydroxyalkyl substituent
in the six membered piperidine iminosugars is the decisive factor
for either 4C1 or 1C4 conformation9,18 that plays a crucial role in
the binding properties of iminosugars with different enzymes
and thus affecting the inhibitory activity.6,19 Therefore, conforma-
tions of 8a–f were determined using the 1H NMR data based on

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P.R. Markad et al. / Bioorg. Med. Chem. 22 (2014) 5776–5782
Table 2
the coupling constant values between the H-2 and H-3 protons. In
case of parent compound 8a, due to the presence of the plane of
symmetry, the C-2/C-6 axial and equatorial protons and C-3/C-5
protons were found to be magnetically equivalent. This led to the
five sets of protons in the 1H NMR spectra. The C-2/C-6 axial
protons were appeared as an apparent triplet with large coupling
constant of 10.7 Hz (J2a,2e = J2a,3a = 10.7 Hz). In analogy with this,
the H-3/H-5 appeared as a doublet of doublet (dd) with coupling
constant of 10.7 and 4.5 Hz (J2a,3a = 10.7 Hz and J2e,3a = 4.5). This
requires an axial orientation of the H-3/H-5 confirming the 5C2
conformation of the compound as shown in Figure 2. An analogous
behaviour was noticed for N-alkylated iminosugars 8b–f (Table 1),
wherein H-3/H-5 appeared as a dd with large coupling constant of
10–11 Hz and equatorial coupling constant of 4–5 Hz indicating
their 5C2 conformation (Fig. 2).
IC50 (mM) values for iminosugars 8a–f and standard Miglitol (NI: no inhibition at
1 mM)
Enzyme
8a
8b
8c
8d
8e
8f
0.252 0.171
NI 1.850
Miglitol
a
a
a
-Glucosidase
-Galactosidase NI
-Mannosidase
0.418 0.360 NI
NI
NI
NI
0.167 0.315 NI
1.826 0.252 0.183 0.143 0.112 0.873 0.379
Table 3
IC50 M) values for iminosugars 8a–f
(l
IC50
(
lM)
8a
8b
8c
8d
8e
8f
MLR
B-cell assay
Jurkat
>10
>50
>10
>10
>50
>10
>10
>50
>10
>10
>50
>10
>10
15
>10
>10
>50
>10
2.2. Biological activity
2.2.2. Immunosuppressive activity
2.2.1. Glycosidase inhibition
The immunosuppressive activity of newly synthesized com-
pounds was evaluated in vitro by investigating their ability to inhi-
bit the proliferation of human lymphocytes in the Mixed
Lymphocyte Reaction (MLR) assay and to inhibit the production
of IgG by human B cells stimulated with a CpG oligonucleotide
(B-cell assay). These two tests target the two major components
of the immune response, the T-cells and B-cell respectively. A cyto-
toxicity counter screen was performed on Jurkat cells and results
are summarized in Table 3. Amongst the compounds studied, imi-
Glycosidase inhibitory activity of 8a–f was studied against
a
a
-glucosidase (E.C. 3.2.1.20),
a-galactosidase (E.C. 3.2.1.22) and
-mannosidase (E.C. 3.2.1.24) with reference to known standard
N-hydroxyethyl DNJ (trade name Miglitol). The IC50 values are
summarized in Table 2. The parent iminosugar 8a showed no inhi-
bition of
cosidase and
which the ring nitrogen is substituted with the n-butyl and n-hexyl
groups, respectively, showed good inhibition of -galactosidase
and -mannosidase as compared to miglitol. Among these
compounds, 8b was found to be a better -galactosidase inhibitor
than 8c while compound 8c was noticed to be better inhibitor of
-mannosidase than 8b. The n-octyl 8d and n-decyl 8e nitrogen
derivatives were found to be selective and moderate inhibitor of
-mannosidase showing IC50 values of 143 M and 112 M,
respectively. The N-hydroxyethyl substituted compound 8f (migli-
tol derivative) showed weaker -glucosidase activity than miglitol.
a-galactosidase but found to be a weak inhibitor of
a-glu-
a-mannosidase while; compounds 8b and 8c in
a
nosugar 8e showed weak activity (IC50 = 15 lM) in the B-cell assay,
while the other compounds were found to be inactive in both
assays.
a
a
a
2.2.3. Molecular docking
In order to understand the interactions of synthesized iminosug-
ars with the amino acid residues of the mannosidase, molecular
docking studies were performed. Since compound 8e showed
a
l
l
a
highest
studies. Glycosidase inhibitory activity of 8e showed its efficiency
as selective -mannosidase inhibitors (isolated from jack bean
a-mannosidase activity, it was considered for the docking
The comparative study of IC50 values in Table 2 indicated an
increase in the hydrophobicity of the molecule with increase in
the N-alkyl chain length resulting into improved inhibition and
a
mannosidase). The complete sequence of jack bean mannosidase
is unknown; hence it is difficult to perform the docking studies with
the amino acid residues in the active site. Therefore, crystal struc-
selectivity towards
a-mannosidase. The selective a-mannosidase
activity in the micromolar range prompted us to substantiate our
results with molecular docking studies.
tures for human a-mannosidase were downloaded from the protein
databank (http://www.rcsb.org) and structure (PDB: 1X9D) having
high resolution was chosen as the target structure to perform the
molecular docking studies and predict the binding efficiency of
compound 8e to the human mannosidase (PDB: 1X9D). Molecular
docking was performed using AUTODOCK 4.2 wizard.20 The molec-
OH
R
H
H
5
N1
R
N1
6
2
6
H
HO
H
3OH
2
4
HO
H
4
ular docking results of
a-mannosidase (PDB: 1X9D) with com-
5
3
HO
OH
H
OH
2C5
pound 8e is depicted in Figure 3. The AUTODOCK binding energy
for the compound 8e is found to be 8.11 kcal/mol. The observed
intermolecular hydrogen bonding and hydrophobic interactions
for human mannosidase–8e complex were determined and
HO
5C2
Figure 2. Conformational analysis of 8a–f.
Table 1
Chemical shift (d, ppm) and coupling constant (J, Hz) values for 8a–f
Compd
8a
8b
8c
8d
8e
8f
H-2a/H-6a
3.10 (t)
2.23 (t)
2.31 (t)
2.35 (t)
2.29 (t)
2.81 (t)
J2a,2e = 11.5
J2a,3a = 11.5
3.25 (dd)
J2a,2e = 11.5
J2e,3a = 4.1
3.85 (dd)
J3a,2a = 11.5
J3a,2e = 4.1
J2a,2e = 10.7
J2a,3a = 10.7
2.68 (dd)
J2a,2e = 10.7
J2e,3a = 4.5
3.47 (dd)
J3a,2a = 10.7
J3a,2e = 4.5
J2a,2e = 10.7
J2a,3a = 10.7
2.73 (dd)
J2a,2e = 10.7
J2e,3a = 4.5
3.49 (dd)
J3a,2a = 10.7
J3a,2e = 4.5
J2a,2e = 10.8
J2a,3a = 10.8
2.81 (dd)
J2a,2e = 10.8
J2e,3a = 4.1
3.59 (dd)
J3a,2a = 10.8
J3a,2e = 4.1
J2a,2e = 10.7
J2a,3a = 10.7
2.72 (dd)
J2a,2e = 10.7
J2e,3a = 4.3
3.49 (dd)
J3a,2a = 10.7
J3a,2e = 4.3
J2a,2e = 11.0
J2a,3a = 11.0
3.10 (dd)
J2a,2e = 11.0
J2e,3a = 4.2
3.65–3.85
(m)
H-2e/H-6e
H-3/H-5

P.R. Markad et al. / Bioorg. Med. Chem. 22 (2014) 5776–5782
5779
hydrophobic environment with Asp463, Glu397, Phe329 and
Phe659 residues of human mannosidase. Compound 8e possesses
high potential binding affinity into the binding site of 3D macro-
molecule and these molecular docking studies were found to be
in agreement with the biological activity.
3. Conclusion
We have exploited carbon skeleton of
otherwise difficult hydroxyethyl functionality at
piperidine to obtain new -hydroxyethyl substituted iminosugar
D
-glucose to introduce
c
-position of
c
8a. The N-alkylations of 8a afforded N-alkyl substituted iminosug-
ars 8b–f. Amongst N-alkylated compounds, 8b and 8c showed
a
-galactosidase inhibition while; 8d and 8e were found to be selec-
tive as well as potent inhibitors of -mannosidase. Compound 8e
was found to be a weak inhibitor of the IgG secretion by CpG oligo-
nucleotide stimulated B-cells. The moderate -mannosidase inhi-
a
a
bition of 8e was further supported by the molecular docking
studies.
Figure 3. Molecular docking study of a-mannosidase with 8e compound.
4. Experimental section
4.1. General experimental methods
depicted in Figure 4 and Table 4. As shown in Figure 4, iminosugar
8e was noticed to be strongly interacting with catalytic residues
such as Glu330, Arg334, Arg597, Glu599 and Glu689 with
intermolecular hydrogen bonding interactions (Table 4). Besides
this, the large hydrophobic side chain of 8e enabled to establish a
Melting points were recorded with melting point apparatus and
are uncorrected. IR spectra were recorded with an FTIR as a thin
film or using KBr pellets and are expressed in cmꢁ. 1 1H (300 MHz)
Figure 4. Intermolecular hydrogen bonding and hydrophobic interactions between a-mannosidase and 8e enzyme–inhibitor complex.
Table 4
The total energy (Etotal), Van der Waals energy (Evdw) and electrostatic energy (Eelec) between active site residues of human mannosidase (PDB: 1X9D) and ligand 8e
Residues
H-bond energy kcal/mol
Evdw/kcal/mol
Eelec/kcal/mol
Etotal/kcal/mol
Glu330
Arg334
Arg597
Glu599
Glu689
ꢁ0.203
ꢁ0.144
ꢁ0.151
ꢁ2.564
ꢁ2.480
ꢁ0.27
ꢁ1.52
ꢁ0.59
ꢁ0.85
ꢁ0.48
1.06
ꢁ1.39
ꢁ1.40
1.01
0.79
ꢁ2.91
ꢁ1.99
0.15
2.06
1.58

5780
P.R. Markad et al. / Bioorg. Med. Chem. 22 (2014) 5776–5782
and 13C (75 MHz) NMR spectra were recorded using CDCl3, MeOD or
D2O as solvent. Chemical shifts were reported in d units (ppm) with
reference to TMS as an internal standard and J values are given in Hz.
Elemental analyses were carried out with a C, H-analyzer. Optical
rotations were measured using a polarimeter at 25 °C. Thin layer
chromatography was performed on pre-coated plates. Column chro-
matography was carried out with silica gel (100–200 mesh). The
reactions were carried out in oven-dried glassware under dry N2.
Methanol and THF were purified and dried before use. Distilled n-
hexane and ethyl acetate were used for column chromatography.
After quenching of the reaction with water, the work-up involves
washing of combined organic layers with water, brine, drying over
anhydrous sodium sulfate and evaporation of the solvent at reduced
pressure.
(s, 3H), 1.52 (s, 3H), 2.16–2.32 (m, 1H), 2.54–2.82 (m, 1H),
2.86–3.10 (m, 1H), 3.12–3.26 (br s, 1H, –OH), 3.75–4.50 (m, 5H),
4.75–5.20 (m, 2H), 5.56 (br s, 1H), 7.10–7.50 (m, 10H), 9.80 (br s,
1H). 13C NMR (75 MHz, CDCl3) d 26.3, 44.6, 45.4, 51.0, 67.5, 78.2,
80.0, 80.7, 103.4, 112.7, 127.3, 127.9, 128.0, 128.5, 135.0, 137.3,
156.4, 200.9. Anal. Calcd for C25H29NO7: C, 65.92; H, 6.42. Found:
C, 66.03; H, 6.22.
4.1.3. 1,2-O-Isopropylidene-3-C-(2-hydroxyethyl)-5-N-benzyl-N-
benzyloxycarbonyl-a-D-ribo-1,4-furanose (13)
To a cooled solution of 12 (0.9 g, 2.89 mmol) in THF/water
(10 ml, 4:1) was added sodium borohydride in portions slowly at
0 °C and then the reaction mixture was stirred at the same temper-
ature for 1.5 h. After completion of the reaction, saturated solution
of ammonium chloride (2 ml) was added carefully. Solvents was
then evaporated under reduced pressure, residue obtained was
extracted using ethyl acetate (10 ml ꢂ 3) and concentrated. Purifi-
cation using column chromatography (n-hexane/ethyl ace-
4.1.1. 1,2-O-Isopropylidene-3-C-(20-propenyl)-5-N-benzyl-N-
benzyloxycarbonyl-a-D-ribo-1,4-furanose (11)
To a solution of benzyl amine (0.42 ml, 3.85 mmol) in dry meth-
anol (10 ml) was added a drop of glacial acetic acid, this reaction
mixture was cooled to ꢁ20 °C. At this cooled conditions then start-
ing 10 (0.8 g, 3.50 mmol) dissolved in dry methanol (5 ml) was
added drop wise over period of 10–15 min. Stir the reaction
mixture at same temperature for 60 min. After this period sodium
cyanoborohydride (0.602 g, 8.75 mmol) was added in two portions
and reaction mixture was stirred at the same temperature for
30 min and then for 2 h at 0 C. After completion of reaction all
solvent was evaporated under reduced pressure. The residue was
extracted with ethyl acetate (20 ml ꢂ 2) and washed with satu-
rated solution of sodium bicarbonate and concentrated. The crude
obtained was used without purification for the next step.
The crude obtained in above step was dissolved in methanol/
water (10 ml, 9:1) and cooled to 0 °C. To this cooled solution then
sodium bicarbonate (0.79 g, 9.42 mmol) was added followed by
slow drop wise addition of carbobenzyloxy chloride 50% soln
(0.74 ml, 4.7 mmol) and resulting reaction mixture was stirred
for 2 h while allowing to come to rt. After completion of reaction
and evaporation of all solvent under reduced pressure, resulting
residue was extracted with chloroform (20 ml ꢂ 2) and concen-
trated. Purification by column chromatography (n-hexane/ethyl
tate = 7:3) gave 13 (0.78 g, 86.6% over two step) as viscous oil: Rf
26.6
0.2 (n-hexane/ethyl acetate = 6:4); [
a]
D
+42.16 (c 1.0, CHCl3); IR
.
(CHCl3) 1678.1 cmꢁ1
,
3100–3600 cmꢁ1 1H NMR (300 MHz,
CDCl3+D2O) d 1.32 (s, 3H), 1.51 (s, 3H), 1.5–1.85 (m, 2H), 3.0–
3.09 (m, 1H), 3.60–4.10 (m, 4H), 4.20–4.35 (m, 2H), 4.35–5.18
(m, 3H), 5.36 (br s, 1H), 7.16–7.37 (10H); 13C NMR (75 MHz,
CDCl3+D2O) d 26.2, 31.7, 45.7, 50.8, 58.2, 67.3, 79.5, 80.0, 80.9,
103.4, 112.2, 127.1, 127.6, 128.4, 137.4, 146.2, 156.2. Anal. Calcd
for C25H31NO7: C, 65.63; H, 6.83. Found: C, 65.64; H, 6.88.
4.1.4. 1,2-O-Isopropylidene-3-C-((2-acetoxy)ethyl)-5-N-benzyl-
N-benzyloxycarbonyl-a-D-ribo-1,4-furanose (14)
To a cooled solution of 13 (0.75 g, 1.63 mmol) in dry pyridine
(3 ml) was added acetic anhydride (0.17 ml, 1.80 mmol) slowly at
0 °C and the reaction mixture was stirred for 4 h while allowing
reaction to come to rt. After completion of reaction extracted with
chloroform (20 ml) and organic layer was washed with 2 N HCl
solution (20 ml) and then concentrated. Purification using column
chromatography (n-hexane/ethyl acetate = 1.5:8.5) gave 14 (0.76 g,
93.82%) as viscous liquid: Rf 0.6 (n-hexane/ethyl acetate = 6:4);
27.7
[a]
+40.37 (c 0.43, CHCl3); IR (CHCl3) 1699.34 cmꢁ1
,
D
acetate = 9:1) gave 11 (1.35 g, 85.4%) as viscous oil: Rf 0.55 (n-hex-
1737.92 cmꢁ1, 3439.19 cmꢁ1 (broad). 1H NMR (300 MHz, CDCl3+
D2O d 1.12 (s, 3H), 1.52 (s, 3H), 1.40–2.0 (m, 2H), 2.00 (br s, 3H),
3.03–3.07 (m, 1H), 3.72–4.47 (m, 5H), 4.88–5.33 (m, 4H), 7.16–
7.38 (m, 10H); 13C NMR (75 MHz, CDCl3+D2O) d 20.8, 26.3, 29.1,
45.5, 50.8, 59.9, 67.3, 78.1, 79.9, 81.2, 103.4, 112.3, 127.2, 127.7,
128.3, 128.4, 137.5, 156.3, 170.7. Anal. Calcd for C27H33NO8: C,
64.92; H, 6.66. Found: C, 64.98; H, 6.60.
27.5
ane/ethyl acetate = 7:3); [
a
]
,
+33.7 (c 1.0, CHCl3); IR (CHCl3)
D
1693.5 cmꢁ1 1637.6 cmꢁ1
,
3443.0 cmꢁ1 (broad). 1H NMR
(300 MHz, CDCl3+D2O) d 1.32 (s, 3H), 1.52 (s, 3H), 1.82–2.12 (m,
1H), 2.20–2.42 (m, 1H), 3.0–3.40 (m, 1H), 3.70–4.10 (m, 2H),
4.20–4.45 (m, 2H), 4.80–5.40 (m, 5H), 5.62–6.0 (m, 2H), 7.10–
7.37 (m, 10H); 13C NMR (75 MHz, CDCl3) d 26.3, 35.3, 45.5, 51.0,
67.15, 78.3, 79.93, 80.19, 103.4, 112.1, 116.8, 127.2, 127.7, 127.6,
128.3, 136.3, 137.5, 132.2, 156.2. Anal. Calcd for C26H31NO6: C,
68.86; H, 6.89. Found: C, 68.94; H, 6.68.
4.1.5. (3R,4s,5S)-4-(2-Hydroxyethyl) piperidine-3,4,5-triol (8a)
Cold TFA/water (3:1, 6 ml) was added drop wise to the com-
pound 14 (0.75 g) at 0 °C, and reaction mixture was stirred at this
temperature for 2 h. After completion of reaction TFA was co-evap-
orated with toluene at reduced pressure. The compound obtained
was dissolved in methanol then charged in parr high pressure reac-
tor along with 10% Pd/C (0.050 g) and reaction was carried out at
150 psi of H2 pressure for 24 h. After completion of reaction the
catalyst was filter off over Celite bed, filtrate was evaporated at
reduced pressure. Purification using column chromatography
(MeOH) gave compound 8a (0.164 g, 80.8%) as white solid; mp
186–188 °C; Rf 0.2 (25% aq NH4OH/MeOH = 1:9), IR (KBr) 3500–
4.1.2. 1,2-O-Isopropylidene-3-C-(2-oxoethyl)-5-N-benzyl-N-
benzyloxycarbonyl-a-D-ribo-1,4-furanose (12)
To a solution of 11 (1.3 g, 2.53 mmol) in acetone/water (15 ml,
8:2) was added NMO (0.59 g, 5.07 mmol) and potassium osmate
(0.01 g, 5 mol %). The reaction mixture was stirred at rt for 24 h.
Sodium sulfite (0.7 g) was added and stirred for 1 h. Acetone was
removed at reduced pressure, residue obtained extracted with
ethyl acetate and concentrated to afford diol as thick liquid. To a
crude diol in acetone/water (20 ml, 9:1) was added sodium
metaperiodate (0.71 g, 3.32 mmol) at 0 °C and stirred for 2 h. Eth-
ylene glycol (0.5 ml) was added and acetone was evaporated under
reduced pressure. The residue obtained was extracted with chloro-
form and concentrated. Purification using column chromatography
2600 (br) cmꢁ1 1H NMR (300 MHz, D2O) d 2.04 (t, J = 6.5 Hz, 2H),
,
3.10 (t, J = 11.5 Hz, 2H, H-2a & H-6a), 3.25 (dd, J = 11.5, 4.1 Hz,
2H, H-2e & H-6e), 3.76 (t, J = 6.5, Hz, 2H, H-8), 3.85 (dd, J = 11.5,
4.7 Hz, 2H, H-3 & H-5); 13C NMR (75 MHz, CDCl3) d 35.7 (C-7),
44.9 (C-2 & C-6), 57.0 (C-8), 69.5 (C3 & C5) 74.3 (C-4). Anal. Calcd
for C7H15NO4: C, 47.45; H, 8.53. Found: C, 47.52; H, 8.33.
(n-hexane/ethyl acetate = 9:1) gave 12 (0.9 g, 69.2% over two step)
26.6
as viscous oil: Rf 0.5 (n-hexane/ethyl acetate = 6:4); [
a]
+50.71
D
(c 0.9, CHCl3); IR (CHCl3) cmꢁ1
;
1H NMR (300 MHz, CDCl3) d 1.33

P.R. Markad et al. / Bioorg. Med. Chem. 22 (2014) 5776–5782
5781
4.1.6. General procedure for alkylation of 8a
(C2 & C6), 57.8 (C9) 58.4 (C8), 60.2 (C10), 69.4 (C3 & C5), 74.1
(C4). Anal. Calcd for C9H19NO5: C, 48.86; H, 8.66. Found: C, 48.52;
H, 8.33.
To the solution of compound 8a (1 mmol) in dry DMF (1 ml)
was added bromo alkane (1.5 mmol) followed by addition of the
K2CO3 (3 mmol) under N2 atmosphere, resulting mixture was then
heated at 80 °C for 10–14 h. After completion of reaction solvent
was evaporated under reduced pressure. Purification using column
chromatography (chloroform/MeOH) gave the desired compound.
4.2. Glycosidase inhibition assay
Glycosidase inhibition assay of derivatives was carried out by
mixing 0.1 unit/ml each of
a
-galactosidase,
a-mannosidase and
4.1.7. Compound (8b)
a-glucosidase with the samples and incubated for 1 h at 37 °C.
Compound obtained as off white solid with 66.5%: mp 114–
Enzyme action for
a-galactosidase was initiated by addition of
116 °C; Rf 0.3 (MeOH/CHCl3 = 1:9), IR (KBr) 3500–2600 (br) cmꢁ1
,
10 mM p-nitrophenyl-a-D-galactopyranoside (pNPG) as a substrate
1H NMR (300 MHz, MeOD) d 0.91 (t, J = 7.2 Hz, 3H, CH3), 1.24–
1.40 (m, 2H, H-11), 1.40–1.56 (m, 2H, C-10), 1.94 (t, J = 6.2 Hz,
2H, H-7), 2.23 (t, J = 10.7 Hz, 2H, H-2a & H-6a), 2.37–2.44 (m, 2H,
H-9), 2.68 (dd, J = 10.7, 4.5 Hz, 2H, H-2e & H-6e), 3.47 (dd,
J = 10.7, 4.5 2H, H-3 & H-5), 3.72 (t, J = 6.2, Hz, 2H, H-8); 13C NMR
(75 MHz, CDCl3) d 14.4 (C12), 21.8 (C11), 29.8 (C1), 38.9 (C7),
55.6 (C2 & C6), 58.6 (C9), 59.0 (C8), 71.3 (C3 & C5), 74.6 (C4). Anal.
Calcd for C11H23NO4: C, 56.63; H, 9.94. Found: C, 56.61; H, 10.10.
in 200 mM sodium acetate buffer followed by an incubation for
10 min at 37 °C and stopped by adding 2 ml of 200 mM borate buf-
fer of pH 9.8.
a-Mannosidase activity was initiated by addition of
10 mM p-nitrophenyl-
a
-D
-mannopyranoside as substrate in
a
100 mM citrate buffer of pH 4.5. The reaction was incubated at
37 °C for 10 min and stopped by adding 2 ml of 200 mM borate
buffer of pH 9.8. Initiation of
out by addition of 10 mM p-nitrophenyl-
100 mM phosphate buffer of pH 6.8 and stopped by adding 2 ml
of 0.1 M Na2CO3 after an incubation of 10 min at 37 °C. -Glycosi-
dase activity was determined by measuring absorbance of the
p-nitrophenol released from pNPG at 420 nm using Shimadzu
Spectrophotometer UV-1601.
a-glucosidase activity was carried
a-D
-glucopyranoside in
4.1.8. Compound (8c)
a
Compound obtained as off white solid with 61.05%: mp 107–
109 °C; Rf 0.4 (MeOH/CHCl3 = 1:9); IR (KBr) 3500–2600 (br)
cmꢁ1 1H NMR (300 MHz, MeOD) d 0.87 (t, J = 6.7 Hz, 3H, H-14),
;
1.18–1.38 (m, 6H, H-11, H-12, H-13), 1.42–1.60 (m, 2H, H-10),
1.94 (t, J = 6.2 Hz, 2H, H-7), 2.31 (t, J = 10.7 Hz, 2H, H-2a & H-6a),
2.40–2.52 (m, 2H, H-9), 2.73 (dd, J = 4.5, 10.7 Hz, 2H, H-2e & H-
6e), 3.49 (dd, J = 4.5, 10.7 2H, H-3 & H-5), 3.73 (t, J = 6.2, Hz, 2H,
H-8); 13C NMR (75 MHz, CDCl3) d 14.4 (C-14), 23.7 (C13), 27.3
(C12), 28.2 (C11), 32.9 (C-10), 38.7 (C-7), 55.3 (C-2 & C-6), 58.5
(C-9), 59.2 (C-8), 70.9 (C-3 & C-5), 74.5 (C-4). Anal. Calcd for
4.3. Immunosuppressive assay
4.3.1. MLR assay
The MLR assay has been described previously.21 Briefly, periph-
eral blood mononuclear cells (PBMCs) were isolated from heparin-
ized blood of healthy donors by density-gradient centrifugation
over Lymphoprep. PBMCs were resuspended at a concentration
of 1.2 ꢂ 106 cells/ml in complete medium (RPMI-1640 containing
10% heat-inactivated fetal calf serum (FCS) and antibiotics).
C13H27NO4: C, 59.74; H, 10.41. Found: C, 59.64; H, 10.47.
4.1.9. Compound (8d)
Compound obtained as solid with 67.82%: mp 112–115 °C; Rf
0.45 (MeOH/CHCl3 = 1:9); IR (KBr) 3500–2600 (br) cmꢁ1 1H NMR
;
RPMI1788 cells were treated with 30 lg/ml mitomycin C for
20 min at 37 °C, washed with medium and finally suspended in
(300 MHz, MeOD) d 0.89 (t, J = 6.0 Hz, 3H, H-16), 1.20–1.40 (m,
10H), 1.45 (m, 2H, H-10), 1.96 (t, J = 5.5 Hz, 2H, H-7), 2.35 (t,
J = 10.8 Hz, 2H, H-2a & H6a), 2.42–2.56 (m, 2H, H-9), 2.81 (dd,
J = 10.8, 4.1 Hz, 2H, C-2e & C-6e), 3.59 (dd, J = 10.8, 4.1 Hz, 2H, H-
3 & H-6), 3.77 (t, J = 5.5, Hz, 2H, H-8); 13C NMR (75 MHz, CDCl3)
d 14.2 (C16), 23.0 (C15), 26.5 (C14), 27.8 (C13), 29.6 (C12), 29.8
(C11), 32.2 (C10), 38.1 (C7), 54.4 (C2 & C6), 57.7 (C9), 58.5 (C8),
70.3 (C3 & C5), 73.7 (C4). Anal. Calcd for C15H31NO4: C, 62.25; H,
10.80. Found: C, 62.32; H, 10.91.
complete RPMI medium to a density of 0.45 ꢂ 106 cells/ml. An
amount of 100 ll of each cell suspension was mixed with 20 ll
of diluted compound. The mixed cells were cultured at 37 °C for
6 days in 5% CO2. DNA synthesis was assayed by the addition of
1
l
Ci (methyl-3H)thymidine per well during the last 18 h in cul-
ture. Thereafter, the cells were harvested on glass filter paper
and the counts per minute (cpm) determined in a liquid scintilla-
tion counter.
4.3.2. IgG production by CpG oligonucleotide stimulated B-cells
B-cells were isolated from PBMC’s of healthy donors by positive
magnetic selection using CD19 MicroBeads technology (Miltenyi
Biotec) according the manufacturer’s protocol. Twenty five thou-
4.1.10. Compound (8e)
Compound obtained as colourless solid with 63.5%: mp 109–
111 °C; Rf 0.5 (MeOH/CHCl3 = 1:9); IR (KBr) 3500–2600 (br)
cmꢁ1 1H NMR (300 MHz, MeOD) d 0.86 (t, J = 6.3 Hz, 3H, H-18),
;
sand cells were incubated in 50
l
l of culture medium in the pres-
l of compound
1.18–1.38 (m, 14H), 1.42–1.58 (m, 2H, H-10), 1.94 (t, J = 6.1 Hz,
2H, H-7), 2.29 (t, J = 10.7 Hz, 2H, H-2a & H-6a), 2.43 (apperent t,
J = 7.7, Hz, 2H, H9), 2.72 (dd, J = 10.7, 4.3 Hz, 2H, H-2e & H-6e),
3.49 (dd, J = 10.7, 4.3, 2H, H-3 & H-5), 3.71 (t, J = 6.1, Hz, 2H, H-
8); 13C NMR(75 MHz, CDCl3) d 14.5 (C18), 23.8 (C17), 27.4 (C16),
28.5 (C15), 30.5 (C14), 30.6 (C13), 30.7 (C12), 33.1 (C10), 38.7
(C7), 55.3 (C2 & C6), 58.6 (C9), 59.2 (C8), 71.0 (C3 & C5), 74.57
(C4). Anal. Calcd for C17H35NO4: C, 64.32; H, 11.11. Found: C,
64.20; H, 11.23.
ence of 0.1 M of ODN2006 (Invivogen) and 5
l
l
dilution. After 7 days incubation at 37 °C in 5% CO2, supernatants
were collected and IgG concentration was determined using the
Human IgG immunoassay kit (PerkinElmer) according to the man-
ufacturer’s protocol.
4.3.3. Cytotoxic assay
Exponential growing Jurkat cells (ATCC, TIB-152) were seeded
at 50 ꢂ 103 per 200
ll of complete medium. Twenty lL of test
compound dilution were added to each well and the plates were
incubated for 48 h at 37 °C, 5% CO2. Untreated cells and positive
control (1% triton X-100, for the last 15 min) served as reference
for maximum and minimum viability. At the end of incubation
4.1.11. Compound (8f)
Compound obtained as thick liquid with 60.38%; Rf 0.8 (MeOH);
IR (KBr) 3500–2600 (br) cmꢁ1; 1H NMR (300 MHz, MeOD) d 1.98 (t,
J = 6.2 Hz, 2H, H-7), 2.81 (t, J = 11 Hz, 2H, H-2a & H-6a), 3.0 (t,
J = 5.2, Hz, 2H, H-9), 3.1 (dd, J = 11.0, 4.2 Hz, 2H, H-2e & H-6e),
3.65–3.85 (m, 6H); 13C NMR (75 MHz, CDCl3) d 37.9 (C7), 54.1
100
ll of supernatant were removed and replaced by 10 ll of
WST-1 solution (Cell Proliferation Reagent WST-1, Roche Applied

5782
P.R. Markad et al. / Bioorg. Med. Chem. 22 (2014) 5776–5782
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4.4. Molecular docking
4.4.1. Computational methodology and Molecular docking
parameters
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Crystal structures for human a-mannosidase were downloaded
from protein databank (http://www.rcsb.org) and structure
(1X9D.pdb) having high resolution was selected for docking stud-
ies. Molecular docking was performed using AUTODOCK 4.2 wiz-
ard.22 From receptor molecule, non-polar hydrogen atoms were
replaced with polar hydrogen atoms by employing Kollman united
atom charges. The obtained receptor structure of
a-mannosidase
was minimized for 5000 steps of steepest descent method using
SpdbViewer22a to remove internal stain. Minimized structure was
finally subjected to docking studies with 8e compound.
Molecular structure of 8e synthetic ligand was constructed
using ‘Spartan’14’ Parallel suit for Windows22b and geometrically
optimized by quantum chemical semi-empirical RM1 method.22e
The Gasteiger charges and hydrogen atoms were added to the
ligand molecule using AUTODOCK 4.2 wizard. Whereas, AutoGrid
was used for calculating the grid maps and centered on the ligand
binding site of human a-mannosidase, in such a way that it would
totally cover the ligand molecule.22 The grid size was set to
68 A ꢂ 70 A ꢂ 68 A with a grid spacing 0.375 A . The step size of
1 Å for translation and the maximum number of energy evaluation
was set to 2,500,000. The 100 runs were performed. For each of the
100 independent runs, a maximum number of 2,70,000 LGA oper-
ations were generated on a single population of 150 individuals.
The operator weights for crossover, mutation and elitism were
maintained as default parameters (0.80, 0.02, and 1, respectively).
The resulting 100 docked conformations were analyzed for
binding energy, intermolecular energy and internal energy using
AUTODOCK 4.2 wizard. Mannosidase–inhibitor complex with low-
est binding energy was subjected for geometrical parameters anal-
ysis. Chimera software was used to generate pictorial presentation
of selected docked conformation.22c LigPlot program was used for
analysis of hydrogen bonding and hydrophobic interactions from
mannosidase–8e complex.22d
Acknowledgments
P.R.M. is thankful to the CSIR, New Delhi and D.P.S. is thankful
to the UGC, New Delhi for the senior research fellowship. We are
thankful to the Department of Science and Technology, New Delhi
(Project File No. SR/S1/OC-20/2010) and to the Industrial Research
Funds of KU Leuven (Kennisplatform KP/12/010) for the financial
support.
16. We have repeated this protocol four times. In each case we isolated de-
acetylated product.
17. Ghisaidoobe, A.; Bikker, P.; de Bruijn, Arjan C. J.; Godschalk, F. D.; Rogaar, E.;
Guijt, M. C.; Hagens, P.; Halma, J. M.; van’t Hart, S. M.; Luitjens, S. B.; van Rixel,
V. H. S.; Wijzenbroek, M.; Zweegers, T.; Donker-Koopman, D. E.; Strijland, A.;
Boot, R.; Marel, G.; Overkleeft, H. S.; Aerts, J. M.; van den Berg, R. ACS Med.
Chem. Lett. 2011, 2, 119.
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