3-Hydroxypyrrolidine and (3,4)-dihydroxypyrrolidine derivatives: Inhibition of rat intestinal α-glucosidase

Article abstract of DOI:10.1016/j.bioorg.2014.04.007

Thirteen pyrrolidine-based iminosugar derivatives have been synthesized and evaluated for inhibition of α-glucosidase from rat intestine. The compounds studied were the non-hydroxy, mono-hydroxy and dihydroxypyrrolidines. All the compounds were N-benzylated apart from one. Four of the compounds had a carbonyl group in the 2,5-position of the pyrrolidine ring. The most promising iminosugar was the trans-3,4-dihydroxypyrrolidine 5 giving an IC₅₀ of 2.97 ± 0.046 and a K_I of 1.18 mM. Kinetic studies showed that the inhibition was of the mixed type, but predominantly competitive for all the compounds tested. Toxicological assay results showed that the compounds have low toxicity. Docking studies showed that all the compounds occupy the same region as the DNJ inhibitor on the enzyme binding site with the most active compounds establishing similar interactions with key residues. Our studies suggest that a rotation of ～90° of some compounds inside the binding pocket is responsible for the complete loss of inhibitory activity. Despite the fact that activity was found only in the mM range, these compounds have served as simple molecular tools for probing the structural features of the enzyme, so that inhibition can be improved in further studies.

Full text of DOI:10.1016/j.bioorg.2014.04.007

Accepted Manuscript
3-Hydroxypyrrolidine and (3,4)-Dihydroxypyrrolidine Derivatives: Inhibition
of rat intestinal α-Glucosidase
Elisabete P. Carreiro, Patricia Louro, Gizé Adriano, Romina A. Guedes,
Nicholas Vannuchi, Ana R. Costa, Célia M.M. Antunes, Rita C. Guedes, A.J.
Burke
PII:
S0045-2068(14)00031-5
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.04.007
YBIOO 1718
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
20 February 2014
Please cite this article as: E.P. Carreiro, P. Louro, G. Adriano, R.A. Guedes, N. Vannuchi, A.R. Costa, C.M.M.
Antunes, R.C. Guedes, A.J. Burke, 3-Hydroxypyrrolidine and (3,4)-Dihydroxypyrrolidine Derivatives: Inhibition
of rat intestinal α-Glucosidase, Bioorganic Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.04.007
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3-Hydroxypyrrolidine and
(3,4)-Dihydroxypyrrolidine Derivatives: Inhibition
of rat intestinal α-Glucosidase
E.P. Carreiro, P. Louro, G. Adriano, R.A. Guedes, R. N. Vannuchi, A.R. Costa, C.M. Antunes, R.C. Guedes,
A.J. Burke*
Departamento de Química and Centro de Química de Evora, Universidade de Evora, Rua Romão Ramalho, 59,
7000 Evora, Portugal.

Bioorganic Chemistry
journal homepage: www.els evier.com
3-Hydroxypyrrolidine and (3,4)-Dihydroxypyrrolidine Derivatives: Inhibition of rat
intestinal α-Glucosidase
Elisabete P. Carreiroa, Patricia Louroa, Gizé Adrianoa, Romina A. Guedesc, Nicholas Vannuchia, Ana R.
Costac, Célia M.M. Antunesb,d, Rita C. Guedesc, A.J. Burkea*
a Departamento de Química and Centro de Química de Evora, Universidade de Evora, Rua Romão Ramalho, 59, 7000 Evora, Portugal.
b Departamento de Química and Instituto de Ciências Agrárias e Ambientais Mediterrânicas (ICCAM), Universidade de Évora.
c Research Institute for Medicines and Pharmaceutical Sciences- iMed.UL, Faculty of Pharmacy, Universidade de Lisboa, Av. Prof. Gama Pinto, 1649-003,
Lisbon, Portugal.
d Center For Neurosciences and Cell Biology, University of Coimbra.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
Thirteen pyrrolidine-based iminosugar derivatives have been synthetized and evaluated for
inhibition of α-glucosidase from rat intestine. The compounds studied were the non-hydroxy,
mono-hydroxy and dihydroxypyrrolidines. All the compounds were N-benzylated apart from
one. Four of the compounds had a carbonyl group in the 2,5-position of the pyrrolidine ring. The
most promising iminosugar was the trans-3,4-dihydroxypyrrolidine 5 giving an IC50 of
2.97 0.046 and a KI of 1.18 mM. Kinetic studies showed that the inhibition was of the mixed
type, but predominantly competitive for all the compounds tested. Toxicological assay results
showed that the compounds have low toxicity. Docking studies showed that all the compounds
occupy the same region as the DNJ inhibitor on the enzyme binding site with the most active
compounds establishing similar interactions with key residues. Our studies suggest that a
rotation of ~90º of some compounds inside the binding pocket is responsible for the complete
loss of inhibitory activity.
Available online
Keywords:
1-benzyl-3-hydroxypyrrolidine
1-benzyl-3,4-dihyrdoxypyrrolidine
small molecule inhibitor
α-glucosidase
Despite the fact that activity was found only in the mM range, these compounds have served as
simple molecular tools for probing the structural features of the enzyme, so that inhibition can be
improved in further studies.
Rat intestinal cells
2009 Elsevier Ltd. All rights reserved.
1. Introduction
potent and specific than DAB [9]. In the literature there exists an
extensive variety of iminosugars with structures based on the
Over the last two decades, glycosidase inhibitors have been a
key target for academic researchers, because of their role in a
variety of ailments [1], such as: diabetes mellitus type II [2],
cancer [3], hepatitis [4], HIV [5] and Gaucher disease [6]. One of
the most studied classes of inhibitor are the polyhydroxylated
pyrrolidines and piperidines [7]. Before the 90s [8] most
glycosidase inhibitors studied were obtained from plants and
micro-organisms, and three important examples are:
pyrrolidine unit, which exhibit α-glucosidase activity. Several
derivatives of DAB were synthetized and tested in different types
of α-glucosidades [10]. Most of the structures were
functionalized with alkyl [11], aryl [12], amide [13], amine [13],
or alcohols [14] units in the 2,5-positions of the pyrrolidine ring.
In 2012 Kato et al. [11a] described a new family of potent
pyrrolidine α-glucosidase inhibitors; this family was basically a
set of analogues of the LAB structure, having alkyl chains of
different lengths in the 2 and 4 positions. The most promising α-
glucosidase inhibitor was the 1-C butyl derivative, which is one
order of magnitude more active than LAB (Figure 1). On the
contrary, the 2,4 dibutyl derivative didn´t show any inhibition,
leading us to conclude that the hydroxyl group in the 4-postion is
important for α-glucosidase inhibition.
deoxynojirimycin
(DNJ),
2R,5R-dihydroxymethyl-3R,4R-
dihydroxypyrrolidine (DMDP) and 1,4-dideoxy-1,4-imino-d-
arabinitol (DAB) (Figure 1). These three molecules since the 90s
have been the prototypes for the synthesis of a wide range of
potential glycosidase inhibitors [1]. For example, miglitol, an N-
alkyl derivative of DNJ used in the treatment of diabetes [2].
Curiously, Miglustat (N-butyl DNJ, Zavesca) is structurally
similar to miglitol and is licensed for substrate reduction therapy
in Gauchers disease. In the case of DAB, it shows strong
inhibition of α-glucosidases, but its enantiomer, LAB is more
Recently we designed and synthesised a small library of
pyrrolidine iminocyclitol inhibitors with a structural similarity to
DAB. This library was specifically designed to gain a better

insight into the mechanism of inhibition of glycosidases by
polyhydroxylated pyrrolidines. These compounds were assayed
for baker’s yeast α-glucosidadse inhibition using acarbose as a
reference and it was the non-benzylated diol 7’ which showed the
highest inhibition (IC50 = 10.9 mM). It seems that the presence of
a free NH group and free hydroxyl groups in the 3 and 4
positions are important for favorable interaction with the enzyme
active site. There were no substituents in the 2,5-position of the
pyrrolidine ring in these compounds, but it appears quite obvious
now that the presence of substituentes in these positions is
important for inhibition [7]. Our results are similar to those
obtained with the iminosugars reported by Bols [15] and Lundt
[16] (Figure 1), although in their case the 3,4-hydroxy groups had
a cis relative configuration.
On the basis of our last publication we observed that O-
acetylated dihydroxypyrrolidines - despite showing good docking
simulations [7a] - seem to suffer hydrolysis during the biological
assays, and for this reason we decided to substitute the acetyl
group for the more robust benzyl group. Besides this the
possibility of the benzyl groups establishing significant non-
covalent π−π interactions with the active site residues was very
likely, and worth investigating.
2. Materials and Methods
2.1. General Chemical
All reagents were obtained from Aldrich, Fluka, Alfa Aesar or
Acros. Solvents were dried using common laboratory methods.
Compounds 5 and its respective enantiomer 5’ were synthesized
using the precursors: (3S,4S)-N-benzyl-3,4-dihydroxy-2,5-
dioxopyrrolidine
and
(3R,4R)-N-benzyl-3,4-dihydroxy-2,5-
dioxopyrrolidine, respectively, both enantiomers had an
enantiomeric purity of 99% ee. TLC was carried out on
aluminium backed Kiselgel 60 F254 plates (Merck) and the plates
were
Figure 2. Library of 5-membered iminosugars synthetized and test.
Figure 1. Examples of natural and synthetic iminosugars.
visualized either by UV light or with phosphomolybdic acid in
1
ethanol. The H NMR and 13C NMR spectra were recorded on a
Bruker Avance instrument (1H: 400 MHz and 13C: 100 MHz)
using CDCl3 as solvent and the signal from residual CHCl3 as an
internal standard (for the measurements made with the Bruker
Avance instrument). Specific rotations were measured on a
Perkin-Elmer 241 polarimeter.
In this publication we report on a study of another library of 1-
benzyl-3-hydroxypyrrolidine derivatives (1, 1’, 2, 2’, 3, 3’, 4, 4’)
and 1-benzyl-3,4-dihydroxypyrrolidne derivatives (5, 5’, 6, 7),
derived from D- and L-malic acid and D- and L-tartaric acid (Fig.
2), respectively. It must be noted that a wide range of important
pharmacologically active compounds contain the 3-
hydroxypirrolidine subunit, one example is Barnidipine, an
antihypertensive agent [17]. In our study, the compounds were
assayed for rat intestine α-glucosidase (EC 3.2.1.20) [18]
inhibition, as well as the mechanism of inhibition and KI
determination. Acarbose and DNJ were used as references.
Cytoxicity studies were also conducted for compounds 5, 5’ and
7. Docking studies were realized with the human α-glucosidase
(intestinal maltase-glucoamylase) and a homology model of rat
2.2. General Biological assays
2.2.1 Enzymatic assays with rat α-glucosidase
The enterocytes were isolated from Wistar rat small intestine
according to Watford et al., 1979 [19a] with few modifications.
Briefly, the α-glucosidase rich homogenates were obtained after
mucus removal with calcium free Krebs-Henseilt buffer (in mM:
120 NaCl, 2 KCl, 26 NaHCO3, 10 MgSO4, 1.18 KH2PO4, 11
glucose, 5 EDTA; supplemented with 0.25% (p/v) BSA; at 37ºC
during 20 min under mild stirring – 60 rpm) and detachment of
mucosa epithelial cells using Krebs-Henseilt buffer (as
previously described without EDTA and with 0.5 mM CaCl2,
supplemented with 2.5% (p/v) BSA). The total protein
glucosidase.
We
wanted
to
compare
both
the
monohydroxypyrrolidine and dihydroxypyrrolidine series with
pyrrolidines lacking an hydroxyl group. The structural diversity
encountered in this library was for the purpose of probing the
mechanism of inhibition of α-glycosidase, so that we can design
and synthesize more potent, non-toxic α-glycosidase inhibitors.

concentration was determined using the Bradford dye-binding
method [19b].
crystallographic structure. The experimental x-ray structure was
obtained from the RCSB Protein Data Bank (PDB ID 3L4U,
resolution 1.9 Å). The preparation of the human α-glucosidase
structure involved the removal of the original crystallographic
ligand (kotalanol) as well as the crystallographic waters. The
protonation and tautomeric states of Asp, Glu, Arg, Lys, and His
were adjusted to match a pH of 7 using the Protonate 3D
algorithm within the Molecular Operating Environment (MOE)
2012.10 program (www.chemcomp.com). These enzyme
structures and the proposed docking protocol were previously
validated by re-docking the co-crystallized ligand. The molecular
structures of the synthesized compounds were built and
optimized with MOE package (2012.10) with the MMFF94x
forcefield as implemented in this software. These compounds
were docked into the homology model of rat intestinal
glucosidase developed previously by this group and to human
glucosidase active sites. For each compound, 500 docking runs
were performed. The following genetic algorithm parameters
were used: population size = 100; selected pressure = 1.1;
number of operations = 1000; number of islands = 5; niche size =
2; migrate = 10; mutate = 95; crossover = 95. Each conformation
was ranked according to its goldscore scoring function. The top
solutions (the ones with the highest goldscore) were visually
inspected and critically evaluated and, for each inhibitor, the
highest scoring conformation was chosen as the actual binding
conformation (Figure 3).
The α-glucosidase activity was determined by monitoring the
p-nitrophenol (p-NP) released from p-nitrophenyl-α-D-
glycopyranoside spectrophotometrically at 405 nm, over 60 min.
The assay mixture had the following composition: 0.1M
phosphate buffer (pH 6.7), 12 mM p-nitrophenyl-α-D-
glucopyranoside (the method was optimized giving KM and Vmax
values of 0.961 mM and 2.51x10-5 mmol p-NP.min-1.mg-1,
respectively) and 30 μg/mL rat enterocyte α-glucosidase rich
homogenates. The test compounds were dissolved in 2% DMSO
in 0.1M phosphate buffer (pH 7) and solutions with concentration
in the range of 0.01 x 10-3 and 92 mM were used. All
experiments were performed in 5 replicates. The IC50 values were
obtained from the inhibition curves.
2.2.2 Toxicity assays
The toxicity of the compounds was measured using two
methods. The first method described consists in the
determination of the cell viability of the cellular line BRIN-BD
11 from pancreatic beta cells. The second method was the
evaluation of the half maximal lethal concentration (LC50) using
Artemia salina.
2.2.2. 1 Cytotoxicity Assay
2.4. Homology modelling of the Rat intestinal
glucosidase:
BRIN BD-11 cells were cultured in 96 well micro plates
(2x104 cells/well) in an incubator at 37ºC with 5% CO2 and 95%
O2. The cells were exposed to the test compounds in the
concentration range of 3 – 50 mM for 24 h. A negative control
(without inhibitors) and a positive control (1% sodium dodecyl
sulfate) tests were performed. Cell viability was determined
using the cell counting kit-8 (CCK-8, Sigma-Aldrich) as
established by the supplier.
To investigate the inhibitory activity of the synthesized
compounds against rat intestinal glucosidase, a homology
modeling of α-glucosidase from Rattus norvegicus was carried
out to predict its 3D-structure (the 3D structure has never been
resolved experimentally). The amino acid sequence of α-
glucosidase from Rattus norvegicus comprises 953 amino acid
residues and was retrieved from the UniProt protein resource data
bank (http://www.uniprot.org/), under the access code Q6P7A9.
Using the Molecular Operating Environment program (MOE)
version 2012.10 (http://www.chemcomp.com/software) we
searched for proper structural templates on the PDB database of
protein structures and sequences and aligned the results obtained
with the MOE-Align feature. MOE-Align implement a modified
version of the alignment methodology originally introduced by
Needleman and Wunsch. All the default settings in the MOE-
Align panel were used for the sequence alignment. Our search
identified the Homo sapiens intestinal maltase-glucoamylase (in
complex with O-sulfonated kotalanol) crystallographic structure
(PDB code 3L4U, 1.90 Å resolution)[21] sharing 45.7% of
sequence identity (calculated with BLAST) with α-glucosidase
enzyme from Rattus norvegicus as the most suitable template.
The catalytic site is highly conserved in both structures. The
crystallographic structure and the homology model were
superposed with MOE software and the RMSD values obtained
were 1.04 Å for the Rattus norvegicus homology model. The 3D
homology models were built with Molecular Operating
Environment (MOE) software using only a single template and a
set of 10 intermediate models were generated and refined with
Amber99 forcefield, resulting in the corresponding homology
model. The stereochemical quality of the enzyme backbone and
side chains was validated by Ramachandran plots. To validate the
model we first docked kotanalol into the structure’s active site
and compared the final docked complexes with the
crystallographic structure obtained by Sim et al.[21] We
confirmed that kotanalol was placed in an identical position to
that adopted in the crystallographic structure (PDB ID: 3L4U)
having similar active site residue interactions.
2.2.2. 2 Artemia salina lethal toxicity assay
The percentage of dyed nauplii of Artemia salina, grown in
the presence of variable concentrations of the inhibitor
compounds, was measured to determine the LC50 values. The
Artoxkit M was used.
2.2.3 Statistical analysis
Results are presented as mean sd for a given number of
observations. Statistical analyses were done using one-way
ANOVA for enzymatic activity. In the case of the toxicological
results Paired-samples t-Test was used for comparison with
samples showing negative control.
2.3. Molecular Modeling
2.3.1.
Molecular docking calculations
Molecular docking calculations of the synthesized compounds
with human and a homology model of rat α-glucosidase were
performed with GOLD 5.1.0 (Genetic Optimization Ligand
Docking) [20] software using the Goldscore scoring function.
This software uses an evolutionary genetic algorithm to optimize
the docked conformation of the flexible inhibitor within the
enzyme. The docking calculations were performed using the
homology model described in the next section for the rat α-
glucosidase and the available human α-glucosidase

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Figure 3. – Docking poses of DNJ and compound 5 inside human α-glucosidase active site.
Besides that, acarbose (a known α-glucosidase inhibitor widely
used in the treatment of diabetes type 2) was also docked into the
homology model active site, and was correctly placed.
to (1:1) Hex:EtOAc)] to give the title compound as a light brown
solid (0.19 g, 52%).
1H NMR (CDCl3, 400 MHz) δ: 2.14 (s, 3H, CH3) ppm, 2.64
(dd, J 4, 20 Hz, 1H, CH2CO), 3.13 (dd, J 8, 20 Hz, 1H, CH2CO),
4.6 (d, J 20 Hz, 1H, ABX system, CH2Ph), 4.7(d, J 12,1H, ABX
system, CH2Ph), 5.42 (dd, J 8, 12 Hz, 1H, CHOAc), 7.33 (m, 5H,
Ph). 13C NMR (100.6 MHz, CDCl3): δ = 20.5 ppm, 35.7, 42.6,
2.5. Synthesis of Pyrrolidine Iminocyclitol Inhibitors
Synthesis of (3R)-1-benzyl-3-hydroxypyrrolidine-2,5-dione
(1): (3R)-1-benzyl-3-pyrrolidine-2,5-dione 1 was synthesized
according to the thermal condensation method [22a,23]. The
enantioselectivity was determined by chiral HPLC (column AD-
H, (60:40) n-hexane:ethanol, 1 mL/min) tR= 15.8 min, 98% ee.
67.5, 128.1, 128.7, 128.9, 135.1, 169.8, 172.9, 173.2. [α]28
+20.6 (c 1.45, CHCl3). {[α]20D = +39 (1%, w/v MeOH)} [24].
=
D
Synthesis of (3S)-1-benzyl-3-acetatepyrrolidine-2,5-dione
(2’): (3S)-1-benzyl-3-acetatepyrrolidine-2,5-dione 2’ was
synthesized according the procedure described previously, the
title compound was obtained as a brown solid (0.29 g, 78%).
Synthesis of (3S)-1-benzyl-3-hydroxypyrrolidine-2,5-dione
(1’): (3S)-1-benzyl-3-hydroxypyrrolidine-2,5-dione 1’ was
synthesized according to the thermal condensation
method[22a,23]. The enantioselectivity was determined by chiral
HPLC (AD-H, (60:40) n-hexane:ethanol, 1 mL/min: tR= 20.6
min, 100% ee.
1H NMR (400 MHz, CDCl3): δ 2.14 (s, 3H, CH3) ppm, 2.64
(dd, J=4, 16 Hz, 1H, CH2CO), 3.14 (dd, J 8, 20 Hz, 1H, CH2CO),
4.65 (d, J 16 Hz, 1H, ABX system, CH2Ph), 4.70 (d, J 16 Hz, 1H,
ABX system, CH2Ph), 5.25 (dd, J 4, 8 Hz, 1H, CHOAc), 7.34
(m, 5H, Ph). 13C NMR (100.6 MHz, CDCl3): δ 20.5 ppm, 35.7,
Synthesis of (3R)-1-benzyl-3-acetatepyrrolidine-2,5-dione
(2): (3R)-1-benzyl-3-hydroxypyrrolidine-2,5-dione 1 (300 mg,
1.5 mmol) was dissolved in pyridine (0.78 mL) and acetic
anhydride (1.23 mL) was then added. The solution was stirred for
23h at rt. The solvents were removed in vacuo, and the crude
product was purified by silica gel column chromatography [(2:1)
42.7, 67.5, 128.2, 128.7, 128.9, 135.2, 169.8, 172.9, 173.2. [α]28
D
= -21.5 (c 1.14, CHCl3). {[α]20D = -40.6 (1%, w/v MeOH)} [22].
Synthesis of (3R)-1-benzyl-3-hydroxypyrrolidine (3): (3R)-
1-benzyl-3-hydroxypyrrolidine 3 was synthesized according to

the method described by Zheng et al.[22a] [α]29 = +56.3 (c
(3R,4R)-pyrrolidine-3,4-diol (7):
D
0.045, CHCl3).
A dry 5mL round-bottomed flask containing a magnetic
stirring bar was charged with (3R,4R)-1-benzyl-3,4-
dihydroxypyrrolidine diol 5 (0.45 g, 2.3 mmol, EtOH (15 mL)
and Pd(0)En cat 3NP (1 g, 0.4 mmol Pd/g). A balloon filled with
hydrogen was attached to the flask, the mixture was warmed to
50ºC and stirred for 26 h. It was then allowed to cool to room
temperature, filtered and washed with CH2Cl2 (3mL), and the
solvent was removed in vacuo giving 7 (0.24 g, 100%) as a white
solid.
Synthesis of (3S)-1-benzyl-3-hydroxypyrrolidine (3’): (3S)-
1-benzyl-3-hydroxypyrrolidine-2,5 3’ was synthesized according
to the method described by Zheng et al.[22a] [α]29D = -3.14 (c
1.08, CHCl3). {[α]25D = -3.145 (c 1.2, CHCl3)}[22a]
Synthesis of (3R)-1-benzyl-3-acetatepyrrolidine (4): (3R)-1-
benzyl-3-acetatepyrrolidine 4 was synthesized according to the
method described for compound 2, the title compound was
obtained as a brown solid (0.14 g, 63%).
1H NMR (400 MHz, D2O): δ 3.36 ppm (d, J 12Hz, CHH, 2H),
3.6 (d, J 12Hz, CHH, 2H), 4.41 (s, 2H, CHO, 2H). 13C NMR
(D2O, 100MHz): δ 49.2 ppm, 72.7.
1H NMR (400 MHz, CDCl3): δ 1.85 (m, 1H, CH2) ppm, 2.03
(s, 3H, CH3), 2.25 (m,1H, CH2), 2.46 (m, 1H, CH2), 2.66 (m, 1H,
CH2), 2.8 (m, 2H, CH2), 3.60 (d, J 12 Hz, 1H, ABX system,
CH2Ph), 3.7(d, J 12 Hz, 1H, ABX system, CH2Ph), 5.17 (m, 1H,
CHOAc), 7.28 (m, 5H, Ph); 13C NMR (100.6 MHz, CDCl3): δ
21.0 ppm, 31.6, 52.5, 59.7, 60.1, 74.1, 127.6, 128.7, 129.4, 138.6,
1-Benzyl pyrrolidine (8):
To a round bottom flask (100 mL) with a magnetic stir bar
was added pyrrolidine (1g, 14 mmol), THF (10 mL) and
triethylamine (1.94 mL, 14 mmol). The mixture was cooled to
0ºC and benzyl bromide (1.67 mL, 21 mmol) was added drop
wise over 15 min. The mixture was stirred until all the substrate
was consumed. The solids were filtered and the filtrate was
concentrated in vacuo. The crude product 8 was dissolved in
CH2Cl2 (2 mL) and washed with water (2 mL). The organic phase
was dried with MgSO4, filtered and concentrated giving 8 (1.0 g,
46%) as yellow oil [27].
1H NMR (400 MHz, CDCl3): δ 1.81 ppm (m, 4H, CH2), 2.53
(m, 4H, CH2), 3.64 (s, 2H, CH2), 7.31 (m, 5H, Ph). 13C NMR
(100 MHz, CDCl3): δ 23.5 ppm, 54.3, 60.9, 126.9, 128.3, 129.0,
139.5.
171.0. [α]28 = +5.01 (c 3.77, CHCl3). {[α]D = +22.0 (c 5,
D
MeOH)}[25]
Synthesis of (3S)-1-benzyl-3-acetatepyrrolidine (4’): (3S)-
1-benzyl-3-acetatepyrrolidine 4’ was synthesized according to
the method described for compound 2, the title compound was
obtained as a brown solid (0.11 g, 49%).
1H NMR (400 MHz, CDCl3): δ 1.86 (m, 1H, CH2) ppm, 2.04
(s, 3H, CH3), 2.27 (m,1H, CH2), 2.40 (m, 1H, CH2, 2.66 (m, 1H,
CH2), 2.78 (m, 2H, CHH), 3.60 (d, J 12 Hz, 1H, ABX system,
CH2Ph), 3.69 (d, J 16 Hz, 1H, ABX system, CH2Ph), 5.18 (m,
1H, CHOAc), 7.29 (m, 5H, Ph); 13C NMR (100.6 MHz, CDCl3):
δ 21.3 ppm, 31.9, 52.7, 59.9, 60.2, 74.1, 127.6, 128.3, 128.9,
138.5, 171.0. [α]28D = -19 (c 2.4, CHCl3). {[α]20 = -23.0 (c 1,
D
3. Results and Discussion
MeOH)}[26]
3.1. Chemistry
Synthesis of (3R,4R)-1-benzyl-3,4-dihydroxypyrrolidine
(5): (3R,4R)-1-benzyl-3-dihydroxypyrrolidine 5 was synthesized
In the case of the synthesis of 1-benzyl-3-hydroxypyrrolidine
derivatives [22] D- or L-malic acid were used as substrates
(Scheme 1). In the case of compounds 1 and 1’, two methods
were used, in the first method only 89% ee was obtained (1st step
formation of hydroxyamide; 2nd step cyclization to form the
malimide). The first method was originally reported by Joullié et
al.[22b] It seems that the second step which involved heating at
high temperature without solvent gave some racemization. In the
second method using a direct thermally induced condensation
between the malic acid and the amine, in xylene, an enantiopurity
of 99%ee was obtained for the product. For the synthesis of 3 and
3’ the method described by Zheng et al.,[22a] was used and the
yields were approximately 50%. The acetylation of 1, 1’, 3, and
3’ with pyridine and acetic anhydride formed the products 2, 2’,
4 and 4’, respectively [7].
according to the method of Nagel [23]. [α]20 = -30.5 (c 4.23,
D
MeOH).
Synthesis of (3S,4S)-1-benzyl-3,4-dihydroxypyrrolidine
(3S,4S)-1-benzyl-3-dihydroxypyrrolidine
(5’):
5’
was
synthesized according to the method of Nagel [23]. [α]20D = +40
(c 3.7, MeOH) [[α]20D = +32.4 (c 4.2, MeOH)] [23a].
Synthesis
bis(benzyloxy)pyrrolidine (6)
of
(3R,4R)-1-benzyl-3,4-
NaH (0.828 g of 60% dispersion in mineral oil, 21 mmol) was
added to a suspension of (3R,4R)-1-benzyl-3,4-pyrrolidinediol 5
(1 g, 5.2 mmol) in anhydrous DMF (2 mL) at room temperature.
The suspension was stirred for 3min then cooled in an ice bath.
Benzyl bromide (1.85 mL, 16 mmol) was added dropwise over a
5-min period, and after 1min the ice bath was removed. The
reaction mixture was stirred overnight, and then 1 mL of MeOH
was added slowly to react with the excess of the NaH. DMF was
removed under reduced pressure at 55ºC. The residue was
dissolved in CH2Cl2 (25 mL) and washed with water and brine,
dried (MgSO4), filtered, and evaporated to give the crude product
6 as a yellow oil which was purified by silica gel column
chromatography [hexane, to (9:1) hexane:EtOAc to EtOAc] (1.16
g, 60%, as a colorless oil).
The non-hydroxylated 1-benzyl pyrrolidine 8 was synthesized
by alkylation of pyrrolidine using triethylamine and benzyl
bromide, furnishing the product in a yield of 46% without any
purification (Scheme 2). This compound was used as a reference.
(3R,4R)-1-Benzyl-3,4-pyrrolidinediol
5
and the respective
enantiomer 5’ were synthetized using the method of Nagel.23 The
dibenzylated derivative 6 was obtained by protecting the
hydroxyl groups of 5 using NaH in DMF and benzylbromide, the
yield was good.
1H NMR (400 MHz, CDCl3): δ 2.74 ppm (dd, J 4, 8 Hz, 2H,
CH2), 2.99 (dd, J 4, 12 Hz, 2H, CH2), 3.66 (d, J 12 Hz, 1H, ABX
system, CH2), 3.73(d, J 12 Hz, 1H, ABX system, CH2), 4.16 (d,
2H, 2xCH), 4.57 (q, 4H, 2x CH2Ph), 7.37 (m, 15H, Ph). 13C
NMR (100MHz, CDCl3): δ 58.5 ppm, 60.4, 71.5, 83.7, 127.1,
127.7, 127.9, 128.3, 128.4, 129.0, 138.2, 138.4. [α]D28 = -28.6 (c
1.13, CHCl3). MS (ESI-TOF) 374.22 (M+1).
(3R,4R)-Pyrrolidine-3,4-diol 7 (Scheme 3) was synthetized
using supported palladium (0) - Pd-EnCat - and this was used in
order to avoid leaching of the metal into the product, which is a
problem with these systems [28].

very poor inhibitory action (with an IC50 of 72 mM), suggesting
the requirement of hydroxyl groups in the pyrrolidine ring. Both
enantiomers 2 and 2’, have an acetoxyl group in the 3-position
of the pyrrolidine ring. In this case, however, it was not possible
to determine an exact IC50 value; instead a value in the range of
10-20 mM was inferred. The main reason for this result was that
probably the acetylated compounds suffered partial hydrolysis
during the inhibition period leading to a mixture of acetylated
and deacetylated products. In the case of the monohydroxylated
compounds 3 and 3’, the IC50 were 4.6 0.1 and 4.2 0.3 mM,
respectively. These compounds are better inhibitors than the
respective acetylated compounds (2 and 2’), perhaps as a result
of better interactions with the active site of the enzyme. The
same was the case for compound 6, with a lower IC50 value
compared to that of compound 5. Another problem with
compound 6 was its poor solubility in the assay solution hence
deviations in the solution concentration might occur. In the case
of the enantiomers 5 and 5’, the IC50 values were 2.97 0.046 and
5.82 0.034 mM, respectively; Interestingly 5 is more active than
5’. In an attempt to achieve better inhibition we decided to
synthetize compound 7, based on the modification of the
structure of enantiomer 5, making it more polar, with a free NH
group for interaction by hydrogen bonding, with the active site of
the enzyme. Unfortunately, the IC50 values increased slightly to
4.07 0.042 mM. On comparing the monohydroxyl with the
dihydroxyl compound series, the inhibition is quite similar.
Based on these results, our best inhibitor was compound 5.
Scheme 1. Reaction conditions: (a)(i) BnNH2, MeOH/H2O,
170ºC; (ii) 160ºC (b) BnNH2, Xylene, 170ºC (c) LiAlH4, THF,
NaOH (20%) (d) py, Ac2O, rt
In order to understand the molecular interactions between the
inhibitors and the enzyme, we carried out some kinetic studies
basically to determine the type of inhibition and the (KI) value
Figure 4 and Table 1.
Scheme 2. Reaction conditions: NEt3, THF, BnBr, 0oC to rt.
Table 1: The concentration of iminosugar giving 50%
inhibition of α-glucosidase from rat intestine mucosa.
Inhibitor
IC50 (mM)
DNJ
1
1.67 x 10-4 0.163x10-4
NI
1’
2
NI
NI
2’
3
NI
4.6 0.1
3’
4
4.2 0.3
10-20
4’
5
10-20
Scheme 3. Reaction Conditions: (a) Pd(0)EnCat, EtOH,
H2;(b) NaH, DMF, BnBr.
2.97 0.046 (KI= 1.18 mM)
5.82 0.034 (KI= 0.27 0.25 mM)
0.01 0.015
5’
3.2. Biological screening and Toxicological studies
The synthetized compounds were assayed for the inhibition of
rat intestine α-glucosidase using inhibitor concentrations between
0 and 46 mM, with the exception of compound 8 which was used
at concentrations of between 0 and 92 mM. DNJ was used as the
positive control. The IC50 for DNJ inhibition of rat α-glucosidase
was found to be in the nM range (IC50=0.167 0.016 μM) (Fig.
3(A)) and consistent with other reports [29]. Table 1 shows the
IC50 of the studied iminosugars, determined by the inhibition
curves (Fig. 4 (A)). Based on the results shown in the table 1, all
the compounds containing a 2,5- carbonyl group (1, 1’, 2, 2’)
failed to evoke any inhibition; on the contrary all the examples
lacking this group have shown inhibition in the concentration
range 0.01 – 72 mM. As expected 1-benzylpyrrolidine 8, showed
6
7
8
7.94
4.07 0.042(KI= 0.44 0.164 mM)
72.0 0.3
NI: no inhibition
Only three compounds were selected, 5, 5’ and 7, which were
the most promising inhibitors. On the basis of the kinetic results,
all the compounds showed a mixed type of inhibition (Figure 4
(B) and (C)). Based on the Dixon plot, the mechanism of
inhibition observed is predominately a mixed competitive type.
The KI values that were determined were different from the
respective IC50 values, again indicating competitive inhibition.

Despite the fact that these compounds were moderate α-
glucosidase inhibitors, it still was considered of interest to
evaluate their toxicological proprieties, particularly as they could
be used to address other therapeutic targets. The methods used
were (i) BRIN-BD11 cell viability assay and (ii) artemia Salina
toxicity assay. In the case of the first method used, Figure 5(A), it
was observed that compounds 5, 5’ and 7 had low toxicity,
compound 5 is the most toxic affecting the cellular viability in
concentration above 12.5 mM and compound 5’ affected the
cellular viability for concentrations above 25 mM whereas 7 did
not affect the cellular viability in the range of concentrations
studied, up to 50 mM. It is noteworthy that these concentration
values are 4 and 10 times higher than the IC50 of compounds 5/5’
and 7, respectively. In the case of the tests with artemia salina
only the enantiomers 5 and 5’ were evaluated. The results
obtained show that the LC50 values were 15.53 and 38.56 mM,
respectively (Figure 5(B)). For both compounds the LC50 is 5
times higher than the IC50. These results are in agreement with
the cellular viability test and show that 5’ is less toxic than 5. All
together, these results suggest that, although moderate inhibitors
of mammalian α-glucosidase, these compounds might have some
potential as pharmacological agents for other therapeutic targets.
In addition, enantiomers 5 and 5’ are more toxic than compound
7, suggesting that benzylated pyrrolidine are more deleterious
than debenzylated ones.
Asp542 and His600 (catalytic site) stabilizing the enzyme ligand
complex, in a similar fashion to acarbose and DNJ. A very
similar pose is observed for compound 7, however, contrary to
the former, this compound cannot establish interactions with
Asp203 due to lack of the N-benzyl group. Compounds 3, 3’, 6
and 8 show an inverted pose inside the binding pocket compared
with compounds 5 and 7. However, it was shown that they can
establish key interactions with Asp203, Arg526 and Asp542.
When we compare, for example compounds 5 and 3 (differing in
the mono and dihydroxypyrrolidine substitution at positions 3
and 4) we observed an opposed pose inside the binding cavity
bearing testimony to the importance of disubstitution for
stabilizing interactions that can improve inhibitory activity.
Compounds 1, 1’, 2 and 2’ showed a rotated pose (~90º) inside
the binding pocket when compared with compound 5. Despite the
fact, that these compounds occupy the same region as the active
ones, their lack of activity might be due to the fact that they have
carbonylic oxygen H-bond acceptors at positions 2 and 5,
apparently forcing them to rotate and lose the important H-
bonding interaction with Asp542.
4. Conclusions
Compounds with carbonyl groups in the 2,5-positions of the
pyrrolidine ring did not show any α-glucosidase inhibition.
Compounds with protected hydroxyl groups and without this
group were shown to be poor inhibitors.
3.3. Molecular Modeling Studies
On
comparing
3-hydroxylpyrrolidine
with
(3,4)-
In order to rationalize the experimental inhibitory activity for
the compounds with different types of substitution in positions 2-
5, the compounds were docked into the two enzymes active site.
All the compounds were protonated when docked at the α-
glucosidase active site. The results obtained for both enzymes
were very similar, thus for simplicity, only the docking results
using human α-glucosidase will be discussed.
dihydroxylpyrrolidine, analogous IC50 values were observed.
However, compound 5, a (3R,4R)-dihydroxypyrrolidine, was the
best inhibitor with a IC50 value of 2.97 0.046 mM. Compound 5
was more potent than its respective enantiomer.
Kinetic studies on compounds 5, 5’ and 7 revealed a
mechanism of inhibition predominately of the mixed competitive
type. Toxicological evaluation has shown that the compounds
studied have, in general, low toxicity, only presenting deleterious
effects for concentrations that are 4 to 10 times higher than the
IC50. Finally, the N-benzylated compounds are more toxic than
the debenzylated compound 7.
As stated in our previous paper, the docking results show that
all the tested compounds occupy the same region of the binding
pocket as the amino group and the corresponding cyclohexenyl
ring of acarbose. The obtained docking poses show all the
compounds lying inside the binding cavity and interacting with
the most important residues, notably, Asp203, Asp542, Asp443,
Asp327 Arg526 and His600. We also docked DNJ into the α-
glucosidase active site and this showed that the most active
compounds show a very similar pose when compared with the
ones obtained for DNJ (Figure 3).
Our docking studies suggest that a rotation of ~90º of
some compounds inside the binding pocket is responsible for the
complete loss of inhibitory activity.
We are currently looking at the synthesis of libraries of novel
sugar-pyrrolidine-diols for glucosidase inhibition.
Compound 5, the most potent compound in the series, is
predicted to form an H-bond between the pyrrolidine nitrogen
and Asp443 (1.5 Å), as well having interactions between the
oxygen atoms from the free hydroxyl groups, with Asp327,

A
120
100
80
60
40
20
0
3’
3
5’
5
6
7
DNJ
-20
-9
-8
-7
-6
-5
-4
Log[Inhibitor(M)]
-3
-2
-1
B
9x103
5x10-4
4x10-4
3x10-4
2x10-4
[p-NPG]=12mM
[p-NPG]=6mM
[p-NPG]=1,5mM
Inhibitor5
6x103
3x103
withoutinhibitor
1,2mM
1x10-4
0
2,6mM
6,2mM
0
0
3
6
9
12
-4
-2
0
2
4
[Inhibitor5](mM)
6
8
[p-NPG](mM)
C
8x10-4
6x10-4
4x10-4
8x103
6x103
4x103
[p-NPG]=12mM
Inhibitor7
[p-NPG]=6mM
[p-NPG]=3mM
[p-NPG]=1,5mM
withoutInhibitor
0,3mM
2x10-4
2x103
1,1mM
2,5mM
0
0
0
3
6
9
12
-2
-1
0
1
2
3
[p-NPG](mM)
[Inhibitor7](mM)
Figure 4 - (A) Inhibitory effects of compounds DNJ (ꢀ), 3 (ꢀ), 3’ (ꢁ), 5 (ꢀ), 5’ (ꢁ), 6 (ꢂ) and 7 (ꢁ) against rat α-glucosidase activity, using p-
nitrophenol-α-D-glucopyranoside as a substrate. Values are expressed as the mean sd (obtained in two independent experiments performed in quintuplicate).

DNJ served as positive control. (B) Michaelis-Menten Plot and Dixon Plot for compound 5 in the presence of several concentrations for determination of KI. (C).
Michaelis-Menten Plot and Dixon Plot for compound 7 in the presence of several concentrations for determination of KI. The values are expressed as mean sd (5
replicates).
Figure 5. (A) Viability of BRIN BD-11 cells with different concentrations of compounds 5, 5’ and 7. Batches of cells without inhibitors (control) and with 1%
SDS, a cell membrane disrupter, were used as negative and positive control, respectively. *indicates statistical significance relative to controls (p<0.05). (B)
Mortality (%) of artemia salina vs log[iminosugar 5 or 5’] – dose-response curve.
3. (a) A. Mitrakou, N. Tountas, A.E. Raptis, R.J. Bauer, H. Schulz,
ACKNOWLEDGEMENTS
S.A. Raptis, Diabetic Med.1998, 15, 657-660; (b) A. Trapero, A.
Llebaria, J. Med. Chem. 2012, 55, 10345-10346.
4. J.R. Jacob, K. Mansfield, J.E. You, B.E. Tennant, Y.H. Kim, J.
Microbiol. 2007, 45, 431-440.
5. G.S. Jacob, Curr. Opin. Struct. Biol. 1995, 5, 605-611.
6. T. Butters, R.A. Dwek, F.M. Platt, Curr. Top. Med. Chem 2003, 3,
561-574.
7. (a) L.R. Guerreiro, EP Carreiro, L. Fernandes, T.A.F. Cardote, R.
Moreira, A.T. Caldeira, R.M. Guedes, A.J. Burke, Bioorg. Med.
Chem. 2013, 21, 1911-1917 and references cited therein. (b) N. S.
H. N. Moorthy, N. F. Bras, M. J. Ramos, Bioorg. Med. Chem.
2012, 20, 6945; (c) C. Bello, M. Cea, G. Dal Bello, A. Garuti, I.
Rocco, G. Cirmena, E. Moran, A. Nahimana, M. A. Duchosal, F.
Fruscione, P. Pronzato, F. Grossi, F. Patrone, A. Ballestrero, M.
Dupuis, B. Sordat, A. Nencioni, P. Vogel, Bioorg. Med. Chem.
2010, 18, 3320; (d) E. Moreno-Clavijo, A. T. Carmona, Y. Vera-
Ayoso, A. J. Moreno-Vargas, C. Bello, P. Vogel, I. Robina, Org.
Biomol. Chem. 2009, 7, 1192.
EPC thanks the Fundação para a Ciência e a Tecnologia
(FCT) for post-doctoral research fellowship
a
(SFRH/BPD/72182/2010). We are very grateful to Dr. Olivia
Furtado Burke of LNEG, Lisbon for the optical activity
measurements. We acknowledge LabRMN at FCT-UNL for the
acquisition of the NMR spectra; the NMR spectrometers are part
of the National NMR Network and were purchased within the
framework of the National Programme for Scientific Re-
equipment (contract REDE/1517/RMN/2005), with funds from
POCI 2010 (FEDER) and FCT Strategic Projects PEst-
OE/QUI/UI0619/2011. PEst-C/SAU/LA0001/2011 (CNC) and
PEst-C/QUI/UI0062/2011 (ICAAM). The University of Vigo
(Spain) is gratefully acknowledged for MS analysis.
8. M.L. Sinnott, Chem. Rev. 1990, 90, 1171-1202 and references
cited therein.
9. (a) G.W.J. Fleet, S.J. Nicholas, P.W. Smith, S.V. Evans, L.E.
Fellows, R.J. Nash, Tetrahedron Lett. 1985, 26, 3127-3130; (b)
A.M. Scofield, L.E. Fellows, R.J. Nash, G.W.J. Fleet, J. Life Sci.
1986, 39, 645-650; (c) G.W.J. Fleet, P.W. Smith, Tetrahedron
1986, 42, 5685-5692; (d) J.R. Behling, A.J. Campbell, K.A.
Babiak, J.S. Ng, J. Medich, P. Farid, G.W.J. Fleet, Tetrahedron
1993, 49, 3359-3366.
10. F.P. Cruz, S. Newberry, S.F. Jenkinson, M.R. Wormald, T.D.
Butters, D.S. Alonzi, S. Nakagawa, F. Becq, C. Norez, R.J. Nash,
A. Kato, G.W.J. Fleet, Tetrahedron Lett. 2011, 52, 219-223.
11. (a) A. Kato, E. Hayashi, S. Miyauchi, I. Adachi, T. Imahori, Y.
Natori, Y. Yoshimura, R.J. Nash, H. Shimaoka, I. Nakagome, J.
Koseki, S. Hirono, H. Takahata, J. Med. Chem. 2012, 55, 10347-
References
.
1. (a) V.H. Lillelund, H.H. Jensen, X. Liang, M. Bols, Chem. Rev.
2002, 102, 515. (b) M. Sugiyama, Z. Hong, P-H. Liang, S.M.
Dean, L.J. Whalen, W.A. Greenberg, C-H. Wong, J. Am.
Chem.Soc. 2007, 129, 14811-17, and references cited therein.
2. (a) N. Zitmann, A.S. Metha, S. Carroueé, T.D. Butters, F.M. Platt,
J. McCauley, B.S. Blumberg, R.A. Dwek, T.M. Block, Proc. Natl.
Acad. Sci. USA 1999, 96, 11878-11882. (b) N.J. Asano, J. Enzyme
Inhib., 2000, 15, 215-234.

10362. (b) T.M. Chapman, S. Courtney, P. Hay, B.G. Davis,
Chem. Eur. J. 2003, 9, 3397, and references cited therein.
12. E-L. Tsou, S-Y. Chen, M-H. Yang, S-C. Wang, T-R.R. Cheng, W-
C. Cheng, Bioorg. Med.Chem. 2008, 16, 10198-10204.
13. (a) P.-H. Liang, W.-C. Cheng, Y.-L. Lee, H.-P. Yu, Y.-T. Wu, Y.-
L. Lin, C.-H. Wong, ChemBioChem 2006, 7, 165. (b) P.-H. Liang,
Y.-L Lin, C.-H. Wong, Patent Application US2012/0046337 A1,
2012.
14. M. Takebayashi, S. Hiranuma, Y. Kanie, T. Kajimoto, O. Kanie,
C-H. Wong, J. Org. Chem. 1999, 64, 5280-91.
15. M. Bols, Tetrahedon Lett. 1996, 37, 2097-100.
16. M. Godskesen, I. Lundt, Tetrahedon Lett. 1998, 39, 5841-4.
17. (a) S.G. Pyne, M. Tang, Curr. Org. Chem., 2005, 9, 1393. (b) K.
Tamazawa, H. Arima, T. Kojima, Y. Isomura, M. Okada, S.
Fujita, T. Furuya, T. Takenaka, O. Inagaki, M. Terai, J. Med.
Chem. 1986, 29, 2504.
18. W. Hakamata, M. Kurihara, H. Okuda, T. Nishio, T. Oku, Curr.
Top. Med. Chem. 2009, 9, 3-12.
19. (a) M. Watford, P. H. Lund, Biochemistry J. 1979, 178, 589-596.
(b) M.M. Bradford, Anal. Biochem. 1976, 72, 248-254.
20. (a) G. Jones, P. Willett, R. C. Glen, J. Mol. Biol., 1995, 245, 43-
53.; G. Jones, P. Willett, R. C. Glen, A. R. Leach, R. Taylor, J.
Mol. Biol., 1997, 267, 727-748. b) GoldScore performs a force
field based scoring function and is made up of four components:
1) Protein-ligand hydrogen bond energy (external H-bond); 2)
Protein-ligand van der Waals energy (external vdw); 3) Ligand
internal van der Waals energy (internal vdw); 4) Ligand
intramolecular hydrogen bond energy (internal- H- bond).
21. L. Sim, K. Jayakanthan, S. Mohan, R. Nasi, B.D. Johnston, B.
Mario Pinto, D.R. Rose, Biochemistry 2010, 49, 443–451.
22. (a) J.-L. Zheng, H. Liu, Y.-F. Zhang, W. Zhao, J.-S. Tong, Y.-P.
Ruan, P.-Q. Huang, Tetrahedron:Asymmetry 2011, 22, 257-263.
(b) K. L. Bhat, D. M. Flanagan, M. M. Joullié, Synth. Commun.
1985, 15, 587–598.
23. (a) U. Nagel, E. Kinzel, J. Andrade, G. Prescher, Chem. Ber.,
1986, 119, 3326. (b) W. Beck, U. Nagel, 1985, US patent,
4,634,775.
24. A. Naylor, D.B. Judd, D.I.C. Scopes, A.G. Hayes, P.J. Birch, J.
Med. Chem. 1994, 37, 2138-2144.
25. P.D. Cesare, D. Bouzard, M. Essiz, J.P. Jacquet, B. Ledoussal,
J.R. Kiechel, Remuzon, R.E. Kessler, J. Fung-Tomc, J. Desiderio,
J. Med. Chem. 1992, 35, 4205-4213.
26. H. Tomori, K. Shibutani, K. Ogura, Bull. Chem. Soc. Jap. 1996,
69, 207-215.
27. J.R. Sundberg, C.P. Walters, J.D. Bloom, J. Org. Chem.1981, 46,
3730-3732.
28. (a) J. Toubiana, M. Chidambaram, A. Santo, Y. Sasson, Adv.
Synth. Catal. 2008, 350, 1230-1234. (b)
http://reaxa.com/applications_Encat_30NP.html (29th Setember
2013).
29. C. Kuriyama, O. Kamiyama, K. Ikeda, F. Sanae, A. Kato, I.
Adachi, T. Imahori, H. Takahata, T. Okamoto, N. Asano, Bioorg.
Med. Chem. 2008, 16, 7330–7336.
•
•
•
•
The synthesis of a library of 13 chiral pyrrolidine
derivatives.
Enzyme kinetics implied a mixed inhibition mode,
corroborating our molecular docking studies.
Toxicological studies showed that they were non-
toxic.
Molecular docking studies indicated the mechanism
of competitive inhibition.