Galantamine derivatives with indole moiety: Docking, design, synthesis and acetylcholinesterase inhibitory activity

Article abstract of DOI:10.1016/j.bmc.2015.07.058

The inhibitors of acetylcholinesterase are the main therapy against Alzheimer's disease. Among them, galantamine is the best tolerated and the most prescribed drug. In the present study, 41 galantamine derivatives with known acetylcholinesterase inhibitor

Full text of DOI:10.1016/j.bmc.2015.07.058

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Galantamine derivatives with indole moiety: Docking, design,
synthesis and acetylcholinesterase inhibitory activity
Mariyana Atanasova , Georgi Stavrakov a, , Irena Philipova b, , Dimitrina Zheleva , Nikola Yordanov ,
Irini Doytchinova
a
a,
a
a
a,
⇑
Faculty of Pharmacy, Medical University of Sofia, 2 Dunav Str., 1000 Sofia, Bulgaria
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. 9, 1113 Sofia, Bulgaria
b
a r t i c l e i n f o
a b s t r a c t
Article history:
The inhibitors of acetylcholinesterase are the main therapy against Alzheimer’s disease. Among them,
galantamine is the best tolerated and the most prescribed drug. In the present study, 41 galantamine
derivatives with known acetylcholinesterase inhibitory activities expressed as IC50 were selected from
the literature and docked into a recombinant human acetylcholinesterase by GOLD. A linear relationship
between GoldScores and pIC50 values was found and used to design and predict novel galantamine
derivatives with indole moiety in the side chain. The four best predicted compounds were synthesized
and tested for inhibitory activity. All of them were between 11 and 95 times more active than
galantamine. The novel galantamine derivatives with indole moiety have dual site binding to the
enzyme—the galantamine moiety binds to the catalytic anionic site and the indole moiety binds to
peripheral anionic site. Additionally, the indole moiety of one of the novel inhibitors binds in a region,
Received 11 June 2015
Revised 20 July 2015
Accepted 25 July 2015
Available online xxxx
Keywords:
Acetylcholinesterase inhibitors
Galantamine
Molecular docking
GoldScore
Indole
close to the peripheral anionic site of the enzyme, where the X-loop of amyloid beta peptide adheres
Amyloid beta peptide
to acetylcholinesterase. This compound emerges as a promising lead compound for multi-target anti-
Alzheimer therapy not only because of the strong inhibitory activity, but also because it is able to block
the amyloid beta deposition on acetylcholinesterase.
Ó 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Alzheimer’s disease (AD) is the most common type of dementia
donepezil, rivastigmine and galantamine, while the NMDA inhibi-
tors are represented by one drug—memantine. Although there
are many successful therapeutic strategies tested in animal mod-
1
2
worldwide. The hallmark pathologies of AD are the progressive
accumulation of the protein fragment beta-amyloid (plaques) out-
side neurons in the brain and twisted strands of the protein tau
els, most of them have failed in humans. Recently, the current sta-
tus of anti-AD therapies has been extensively reviewed.2
–6
The neurotransmitter acetylcholine (ACh) plays a key role in
memory and cognition. According to the ‘cholinergic hypothesis’,7
the death of neurons strongly diminishes the levels of ACh in
cholinergic brain synapses. The inhibition of the enzyme acetyl-
cholinesterase (AChE) which hydrolyses ACh directly enhances
the levels of ACh and improves the cholinergic transmission.
Additionally, it was found that AChE accelerates the aggregation
of amyloid-b peptide (Ab) which is the main constituent of senile
(
tangles) inside neurons. These changes are eventually accompa-
nied by the damage and death of neurons. Early symptoms of AD
are difficulty remembering recent conversations, names or events,
apathy and depression. Later symptoms include impaired commu-
nication, disorientation, confusion, poor judgment, behavior
changes, and difficulty speaking, swallowing and walking. AD is
ultimately fatal.
The current treatment of AD reduces dementia symptoms but
does not arrest or reverse the underlying neurodegenerative disor-
der. There are only two classes of drugs approved for AD treat-
ment: acetylcholinesterase inhibitors (AChEIs) and inhibitors of
8
–10
plaques and a major neurotoxic agent.
AChE forms stable neu-
rotoxic complexes with Ab. The complexes induce Ab-dependent
2
+
deregulation of intracellular Ca
in hippocampal neurons,
mitochondrial dysfunction, neurite network dystrophia and
1
0
N-methyl-
D
-aspartate (NMDA) receptors. The available AChEIs are
apoptosis.
The binding site of recombinant human AChE (rhAChE) consists
1
1–15
of several domains.
0 Å deep and narrow binding gorge. It consists of the catalytic
triad: Ser203, Glu334 and His447. The anionic domain binds the
The catalytic site lies on the bottom of
⇑
Corresponding author.
E-mail address: idoytchinova@pharmfac.net (I. Doytchinova).
Authors with equal contributions.
2
http://dx.doi.org/10.1016/j.bmc.2015.07.058
968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
0
Please cite this article in press as: Atanasova, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.058

2
M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
quaternary trimethylammonium choline moiety of ACh. Despite its
name, it does not contain any anionic residues but only aromatic
ones: Trp86, Tyr130, Tyr337 and Phe338. They are involved in
interactions between the ligand and AChE and makes possible
the inhibition of Ab peptide binding to PAS.
cation–
p interactions with the protonated head of ACh. The acyl
2
2
. Results and discussion
pocket consists of two bulky residues: Phe295 and Phe297, and it
determines the selective binding of ACh by preventing the access
of larger choline esters. The oxyanion hole hosts one molecule of
structural water and consists of Gly121, Gly122 and Ala204. The
water molecule bridges the binding between enzyme and sub-
strate by hydrogen-bond networking and stabilizes the substrate
tetrahedral transition state. Finally, the peripheral anionic site
.1. Molecular docking of galantamine derivatives on rhAChE
Two series (A and B) of 41 GAL derivatives in total with AChE
inhibitory activities expressed as IC50 values were selected from
the literature
ing scores and activities. The structures and IC50 values of the stud-
ied compounds are given in Supplementary data. The compounds
were docked into the rhAChE (pdb code: 4EY6) by GOLD v.
56,57
and used to derive a relationship between dock-
(
PAS) lies at the entrance to binding gorge. It is composed of five
residues: Tyr72, Asp74, Tyr124, Trp286 and Tyr341. PAS allosteri-
27
cally modulates catalysis16 and is implicated in non-cholinergic
58
5
.1. The molecular docking protocol was optimized in several
17
functions as amyloid deposition, cell adhesion and neurite out-
growth.
steps as described in Methods. At each step of the docking protocol
optimization, the correlation between the docking scores and the
pIC50 (ꢀlogIC50) values of the compounds was evaluated by the
Pearson’s correlation coefficient r. The highest correlation coeffi-
cient r = 0.826 was achieved at the following settings: GoldScore
fitness function, flexible binding site with radius of 10 Å and a
water molecule inside. The relationship between GoldScores and
pIC50 is given below and illustrated in Figure 1.
1
8,19
It was found that the Ab peptide binds close to PAS
and the blockade of PAS prevents the AChE-induced Ab aggrega-
1
7
tion. This pivotal finding prompted the design of novel AChEIs
with dual binding moieties—one blocking the catalytic site at the
20–25
bottom of the gorge and one blocking the PAS.
The BLAST alignment of AChEs from human, rat, rabbit and
Torpedo californica showed that the main residues forming the
binding site are conserved (data not shown). The only difference
concerns Tyr337 which in T. californica mutates to Phe
pIC50 ¼ 0:083GoldScore ꢀ 1:723:
2
6
(
Phe330). The X-ray data show that the binding of galantamine
in rhAChE is similar to that observed in tAChE with one additional
2
.2. Design of novel galantamine derivatives and AChE
2
7
hydrogen bond formed between the tertiary amine and Tyr337.
Donepezil, however, binds differently to rhAChE than it does to
inhibitory activity prediction
2
7
tAChE.
Ten novel galantamine derivatives with indole moiety ending
the side chain attached to N-atom were designed and docked on
the rhAChE using the optimized docking protocol derived previ-
ously. The indole moiety was selected as suitable for binding to
the aromatic residues in PAS. The average GoldScores of three runs
were used to predict the pIC50 values according to the above rela-
tionship. The top four best predicted compounds were synthesized
and tested for AChE inhibitory activity. The structures of the newly
designed compounds are given in Figure 2, while their GoldScores
and predicted pIC50 values—in Table 1.
Galantamine (GAL) is an alkaloid initially isolated from the
bulbs and flowers of Galanthus caucasicus, Galanthus woronowii
(
Amaryllidaceae) and related genera.28 Paskov first developed
GAL as an industrial drug under the trade name Nivalin
2
9
(
Sopharma, Bulgaria). It has been used for treatment of myasthe-
nia gravis, myopathy, residual poliomyelitis paralysis syndromes,
3
0–34
sensory and motor disorders of CNS, and decurarization.
Because of its ability to cross the blood-brain barrier and to affect
the central cholinergic function, in 1980s GAL was investigated for
treatment of Alzheimer’s disease (AD) and in 2000 was approved
35–37
for use in Europe, United States and Asia.
In addition to its AChE inhibiting ability, GAL has been identified
as an allosteric modulator of nicotinic acetylcholine receptors
2.3. Synthesis of the best predicted galantamine derivatives
3
8–41
(
Ca
nAChRs).
The stimulation of nAChRs can increase intracellular
Our synthetic strategy towards the target molecules focused on
the preparation of bromine-containing building blocks and their
subsequent substitution with N-demethylated galantamine.
Construction of the required indole based building blocks was
performed by applying simple and effective synthetic procedures
(Scheme 1). Compound 3a was prepared by reaction of reductive
alkylation of 1H-indol-5-amine with readily available 4-(6-bromo-
2
+
levels and facilitate noradrenaline release; both effects
42
enhance the cognitive brain function. Recently, Takata et al. have
demonstrated that treatment of rat microglia with GAL significantly
enhanced microglial amyloid-b (Ab) phagocytosis and facilitated Ab
clearance in brains of rodent AD models.43 This multiple-target
action of GAL makes it a most valuable drug for AD treatment
2
2
and prompts the synthesis of novel GAL derivatives with
hexyloxy)benzaldehyde
3
in the presence of NaBH(OAc) . The
improved binding to AChE.4
4–50
Several series of GAL derivatives
desired product was obtained in quantitative yield after flash col-
umn chromatography. Treatment of 1H-indol-5-ol with commer-
cially available 1,6-dibromohexane in the presence of potassium
with dual site binding to the enzyme have been prepared and
4
6,51–53,24
tested.
All of them showed good AChE inhibitory activities.
In the present study, we applied molecular docking on two ser-
ies of GAL derivatives binding to AChE and we derived a quantita-
tive relationship between the docking-based scores and AChE
inhibitory activities. This relationship was used to predict the
activities of newly designed galantamine derivatives with indole
moiety ending the side chain attached to N-atom. The indole moi-
ety was selected as suitable for binding to the aromatic residues in
3
carbonate in CH CN at 60 °C for 24 h gave rise to compound 3b
in 68% yield after purification by flash chromatography. The forma-
tion of the products 3c and 3d was accomplished by procedures
developed for peptide synthesis. The reaction of 1H-indol-5-amine
with commercially available 6-bromohexanoic acid in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and
1-Hydroxybenzotriazole hydrate (HOBT) as coupling reagents
PAS because of its ability to take part in hydrophobic and
p
–
p
afforded 3c. Following the same protocol for L-tryptophan methyl
5
4,55
interactions.
The best four predicted compounds were synthe-
ester hydrochloride and in the presence of N,N-diisopropylethy-
lamine (DIPEA) we synthesized amide 3d. The products were
obtained in excellent yields and purity after flash column
chromatography.
sized and tested. All of them showed activity higher than that of
GAL. The most active derivative was 95 times more active than
GAL. The prolongation of the side chain at N-atom increases the
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M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
9
8
7
6
5
4
considered: hydrogen bonds, hydrophobic,
p
–
p
and cation–
p
interactions. They are visualized on the highest-scored pose of
the complex compound 1a—rhAChE. The compound 1a is the
ligand with the highest AChE affinity among the newly synthesized
ligands (IC50 = 0.011 lM).
The single water molecule in binding site is situated deep in the
bottom of the gorge and is involved in a hydrogen-bond network
connecting ligand and enzyme (Fig. 3a). It acts as hydrogen-bond
acceptor with Gly122 and Ala204 from the oxyanion hole and as
a hydrogen-bond donor with the ligand hydroxyl and methoxy
group. The ligand itself is involved in additional three hydrogen
bonds. The protonated nitrogen is a donor in the hydrogen bond
with Tyr337 from the anionic site; the hydroxyl group acts as a
donor in the bonding with Glu202; the oxygen atom from the
methoxy group is an acceptor in the bonding with Ser203 from
the catalytic domain. No hydrogen bonds exist along the N-side
chain.
7
0
80
90
100
110
120
GoldScore
Figure 1. Relationship between GoldScores and pIC50 of 41 galantamine deriva-
tives, r = 0.826. The compounds from series A and B are given with black and grey
diamonds, respectively.
An efficient N-selective demethylation of galantamine to nor-
galantamine 2 was accomplished by a non-classical Polonovski
The hydrophobic interactions inside the binding gorge are visu-
alized in Figure 3b. The GAL moiety makes hydrophobic interac-
tions with Trp86 and Phe295. The side chain interacts with
5
9
reaction using iron(II) sulfate heptahydrate (Scheme 2).
Alkylation of norgalantamine 2 with the indole-based bromides
a–d was realized via nucleophilic substitutions.57 Thus, heating
of 2 with bromides 3a–d in acetonitrile in the presence of K CO
Tyr72. The aromatic ring from the GAL moiety is involved in
p–p
3
interactions with Tyr124 and Phe297 (Fig. 3c). Trp286 is in a good
position of stacking with the phenyl ring from GAL side chain and
2
3
as a base for 24 h afforded the targeted compounds 1a–d in good
to make a p–p interaction (Fig. 3c). Finally, the protonated N-atom
yields and excellent purity after flash column chromatography
of GAL moiety interacts with Trp86 (Fig. 3d).
(
Scheme 2). The structures of the newly synthesized compounds
View of the complex 1a–AChE in profile of the binding gorge is
given in Figure 4 (1a is given in blue). The binding gorge is totally
filled by the ligand and even part of the indole moiety protrudes
outside the gorge. Recent studies reveal that N-substituents with
long chains are favourable for AChE inhibitory activity because of
were proven by NMR spectroscopy and elemental analysis.
2
.4. Assessment of AChE inhibitory activity
4
6,51–53,24,54–57
The inhibitory potential of the novel GAL derivatives against T.
the ability to interact with the PAS.
californica AChE (tAChE) was tested according to the methodology
developed by Ellman et al.62 with some modifications. GAL was
used as a positive control and the enzyme activity was calculated
In order to explore the importance of PAS for amyloid beta (Ab)
61
deposition on AChE, a complex was generated between AChE and
6
3
Ab peptide by RosettaDock as described in Materials and meth-
at IC50 = 1.07
l
M for GAL. The IC50 values (lM) of the novel com-
ods. The complex with the lowest score was selected as the most
pounds are shown in Table 1. Compounds 1a–c demonstrated the
probable. In this complex, the X-loop of Ab is bound in a region,
highest acetylcholinesterase inhibitory activity, with similar IC50
close to the PAS of AChE (Fig. 4). This pose coincides with the
AChE–Ab binding mode, identified by Inestrosa in 2001.17 The
superposed complexes 1a–AChE, 1c–AChE and AChE–Ab are given
in Figure 4. It is evident that the phenyl ring of 1a and the indole
ring of 1c bind inside the PAS, while the indole moiety of 1a and
values (0.011
compound 1d was less active (0.094
derivatives showed AChE inhibitory activities between 11 and 95
times higher than this of GAL (IC50 = 1.070 M). The differences
lM, 0.012
lM and 0.015
lM, respectively), while
l
M). All newly synthesized
l
between predicted and experimental pIC50 values of the novel
the
X-loop of Ab bind in a region, proximal to PAS.
compounds were less than one log unit.
In Figure 5 are given superposed main residues of PAS (Tyr72,
Asp74, Tyr124, Trp286 and Tyr341) from the complexes AChE–Ab
(in yellow) and 1a–AChE (in cyan). Four of the five residues, that
is, Tyr72, Asp74, Tyr124 and Tyr341, adopt almost the same
conformations in both complexes. Only residue Trp286 makes a
significant shift. In the complex AChE–Ab, Trp286 takes an ‘open’
2
.5. Interactions between the new compounds and rhAChE
The interactions between the novel GAL derivatives and rhAChE
were analyzed by YASARA.62 Four types of interactions were
OH
OH
NH
O
HN
O
NH
N
(CH2)6
O
N
(CH2)6
O
H CO
H CO
3
3
1a
1b
OH
OH
O
O
O
O
N
(CH2)5
N
(CH2)5
NH
NH
HN
HN
H3CO
H3CO
CO2Me
1c
1d
Figure 2. Structures of the newly designed GAL derivatives.
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4
M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Table 1
3
. Conclusion
GoldScores, predicted and experimental pIC50 values of the newly designed and
synthesized GAL derivatives
The molecular docking study on GAL derivatives binding to
Compd
GoldScore pIC50
IC50
l
M (exp) Times pIC50 pIC50 (pred)–
AChE shows that the GoldScores of the inhibitor–AChE complexes
derived by an optimized docking protocol correlate well with the
inhibitory activities of the studied compounds. This docking proto-
col is able to predict the activities of newly designed AChE inhibi-
tors with indole moiety ending the side chain attached to N-atom.
The synthesized and tested novel compounds confirm the predic-
tions. The docked complexes reveal that the novel inhibitors show
dual site binding to the enzyme—the galantamine moiety binds to
the catalytic anionic site and the indole moiety binds to peripheral
anionic site. The indole moiety of one of the inhibitors binds in the
same region, where Ab adheres to AChE.
*
(pred) tAChE
more (exp) pIC50 (exp)
active
than
GAL
1
a
119.15
112.32
109.06
113.89
73.59
8.166 0.011 ± 0.0004 95
7.599 0.012 ± 0.0021 93
7.329 0.015 ± 0.0003 72
7.730 0.094 ± 0.0118 11
7.949
0.218
1
b
7.938 ꢀ0.339
1
c
7.829 ꢀ0.501
1
d
7.027
0.703
GAL HBr
4.385 1.070 ± 0.1559
1
5.971 ꢀ1.586
*
Recalculated at GAL IC50 = 1.07
l
M.
4
. Materials and methods
OHC
O(CH2)6 Br
NaBH(OAc)3
O(CH2)6Br
H
NH2
OH
N
4.1. Datasets and molecular docking protocol
N
H
DCE
4 h, r.t.
N
H
Two series of 41 GAL derivatives with AChE inhibitory activities
2
6,57
3a
expressed as IC50 values were selected from the literature5
and
99%
used as a training set to derive a relationship between docking
scores and IC50s. The structures are given in Supplementary mate-
rial. All IC50 values are used in p-units (ꢀlog).
Br(CH2)6Br
K2CO3
O(CH ) Br
2 6
Recently published X-ray structure of rhAChE in complex with
N
H
CH CN
N
H
3
27
galantamine (GAL) (pdb id: 4EY6, R = 2.15 Å) was used as a tem-
2
4 h, 60 oC
3
b
58
6
8%
plate for docking calculations by GOLD v. 5.1. GOLD is a docking
tool based on a genetic algorithm and it has proven successful in
virtual screening, lead optimisation, and identification of the cor-
Br(CH2)5CO2H
EDC / HOBT
H
N
6
4,65
NH2
(CH ) Br
rect binding mode of active molecules.
GOLD takes into
2
5
account the flexibility of the ligand as well as the flexibility of
the residues in the binding site.
O
N
H
24 h, r.t.
CH2Cl2
N
H
8
4%
3c
The molecular docking protocol was optimized in several steps.
Initially, the protocol consisted of rigid protein, flexible ligand,
0
radius of the binding site 6 ÅA and one water molecule (HOH846)
kept within the binding site. According to the X-ray structure of
Br(CH2)5CO2H
EDC / HOBT
DIPEA
O
MeO2C
MeO2C
(CH2)5Br
2
7
NH2.HCl
NH
the complex GAL–rhAChE, only one water molecule was left in
the binding gorge, making a hydrogen bond network between
GAL, Ser203, Gly121 and Gly122. This initial protocol was used
to select a scoring function (SF) among the four available SFs in
GOLD: ChemPLP, ChemScore, GoldScore and ASP. Next, the influ-
ence of structural water and flexible protein binding site were
examined. Ten amino acids from the binding site situated in a close
proximity to the binding ligands were selected as flexible. These
were Tyr72, Asp74, Trp86, Tyr124, Ser125, Trp286, Phe297,
Tyr337, Phe338 and Tyr341. Finally, the optimal radius of the bind-
ing site was adjusted. Each run generated 10 docking poses. The
poses were ranked by two criteria: (1) highest fitness score and
N
H
CH2Cl2
N
H
2
4 h, r.t.
3d
99%
Scheme 1. Synthesis of indole based building blocks.
position and makes
AbPhe4). In the complex 1a–AChE, Trp286 moves toward the phe-
nyl ring of 1a because of the stacking and reduces the cavity of
p–p interaction with Phe4 from Ab (given as
p–p
PAS (‘closed’ position). The ‘closed’ position of Trp286 prevents
from interaction with Ab Phe4. Additionally, the indole moiety of
a fills the region, proximal to PAS, where the
1
X-loop of Ab adheres
(
2) rmsd (root mean square deviation) lower than 1.5 Å. These
to AChE.
poses that did not meet these criteria were assigned as ‘Non-
docked’. Each docking run was repeated tree times and the average
Although the novel galantamine derivatives have close AChE
inhibitory activities, compound 1a emerges as the most promising
lead compound for multi-target anti-Alzheimer therapy not only
because of the strong AChE inhibitory activity, but also because it
is able to block the Ab deposition on AChE. The experimental con-
firmation of this hypothesis is in progress.
fitness score was used for correlation with the pIC50
.
At each step of the docking protocol optimization, the correla-
tion between the docking scores and the pIC50 (ꢀlog IC50) values
of the training compounds was evaluated by the Pearson’s
Scheme 2. Synthesis of the target molecules via norgalantamine.
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M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
Figure 3. (a) Hydrogen bonds (yellow dotted lines) between ligand (blue sticks), water molecule (purple sticks) and AChE residues (sticks colored by element). (b)
Hydrophobic interactions (green lines). (c)
p–p
interactions (red lines). (d) Cation–
p
interaction (blue lines).
correlation coefficient r. Some compounds were not docked at the
tested conditions. Apart from r, the number of docked structures
was considered during the optimization.
for 13C NMR) spectrometer with TMS as internal standards for
1
13
chemical shifts (d, ppm). H and C NMR data are reported as fol-
lows: chemical shift, multiplicity (s = singlet, d = doublet, t = tri-
plet, q = quartet, br = broad, m = multiplet), coupling constants
6
3
RosettaDock server was used to generate the complex AChE–
1
13
Ab peptide. The docking partners were uploaded as two separate
(Hz), integration, identification. The assignment of the H and
C
2
7
66
pdb files: 4EY6 for AChE and 1AML for Ab. The complex with
NMR spectra was made on the basis of DEPT, COSY and HSQC
experiments. The spectra are given in Supplementary material.
Elemental analyses were performed by Microanalytical Service
Laboratory of Faculty of Pharmacy, Medical University of Sofia,
using Vario EL3 CHNS(O).
the lowest score was selected as the most probable.
4
4
.2. Synthesis
.2.1. General
Reagents were commercial grade and used without further
4.2.2. Synthesis of indole based building blocks
purification. Thin layer chromatography (TLC) was performed on
aluminum sheets pre-coated with Merck Kieselgel 60
.25 mm (Merck). Flash column chromatography was carried out
using Silica Gel 60 230–400 mesh (Fluka). Commercially available
solvents for reactions, TLC and column chromatography were used
after distillation (and were dried when needed). Melting points
were determined in a capillary tube on SRS MPA100 OptiMelt
4.2.2.1.
3a.
N-(4-(6-Bromohexyloxy)benzyl)-1H-indol-5-amine
A mixture of 1H-indol-5-amine (0.100 g, 0.757 mmol)
F
254
0
and 4-(6-bromohexyloxy)benzaldehyde (0.216 g, 0.757 mmol) in
8 ml 1,2-dichloroethane was stirred at rt for 30 min. Then
NaBH(OAc)
mixture was stirred at rt for 24 h. The reaction was quenched with
sat. aq NaHCO , the product was extracted with CH Cl , dried over
Na SO and the solvent was evaporated under reduced pressure.
Purification by flash-column chromatography on silica gel
(hexane/EtOAc/Et N = 2:1:0.5) gave 0.303 g (99%) of the product
as white crystals, mp 86–89 °C. H NMR (CDCl
d = 7.96 (br s, 2H, NH), 7.33–7.31 (m, 2H, arom.), 7.21–7.19 (m,
3
(0.240 g, 1.136 mmol) was added and the reaction
3
2
2
(
Sunnyvale, CA, USA) automated melting point system (uncor-
2
4
20
rected). Optical rotation ([
a]
D
) were measured on Perkin–Elmer
2
41 polarimeter. The NMR spectra were recorded on a Bruker
3
1
13
1
Avance II+ 600 (600.13 for H MHz and 150.92 MHz for C NMR)
and Bruker Avance DRX-250 (250.13 for H MHz and 62.92 MHz
3
, 600 MHz)
1
Please cite this article in press as: Atanasova, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.058

6
M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
6
(
7.69 (OCH
CH ), 27.90 (CH
C 62.85, H 6.28, N 6.98, found C 63.18, H 6.47, N 6.83.
2
), 49.21 (HNCH
2
), 33.85 (CH
2
Br), 32.65 (CH
2
), 29.07
2
2
), 25.28 (CH
2
) ppm. C21
H
25BrN O (401.34): calcd.
2
4
.2.2.2. 5-(6-Bromohexyloxy)-1H-indole 3b.
To a solution of
1
H-indol-5-ol (0.100 g, 0.750 mmol) in 15 ml CH CN was added
3
2
K CO
3
(0.310 g, 2.250 mmol) and 1,6-dibromohexane (0.220 g,
0
.900 mmol). The mixture was heated at 60 °C for 24 h.
Concentration of the solvent and purification by flash-column
chromatography on silica gel (hexane/EtOAc = 4:1) gave 0.151 g
1
(
68%) of the product as colourless oil. H NMR (CDCl
3
, 600 MHz)
d = 8.03 (br s, 1H, NH), 7.29–7.25 (m, 1H, arom.), 7.18–7.16 (m,
1
2
H, arom.), 7.10 (d, Jh,h = 2.4 Hz, 1H, arom.), 6.85 (ddd, Jh,h = 8.8,
.4, 0.4 Hz, 1H, arom.), 6.48–6.45 (m, 1H, arom.), 4.00 (t,
J
1
(
h,h = 6.4 Hz, 2H, OCH
2
), 3.42 (t, Jh,h = 6.8 Hz, 2H, CH
), 1.55–1.49 (m, 4H, 2 CH ) ppm. C NMR
, 150.9 MHz) d = 153.59 (1 arom. C), 131.00 (1 arom. C),
2
Br), 1.93–
13
.79 (m, 4H, 2 CH
2
2
CDCl
3
1
1
6
28.32 (1 arom. C), 124.79 (1 arom. CH), 112.93 (1 arom. CH),
11.61 (1 arom. CH), 103.54 (1 arom. CH), 102.39 (1 arom. CH),
8.57 (OCH
), 25.40 (CH
.13, N 4.73, found C 56.48, H 6.39, N 5.02.
2 2
), 33.86 (CH Br), 32.75 (CH
2 2
), 29.31 (CH ), 27.99
(
CH
2
2
) ppm. C14
H18BrNO (296.20): calcd. C 56.77, H
6
4
.2.2.3. 6-Bromo-N-(1H-indol-5-yl)hexanamide 3c.
To a
solution of 6-bromohexanoic acid (0.174 g, 0.832 mmol) in 7 ml
CH Cl was added 1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide (0.160 g, 0.832 mmol), 1-Hydroxybenzotriazole (0.112 g,
.832 mmol) and 1H-indol-5-amine (0.100 g, 0.757 mmol). The
mixture was stirred at rt for 24 h. The reaction was quenched with
water, and the product was extracted with CH Cl . The combined
organic extracts were dried over Na SO and the solvent was evap-
orated under reduced pressure. Purification by flash-column chro-
matography on silica gel (CH Cl /EtOAc = 10:1) gave 0.205 g (84%)
of the product as colourless oil. H NMR (CDCl
d = 7.78–7.77 (m, 1H, arom.), 7.33–7.30 (m, 1H, arom.), 7.23–7.19
m, 2H, arom.), 6.46 (dd, Jh,h = 3.2, 0.8 Hz, 1H, arom.), 3.42 (t,
h,h = 6.7 Hz, 2H, CH Br), 2.37 (t, Jh,h = 7.2 Hz, 2H, COCH ), 1.96–
), 1.82–1.70 (m, 2H, CH ), 1.60–1.49 (m, 2H,
) ppm. C NMR (CDCl /CD OD, 150.9 MHz) d = 172.08 (CO),
Figure 4. AChE binding gorge viewed in profile. AChE molecular surface is given in
grey. Compounds 1a (in blue) and 1c (in magenta) are docked inside the gorge. The
Ab peptide bound in the PAS is given in dark orange.
2
2
0
2
2
2
4
2
2
1
3 3
/CD OD, 600 MHz)
(
J
1
2
2
.84 (m, 2H, CH
2
2
1
3
CH
2
3
3
1
1
1
33.51 (1 arom. C), 130.14 (1 arom. C), 128.00 (1 arom. C),
25.39 (1 arom. CH), 116.42 (1 arom. CH), 112.88 (1 arom. CH),
11.25 (1 arom. CH), 102.16 (1 arom. CH), 37.10 (COCH
Br), 32.56 (CH ), 27.85 (CH ), 25.00 (CH ) ppm. C14
309.20): calcd. C 54.38, H 5.54, N 9.06, found C 54.01, H 5.87, N
2
), 33.77
(
CH
2
2
2
2
2
H17BrN O
(
9
.13.
4
.2.2.4. (S)-Methyl 2-(6-bromohexanamido)-3-(1H-indol-2-
yl)propanoate 3d.
To a solution of 6-bromohexanoic acid
0.115 g, 0.550 mmol) in 10 ml CH Cl was added 1-ethyl-3-
3-dimethylaminopropyl)carbodiimide (0.105 g, 0.550 mmol),
-Hydroxybenzotriazole (0.074 g, 0.550 mmol), -tryptophan methyl
(
(
1
2
2
L
ester hydrochloride (0.127 g, 0.550 mmol) and N,N-diisopropy-
lethylamine (0.1 ml, 0.550 mmol). The mixture was stirred at rt
for 24 h. The reaction was quenched with water and the product
Figure 5. Superposed residues of PAS. The complex AChE–Ab is given in yellow
(
AChE) and dark orange (Ab). The complex 1a–AChE is given in cyan (AChE) and
2 2
was extracted with CH Cl . The combined organic extracts were
blue (1a).
dried over Na SO and the solvent was evaporated under reduced
2
4
pressure. Purification by flash-column chromatography on silica
gel (CH Cl /EtOAc = 10:1) gave 0.195 g (99%) of the product as
1
H, arom.), 7.11–7.10 (m, 1H, arom.), 6.88–6.86 (m, 3H, arom.),6.65
2
2
2
0
1
(
dd, Jh,h = 8.6, 2.2 Hz, 1H, arom.), 6.38–6.37 (m, 1H, arom.), 4.28 (s,
colourless oil. [
a]
D
= +42.7 (c 0.400, CHCl3). H NMR (CDCl3,
2
2
1
H, HNCH
H, CH Br), 1.90–1.88 (m, 2H, CH
.51–1.50 (m, 4H, 2 CH
) ppm. 13C NMR (CDCl
2
), 3.95 (t, Jh,h = 6.4 Hz, 2H, OCH
), 1.80–1.78 (m, 2H, CH
, 150.9 MHz)
2
), 3.42 (t, Jh,h = 6.8 Hz,
250 MHz) d = 8.17 (br s, 1H, NH-indole), 7.53 (d, Jh,h = 7.7 Hz, 1H,
arom.), 7.36 (d, Jh,h = 7.7 Hz, 1H, arom.), 7.23–7.09 (m, 2H, arom.),
6.98 (d, Jh,h = 2.3 Hz, 1H, arom.), 5.94 (d, Jh,h = 7.7 Hz, 1H, HNCO),
5.00–4.93 (m, 1H, COCHCH ), 3.71 (s, 3H, OCH ), 3.38–3.30 (m, 4H,
2
2
2
),
2
3
d = 158.17 (1 arom. C), 142.28 (1 arom. C), 131.81 (1 arom. C),
2
3
1
1
1
30.09 (1 arom. C), 128. 95 (2 arom. CH), 128. 71 (1 arom. C),
24.39 (1 arom. CH), 114.47 (2 arom. CH), 112.17 (1 arom. CH),
1.58 (1 arom. CH), 102.35 (1 arom. CH), 101.79 (1 arom. CH),
2 2 h,h 2
CH Br, CHCH -indole), 2.15 (t, J = 7.1 Hz, 2H, COCH ), 1.87–1.76
(m, 2H, CH ), 1.65–1.53 (m, 2H, CH ), 1.45–1.33 (m, 2H, CH ) ppm.
2
2
2
1
3
C NMR (CDCl , 62.92 MHz) d = 172.59 (CO), 172.28 (CO), 136.11
3
Please cite this article in press as: Atanasova, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.058

M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
(
1 arom. C), 127.75 (1 arom. C), 122.65 (1 arom. CH), 122.33 (1 arom.
ppm. C30
36 2 4
H N O (488.62): calcd. C 73.74, H 7.43, N 5.73, found C
CH), 119.75 (1 arom. CH), 118.57 (1 arom. CH), 111.30 (1 arom. CH),
74.02, H 7.72, N 5.68.
1
3
10.17 (1 arom. C),52.89 (COCHCH
3.59 (CH Br), 32.42 (CH ), 27.68 (CH
) ppm. C18 23BrN (395.29): calcd. C 54.69, H 5.86, N 7.09,
found C 54.36, H 6.21, N 7.23.
2
), 52.38 (OCH
3
), 36.24 (COCH
2
),
2
2
2
-indole), 27.64 (CH
2
), 24.15
4.2.3.3.
N-(1H-Indol-5-yl)-6-((4aS,6R,8aS)-6-hydroxy-3-meth-
(
CH
2
H
2
O
3
oxy-5,6,9,10-tetrahydro-4aH-benzo[2,3]-benzofuro[4,3-cd]aze-
pin-11(12H)-yl)hexanamide 1c.
Yield: 47%; white crystals;
2
0
1
mp 101–104 °C.
[
a]
D
= ꢀ47.6 (c 0.250, CHCl
3
).
H
NMR
4
.2.3. General procedure for preparation of compounds 1a–d
(CDCl /CD OD, 600 MHz) d = 7.80 (d, J = 1.8 Hz, 1H, arom.), 7.33
3
3
h,h
To a solution of norgalanthamine 2 (1 equiv) in anhydrous ace-
(d, Jh,h = 8.6 Hz, 1H, arom.), 7.23–7.21 (m, 2H, arom.), 6.66 (d,
Jh,h = 8.2 Hz, 1H, H-2), 6.63 (d, Jh,h = 8.2 Hz, 1H, H-1), 6.45 (d,
Jh,h = 2.6 Hz, 1H, arom.), 6.07 (d, Jh,h = 10.2 Hz, 1H, H-8), 5.99 (dd,
tonitrile (2 mL), was added 3 (1.1 equiv) and anhydrous K
2 3
CO
(
3 equiv) under argon atmosphere. After stirring at 60 °C for 24 h,
the solvent was removed in vacuo. The residue was directly sub-
J
h,h = 10.2, 5.1 Hz, 1H, H-7), 4.60 (br s, 1H, H-6), 4.16–4.14 (m, 1H,
H-4a), 4.15 (d, Jh,h = 15.4 Hz, 1H, H-12), 3.85 (d, Jh,h = 15.4 Hz, 1H,
H-12), 3.83 (s, 3H, OCH ), 3.38–3.33 (m, 1H, H-10), 3.20–3.19 (m,
H, H-10), 2.65–2.62 (m, 1H, H-5), 2.57–2.47 (m, 2H, NCH ), 2.36
t, Jh,h = 7.4 Hz, 2H, CH CO), 2.08–2.00 (m, 2H, H-5, H-9), 1.76–1.71
m, 2H, CH ), 1.57–1.55 (m, 3H, H-9, CH ), 1.40–1.34 (m, 2H, CH
/CD OD, 150.9 MHz) d = 172.66 (CO), 145.93
C-3a), 144.40 (C-3), 133.61 (1 arom. C), 133.59 (C-12b), 130.10 (C-
jected to purification by flash column chromatography on silica
1
gel (CH
and
2
Cl
2
/MeOH/NH
4
OH = 20/1/0.02) to give product 1. The
H
3
1
3
C NMR spectra of compounds 1a–d are given in
1
(
(
2
Supplementary data.
2
2
2
2
)
4
.2.3.1. (4aS,6R,8aS)-11-(6-(4-((1H-Indol-5-ylamino)methyl)phe-
noxy)hexyl)-3-methoxy-5,6,9,10,11,12-hexahydro-4aH-benzo[2,3]-
benzofuro[4,3-cd]azepin-6-ol 1a. Yield: 73%; white crystals;
13
ppm.. C NMR (CDCl
(
3
3
1
1
2a), 130.32 (1 arom. C), 128.01 (1 arom. C), 127.62 127.56 (C-8),
26.99 (C-7), 125.52 (1 arom. CH), 122.65 (C-1), 116.29 (1 arom.
2
0
1
mp 73–76 °C. [
a
]
D
= ꢀ45.9 (c 0.290, CHCl
00 MHz) d = 7.99 (br s, 2H, NH), 7.32–7.31 (m, 2H, arom.), 7.21–
.20 (m, 1H, arom.), 7.12–7.11 (m, 1H, arom.), 6.97–6.85 (m, 3H,
3 3
). H NMR (CDCl ,
6
7
CH), 112.74 (1 arom. CH), 111.51 (C-2), 111.30 (1 arom. CH),
01.98 (1 arom. CH), 88.57 (C-6), 61.97 (C-4a), 57.61 (C-12), 55.98
OCH ), 51.51 (C-10), 51.44 (NCH ), 48.34 (C-8a), 36.89 (CH CO),
2.56 (C-9), 29.99 (C-5), 26.81 (CH ), 25.45 (CH ), 25.65 (CH
(501.62): calcd. C 71.83, H 7.03, N 8.38, found C
1.97, H 6.78, N 8.49.
1
arom.), 6.65–6.64 (m, 1H, arom.), 6.65 (d, Jh,h = 8.2 Hz, 1H, H-2),
.62 (d, Jh,h = 8.2 Hz, 1H, H-1), 6.39–6.38 (m, 1H, arom.), 6.09 (d,
h,h = 10.2 Hz, 1H, H-8), 6.00 (dd, Jh,h = 10.1, 5.0 Hz, 1H, H-7), 4.62
(
3
2
2
6
J
(
(
3
3
3
2
2
2
)
35 3 4
ppm. C30H N O
br s, 1H, H-6), 4.28 (s, 2H, PhCH
d, Jh,h = 15.1 Hz, 1H, H-12), 3.93 (t, Jh,h = 6.5 Hz, 2H, OCH
H, OCH
3
2
), 4.15–4.12 (m, 1H, H-4a), 4.13
), 3.83 (s,
), 3.83–3.80 (m, 1H, H-12), 3.38–3.33 (m, 1H, H-10),
.19–3.16 (m, 1H, H-10), 2.70–2.67 (m, 1H, H-5), 2.54–2.44 (m, 2H,
), 2.41 (br s, 1H, OH), 2.07–1.98 (m, 2H, H-9, H-5), 1.79–1.73
m, 2H, CH ), 1.53–1.43 (m, 5H, H-9, 2 CH ), 1.38–1.32 (m, 2H,
, 150.9 MHz) d = 158.20 (2 arom. C),
7
2
4.2.3.4. (S)-Methyl-3-(1H-indol-3-yl)-2-(6-((4aS,6R,8aS)-6-hydroxy-
3-methoxy-5,6,9,10-tetrahydro-4aH-benzo[2,3]-benzofuro[4,3-
NCH
2
(
CH
1
1
2
1
2
cd]azepin-11(12H)yl)hexanamido)propanoate 1d.
Yield: 76%;
3
). H NMR
3
20
1
) ppm. C NMR (CDCl
3
white crystals; mp 86–89 °C. [
a]
D
= ꢀ18.6 (c 0.290, CHCl
2
45.72 (C-3a), 144.03 (C-3), 142.44 (1 arom. C), 133.12 (C-12b),
31.87 (C-12a), 130.04 (1 arom. C), 128.90 (2 arom. CH), 128.72 (1
(CDCl , 600 MHz) d = 8.32 (br s, 1H, NH-indole), 7.51 (d, Jh,h = 8.0 Hz,
3
1H, arom.), 7.35 (d, Jh,h = 8.2 Hz, 1H, arom.), 7.19 (dt, Jh,h = 7.0, 1.1 Hz,
1H, arom.), 7.11 (dt, Jh,h = 7.1, 1.0 Hz, 1H, arom.), 6.96 (d, Jh,h = 2.3 Hz,
1H, arom.), 6.65 (d, Jh,h = 8.2 Hz, 1H, H-2), 6.61 (d, Jh,h = 8.2 Hz, 1H, H-
1), 6.07 (d, Jh,h = 10.2 Hz, 1H, H-8), 6.01 (dd, Jh,h = 10.2, 5.0 Hz, 1H, H-
7), 5.97 (d, Jh,h = 7.9 Hz, 1H, NH), 4.95 (dq, Jh,h = 7.9, 5.4 Hz, 1H,
arom. C), 127.53 (C-8), 126.92 (C-7), 124.38 (1 arom. CH), 122.01
C-1), 114.47 (2 arom. CH), 112.11 (1 arom. CH), 111.57 (1 arom.
CH), 111.03 (C-2), 102.21 (1 arom. CH), 101.79 (1 arom. CH), 88.72
C-6), 67.84 (OCH ), 62.08 (C-4a), 57.74 (C-12), 55.84 (OCH ),
1.49 (C-10), 51.35 (NCH ), 49.15 (PhCH ), 48.40 (C-8a), 32.84 (C-
), 29.90 (C-5), 29.21 (CH ), 27.34 (CH ), 27.12 (CH ), 26.01 (CH
(593.76): calcd. C 74.84, H 7.30, N 7.09, found C
4.93, H 7.47, N 7.13.
(
(
5
9
2
3
2
2
CHCH ), 4.61 (br s, 1H, H-6), 4.14 (br s, 1H, H-4a), 4.13 (d,
2
2
2
2
2
)
J
h,h = 15.4 Hz, 1H, H-12), 3.83 (s, 3H, OCH
H-12), 3.70 (s, 3H, COOCH ) 3.37–3.27 (m, 3H, H-10, CHCH
h,h = 14.0 Hz, 1H, H-10), 2.70–2.67 (m, 1H, H-5), 2.51–2.40 (m, 2H,
NCH ), 2.14–2.11 (m, 2H, CH CONH), 2.05–1.98 (m, 2H, H-9, H-5),
.60–1.55 (m, 2H, CH ), 1.51 (d, Jh,h = 13.6 Hz, 1H, H-9),1.48–1.43 (m,
H, CH ), 1.27–1.22 (m, 2H, CH
d = 172.52 (2 CO), 145.78 (C-3a), 144.16 (C-3), 136.09 (C-12b), 133.11
C-12a), 127.69 (C-8), 127.67 (C-7), 126.82 (1 arom. C), 126.78 (1 arom.
), 3.81 (d, Jh,h = 15.4 Hz, 1H,
3
43 3 4
ppm. C37H N O
3
2
), 3.16 (d,
7
J
2
2
4
.2.3.2.
(4aS,6R,8aS)-11-(6-(1H-Indol-5-yloxy)hexyl)-3-meth-
1
2
2
oxy-5,6,9,10,11,12-hexa
cd]azepin-6-ol 1b.
hydro-4aH-benzo[2,3]benzofuro[4,3-
13
2
2 3
) ppm. C NMR (CDCl , 150.9 MHz)
Yield: 63%; white crystals; mp 64–67 °C.
2
0
1
[
(
a
]
D
= ꢀ74.6 (c 0.260, CHCl
3
). H NMR (CDCl
3
, 600 MHz) d = 8.15
(
br s, 1H, NH), 7.26 (s, 1H, arom.), 7.17 (t, Jh,h = 5.5 Hz, 1H, arom.),
.09 (d, Jh,h = 2.4 Hz, 1H, arom.), 6.84 (dd, Jh,h = 8.8, 2.4 Hz, 1H,
arom.), 6.65 (d, Jh,h = 8.2 Hz, 1H, H-2), 6.62 (d, Jh,h = 8.2 Hz, 1H, H-
), 6.47–6.46 (m, 1H, arom.), 6.08 (d, Jh,h = 10.2 Hz, 1H, H-8), 6.00
dd, Jh,h = 10.2, 5.1 Hz, 1H, H-7), 4.61 (br s, 1H, H-6), 4.16–4.13 (m,
C), 122.74 (1 arom. CH), 122.21 (C-1), 122.15 (1 arom. CH), 122.04 (1
arom. C), 119.63 (1 arom. CH), 118.55 (1 arom. CH), 111.30 (1 arom.
CH), 110.05 (1 arom. C), 88.72 (C-6), 62.06 (C-4a), 57.60 (C-12), 55.88
7
1
(
1
1
3
1
(
4
2
OCH
8.35 (C-8a), 36.40 (CH
6.94 (CH ), 26.82 (CH ), 25.26 (CH
3
), 52.76 (CHCH
2
), 52.38 (COOCH
CO), 32.73 (C-9), 29.91 (C-5), 27.62 (CHCH
) ppm. C34 (587.71): calcd.
3
), 51.45 (C-10), 51.08 (NCH
2
),
),
2
2
H, H-4a), 4.14 (d, Jh,h = 15.2 Hz, 1H, H-12), 3.98 (t, Jh,h = 6.5 Hz,
H, OCH ), 3.83 (d, Jh,h = 15.2 Hz, 1H, H-12), 3.82 (s, 3H, OCH ),
.39–3.34 (m, 1H, H-10), 3.20–3.18 (m, 1H, H-10), 2.70–2.66 (m,
H, H-5), 2.55–2.45 (m, 2H, NCH ), 2.41 (br s, 1H, OH), 2.07–1.98
), 1.53–1.46 (m, 5H, H-9,
) ppm. 13C NMR (CDCl
, 150.9 MHz)
d = 153.53 (2 arom. C), 145.72 (C-3a), 144.07 (C-3), 133.09 (C-12b),
2
2
2
41 3 5
H N O
2
3
C 69.48, H 7.03, N 7.15, found C 69.32, H 7.24, N 7.21.
2
(
2
m, 2H, H-9, H-5), 1.81–1.77 (m, 2H, CH
CH ), 1.40–1.31 (m, 2H, CH
2
4.3. Assessment of AChE inhibitory activity
2
2
3
60
AChE activity was assayed as described by Ellman et al. with
6
1
1
1
30.90 (C-12a), 128.24 (1 arom. C), 127.56 (C-8), 126.87 (C-7),
24.79 (1 arom. CH), 122.09 (C-1), 112.84 (1 arom. CH), 111.59 (1
some modifications.
Aldrich) in buffer phosphate (pH 7.6) and 50
pounds (4–500 M in methanol) dissolved in 700
buffer. The mixtures were incubated for 30 min at room tempera-
ture before the addition of 100 L of the substrate solution (0.5 M
DTNB, 0.6 mM ATCI in buffer, pH 7.6). The absorbance was read in
Fifty
l
L of T. californica AChE (Sigma–
L of the tested com-
L in the same
l
arom. CH), 111.06 (C-2), 103.36 (1 arom. CH), 102.27 (1 arom. CH),
8.70 (C-6), 68.59 (OCH ), 62.06 (C-4a), 57.68 (C-12), 55.83
), 51.46 (C-10), 51.33 (NCH ), 48.36 (C-8a), 32.76 (C-9),
), 27.27 (CH ), 27.14 (CH ), 26.06 (CH
l
l
8
(
2
2
OCH
3
2
l
9.89 (C-5), 29.38 (CH
2
2
2
2
)
Please cite this article in press as: Atanasova, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.058

8
M. Atanasova et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
23. Leon, R.; Marco-Contelles, J. Curr. Med. Chem. 2011, 18, 552.
Shimadzu spectrophotometer at 405 nm after three minutes.
Enzyme activity was calculated as a percentage compared to an
assay using a buffer without any inhibitor using nonlinear regres-
sion. IC50 values are means ± SD of three individual determinations
each performed in triplicate.
2
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This work was supported by the Medical Science Council (Grant
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-S/2013).
Supplementary data
4
4
Supplementary data (the structures and IC50 values of the GAL
1
13
derivatives used in the docking study. The H and C NMR spectra
of compounds 1a–d) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2015.07.058.
4
4
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Please cite this article in press as: Atanasova, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.07.058