Synthesis of novel vanillin derivatives: Novel multi-targeted scaffold ligands against Alzheimer's disease

Article abstract of DOI:10.1039/c9md00048h

Alzheimer's disease (AD) is the most common cause of dementia worldwide, normally affecting people aged over 65. Due to the multifactorial nature of this disease, a "multi-target-directed ligands" (MTDLs) approach for the treatment of this illness has generated intense research interest in the past few years. Vanillin is a natural antioxidant and it provides a good starting point for the synthesis of new compounds with enhanced antioxidant properties, together with many biological activities, including β-amyloid peptide aggregating and acetylcholinesterase inhibiting properties. Here we report novel vanillin derivatives, bearing a tacrine or a naphthalimido moiety. All compounds exhibited improved antioxidant properties using DPPH assay, with IC₅₀ as low as 19.5 μM, FRAP and ORAC assays, with activities up to 1.54 and 6.4 Trolox equivalents, respectively. In addition, all compounds synthesized showed inhibitory activity toward acetylcholinesterase enzyme at μmolar concentrations using the Ellman assay. Computational docking studies of selected compounds showed interactions with both the catalytic anionic site and the peripheral anionic site of the enzyme. Furthermore, these compounds inhibited Aβ_(1-42) amyloid aggregation using the fluorometric ThT assay, with compound 4 showing comparable inhibitory activity to the positive control, curcumin. At cellular level compound 4 (1 μM) showed significant protective effects in neuroblastoma SH-SY5Y cell line when treated with hydrogen peroxide (400 μM). In our opinion, vanillin derivatives could provide a viable platform for future development of multi-targeted ligands against AD.

Full text of DOI:10.1039/c9md00048h

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Page 1 of 27
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Synthesis of novel vanillin derivatives: novel multi-targeted scaffold ligands against Alzheimer’s
DOI: 10.1039/C9MD00048H
disease
Matteo Scipioni1, Graeme Kay1, Ian L. Megson2, Paul Kong Thoo Lin*1
1School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen, UK
2Institute of Health Research & Innovation, University of the Highlands and Islands, Inverness, UK
Abstract
Alzheimer’s Disease (AD) is the most common cause of dementia worldwide, normally affecting people
aged over 65. Due to the multifactorial nature of this disease, a “multi-target-directed ligands” (MTDLs)
approach for the treatment of this illness has generated intense research interest in the past few years.
Vanillin is a natural antioxidant and it provides a good starting point for the synthesis of new compounds
with enhanced antioxidant properties, together with many biological activities, including -amyloid peptide
aggregating and acetylcholinesterase inhibiting properties. Here we report novel vanillin derivatives, bearing
a tacrine or a naphthalimido moiety. All compounds exhibited improved antioxidant properties using DPPH
assay, with IC50 as low as 19.5 M, FRAP and ORAC assays, with activities up to 1.54 and 6.4 Trolox
equivalents, respectively. In addition, all compounds synthesized showed inhibitory activity toward
acetylcholinesterase enzyme at molar concentrations using the Ellman assay. Computational docking
studies of selected compounds showed interactions with both the catalytic anionic site and the peripheral
anionic site of the enzyme. Furthermore, these compounds inhibited Aβ(1-42) amyloid aggregation using the
fluorometric ThT assay, with compound 4 showing comparable inhibitory activity to the positive control,
curcumin. At cellular level compound 4 (1 M) showed significant protective effects of neuroblastoma SH-
SY5Y cell line when treated with hydrogen peroxide (400 M). In our opinion, vanillin derivatives could
provide a viable platform for future development of multi-targeted ligands against AD.
Keywords: Alzheimer’s Disease, Synthetic antioxidants, AChE inhibitors, Multitarget-directed ligands,
Vanillin derivatives.

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DOI: 10.1039/C9MD00048H
Introduction
Amongst all neurodegenerative diseases, Alzheimer’s disease (AD) is the most common cause of dementia,
mostly affecting people over the age of 65 1. In developed countries, AD is the fifth cause of death 2. AD is
characterized by memory loss, confusion with space and time, speaking problems, poor judgment and
changes in mood and personality. Due to increased life expectancy, 1 million new cases per year are
expected by 2050 3. For this reason, there is an urgent need for effective therapies.
AD is a multifactorial disease normally characterized by neuronal loss and -amyloid deposition that leads to
4
extracellular amyloid plaques in the cerebral cortex . Although the cause of these events is not fully
2
understood, the “amyloid hypothesis” is the most recognized for development of AD . More recently,
oxidative injury to macromolecules, including proteins, lipids and nucleic acids has been identified as a key
5
feature in the development of AD, thus leading to the “oxidative stress hypothesis” . Finally, a decrease in
2
acetylcholine availability at neuronal synapses is a common hallmark in AD patients , making this a
possible therapeutic target.
Due to the many contributing factors involved in the development and progression of AD, the current trend
of drug development based on a “one target-one molecule” point of view is no longer favored. A multi-
targeted approach, aimed at targeting different steps of the neurotoxic cascade, has started to attract much
interest among the research community 6. In particular, the approach of designing multi-target agents against
neurodegenerative diseases has shown an increase 7–9. For example, recent reports include multifunctional
10
agent chromone-2-carboxyamidoalkylbenzylamines
and tacrine-8-hydroxyquinoline hybrids 11. Both
groups of compounds target the cholinesterase enzymes acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE), act as chelating agent with copper ion and inhibit A42) aggregation. All
these events play a crucial role in the onset and progression of AD.
Multi-target compounds acting as antioxidants and acetylcholinesterase inhibitors including tacrine, a well-
12
13
known acetylcholinesterase inhibitor and resveratrol, a strong natural antioxidant have been recently
reported 14. The compounds described in this work were strong inhibitors of acetylcholinesterase (with IC50 at
nanomolar concentrations) and Aβ(1-42) aggregation, while showing antioxidant activities in the 2,2-diphenyl-
1-picrylhydrazyl (DPPH) assay comparable to Trolox 14. Furthermore several hybrids between tacrine and
Trolox, a common synthetic antioxidant, were previously reported; the compounds showed inhibitory
activity toward AChE comparable to tacrine, with a two-fold decrease in the antioxidant activities in DPPH
assay when compared to Trolox standard 15.
Previously, we reported novel vanillin derivatives with strong antioxidant properties; in particular,
compound 2c (4,4',4'',4'''-((1,4-phenylenebis(azanetriyl))tetrakis(methylene))tetrakis(2-methoxyphenol))
exhibited remarkable scavenging activity toward DPPH free radical, as well as reducing activity in FRAP
assay. In addition, the latter compound protected the SH-SY5Y neuroblastoma cell line against hydrogen
peroxide (400 M) treatment, increasing the cell viability by 30% at compound concentrations as low as 10
M 16.

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Here we report the design and synthesis of four novel vanillin derivatives with multi-target functionalities
DOI: 10.1039/C9MD00048H
that would be expected to exhibit antioxidant properties, as well as inhibitory AChE and amyloid Aβ(1-42)
aggregation activities (Figure 1). Among the vanillin derivatives synthesized, three contained a
naphthalimido moiety and one has a tacrine group. It is noteworthy to mention previous work reporting a
naphthalimido moieties linked with ranitidine to produce potential multi-targeted AD agents 17. The
antioxidant properties, inhibitory AChE activity and -amyloid aggregation inhibitory properties and their
protective effect on peroxide-treated neuroblastoma SH-SY5Y cells of all the compounds synthesized in this
work will be discussed.
Figure 1. Rationale for the synthesis of vanillin derivatives 18,19
.
The phenolic moiety, important for the antioxidant activity, was linked to the tacrine moiety, a well-known
AChE inhibitor. Tacrine was substituted with a naphthalimido structure to determine the impact of the latter
on AChE and -amyloid inhibitory activity. In addition, the aromatic linker was substituted with a propyl
moiety, which shows similar distance between the two nitrogens (5.2 and 4.9 Å, respectively, calculated
using PyMOL software) and more flexibility for the evaluation of its impact on AChE and amyloid Aβ(1-42)
peptide inhibitory activities.

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DOI: 10.1039/C9MD00048H
Results and discussion
Chemical Synthesis
The synthesis of novel naphthalimido vanillin derivatives 1, 2, and 3 was achieved as depicted in Scheme 1
using 2-(3-aminopropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (I-1) as a precursor. The latter was
synthesised from the reaction between naphthalic anhydride and 1,3-diamino propane in ethanol according
to previous work 20. Vanillin derivatives (1, 2, and 3) were then prepared by reacting vanillin or
syringaldehyde with I-1 to afford the corresponding imines. The latter were then reduced in the presence of
sodium borohydride in methanol (2, 86%) or propan-2-ol (3, 38%).
O
O
1h, 78°C
EtOH
O
N
NH2
+
H2N
NH2
O
O
I-1
O
O
X
O
O
OH
+
MeOH
N
NH2
N
N
HO
X
O
O
O
1
. X= -H
MeOH or
propan-2-ol
NaBH4
X= -H
-OCH3
O
O
OH
X
H
N
N
O
2
. X= -H
3
. X= -OCH3
Scheme 1. Chemical strategy for the synthesis of derivatives 1-3.
1
The naphthalimido compounds (1, 2, 3) showed characteristic features in their H NMR spectra. The latter
showed three sets of peaks between 8.65 and 7.74 ppm due to the benzo[de]isoquinoline-1,3-dione
(naphthalimido) moiety along with two sets of peaks or a singlet between 7.01 and 6.50 ppm attributed to the
vanillin or the syringaldehyde moieties, respectively. A singlet between 3.9 and 3.7 ppm is due to the
methoxy group (in both vanillin and syringaldehyde structures) and the methylene (Ar-CH2-N) group
respectively. The propyl groups in both compounds were identified by the presence of two triplets and a
multiplet between 4.4 and 1.9 ppm.

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The reduction of compound 1, to generate compound 2, was confirmed by the disappearance of the singlet at
DOI: 10.1039/C9MD00048H
8.2 ppm with the appearance of new singlet (CH2) at 3.76 ppm.
The presence of an extra methoxy group in compound 3 compared to compound 2 was also confirmed by the
integration of the peak at 3.79 ppm (6 protons instead of 3).
The synthesis of compound 4 (Scheme 2) involved intermediates I-2 and I-3. Compound I-2 (67% yield)
was prepared following the procedure described by Szymański, Zurek and Mikiciuk-Olasik, by reacting
anthranilic acid with 2 equivalents of cyclohexanone in POCl3 (7.5mL) 21. Intermediate I-3 (39 % yield) was
synthesized by the N-alkylation reaction between I-2 and of 2.5 equivalents of p-phenylenediamine in 1-
pentanol in the presence of catalytic amount of potassium iodide (KI).
O
OH
Cl
N
O
NH2
+
I-2
24 hrs, 155°C
POCl3
O
OH
H2N
Cl
N
NH2
NH2
i) Vanillin
ii) NaBH4
NH
N
KI
HN
+
24 hrs, 138°C
1-pentanol
24 hrs, 82°C
2-propanol
NH
N
I-3
4
Scheme 2. Chemical strategy for the synthesis of derivative 4.
1
The H NMR spectrum of compound 4 showed seven sets of peaks in the region between 7.6 and 6.5 ppm
attributed to the aromatic protons of the tacrine, p-phenylenediamine and phenolic rings. Two multiplets and
two triplets between 2.6 and 1.8 ppm of the tetrahydroacridine motif from the tacrine ring system and the
singlet at 3.8 ppm were due to the methoxy group from the phenolic moiety. (See experimental section and
Supplementary Information, SI).
Antioxidant Activity
All compounds were evaluated for their antioxidant properties using three different assays (DPPH, FRAP
and ORAC) with differing oxidative potential evaluating mechanisms (Table 1). DPPH assay was
22
employed to evaluate the scavenging activity of the novel vanillin derivatives whereas FRAP
23
assay was selected in order to measure the electron transfer properties of the latter. Finally, the

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ORAC assay was utilized for the evaluation of the hydrogen atom transfer (HAT) abilities of our
DOI: 10.1039/C9MD00048H
novel compounds emulating a more relevant environment to biological system 24.
Table 1. Antioxidant properties of vanillin derivatives.
Compound
FRAP (TE)
0.07 ± 0.002
0.26 ± 0.04
1.45 ± 0.02
1.54 ± 0.15
1
ORAC (TE)
DPPH (IC50 M)
1
2.1 ± 0.5
3.9 ± 1.3
2.0 ± 0.5
6.4 ± 1.6
1
> 250 M
2
3
50.7 ± 0.8
19.5 ± 0.3
20.5 ± 0.3
24.4 ± 0.9
Inactive a
4
Trolox
Tacrine
Inactive a
<0.01b
Results from each assay are expressed as a mean ± SD of three independent experiments.
a Compounds were tested up to 250 M. Trolox was used as a positive control
b Reported by 25
.
2,2-diphenyl-1-picrylhydrazyl (DPPH) Assay
Diphenyl-1-picrylhydrazyl (DPPH) is a stable organic nitrogen radical with an absorption maximum at 515
nm. Its reduction in the presence of antioxidants, acting as free radical scavengers, was monitored
spectrophotometrically 22. The IC50 value of each compound was determined (see Table 1). The imine
compound 1 showed no IC50 up to concentration of 250 M whereas its corresponding reduced amine
showed an IC50 of 50.7 M, confirming our previous finding that an imine exhibits lower activity in free
radical scavenging compared with the corresponding amine 16. In addition, compound 3, bearing an extra
methoxy group in the phenolic moiety compared to compound 2, turned out to be ~two-fold more active
(19.5 M), confirming the important role of an extra methoxy group for the antioxidant activity.
Interestingly, compound 4 showed similar activity to compound 3, although bearing only one methoxy group
in its phenolic moiety (20.5 and 19.5 M, respectively). This confirms our previous finding regarding the
increase of antioxidant activity due to the electronic conjugation between the two nitrogens and an aromatic
ring.

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The compounds showed greater antioxidant activities compared to the tacrine-resveratrol hybrids reported by
DOI: 10.1039/C9MD00048H
Jeřabek et al. which showed weak DPPH scavenging abilities due to the absence of phenolic moieties in their
chemical structures 14.
Interestingly, tacrine turned out to be completely inactive at concentrations as high as 250 M, despite the
fact it has a nitrogen atom linked with an aromatic moiety. The lack of activity can be explained by the
absence of a phenolic moiety in the aromatic structure.
Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay is based on the reduction of the ferric-tripyridyltriazine complex by antioxidants and it was
performed at pH 3.5. The ferrous-tripyridyltriazine complex that is formed after the reduction of the iron
core leads to a measurable blue color (593 nm) 23. The results are expressed as Trolox Equivalent (TE) after
comparison with the standard Trolox calibration curve. Again, compound 1 showed the lowest activity (0.07
TE) (see table 1), almost 15 times less active than the standard Trolox. Its reduced derivative, compound 2,
was almost 4 times less active than Trolox (0.26 TE), showing better performance compared to the
corresponding imine (compound 1). In contrast, compound 3 showed a 5-fold increase in activity (1.45 TE)
compared to compound 2 confirming the importance of the extra methoxy group in the phenolic moiety for
improved activity in the FRAP assay. Compound 4 turned out to be the most active among the four vanillin
derivatives (1.54 TE), confirming the role of the electronic delocalisation of the nitrogen electron in the
antioxidant activity 16. Again, tacrine on its own showed to be completely inactive in this assay at
concentrations as high as 250 M.
Oxygen Radical Absorbance Capacity (ORAC) Assay
ORAC assay is based on hydrogen atom transfer (HAT) mechanism and measures the ability of antioxidants
to inhibit the oxidation of the fluorescent probe fluorescein caused by peroxyl free radical generated by
thermal decomposition of the 2,2’-azobis(2-amidinopropane) (AAPH) 26–28. The use of peroxyl free radical,
which is commonly found in the body, makes this assay more relevant to biological systems 24.
All the novel vanillin derivatives showed improved antioxidant activities in this assay compared to the
standard Trolox. Unlike the previous assays, the vanillin derivative 2 showed better activity compared to its
corresponding syringaldehyde derivative 3 (3.9 and 2.0 TE, respectively) highlighting the deleterious impact
of the extra methoxy moiety in the phenolic ring on ORAC assay. It is worth mentioning that vanillin itself is
more active than syringaldehyde in this assay (2.2 and 1.5 TE, respectively) 16.
The imine 1 showed weaker scavenging activity compared to the amine 2 (2.1 and 3.9 TE, respectively) in
this assay, confirming the role of the nitrogen’s electron availability in the antioxidant activity.
Finally, compound 4 showed the highest ORAC value (6.4 TE) highlighting the predominant role of
electronic delocalisation in the peroxyl free radical scavenging activity.
The latter showed higher activities compared to the series of melatonin-tacrine hybrid reported by
Rodriguez-Franco et al., which ORAC values were ranging from 1.7 - 4.0 TE 25.

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DOI: 10.1039/C9MD00048H
Cellular Protection
MTT Assay
The production of reactive oxygen species is linked with cell death and it is a peculiar hallmark of many age-
related diseases 29. Based on all the above results, derivative 4 was found be the most active compound with
regard to antioxidant properties. Therefore, the latter was chosen to study its ability to protect stressed cells.
Neuroblastoma SH-SY5Y cell line was used for this purpose and the cell viability was determined
spectrophotometrically using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay.
Hydrogen peroxide was used as a stressor to evaluate the protective effect of compound 4 against oxidative
damage; cells were exposed for 24 hours with hydrogen peroxide (400 M) after pre-treatment (24 hours)
with compound 4 at different concentrations, ranging from 0.01 to 5 M.
The choice of the working concentration in the protective effect of compound 4 was based on its toxicity,
after 24 hours, exposure towards SH-SY5Y neuroblastoma cell line. Compound 4 exhibited an IC50 of 45.1 ±
3.5 M, although some toxic effect was observed at concentration of 25 M. No significant toxicity was
however found at 12.5 M, hence the decision to work with 5 M which is significantly below the toxic
concentrations (see Figure 2). Compound 4 showed strong protective effect against oxidative insult and the
results are reported in Figure 2.
ns
ns
ns
***
100
50
0
100
50
***
***
ns
*
***
ns
***
***
0
1
5
0.1
2.5
0.01
25
50
100
6.25
12.5
3.125
Media Control
Peroxide Control
Media Control
Compound 4 conc. (M)
compound 4 conc. (M)
Figure 2. (Left) Toxicity of Compound 4 toward SH-SY5Y cells after 24 hours exposure. (Right)
Protective effects of Compound 4 in hydrogen peroxide (400 M)- stressed cells. Cells were
incubated for 24 hours with compound 4 at different concentrations before the addition of the
stressor. After 24 hours, cell viability was measured through MTT assay. Values are expressed as the
percentage of the untreated control and represented as mean ± SD of three independent experiments
in each group. ***p < 0.001, **p < 0.01, *p < 0.05, ns no significantly different compared to the
control.

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Compound 4 almost completely reversed peroxide-induced cell death at the highest concentration tested (5
DOI: 10.1039/C9MD00048H
M); significant protection was seen at concentrations as low as 1 M.
No statistical protection was observed by the compound at the lowest concentrations tested (0.01 and 0.1
M).
It is worth noting that the protective effects of compound 4 are comparable to our previously reported
compound 2c, which showed an increase in peroxide-treated cell viability by 26% at 5 M, although 2c
showed better antioxidant properties in both the DPPH, FRAP and ORAC assays 16.
AChE Inhibitory Activity
All the compounds were evaluated for their AChE inhibition properties according to Ellman’s method with
minor modifications 30. Tacrine was used as reference standard and the results are reported in table 2.
Table 2. Cholinesterase AChE inhibitory activity of vanillin derivatives.
Compound
AChE inhibition (IC50 M)
1
128.3 ± 6.1
2
10.1 ± 1.15
3
4
24.25 ± 1.35
2.13 ± 0.1
Tacrine
0.93 ± 0.09
Results are expressed as a mean ± SD of three independent experiments.
All compounds tested showed weak to good inhibitory activity, with compound 4 the most active, with an
IC50 value ~2 times higher than the standard tacrine (IC50 of 2.13 and 0.93 M, respectively). The results
obtained here for the positive control tacrine agree with the data previously reported (IC50 value of 0.9 µM
for tacrine) 31. However, compound 4 showed lower activity compared to related tacrine derivatives reported
32
by Luo et al. which turned out to be several fold-times more active than tacrine . This could be explained
by the presence of long alkyl chains in the compounds reported by the authors, which show great flexibility
and adaptability within the narrow gorge of AChE compared to compound 4 that shows lower flexibility, on
account of the aromatic ring linked to the tacrine moiety. The imine derivative (compound 1) was found to

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be the weakest inhibitor, with an IC50 of 128.3 M. By contrast, its reduced form (compound 2) shVoieww eArdticlaenOnline
DOI: 10.1039/C9MD00048H
increase in activity of 13-fold (IC50 of 10.1 M). This could be explained by the fact that compound 1, which
bears an imino group, will show less flexibility compared to compound 2 (amine) due to the rigidity of the
double bond, thus showing reduced flexibility in the narrow gorge of the AChE enzyme. Finally, the
presence of an extra methoxy group in the phenolic moiety, as in the case of compound 3, caused a two times
fold reduction in the AChE inhibition when compared with compound 2 (24.25 and 10.1 M, respectively).
Molecular Modelling
To understand better the molecular elements that contribute to the AChE inhibitory activities of these
33
vanillin derivatives, molecular binding studies of compounds 2 and 4 with TcAChE (PDB code: 2CMF)
were performed. Several studies reported the crystal structure of AChE from Torpedo Californica. The active
site lies at the bottom of a 20 Å deep gorge and it is characterized by the presence of three main residues
(SER-200, GLU-327, and HIS-440), which form the esteratic site involved in the hydrolysis of acetylcholine,
and a catalytic anionic site (CAS), characterized by the presence of TRP-84, which plays a fundamental role
34
in binding the acetylcholine molecule through a cation-π interaction with its positive quaternary nitrogen ,
and PHE-330 35. Finally, the peripheral anionic site (PAS) lies at the top of the gorge, approximately 20 Å
above the active site. The PAS is involved in binding acetylcholine at the first step of the catalytic pathway.
It is composed of residues TYR-70, ASP-72, TYR-121, TRP-279, and TYR-334 36.
Ligand optimization was performed using Chemdraw 16.0 (Cambridgesoft, Waltham, MA) and Chem3D
Ultra 16 version (Cambridgesoft, Waltham, MA) using a MM2 force field energy minimization tool. Protein
optimization was performed using Autodock Vina 1.1.2 37. The results were then visualized using PyMOL
(the PyMOL Molecular Graphics System, Version 2.0.7 Schrödinger, LLC).
For the validation of the docking model, the original ligand in the 2CMF structure was redocked. The
binding energy obtained was -14.6 kcal/mol, indicating high affinity of the latter for the AChE molecule. In
addition, the comparison of the positions of the original and redocked ligands resulted in a RMSD score of
0.598 (see Figure 3), confirming the effectiveness of the Autodock Vina software.

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DOI: 10.1039/C9MD00048H
Figure 3. Redocking of the bis-tacrine derivative in Torpedo Californica AChE (2CMF) for the
docking validation; in yellow, the ligand from the original crystal and in red, the ligand generated
by redocking.
The binding scores for compounds 2 and 4 were -12.0 and -13.3 kcal/mol, respectively, matching the in vitro
results obtained in the Ellman assay with the highest AChE inhibitory activity displayed by compound 4.
Figure 4 shows the interactions of compound 2 and 4 within the gorge of the enzyme.

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DOI: 10.1039/C9MD00048H
a
b
c
d
Figure 4. Predicted binding model of compounds 2 (a and b) and 4 (c and d) within the
Acetylcholinesterase gorge. Compounds 2 and 4 are in green sticks whereas the amino acids involved
in the interactions are in grey. Hydrogen bonds are shown as yellow dashed lines. In both models,
the aromatic moieties of the compounds are stacked between the residues PHE-330 and TRP-84 (a
and c). The two carbonyl groups from compound 2 for hydrogen bonds with the residues ASP-72,
TYR-334, and SER-122 whereas the protonated nitrogen in the quinoline moiety of compound 4 forms
hydrogen bond with the oxygen of the carbonyl group in HIS-440. In addition, the two OH-π
interactions occur between the hydroxy groups of TYR-334 and SER-122 and the aromatic linker of
compound 4. Finally, both compounds bind to TYR-121 and TRP-279 in the PAS to ILE-287 (b and
d).
Interestingly, the napthalimido moiety of compound 2 is situated between amino acids PHE-330 and TRP-84
through π-π stacking, whereas the two carbonyl groups are involved in hydrogen bonds with hydroxy groups
of residues amino acids ASP-72, TYR-334 (3.2 and 1.9 Å, respectively) and SER-122 (2.5 Å) (Figure 4a). In
contrast, the molecular modelling studies reported by Gao et al. on their ranitidine derivatives bearing
naphtalimido moieties demonstrated strong interactions of the latter structure with the residue TRP-286 of
17,38
mouse AChE, equivalent to tcAChE, TRP-279 at the entrance of the gorge, in the PAS of the AChE
.
Furthermore, hydrogen bonds are established between the phenolic group of the phenolic moiety and the
carbonyl group of ILE-287 (2.7 Å) and between the nitrogen and the hydroxy group of TYR-121 (2.1 Å).

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Finally, hydrophobic interactions between the phenolic moiety and TRP-279 are apparent (Figure 4b).
DOI: 10.1039/C9MD00048H
Similarly, the tacrine moiety of compound 4 is stacked between PHE-330 and TRP-84. However, the
charged nitrogen of tacrine provides a cation-π interaction with TRP-84, whereas the protonated nitrogen in
the quinoline ring establishes a hydrogen bond (2.0 Å) with the carbonyl group of the main chain of HIS-440
(Figure 4c). Two OH-π interactions are established between the hydroxy group of SER-122 and TYR-334
and the aromatic linker of compound 4. In addition, the phenolic moiety is involved in hydrogen bonding
with two amino acids of the PAS (TYR-121, 2.2 Å and TYR-334, 3.8 Å), along with another hydrogen bond
with ILE-287 (3.9 Å) and hydrophobic interactions with TRP-279 (Figure 4d).
Inhibition of Self-Mediated Aβ(1-42) Aggregation
Aβ(1-42) amyloid aggregation is a hallmark of AD, contributing to the deposition of extracellular amyloid
2
plaques in the cerebral cortex . The ability of the compounds to inhibit the self-mediated aggregation of
amyloid Aβ(1-42) peptide was tested using Thioflavin T (ThT) fluorescence assay 32 with some modifications.
The assay is based on the enhanced fluorescence of ThT upon binding to amyloid fibrils 39. All the
compounds were tested at concentration of 10 M using a final concentration of Aβ(1-42) peptide of 10 M as
well. Curcumin was used a positive control. The results are shown in Figure 5.
ns
100
ns
*
*
50
0
***
***
1
2
3
4
Tacrine
Curcumin
42 Control

A
Compounds (10 M)
Figure 5. Aggregation inhibitory effects of vanillin derivatives towards amyloid Aβ(1-42) peptide. All the
compounds were tested at concentration of 10 M. Values are expressed as the percentage of the control and
represented as mean ± SD of three independent experiments in each group. ***p < 0.001, **p < 0.01,
*p < 0.05, ns no significantly different compared to the control.
All compounds showed good to excellent inhibitory properties toward peptide aggregation, with the
exception of compound 1, which showed no significant inhibition compared with the amyloid control.
Compounds 2 and 3 showed similar activities, inhibiting Aβ(1-42) peptide aggregation by 30.4 and 33.2%,

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respectively. Compound 4 inhibited the Aβ(1-42) peptide aggregation by 77.1% showing similar activity to the
DOI: 10.1039/C9MD00048H
32
positive control curcumin (75.9%) and related tacrine-based derivatives reported by Luo et al.
.
Interestingly, tacrine alone did not show significant inhibition toward Aβ(1-42) peptide aggregation. The Aβ(1-
peptide aggregation inhibitory activities of compounds 2, 3, and 4 could be explained by the work of
42)
Reinke and Gestwicki, which suggested the importance of two aromatic end groups capable of taking part of
hydrogen bonding 40. All of the compounds described in this work bear a hydroxy group in the phenolic
moiety that can be involved in hydrogen bonding. However, compound 1 did not show significant Aβ(1-42)
peptide aggregation inhibition compared to the control, despite bearing two aromatic end groups and the
hydroxy substituent. This may be explained by the length of the linker between the two aromatic end groups,
which is shorter in compound 1 compared to compounds 2 and 3 due to the imino double bond. In fact,
Reinke and Gestwicki reported that the optimal length of the linker lies between 8 and 16 Å. The length of
the linker in compound 1 is 6.9 Å compared to compounds 2, 3, 4, and positive control curcumin (7.3, 7.3,
8.7, and 9.2 Å respectively). Finally, the remarkable activity of compound 4 in this assay could be explained
by the presence of the aromatic linker, which confers low flexibility important for the aggregation inhibitory
activity 40. We would like note that all the biological activities observed with our most active
compounds are within the same range of activities reported by others working in similar field 41–43
.
Conclusions
A series of vanillin derivatives was synthesized and found to have similar antioxidant activity to the
reference antioxidant, Trolox, except for compound 1. The results obtained in antioxidant assays
agree with our previous study regarding the structure activity relationship for this class of
antioxidants 16. Compound 4 displayed the highest antioxidant properties among the vanillin
derivatives, showing similar activity to Trolox in DPPH and FRAP assays. However, the latter
vanillin derivative turned out to be six times more active than Trolox in the ORAC assay, which is
more relevant to the biological systems, involving the use of peroxyl free radicals. To date, there
has been limited evidence of multi-target-directed ligands with antioxidant activity in ORAC assay
44
as strong as this vanillin derivative for example. . Furthermore, compound 4 exhibited protection
of SH-SY5Y cell line against hydrogen peroxide; at concentrations of 5 M, which turned out to be
completely safe in this cell line, the compound reversed peroxide-induced death by 30% after
treatment with hydrogen peroxide (400 M) for 24 hours and showed significant protection at
concentration as low as 1 M. In addition, the compounds showed inhibitory properties toward eel
AChE, with IC50 ranging from 2.13 to 128.3 M with the most active compound (4) 2-fold less
active than the reference agent, tacrine. Computational docking studies suggested that compounds 2
and 4 could bind both the CAS and the PAS of the AChE enzyme through interaction with different

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amino acids residues. Although a reduced activity of AChE inhibitory activity waDsOoI:b10s.e10r3v9e/Cd9MwD0it0h048H
compounds 4, the overall activity is well compensated by its enhanced antioxidant properties (due
to the p-phenylenediamine linker). All the derivatives, except compound 1, showed inhibitory
activities against the self-mediated aggregation of amyloid peptide, with compound 4 being the
most active, reducing Aβ(1-42) peptide aggregation by 77.1% and showing similar activity to
curcumin (75.9%). Taken together, the vanillin derivatives reported in this work could provide a
viable platform in the development of multi targeted AD therapy.
Experimental section
All the reagents were purchased from Sigma-Aldrich and Fisher Scientific unless otherwise stated. MTT was
purchased from ACROS organics. Amyloid Aβ(1-42) was purchased from Calbiochem. All 1H and 13C spectra
were collected using a Magnet Ultrashield Bruker 400 MHz spectrometer. Low-resolution mass spectrometry
was performed using Agilent Technologies 1200 series. High-resolution mass spectrometry was performed at
EPSRC National Mass Spectrometry Service Centre at Swansea University, Swansea, using Thermo
Scientific LTQ Orbitrap XL or Waters Xevo G2-S spectrometers. The progress of each reaction was
monitored by thin layer chromatography (TLC aluminium foil silica gel 60 with fluorescence indicator 254
nm, Sigma-Aldrich) through UV light (254-265 nm). Column chromatography was carried out using silica
gel (Alfa Aesar 70-230 mesh) as the stationary phase and chloroform/methanol as the mobile phase. The
chemical drawing and nomenclature of the compounds were applied according to ChemBioDraw Ultra
version 16.0 (CambridgeSoft). Neuroblastoma SH-SY5Y cells were from the European Collection of
Authenticated Cell Cultures (ECACC). Cells were maintained at 37 °C (5% CO2) in DMEM medium
(containing GlutaMAX-1 with 25 mM HEPES), supplemented with 10% (v/v) Foetal Bovine Serum (FBS)
and 1% Penicillin/Streptomycin (10 mg/mL).
Synthesis of 2-(3-aminopropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione – I-1
1,8-Naphthalic anhydride (2 g, 10 mmol) was dissolved in ethanol (100 mL); 1,3-Diaminopropane (1.48 g,
20 mmol) was carefully added to the solution and stirred under reflux for 1 hour. The resulting precipitate
was filtered and the filtrate concentrated using a rotary evaporator. The solid obtained was washed several
times with diethyl ether to give a pale-yellow solid as the product (yield: 56%).
1HNMR: (CDCl3 solvent peak δ:7.27), 8.63-7.76 (m, Ar-H, 6H), 4.32-4.29 (t, N-CH2-CH2, J = 6.8 Hz, 2H),
2.80-2.77 (t, CH2-NH2, J = 5.6 Hz, 2H), 1.95-1.89 (m, CH2-CH2-CH2, J = 13.6 Hz, 2 H), 1.52 (b, NH2, 2H).
13CNMR: (CDCl3 solvent peak δ: 77.4-76.8) 164.4, 134.0-122.65, 39.48, 37.77, 32.19. LRMS calcd for
C15H15N2O2 [M+H]+ 255.1, m/z found 255.0.

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DOI: 10.1039/C9MD00048H
Synthesis of 2-(3-((4-hydroxy-3-methoxybenzylidene)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-
dione – 1
Vanillin (0.5 g, 3.3 mmol) was dissolved in methanol followed by the addition of 2-(3-aminopropyl)-1H-
benzo[de]isoquinoline-1,3(2H)-dione (0.83 g, 3.3 mmol). The reaction was left to stir overnight at RT. The
solution obtained was concentrated through rotary evaporation and the solid obtained was dissolved in DCM
(25 mL) and extracted 3 times with saturated NaHCO3 (20 mL). The organic layer was collected and dried
with anhydrous sodium sulfate and concentrated through rotary evaporator to afford an orange solid (yield:
73%).
1HNMR: (CDCl3 solvent peak δ:7.30), 8.61-6.85 (m, Ar-H, 9H), 8.22 (s, Ar-CH=N, 1H), 4.39-4.35 (t, N-
CH2-CH2, J = 6.8 Hz, 2H), 3.81 (s, -OCH3, 3H), 3.79-3.77 (t, CH2-N=CH, J = 6.8 Hz, 2H), 3.77-3.75 (t, N-
CH2-CH2-, J = 6.8 Hz, 2H), 2.26-2.23 (m, CH2-CH2-CH2, J = 14.0 Hz, 2H). 13CNMR: (CDCl3 solvent peak δ:
77.4-76.7) 164.3, 161-107.8, 59.47, 55.87, 38.95, 29.17. HRMS calcd for C23H21N2O4 [M+H]+ 389.1497, m/z
found 389.1497.
Synthesis of 2-(3-((4-hydroxy-3-methoxybenzyl)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione - 2
0.4 g of 2-(3-((4-hydroxy-3-methoxybenzylidene)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1
mmol) were dissolved in methanol, then 0.057 g of NaBH4 (1.5 mmol) added. The reaction was stirred for 2
hour and concentrated through rotary evaporation. The solid obtained was dissolved in DCM and extracted 3
times with NaHCO3 (20 mL). The organic layer was collected and dried with anhydrous sodium sulfate and
concentrated through rotary evaporation to afford a pale-yellow solid (yield: 86%). 1HNMR: (CDCl3 solvent
peak δ:7.31), 8.65-6.82 (m, Ar-H, 9H), 4.34-4.31 (t, N-CH2-CH2, J = 7.2 Hz, 2H), 3.91 (s, -OCH3, 3H), 3.76
(s, Ar-CH2-N, 2H), 2.78-2.74 (t, CH2-CH2-N, J = 6.8 Hz, 2H), 2.05-2.01 (t, CH2-CH2-CH2, J = 6.8 Hz, 2H).
13CNMR: (CDCl3 solvent peak δ: 77.4-76.7) 164.3, 146.61-110.9, 55.9, 53.8, 46.4, 38.3, 28.3. HRMS calcd
for C23H23N2O4 [M+H]+ 391.1652, m/z found 391.1651.
Synthesis of 2-(3-((4-hydroxy-3,5-dimethoxybenzyl)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione
- 3
Syringaldehyde (0.29g, 1.57 mmol) was mixed with methanol (8 mL) followed by the addition of 2-(3-
aminopropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (0.40g, 1.57 mmol). The solution was refluxed for 2
hours and left stirring overnight at RT to yield a red solution. The solvent was evaporated under reduced
pressure to yield a red solid which was suspended in propan-2-ol followed by the addition of NaBH4 (3.0
mmol). The solution was refluxed for 48 hours. At the completion of the reaction, the solvent was removed
under vacuo to afford a solid. The latter was collected by filtration, washed thoroughly with water and
methanol to yield the final product (38%) . 1HNMR: (CDCl3 solvent peak δ:7.20), 8.53-6.50 (m, Ar-H, 8H),

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4.23-4.20 (t, N-CH2-CH2, J = 6.8 Hz, 2H), 3.80 (s, -OCH3, 6H), 3.64 (s, Ar-CH2-N, 2H), 2.68-2.64 (t, CH2-
DOI: 10.1039/C9MD00048H
CH2-N, J = 6.8 Hz, 2H), 1.95-1.91 (t, CH2-CH2-CH2, J = 6.8 Hz, 2H). 13CNMR: (CDCl3 solvent peak δ: 77.4-
76.7) 164.3, 147.1-104.9, 56.2, 54.2, 46.5, 38.3, 28.3. HRMS calcd for C24H25N2O5 [M+H]+ 421.1758, m/z
found 421.1756.
Synthesis of 9-chloro-1,2,3,4-tetrahydroacridine - I-2
Intermediate I-2 was prepared following the procedure reported in literature 21. To a mixture of anthranilic
acid (1.85 g, 1.3 mmol) and cyclohexanone (2.6 mL, 2.6 mmol), 15 mL of POCl3 (0.16 mol) was added in an
ice bath. The mixture was heated under reflux and stirred for 24 hours, then cooled and concentrated under
reduced pressure. The residue was diluted with ethyl acetate (50 mL), neutralized with saturated Na2CO3 (30
mL) and washed 3 times with brine (30 mL). The organic layer was dried and the product was recrystallized
from acetone (yield: 67%).
1HNMR: (CDCl3 solvent peak δ:7.20), 8.11-7.45 (m, Ar-H, 4H), 3.07-3.04 (t, Ar-CH2-CH2, J = 5.2 Hz, 2H),
2.97- 2.94 (t, Ar-CH2-CH2, J = 6.4 Hz, 2H), 1.92- 1.84 (m, CH2-CH2, 4H). 13CNMR: (CDCl3 solvent peak δ:
77.8-76.7) 159.6- 123.7, 34.3, 27.6, 22.72, 22.68. LRMS calcd for C13H13ClN [M+H]+ 218.7, m/z found
218.1.
Synthesis of N1-(1,2,3,4-tetrahydroacridin-9-yl)benzene-1,4-diamin - I-3
9-chloro-1,2,3,4-tetrahydroacridine (107 mg, 0.5 mmol) was dissolved in 1-pentanol (5 mL) followed by the
addition of KI (80 mg, 0.5 mmol) and heated under reflux. Then, p-phenylenediamine (135 mg, 1.25 mmol)
was added and the mixture stirred for 24 hours. The solution was concentrated under pressure and the solid
obtained dissolved in 50 mL of DCM and extracted 3 times with Na2CO3 (50 mL). The organic layer was
dried through rotary evaporation and the crude was purified through column chromatography (DCM/MeOH
99:1) to afford a brown solid (yield 39%).
1HNMR: (CDCl3 solvent peak δ:7.30), 8.04-6.61 (m, Ar-H, 8H), 5.95 (s, Ar-NH-Ar, 1H), 3.59 (s, Ar-NH2,
2H), 3.20-3.17 (t, Ar-CH2-CH2, J = 6 Hz, 2H), 2.72-2.69 (t, Ar-CH2-CH2, J = 6.4 Hz, 2H), 1.98- 1.95 (m, -
CH2-CH2-, J = 5.6 Hz, 2H), 1.95- 1.89 (m, -CH2-CH2-, J = 5.6 Hz, 2H). 13CNMR: (CDCl3 solvent peak δ:
77.4-76.7) 141.7- 116.1, 33.8, 25.2, 24.9, 22.8, 22.7. LRMS calcd for C19H20N3 [M+H]+ 290.2, m/z found
290.2.
Synthesis of 2-methoxy-4-(((4-((1,2,3,4-tetrahydroacridin-9-yl)amino)phenyl)amino)methyl)phenol - 4
I-3 (50 mg, 0.17 mmol) was dissolved in 2-propanol (10 mL) followed by the addition of vanillin (20 mg,
0.13 mmol). The reaction was stirred under reflux and monitored through TLC; when the vanillin spot had
disappeared, the solution was cooled down and NaBH4 (25mg, 0.7 mmol) was added. The solvent was dried

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and the solid obtained was purified through column chromatography (DCM/MeOH 99:1) to afford an orange
DOI: 10.1039/C9MD00048H
solid (yield 46%).
1HNMR: (CDCl3 solvent peak δ:7.19), 8.05-6.50 (m, Ar-H, 11H), 4.14 (s, Ar-CH2-N, 2H), 3.81 (s, -OCH3,
3H), 3.13-3.11 (t, Ar-CH2-CH2, J = 5.6 Hz, 2H), 2.95-2.93 (t, Ar-CH2-CH2, J = 7.2 Hz, 2H), 2.60- 2.56 (m, -
13
CH2-CH2-, J = 6.4 Hz, 2H), 1.86- 1.80 (m, -CH2-CH2-, J = 12.4 Hz, 2H). CNMR: (CDCl3 solvent peak δ:
77.4-76.7) 146.8- 110.3, 56.0, 48.8, 45.82, 25.0, 22.7, 22.3. HRMS calcd for C27H28N3O2 [M+H]+ 426.2176,
m/z found 426.2172.
DPPH Assay
The ability of the compounds to scavenge DPPH free radical was determined following the procedure
reported by Payet, Sing and Smadja with some modifications in a 96-well set-up 22. A dilution series of
antioxidants ranging from 1.5 to 750 M was made in Eppendorf® tubes, then 50 L were transferred to the
corresponding well, while 50 L of vehicle (methanol) were used as negative control wells. 100 L of DPPH
solution (0.1 mM) were added to each well followed by incubation in the dark for 30 min. Absorbance was
then measured at 517 nm using a Bio-Rad iMark microplate reader.
FRAP Assay
The reducing properties of the compounds was tested through the FRAP assay following the method
described by Firuzi et al. with minor modifications in a 96-well plate 23. FRAP reagent was prepared by
mixing 2.5 mL of 10 mM TPTZ (in 40 mM HCl) with 2.5 mL of 20 mM FeCl3 (in deionized water) and the
volume brought to 30 mL with 300 mM sodium acetate buffer (pH 3.6). A dilution series of antioxidants and
Trolox ranging from 500 to 5000 M was made in Eppendorf® tubes, then 10 L of each was pipetted in the
corresponding well in a 96 well-plate along with 190 L of FRAP reagent. The plate was stored in the dark
for 30 minutes before the absorbance was measured at 593 nm using a Bio-Rad iMark microplate reader.
ORAC Assay
The ability of the compounds to prevent oxidative degradation of fluorescein was measured using ORAC
assay following the previously protocols reported with minor modifications on a black-walled 96-well plate
16,22,45
.
A dilution series of antioxidants and Trolox was made in phosphate buffer (75 mM, pH 7.4) in Eppendorf®
tubes and 25 L of each was transferred into the corresponding well whereas 25 L of phosphate buffer was
added in the control wells.
150 L of 25 nM sodium fluorescein solution was added in each well and the plate was incubated at 37°C for
30 minutes.

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Finally, 25 L of 0.15 M AAPH solution was added in the positive control and in the DaOnIt:i1o0x.1i0d3a9/nCt9MwDe0l0l0s48H
whereas 25 L of phosphate buffer was added in the fluorescein control.
The fluorescence was measured every 2 minutes over a period of 2 hours (485/20 nm excitation, 525/20 nm
emission) using a BioTek Synergy HT microplate reader.
MTT Assay
Cell survival after treatment with H2O2 (400 M) was assessed using the MTT cell assay, using a procedure
similar to previous publications 16,46. SH-SY5Y cells were seeded (7000 cells/well) in a 96-well plate. After
adhesion (24 h), cells were treated with different concentrations of compound 4, ranging from 0.01 to 5 M,
and incubated for 24 h.
Subsequently H2O2 solution was added to the positive control and the test drug wells, followed by incubation
for another 24 h. After incubation, all the solutions were removed from all the wells and 100 L of MTT
solution (1 mg/mL) was added in each well. The plate was wrapped in aluminum foil and incubated for 4 h at
37 °C.
The solutions in each well were removed by pipette and 100 L of DMSO added to each well to dissolve the
formazan crystals. The plates were gently shaken for 20 minutes and the absorbance measured at 490 nm
with a Bio-Rad iMark microplate reader. Cell viability was expressed as a percentage of the absorbance from
control cells.
AChE Inhibition Assay
The inhibitory properties of compounds toward AChE was determined through Ellman method with some
modifications 30. A 22 U/mL stock solution of AChE from Electrophorus electricus was prepared in 20 mM
tris HCl pH 7.5 and diluted 1/100 before use. A 3 mM (5,5'-dithiobis-(2-nitrobenzoic acid)) solution was
prepared by dissolving 0.1189 g of DTNB in 0.05 M phosphate/ 0.09 M hepes buffer (pH 7.5). A 15 mM
acetylthiocholine iodide solution was prepared by dissolving 0.1084 g of compound in deionized water. A
dilution series of compounds (in methanol) was made in Eppendorf® tubes and 25 L of the latter was
pipetted in the corresponding well in a 96-well plate along with 125 L of DTNB solution and 25 L of
diluted AChE solution. 25 L of methanol were added in the control wells. The plate was incubated for 10
minutes at 37°C then 25 L of acetylthiocholine iodide solution were added to each well and incubated for
another 10 minutes. The absorbance was measured at 415 nm using a Bio-Rad iMark microplate reader.
Molecular Modelling
For the docking procedure, the pdb structure of 2CMF (Torpedo californica AChE in complex with a bis-
tacrine linked by a five-carbon spacer) was taken from Protein Data Base (http://www.rcsb.org). The choice
of the TcAChE instead of an electrophoresus electricus model of AChE (which was employed for the

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Ellman assay) was based on the good resolution of the structure (2.5 Å), the similarity of the ligand in the
DOI: 10.1039/C9MD00048H
structure with compound 4 and the unavailability of high resolution eeAChE structures 47. It is worth noting
48
the high degree of identity between TcAChE and EeAChE . Water molecules and the original ligand were
removed from the protein structure and polar hydrogens and charges were added. The protein was saved as
pdbqt file. The 3D structures of compounds 2 and 4 were built using ChemDraw 16.0 (Cambridgesoft,
Waltham, MA) and Chem3D Ultra 16 version (Cambridgesoft, Waltham, MA) and its MM2 force field
energy minimization tool. The structures were saved as pdb file. The ligands and the macromolecule were
then loaded into AutoDock Vina 1.1.2 (Molecular graphics laboratory, The Scripps research group, La Jolla,
CA) and prepared for docking. Docking was performed on a grid box (40x40x40 Å) centered on the active
site of AChE (residue TRP-84). The lowest energy conformation of each ligand enzyme complex was
selected for analyzing the interactions between AChE and the inhibitor and the results were visualized using
PyMOL (the PyMOL Molecular Graphics System, Version 2.0.7 Schrödinger, LLC).
ThT Assay
The ability of the compounds to inhibit self-mediated amyloid Aβ(1-42) aggregation was determined through
the fluorometric ThT assay following the method described by Luo et al. with some modifications 32. A 500
M stock solution of amyloid Aβ(1-42) peptide was prepared by dissolving 0.25 mg of the peptide in 110 L
of DMSO. The solution was aliquoted in Eppendorf tubes and stored at -20°C.
Briefly, 2 L of peptide solution was pipetted in 96 L of 10 mM phosphate/ 10 mM NaCl buffer (pH 8)
along with 2 L of inhibitor solution (dissolved in DMSO) or 2 L of DMSO (for the control). The final
concentrations of both the amyloid peptide and inhibitors were 10 M. The solutions were incubated for 24
hours at 37°C. Then, 300 L of 50 mM glycine/ NaOH buffer (pH 8.5) containing 5 M of ThT was added
to all the samples.
Each solution was transferred into a cuvette and the fluorescence was measured using a Perkin Elmer LS55
luminescence spectrometer (excitation 446 nm, emission 490 nm).
Statistical Analysis
Data are shown as mean ± standard deviation (SD) and all the experiments were conducted on at least 3
separate occasions. Statistical analysis was performed using GRAPH PAD prism (6.00 for Windows,
GraphPad Software, La Jolla California USA, www.graphpad.com) using ONE WAY ANOVA and
Bonferroni's multiple comparison test. Significant differences are labelled accordingly (ns - not significant, p
< 0.05*, p < 0.01**, p < 0.001 ***).
Conflicts of interest

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DOI: 10.1039/C9MD00048H
There are no conflicts of interest to declare
Acknowledgments
The authors would like to thank the Robert Gordon University for financial support (studentship to MS), the
EPSRC UK National Mass Spectrometry Service Centre at Swansea University, Swansea, UK for the HR
mass spectral analysis and Professor Andrey Shiryaev (Samara Technical University, Russia) for his advice
with the molecular modelling analysis.

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DOI: 10.1039/C9MD00048H
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