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CNS & Neurological Disorders - Drug Targets, 2017, 16, 000-000 1

RESEARCH ARTICLE

Securinine Derivatives as Potential Anti-amyloid Therapeutic Approach

Margarita E. Neganova1, Sergey G. Klochkov1, Ludmila N. Petrova1, Elena F. Shevtsova1,

Svetlana V. Afanasieva1, Ekaterina S. Chudinova1, Vladimir P. Fisenko2, Sergey O. Bachurin1,
George E. Barreto3,4 and Gjumrakch Aliev1,5,6,*

1
Institute of Physiologically Active Compounds of Russian Academy of Science, Chernogolovka, Russia; 2Department of
Pharmacology, Therapeutic Faculty, I.M. Setchenov Moscow Medical Academy, ul. Bol'shaya Pirogovskaya 2/6, Mos-
cow, 119881 Russia; 3Departamento de Nutrición y Bioquímica, Facultad de Ciencias, Pontificia Universidad Javeri-
ana, Bogotá D.C., Colombia; 4Instituto de Ciencias Biomédicas, Universidad Autónoma de Chile, Santiago, Chile;
5
GALLY International Biomedical Research Consulting LLC, San Antonio, Texas, 78229, USA; 6School of Health Sci-
ence and Healthcare Administration, University of Atlanta, Johns Creek, Georgia, 30097, USA

ARTICLE HISTORY
Received: August 10, 2016
Revised: October 13, 2016
Accepted: October 25, 2016
DOI:
10.2174/18715273156661611070905
25
Abstract: Background: Oxidative stress and amyloid deposition are tightly interconnected pathological
features of Alzheimer disease. In this respect, both amyloid production and aggregation may be stimu-
lated by oxidative stress and also the increase of pathogenic β-amyloid and its aggregated form lead to
oxidative stress progression. Therefore, the search for potential drugs with both antioxidant and anti-
aggregation properties are of great interest.
Methods: In this study, we described the stereospecific synthesis of alkaloid securinine aminoderivatives.
Results: We showed that the newly synthesized compounds possess antioxidant and metal-chelating
properties. Indeed, we report that one compound has inhibitory effects towards β-amyloid aggregation.
Conclusion: Based on these results, aminoderivatives of securinine scaffold are promising compounds
for development of new drugs for the treatment of neurodegenerative diseases.

Keywords: Alkaloids, antioxidant, lipid peroxidation, neurodegenerative diseases, neuroprotection, securinine, β-amyloid.

INTRODUCTION pathway of such compounds, and it also formed additional

Wild plant constitutes a wide source of potential drugs
that has been intensively overlooked during the last decades
[1-6]. The well-known alkaloid securinine (Fig. 1), which
was isolated from Securinega suffruticosa (Pall.) Rehd.
(Euphorbiaceae), has actions on the human central nervous
system and is used in traditional medicine as a spinal cord
stimulant [7]. Also, it was shown that securinine could be
used for the treatment of some neurological diseases such as
amyotrophic lateral sclerosis [8], and prevents amyloid-beta
(Aβ25-35)-induced spatial cognitive dysfunction [9]. In this
context, our group has previously demonstrated the neuro-
protective effects of securinine derivatives [10-12].
The structure of securinine includes a highly reactive
endocyclic diene system that is activated by an ester car-
bonyl. The Michael reaction can act as one modification
*Address correspondence to this author at the “GALLY” International Bio-
medical Research Consulting LLC, 7733 Louis Pasteur Drive, #330, San An-
tonio, TX 78229, USA; Tel: +1(440) 263-7461; E-mails: aliev03@gmail.com,
cobalt55@gallyinternational.com
asymmetric center in the 9-position. To receive only one
isomer, we used Lewis catalyst Yb(OTf)3. In the present
study we report the synthesis and biological evaluation of an
original series of securinine aminoderivatives.
MATERIALS AND METHODS
Synthesis of Securinine Derivatives
The aminoderivatives of securinine (Fig. 1) were synthe-
sized using the reported procedure [10]; the reaction of the
securinine with a series of amines in methanol in the pres-
ence of the Lewis acid catalyst ytterbium triflate, proceeded
stereospecifically to generate compounds 2-8 (Fig. 1).
Compound 8 - (6S,11aR,11bS)-5-[4-(4-fluorophenyl)
piperazin-1-yl]-5,6,9,10,11,11a-hexahydro-8H-6,11b-metha-
nofuro [2,3-c]pyrido[1,2-a]azepin-2(4H)-one - was synthesized
by reacting 1 (0.217g, 1 mmol) and 1-(4-fluorophenyl)
piperazine (0.193 g, 1.07 mmol) in MeOH (5 mL) in the
presence of ytterbium(III) trifluoromethanesulfonate (tri-
flate) hydrate (0.062 g, 0.1 mmol) at room temperature for

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2 CNS & Neurological Disorders - Drug Targets, 2017, Vol. 16, No. 1
Fig. (1). Structures of compounds 1-8 and their synthesis.
1 day. The course of the reaction was monitored by TLC on
Silufol UV-254 plates. When the reaction was finished, the
mixture was evaporated at reduced pressure. The dry residue
was dissolved in EtOAc, passed over a column of neutral
Al2O3 (5 g) in order to remove the catalyst, and evaporated at
reduced pressure. The residue was dissolved in MeCN and
purified by semi-preparative HPLC [Turbo LC 200 chro-
matograph, PerkinElmer, USA; UV detector at 254 nm; col-
umn 10 x 250 mm with Kromasil C18, 5 µm; gradient elu-
tion, eluent A, trifluoroacetic acid (TFA) (0.1%) in distilled
H2O (pH 2.0), eluent B, MeCN; flow rate 4 mL/min to
afford 8 (0.156 g, 40%), mp 196-197°C. Found: m/z
[M+H] +=398.2244. C23H28FN3O2. Calcd: [M+H]+=398.2238.
PMR spectrum (500 MHz, CDCl3, δ, ppm, J/Hz) (lower
indices a and b denote nonequivalent protons): 1.32-1.42 (m,
2 H, H-6α, 4α); 1.42-1.50 (m, 1 H, H-3α); 1.47-1.52 (m, 1
H, H-5β); 1.60-1.65 (m, 1 H, H-3β); 1.88-1.93 (m, 1 H, H-
4β); 2.01 (d, J=10.27 Hz, 1 H, H-15α); 2.55 (dd, J=10.30,
5.90 Hz, 1 H, H-15β); 2.57-2.60 (m, H-8); 2.59-2.63 (m, 2
H, H-3’eq, H-5’eq); 2.64-2.70 (m, 2 H, H-2’ax, 6’ax); 2.81
(ddd, J=15.80, 6.30, 2.10 Hz, 1 H-10α); 2.89-2.93 (m, 1 H,
H-6α); 2.94-2.99 (m, 3 H, 6β, 2, 10β); 3.07 (t, J=4.84 Hz, 4
H, H-2’, 6’); 3.42 (br. t, J=4.80, 4.80 Hz, 1 H, H-9); 5.62 (d,
J=2.35 Hz, 1 H, H-12); 6.86 (dd, J=9.24, 4.55 Hz, 2 H; H-6”,
2”); 6.96 (t, J=9.20 Hz, 2 H, H-5”, 4”). 13C NMR spectrum
(126 MHz, CDCl3, δ, ppm): 50.26 (C-6’); 51.04 (C-3’);
51.04 (C-5’); 60.10 (C-2); 61.60 (C-9); 66.14 (C-8); 91.16
(C-1); 110.52 (C-12); 115.47 (C-3”); 115.65 (C-5”); 117.75
(C-2”); 117.81 (C-6”); 147.76 (C-1”); 157.26 (d, J=230.70
Hz, C-4”); 173.23 (C-11); 174.57 (C-13).
Biological Evaluation
Experiments were carried out on outbred albino male rats
weighing 200-300 g and aged 3-4 months. The animals had
free access to food and water. All procedure is in compliance
with the Guidelines for Animal Care and Experiments at
IPAC RAS.
Following all experimental procedures, animals were
narcotized with carbon dioxide and decapitated using a
guillotine. The brain was homogenized in 120 mM KCl/20
mM HEPES on ice. After centrifuged at 4000 rpm
supernatant was used in experiments on the same day. At
least three different brain homogenates and three replicates
of each point were used. The protein content in brain
homogenate was measured using a microbiuret method [13].
Neganova et al.
Lipid peroxidation intensity in brain homogenate was
assayed using a modified TBA test [14] and data were nor-
malized between control and blank probe. Fe(II) chelating
activity was evaluated using a modified method described
elsewhere [15] and also were normalized.
Antiamyloidogenic activity was assayed as previously
described [16]. Shortly, Aβ1-42 (Bachem, Switzerland) was
dissolved in dimethyl sulfoxide (DMSO) to a concentration
of 100 µM and sonicated for 1 min to prevent preaggrega-
tion. Immediately before the experiment Aβ solutions were
prepared by dilution with 10 mM phosphate-buffered saline
with 0.5 mM EDTA (pH 7.4) to a final concentration of 10
µM. All compounds were dissolved in DMSO. The final
experimental samples containing 0,4% (v/v) DMSO were
incubated at 37 °C up to 24 hours, and the fibrillogenesis
was monitored using ThT fluorescence analysis on Cary
spectrofluorimeter (Varian) at 0, 5 and 24 hours.
Statistics
Data are presented as the mean ± SEM from 3 independ-
ent experiments. Statistical comparisons by one-way analysis
of variance (ANOVA) followed by Dunnett’s multiple com-
parison with control test were performed using GraphPad
Prism software, version 5.01 (GraphPad Software, San
Diego, CA, USA). In all cases, significance was accepted at
p ≤ 0.05.
RESULTS AND DISCUSSION
Test Compounds
Compounds 2-7 were prepared from securinine scaffold
(Fig. 1) using the stereospecific catalyst ytterbium triflate as
described earlier [10, 17, 18]. Compound 8 was produced by
reacting of securinine scaffold and 1-(4-fluorophenyl)
piperazine in the presence of ytterbium(III) trifluoro-
methanesulfonate (triflate) hydrate (Fig. 2). Structure of this
compound, and asymmetric configuration of 9-th carbon
atom were fixed with NMR-spectroscopy and two-
dimentional analysis COSY, HSQC, NOESY. The presence
of an α,β-unsaturated lactone ring was confirmed by the cor-
responding resonances in 13C NMR spectra (ppm) 174.57 for
the carbonyl C atom, 110.52 for C-12 atom. The PMR spec-
trum showed doublet for the olefinic proton on C-12 (δH =
5.62, J = 2.35 Hz). The NMR spectra also contained reso-
Securinine Derivatives Therapeutic Approach CNS & Neurological Disorders - Drug Targets, 2017, Vol. 16, No. 1 3

[4-(4-fluorophenyl)piperazin-1-yl]-5,6,9,10,11,11a-hexahydro-
8H-6,11b-methanofuro[2,3-c]pyrido[1,2-a]azepin-2(4H)-one.

Fig. (2). Structurally significant NOE correlations for compounds 8.
nances for protons of a 1-(4-fluorophenyl)piperazine (for
example, the characteristic doublet in 13C NMR spectra
(ppm) 157.26, J=230.70 Hz for C-4”). In the NOESY spec-
trum of compound 8, there are clear NOE correlations be-
tween the H-9 and H-10α, the H-8 and H-10β protons. It was
established the structure of compound 8-(6S,11aR,11bS)-5-
Antioxidant Activity
Oxidative stress is one of the main pathogenic mecha-
nisms for the development of many human diseases, includ-
ing age-related neurodegenerative diseases [2, 19]. At the
first stage of this study we investigated the effect of test
compounds on the lipid peroxidation of the rat brain
homogenates initiated by Fe(III). The alkaloid securinine
(Fig. 1) showed no activity up to 0.1 mM. However, its ad-
ducts with pharmacophoric amines showed significantly
higher inhibition activity of the lipid peroxidation induced by
Fe(III) (Fig. 3), except compounds 5 and 6 which are non-
active and not present on diagram. The antioxidant activity
increases steadily with increasing concentration of these
compounds. The IC50 values for compounds 2-4 and 8 were
greater than 50µM and only for compound 7 the IC50 was
7.7 ± 0.1µM.
In order to elucidate the mechanism of antioxidant action
of the test compounds, we have assessed their metal ion
chelating properties (Fig. 3). Compounds 2 and 3 exhibited

Fig. (3). Antioxidant and Fe(II)-chelating activities of the test compounds (0.1 mM). For all compounds LP inhibition and Fe2+-chelating
activity were significant (* p<0.05) according to Dunnett's test (ANOVA).

Fig. (4). A - Effect of test compounds (0.1 mM) on aggregation β-amyloid 1-42 (Ab). B - The concentrations dependence of Ab anti-
aggregation activity of compound 2. Levels of TfT fluorescence for each points were normalized to control probe at 24 hours and significant
differences (p<0.05) were observed for compounds 1, 2 (A) and for 10 and 20µM of compound 2 (B) at 5 hours and for all compounds (A)
and all concentrations of compound 2 (B) at 24 hours according to Dunnett's test (ANOVA).
4 CNS & Neurological Disorders - Drug Targets, 2017, Vol. 16, No. 1
iron-chelating properties, which may be the main mechanis-
tic pathway for inhibiting Fe (III)-induced lipid peroxidation.
In this regard, the chelation of Fe(III), presumably through
the secondary amino group of these compounds, attenuated
the oxidative stress at the initial stage of Fenton reaction.
Influence on β-amyloid Aggregation
Accumulating evidence has shown that oxidative stress-
induced damage may play an important role in the initiation
and progression of neurodegenerative diseases [2, 19-21],
especially AD pathogenesis [22, 23]. Due to an imbalance
between the production and quenching of free radicals, oxy-
gen reactive species are formed and aggregates of β-amyloid
as well [24]. Therefore, antioxidant substances can exhibit
not only neuroprotective properties, but also inhibit the for-
mation and extension of β-amyloid fibrils (fAβ), as well as to
destabilize preformed fAβ in vitro [25].
In this work it has been shown that the effective antioxi-
dant compound 2 concentrations-dependently inhibits the
aggregation of β-amyloid 1-42 during the 24-hours incuba-
tion at 37°C (Fig. 4A-B). Its precursor, compound 1 (secur-
inine), which has no antioxidant properties, was not able to
inhibit β-amyloid aggregation; however, its weak antioxidant
tryptamine reveals significant lower activity as inhibitor of
β-amyloid aggregation.
CONCLUSION
Series of stereospecific analogs of the naturally occurring
alkaloid securinine (1) were synthesized as potential neuro-
protective agents. It was shown that these compounds pos-
sess antioxidant and metal-chelating properties, and for
compound (2) allomargaritarine the properties of inhibitor of
β-amyloid aggregation were also revealed. Based on these
results, aminoderivatives of securinine scaffold (1) are prom-
ising compounds for development of new drugs for the
treatment of neurodegenerative diseases at the early stages of
the disease.
LIST OF ABBREVIATIONS
Ab = β-amyloid
AD = Alzheimer Disease
LP = Lipid Peroxidation
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The equipment of Center for Collective Use IPAC RAS,
Chernogolovka, Russia was used for this study. This study
was partially supported by the "GALLY" International
Biomedical Research Consulting LLC, San Antonio, TX,
USA.
Neganova et al.
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PMID: 27823572