G. Tommonaro et al. / European Journal of Medicinal Chemistry 122 (2016) 326e338
335
fitted with a gradient along the Z axis and on Bruker instruments at
00 MHz. Samples for NMR analysis were dissolved in the appro-
priate solvent (CDCl ); a downfield shift of the signal of the solvent
was used as the internal standard.
AChE activity inhibition. Logistic curves for IC50 determinations
were performed according to the program SigmaPlot v.11 (Systat
Software INC., 2012). Data are expressed as means ± s.e.m. of at
least three different experiments in triplicates.
4
3
4
.1.1.1. 2-[2,5-Dihydroxy-3-(3-methylbut-2-enyl)phenylthio]benzoic
4.3. Kinetic analysis of the EeAChE inhibition by TPHs and TAVA
1
acid (TPH1). H NMR
d: 9.95 (1H), 7.93 (1H), 7.41 (1H), 7.39 (1H),
7
.21 (1H) 6.95 (1H), 6.85 (2H), 5.71 (1H), 4.42 (1H), 3.95 (1H), 3.48
A study on the enzymatic mechanism of action of TPHs and
TAVA was carried out. To obtain estimates of the Km’s and Vmax’s,
of EeAChE, reciprocal plots of 1/V versus 1/[S] were constructed at
different concentrations of the substrate ATCh (0.05e1 mM) and
13
(
2H), 1.69 (3H), 1.63 (3H); C NMR
d: 169.4, 150.8, 137.2, 134.6,
148.3, 131.4, 131.0, 132.1, 130.9, 129.3, 127.2, 123.6, 116.9, 116.1, 113.5,
2
8.9, 25.8, 17.5.
different drug concentrations (0.01e10 mM), using the enzymatic
4
.1.1.2. (E)-2-((3-(3,7-Dimethylocta-2,6-dien-1-yl)-2,5-dihydroxy-
activity assay described earlier [31]. Km’s were then plotted against
indicated compound concentrations, for Ki’s calculations.
1
phenyl)thio)benzoic acid (TPH2). H NMR
.41 (1H), 7.39 (1H), 7.21 (1H), 6.62 (1H), 6.55 (1H), 6.52 (1H), 5.24
1H), 5.0 (1H), 3.24 (2H), 2.03 (2H), 2.06 (2H), 1.70 (3H), 1.63 (3H),
.54 (3H); 13C NMR
: 169.0, 147.7, 148.6, 148.2, 137.2, 134.6, 131.4,
31.0, 130.9, 128.1, 127.2, 123.6, 122.0, 120.8, 116.6, 116.2, 114.3, 40,
d: 9.93 (1H), 7.91 (1H),
7
(
1
1
3
4.4. Neuroprotection of TPHs and TAVA
d
4.4.1. Cell culture and treatment
0, 26.7, 26.6, 18.1, 15.5.
Cells from the human neuroblastoma cell line SHSY5Y were
cultured in Dulbecco’s: Ham’s F12, 1:1 [vol/vol] containing 3.15 mg/
mL glucose, 2.5 mM Glutamax, and 0.5 mM sodium pyruvate
DMEM/F-12, GlutaMAX™; GIBCO, Life Technologies, Madrid
(Spain), 1% Antibiotic-Antimycotic (Gibco; Life Technologies,
4
2
.1.1.3. 2-((2,5-Dihydroxy-3-((2E,6E)-3,7,11-trimethyldo-deca-
1
,6,10-trien-1-yl)phenyl)thio)benzoic acid (TPH3). H NMR
d: 9.97
(
1H), 7.93 (1H), 7.40 (1H), 7.35 (1H), 7.21 (1H), 6.61 (3H), 5.32 (1H),
13
5
.13 (2H), 3.32 (2H), 1.77 (3H), 1.70 (3H), 1.61 (6H); C NMR
d:
Madrid, Spain) at 100 units of penicillin, 100
mg of streptomycin,
1
1
69.2,149.4,148.2,138.7,137.2,135.6,134.6,131.5,131.4,131.0,130.9,
28.5, 127.2, 124.5, 123.8, 121.4, 116.7, 113.8, 39.8, 29.8, 26.8, 26.5,
and 0.25 g of amphotericin B final concentrations, and supple-
mented with 10% Foetal Calf Serum (FCS) [Gibco; Life Technologies,
m
2
5.8, 17.8, 16.4, 16.2.
Madrid (Spain)]. Cultures were seeded into flasks containing sup-
plemented medium and maintained at 37 C in a humidified at-
ꢁ
4
.1.1.4. 2-((2,5-Dihydroxy-3-((2E,6E,10E)-3,7,11,15-tetramethyl-hex-
2
mosphere of 5% CO and 95% air. Culture media were changed every
adeca-2,6,10,14-tetraen-1-yl)phenyl)thio)benzoic
acid
: 9.94 (1H), 7.93 (1H), 7.42 (1H), 7.38 (1H), 7.20 (1H), 6.62
3H), 5.32 (1H), 5.14 (2H), 5.20 (1H), 3.32 (2H), 2.00 (2H), 2.02 (2H),
(TPH4).
2 d. Cells were sub-cultured after partial digestion with 0.25%
trypsin-EDTA. For assays, SHSY5Y cells were subcultured in 96-well
1
H NMR
d
5
(
1
1
1
plates at a seeding density of 2.5 ꢂ 10 cells per well. When the
13
.77 (3H), 1.71 (6H), 1.70 (3H), 1.60 (6H); C NMR
48.0, 139.63, 138.7, 137.0, 135.6, 134.4, 132.1, 131.5, 131.2, 131.0,
30.9, 128.5, 127.2, 124.5, 123.8, 121.4, 116.7, 113.8, 39.8, 29.8, 26.8,
d: 169.4, 149.2,
SHSY5Y cells reached 80% confluence, the medium was replaced
with fresh medium containing 1e100 mM compounds or 0.1%
DMSO as a vehicle control.
2
6.5, 25.8, 25.6, 24.3, 19.6, 17.8, 17.5, 16.2, 16.0.
4.4.2. Measurement of cell viability
4
1
.1.1.5. (E)-2-((2,5-Dihydroxy-3-(3,7,11,15-tetramethylhexadec-2-en-
This was determined by the XTT [2,3-bis-(2-methoxy-4-nitro-5-
-yl)phenyl)thio)benzoic acid (TPH5). 1H NMR
d: 9.95 (1H), 7.90
sulfophenyl)-2H-tetrazolium-5-carboxanilide] method. This assay
is based on the ability of living metabolically active cells to reduce
the yellow tetrazolium salt (XTT) to form an orange formazan dye.
Therefore, the conversion only occurs in living cells. The formazan
dye formed is directly quantified using a scanning multi-well
spectrophotometer at wavelength 492 nm (reference wavelength
(
1H), 7.41 (1H), 7.38 (1H), 7.21 (1H) 6.95 (1H), 6.85 (2H), 5.71 (1H),
.42 (1H), 3.95 (1H), 3.48 (2H), 1.69 (3H), 1.63 (3H) 1.06 (6H), 1.01
6H), 1.25 (10H); 13C NMR
: 169.1, 150.6, 148.0, 137.2, 134.4, 132.1,
31.4, 131.2, 130.9, 127.0, 129.3, 123.6, 116.9, 116.1, 113.5, 28.9, 25.8,
4
(
1
2
d
5.0, 24.4, 24.7, 28.2, 23.2, 33.2, 33.4, 39.9, 37.7, 37.8, 39.7, 17.5.
690 nm). The amount of orange formazan formed, as monitored by
4
.2. Determination of EeAChE/EqBuChE inhibition (IC50) by TPHs
its absorbance, directly correlates with the number of living cells.
Control and treated neuroblastoma cells were washed with PBS and
incubated with the XTT solution (final concentration 0.3 mg/mL)
according to the Kit specifications. After this incubation period,
orange dye solution was spectrophotometrically quantified. Results
are expressed as percentages with respect to the control cells.
and TAVA
IC50’s were determined using 0.036 U/mL of EeAChE. Enzymatic
activity was evaluated by the Ellman’s method [24] with some
modifications as described in Oset-Gasque et al., 2014 [55]. The
reaction was performed in multi-well Petri dishes of 48 wells in a
final volume of 500
BuChE (Sigma Aldrich, Madrid, Spain) in 0.1 M pH 8 phosphate
m
L. 0.036 U/mL of EeAChE or eqBuChE or human
4.4.3. Assessment of cell viability after exposure of cell cultures to
oligomycin A-Rotenone (OR) and okadaic acid (OKA) treatments
To investigate the neuroprotective effect of synthesized com-
pounds, several concentrations of these compounds between 0.01
buffer was incubated for 15 min at different drug concentrations at
ꢁ
37
C. Afterwards, the reaction was triggered by the addition of
0
.35 mM acetylthiocholine iodide (ATCh) or 0.5 mM butyrylth-
and 100
the increase in cell viability after 24 h treatment with a mixture of
30 M rotenone and 10 M Oligomycin-A (OR) or 20 nM okadaic
mM were used. Neuroprotection was assayed by measuring
0
iocholine iodide and 0.35 mM 5,5 -ditiobis-2-nitrobenzoico
DTNB), where the DTNB produces the yellow anion 5-thio-2-
(
m
m
nitrobenzoic acid along with the enzymatic degradation of ace-
tylthiocholine or butyrylthiocholine. Changes in absorbance were
measured at 410 nm in a Biotek PowerWave XS spectrophotometer
microplate-reader. Inhibition curves were plotted by using at least
acid (OKA), which induced neuronal cell death. The mixture of
Oligomycin-A plus rotenone blocks mitochondrial electron trans-
port chain complexes V and I, respectively, thus inducing cell death
by oxidative stress, and OKA actívate tau-phosphorylation [56].
Measurements were carried out on human neuroblastoma cells
SHSY5Y seeded into 96-well culture plates as described [28].
nine concentrations (10nM-100
mM) of each compound. IC50 values
were calculated as the concentration of the compound yielding 50%