4
F. Mao et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
N1,N1-di(prop-2-yn-1-yl)-N2-(1,2,3,4-tetrahydroacridin-9-
yl)ethane-1,2-diamine (7b)
at 37 ꢁC in a humidified incubator with 5% CO2 in Dulbecco’s
modified Eagle’s medium/Nutrient Mixture F-12 (DMEM/
F12 ¼ 1:1, GIBCO) supplemented with 10% fetal calf serum
(FCS, GIBCO), 1 mM glutamine, 100 U/mL penicillin and
100 mg/mL streptomycin. SH-SY5Y cells were seeded at a density
of 3 ꢀ 103 cells/well in 96-well plates. After 24 h, the medium was
removed and replaced with the test compounds at different
concentrations for another 24 h at 37 ꢁC. All compounds were
dissolved in DMSO and diluted with fresh medium. The DMSO
concentration of every well in final dilutions was 1% (including
the controls). The cells were then incubated with MTT (0.5 mg/
mL, final concentration) in PBS for 4 h. After the removal of
MTT, the formazan crystals were dissolved in DMSO. The
amount of formazan was measured at 570 nm. Cell viability was
expressed as the percentage of viable cells compared with
untreated control cells.
Compound 6 was treated with 2.1 eq propargyl bromide and 2.1 eq
potassium carbonate in acetonitrile according to the general
procedure to give the desired product as a grey yellow solid (82%
1
yield). H NMR (400 MHz, CDCl3) ꢁ 8.18 (d, J ¼ 8.4 Hz, 1H),
8.08 (d, J ¼ 8.5 Hz, 1H), 7.62 (t, J ¼ 7.6 Hz, 1H), 7.39 (t,
J ¼ 7.6 Hz, 1H), 3.76 (s, 2H), 3.53 (d, J ¼ 2.1 Hz, 4H), 3.18 (s,
2H), 2.99–2.88 (m, 2H), 2.71 (s, 2H), 2.30 (s, 2H), 1.92 (s, 4H).
13C NMR (101 MHz, CDCl3) ꢁ 154.74, 151.59, 142.88, 128.93,
124.68, 123.22, 122.25, 117.47, 113.12, 77.23 (2C), 72.72 (2C),
50.64, 44.11, 41.19 (2C), 28.68, 23.20, 21.58, 20.99. Purity:
98.9% by HPLC; LC/MS (ESI) m/z: [M+H]+ 318.2.
Biological activity
Enzyme inhibition assays
Determination of hepatotoxicity
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from the electric eel),
human acetylcholinesterase (hAChE, E.C. 3.1.1.7, from human
erythrocytes), butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from
equine serum), 5,50-dithiobis-(2-nitrobenzoic acid) (Ellman’s
reagent, DTNB), acetylthiocholine chloride (ATC) and butylthio-
choline chloride (BTC) were purchased from Sigma Aldrich.
Tacrine–propargylamine derivative hybrids were dissolved in
DMSO and then diluted in PBS (0.1 M KH2PO4/K2HPO4 buffer
solution, pH 8.0) to the final concentration.
Cell viability of human hepatic stellate cells (HSCs) was
determined by the MTT assay. HSC were routinely cultured at
37 ꢁC in a humidified incubator with 5% CO2 in Dulbecco’s
modified Eagle’s medium (DMEM, GIBCO) supplemented with
10% foetal calf serum (FCS, GIBCO), 1 mM glutamine, 100 U/
mL penicillin and 100 mg/mL streptomycin. HSC cells were
seeded at a density of 3 ꢀ 103 cells/well in 96-well plates. After
24 h, the medium was removed and replaced with the test
compounds at different concentrations for another 24 h at 37 ꢁC.
All compounds were dissolved in DMSO and diluted with fresh
medium. The DMSO concentration of every well in final dilutions
was 1% (including the controls). The cells were then incubated
with MTT (0.5 mg/mL, final concentration) in PBS for 4 h. After
the removal of MTT, the formazan crystals were dissolved in
DMSO. The amount of formazan was measured at 570 nm. Cell
viability was expressed as the percentage of viable cells compared
with untreated control cells.
All in vitro AChE assays were carried out in PBS, using a
multifunctional microplate reader (Molecular Devices, Flex
Station 3). The assay medium (PBS, 200 mL) consisted of the
diluted compound (90 mL), enzyme (10 mL), 0.002 M substrate
(50 mL) (ACh chloride solution) and 0.002 M DTNB (50 mL). The
test compounds were added to the assay solution and preincubated
at 37 ꢁC with the enzyme for 15 min, followed by the addition of
the mixture of substrate and DTNB. The inhibitory activity was
determined by measuring the increase in absorbance at 412 nm at
1 min intervals at 37 ꢁC. Calculations were performed according
to the method of Ellman et al30. Each concentration was assayed Results and discussion
in triplicate. Data from concentration-inhibition experiments with
the inhibitors were subjected to nonlinear regression analysis
In vitro inhibition studies of AChE and BuChE
using GraphPad Prism 5.0 Software, which gave estimates of the To evaluate the potential application of target compounds for the
IC50 (concentration of drug resulting in 50% inhibition of the treatment of AD, the AChE (electric eel) and BuChE (equine
enzyme activity).
serum) inhibitory activities of the tacrine–propargylamine deriva-
The in vitro BuChE (BuCh as the enzyme substrate) and tives 3a–3c, 7a and 7b were examined with tacrine as the
hAChE assay (hAChE from human erythrocytes as the enzyme) reference standard by Ellman et al.’s spectroscopic method29. The
was performed according to a method similar to that described IC50 values of eeAChE and BuChE inhibition are summarised in
above.
Table 1. The results of eeAChE inhibition of compounds showed
that a propargyl substituted amino group in tacrine was beneficial
to the inhibitory activities of eeAChE, for example, compound 3a
(IC50 ¼ 51.3 nM) was more potent than tacrine (IC50 ¼ 105.8 nM)
by 2-fold improvement and 3b (IC50 ¼ 11.2 nM) was also more
potent than 6-chlorotacrine (IC50 ¼ 23.5 nM). However,
di-propargyl group tacrine derivatives, such as 3c and 7b,
Kinetic characterisation of AChE inhibition
The kinetic characterisation of the AChE inhibitory activity was
performed according to a published protocol30. Briefly, the test
compound was added to the assay solution and incubated with the
enzyme at 37 ꢁC for 15 min, and then the mixture of substrate (the
final concertrations of substrate were 0.05 mM, 0.0625 mM,
0.10 mM, 0.125 mM, 0.25 mM, 0.50 mM, respectively) and
DTNB was added. The kinetic characterisation of the hydrolysis
of ATC catalysed by AChE was performed spectrophotometric-
ally at 412 nm. The parallel control experiments were carried out
without inhibitor in the mixture.
provided
relatively
poor
inhibitory
activities
(3c,
IC50 ¼ 883.7 nM; 7b, IC50 ¼ 339.4 nM, respectively). Compared
with the IC50 values of compounds 3a and 7a (51.3 and
225.6 nM), we realised that the chain between tacrine and the
propargylamine moiety decreased the inhibitory activities.
Most of the tacrine-propargyl derivatives substantially inhib-
ited BChE. Compounds 3a and 3b, which had excellent AChE
inhibitory activities, also had very good BuChE inhibitory
activities (IC50 values: 77.6 and 83.5 nM, respectively). Similar
Determination of neurotoxicity
Cell viability in the human neuroblastoma cell line, SH-SY5Y, to AChE inhibitory activities, compounds 3c and 7b also had
was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl relatively weaker BuChE inhibitory activities (3c:
tetrazolium bromide (MTT) assay according to previously IC50 ¼ 5375.3 nM; 7b: IC50 ¼ 352.7 nM) when compared with
described procedures31. SH-SY5Y cells were routinely cultured the mono-propargyl substituted tacrine derivatives 3a and 3b.