S. F. da Cunha Xavier Soares et al. / Bioorg. Med. Chem. 21 (2013) 5923–5930
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2.2. NMR experiments
oxime was confirmed by 1H and 13C NMR spectra (See Supplemen-
tary Material).
All NMR experiments were performed at 20.0 0.1 °C in the
Varian Inova 500 MHz spectrometer of the LNBio (National Biosci-
ence Laboratory) in Campinas/SP (Brazil). The samples were pre-
pared in 5 mm NMR tubes using phosphate buffer in D2O (pH
7.4) as solvent. All samples were prepared exactly 1 min before
their introductions into the magnet, and the lock and shimming
process was carried in all samples during 4 min, in order to have
exactly the same processing time in all experiments. The number
of transients for each spectrum was 16 and the acquisition time
was 3.75 s for each transient.
2.4. Molecular modeling
All oximes structures were optimized on Intel dual 2.66 GHz
processor with 2048 Mb RAM using the Spartan06 package with
the functional density (DFT) B3LYP method and the 6-311++G⁄⁄ ba-
sis set. The obtained molecular properties were relative energy (kJ/
mol), molecular mass (atomic units – amu), molecular area (MA),
PSA (polarization surface area), LogP and dipole moment (Debye).
3. Results and discussion
2.2.1. NMR monitoring of EeAChE inhibition by neutral oximes
Monitoring ACh hydrolysis by EeAChE was performed with
NMR by integration of the singlet signal of the ACh methyl group
at 2.24 ppm and of the produced acetic acid at 2.16 ppm. The signal
intensity in all tests was calculated by integration of a 0.3 ppm
zone centralized on each methyl signal.
1H NMR spectroscopy was chosen to monitor the inhibition and
reactivation of AChE in order to develop a simple and more effec-
tive method, which could be applied on further studies for the
development of potential compounds for treatment of Alzheimer
disease (AD) and defense agents against organophosphorus chem-
ical weapons. Inhibition and reactivation o AChE were executed in
order to determine the efficiency of NMR in both methods. The
other motive for working in these two topics is that development
of AChE inhibitors for Alzheimer disease and reactivators of AChE
inhibited by organophosphorus compounds, are two important
topics for health problems.
In each case, the different solution components were added in
the following order: firstly 2.00
followed by 5.00 L of the respective oxime solution, then 5.00
of ACh solution and the necessary volume of phosphate buffer to
adjust the total solution volume to 600 L. Each experiment was
lL of the basic solution of EeAChE,
l
lL
l
performed with the following concentrations: 2.5 mM of ACh,
1.09 pM of EeAChE and 0.1 mM of oxime. The hydrolysis process
was monitored by collection of 1H NMR spectra in intervals of
5 min. Each spectrum was obtained using 30° pulses and 16 scans,
with d1 of 0.5 s and at 20 °C (NMR room temperature). The process
was also executed with EeAChE and ACh without oximes, in order
to use these results as the positive comparative data. All experi-
ments were executed in duplicate.
The fact that cationic oximes, which are usually pyridine deriv-
atives, are used as reactivators of AChE confirms that they interact
with AChE active site, suggesting that they could also act as AChE
competitive inhibitors. One of the problems of AD is the small pro-
duction of the neurotransmissor acetylcholine (ACh) on the pa-
tients. For this reason, AChE inhibitors are used to increase ACh
concentration, resulting in better brain performance.64,65 Accord-
ingly, oximes could also have potential for the treatment of AD,
especially the neutral ones, which display a better permeation
through the hematoencephalic barrier than the cationic oximes.
Some simple neutral oximes were reported as potential reacti-
vators of human AChE inhibited with paraoxon.41 Some of these
compounds, shown in Figure 1, were selected for NMR testing in
this work, in order to compare this method with the Ellman test
and to evaluate the effect of the para-substituent of benzaldoximes
(4–8) on their capacity to inhibit or reactivate AChE. For example,
thiophene-2-aldoxime (3) was selected because, when tested as
human AChE reactivator using the Ellman test, it gave better re-
sults (93% reactivation) than 2-PAM (83% reactivation).41 As the
positive active reference compound we selected pralidoxime (2),
which is the simplest commercial cationic oxime with reasonable
AChE reactivating capacity, but usually insufficient against very
toxic organophosphorus warfare agents.66 There are other cationic
oximes with better reactivation capacity than pralidoxime,67 but
this compound was selected as control for two reasons. The first
motive for selecting 2-PAM is its structural similarity to the tested
neutral oximes, a condition that allows a better comparison of the
obtained results. The second motive is the comparison of obtained
NMR results with the Ellman test previously executed with these
compounds, a process that was also executed using 2-PAM as po-
sitive control.41 Also, because the Ellman test was executed with
AChE inhibited with paraoxon, this compound was selected as
organophosphorus inhibitor in this work.
2.2.2. Inhibition of EeAChE with ethyl-paraoxon
AChE inhibition was first conducted by treatment of 2.0
lL of
the AChE solution (0.33 nM) with 1.5 L of the ethylparaoxon
l
(diethyl-4-nitrophenyl phosphate) solution (3.70 nM) for 1 h at
25 °C. After 1 h, the inhibited enzyme solution was mixed with
5.0 lL of ACh solution and monitored by NMR for 60 min, indicat-
ing that ACh was completely not hydrolyzed, confirming the AChE
inhibition. Monitoring the reactivation process of the inhibited
AChE was conducted with the same conditions used for the inhibi-
tion studies of this enzyme (Section 2.2.1), but 5.00
lL of the
respective oxime solution were added before adjusting the final
volume to 600
control.
lL. Pralidoxime (2-PAM) was used as positive
2.2.3. NMR monitoring neutral oximes reactivation of EeAChE
inhibited with paraoxon
To analyze the process of reactivation of EeAChE inhibited with
ethyl-paraoxon by NMR, it was used the previously described pro-
cedure applied for the oxime inhibition monitoring (Section 2.2.1),
with the pure EeAChE fraction (2.0 lL) replaced by the ethyl-
paraoxon inhibited EeAChE solution (2.0
lL) on the same
concentration.
2.3. Synthesis of oximes
All used oximes, which are known compounds, were prepared
by reaction of the respective aldehydes (2.0 mmol) with hydroxyl-
amine hydrochloride (4.0 mmol) in a 50 mL round bottom flask,
using a mixture of 3.0 mL of water and 10 mL of 95% ethanol as sol-
vent.41 The reaction mixture was kept under stirring at 25 °C for
24 h. The precipitated products were separated by filtration and
recrystallized from methanol, leading to the pure oximes with
yields from 60% to 85%. The structure and purity of each prepared
3.1. Inhibition of EeAChE
The main problem of monitoring enzyme activity by NMR is the
necessity of manipulation of the samples after mixing all compo-
nents in a 5 mm NMR tube. For this reason, the addition of the en-
zyme substrate needs to be performed as the last sample