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D. A. S. KITAGAWA ET AL.
as Alzheimer’s disease. Therefore, we are also carrying out studies acquired from Biotage (Charlotte, NC). NMR spectra were obtained
on the inhibitory potential of such derivatives.
from Varian Unity 400 MHz (Santa Clara, CA, USA) and Bruker
Compound 13c presented the best performance in the in vitro Avance 400 MHz (Palo Alto, CA, USA) and referred to tetramethyl-
silane for 1H and 13 C NMR spectra. GC-MS (Gas Chromatography-
assay, for both OP at both reactivator concentrations used (see
Mass Spectrometry) data were obtained from Agilent 6890 GC sys-
tem equipped with 5975 C mass spectrometer detector (Billerica,
MA, USA). LCMS (liquid chromatography–mass spectrometry) data
were obtained from Agilent 1210 LC system equipped with 6410B
triple quadrupole mass spectrometer detector (Billerica, MA, USA).
SpectraMax Plus 384 microplate reader (Molecular Devices, San
Jose, CA, USA) was used in all in vitro assays. 96-wells microplates
Table 5), indicating that the linker 1,5-pentanediyl is the best
choice to achieve optimal interaction with the AChE binding sites
(Figure 3), even at the lowest concentration tested (1 mM). Even
exhibiting an estimated drug score relatively lower than 2-PAM
(Table 1), reactivation values were similar to 2-PAM, and presented
favourable pharmacokinetic properties, such as higher lipophilicity,
compared to 2-PAM (Table 1). Based on these results, we specu-
late that 13c might be used in lower doses than 2-PAM, making it
a promising reactivator for AChE inhibited by PXN and VX.
Compound 13e showed reactivation values similar to 2-PAM at
both concentrations used for AChE inhibited by NEMP (Table 5),
and also had an estimated toxicity lower than 2-PAM (see Table
1). Thus, this compound may be administered at concentrations
higher than that of conventional oximes, being an interesting
reactivator candidate for VX-inhibited AChE.
~
ꢁ
ꢁ
were purchased from Kasvi Brasil (Sao Jose dos Pinhais, Parana,
Brazil). Gilson single channel pipettes were purchased from Gilson
Inc. (Middleton, WI, USA) and Eppendorf 8-channel pipettes were
~
acquired from Eppendorf Brasil (Sao Paulo, Brazil). Ellman’s tests
were performed in triplicate, in three different assays, by at least
40
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V
three different operators, measured at 25 2 C . Microsoft Excel
2010 was used for all calculations. All disposable materials and
glassware in contact with OP compounds were decontaminated
with aqueous solution containing 10% w/v NaOH and 10% w/v
NaClO for 48 h at room temperature in a fume hood before cor-
rect destination. Estimations of pKa and logP for clinical antidotes
and test compounds were obtained from ChemAxon Online
Suite34. Estimations of DS were obtained from OSIRIS Property
Explorer36. Agilent Chemstation E.02.02.1431 was used for area
integration of GC data for purity calculations. Pyridine-4-aldoxime
and NEMP were synthesised in accordance to literature9,10. Full
spectroscopic data of all synthesised compounds can be found at
the Supplemental material.
4. Conclusions
Our theoretical and experimental studies showed that the isatin
derivatives 13b and 13e proved to be promising candidates to
address AChE inhibition caused by PXN and NEMP, respectively.
Derivative 13c presented the best in vitro performance for both
OP at both reactivator concentrations used, being better than 2-
PAM for PXN and comparable for VX. Besides, all compounds pre-
sented similar reactivation results for PXN. We believe that our
results indicate that these novel isatin derivatives are potential
scaffolds to be further explored in the design of novel reactivators
for OP-inhibited AChE. We are now carrying out studies using the
human isoform of AChE to compare results, as well working on
improvement of current design to achieve better reactivation rates
at the lowest concentrations, proving the potential of the com-
pounds to be prophylactic countermeasures towards such toxi-
cants and analysing the results to find novel valuable
AChE inhibitors.
5.2. General GC-MS and HPLC-MS methods
For GC-MS analysis of N-(x-haloalkyl)isatins 14a–e, 100 ppm solu-
tion of pure compounds in dichloromethane (for reactional mix-
tures, samples were at same concentration and filtered through a
syringe filter 0.22 lm before injection) were prepared. Column:
Agilent J&W HP5-MS ((5%-phenyl)-methylpolysiloxane, 30 m ꢂ
0.25 mm, 0.25 l); injection volume, 1 lL; carrier gas: helium; flow
rate: 1.8 m–/min; inlet: 170 ꢁC; temperature program: held at 40 ꢁC
for 1 min, ramp: 10 ꢁC/min, held at 280 ꢁC for 6 min; MS interface:
250 ꢁC; ionisation energy: 70 eV (electron impact mode).
5. Materials and methods
For LC-MS analysis of isatin-pyridine 4-oxime monocationic
hybrids 13a–e, 10 ppm solution of pure compounds 13a–e in
methanol for reactional mixtures, samples were at same concen-
tration and filtered through a syringe filter 0.22 lm before injec-
tion). Column: Agilent Zorbax HILIC (100 ꢂ 2.5 ꢂ 3.5 lm); injection
volume: 1 lL; isocratic elution: 80% acetonitrile (with 0.1% formic
acid) and 20% water (with 0.1% formic acid), flow: 0.25 mL/min;
ESI-MS mode (positive ion mode); UV at 254 nm (detection:
254 nm/4, reference: 360 nm/100); fragmentator: 70 V; voltage:
4000 V; mass range: m/z 50–500; gas pressure: 40 psi; vaporiser:
37 ꢁC; gas flow: 10 L/min. MS-MS spectra were acquired in similar
conditions to MS spectra: method “Product Ion”; ESI-MS mode
5.1. General information
All chemicals used in this work were purchased from commercial
suppliers and used as received. Isatin, EeAChE (C2888, Type V-S,
1000 U/mg protein), pralidoxime iodide, PXN, 1,x-dihaloalkanes
(1,3-dibromopropane, 1,4-dichlorobutane, 1,5-dibromopentane,
1,6-dichlorohexane, bischloroethyl ether), DMF (dry, sealed bottle)
and inorganic compounds (anhydrous potassium carbonate, phos-
phate salts for buffer solutions, anhydrous sodium sulphate) were
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purchased from Sigma Aldrich Brazil (Sao Paulo, Brazil).
Acetonitrile and methanol for HPLC-DAD-MS were purchased from
~
Merck Brasil (Sao Paulo, Brazil). Deuterated solvents (chloroform-d,
(positive
ion
mode);
fragmentator:
70–135 V;
collision
DMSO-d6, methanol-d4, acetone-d6) were purchased from
Cambridge Isotopes Laboratories (Tewksbury, Massachusetts, USA).
Purified water was obtained from Millipore Milli-Q system (18.2
energy: 20–30 V.
ꢁ
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MX cm at 25 C, Millipore Brazil, Sao Paulo, Brazil). Isolera ACI
Chromatography System (Charlotte, NC, USA) was used for flash
chromatography. Sealed tubes (Q-Tube) were purchased from Q-
Labtech (East Lyme, CT, USA). TLC aluminium plates coated with
5.3. Synthesis of N-(x-haloalkyl)isatins
In a dry flask (30 min at 130 ꢁC in an oven), under an inert atmos-
phere and room temperature, isatin 16 (1 mmol) and K2CO3
(2.5 mmol) were added in dry DMF (4 mL). After 10 min of stirring
~
silica gel F254 were purchased from Merck Brazil (Sao Paulo,
Brazil). Preparative glass TLC coated with silica gel (type KP-Sil, at room temperature, 1,x-dihaloalkanes 17a–d (3 ꢃ x ꢃ 6) or bis-
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Biotage HP-Sphere, 25 l spherical silica, 5 cm ꢂ 5 cm) were chloroethylether 17e (5 mmol, Table 4) were added. The mixture