Design, synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase

Article abstract of DOI:10.3109/14756366.2015.1094468

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman’s test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.

Full text of DOI:10.3109/14756366.2015.1094468

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Design, synthesis, and evaluation of
guanylhydrazones as potential inhibitors or
reactivators of acetylcholinesterase
Elaine da Conceição Petronilho, Magdalena do Nascimento Rennó, Newton
Gonçalves Castro, Fernanda Motta R. da Silva, Angelo da Cunha Pinto & José
Daniel Figueroa-Villar
To cite this article: Elaine da Conceição Petronilho, Magdalena do Nascimento Rennó, Newton
Gonçalves Castro, Fernanda Motta R. da Silva, Angelo da Cunha Pinto & José Daniel Figueroa-
Villar (2015): Design, synthesis, and evaluation of guanylhydrazones as potential inhibitors or
reactivators of acetylcholinesterase, Journal of Enzyme Inhibition and Medicinal Chemistry,
DOI: 10.3109/14756366.2015.1094468
To link to this article: http://dx.doi.org/10.3109/14756366.2015.1094468
Published online: 11 Nov 2015.
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, Early Online: 1–10
2015 Taylor & Francis. DOI: 10.3109/14756366.2015.1094468
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RESEARCH ARTICLE
Design, synthesis, and evaluation of guanylhydrazones as potential
inhibitors or reactivators of acetylcholinesterase
1
2
Elaine da Conceic¸a˜o Petronilho , Magdalena do Nascimento Renno , Newton Gonc¸alves Castro , Fernanda Motta R.
3
´
da Silva , Angelo da Cunha Pinto , and Jose Daniel Figueroa-Villar
3
1
1
´
1Department of Chemistry, Military Institute of Engineering, Rio De Janeiro, Brazil, 2Laboratory of Molecular Modeling and Pharmaceutical Sciences,
3
Federal University of Rio De Janeiro, Macae, Brazil, and, Institute of Biomedical Sciences, Federal University of Rio De Janeiro, Rio De Janeiro, Brazil
´
Abstract
Keywords
Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic
organophosphorus compounds, were designed, synthesized, characterized, and tested as
potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman’s test,
nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-
2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six
other guanylhydrazones obtained from different benzaldehydes. The results indicate that all
compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective
AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and
was compared with tacrine, displaying similar activity to this reference material. These results
indicate that guanylhydrazones as well as future similar derivatives may function as drugs for
the treatment of Alzheimer’s disease.
Acetylcholinesterase, Alzheimer’s disease,
guanylhydrazones, molecular docking,
NMR plus Ellman’s test
History
Received 8 May 2015
Revised 3 September 2015
Accepted 4 September 2015
Published online 9 November 2015
Introduction
sometimes causes several toxic problems. To reactivate inhibited
and phosphorylated AChE, it is necessary to use agents with the
appropriate capacity to execute a nucleophilic attack on the
phosphorus atom bound to Ser203, leading to the reactivation of
the function of this serine. The AChE reactivator agents are
mainly cationic oximes7, which are effective but, unfortunately,
not appropriate against all neurotoxic organophosphorus agents8,
thereby necessitating the development of new and more effective
agents for the protection of all humanity.
Some reversible inhibitors of AChE have medical applications
and are particularly important for the treatment of Alzheimer’s
disease (AD). When people develop AD, their neurons degener-
ate, leading to the low production of neurotransmitters, a process
that induces serious memory problems. In this case, the inhibition
of AChE increases the concentration of ACh in synaptic clefts,
improving the neurotransmission process and brain function. For
this reason, AChE inhibitors are very important agents for the
treatment of AD, but some of these inhibitors are toxic, such as
tacrine, requiring the development of new agents. Interestingly,
some AChE reactivators also display competitive inhibition of the
enzyme8, and the reversible inhibitor and AD drug galantamine
protects animals from soman, sarin, and paraoxon intoxication9,
suggesting that novel compounds may have dual application, for
AD and organophosphorus intoxication.
Acetylcholinesterase (AChE) is a very important enzyme used to
control transmission between neurons1,2, when the process is
either mediated or modulated by the neurotransmitter acetylcho-
line (ACh). ACh is released by the axon terminal or varicosities of
the transmitter neuron into the extracellular spa
ce to interact with the receptors of the other neuron. To maintain
control of neurotransmission, it is necessary for AChE, after ACh
executes its function, to catalyze ACh hydrolysis, converting ACh
to choline (Ch) and acetate (Ac). After ACh hydrolysis, Ch is re-
absorbed by the axon terminal to produce more ACh. If AChE is
inhibited in the central nervous system, the concentration of ACh
increases in the synaptic cleft, leading to cholinergic crisis, which
affords several dangerous effects, such as convulsion and
respiratory problems, which could lead to death.
AChE can be inhibited with reversible and irreversible
inhibitors. The irreversible inhibitors are basically neurotoxic
organophosphorus compounds, normally used as pesticides,
insecticides, and chemical warfare agents3,4. These agents lead
to the phosphorylation of Ser203, which is the most important
active amino acid involved in ACh hydrolysis, at the active site of
AChE, leading to complete inhibition of this enzyme5. Despite the
use of chemical warfare agents being prohibited by the United
Nations, these agents constitute one of the greatest threats in the
modern world6. In parallel, many organophosphorus pesticides are
used by all countries for agricultural production, a process that
Therefore, the development of new agents that interact with
AChE as potential drugs for the treatment of AD or as reactivators of
this enzyme for the detoxification of neurotoxic organophosphorus
compounds is very important. This work describes the preparation of
compounds with a certain similarity to pralidoxime and their
evaluation as AChE inhibitors or reactivators using the Ellman’s test,
nuclear magnetic resonance (NMR) and molecular docking.
´
Address for correspondence: Dr. Jose Daniel Figueroa-Villar, Department
of Chemistry, Military Institute of Engineering, Prac¸a General Tiburcio,
80, Rio de Janeiro 22290-270, Brazil. E-mail: jdfv2009@gmail.com
´

2
E. da Conceic¸a˜o Petronilho et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Materials and methods
IR (cmꢀ1): 3291 (amine N-H), 3119 (aromatic C-H), 1628-
1505 (C ¼ N and C ¼ C), 1293 and 1247 (C-N), 780 (C-H out of
plane (Di-1,2)).
Chemistry
Solvents (ethyl alcohol 95%, diethyl ether, methanol, chloroform)
1H NMR (300 MHz, DMSO-d6) d (ppm): 4.17 (s, 3H, CH3);
were purchased from VETEC (Duque de Caxias, Brazil) (used 7.65 (dt, J ¼ 2.3 and 6.4 Hz, 1H, Ar-H); 7.89 (s, 1H, H-C ¼ N-
with further purification), and reagents were purchased from NH2); 8.22 (m, 2H, Ar-H); 8.67 (d, 6.3 Hz, 1H, Ar-H); 9.16 (s,
Merck and Sigma-Aldrich (Sa˜o Paulo, Brazil) (used without 2H, C ¼ N-NH2).
further purification). Reactions were monitored by TLC using DC-
13C NMR (75 MHz, DMSO-d6) d: 44.8 (CH3); 121.9 (CH),
Alufolien Kieselgel 60 F254 (Merck, Darmstadt, Germany). The 122.6 (CH), 123.4 (CH), 142.5 (CH), 144.2 (CH), 151.2 (C).
NMR spectra were determined on a Varian 600 MHz spectrometer, For C7H10N3 calculated: (31.9%) C, (15.9%) N, (3.8%) H;
using dimethyl sulfoxide-d6 (DMSO-d6) as solvent and tetra- found (32.0%) C, (15.4%) N, (3.8%) H.
methylsilane as internal standard. The infrared (IR) spectra were
measured using a Spectrum 100 spectrometer. The absorption found 136.0868.
values are expressed in wave number, using inverse centimeters
(cmꢀ1) as units. The mass spectra were obtained on a Waters
HRMS (ESI) m/z: Calcd for C7H10N3 [M + H]+ 136.0869;
General procedure for guanylhydrazones (3–12)
(Milford, MA) spectrometer, model Q-Tof micro, using positive
mode detection (MS ES+) and acetonitrile as solvent. The IR,
HRMS and NMR spectra are available in the Supplemental file.
Aminoguanidine hydrochloride (1.4 mmol) dissolved in 20 mL of
95% ethanol, the corresponding aldehyde (1 mmol) and two drops
of HCl (0.6 M) were added to a 50.0 mL round-bottom flask. The
solution was stirred and heated under reflux. Evaporation of the
solvent gave a precipitate that was solubilized in distilled water
and extracted with dichloromethane (5 ꢂ 20 mL). The aqueous
phase was evaporated, and the product was obtained as crystals.
The crystals were recrystallized from ethanol.
Synthesis of the compounds
N-methylation method for pyridine-2-carboxaldehyde
2-Formyl-1-methylpyridinium iodide was prepared using 5.0 g
pyridine-2-carboxaldehyde (0.05 mol) and CH3I (14 g) with stirring
and heating under reflux for 2 h. The orange crystals formed were
filtered under nitrogen and washed with dry diethyl ether.
1-Methylpyridine-2-carboxaldehyde guanylhydrazone (3)
Yield 44%. Yellow solid. mp: 220–221 ꢁC.
IR (cmꢀ1): 3294 (amine N-H), 3086 (aromatic C-H), 1679-
1509 (C ¼ N and C ¼ C), 1290 and 1251 (C-N), 782 (C-H out of
plane (Di-1,2)).
Method of synthesis for 2-PAM (1)
The method for the preparation of 1-methylpyridine-2-oxime
iodide (1) was developed in two stages. First prepared was the
iodide of 2-formyl-1-methylpyridine. Then, the iodide of 2-
formyl-1-methylpyridine (1.4 mol) diluted in ethanol was reacted
with hydroxylamine (0.0014 mol) in the presence of pyridine
(1 mL) as catalyst. This reaction was stirred and heated under
reflux for 40 min. Afterwards, distilled water (1 mL) was added,
and the product was filtered.
1H NMR (300 MHz, DMSO-d6) d (ppm): 4.41 (s, 3H, CH3);
8.19 (t, J ¼ 6.1 Hz, 1H, Ar-H); 8.37 (s, 4H, C ¼ NHCN2H4); 8.70
(t, J ¼ 7.9 Hz, 1H, Ar-H); 8.75 (s, 1H, HC ¼ N); 9.06 (d,
J ¼ 7.9 Hz, 1H, Ar-H); 9.13 (d, J ¼ 6.1 Hz, 1H, Ar-H); 12.88 (s,
1H, C ¼ NNH).
13C NMR (75 MHz, DMSO-d6) d: 44.8 (CH3), 125.3 (CH),
127.5 (CH), 137.1 (CH), 144.6 (CH), 146.5 (CH), 147.6 (C),
155.3 (C).
Pralidoxime (1)
For C8H12N5 calculated: (30.3%) C, (21.8%) N, (4.3%) H;
found (28.1%) C, (20.5%) N, (3.8%) H.
HRMS (ESI) m/z: Calcd for C8H12N5 [M + H]+ 178.1087;
found 178.1090.
Yield 26%; Yellow solid. mp: 212–214 ꢁC (Lit. 224–225 ꢁC)10.
1H NMR (300 MHz, DMSO-d6) d (ppm): 4.37 (s, 3H, CH3);
8.06 (ddd, J ¼ 1.6; 7.6 e 6.3 Hz, 1H, Ar-H); 8.38 (dd, J ¼ 1.4 e
8.1 Hz, 1H, Ar-H); 8.53 (bt, 7.9 Hz, 1H, Ar-H); 8.67 (s, 1H, H-
C ¼ N-OH); 8.97 (bd, 6.3 Hz, 1H, Ar-H); 13.10 (s, 1H,
C ¼ N-OH).
Pyridine-2-carboxaldehyde guanylhydrazone (4)
Yield 45%. Yellow solid. mp: 213–215 ꢁC (Lit. 190–192 ꢁC)11
IR (cmꢀ1): 3460 (amine N-H); 3047(aromatic C-H), 1662-
1465 (C ¼ N and C ¼ C), 1341 and 1174 (C-N), 770 (C-H out of
plane (Di-1,2)).
1H NMR (300 MHz, DMSO-d6) d (ppm): 7.79 (t, J ¼ 5.2 Hz;
1H, Ar-H), 8.19 (s, 4H, C ¼ NH-C-N2H4), 8.36 (m, 3H, HC ¼ N
and Ar-H), 8.77 (d, J ¼ 5.2 Hz, 1H, Ar-H), 12.80 (s, 1H, C ¼ NH-
C-N2H4).
13C NMR (75 MHz, DMSO-d6) d: 46.4 (CH3), 125.1 (CH),
127.2 (CH), 141.8 (CH), 145.0 (CH), 146.6 (CH), 147.5 (C).
For C7H9N2 calculated: (31.7%) C, (10.5%) N, (3.7%) H; found
(31.9%) C, (10.4%) N, (3.4%) H.
MS (ESI) m/z 137.0.
General procedure for hydrazone (2)
13C NMR (75 MHz, DMSO-d6) d: 157.4 (C), 148.5 (CH);
146.8 (C), 144.2 (CH); 138.9 (CH); 128.8 (CH); 128.2 (CH).
For C7H10N5 calculated: (42.1%) C, (35.0%) N, (5.0%) H;
found (50.3%) C, (41.7%) N, (5.1%) H.
The method for the preparation of 1-methylpyridine-2-hydrazone
iodide (2) was developed in two stages. First prepared was the
iodide of 2-formyl-1-methylpyridine. Then, the iodide of 2-
formyl-1-methylpyridine (0.004 mol) diluted in methanol was
reacted with hydrazine hydrate (0.004 mol) in the presence of HCl
(0.6 M) as catalyst. This reaction was stirred and heated under
reflux for 30 min and then was cooled to 5 ꢁC and filtered. The red
liquid filtrate was evaporated, and the product was recrystallized
from diethyl ether.
HRMS (ESI) m/z: Calcd for C7H10N5 [M + H]+ 164.0920;
found 164.0931.
Benzaldehyde guanylhydrazone (5)
Yield 65%. White solid. mp: 45–47 ꢁC (Lit. 145–147 ꢁC)12.
IR (cmꢀ1): 3316 (amine N-H), 3167 (aromatic C-H), 1676-
1446 (C ¼ N and C ¼ C), 1229; 1156 (C-N), 748 and 686 (C-H
out of plane).
1-Methylpyridine-2-carboxaldehyde hydrazone (2)
Yield 36%. Yellow solid. mp: 174–176 ꢁC.

DOI: 10.3109/14756366.2015.1094468
Design, synthesis, and evaluation of guanylhydrazones
3
1H NMR (300 MHz, DMSO-d6) d (ppm): 7.43 (m, 3H, Ar-H); C ¼ NH-CN2H4); 6.86 (d, J ¼ 8.8 Hz, 2H, Ar-H); 8.04 (s, 1H,
7.85 (m, 5H, Ar-H and C ¼ NH-CN2H3); 8.19 (s, 1H, HC ¼ N); HC ¼ N); 11.89 (s, 1H, C ¼ NH-CN2H4).
12.16 (s, 1H, C ¼ NH-CN2H3).
13C NMR (75 MHz, DMSO-d6) d: 155.2 (C), 150.8 (C), 147.1
13C NMR (75 MHz, DMSO-d6) d: 155.5 (C); 146.7 (CH), (CH), 128.9 (2CH), 122.4 (C), 112.8 (2CH), 40.4 (2CH3).
133.4 (CH), 130.5 (C), 128.7 (CH), 127.6 (CH). For C10H16N5 calculated: (49.7%) C, (28.9%) N, (6.7%) H;
For C8H11N4 calculated: (48.3%) C, (28.2%) N, (5.6%) H; found (48.0%) C, (28.3%) N, (6.8%) H.
found (37.2%) C, (26.7%) N, (6.9%) H.
HRMS (ESI) m/z: Calcd for C10H16N5 [M + H]+ 206.1406;
HRMS (ESI) m/z: Calcd for C8H11N4 [M + H]+ 163.0978; found 206.1357.
found 163.0881.
4-Hydroxybenzaldehyde guanylhydrazone (10)
4-Nitrobenzaldehyde guanylhydrazone (6)
Yield 70%. White solid. mp: 245–246 ꢁC (Lit. 245–247 ꢁC)14.
IR (cmꢀ1): 3425 (amine N-H), 3259 (O-H) 3102 (aromatic C-
Yield 60%. Yellow solid. mp: 244–245 ꢁC (Lit. 244–245 ꢁC)12.
IR (cmꢀ1): 3272 (amine N-H), 3101 (aromatic C-H), 1676- H), 1682-1515 (C ¼ N and C ¼ C), 1268 (C-O) 1216 (C-N), 832
1583 (C ¼ N and C ¼ C), 1510 and 1339 (NO2), 1231 (C-N), 835 (C-H out of plane (Di-1,4)).
(C-H out of plane (Di-1,4)).
1H NMR (300 MHz, DMSO-d6) d (ppm): 6.83 (d, J ¼ 8.6 Hz,
1H NMR (300 MHz, DMSO-d6) d (ppm): 8.01 (s, 4H, C ¼ NH- 2H, Ar-H); 7.67 (s, 4H, C ¼ NH-CN2H4); 7.67 (d, J ¼ 8.6 Hz, 2H,
CN2H4); 8.16 (d, J ¼ 8.9 Hz, 2H, Ar-H); 8.27 (d, J ¼ 8.9 Hz, 2H, Ar-H); 8.05 (s, 1H, HC ¼ N); 10.09 (s, 1H, C ¼ NH-CN2H4),
Ar-H); 8.32 (s, 1H, HC ¼ N); 12.48 (s, 1H, C ¼ NH-CN2H4).
13C NMR (75 MHz, DMSO-d6) d: 155.6 (C), 148.0 (C), 144.3
(C), 139.7 (CH), 128.5 (2CH), 123.7 (2CH).
11.85 (s, 1H, OH).
13C NMR (75 MHz, DMSO-d6) d: 159.8 (C), 155.2 (C), 146.9
(CH), 129.3 (CH), 124.3 (C), 115.5 (CH).
For C8H11N4O calculated: (44.7%) C, (26.1%) N, (5.1%) H;
found (44.7%) C, (26.0%) N, (4.8%) H.
HRMS (ESI) m/z: Calcd for C8H11N4O [M + H] + 179.0933;
found 179.0865.
For C8H10N5O2 calculated: (39.4%) C, (28.7%) N, (4.1%) H;
found (37.6%) C, (27.1%) N, (4.2%) H.
HRMS (ESI) m/z: Calcd for C8H10N5O2 [M + H]+ 208.0834;
found 208.0791.
4-Chlorobenzaldehyde guanylhydrazone (7)
2,4-Dinitrobenzaldehyde guanylhydrazone (11)
Yield 94%. White solid. mp: 70 ꢁC dp (Lit. 156–158 ꢁC)12.
IR (cmꢀ1): 3251 (amine N-H), 3105 (aromatic C-H), 1678-
Yield 80%. White solid. mp: 190–191 ꢁC.
IR (cmꢀ1): 3386 (amine N-H), 3107 (aromatic C-H), 1685-
1492 (C ¼ N and C ¼ C), 1276 (C-N), 832 (C-Cl), 750 (C-H out of 1558 (C ¼ N and C ¼ C), 773 (NO2), 728 (C-H out of plane).
plane (Di-1,4)).
1H NMR (300 MHz, DMSO-d6) d (ppm): 8.12 (s, 4H, C ¼ NH-
1H NMR (300 MHz, DMSO-d6) d (ppm): 7.89 (s, 4H, C ¼ NH- CN2H4); 8.50 (s, 1H, HC ¼ N); 8.51 (d, J ¼ 2,3 Hz; 1H, Ar-H);
CN2H4); 7.89 (d, J ¼ 8.4 Hz, 2H, Ar-H); 7.48 (d, J ¼ 8.4 Hz, 2H, 8.73 (dd, J ¼ 2.3 e 8.8 Hz; 1H, Ar-H); 8.76 (d, J ¼ 8.8 Hz; 1H, Ar-
Ar-H); 8.19 (s, 1H, HC ¼ N); 12.26 (s, 1H, C ¼ NH-CN2H4).
13C NMR (75 MHz, DMSO-d6) d: 155.5 (C), 148.0 (C), 145.4
(CH), 132.4 (C), 129.2 (2CH), 128.7 (2CH).
H); 12.76 (s, 1H, C ¼ NH-CN2H4).
13C NMR (75 MHz, DMSO-d6) d: 155.5 (C), 147.8 (C), 147.5
(C), 140.6 (CH), 133.3 (C), 130.0 (CH), 127.2 (CH), 120.3 (CH).
MS (ESI) m/z 253.0.
For C8H10N4Cl calculated: (41.2%) C, (24.0%) N, (4.3%) H;
found (39.5%) C, (23.1%) N, (5.1%) H.
HRMS (ESI) m/z: Calcd for C8H10N4Cl [M + H]+ 197.0594; 2,4-Dichlorobenzaldehyde guanylhydrazone (12)
found 197.0559.
Yield 50%. White solid. mp: 246–248 ꢁC (Lit. 222–223 ꢁC)13.
IR (cmꢀ1): 3308 (amine N-H), 3143 (aromatic C-H), 1677-
4-Methylbenzaldehyde guanylhydrazone (8)
1519 (C ¼ N and C ¼ C), 823 (C-Cl), 775(C-H out of plane).
1H NMR (300 MHz, DMSO-d6) d (ppm): 7.95 (s, 4H, C ¼ NH-
Yield 40%. White solid. mp: 155–156 ꢁC (Lit. 170–171 ꢁC)13.
IR (cmꢀ1): 3343 (amine N-H); 3153 (aromatic C-H), 1682-
1483 (C ¼ N and C ¼ C), 1231 and 1178 (C-N), 810 (C-H out of
plane (Di-1,4)).
CN2H4); 8.50 (s, 1H, HC ¼ N); 7.70 (d, J ¼ 2.0 Hz; 1H, Ar-H);
7.51 (dd, J ¼ 2.0 e 8.6 Hz; 1H, Ar-H); 8.33 (d, J ¼ 8.6 Hz; 1H, Ar-
H); 11.83 (s, 1H, C ¼ NH-CN2H4).
13C NMR (75 MHz, DMSO-d6) d: 155.3 (C), 141.5 (CH),
135.5 (C), 133.9 (C), 129.7 (C), 129.3 (CH), 128.9 (CH), 127.7
(CH).
1H NMR (300 MHz, DMSO-d6) d (ppm): 2.34 (s, 3H, CH3);
7.25 (d, J ¼ 8.4 Hz, 2H, Ar-H); 7.74 (d, J ¼ 8.0 Hz, 2H, Ar-H);
7.76 (s, 4H, C ¼ NH-CN2H4); 8.14 (s, 1H, HC ¼ N); 12.06 (s, 1H,
C ¼ NH-CN2H4).
For C8H9N4 calculated: (35.9%) C, (20.9%) N, (3.4%) H; found
(33.0%) C, (19.1%) N, (3.9%) H.
MS (ESI) m/z 230.9.
13C NMR (75 MHz, DMSO-d6) d: 155.4 (C), 146.7 (CH),
140.3 (C), 130.7 (C), 129.2 (2CH), 127.5 (2CH), 21.0 (CH3).
For C9H13N4 calculated: (50.8%) C, (26.3%) N, (6.1%) H;
found (52.0%) C, (27.1%) N, (6.5%) H.
Biological evaluation
HRMS (ESI) m/z: Calcd for C9H13N4 [M + H]+ 177.1140;
found 177.1058.
AChE inhibition assay by Ellman’s method
Inhibition of AChE by guanylhydrazones and hydrazone was
performed by Ellman’s colorimetric method modified for reading
in a microplate spectrophotometer (SpectraMax 250, Molecular
Devices, Sunnyvale, CA, USA) and at pH 7.415,16. In this study,
instead of using the homogenate of rat brain, enzyme purified
from Electrophorus electricus was used. Equine serum butyr-
ylcholinesterase was used to evaluate selectivity among the
enzymes. To evaluate the inhibitory capacity of the compounds, a
final volume of 200 mL was used and to a microplate were added
4-Dimethylaminobenzaldehyde guanylhydrazone (9)
Yield 85%. Pale pink solid. mp: 212–214 ꢁC (Lit. 179–182 ꢁC)13.
IR (cmꢀ1): 3438 (amine N-H), 3100 (aromatic C-H), 1675-
1530 (C ¼ N and C ¼ C), 1233 and 1181 (C-N), 818 (C-H out of
plane (Di-1,4)).
1H NMR (300 MHz, DMSO-d6) d (ppm): 2.98 (s, 6H,
N(CH3)2); 7.68 (d, J ¼ 8.8 Hz, 2H, Ar-H); 7.68 (s, 4H,

4
E. da Conceic¸a˜o Petronilho et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
20 mL of cholinesterase (1 IU/mL), 5 mL of 5,50-dithiobis-(2- Docking
nitrobenzoic acid) (DTNB) (0.01 M), 100 mL of inhibitor
The ligands’ three-dimensional structures were built using the
Spartan’10 program19, and the conformer distribution with
molecular mechanics using 100 conformers were examined.
Their partial atomic charges were calculated using the RM1 semi-
empirical method, and the molecular energies were calculated
using Hartree-Fock 6–31 G*. The ligands’ rotatable bonds and
atomic charges were defined. The 3D coordinates of TcAchE
(PDB Code: 1ACJ) were obtained from the Protein Data Bank20.
AutoDock 4.221 was used for the docking study of the ligands in
the active site of TcAchE. To validate the docking protocol, we
first performed the docking simulation of tacrine against the
active site of TcAchE and compared it to the crystallographic
structure. The interactions of ligands with the TcAChE active site
(200 mM) (final concentration of 100 mM), and 55 mL of pH 7.4
phosphate buffer (0.1 M). Inhibitors, DTNB, and acetylthiocho-
line iodide were dissolved in phosphate buffer, pH 7.4. After 10
min, 20 mL of acetylthiocholine iodide (0.005 M) was added. This
analysis was performed at room temperature (22–25 ꢁC) and
followed at 412 nm for 5 min to determine reaction velocities, in
triplicate. The compounds that showed inhibition above 50% were
reevaluated at final concentrations of 3, 10, 30, 100, 300, and
1000 mM. The mean inhibitory concentration (IC50) was obtained
by nonlinear regression with GraphPad Prism software.
AChE reactivation assay by Ellman’s method
For evaluating the reactivation of AChE by hydrazones, we used a were performed using AutoDock 4.2, using each ligand with 50
˚
final volume of 200 mL. To a microplate were added 20 mL of E. poses and grid points of 48 ꢂ 38 ꢂ 42 with 0.375 A spacing. The
electricus AChE (1 IU/mL), 5 mL of DTNB solution, 100 mL of a best conformation of each ligand was selected according to the
solution of ethyl paraoxon 200 nM (final concentration of 100 nM) evaluation of the lowest energy of interaction with the enzyme.
and 35 mL of pH 7.4 phosphate buffer. After 1 h, the time required The figures were generated using a PyMOL Molecular Graphics
for enzyme inhibition (established in preliminary experiments), System22, AutoDock Tools and Python Molecular Viewer (ADT/
20 mL of hydrazones (final concentration in plate 10 mM, 100 mM, PMV/Viewer)23.
and 500 mM) were added, and after 20, 30, and 60 min (to
allow the reactivation of the enzyme), 20 mL of acetylthiocholine Results and discussion
iodide (0.005 M) was added at the moment that sample was
Synthetic aspects
read in the spectrophotometer, i.e. when then the enzymatic
reaction is triggered. The analysis was performed in a spectro- One previous study using molecular modeling with ab initio
photometer at 412 nm in triplicate and reagents were used at room methods suggested the evaluation of anionic nucleophiles differ-
temperature.
ent from oximates, for example hydrazonates, as AChE
reactivators24. According to this molecular modeling study, we
decided to prepare a hydrazone with a structure similar to
pralidoxime to test its capacity as an AChE reactivator or
inhibitor. Because hydrazones normally display low acidity, the
binding of hydrazonates in the active site of AChE may be
relatively difficult, indicating that these compounds would not be
very effective reactivators of this enzyme when inhibited by
organophosphorus agents. However, other hydrazones could
display more acidity than the NH2 group but certainly would be
less acidic than the respective oximes. To test this possibility, the
only hydrazone selected for preparation and evaluation was
1-methylpyridine-2-carboxaldehyde hydrazone (2) (Figure 1),
with a structure very similar to pralidoxime (2-PAM, 1), which
is one of the simplest cationic oximes used commercially for the
reactivation of phosphorylated AChE and which certainly inter-
acts with the active site of this enzyme.
AChE inhibition assay by the NMR method
The EeAChE activity and inhibition determination by NMR was
performed on a 600 MHz spectrometer using 5-mm NMR tubes,
as described in our previous article on this topic17. To determine
EeAChE activity, 2 mL of its solution of 0.2 mM concentration in
pH 7.4 phosphate buffer with D2O and in the presence of 1%
bovine serum albumin was used. At the starting time of the
reaction, this solution was mixed with 30 mL of acetylcholine
(0.1 M) in the same solvent, and the complete mixture was diluted
to a volume of 600 mL with the D2O pH 7.4 phosphate buffer in
the 5-mm NMR tube. This sample was immediately introduced to
the magnet for locking and shimming, allowing for the observa-
1
tion of the first H spectra exactly 5 min after the introduction of
1
acetylcholine. All of the next H spectra were obtained every 5
min with a single scan over 80 min. The intensity of the methyl
signals of acetylcholine (2.24 ppm) and acetic acid (2.16 ppm)
was determined by scanning, affording information about
EeAChE kinetics. The AChE inhibition test was executed with
the same procedure, including the addition of 5 mL of each
potential EeAChE inhibitor (12 mmol), always before the addition
of acetylcholine, using exactly the same time periods. The
concentration of acetylcholine and Ac from each 1H spectrum was
determined by the integration of their methyl signals, using
exactly the same integration length in all cases. The time used for
the pure enzyme to afford 50% of Ac was also used to determine
the respective Ac production (%) in the presence of the tested
inhibitors. The comparison of the Ac concentration at this time
with the pure AChE allows the determination of the percent
inhibition of each tested compound. These results were obtained
in triplicate.
Considering the possibility that other cationic or neutral
compounds similar to pralidoxime could interact well with the
AChE active site and with inhibition capacity, we decided
to prepare and test the compounds 1-methylpyridine-2-carbox-
aldehyde guanylhydrazone (3), pyridine-2-carboxaldehyde
guanylhydrazone (4), benzaldehyde guanylhydrazone (5), 4-
nitrobenzaldehyde guanylhydrazone (6), 4-chlorobenzaldehyde
guanylhydrazone (7), 4-methylbenzaldehyde guanylhydrazone
(8), 4-dimethylaminobenzaldehyde guanylhydrazone (9), and
4-hydroxybenzaldehyde guanylhydrazone (10), as shown in
Figure 1. Guanylhydrazones were selected because they contain
one cationic site and therefore are appropriate to interact with the
anionic region of the AChE active site and compete with
acetylcholine. Several guanylhydrazones have been used as
pharmacological agents for diverse diseases, such as cancer25,26
,
Chagas disease27,28, malaria and others29. These compounds were
also studied by NMR and theoretical chemistry30.
To determine if hydrazones could act as inhibitors or
Molecular modeling
The structure conformation, dipole moment, and determination of reactivators of AChE, the first step was the N-methylation of
atomic electronic charges were obtained with Spartan’06 using pyridine-2-carboxaldehyde using methyl iodide as solvent with
the B3LYP system and the 6–311++G** basis set18.
stirring and under reflux for 2 h. This compound was used to

DOI: 10.3109/14756366.2015.1094468
Design, synthesis, and evaluation of guanylhydrazones
5
Figure 1. Preparation of pralidoxime
(2-PAM) (1); 1-methylpyridine-2-carboxal-
dehyde hydrazone (2); 1-methylpyridine-
2-carboxaldehyde guanylhydrazone (3), pyri-
dine-2-carboxaldehyde guanylhydrazone (4),
benzaldehyde guanylhydrazone (5), 4-nitro-
benzaldehyde guanylhydrazone (6), 4-chlor-
obenzaldehyde guanylhydrazone (7),
4-methylbenzaldehyde guanylhydrazone (8),
4-dimethylaminobenzaldehyde guanylhydra-
zone (9), 4-hydroxybenzaldehyde guanylhy-
drazone (10).
prepare 2-PAM (1) in 26% yield by reaction with hydroxylamine
hydrochloride in ethanol with pyridine under reflux for 40 min.
The cationic hydrazone 2 was prepared by the reaction of
N-methylpyridine-2-carboxaldehyde with hydrazine hydrochlor-
ide under reflux in 95% ethanol for 2 h, affording the compound
in 36% yield. The N-methylpyridine-2-carboxaldehyde was also
used to prepare the bis-cationic guanylhydrazone (3) by reaction
with aminoguanidine hydrochloride in ethanol and HCl under
reflux for 2 h, affording the compound in 44% yield. All other
guanylhydrazones (4–10) were prepared by reaction of the
respective aldehydes with aminoguanidine hydrochloride using
95% ethanol as solvent and heating under reflux for 2–7 h, leading
to mono-cationic compounds obtained in 40–94% yields. All
compounds were characterized by IR, NMR and mass
spectrometry.
Table 1. Results of inhibition of EeAChE and EqBuChE.
Ellman’s test
NMR test
EeAChE
inhibition (%)
EeAChE
inhibition (%)
EqBuChE
inhibition (%)
Compounds
1
2
3
4
5
6
7
8
9
10
24.34 0.94
55.35 1.43
39.07 3.01
23.56 2.37
14.06 2.12
52.99 0.30
25.62 1.61
20.97 1.71
16.49 1.19
17.25 1.05
1.17 1.6
15.99 1.4
ꢀ1.50 1.6
42.45 1.4
24.11 1.5
20.01 1.9
21.92 0.6
27.94 1.0
40.06 0.8
34.05 1.4
7.9 1.4
41.0 2.3
29.4 1.2
27.0 2.1
13.9 1.1
60.8 3.2
32.7 2.1
25.0 1.3
10.4 2.1
12.2 2.2
The only previous attempt to synthesize compound 2 was
made in 1961 by Poziomek, who reported that at the end of the
synthesis procedure, they obtained incomplete dehydration of the
molecule and not the desired product31. Our procedure led to the
desired hydrazone after recrystallization from diethyl ether, and
this compound has not been described in the literature. Compound
3 is a new unpublished agent. Compounds 4–10 have been
described in the literature11,12,32,33, but never for their application
in AChE inhibition or reactivation, and thus, this is a new type of
application for guanylhydrazones.
displays an identical active site and very similar activity to that of
the human enzyme34,35. We have also used the Ellman’s assay for
EqBuChE inhibition, to assess the compounds’ selectivity. All
results obtained for cholinesterase inhibition using the Ellman’s
test and NMR are shown in Table 1.
Analysis of the Ellman’s test indicates that all compounds
display EeAChE inhibition activity, confirming that guanylhy-
drazones and hydrazones are potential inhibitors, especially
because all contain cationic groups that are capable of interacting
with the active site of this enzyme.
Assessment of inhibition of EeAChE
The tests of AChE inhibition with these compounds were carried
The most effective EeAChE inhibitors were 1-methylpyridine-
out with two methods – the Ellman’s test15,16 and the recently 2-carboxaldehyde hydrazone (2) (55.35%) and 4-nitrobenzalde-
published NMR method16 using tacrine as a reference standard. hyde guanylhydrazone (6) (52.99%). It is observed that the two
The selected AChE was from E. electricus (EeAChE), which compounds containing a N-methylpyridine ring are effective

6
E. da Conceic¸a˜o Petronilho et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
(2 and 3), but compound 2, with a single cationic group low concentration of enzyme (1.0 pM) in phosphate buffer at
(N-methylpyridine), is more effective than 3, which contains pH 7.4 and with the presence of 5% albumin to maintain the
two cationic groups (guanidine and N-methylpyridine). However, enzyme activity for a long time because EeAChE is a very fast
pyridine-2-carboxaldehyde guanylhydrazone (4) is less effective enzyme in its hydrolysis of ACh. To perfectly control the time for
1
(23.56%); its only cationic group is the guanidine. Furthermore, each H NMR spectrum, it was also necessary to introduce the
2-PAM (1) displays a low effect (24.34%) similar to compound 4, ACh into the 5-mm NMR tube as the last ingredient and then take
despite containing an N-methylpyridine ring as a single cationic 5 min to lock, gradient auto-shim and obtain the first spectra with
group. These results indicate that for the pyridine derivatives one single transient. Continuing in this manner with additional
(1, 2, 3, and 4), the best situation is where the cationic spectra taken every 5.0 min led to the acquisition of 20 spectra
N-methylpyridine ring is associated with a hydrazone group (2) during 100 min. The substrate (ACh) and product (Ac) concen-
and not with
a
guanylhydrazone group. However, as trations from each spectrum were obtained from the integration of
4-nitrobenzaldehyde guanylhydrazone (6), the second most the corresponding methyl signals at 2.15 and 1.91 ppm, respect-
effective compound, shows, it is clear that guanylhydrazones ively. These experiments, which were executed first with pure
not associated with pyridine rings could be effective.
EeAChE and followed with compounds 2–10, are shown in
The IC50 of the most active compounds at EeAChE from Table 1. In general terms, despite the results of both methods
Table 1 were 49.55 mM (compound 2) and 45.64 mM (compound being similar, there are also important differences with some
6). The inhibition curves are available in the Supplemental file. compounds. For example, the results for compound 5 are very
The EqBuChE assay showed that most compounds were not very similar (14.06 by Ellman’s and 13.9 by NMR), but for compound
selective, except for 2-PAM (1) and 3, which did not inhibit the 9, the results are different (16.59 by Ellman and 10.4 by NMR).
enzyme at 100 mM.
However, because the NMR test allows for perfect signal
To confirm the results obtained by the Ellman’s test, 1H NMR determination of the AChE substrate (ACh) and products (Ch
experiments were also carried out17. The NMR method required a and HOAc), as well as their correct concentration by integration,
this method is as appropriate as the Ellman’s test; because the
NMR method includes the structural determination of all agents,
substrates and products, it is also a very good method.
Comparing the effects on AChE for the guanylhydrazones
prepared from benzyl aldehydes in Table 1, it is observed that the
compound containing one para-NO2 group (6) is the most effective,
and the second most effective is compound 7, which is para-
chlorinated. The third most effective compound is the para-
methylated compound (8), and the less effective compounds are 5,
which does not contain an R group, and 9 and 10, whose para-R
groups, Me2N and HO, respectively, are electropositive.
This information indicates that the best benzylidene guanylhydra-
zones for the inhibition of AChE should contain strong electron
withdrawing R groups. To confirm this effect, all guanylhydrazones
were studied by molecular modeling (B3LYP 6–311+ G**) to
determine the correlation between some calculated properties of
each compound and their percentage of AChE inhibition. In this
Figure 2. Correlation of the dipole moment calculated by molecular
modeling with the AChE inhibition of compounds 5–10.
case, the correlation of their dipole moment (Debye) with inhibition
afforded an R2 of 0.9361, as shown in Figure 2.
Figure 3. Structure of the two selected more
electronegative guanylhydrazones, 11 and 12,
and tacrine (13).
Table 2. Results of EeAChE inhibition with compounds 11, 12, and 13 by NMR and the Ellman’s test.
Ellman’s test
EqBuChE inhibition (%)
NMR test
Compound
EeAChE inhibition (%)
EeAChE inhibition (%)
11
12
13
63.79 0.48
45.49 0.15
100 0.42
5.74 2.3
34.90 7.3
100 0.4
75.6 3.5
41.3 2.1
93.7 3.4

DOI: 10.3109/14756366.2015.1094468
Design, synthesis, and evaluation of guanylhydrazones
7
The correlation of the electronic charge of the nitrogen atom
The results are shown in Table 2, and Figure 4 is the graphic of
bound to the carbonyl aldehydes with AChE inhibition also their NMR tests. The inclusion of the dipole moments of two
afforded an R2 ¼ 0.8310. These results confirm that the electron additional compounds 11 and 12 with their AChE activities in the
withdrawing characteristics of the R groups at the para-position correlation graph of compounds 5–10 afforded an R2 of 0.9119.
on the benzene ring affect the AChE-inhibiting capacity of these These results confirm that including more electronegative groups
guanylhydrazones.
To test this possibility after obtaining the previous results,
on the benzylidene ring increases the EeAChE inhibition capacity.
These results obtained by NMR are very similar to those
the two compounds with more electronegative groups on obtained by the Ellman’s test, confirming their capacity for
the benzylidene ring, 2,4-dinitrobenzaldehyde guanylhydrazone
(11) and 2,4-dichlorobenzaldehyde guanylhydrazone (12), were
Table 3. Docking results of compounds 2, 6, 11, 12, and tacrine with
TcAChE.
prepared and tested (Figure 3). The most electronegative
compound, 11, displayed EeAChE inhibition of 63.79% by
the Ellman’s test, with an IC50 of 11.25 mM, and 75.6% by
NMR, thus being the most effective compound, being superior
to compound 2. Compound 11 was also more selective for
EeAChE over EqBuChE. However, the bis-chlorinated com-
pound, (12), which is less electronegative, affords 45.49%
inhibition by the Ellman’s test and 41.3% inhibition by
NMR, therefore being the fourth most effective inhibitor. In this
case, tacrine (13) was used as the reference AChE inhibition
agent.
Intermolecular
energy (kcal/mol)
Binding
energy
Compound
2
6
11
ꢀ6.11
ꢀ5.16
ꢀ5.56
ꢀ5.59
ꢀ8.08
ꢀ5.52
ꢀ4.27
ꢀ4.37
ꢀ5.00
ꢀ8.08
12
Tacrine (13)
Figure 4. Kinetics and inhibition of EeAChE
with benzaldehyde guanylhydrazone (5), 2,4-
dinitrobenzaldehyde guanylhydrazone (11)
and tacrine (13) by NMR.
Figure 5. Correlation between the AChE
inhibition results using the Ellman or NMR
tests.

8
E. da Conceic¸a˜o Petronilho et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 6. Interaction superposition of tacrine
(13, green) and compound 11 (blue) in the
active site of TcAChE. For references to
color, see the online version of the article.
Figure 7. Docking interaction of tacrine (A),
compounds 2 (B), 6 (C), and 11 (D) in the
active site of TcAChE using carbon in
magenta. H-bond distances in angstroms. For
references to color, see the online version of
the article.
EeAChE inhibition. It is observed that 2,4-dinitrobenzaldehyde obtained from the Protein Data Bank under the code PDB
guanylhydrazone (11) displays a very efficient capacity to inhibit ID:1ACJ20, for docking. To determine the effectiveness of
EeAChE (75.6%), indicating that other similar derivatives could AutoDock 4.2, the re-docking of tacrine in the TcAChE site was
be more effective and that guanylhydrazones and their analogs executed to compare with the reported X-ray crystallographic
can be used as prototypes in the preparation of new drugs.
The correlation of AChE inhibition by the Ellman’s test with deviation obtained for the re-docking of tacrine inside of the 3D
that using NMR, as shown in Figure 5, indicate that these methods structure of TcAChE was 0.53, confirming the effectiveness of
structure of this enzyme-tacrine complex36. The root mean square
afford relatively similar results (R2 ¼ 0.8914).
AutoDock 4.2. After this, the docking of compounds 2, 6, 11, and
12 was performed, and their intermolecular binding energies were
determined. The data obtained, in comparison with tacrine, are
shown in Table 3.
Molecular docking studies
To confirm this information, the interaction of compounds 2 and 6
These docking results indicate that these compounds are
in comparison with tacrine (13) was studied using AutoDock relatively effective as AChE inhibitors and demonstrate that
4.2 software21. Because the structure of E. electricus AChE compounds 2 and 12 have similar action but are less effective than
(EeAChE) has not been reported as a complex with any ligand, we tacrine. Despite the docking results being relatively different from
used the structure of Torpedo californica AChE (TcAChE), the experimental results obtained by the Ellman and NMR tests

DOI: 10.3109/14756366.2015.1094468
Design, synthesis, and evaluation of guanylhydrazones
9
Table 4. Ellman’s test results for the reactivation of AChE inhibited with
paraoxon and incubated with the compounds for 20 min.
the phosphate group bound to Ser200, leading to enzyme
reactivation. Hydrazones are usually not acidic compounds, but
compound 2, which contains the N-methyl group on the pyridine
ring, is more acidic than average. Compound 3 also failed to
reactivate, in spite of the cationic pyridine ring, which would also
increase the acidity and nucleophilicity of the NH group. The
expected reactivation mechanism for the cationic compounds 4–
12 relied on deprotonation of their guanidine groups by active site
anionic amino acid residues. This would lead to neutral
intermediates with some nucleophilicity at the nitrogen atoms;
however, the mechanism was not effective. These negative results
are relevant for the design of new agents with more activity for
defense against neurotoxic organophosphorus compounds.
EeAChE activity (%)
Compounds
10 mM
100 mM
500 mM
1
2
3
4
5
6
7
8
74.42 1.69
1.32 0.52
1.16 0.95
0.61 0.17
0.21 0.19
0.28 0.73
0.19 0.20
0.03 0.13
0.38 0.31
1.02 1.27
ꢀ3.53 1.33
ꢀ1.68 1.54
75.41 2.45
ꢀ0.65 0.05
0.05 0.04
ꢀ0.13 0.04
0.07 0.01
ꢀ0.66 0.11
ꢀ0.04 0.13
ꢀ0.16 0.04
ꢀ0.17 0.12
0.28 0.08
–
73.28 4.82
ꢀ1.27 0.22
0.03 0.01
ꢀ0.88 0.42
ꢀ0.22 0.17
ꢀ8.14 1.47
ꢀ0.51 0.50
ꢀ0.77 0.18
ꢀ1.17 0.19
1.05 0.27
–
9
10
11
12
Conclusions
–
–
Our test for the reactivation of paraoxon-inhibited AChE with 1-
methylpyridine-2-carboxaldehyde hydrazone (2) indicates that in
general hydrazones may be ineffective. This result suggests that
for the reactivation of phosphorylated AChE, it is necessary to use
better nucleophiles, especially compounds that could be easily
deprotonated by the diverse basic groups of the amino acids in
this enzyme.
The analysis of guanylhydrazones by the Ellman’s test
indicates that they are also very poor AChE reactivators.
However, these agents display interesting AChE inhibition
capacity, being more effective when they possess electronegative
groups on their benzylidene ring. The polarization of guanylhy-
drazones increases in the presence of electronegative groups, with
the most effective being nitro groups, an effect that was confirmed
by the Ellman’s test, NMR and molecular modeling. This
condition indicates that guanylhydrazones with high polarization
interact better with the active site of AChE. With these results,
more effective AChE inhibitors are being designed as new
potential agents for the treatment of AD, indicating that
guanylhydrazones and analogs could be good effective agents
for AD.
(Tables 1 and 2), these results confirm that one hydrazone and
several guanylhydrazones are potential AChE inhibitors. The
superposition of the lowest energy poses of tacrine and compound
11 interacting in the active site of TcAChE is shown in Figure 6.
These results show that tacrine and compound 11, which is the
most effective experimental AChE inhibitor of the tested com-
pounds, interact with the active site of this enzyme, indicating that
both display a very similar inhibitory capacity.
Despite the importance of docking studies, these results
indicate that theoretical calculations usually are not in parallel
with experimental information. The interaction of compounds 2,
6, 11, and tacrine (13) are shown in Figure 7.
We have also performed a preliminary in silico screening of
drug properties of the most active compounds, using the web tools
Osiris Property Predictor (http://www.organic-chemistry.org/prog/
peo/) and lazar (http://lazar.in-silico.ch/predict). Compounds 2, 6,
and 11 all obey the rule-of-five principle and showed low toxicity
risk, with confidence values below 0.25 in all assessments of
carcinogenicity and mutagenicity. The pyridinium compounds,
including pralidoxime (1) and 2, are not expected to permeate the
blood-brain-barrier efficiently. However, compounds 6 and 11
showed CLogP of 0.11 and ꢀ0.81, respectively, which is
compatible with brain penetration and eventual application in AD.
Acknowledgements
We would like to thank the following Brazilian funding agencies for
financial support: INBEB, CAPES, CNPq and FAPERJfor the financial
support.
Declaration of interest
EeAChE reactivation
The authors report no conflict of interest.
To perform the AChE reactivation tests, EeAChE was inhibited
with paraoxon (100 nM) by the phosphorylation of Ser200. The
complete inhibition of this enzyme was also confirmed by the
Ellman’s test. To test the activity of these compounds,
pralidoxime (2-PAM, 1) was used as a reference material. The
Ellman’s tests for the reactivation of EeAChE inhibited with
paraoxon confirmed that pralidoxime (1) is the only good
reactivator, with the guanylhydrazones being ineffective, as
shown in Table 4. As expected, 1 completely reactivated
EeAChE, considering that it directly inhibited the enzyme by
24.3% at 100 mM (Table 1), so that the maximum expected
activity in the reactivation assay would be 75.7%. Likewise, for
compounds 2–12, only partial recovery of activity should be
expected after reactivation, due to their own inhibitory properties.
The reactivation of EeAChE inhibited with paraoxon was also
assayed with incubation times of 30 and 60 min but did not show
significant alteration of the results.
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Supplementary material available online