Synthesis of new quinoline-piperonal hybrids as potential drugs against Alzheimer’s disease

Article abstract of DOI:10.3390/ijms20163944

Six quinoline-piperonal hybrids were synthesized and evaluated as potential drugs against Alzheimer’s disease (AD). Theoretical analysis of the pharmacokinetic and toxicological properties of the compounds suggest that they present good oral bio-availability and are also capable of penetrating the blood–brain barrier, qualifying as leads for new drugs against AD. Evaluation of their inhibitory capacity against acetyl-and butyrilcholinesterases (AChE and BChE) through Ellmann’s test showed that three compounds present promising results with one of them being capable of inhibiting both enzymes. Further docking studies of the six compounds synthesized helped to elucidate the main interactions that may be responsible for the inhibitory activities observed.

Full text of DOI:10.3390/ijms20163944

International Journal of
Molecular Sciences
Article
Synthesis of New Quinoline-Piperonal Hybrids as
Potential Drugs against Alzheimer’s Disease
Juliana de Oliveira C. Brum 1,
* ,
, Denise Cristian F. Neto 1, Joyce Sobreiro F. D. de Almeida 2
Josélia Alencar Lima 2,3, Kamil Kuca 4,*, Tanos Celmar C. França 2,4 and
José D. Figueroa-Villar 1,
*
1
Medicinal Chemistry Group, Department of Chemistry, Military Institute of Engineering, Praça General
Tibúrcio 80, 22290-270 Rio de Janeiro, RJ, Brazil
2
3
4
Laboratory of Molecular Modeling Applied to the Chemical and Biological Defense (LMCBD), Military
Institute of Engineering, Praça General Tibúrcio 80, 22290-270 Rio de Janeiro, Brazil
Graduate Program in Adult Health, Federal University of Maranhão, Avenida dos Portugueses, 1966. Vila
Bacanga, 65080-805 São Luís, MA, Brazil
Department of Chemistry, Faculty of Science, University of Hradec Kralove, Rokitanskeho 62, 50003 Hradec
Kralové, Czech Republic
*
Correspondence: juliana_brum2005@yahoo.com.br (J.d.O.C.B.); kamil.kuca@uhk.cz (K.K.);
jdfv2009@gmail.com (J.D.F.-V.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 June 2019; Accepted: 9 August 2019; Published: 14 August 2019
Abstract: Six quinoline-piperonal hybrids were synthesized and evaluated as potential drugs against
Alzheimer’s disease (AD). Theoretical analysis of the pharmacokinetic and toxicological properties of
the compounds suggest that they present good oral bio-availability and are also capable of penetrating
the blood–brain barrier, qualifying as leads for new drugs against AD. Evaluation of their inhibitory
capacity against acetyl- and butyrilcholinesterases (AChE and BChE) through Ellmann’s test showed
that three compounds present promising results with one of them being capable of inhibiting both
enzymes. Further docking studies of the six compounds synthesized helped to elucidate the main
interactions that may be responsible for the inhibitory activities observed.
Keywords: piperonal; quinolines; guanil-hydrazones; acetylcholinesterase; Alzheimer’s disease
1. Introduction
Alzheimer’s disease (AD) is a progressive and neurodegenerative disease recognized today as
the most common cause of dementia worldwide [1–3]. During the manifestation of this disease
an atrophy of the Meynert nucleus basalis occurs in the brain, resulting in lowering levels of the
neurotransmitter acetylcholine (ACh). This compromises the transmission of nerve impulses in the
patient body, causing serious damages on his cognitive functions, characteristic of AD. The therapeutic
approach to counteract this effect is raising the concentration of ACh through the inhibition of the
enzymes acetyl- and butyrilcholinesterase (AChE and BChE), which are responsible for the hydrolysis
of ACh in the synaptic clefts [4]. For this reason the cholinesterase inhibitors have been the first line
drugs for treatment of light to moderate manifestations of AD. These drugs are the majority of the few
drugs approved so far for the treatment of AD [5].
Donepezil, rivastigmine and galantamine are the cholinesterase inhibitors approved today for
clinical use against AD. Donepezil and galantamine are selective inhibitors of AChE, while rivastigmine
is a dual inhibitor of both AChE and BChE [6]. However, these drugs can cause side effects like nausea,
vomiting and diarrhea, in addition to cardiovascular and neurologic effects.
Tacrine is also an effective cholinesterase inhibitor for the symptomatic treatment of AD. It is
capable of avoiding the excessive degradation of ACh, increasing the cholinergic neurotransmission [7].
Int. J. Mol. Sci. 2019, 20, 3944; doi:10.3390/ijms20163944
www.mdpi.com/journal/ijms

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However, this drug is also capable of raising the serum levels of the enzyme alanine aminotransferase,
provoking considerable hepatoxicity. For this reason the use of tacrine was discontinued in USA in
2013 [8]. Its quinolinic nucleus, however, presents a wide range of pharmacologic activities, being for
this reason incorporated into a wide variety of compounds [
9–
11]. Considering the pharmacological
potential of the quinolinic nucleus of tacrine we investigated in this work hybrid compounds, merging
it with piperonal, and evaluated their cholinesterase activities. Our compounds contain the 3,4-dioxole
bridge, known as a good acceptor of hydrogen bonds—a fact that may increase their capacity of
inhibiting AChE [12]. We also evaluated the inclusion of a guanylhydrazone group in order to
investigate the effects of this group inside AChE. This functional group is still little investigated in the
literature as a cholinesterase inhibitor. Our group has pioneered this research in a previous work [13
]
where we report the results of the guanylhydrazones shown in Figure 1 as potential cholinesterase
inhibitors. These compounds were evaluated against AChE through Ellman’s and NMR studies
and presented inhibitory activity of AChE from Electrophorus electricus (EeAChE), confirming the
guanylhydrazones as potential inhibitors of AChE. We believe that this activity is due to the presence
of the cationic groups, able to interact with the peripheral anionic site (PAS) of AChE [13].
O2N
O2N
NH2
_
NH2
_
+
+
Cl
N
Cl
N
N
H
NH2
N
H
NH2
NO2
Figure 1. Structures of guanylhydrazones capable of inhibiting acetylcholinesterase (AChE) reported
by Petronilho et al. [13].
2. Results and Discussion
2.1. Synthesis of the Quinoline-Piperonal Hybrids
The synthetic route of this work started with the transformations of piperonal into intermediates
6-nitro-1,3-benzodioxole-5-carbaldehyde (1) and 6-amino-1,3-benzodioxole-5-carbaldehyde (2), as
shown in Scheme 1 [14–16].
CHO
NH2
CHO
NO2
CHO
O
O
O
O
O
O
1) FeSO4, H2O, 1 min
HNO3
0 oC, 5h
2) NH4OH, 200 oC, 10 min
(2)
(1)
Scheme 1. Synthesis of intermediates (1) and (2).
The hybrids piperonal-quinoline were synthesized from condensations via Friedländer
reaction [14], between intermediate (2) and the appropriate di-carbonyl compound as shown in
Schemes 2 and 3 for the formation of compounds 7,8-dihydro-7,7-dimethyl-1,3-dioxolo[4,5-b]acridin-
9(6h)-one (3) and 6-methyl-[1,3]dioxolo[4,5-g]quinone-7-carboxilate (4). Due to the characteristics of
the dicarbonyl compound used, the synthesis of compound (3) was performed under acid media. The
acid catalysis promotes the protonation of one of the carbonyl groups of the di-carbonyl compound,
allowing the formation of a double bond inside the ring and favoring the nucleophile attack to
intermediate (2). This way the aldol bond is formed, followed by the imine formation (see mechanism
in Figure S1 of Supplementary Materials). Through this reaction, compound (3) was obtained as a
brown solid with melting point ranging between 176–178 ◦C, and 68% of yield [17].

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O
O
CHO
O
O
O
Dioxane, HCl 6M
Reflux, 120 h
+
O
N
O
NH2
(3)
(2)
Scheme 2. Synthesis of compound (3).
O
O
O
CHO
O
O
O
MeOH, Et3N
Reflux, 24 h
O
+
O
O
N
NH2
(2)
(4)
Scheme 3. Synthesis of compound (4).
The synthesis of compound (4) was performed under basic media. In this case the basic catalysis
allowed the formation of an efficient nucleophile from the di-carbonyl compound (Scheme 3), favoring
the nucleophile attack to compound (2) (see proposed mechanism for this reaction in Figure S2 of
Supplementary Materials). This reaction produced compound (4) as a white solid with melting point
around 207–209 ◦C and 53% of yield [18].
The acid 5,6-diidro-6-oxo-1,3-dioxolo [4,6-g] quiniline-7-carboxilic (5) was obtained from the
reaction between (2) and the Meldrum acid, as shown in Scheme 4. For production of (5) after aldol
condensation and formation of the hydroxyl-imine, the hydrolysis of the Meldrum acid ring with
elimination of one molecule of acetone is necessary. A possible mechanism for this reaction is shown
in Figure S3 of Supplementary Materials.
O
O
CHO
NH2
O
O
O
O
EtOH, HCl 6M
Reflux, 96 h
O
+
N
H
(5)
O
O
O
(2)
Scheme 4. Synthesis of compound (5).
Compound (6) (1,3-dioxolo [4,5-g] pirimido [4,5-b] quinoline-7,9 (6H,8H)-dione [17]) was
synthesized from the reaction between compound (2) and pyrimidine-2,4,6 (1H, 3H, 5H)-trione
as shown in Scheme 5. Dioxane and ethanol were used as solvents in the early attempt of synthesis of
this compound but both solvents led to low yields. The use of the higher boiling point solvent butanol
allowed rising the reaction temperature and achieving 62% of yield.
O
O
CHO
NH2
O
O
O
O
BuOH, HCl 6M
Reflux, 72 h
NH
+
N
H
(6)
O
N
O
N
H
O
(2)
Scheme 5. Synthesis of compound (6).
Compound (7) (ethyl 5,6-dehidro-6-oxo-[1,3] dioxolano [4,5-g] quinoline-7-carboxilate) [19] was
synthesized from the reaction between compound (2) and diethyl propanedioate, as shown in Scheme 6.
The replacement of butanol by dioxane as solvent allowed the obtainment of a sole product. As dioxane
is an inert solvent, its use avoided the formation of secondary products due to transterification.

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O
O
O
O
CHO
O
O
O
Dioxane, Et3N
Reflux, 72 h
O
+
O
O
O
N
H
NH2
(2)
(7)
Scheme 6. Synthesis of compound (7).
Compound (8) (guanylhydrazone of 7,7-dimethyl-7,8-dihydro-[1,3]dioxolo[4,5-b]acridin-9
(6h)-one) was synthesized from the reaction between compound (3) and hydrazinecarboximidamide,
as shown in Scheme 7. This reaction was also performed under acid media because the protonation of
the carbonyl oxygen of (3) makes the subsequent nucleophile substitution more efficient, allowing the
formation of the guanylhydrazone. Compound (8) was obtained as a dark yellow solid with melting
point around 269–271 ◦C and 51% of yield [20]. This compound was also found to be never reported
before in the literature.
H2N
N
NH
NH
O
NH
NH2
NH
O
O
O
O
EtOH, HCl 6M
Reflux, 72 h
H2N
+
N
N
(3)
(8)
Scheme 7. Synthesis of compound (8).
Compound (8) represents the junction of three different chemical classes in the same molecule,
where it is possible to find the piperonal, quinoline and guanylhydrazone moieties (see Figure 2). It
was expected that these different groups enable interactions with the active site of AChE, like tacrine
does, as well as the PAS, due to the positive charges of the guanidine group. It was also expected that
the oxole group, from the piperonal moiety, enables H-bond interactions with residues of the active
site, increasing the affinity of this compound for the enzyme.
Figure 2. Different chemical classes present in the structure of compound (8).
2.2. Calculation of Pharmacokinetic and Toxicological Properties
The tridimensional structures of the compounds studied and the values of weight (amu), area
(Å2), volume (Å3), and polar surface area (PSA, Å2) were obtained with the software Spartan’08, using
Density Functional with M06 and the basis set 6-311++G** [21]. Some pharmacological properties
were calculated by the online program Pre ADMET (https://preadmet.bmdrc.kr/adme-prediction/):
toxicity, Lipinski’s rule (rule of five) [22
,23] and blood–brain barrier (BBB) penetration. The values
obtained for compounds (3)–(8) and tacrine are shown in Table 1.

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Table 1. Values of weight (amu), area (Å2), volume (Å3), polar surface area (PSA, Å2), acute toxicity,
Lipinski’s rule and blood–brain barrier (BBB) calculated for compounds (3)–(8).
Weight
(amu)
Area
(Å2)
Volume
(Å3)
Acute Toxicity Carcinog.
Meet the
Lipinski’s Rule?
BBB
Penetration
PSA (Å2)
Comp
(algae)
(mouse)
3
4
5
6
7
8
269.30
245.23
233.18
257.21
261.23
325.37
198.27
275.40
250.08
216.47
235.21
260.62
334.47
225.26
266.12
233.59
202.36
221.47
242.00
317.07
213.94
38.55
45.63
70.70
75.45
63.40
89.56
30.24
0.05
0.09
0.15
0.19
0.12
0.03
0.05
Negative
Negative
Positive
Positive
Negative
Negative
Positive
Yes
Yes
Yes
Yes
Yes
Yes
Yes
2.69
1.37
0.29
0.39
0.04
0.11
0.86
Tacrine
According to the Lipinski’s rule of five [22
,
23], the PSA must present values
≤
140 and
≤
90 Å2,
respectively, to provide good oral bioavailability and good penetration into the BBB. Therefore, our
results (Table 1) suggest both a good oral bio-availability, in addition to capability of penetrating
the BBB and entering the central nervous system (CNS), for all compounds studied. Among them,
according to the model proposed by Ma et al. [24], compounds (3), (4), (6) and tacrine would be the ones
with higher CNS uptake, while compounds (5), (7) and (8) would show average uptake by the CNS.
Regarding acute toxicity, (algae) compound (8) was the only one presenting a calculated value
lower than tacrine’s. This result suggests that this compound can be a suitable lead for the design
of less toxic anti-AD drugs. Together with (3), (4) and (7), compound (8) also showed a negative
prediction for rodent carcinogenicity.
2.3. Cholinesterase Inhibition Assay through Ellman’s Method
The inhibitory activities of compounds (3) (8) were evaluated through the modified
spectrophotometric Ellman’s method [25 26] on EeAChE and butyrylcholinesterase from equine
–
,
serum (EqBChE). Results (Table 2) show that while compound (4) is not able to inhibit any of the
enzymes, compound (8) is a non-selective inhibitor of both enzymes, being twice more potent in
inhibiting EeAChE (IC50 = 8.50
±
0.39
µ
M ) than EqBChE (IC50 = 15.16
±
0.75 µM). The other compounds
(
(3), and (5) (7)) selectively inhibited EeAChE. Tacrine, a non-selective inhibitor of both enzymes, with
–
major selectivity towards BChE, instead of AChE [27], was the reference compound.
Table 2. IC50 values, in µM, of compounds (3)–(8) compared to tacrine.
Compound
EeAChE
EqBChE
(3)
(4)
51.51 ± 2.47
-
-
-
(5)
(6)
(7)
(8)
14.15 ± 1.15
32.06 ± 3.53
62.76 ± 2.02
8.50 ± 0.39
0.0414 ± 0.0002
-
-
-
15.16 ± 0.75
Tacrine
0.0045 ± 0.0005
The compounds synthesized in this work were designed with a dioxole ring because according to
the literature this ring is important for AChE inhibition, due to the capacity of accepting H-bonds of
the 3,4-dioxole moiety [12
molecule. This confers rigidity and planarity, allowing
of the AChE gorge [20 30 31]. Comparing compounds (3) and (8) it is possible to see that they differ
only by the substitution of a carbonyl group in compound (3) by an amino-guanidine in compound (8)
,
28,
29]. Our compounds also present two aromatic rings in the middle of the
π–π
interactions with the aromatic residues
,
,
.
This structural change conferred to (8) an inhibitory activity over EeAChE six times higher than (3)
,
besides making this compound also active towards EqBChE.
According Sugimoto et al. [30], carbonyl groups conjugated to aromatic rings are important for
inhibition of AChE. However, Ferreira Neto et al. [25] showed that the presence of a guanyl-hydrazone

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allows interactions with the catalytic anionic site (CAS) of AChE, improving the activity, as also
shown here.
2.4. Kinetic Study of Compound (8) by NMR
As compound (8) presented the lowest IC50 value in Ellman’s test, it was selected for an additional
kinetic study by NMR [32] in order to verify the percentage of inhibition when compared to the
reference drug (tacrine). The enzyme selected for this study was EeAChE because it displays an
identical active site and a very similar activity to the human enzyme [33,34].
NMR is a good method for the biological evaluation of AChE inhibitors. Recent published works
have indicated results consistent with Ellman’s method [25,32–36]. In our study we used the NMR
method [29] to determine the concentrations of the substrate (ACh) and the product acetate (Ac),
which are obtained from direct integration of the corresponding absorption peaks for methyl groups
(2.24 ppm for ACh and 2.16 ppm for Ac) [32,35]. The comparison between the integration of the Ac
peak in pure AChE (no inhibitor) and in the presence of the inhibitor allowed the determination of the
percentages of inhibition shown in the plot of Figure 3.
Figure 3. AChE inhibition by compound (8) using the NMR test.
Results showed that compound (8) presented 73.90
±
6.26 of AChE inhibition versus 93.50
±
5.42
of tacrine. This corroborates recent studies [13 25 36] showing that the presence of an electrophilic
,
,
region in the molecule (the guanylhydrazone moiety) may favor interactions with the enzyme and thus
allow its inhibition to be more effective. This result reaffirms the importance of this class as promising
for the inhibition of AChE and suggests that similar derivatives should be developed as prototypes for
the design of more efficient drugs.
2.5. Docking Studies
2.5.1. Docking Studies on EeAChE
The root mean square deviation (RMSD) values obtained for tacrine inside EeAChE and EqBChE
were 0.28 and 0.30 respectively. These values are enough to validate our docking protocol because,
according to the literature [37], RMSD values < 2.50 Å are considered acceptable. The re-docking poses
obtained for tacrine inside EeAChE and EqBChE are shown in Figure S4 of Supplementary Materials.
Table 3 presents the values of energy for the enzyme–ligand interactions and H-bonds, as well
as the corresponding interacting residues, for the best poses of each ligand inside EeAChE, obtained
from the docking studies (Figure 4). As can be seen, compounds (3)–(8) presented negative values
of intermolecular energy inside EeAChE. This suggests that they present stabilization in the region
between the CAS and PAS. Residues Tyr124 (PAS residue), Tyr133 (CAS residue) and His447 (which is
a residue of the catalytic triad) contributed to the stabilization of the ligands, with compound (8) as the
one presenting the lowest intermolecular energy. This is in accordance with the experimental results
pointing to this compound as the best AChE inhibitor among the studied compounds.

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Table 3. Docking results inside AChE from Electrophorus electricus (EeAChE).
Einter (Kcal.mol−1
)
EH-bond (Kcal.mol−1
)
Ligand
H-bond Interactions
RMSD (Å)
Redocking Tacrine
−132.39
−132.69
−135.51
−140.88
−145.33
−119.49
−155.88
−0.20
−0.13
−0.18
−0.22
−1.76
−0.46
−2.35
His447
Tyr133
Trp439, Tyr449
Tyr133
Tyr133, His447
Tyr124, Ser125
Tyr133, His447
0.28
(3)
(4)
(5)
(6)
(7)
(8)
-
-
-
-
-
-
Figure 4. Best docking poses of the ligands inside EeAChE.
2.5.2. Docking Studies on EqBChE
Table 4 presents the values of energy for the enzyme-ligand interactions and H-bonds, as well
as the corresponding interacting residues, for the best poses of each ligand inside EqBChE, obtained
from the docking studies (Figure 5). All the compounds presented negative values of intermolecular
energy, suggesting that they present stabilization inside EqBChE. Compound (8) presented the lowest
intermolecular energy, pointing to a better stabilization than other compounds. The residues Asp70,
Tyr332 (PAS residues), Tyr128 (CAS residue) and His438 (which is a residue of catalytic triad) contributed
to the stabilization of the ligands. Regarding the experimental results, it is important to notice that,
besides tacrine, (8) was the only compound that was able to also inhibit EqBChE.
Table 4. Docking results on butyrylcholinesterase from equine serum (EqBChE).
Einter (Kcal.mol−1
)
EH-bond (Kcal.mol−1
)
Ligand
H-bond Interactions
RMSD (Å)
Redocking Tacrine
−133.63
−153.23
−152.11
−155.08
−151.08
−158.06
−196.99
−2.08
−2.50
−5.27
−5.73
−3.96
−4.26
−5.43
His438
Ser198
Gly116, Tyr128, Tyr332
Tyr128, Tyr332, His438
Tyr128, Tyr332
0.30
(3)
(4)
(5)
(6)
(7)
(8)
-
-
-
-
-
-
Tyr128, Tyr332
Asp70, Ser198, Tyr332

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Figure 5. Best docking poses of the ligands inside EqBChE.
3. Materials and Methods
3.1. Chemistry
Solvents (dioxane, ethyl alcohol, methanol, chloroform), used with further purification, were
purchased from VETEC, and reagents were purchased from Sigma-Aldrich. Reactions were monitored
by TLC using DCAlufolien Kieselgel 60 F254 (Merck, Kenilworth, NJ, USA).
3.2. Synthesis of The Compounds
3.2.1. 6-Nitro-1,3-benzodioxole-5-carboxaldehide [6-nitropiperonal, (1)]
The synthesis was done following the procedure described in literature [14,15] with some
modifications to improve the yield. Firstly, 20.0 g of piperonal powdered to a fine dark brown powder
was added to 100 mL of HNO3 65% PA, over an ice bath, under constant stirring. After 3 h of reaction
the light yellow precipitate that formed was filtered and washed with cold water till total removal of
the HNO3 [11
/ppm 10.08 (1H,s); 7.74 (1H,s); 7.32 (1H,s); 6.33 (2H,s); NMR 13C (DMSO-d6, 125 MHz):
δ/ppm 188.18;
,
12]. Physical aspect: light yellow solid, yield: 91%. NMR 1H (DMSO-d6, 600 MHz):
δ
151.89; 151.31; 145.67; 127.47; 107.04; 104.99; 104.40.
3.2.2. 6-Amino-1,3-benzodioxole-5-carboxaldehide [(6-aminopiperonal, (2)]
Firstly, 1.00 g of compound (1) was first solubilized in 50.0 mL of boiling ethanol 50%. Next, a
solution of 10 g of ferrous sulfate heptahydrate, in 50.0 mL of distilled water, was added to the flask
and left under heating for 1 min. After this time, 1.00 mL aliquots of NH4OH concentrated were added
at intervals of 30 s till achieving 13.0 mL, and left at 100 ◦C for 10 min. The yellow solution obtained
after filtration produced a dark yellow precipitate after cooling for some hours in an ice bath [16,38].
This precipitate was recrystallized in distilled water, producing dark yellow needles. Physical aspect:
dark yellow solid; yield: 77%, NMR 1H (DMSO-d6, 600 MHz):
(1H,s); 6.31(1H,s); 5.96 (2H,s); NMR 13C (DMSO-d6, 125 MHz):
110.95; 110.66; 101.26; 95.18.
δ
/ppm 9.54 (1H,s); 7.24 (2H,bs); 6.98
/ppm 190.49; 153.55; 150.12; 138.10;
δ

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3.2.3. 7,8-Dihydro-7,7-dimethyl-1,3-dioxolo[4,5-b]acridin-9(6h)-one (3)
Firstly, 1 mmol of dimedone (0.140 g), 20.0 mL of dioxane and 1 mmol of compound (2) (0.165 g)
solubilized in 20.0 mL of dioxane were mixed in a tritubed flask and left under reflux for 72 h [17]. The
product was purified through chromatographic column. Physical aspect: brown solid; yield: 68%;
NMR 1H (DMSO-d6, 600 MHz):
(2H,s); 0.96 (6H,s); NMR 13C (DMSO-d6, 125 MHz):
123.70; 104.76; 104.47; 102,86; 52.04; 46.21; 32.89; 28.26.
δ
/ppm 8.56 (1H,s); 7.44 (1H,s); 7.25 (1H,s); 6.17 (2H,s); 2.99 (2H,s); 2.50
/ppm 197.84; 159.23; 153.27; 148.91; 148.01; 134.32;
δ
3.2.4. Methyl 6-Methyl-[1,3]dioxolo[4,5-g]quinone-7-carboxilate (4)
Firstly, 1 mmol of compound (2) (0.165 g) was solubilized in 10.0 mL of methanol and mixed with
1 mmol of ethyl acetoacetate and a catalytic amount of triethylamine. The solution was maintained
under stirring and reflux for 120 h [18]. The white precipitate obtained after this time was filtered,
◦
1
washed and dried. Physical aspect: white solid; melting point: 207-209 C; yield: 53%. NMR H
(DMSO-d6, 600 MHz): δ/ppm 8.65 (1H,s); 7.45 (1H,s); 7.30 (1H,s); 6.23 (2H,s); 3.88 (3H,s); 2.78 (3H,s).
NMR 13C (DMSO-d6, 125 MHz):
104.30; 103.33; 102.37; 52.18; 24.79.
δ/ppm 166.49; 155.40; 152.53; 147.63; 146.93; 138.06; 122.25; 121.04;
3.2.5. Acid 5,6-Diidro-6-oxo-1,3-dioxolo[4,6g] quiniline-7-carboxilic (5)
Firstly, 1 mmol of compound (2) (0.165 g) were added to a tritubed flask and solubilized under
heating in 20.0 mL of dioxane. Then, 1 mmol of meldrum acid (0.144 g) solubilized in 20.0 mL of
dioxane was added and the resulting solution kept under reflux. Next, 16 drops of HCl 6 M were added
to this solution to promote an acid catalysis. After 16 h the formation of the product was observed by
TLC. Physical aspect: brown solid; melting point > 300 ◦C; yield: 56 %. NMR 1H (DMSO-d6, 600 MHz):
δ
/ppm 14.85 (1H,s); 13.14 (1H,bs); 8.81 (1H,s); 7.51 (1H,s); 6.98 (1H,s); 6.21 (2H,s). NMR 13C (DMSO-d6,
125 MHz): δ/ppm 164.99; 163.73; 153.60; 145.37; 145.10; 138.01; 114.44; 113.94; 106.23; 102.84; 95.41.
3.2.6. 1,3-Dioxolo [4,5-g] pirimido [4,5-b] quinoline-7,9 (6H,8H)-dione (6)
Firstly, 20.0 mL of butanol and 1 mmol of compound (2) (0.165 g) were added to a tritubed flask.
Then, a solution containing 1 mmol of barbituric acid (0.128 g) in 20.0 mL of buthanol was added to this
solution. Next, 15 drops of HCl 6M were added, and the resulting solution kept under reflux for 4 h.
The rose-colored precipitate formed was filtered and dried. Physical aspect: pink solid; melting point >
300 ◦C; yield: 62%; NMR 1H (DMSO-d6, 600 MHz):
δ/ppm 11.55 (1H,bs); 11.39 (1H,s); 8.74 (1H,s); 7.50
(1H,s); 7.19 (1H,s); 6.24 (2H,s); NMR 13C (DMSO-d6, 125 MHz):
148.57; 146.62; 136.61; 121.18; 108.28; 103.88; 103.04; 102.42.
δ/ppm 162.25; 153.53; 150.49; 148.94;
3.2.7. Ethyl 5,6-Dehidro-6-oxo-[1,3] dioxolano [4,5-g] quinoline-7-carboxilate (7)
Firstly, 1 mmol of compound (2) (0.165 g) solubilized in 10.0 mL of dioxane was added to 1 mmol
of diethyl malonate in 10.0 mL of dioxane in a tritubed flask. After 72 h of reflux, the light green
precipitated obtained was filtered, washed and dried. Physical aspect: light green solid; melting point;
241–243 ◦C; yield: 71 %. NMR 1H (DMSO-d6, 600 MHz):
δ/ppm 11.92 (1H,s); 8.39 (1H,s); 7.31 (1H,s);
6.80 (1H,s); 6.13 (2H,s); 4.24 (2H,q, 7.0Hz); 1.28 (3H,t, 7.0Hz). NMR 13C (DMSO-d6, 125 MHz):
164.45; 158.34; 152.30; 143.82; 143.61; 138.38; 119.09; 112.02; 105.98; 102.19; 94.55; 60.32; 14.18.
δ/ppm
3.2.8. Guanylhydrazone of 7,7-Dimethyl-7,8-dihydro-[1,3]dioxolo[4,5-b]acridin-9(6h)-one (8)
Firstly, 1 mmol of aminoguanidine chloridrate (0.111 g), 10.0 mL of ethanol, and 10 drops of HCl 6
M were mixed in a tritubed flask and kept under reflux for 10 min. Then, 1 mmol of compound (3)
(0.269 g) and 10 mL of ethanol were added, and the resulting solution kept under reflux for 72 h [20].
The dark yellow precipitate obtained was filtered, washed and dried. Physical aspect: dark yellow
solid; melting point: 269–271 ◦C; yield: 51 %. NMR 1H (DMSO-d6, 500 MHz):
δ/ppm 11.63 (1H,s);

Int. J. Mol. Sci. 2019, 20, 3944
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9.74 (1H,s); 8.03 (4H,bs); 7.68 (1H,s); 7.45 (1H,s); 6.39 (2H,s); 3.18 (2H,s); 2.73 (2H,s); 1.06 (6H,s). NMR
13C (DMSO-d6, 125 MHz):
δ/ppm 156.08; 154.42; 152.58; 149.27; 146.85; 139.51; 137.40; 124.92; 123.84;
103.82; 103.39; 98.28; 56.02; 38.50; 30.49; 27.87. IV (KBr, cm−1): 3318; 2963; 2700; 1682; 1597; 1466; 1366;
1265; 1142; 1034; 933.
3.3. NMR Analysis
The NMR analysis of compounds was executed using a Varian 600 MHz (Premium compact TM)
using tubes of 5 mm and DMSO-d6 as solvent. All spectra were executed at 25 ◦C and using 20 mg of
each sample. The NMR spectra produced were 1H, 13C, APT, Gated Decoupling 13C, gHSQC, gHMBC
and ROESY-1D.
3.4. Structure Optimization and Calculation of Pharmacokinetic and Toxicological Properties
As before [32], the structures of the new compounds were built and optimized, using the
density functional theory (DFT) with M06 and 6-311++G** of Spartan08®
[21]. In addition, the
values of Weight (amu), Polar Surface Area (PSA, Å2), Area (Å2) and their Volume (Å3) were
obtained. Some pharmacological properties were calculated through the online program Pre ADMET
(https://preadmet.bmdrc.kr/adme-prediction/): toxicity, Lipinski’s rule (rule of five) and blood–brain
barrier penetration (BBB). These predictions are important for the characterization of these compounds
and to evaluate if they have the capacity as pharmacological agents.
3.5. Cholinesterase Inhibition Assay by Ellman’s Method
In order to determine the anticholinesterase activity, we used Ellman’s method [26] modified
for a 96-wells plate [39]. The velocities of substrate hydrolysis by AChE and BChE as function of
sample concentration were evaluated for EeAChE and EqBChE. AChE, BChE and Ellman’s reagent
(DTNB) were prepared in phosphate buffer (100 mM, pH 7.4). Acetylthiocholine iodide (ATCI), and
butyrylthiocholine iodide (BTCI) were prepared in distilled water. Stock samples (100 mM) were
prepared in dimethyl sulphoxide (DMSO) and appropriately diluted in distilled water to the desired
concentrations immediately before use. All solutions were kept on ice during the experiment. All
experiments were performed at 37 ± 1 ◦C.
All experimental wells received EeAChE (0.01 U/mL) or EqBChE (0.05 U/mL), DTNB (0.25 mM),
and phosphate buffer (control—enzyme activity) or sample solutions (1 to 100 µM). The mixture was
incubated for 10 min. Then, ATCI (0.5 mM) or BTCI (1.0 mM) was added to all wells and the plate
was read immediately for 5 min in a spectrophotometer (Spectramax 340PC, Molecular Device®). The
spontaneous and sample (100 µM) induced hydrolysis of the substrate were evaluated by replacing
enzyme for buffer. No sample-induced hydrolysis was observed for the compounds studied. The
solvent (DMSO) was evaluated at the highest concentration (0.2%) used in the experiment. All
concentrations refer to final concentrations. The samples were tested in at least 5 concentrations. The
enzyme activity (absorbance/min) in the samples was determined by comparison with the control
(mixture without sample) and expressed as the change in the optic deviation at 412 nm. The values of
absorbance/min were calculated by the software Softmax Pro 6.4®. Inhibition values were calculated
through non-linear regression with the software GraphPadPrism 5 (GraphPad software®, San Diego,
CA, USA). For each sample, results correspond to average
each one being performed in triplicate. EeAChE (EC.3.1.1.7), EqBChE (EC3.1.1.8), ATCI, BTCI and
DTNB were purchased from Sigma-Aldrich (São Paulo, Brazil).
±
standard deviation of three experiments,
3.6. AChE Inhibition Assay by NMR Method
All NMR analyses were performed using an NMR method formerly used in our research group [32
on a Varian Premium COMPACT TM 600 MHz spectrometer using a 5-mm probe at 25 C. Two
]
◦
microliters of EeAChE 10
µM in phosphate buffer (100% D2O, pH 7.4) in the presence of 1% bovine
serum albumin was used. This enzyme solution was mixed with 30
µL of ACh (100 mM in D2O),

Int. J. Mol. Sci. 2019, 20, 3944
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and then it was diluted to 600
µL using phosphate buffer (100 mM, 100% D2O, pH 7.4) in the NMR
tube (final concentration = 5 mM for ACh and 33 nM for the enzyme). This sample was immediately
inserted in the magnet for locking and shimming, allowing for the observation of the first 1H spectra
exactly 5 min after the addition of ACh. The next 1H spectra were acquired at every 5 min with a single
scan over 75 min. For testing the new compound, the same procedure was performed, including the
addition of 5 µL of the potential EeAChE inhibitor (final concentration of the inhibitor in the NMR
tube = 10 mM) before the addition of ACh. The concentrations of ACh and Ac were determined by the
integration of the methyl signals (ACh at 2.24 ppm and Ac at 2.16 ppm). The values generated from
the integration of the signals of ACh (consumed during the reaction) and Ac (produced during the
reaction) are compared between each other and to the values of Ac produced in the presence and in
the absence of the inhibitor. This enables the calculation of the percentages of inhibition, as described
before [32]. All analyses were performed in triplicate.
3.7. Docking Studies
The protonation states of the ligands at pH = 7.4 were obtained using the server Chemicalize
web-based resource supported by ChemAxon Ltd. at https://chemicalize.com/welcome [40]. This
server predicts aqueous ionization constants (pKa) of organic molecules based mainly on empirically
calculated partial charges and parameterized H-bonds. The tridimensional structures of the most
abundant chemical species of each ligand as calculated by the Chemicalize server [40] were constructed
with the software PC Spartan Pro®
[21] and had their partial atomic charges calculated by the RM1
(Recife Model 1) semi-empirical method [41].
The model of EeAChE inhibited by tacrine was constructed from the crystallographic structure
of AChE from Torpedo californica (TcAChE) inhibited by tacrine available in the RCSB PDB Protein
Data Bank (http://www.rcsb.org/pdb/home/home.do), under the code 1ACJ, and the crystallographic
structure of EeAChE, available in PDB under the code 1C2B. These two structures were aligned using
the software spdbviewer [42] and the coordinates of tacrine were copied from the TcAChE to the
EeAChE file. The model of EqBChE inhibited by tacrine was constructed from the crystallographic
structure of BChE from Homo sapiens sapiens (HssBChE) complexed with tacrine in the active site,
available in the RCSB PDB Protein Data Bank (http://www.rcsb.org/pdb/home/home.do), under the
code 4BDS. EqBChE and HssBChE present 90% of identity and the active site 100% conserved. The Fasta
sequence of EqBChE was aligned to HssAChE and further submitted to the Swiss Model server [43] for
construction of the model. Then, the structures of template (HssBChE) and model were aligned using
the software spdbviewer [42] and the coordinates of tacrine were copied from HssBChE to EqBChE.
The models EeAChE/tacrine and EqBChE/tacrine obtained were validated using the server
PDBSum (www.ebi.ac.uk/pdbsum/), where Ramachandran plot and G-factors such as dihedral angles
and covalent forces related to bond angles and length were analyzed. After validation, the ligands
were docked inside each model using the software Molegro Virtual Docker (MVD)®
[44]. The binding
site inside EeAChE was restricted into a sphere with a radius of 10 Å, centered on the coordinates
x = 8.95, y = 68.42 and z = 64.80, while for EqBChE it was centered on the coordinates x = 133.17;
y = 116.22 and z = 40.98. For both models the residues 6 Å closest to tacrine were considered flexible.
The best conformation of each compound was selected from a set of 300 results (poses) obtained for
each ligand, according to the binding energies and hydrogen bonds (H-bond) interactions criteria. The
docking protocol used was validated through re-docking of tacrine inside EeAChE and EqBChE.
4. Conclusions
The reactions among compound (2) and the carbonyl compounds were confirmed to be very
efficient for the synthesis of quinolines. The synthesis of the guanylhydrazone (8) through the reaction
between a quinoline and chloridrate of amino guanidine also showed to be efficient, presenting
good yields.

Int. J. Mol. Sci. 2019, 20, 3944
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The compounds derived from 1,3-benzodioxole-5-carboxaldheide synthesized were evaluated
as inhibitors of AChE. The results obtained were already expected because quinolines and guanyl
hydrazones are molecules with a wide range of applications in medicinal chemistry and bear structural
characteristics essential to enable interactions with the active site of AChE. The presence of the aromatic
rings resembling the base structure of tacrine in the quinolines synthesized enables π–π with the amino
acids present in the active site, while the guanyl hydrazone was capable of interacting with the CAS
of AChE through its guanidine group. Compounds (6) and (8) presented promising results as AChE
inhibitors with results for compound (8) being similar to the known AChE inhibitor rivastigmine. To
the best of our knowledge, the synthesis of this compound was reported here for the first time in
the literature.
Comparison between structures of compounds (3) and (8) allowed to observe that the presence of
a guanylhydrazone contributes to enhancing the inhibitory capacity of this kind of compound.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/20/16/
3944/s1.
Author Contributions: Conceptualization, J.d.O.C.B.; J.D.F.-V.; methodology, J.d.O.C.B.; J.A.L.; D.C.F.N.; T.C.C.F.;
software, J.S.F.D.d.A.; J.A.L.; D.C.F.N.; validation, J.d.O.C.B.; J.A.L.; D.C.F.N.; formal analysis, J.d.O.C.B.; T.C.C.F.;
investigation, J.d.O.C.B.; J.A.L.; D.C.F.N.; J.S.F.D.d.A.; resources, J.D.F.-V.; T.C.C.F.; K.K.; data curation, J.d.O.C.B.;
T.C.C.F.; writing—original draft preparation, J.d.O.C.B.; J.A.L.; D.C.F.N.; J.S.F.D.d.A. and T.C.C.F.; writing—review
and editing, T.C.C.F.; K.K.; visualization, T.C.C.F.; supervision, J.D.F.-V.; project administration, J.D.F.-V.; funding
acquisition, J.D.F.-V.; T.C.C.F.; K.K.
Funding: This research was funded by Brazilian financial agencies: Conselho Nacional de Pesquisa (CNPq) (Grant
No. 308225/2018-0), Fundaç o de Amparo a Pesquisa do Rio de Janeiro (FAPERJ) (Grant No. E-02/202.961/2017),
ã
and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).
Acknowledgments: Authors would like to thank to the Military Institute of Engineering, Fundação Oswaldo
Cruz (FIOCRUZ) for workspace facilities and the group of theoretical chemistry of the Federal University of
Lavras for software facilities. Also supported by University of Hradec Kralove (Faculty of Science, VT2019-2021).
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ACh
Acetylcholine
AChE
AD
Acetylcholinesterase
Alzheimer Disease
ATCI
BBB
Acetylthiocholine Iodide
Blood Brain Barrier
BChE
BTCI
CAS
CNS
DFT
DMSO
DMSO-d6
DTNB
EeAChE
EqBChE
IR
Butyrilcholinesterase
Butyrylthiocholine Iodide
Catalytic Anionic Site
Central Nervous System
Density Functional Theory
Dimethylsulfoxide
Deuterated DMSO
5,5-dithio-bis-(2-nitrobenzoic acid)
Electrophorus electricus AChE
Equine serum BChE
Infrared
MHz
NMR
PAS
Megahertz
Nuclear Magnetic Ressonance
Peripheral Anionic Site
Protein Data Bank
PDB
PSA
Polar Surface Area
RMSD
TLC
Root Mean Square Deviation
Thin Layer Chromatography

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