Antioxidant, antiproliferative, and acetylcholinesterase inhibition activity of amino alcohol derivatives from 1,4-naphthoquinone

Article abstract of DOI:10.1007/s00044-020-02617-1

Natural and synthetic naphthoquinones have demonstrated numerous biological activities; therefore, they provide an interesting scaffold for medicinal chemists in the search for new drugs. A series of amino alcohol derivatives from 1,4-naphthoquinone with a free (2a–e) and acetylated (3a–d) hydroxyl group were synthesized, characterized, and evaluated as antioxidant agents employing 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS·⁺) assays, as acetylcholinesterase (AChE) inhibitors and antiproliferative compounds. In the DPPH assay, compound 3d showed the better result with 51.52% of antioxidant activity at 1 mg/mL, while in ABTS·⁺ was 2e (47.12%). The antiproliferative activity was evaluated against six different tumor cell lines, where the particular best results were for products 2a, 2c, and 2e against cervix line HeLa, with 50% growth inhibition (GI₅₀) of 5.6, 15.0, and 17.0 μM, respectively. All synthesized compounds presented varying degrees of response, some of them with similar results compared with the positive control 5-fluorouracil. AChE inhibition of the products was not as strong as the positive control galantamine; the most potent compound was 2e with a 50% inhibitory concentration (IC₅₀) of 0.0586 mM, followed by 2a (0.0902 mM). No inhibition in the evaluated concentrations was observed for products 2d and 3a-d. Docking of 2a and 2e was realized against AChE in order to gain insight into the interactions made between them and the enzyme residues in its catalytic gorge. In silico calculations for all synthesized products showed their drug-like properties. Alcohol derivatives, specially 2a and 2e, could be further derivatized in the search for new and improved drugs.

Full text of DOI:10.1007/s00044-020-02617-1

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02617-1
ORIGINAL RESEARCH
Antioxidant, antiproliferative, and acetylcholinesterase inhibition
activity of amino alcohol derivatives from 1,4-naphthoquinone
Arturo Estolano-Cobián1 Eduardo Noriega-Iribe Laura Díaz-Rubio José M. Padrón Mirna Brito-Perea
1 ●
1 ●
2 ●
1 ●
●
1 ●
3 ●
1 ●
1 ●
José Manuel Cornejo-Bravo Daniel Chávez Raúl Romero Rivera Juan Manuel Quintana-Melgoza
J. Cruz-Reyes1 Iván Córdova-Guerrero
1
●
Received: 2 July 2020 / Accepted: 13 August 2020
©
Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Natural and synthetic naphthoquinones have demonstrated numerous biological activities; therefore, they provide an
interesting scaffold for medicinal chemists in the search for new drugs. A series of amino alcohol derivatives from 1,4-
naphthoquinone with a free (2a–e) and acetylated (3a–d) hydroxyl group were synthesized, characterized, and evaluated as
antioxidant agents employing 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2-2′-azino-bis-(3-ethylbenzothiazoline-6-sulfo-
+
nate) (ABTS· ) assays, as acetylcholinesterase (AChE) inhibitors and antiproliferative compounds. In the DPPH assay,
+
compound 3d showed the better result with 51.52% of antioxidant activity at 1 mg/mL, while in ABTS· was 2e (47.12%).
The antiproliferative activity was evaluated against six different tumor cell lines, where the particular best results were for
products 2a, 2c, and 2e against cervix line HeLa, with 50% growth inhibition (GI ) of 5.6, 15.0, and 17.0 μM, respectively.
5
0
All synthesized compounds presented varying degrees of response, some of them with similar results compared with the
positive control 5-fluorouracil. AChE inhibition of the products was not as strong as the positive control galantamine; the
most potent compound was 2e with a 50% inhibitory concentration (IC ) of 0.0586 mM, followed by 2a (0.0902 mM). No
5
0
inhibition in the evaluated concentrations was observed for products 2d and 3a-d. Docking of 2a and 2e was realized against
AChE in order to gain insight into the interactions made between them and the enzyme residues in its catalytic gorge. In
silico calculations for all synthesized products showed their drug-like properties. Alcohol derivatives, specially 2a and 2e,
could be further derivatized in the search for new and improved drugs.
●
●
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Keywords Naphthoquinone Amino alcohol Antioxidant Antiproliferative Acetylcholinesterase inhibition Docking
Introduction
Alzheimer’s disease (AD) is a multifactorial neurode-
generative disease, and globally is the most common
cause of dementia in elder people (Tran et al. 2016). In
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02617-1) contains supplementary
material, which is available to authorized users.
2019, only in the United States of America the pre-
valence of AD was 5.8 million, from which 5.6 million
were people over 65 years, and by the year 2050 the
projection for this country is that 13.8 million people
over 65 will have Alzheimer (Alzheimer’s Association
*
Iván Córdova-Guerrero
icordova@uabc.edu.mx
2
019). As cholinergic pathways have been related to AD
1
2
Facultad de Ciencias Químicas e Ingeniería, Universidad
Autónoma de Baja California, 22390 Tijuana, B.C., Mexico
progression, and the cholinergic loss has a correlation to
the severity of dementia in AD, some of the current
pharmacotherapy for this disease include acet-
ylcholinesterase inhibitors (AChEI), which are used for
symptoms treatment (Ferreira-Vieira et al. 2016; Hampel
et al. 2019).
BioLab, Instituto Universitario de Bio-Orgánica “Antonio
González” (IUBO-AG), Universidad de La Laguna,
c/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
3
Centro de Graduados e Investigación en Química,
Tecnológico Nacional de México/Instituto Tecnológico de
Tijuana, 22510 Tijuana, B.C., Mexico

Medicinal Chemistry Research
Additionally, oxidative damage, developed by the loss of
balance between free radicals such as reactive oxygen
species (ROS) and natural antioxidant systems in tissues as
the neuronal one, has also been related to the pathogenesis
and progression of AD (Díaz-Hung and González Fraguela
Material and methods
Chemistry
All commercial reagents and solvents were purchased and
used without previous purification. After the synthetic
process, column chromatography purifications were carried
for the products with silica gel 60 Å, 230–400 mesh (Sigma-
Aldrich). Compound purity was first checked by thin-layer
chromatography (TLC) carried out on aluminum backed
silica plates (Merck) revealed employing a UV light at
254 nm. Further confirmation of purity and chemical char-
acterization was made through gas chromatography-mass
spectrometry on a TRACE 1310 chromatograph (Thermo
Scientific) and single quadrupole ISQ LT mass spectrometer
(Thermo Scientific). Melting points were taken on a Mel-
Temp melting point apparatus (Thermo Scientific). Fourier-
transform infrared spectroscopy was performed on a Nicolet
iS 5 spectrometer (Thermo Scientific). Nuclear magnetic
resonance (NMR) spectra were obtained on a 400 MHz
Avance DPX spectrometer (Bruker). The chemical shifts in
NMR spectra (δ) are shown using tetramethylsilane as an
internal standard (δ: 0.00). In the case of UV–Vis spectra, a
Genesys 20 (Thermo Scientific) spectrophotometer was
used for the antioxidant assays, a Microplate reader Mul-
tiskan FC (Thermo Scientific) for the acetylcholinesterase
assay, a fetal bovine serum was purchased from biowest
(Riverside, MO), and a Microplate reader PowerWave XS
(BioTek) for the antiproliferative assay.
2
014; Butterfield and Halliwell 2019). Furthermore, oxi-
dative stress has been associated with the generation and
development of several cancers, therefore the antioxidant
properties of different compounds have been proposed for
cancer prevention and treatment (Kudryavtseva et al. 2016;
Chikara et al. 2018).
Moreover, despite the primary neuronal role of acet-
ylcholinesterase, recent studies point to a role of this
enzyme in cell proliferation and differentiation, along
with apoptosis. Although further studies are proposed to
fully explore this relationship, it is suggested that the
development of AChEI could lead to some agents for
tumor suppression and cancer treatment therapy for some
cancers (Kocanci et al. 2017; Lazarević-Pašti et al.
2
017).
Naphthoquinones are naphthalene dione derivatives, that
can have their ketones in 1,2 or 1,4 positions. Natural naph-
thoquinones usually are pigments; besides this, both natural
and synthetics naphthoquinones have shown different biolo-
gical properties, such as antihypertensive, antibiotic, anti-
fungal, anticancer, antiparasitic, and antituberculosis amongst
others (Widhalm and Rhodes 2016; Shikov et al. 2018;
Halicki et al. 2018; Jentzsch et al. 2020).
As naphthoquinones undergo redox cycling, their
redox transitions can be related to many of their biolo-
gical properties (Cadenas et al. 1992), developing in
some cases both sides of a pharmacological response; for
example, depending on the conditions of their biological
environment they can act as antioxidants or pro-oxidants
General process for the synthesis
of 1,4-naphthoquinone derivatives
In a round bottom flask, 1 mmol of cupric acetate mono-
hydrate was dissolved in 20 mL of methanol under mag-
netic stirring at 60 °C, while air was being pumped in the
reaction flask. After complete dissolution, 4 mmol of the
corresponding amine was added, followed by 1 mmol of
1,4-naphthoquinone (Scheme 1). The reaction was stirred
till completion, being monitored by TLC. To purify the
product, column chromatography was employed, using
eluent gradients of n-hexane/ethyl acetate from lower to
higher polarity. The structures of the products were con-
firmed by mass spectrometry and NMR spectra, as dis-
cussed below.
(
de Oliveira et al. 2017), anti-inflammatory or pro-
inflammatory (Kumagai et al. 2012). Recently, naph-
thoquinones have been merged with tacrine, a hepato-
toxic acetylcholinesterase inhibitor, in order to produce
new less-toxic treatments for Alzheimer’s disease
(
Nepovimova et al. 2014; Chioua et al. 2018).
In this paper a series of naphthoquinone derivatives
were synthesized, including in their quinone nucleus dif-
ferent amino alcohol chains, as this fragment has been
incorporated in different molecular frames due to the
properties they present, such as AChE inhibition and
improved antiproliferative activities (Córdova et al. 2006;
Vanguru et al. 2017; Mahendran et al. 2018; Zheng et al.
2-((2-hydroxyethyl)amino)naphthalene-1,4-dione (2a):
red crystals (yield 96%); C H O N; mp 146–148 °C; IR
1
2
11
3
−
1
2
018). The synthesized compounds were analyzed in vitro
(ATR diamond, cm ) 3305 (N-H), 3034 (C-H), 1656
−
1
1
in order to conduct an initial screening of their AChE
inhibition, antioxidant and antiproliferative activities, and
these results are supported with in silico analysis to fur-
ther select lead compounds in the synthesis and devel-
opment of new improved drugs.
(C=O), 2907 (C-H aliphatic) cm ; H NMR (400 MHz,
CDCl ): δ = 7.99 (1H, dd, J = 1.2, 7.6 Hz, H-8), 7.95 (1H,
3
dd, J = 1.2, 7.6 Hz, H-5), 7.83 (1H, ddd, J = 1.1, 7.5,
7.5 Hz, H-6), 7.72 (1H, ddd, J = 1.0, 7.5, 7.5 Hz, H-7), 7.28
(1H, t, J = 5.7 Hz, NH), 5.74 (1H, s, H-3), 4.88 (1H, t,

Medicinal Chemistry Research
3
.07 (1H, m, H-1′), 1.12 (3H, d, J = 6.2 Hz, H-3′); 13C
NMR (100 MHz, CDCl ): δ = 181.5 (C-1), 181.3 (C-4),
3
148.6 (C-2), 134.8 (C-6), 133.1 (C-4a), 132.1 (C-7), 130.3
(
C-8a), 125.8 (C-8), 125.3 (C-5), 99.7 (C-3), 64.0 (C-2′),
+
4
1
8
9.5 (C-1′), 21.1 (C-3′); EIMS m/z (rel. int.) 231 [M ] (7),
86 (33), 158 (13), 146 (19), 131 (31), 115 (18), 101 (70),
9 (48), 76 (100), 50 (60), 45 (64), 28 (39).
Compound
a, 2a
b, 2b
c, 2c
R
1
2-((1-hydroxybutan-2-yl)amino)naphthalene-1,4-dione
(
1
2d): red orange solid (yield 93%); C H O N; mp
14 15 3
−1
1
51.5–154 °C; IR (ATR diamond, cm ) 3387 (N-H), 3246
−
1
(
O-H), 3028 (C-H), 2987 (C-H aliphatic), 1671 (C=O) cm ;
1
1
H NMR (400 MHz, CDCl ): δ = 7.97 (1H, dd, J = 0.9,
3
7.7 Hz, H-8), 7.91 (1H, dd, J = 1.0, 7.7 Hz, H-5), 7.62 (1H,
ddd, J = 1.2, 7.5, 7.5 Hz, H-6), 7.50 (1H, ddd, J = 1.2, 7.6,
1
d, 2d
7.6 Hz, H-7), 5.94 (1H, d, J = 8.4 Hz, NH), 5.72 (1H, s, H-
3
), 3.70 (2H, m, H-1′), 3.36 (1H, m, H-2′), 2.33 (1H, bs,
13
OH), 1.65 (2H, m, H-3′), 0.92 (3H, t, J = 7.4 Hz, H-4′);
C
1e, 2e
NMR (100 MHz, CDCl ): δ = 183.3 (C-1), 181.9 (C-4),
3
1
48.0 (C-2), 134.9 (C-6), 133.6 (C-4a), 132.1 (C-7), 130.6
(C-8a), 126.4 (C-8), 126.2 (C-5), 101.1 (C-3), 63.2 (C-1′),
5.8 (C-2′), 24.0 (C-3′), 10.6 (C-4′); EIMS m/z (rel. int.) 245
Scheme 1 Synthesis of 1,4-naphthoquinone amino alcohol derivatives
5
+
[M ] (4), 186 (7), 158 (4), 115 (18), 102 (45), 89 (44), 76
J = 5.4 Hz, OH), 3.62 (2H, c, J = 5.5 Hz, H-2′), 3.26 (2H, c,
(100), 50 (52), 41 (99), 31 (71).
2-((4-hydroxyphenethyl)amino)naphthalene-1,4-dione
(2e): orange crystals (yield 95%); C H O N; mp
13
J = 5.8 Hz, H-1′); C NMR (100 MHz, CDCl ): δ = 181.5
3
(
C-1), 181.3 (C-4), 148.7 (C-2), 134.8 (C-6), 133.1 (C-4a),
1
8
15
3
−
1
1
32.1 (C-7), 130.3 (C-8a), 125.8 (C-8), 125.3 (C-5), 99.6
156–158 °C; IR (ATR diamond, cm ) 3349 (N-H), 3323
(
[
C-3), 58.4 (C-2′), 45.6 (C-1′); EIMS m/z (rel. int.) 217
M ] (8), 199(7), 186 (33), 158 (10), 129 (23), 115 (16),
(O-H), 3012 (C-H), 2925 (C-H aliphatic), 1718 (C=O)
+
−1 1
cm ; H NMR (400 MHz, CDCl ): δ = 9.17 (1H, s, OH),
3
1
01 (52), 89 (46), 76 (100), 50 (84), 39 (23), 31 (71).
-((3-hydroxypropyl)amino)naphthalene-1,4-dione (2b):
orange crystals (yield 84%); C H O N; mp 122–125 °C;
7.96 (1H, d, J = 7.6 Hz, H-8), 7.93 (1H, d, J = 7.6 Hz, H-5),
7.81 (1H, t, J = 7.3 Hz, H-6), 7.71 (1H, t, J = 7.4 Hz, H-7),
7.41 (1H, t, J = 5.6 Hz, NH), 7.06 (2H, d, J = 8.2 Hz, H-2′′,
H-6′′), 6.68 (2H, d, J = 8.3 Hz, H-3′′, H-5′′), 5.70 (1H, s,
H-3), 3.35 (2H, c, J = 7.4 Hz, H-1′), 2.78 (2H, t, J = 7.4 Hz,
2
13
13
3
−
1
IR (ATR diamond, cm ) 3464 (O-H), 3331 (N-H), 2932
−
1
1
(
C-H aliphatic), 1669 (C=O) cm ; H NMR (400 MHz,
1
3
CDCl ): δ = 7.96 (1H, d, J = 7.6 Hz, H-8), 7.93 (1H, d, J =
H-2′); C NMR (100 MHz, CDCl ): δ = 181.5 (C-1), 181.2
3
3
8
7
.0 Hz, H-5), 7.81 (1H, t, J = 7.5 Hz, H-6), 7.70 (1H, t, J =
.6 Hz, H-7), 7.51 (1H, bt, NH), 5.67 (1H, s, H-3), 4.63
(C-4), 155.8 (C-4′′), 148.2 (C-2), 134.8 (C-6), 133.1 (C-4a),
132.1 (C-7), 130.3 (C-8a), 129.6 (C-2′′, C-6′′), 128.8
(C-1′′), 125.8 (C-8), 125.3 (C-5), 115.2 (C-3′′, C-5′′), 99.5
(C-3), 43.6 (C-1′), 32.5 (C-2′); EIMS m/z (rel. int.) 293
(
1H, t, J = 5.0 Hz, OH), 3.50 (2H, c, J = 5.6 Hz, H-3′), 3.24
2H, c, J = 6.6 Hz, H-1′), 1.74 (2H, q, J = 6.3 Hz, H-2′);
(
1
3
+
C NMR (100 MHz, CDCl ): δ = 181.5 (C-1), 181.2 (C-4),
[M ] (1), 186 (14), 107 (100), 104 (8), 77 (91), 53 (23),
3
1
48.5 (C-2), 134.8 (C-6), 133.2 (C-4a), 132.0 (C-7), 130.3
39 (19).
(
3
2
C-8a), 125.8 (C-8), 125.3 (C-5), 99.2 (C-3), 58.7 (C-3′),
9.6 (C-1′), 30.4 (C-2′); EIMS m/z (rel. int.) 231 [M ] (4),
13 (2), 186 (13), 104 (18), 76 (76), 50 (42), 41 (37), 31
Compounds 2a–d, bearing an aliphatic hydroxyl, were
subjected to an acetylation. For the corresponding alcohol
derivative, 1 mmol was dissolved in a round bottom flask
with 10 mL acetic acid, along with acetyl chloride
(1.5 mmol) and potassium bisulfate (1 mmol). The mixture
was kept under magnetic stirring and reflux, and was
monitored with TLC (Scheme 2). After completion of the
starting material transformation, the solvent was neutralized
and the product was extracted with ethyl acetate, which was
then eliminated under reduced pressure. Purification was
made using column chromatography, employing eluent
gradients of n-hexane/ethyl acetate from lower to higher
polarity. The structures of the products (3a–d) were
+
(
100), 28 (37).
-((2-hydroxypropyl)amino)naphthalene-1,4-dione (2c):
orange solid (yield 97%); C H O N; mp 132–134 °C; IR
2
1
3
13
3
−
1
(
ATR diamond, cm ) 3355 (N-H), 3269 (O-H), 3028 (C-
−
1
1
H), 2946 (C-H aliphatic), 1668 (C=O) cm ; H NMR
400 MHz, CDCl ): δ = 7.99 (1H, dd, J = 0.9, 7.6 Hz, H-8),
(
3
7
7
7
.95 (1H, dd, J = 1.0, 7.7 Hz, H-5), 7.83 (1H, ddd, J = 1.3,
.5, 7.5 Hz, H-6), 7.72 (1H, ddd, J = 1.3, 7.5, 7.5 Hz, H-7),
.22 (1H, t, J = 5.8 Hz, NH), 5.75 (1H, s, H-3), 4.94 (1H, d,
J = 4.6 Hz, OH), 3.91 (1H, m, H-2′), 3.17 (1H, m, H-1′),

Medicinal Chemistry Research
+
[
M ] (1), 213 (4), 186 (7), 158 (3), 101 (15), 89 (21), 76
30), 50 (13), 43 (100).
(
1-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)pro-
pan-2-yl acetate (3c): orange solid (yield 93%); C H O N;
1
5
15
4
−
1
mp 111–113 °C; IR (ATR diamond, cm ) 3302 (N-H),
031 (C-H), 2957 (C-H aliphatic), 1715 (C=O) cm ; H
−
1 1
Compound
R
Compound
3a
R
3
NMR (400 MHz, CDCl ): δ = 8.01 (1H, dd, J = 0.9,
3
2a
7
.7 Hz, H-8), 7.97 (1H, dd, J = 0.9, 7.6 Hz, H-5), 7.65 (1H,
ddd, J = 1.3, 7.5, 7.5 Hz, H-6), 7.54 (1H, ddd, J = 1.3, 7.6,
.6 Hz, H-7), 6.07 (1H, bs, NH), 5.70 (1H, s, H-3), 5.11
1H, m, H-2′), 3.25 (2H, m, H-1′), 2.00 (3H, s, H-2′′), 1.26
7
(
(
2
b
3b
3c
1
3
3H, d, J = 6.4 Hz, H-3′); C NMR (100 MHz, CDCl3):
δ = 183.2 (C-1), 181.8 (C-4), 170.8 (C1′′), 148.1 (C-2),
34.9 (C-6), 133.6 (C-4a), 132.2 (C-7), 130.6 (C-8a), 126.4
C-8), 126.3 (C-5), 101.5 (C-3), 68.7 (C-2′), 47.2 (C-1′),
2
c
1
(
2
2
+
1.3 (C-3′), 18.0 (C-2′′); EIMS m/z (rel. int.) 273 [M ] (<1),
2d
3d
13 (9), 186 (13), 158 (4), 146 (5), 131 (7), 101 (18), 89
(
13), 76 (22), 51 (11), 43 (100), 28 (7).
-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)butyl
acetate (3d): orange solid (yield 95%); C H O N; mp
Scheme 2 Acetylation of the aliphatic alcohol derivatives
2
1
6
17
4
−
1
1
22–124 °C; IR (ATR diamond, cm ) 3331 (N-H), 3025
−
1 1
confirmed by mass spectrometry and NMR spectra, as
(C-H), 2940 (C-H aliphatic), 1712 (C=O) cm ; H NMR
discussed below.
(400 MHz, CDCl ): δ = 8.02 (1H, dd, J = 1.1, 7.7 Hz, H-8),
3
2
-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)ethyl
7.98 (1H, dd, J = 1.0, 7.7 Hz, H-5), 7.66 (1H, ddd, J = 1.2,
7.5, 7.5 Hz, H-6), 7.55 (1H, ddd, J = 1.2, 7.6, 7.6 Hz, H-7),
5.83 (1H, d, J = 8.5 Hz, NH), 5.73 (1H, s, H-3), 4.12 (2H,
m, H-1′), 3.50 (1H, m, H-2′), 2.00 (3H, s, H-2′′), 1.63 (2H,
acetate (3a): dark orange crystals (yield 96%); C H O N;
mp 118–120 °C; IR (ATR diamond, cm ) 3261 (N-H),
058 (C-H), 2902 (C-H aliphatic), 1712 (C=O) cm ; H
NMR (400 MHz, CDCl ): δ = 8.02 (1H, dd, J = 0.9,
14
13
4
−
1
−
1 1
3
1
3
m, H-3′), 0.93 (3H, t, J = 7.4 Hz, H-4′); C NMR
3
7
.7 Hz, H-8), 7.98 (1H, dd, J = 1.0, 7.6 Hz, H-5), 7.66 (1H,
ddd, J = 1.3, 7.5, 7.5 Hz, H-6), 7.55 (1H, ddd, J = 1.3, 7.6,
.6 Hz, H-7), 6.06 (1H, bs, NH), 5.70 (1H, s, H-3), 4.26
2H, t, J = 5.2 Hz, H-1′), 3.40 (2H, c, J = 5.6 Hz, H-2′),
.03 (3H, s, H-2′′); 13C NMR (100 MHz, CDCl ): δ = 183.1
(100 MHz, CDCl ): δ = 183.2 (C-1), 181.9 (C-4), 170.9 (C-
3
1′′), 147.6 (C-2), 134.9 (C-6), 133.6 (C-4a), 132.2 (C-7),
130.6 (C-8a), 126.5 (C-8), 126.3 (C-5), 101.5 (C-3), 64.7
(C-1′), 53.1 (C-2′), 24.4 (C-3′), 20.9 (C-2′′), 10.4 (C-4′);
7
(
2
+
EIMS m/z (rel. int.) 287 [M ] (1), 214 (12), 158 (2), 129
3
(
1
(
C-1), 181.8 (C-4), 171.1 (C-1′′), 148.0 (C-2), 134.9 (C-6),
33.6 (C-4a), 132.2 (C-7), 130.6 (C-8a), 126.4 (C-8), 126.3
C-5), 101.5 (C-3), 61.7 (C-1′), 41.8 (C-2′), 20.9 (C2′′);
(5), 115 (5), 102 (14), 89 (11), 76 (22), 50 (9), 43 (100),
28 (11).
+
EIMS m/z (rel. int.) 259 [M ] (1), 199 (8), 186 (9), 158 (3),
29 (6), 115 (6), 101 (17), 89 (14), 76 (31), 50 (16), 43
100), 28 (13).
((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)propyl
acetate (3b): orange crystals (yield 94%); C H O N; mp
DPPH antioxidant assay
1
(
The DPPH radical-scavenging activity was performed
employing Salazar-Aranda methodology with slight mod-
ifications (Salazar-Aranda et al. 2015). Solutions of the
synthesized compounds were prepared with methanol at a
1 mg/mL concentration. A volume of 0.5 mL of each one of
these dilutions was mixed with 0.5 mL of a methanolic
DPPH solution (7.5 mg/250 mL). The resulting mixtures
were incubated at room temperature for 30 min in the dark.
Spectrophotometric measurements were performed at λ =
517 nm, using methanol as a blank, for the compound and
3
1
5
15
4
−
1
1
12–115 °C; IR (ATR diamond, cm ) 3308 (N-H), 3040
−
1 1
(
C-H), 2957 (C-H aliphatic), 1709 (C=O) cm ; H NMR
(
400 MHz, CDCl ): δ = 8.09 (1H, dd, J = 1.0, 7.7 Hz, H-8),
3
8
7
6
5
2
.03 (1H, dd, J = 1.2, 7.6 Hz, H-5), 7.72 (1H, ddd, J = 1.4,
.5, 7.5 Hz, H-6), 7.61 (1H, ddd, J = 1.3, 7.6, 7.6 Hz, H-7),
.10 (1H, bs, NH), 5.74 (1H, s, H-3), 4.20 (2H, t, J =
.9 Hz, H-1′), 3.31 (2H, c, J = 6.4 Hz, H-3′), 2.11 (3H, s, H-
13
′′), 2.03 (2H, q, J = 6.6 Hz, H-2′); C NMR (100 MHz,
DPPH mixtures (A ). A mixture of 0.5 mL DPPH solution
1
CDCl ): δ = 183.1 (C-1), 181.9 (C-4), 171.0 (C-1′′), 148.0
with 0.5 mL methanol was employed as a control (A ), and a
3
0
(
1
(
C-2), 134.9 (C-6), 133.7 (C-4a), 132.1 (C-7), 130.7 (C-8a),
26.4 (C-8), 126.3 (C-5), 101.1 (C-3), 62.2 (C-1′), 40.1
C-3′), 27.4 (C-2′), 21.0 (C-2′′); EIMS m/z (rel. int.) 273
mixture of 0.5 mL compound solution with 0.5 mL metha-
nol was employed as a color correction of the lectures (A2)
(Olalere et al. 2017). All these lectures were made by

Medicinal Chemistry Research
triplicate. The percentage of inhibition was calculated using
the formula:
cells/well; for lines T-47D and WiDr it was 5000 cells/well.
After 24 h, the synthesized compounds were added at a
concentration range of 0.01–100 μM. As positive control
cisplatin and 5-fluorouracil were employed, while DMSO
ꢀ
ꢁ
A À A
1
2
DPPHð%Þ ¼ 1 À
 100:
A
0
0.25% v/v was used as the negative control. The drug
Vitamin C was used as a positive control. The assay was
made by triplicate.
incubation times were 48 h. Afterward, cells were fixed
using 25 μL of ice-cold trichloroacetic acid solution (50%
w/v) for 60 min at 4 °C; after this time the plates were rinsed
with water. Then 25 μL of a solution of sulforhodamine B
ABTS antioxidant assay
(
0.4% w/v in 1% acetic acid) was added for 15 min.
+
The ABTS radical cation (ABTS· ) scavenging assay was
Unbound sulforhodamine B was rinsed with 1% acetic acid.
The remaining dye was dissolved with 150 μL of a 10 mM
Tris solution (pH 10.5). Employing a microplate reader, the
optical density of each well was determined (λ = 530 and
620 nm). According to NCI formulas (Monks et al. 1991),
the antiproliferative activity was calculated and expressed as
50% growth inhibition (GI ).
analyzed based on reported methodologies (Re et al. 1999;
Kuskoski et al. 2005) with slight modifications. A 7 mM
water solution of ABTS was prepared, and the ABTS
radical cation was made reacting this solution with 2.45 mM
potassium persulfate. The resulting mixture was kept at
room temperature in the dark for 16–18 h before being used.
5
0
+
Then ABTS· solution (150 μL) was diluted with methanol
to obtain an absorbance of 0.7 ± 0.02 (λ = 754 nm), taking
Acetylcholinesterase inhibitory activity
this as the initial absorbance (A ). A volume of 980 μL
1
+
ABTS· solution was mixed with 20 μL of the evaluated
AChE inhibitory activity was determined by employing a
previously reported methodology (Adewusi et al. 2011). A
96-well plate was used, adding to each well 75 μL of
Trizma-HCl buffer (50 mM, pH 8), along with 75 μL of a
dilution of the synthesized compounds, which were pre-
pared as serial dilutions, having in the end concentrations
for the compounds between 187.5 and 2.4 μg/mL (with
DMSO at a maximum concentration of 0.15%). Afterward,
25 μL of a buffer solution of 15 mM acetylthiocholine
chloride and 125 μL of a 3 mM Ellman’s reagent in buffer
solution (DTNB) were added to each well, having both
reagents final concentrations of 1.5 mM. A microplate
reader was employed to measure the absorbance every 45 s,
three consecutive times, at λ = 405 nm. Then, 25 μL of a
2 U/mL AChE buffer solution enriched with bovine serum
albumin was added to each well, obtaining a final well
concentration of 0.2 U/mL for the enzyme. After this, the
absorbance was read every 45 s for five consecutive times.
From each plate, six wells were employed without a com-
pound to test in order to act as a 100% enzymatic activity
control. A correction was made for the increase of absor-
bance caused by spontaneous substrate hydrolysis, sub-
tracting the absorbance before enzyme addition from the
absorbance obtained after. The percentage of inhibition was
calculated using the formula:
analyte (1 mg/mL). Each mixture was stirred, incubated at
room temperature in the dark for 7 min, and the final
absorbance (A ) read by triplicate. The percentage of inhi-
2
bition was calculated using the formula:
ꢀ
ꢁ
A1 À A2
A1
%
of inhibition ¼
 100:
Vitamin C was used as a positive control. The assay was
made by triplicate.
Cell lines and culture conditions
The antiproliferative activity of the synthesized compounds
was evaluated against six human solid tumor cell lines. As
drug-sensitive lines, A549 (non-small cell lung), HBL-100
(
breast), HeLa (cervix), and SW1573 (non-small cell lung)
were employed. As drug-resistant lines were used T-47D
breast) and WiDr (colon). The cell lines were kindly pro-
(
vided by Prof. G. J. Peters (VU Medical Center, Amster-
dam, The Netherlands). The cells were maintained in
2
2
5
3
5 cm culture flasks in RPMI 1640 media enriched with
% fetal calf serum (FCS) and 2 mM L-glutamine, at a
7 °C, 5% CO , and 95% humidified air incubator.
2
Antiproliferative activity
ꢂ
ꢃ
Asample
Acontrol
%
of inhibition ¼ 1 À
 100:
The cells were trypsinized and resuspended in DMEM
medium containing 5% FCS, 100 U/mL of penicillin G and
Where Asample is the absorbance difference between time
0 and 225 s in the presence of the compound to test or
inhibitor, and Acontrol is the corresponding absorbance dif-
ference of the 100% enzymatic activity control. Interpolat-
ing from the graphic of inhibition percentages against the
0
.1 mg/mL of streptomycin, counted on a Moxi Z auto-
mated cell counter, and diluted to reach appropriate cell
densities in order to inoculate in 96-well plates. For lines
A549, HBL-100, HeLa, and SW1573, the density was 2500

Medicinal Chemistry Research
employed concentrations, the 50% inhibitory concentration
IC ) was calculated. Galantamine was used as a positive
chiral center in their structures, coming from the respective
amino alcohol employed in their synthesis.
(
5
0
control. The assay was made by triplicate.
The nine synthesized compounds were obtained with
good yields (84–97%) and their structures (Scheme 1 and
Scheme 2) were confirmed through IR and mass spectro-
Molecular docking
1
13
scopy, and with H- and C-NMR. In IR spectroscopy, the
bands for the stretch of N-H and O-H bonds were observed,
as the one for the C=O in the acetylated compounds with
For each of the analyzed compounds, a molecular model
was generated through their SMILES string using UCSF
Chimera (Pettersen et al. 2004). Energy minimization of the
models was done employing Chimera’s default conditions
with MMTK and Antechamber parameters (Wang et al.
1
13
the O-H one disappearing. In H- and C-NMR all the
expected signals were observed in their expected shifts and
multiplicities, such as the aromatic hydrogens in the naph-
thoquinone core (δ = 8.09–7.50 ppm) and the hydrogen
from the amine (δ = 7.51–5.83 ppm). For the acetylated
compounds, their corresponding singlets were observed in
2
006). The rotatable bonds and atomic charges for each
ligand model were defined with AutoDock Tools 1.5.6
Sanner 1999). From Protein Data Bank (http://www.rcsb.
(
1
org/) was retrieved the crystallographic structure of the
AChE receptor (PDB ID: 1EVE). Using AutoDock Tool the
receptor was prepared, which included remotion of the co-
crystalized ligand (donepezil) and water molecules, the
addition of hydrogens, and Gasteiger charges calculation.
AutoDock Vina (Trott and Olson 2010) was employed as
the docking algorithm, using a grid box of 30 × 30 × 30 Å,
with x = 2.27731, y = 63.7499 and z = 65.4998 as center
coordinates. For each analyzed molecular model, five
docking runs with ten poses each were made, with an
exhaustiveness of eight. Validation of the docking protocol
was realized re-docking the co-crystallized donepezil, and
comparing the resulting pose to the crystallographic one
through their RMSD. Visualization of the docked poses for
their analysis was made with UCSF Chimera.
H-NMR (δ = 2.11–2.00 ppm) along with their carbonyl
1
3
signals (δ = 171.1–170.8 ppm) in C-NMR.
Antioxidant evaluation
As mentioned in the introduction, naphthoquinones can act
as pro- or anti-oxidants, due to redox and electrophile
properties, by their direct molecular action or by interaction
with different enzymes which can regulate the oxidative
conditions of the cells (Cadenas et al. 1992; Kumagai et al.
2012). Several natural naphthoquinones have demonstrated
antioxidant activities (Utkina and Pokhilo 2012; Thanh
et al. 2015) although they commonly present alcohol groups
as phenols or over the α-β unsaturated bond of the quinone,
to which their activity tends to be explained as phenolic
compounds show antioxidant properties by mechanisms as
the radical scavenging one (Talalaeva et al. 2016); however,
the presence of phenol fragments is related but not always
directly proportional to the scavenging capacity (Ilyasov
et al. 2020).
For these reasons, and because antioxidant properties
could support the AChE inhibition and antiproliferative
profiles of potential drugs, the potential antioxidant activity
of the synthesized compounds was measured with the
DPPH and ABTS·+ assays. The products analyzed have
amino alcohol fragments, though these alcohols are ali-
phatic instead of aromatic, along with their acetylated ver-
sions to determine which group of products show better
antioxidant properties. Due to the fluctuating antioxidant
activity of the naphthoquinone nucleus, two more com-
pounds were analyzed, 1,4-naphthoquinone and a derivative
with a phenolic fragment (2e), which should improve the
antioxidant results, to search how much of the activity of the
products can be related to their quinone nucleus.
ADME calculations
Drug-likeness prediction of the synthesized compounds was
made obtaining their pharmacokinetic and physicochemical
properties employing the SwissADME web server (Daina
et al. 2017).
Results and discussion
Chemistry
The synthesis of 1,4-naphthoquinone derivatives was
accomplished by reacting this starting material with amines
as nucleophilic reagents in the position 2 of the naphtho-
quinone core. As seen in Scheme 1, different aliphatic
amino alcohols were employed, with variations in the
number and disposition of carbons in the aliphatic chain.
Also, as opposed to these structures, an amino alcohol
where the hydroxyl was in an aromatic ring was introduced
In the DPPH radical scavenging activity assay (Table 1)
the positive control employed, vitamin C, showed the best
antioxidant percentage (97%). Amongst the most active
compounds is 1,4-naphthoquinone, the precursor of the
synthesized products, with 44.73%. It is only surpassed by
(
compound 2e). In the case of the aliphatic alcohols, the
hydroxyl was acetylated as shown in Scheme 2. Com-
pounds 2c and 2d, as their acetylated derivatives, present a

Medicinal Chemistry Research
Table 1 Percentage antioxidant activity of synthesized compounds
fragment, and as DPPH radical stabilizes with a hydrogen
transfer from antioxidants (Kedare and Singh 2011) while
ABTS· can also be neutralized through different mechan-
2
a–e and 3a–d
+
Compound
% antioxidant activity (1 mg/mL)
isms with hydrogen transfers (Ilyasov et al. 2020), the
antioxidant activity of the products 2a–d and 3a–d could
be related to the acidic hydrogen in position 3 of the
naphthoquinone.
DPPH
ABTS
2
3
2
3
2
3
2
3
2
1
a
11.86 ± 0.80
34.03 ± 0.40
46.33 ± 0.54
15.91 ± 1.28
28.52 ± 1.13
33.53 ± 1.04
24.09 ± 1.29
51.52 ± 0.62
19.18 ± 1.18
44.73 ± 0.88
97.00 ± 0.08
0.357 ± 0.123
a
4.503 ± 0.298
7.435 ± 0.148
−0.886 ± 0.606
1.248 ± 0.030
1.709 ± 0.253
0.913 ± 0.108
1.549 ± 0.055
47.125 ± 0.726
10.267 ± 0.059
99.848 ± 0.000
b
b
Antiproliferative activity
c
c
The antiproliferative activity of the synthesized compounds
2a–e and 3a–d was analyzed against six cell lines from
different origins, as stated in the methodology section.
Several references point out the importance of amino
alcohol fragments in medicinal chemistry due to their
properties, one of them being antiproliferative, although this
activity is mostly related to β-amino alcohols (Córdova
et al. 2006; Vanguru et al. 2017; Mahendran et al. 2018). In
the present work, the first naphthoquinone derivative 2a
presents the β-amino alcohol fragment, and most of the
subsequent derivatives, with only the 2b compound having
an extra carbon atom separation between these groups.
Likewise, the analyzed products have their aliphatic alcohol
in its free and acetylated form to allow further exploration
of the structure effect on the biological result.
d
d
e
,4-naphthoquinone
aVitamin C
a
Served as reference compound. Values are mean ± SD, n = 3
product 2b (46.33%) and the most active 3d (51.52%). This
suggests that, to some extension, the antioxidant activity of
the compounds relies greatly on the naphthoquinone
nucleus. The activity related to the fragment incorporated
can be seen between compounds 2a–d and 3a–d; in the
majority of cases the acetylated derivatives presented a
better activity than their alcohol counterparts, the exception
is the set of 2b and 3b, with 46.33 and 15.91% activity,
respectively.
The 50% growth inhibition (GI ) of the nine synthesized
5
0
products are shown in Table 2. All the synthesized products
showed varying degrees of response, but were active against
all cell lines in the evaluated concentrations. The drug
resistant cell lines T-47D and WiDr were accordingly, in
general, the most resistant ones with higher GI50 from the
nine compounds, and even the two positive controls cis-
platin and 5-fluorouracil were not as active as in the other
lines. On the other hand, the more sensitive lines for the
synthesized compounds were HeLa and SW1573. Positive
control cisplatin was the most active compound against the
six cell lines, only being surpassed by the other control
5-fluorouracil in the A549 line. Remarkably, some of the
naphthoquinone derivatives were as active as 5-fluorouracil,
and in some cases even more, depending on the cell line
evaluated.
In the A549, HBL-100 and SW1573 cell lines the nine
products 2a–e and 3a–d showed antiproliferative activity,
although their GI50 were not as low as the positive controls.
Against A549, the GI50 for the nine compounds were
relatively close to each other, in the range 33–47 μM. The
most active product was 2e with GI50 of 33 μM, closely
followed by 2d (34 μM). With the HBL-100 line, the most
active compound was again 2e with GI50 25 μM, though in
this case the next active product 3c had a greater difference
with it (GI50 of 43 μM). The least active product in this line
was 2d (73 μM). In the SW1573 cell line, again all the
compounds achieved similar results, as the most active
+
In the ABTS· scavenging assay (Table 1) the percen-
tages were lower than those obtained in the DPPH techni-
que. The positive control vitamin C surpassed again the
activity of the rest of the compounds (99.8%). From the
naphthoquinones, the best result was 2e with 47.12%,
while the next active product was 1,4-naphthoquinone with
1
0.27%, and the other derivatives did not exceed the 10% of
activity. As in the DPPH assay, this suggests that the
alcohol and acetylated aliphatic chains incorporated mod-
ulate in a subtler way the antioxidant activity of these
products than the naphthoquinone core. Comparing the
acetylated products to the ones with the free alcohol group,
only the acetyl derivative 3b had a lower result than his
alcohol counterpart 2b. The result of 3b could even be
related to the ambivalent properties of naphthoquinones, as
its result was nearer to the null antioxidant activity, being a
slightly negative one. It coincides that 3b is amongst the
less active products in the DPPH assay. Furthermore, as it
+
was the case with DPPH, in the ABTS· assay it concurs
that the set 2b and 3b was the one where the acetyl product
was the less active, as the acetyl derivatives 3a, 3c, and 3d
were better antioxidants than the alcohol derivatives 2a, 2c,
and 2d.
As these results suggest the antioxidant activity of the
synthesized products relies mostly on the naphthoquinone

Medicinal Chemistry Research
Table 2 Antiproliferative
activity of compounds 2a–e and
Cell lines (GI50, μM)a
Compound
3
a–d against six tumor cell lines
A549
HBL-100
HeLa
SW1573
T-47D
WiDr
2
3
2
3
2
3
2
3
2
a
a
b
b
c
39 ± 8.1
39 ± 8.2
36 ± 3.1
47 ± 14
44 ± 13
44 ± 14
34 ± 0.5
46 ± 11
33 ± 11
4.9 ± 0.3
2.2 ± 0.3
70 ± 4.3
62 ± 15
48 ± 3.1
53 ± 2.1
57 ± 5.0
43 ± 4.3
73 ± 10
55 ± 4.3
25 ± 2.8
1.9 ± 0.2
4.4 ± 0.7
5.6 ± 0.9
23 ± 3.5
23 ± 6.0
30 ± 0.5
15 ± 4.2
35 ± 0.7
41 ± 10
35 ± 7.9
17 ± 2.8
1.9 ± 0.4
16 ± 4.5
28 ± 5.2
30 ± 2.3
27 ± 6.7
28 ± 0.6
28 ± 10
27 ± 1.9
30 ± 10
26 ± 4.4
24 ± 4.0
2.7 ± 0.4
3.8 ± 1.8
38 ± 11
85 ± 25
35 ± 9.6
83 ± 27
82 ± 26
76 ± 33
50 ± 0.6
56 ± 6.9
56 ± 10
17 ± 2.3
47 ± 18
41 ± 8.1
78 ± 23
39 ± 7.9
72 ± 26
80 ± 28
72 ± 39
50 ± 5.5
76 ± 34
50 ± 13
23 ± 4.3
45 ± 13
c
d
d
e
bCDDP
b5-FU
aGI50 values are given in μM. Standard deviation was calculated from two to four independent experiments
bCisplatin (CDDP) and 5-fluorouracil (5-FU) were used as reference antiproliferative drugs
product was 2e with GI50 24 μM and the least were both 3a
and 2d (30 μM) with the rest of compounds in between this
small range, being this one of the most sensitive lines.
The HeLa line could be considered the most sensitive
one against the naphthoquinones. Although the least active
product 2d (41 μM) had a higher GI50 compared to the
SW1573 results, the best result of the antiproliferative
activity was obtained in HeLa, as 2a was the best one for all
the naphthoquinones against one of the six evaluated cell
lines. Compounds 2a and 2c, with GI50 of 5.6 and 15 μM
respectively, were even more active than 5-fluorouracil
(
16 μM), with 2e almost as active (17 μM).
Other remarkable results can be seen in the cell line T-
4
7D, as 2b and 2a were more active than 5-fluorouracil (35
and 38 μM, respectively), with 2d (50 μM), 3d, and 2e (both
6 μM) almost as active as this control. Finally, against
5
WiDr again 2b (39 μM) and 2a (41 μM) were more active
than the same control, with 2d and 2e (50 μM) having close
results to it. Surprisingly, these two cell lines are the most
drug resistant evaluated ones, but against them, more of the
synthesized products showed analogous antiproliferative
activity compared to the one of the clinically used drugs.
The antiproliferative activity of compound 2a is also
reported in a previous work (Delarmelina et al. 2015)
against three cell lines, amongst them is H460 (a non-small
cell lung line), showing a GI of 233.4 μM (2.334 × 10−
Fig. 1 GI50 range plot of the tested compounds
the assay compared to their acetyl counterparts. The four
acetyl derivatives 3a–d demonstrated a very similar range
of results. In the alcohol derivatives, their result ranges are
not as consistent. While 2a and 2c presented the two best
particular results, 2a and 2b showed more GI50 results lower
than one of the employed positive controls. Although the set
is small, it appears preliminarily that a shorter, and perhaps
linear over branched, aliphatic chain provides good anti-
proliferative results. As mentioned earlier, 2a has a β-amino
alcohol, but the additional carbon distance in 2b did not
diminish this activity in comparison, even though more
compounds with linear and branched aliphatic chains
should be synthesized for better understanding of these
results. Compound 2e, which was introduced in the
4
50
−
1
mol L ). In the present work, 2a is evaluated against two
non-small cell lung lines, A549 and SW1573, with GI50 of
3
9 and 28 μM respectively. Although the results shown are
against different cell lines and is not possible a direct
comparison, all of them share a common origin, and the cell
lines presented in this work show a higher sensibility
against 2a, with almost tenfold lower GI50 concentrations.
These results, as seen in the range plot in Fig. 1, show a
slight tendency of the alcohol derivatives as more active in

Medicinal Chemistry Research
Table 3 Acetylcholinesterase inhibition of compounds 2a–e and 3a–d
antioxidant evaluation for analysis of the naphthoquinone
nucleus activity, was also incorporated in the anti-
proliferative assay, and as discussed beforehand, it was also
one of the most active compounds. It has an amino alcohol
with an aromatic ring, so another set of derivatives could
also be synthesized from it for further explorations.
Compound
IC50 (μg/mL)
IC50 (mM)
2
3
2
3
2
3
2
a
a
b
b
c
19.6 ± 1.3
>187.5
0.0902
–
34.3 ± 3.6
>187.5
0.1483
–
Consulted literature on naphthoquinones mostly relates
their antiproliferative properties to the production of ROS in
the cancerous cells through different enzymes (de Carvalho
da Silva and Ferreira 2016; Widhalm and Rhodes 2016).
Nevertheless, there are other proposed mechanisms of
action, such as the naphthoquinones induction of apoptosis,
the inhibition of topoisomerase II-α, and the regulation of
tumor suppressor factors p53 and p73, to name some of
them (de Moraes et al. 2014; Pereyra et al. 2019). As stated
at the beginning of this work, a few cancers have shown
abnormalities in their AChE expression and activities, and
even though more studies are still needed regarding this
relationship, the inhibition of AChE has been proposed for
the treatment of some cancers. Examples of some cancers
that show increased AChE activity are the brain, non-small
cell lung, breast, colon, renal and ovarian, amongst others
42.2 ± 2.3
>187.5
0.1825
c
–
d
>187.5
–
3d
>187.5
–
2e
17.2 ± 3.6
0.313 ± 0.014
0.0586
0.0011
aGalantamine
a
Served as reference compound. Values are mean ± SD, n = 3
IC50 of 0.0902 mM. Compounds 2b and 2c are structural
isomers with three carbon atoms in their amino alcohol
fragment, and with higher hydrophobicity (Log P 1.38 and
1.28, respectively) than 2a, there is a slight decrease in their
AChE inhibition, with an IC50 of 0.1483 mM for 2b and
0.1825 mM for 2c. Product 2d has a higher hydrophobic
characteristic (Log P 1.5) and it showed no activity in the
evaluated concentrations, and its IC50 could not be deter-
mined in the molar range (IC > 187.5 μg/mL, Fig. 2).
(
Kocanci et al. 2017; Lazarević-Pašti et al. 2017). As the
majority of the employed cell lines in the antiproliferative
assay fall in these categories, similar tendencies in the
results in the antiproliferative and AChE inhibitory activity
of the synthesized products could be expected. As men-
tioned, in the antiproliferative results the alcohol products
are slightly more active than their acetyl counterparts, and
in the AChE inhibition this tendency is also present and is
even more acute, as discussed in the next section. Amongst
the more active antiproliferative compounds are 2a, 2b, and
5
0
Furthermore, the acetylated derivatives 3a–d lacked the
inhibition activity their alcohol counterparts showed; these
results suggest that in the aliphatic synthesized products, the
better activities come from a lower hydrophobicity and the
presence of a free hydroxyl group.
In the case of the aromatic amine derivative 2e, even
though the total carbon atom count is greater than the ali-
phatic products, and the benzene ring increases the product
Log P, this aromatic derivative showed inhibitory activity in
the evaluated concentrations. A possible explanation to this
could be that the presence of the extra aromatic ring in its
structure can supply with more π–π interactions with the
different aromatic amino acids located in the enzyme active
gorge, as these interactions are amongst the most observed
ones for AChE inhibitors (Kryger et al. 1999; Bartolucci
et al. 2001; Zhao et al. 2017).
In addition to the beforementioned references results,
pharmacophore modeling (Jang et al. 2018; Jana et al. 2018)
shows that alongside the π–π interactions, other important
ones are the H bonds and the π-cation interactions. This
could also explain why in the first set of synthesized com-
pounds, the amino alcohols showed better results, as their
acetylated versions can offer only H bonds acceptors in the
acetyl group, while the alcohols can behave as acceptors
and donors. All this could also support why 2e presented the
best result from all the synthesized compounds, by having
the free hydroxyl and the additional aromatic ring which
2
e, and they also were the more active inhibitors of AChE.
Even though preliminarily, there are some similarities in
both activities results, so a relationship between them could
be possible.
Acetylcholinesterase inhibitory activity
The results of the enzymatic inhibition assay can be seen in
Table 3. In the inhibition of AChE, none of the synthesized
products reached the activity of the positive control galan-
tamine, which is an AChEI with clinical use, that showed an
IC50 of 0.0011 mM (0.313 μg/mL). The aliphatic amino
alcohols employed in the derivatization of the naphthoqui-
none are structurally similar, with the number of carbon
atoms in their chain the main difference between them,
which can be related to hydrophobicity of the products as
shown in Table 4 with their Log P values. Preliminarily, a
relation is hinted between the hydrophobicity of the alcohol
derivatives with their AChE inhibition. Compound 2a has
two carbon in the amino alcohol fragment incorporated, and
a Log P of 0.95; in the AChE inhibition assay it showed an

Medicinal Chemistry Research
Table 4 Pharmacokinetics and drug-likeness of compounds 2a–e and 3a–d, calculated by SwissADME
Descriptors
Compounds
2
a
3a
2b
3b
2c
3c
2d
3d
2e
MW
217.22
3
259.26
4
231.25
3
273.28
4
231.25
3
273.28
4
245.27
3
287.31
4
293.32
#
#
H-bond acceptors
H-bond donors
3
2
1
2
1
2
1
2
1
2
TPSA
66.4
0.95
−2.22
Soluble
High
No
72.47
1.49
−2.94
Soluble
High
No
66.4
1.38
−3.10
Soluble
High
No
72.47
1.79
−3.26
Soluble
High
No
66.4
1.28
−2.67
Soluble
High
No
72.47
1.73
−3.40
Soluble
High
No
66.4
1.5
72.47
1.95
−3.76
Soluble
High
Yes
No
66.4
Consensus Log P
Log S (Ali)
2.56
−3.04
Soluble
High
Yes
No
−4.59
Class (Ali)
Moderately soluble
GI absorption
High
Yes
No
BBB permeant
P-gp substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Lipinski #violations
No
No
No
No
No
No
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
No
Yes
No
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
0
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
0
0
0
0
0
0
0
0
MW molecular weight, TPSA topological polar surface area, Log P logarithm of the partition coefficient, Log S (Ali) Ali et al. 2012 model
logarithm of molar solubility in water, GI gastrointestinal, BBB blood–brain barrier, P-gp permeability glycoprotein, CYP cytochrome
2
1
1
00
80
60
>187.5
>187.5
>187.5
>187.5
>187.5
Fig. 2 Acetylcholinesterase
inhibitory activity of synthesized
compounds 2a–e and 3a–d.
Galantamine served as reference
compound. Values are mean ±
SD, n = 3
140
120
100
80
60
40
20
0
4
2.2 ± 2.3
3
4.3 ± 3.6
1
9.6 ± 1.3
17.2 ± 3.6
.31 ± 0.0014
0
Compounds
can be fragments accounted for these important interactions
with AChE.
et al. 1999) and it was the one employed in the in vitro
assay. The crystallographic model was retrieved from the
Protein Data Bank (PDB ID: 1EVE) and the co-crystallized
ligand (donepezil) was removed. In order to evaluate the
effectiveness of the analysis, donepezil was re-docked in
the AChE model, and the best result pose was compared to
the original donepezil one through their RMSD. The
obtained RMSD of 1.1 is lower to the 1.5–2 Å proposed to
consider the docking parameters suitable for predicting
ligand poses (Hevener et al. 2009). After this, the molecular
models of the most active compounds 2e and 2a were
docked to TcAChE to analyze the binding made with the
enzyme.
Molecular docking
In silico docking studies were performed to further explore
the in vitro results in the AChE inhibition assay, as the
activity evaluated has a clearly defined molecular target
(
acetylcholinesterase), as opposed to the antiproliferative
assay where, due to the methodological conditions where
the assay runs against cell lines, no particular target is
defined at the moment.
For the docking, the acetylcholinesterase enzyme from
Tetronarce californica (TcAChE) was selected, as this
enzyme is highly similar to the human AChE one (Kryger
In Table 5 can be seen the binding scores of the docked
compounds, where a more negative result indicates a better

Medicinal Chemistry Research
binding of the ligand with the target. Compound 2a showed
a binding energy of −8.06 kcal/mol, while in the in vitro
AChE inhibition presented an IC50 of 0.0902 mM. As a
better inhibition was made by 2e (IC50 of 0.0586 mM), the
in silico result agrees with this, as this compound showed a
better docking score (−10.5 kcal/mol). Galantamine, which
was employed as positive control in the in vitro assay, was
also docked, with a score of −9.56 kcal/mol, which
although is not as good as the one for 2e, the pose and H
bonds formed by galantamine could explain its best in vitro
result (IC50 of 0.0011 mM), as it is further explained below.
Donepezil, the co-crystallized ligand for TcAChE presented
accordingly the best docking score, with −11.00 kcal/mol.
The analysis of the resulting poses docked in the enzyme
catalytic gorge shows the residues of TcAChE that could
interact with 2a and 2e (Fig. 3). Compound 2a makes with
its free hydroxyl a H bond with the carbonyl of Gln69,
while 2e shows one H bond with Asp72. As galantamine
showed a lower docking score than 2e, but in vitro was
more active, this could be explained with its docked pose, as
it is very similar to the one obtained by Bartolucci et al.
explain the in vitro result as 2a and 2e are not predicted to
interact with the AChE catalytic triad residues.
Another important residue for ligand interaction is
Trp84, which although is part of the oxyanion AChE hole
and for natural ligand acetylcholine and other compounds
forms a π-cation interaction, can also form classical π–π
interactions, such as in the case of the AChEI donepezil,
tacrine and galantamine (Harel et al. 1993; Bartolucci et al.
2001; Kryger et al. 1999; Dvir et al. 2010). The different π–
π interactions in the case of the synthesized compounds
appear to be the ones ruling their in vitro enzymatic inhi-
bition. The naphthoquinone core of compound 2a stacks
with Trp84 at the bottom of the enzyme catalytic gorge. In
the case of 2e, which was the more active inhibitor syn-
thesized, its activity can be related as the benzyl fragment
has a π–π stacking with Trp84, while the naphthoquinone
nucleus also presents this interaction with the residue
Tyr334, having an extra π–π interaction than compound 2a.
ADME calculations
(
2001) by crystallography. In both cases galantamine shows
The prediction with SwissADME of the pharmacokinetics
and drug-like properties of the synthesized products are
shown in Table 4.
H bonds with the catalytic residues of the enzyme Ser200
and His440, an another one with Glu199, which could
One of the most commonly used guidelines for deter-
mining the drug-like properties of a molecule is Lipinski’s
rule of five (Lipinski et al. 2001), which relates this property
to a series of descriptors, such as lipophilicity, molecular
weight and the H bonds donors and acceptors of the
molecule. All the synthesized compounds in this work
comply with this rule according to each one of these para-
meters, and also show high gastrointestinal absorption. One
observation made to the SwissADME predictions is that in
our products the nitrogen in the amine group, which nor-
mally is counted as a hydrogen bond acceptor, is not being
counted due to the adjacent double bond. Even in the
consideration of this nitrogen atom in the analyzed
Table 5 Docking scores of selected compounds with AChE (PDB
ID: 1EVE)
Compound
Binding energy
Principal residues interactions
(
kcal/mol)
2
a
e
−8.06
Gln69, Trp84
2
−10.50
−11.00
−9.56
Asp72, Trp84, Tyr334
Trp84, Trp279, Phe288
aDonepezil
aGalantamine
Trp84, Ser200,
His440, Glu199
aServed as reference compound
Fig. 3 Docking poses and
interactions of compounds (a) 2a
in yellow and (b) 2e in green
with the catalytic gorge of
TcAChE. H bonds are
represented by black lines

Medicinal Chemistry Research
compounds as a hydrogen bond acceptor, the Lipinski’s rule
for this descriptor (not having more than ten H bond
acceptors) is not violated in any case.
most alcohol derivatives showed enzymatic inhibition,
while the acetyl products lacked to show inhibition activity.
In silico techniques were incorporated, by docking the most
active AChE inhibitors 2a and 2e to acetylcholinesterase,
showing important π–π interactions with the enzyme resi-
dues. The calculation of ADME parameters for the products
was conducted, showing promising drug-like properties.
In both the employed antioxidant techniques, the acetyl
derivatives were generally more active than the alcohol
derivatives. However, both in the antiproliferative activity and
in the acetylcholinesterase inhibition the alcohol derivatives
showed more promising results than the acetyl ones; also, in
the AChE inhibition a tendency regarding the hydrophobicity
of the derivatives was observed, as better inhibitions came
from lower Log P values in the aliphatic amino alcohol
derivatives. This suggests that amino alcohol derivatives from
naphthoquinone can be lead compounds for further derivati-
zation. As the aromatic amino alcohol product 2e showed also
good results both in the antiproliferative and AChE inhibition
assays, it can be selected along with product 2a for the
synthesis of two new groups of 1,4-naphthoquinone amino
alcohol derivatives, aliphatic and aromatic, with new func-
tional groups incorporated in order to search for new and
improved potential Alzheimer and cancer treatment drugs.
The nine products also show a topological polar surface
area (TPSA) inside the limits suggested for good bioavail-
ability (20–130 Å), with the acetylated products 3a–d
having a higher TPSA than their alcohol counterparts 2a–d,
as the additional oxygen in the acetyl group contributes to
more polar area. However, the acetyl group is more
hydrophobic as a whole than the alcohol group, so the
acetyl derivatives have also a higher Log P than the alcohol
ones. In the prediction of cytochrome enzymes inhibition,
which is related to the metabolism and therefore possible
drug-drug interactions (Daina et al. 2017) the alcohol
derivatives 2a–e are listed as inhibitors of the CYP1A2,
while the acetyl derivatives 3a–d are listed also for
CYP2C19.
All these mentioned parameters show the overall drug
characteristics of the synthesized compounds. Regarding
the parameters related to the biological properties analyzed
in this work, it can be highlighted the blood–brain barrier
(
BBB) permeation of the products, which can be a desired
characteristic for the AChE inhibition activity and therefore
the anti- Alzheimer profile of a drug. Of the synthesized
compounds, only 2d, 3d, and 2e are classified as BBB
permeant. However, all of them are no P-gp substrates. As
this glycoprotein is related to the efflux of xenobiotics from
barriers as the BBB, the synthesized products and future
derivatives that can cross this barrier would not be pumped
out, helping with their AChE inhibition activities. Further-
more, as P-gp is also expressed in several human tumors
and can contribute to chemotherapy treatment resistance
Acknowledgements We gratefully acknowledge the Facultad de
Ciencias Químicas e Ingeniería, Universidad Autónoma de Baja
California for the financing given for the realization of this project.
Additionally, to the Consejo Nacional de Ciencia y Tecnología
(CONACYT) for ITT NMR facilities (Grant INFR-2011-3-173395).
A.E.C. thanks CONACYT for the scholarship received for his doctoral
studies (CVU: 764732). J.M.P. thanks the Spanish Government for
financial support through project PGC2018-094503-B-C22 (MCIU/
AEI/FEDER, UE).
(
Sharom 2008), is promising for the synthesized products to
exert their anti-proliferative activities as shown in Table 2,
specially the more active ones, with a lower risk of devel-
oping resistance.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Conclusion
In the present work, a series of amino alcohol (2a–e) and
acetyl (3a–d) derivatives from 1,4-naphthoquinone were
synthesized and characterized. Their antioxidant activity
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