Synthesis of amino acid-naphthoquinones and in vitro studies on cervical and breast cell lines

Article abstract of DOI:10.3390/molecules24234285

We performed an extensive analysis about the reaction conditions of the 1,4-Michael addition of amino acids to 1,4-naphthoquinone and substitution to 2,3-dichloronaphthoquinone, and a complete evaluation of stoichiometry, use of different bases, and the p

Full text of DOI:10.3390/molecules24234285

molecules
Article
Synthesis of Amino Acid–Naphthoquinones and In
Vitro Studies on Cervical and Breast Cell Lines
Ernesto Rivera-Ávalos 1, Denisse de Loera 1, Jorge Gustavo Araujo-Huitrado 2,
Ismailia Leilani Escalante-García 3 , Miguel Antonio Muñoz-Sánchez 3, Hiram Hernández 3
,
Jesús Adrián López 2,
*
and Lluvia López 4,
*
1
School of Chemistry, Universidad Aut
ónoma de San Luis Potosí, Av. Manuel Nava No. 6, SLP 78210, Mexico;
neto_riava@hotmail.com (E.R.-Á.); atenea.deloera@uaslp.mx (D.d.L.)
Laboratorio de microRNAs y C ncer, Unidad Acad mica de Ciencias Biológicas, Universidad Autónoma de
Zacatecas, Av. Preparatoria No.301, ZAC 98068, Mexico; jalopez2210@gmail.com
Unidad Académica de Ciencias Químicas, Universidad Autónoma de Zacatecas, Carr.
Zacatecas–Guadalajara km 6, ZAC 98160, Mexico; ileg@uaz.edu.mx (I.L.E.-G.);
27804270@uaz.edu.mx (M.A.M.-S.); hiram.hernandez.lopez@uaz.edu.mx (H.H.)
2
3
á
é
4
Instituto de Investigación de Zonas Desérticas, Universidad Autónoma de San Luis Potosí, Altaír No. 200,
SLP 78377, Mexico
*
Correspondence: jalopez@uaz.edu.mx (J.A.L.); lluvia.lopez@uaslp.mx (L.L.); Tel.: +52-492-149-2648 (J.A.L.);
+52-444-826-2300 (L.L.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 16 October 2019; Accepted: 18 November 2019; Published: 25 November 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: We performed an extensive analysis about the reaction conditions of the 1,4-Michael
addition of amino acids to 1,4-naphthoquinone and substitution to 2,3-dichloronaphthoquinone,
and a complete evaluation of stoichiometry, use of different bases, and the pH influence was
performed. We were able to show that microwave-assisted synthesis is the best method for the
synthesis of naphthoquinone–amino acid and chloride–naphthoquinone–amino acid derivatives
with 79–91% and 78–91% yields, respectively. The cyclic voltammetry profiles showed that
both series of naphthoquinone–amino acid derivatives mainly display one quasi-reversible redox
reaction process. Interestingly, it was shown that naphthoquinone derivatives possess a selective
antitumorigenic activity against cervix cancer cell lines and chloride–naphthoquinone–amino acid
derivatives against breast cancer cell lines. Furthermore, the newly synthetized compounds with
asparagine–naphthoquinones (3e and 4e) inhibited ~85% of SiHa cell proliferation. These results
show promising compounds for specific cervical and breast cancer treatment.
Keywords: naphthoquinone; amino acids; alternative methods; microwave; ultrasound; anticancer
1. Introduction
Naphthoquinone (NQ) is a nucleus found in several natural and synthetic compounds that
offers several applications, such as pigments, and many biological properties, including antibacterial,
antifungal, anticancer, antimalarial, and antiviral, among others [1,2]. Regarding cytotoxic activity,
several studies have reported that naphthoquinones with different substituents like paclitaxel, esters,
metals, furans, carbazoles, or inclusive with carbohydrates in their structure, among others, present
effects of diminishing cell proliferation [3–5].
These properties are principally attributed to the oxidant-reductive characteristics of the
naphthoquinones, which allow the generation of dianions or semiquinone radicals. In the last
decades, some publications have been dedicated to finding an explanation for the formation of these
intermediates in the synthetic mechanism, and their properties that have produced different compounds
with a plethora of applications of biological importance and effects that involve intra- or intermolecular
Molecules 2019, 24, 4285; doi:10.3390/molecules24234285
www.mdpi.com/journal/molecules

Molecules 2019, 24, 4285
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interactions. One of the principal effects of these compounds is the generation of reactive oxygen species
(ROS), producing cytotoxicity in different cell lines [ 13]. ROS generation with naphthoquinones
6
–
represents a challenge in the design of new active compounds, principally with anticancer effect. In
this context, the addition/substitution on naphthoquinone moiety by atoms or groups such as flour,
oxygen, or amine can be modulated by redox properties, decreasing the toxicity levels and maintaining
or potentiating the biological effect [14–16]. Several quinones from natural origin, like β-laphachone
and menadione, among others, have been well characterized by their specific selectivity for cell lines,
responding to temporal curse and dose response. Cell proliferation inhibition could be achieved
by the induction of apoptosis, topoisomerase II-
Furthermore, it has been shown that -lapachone inhibits epidermal growth factor (pEGFR), protein
kinase B (pAKT/PKB), glycogen synthase kinase (pGsk-3 ), cyclin D1, and cyclooxygenase-2 (COX-2)
α inhibition, and ROS generation, among others [4].
β
β
protein expression in a dose-dependent manner [17]. Interestingly, the naphthoquinone NSC 95,397 is
used as a general potent inhibitor of cell cycle division 25C (Cdc25) [18]. Naphthoquinone regulation
depends of the composition of its substituents, as well as targets and cellular components that could
be inhibited and/or activated. Several naphthoquinones have been produced; however, they present
non-desirable effects in cancer therapy use. Therefore, new compounds produced by alternative
methods are imperative for cancer treatment.
In this regard, the synthesis and biological activities of some naphthoquinone–amino acids
have been reported in several publications; nonetheless, all of these methodologies present different
drawbacks, like low yields and/or long reaction times [19–29]. To our knowledge, the synthesis of
naphthoquinone–amino acid derivatives has not been reported using microwave and ultrasound
irradiation, which offer diverse advantages to conventional synthesis. In previous works, our group
reported the synthesis of some Juglone and Lawsone derivatives using ultrasound and microwave
irradiation under mild reaction conditions [30–32].
In this paper, we present the synthesis of a series of 1,4-naphthoquinone–amino acid (3a
–e)
and 2,3-dichloronaphhtoquinone (4a 4e) derivatives under several activation methods, such as
–
room temperature synthesis (RTS), reflux synthesis (RS), microwave-assisted synthesis (MAS), and
ultrasound-assisted synthesis (UAS), and determined the effectivity on the system. Newly synthesized
compounds are promising in the cancer field. In the present work, we were able to incorporate alanine,
phenylalanine, methionine, glycine, and asparagine to both naphthoquinones (1a,b) by MAS and
evaluated their effect in the cervical cancer cell line, SiHa, and the breast cancer cell line, MCF-7. The
incorporation of amino acids to naphthoquinones could enhance their cytotoxicity capacity, as well as
their specificity.
2. Results
2.1. Chemistry
In a general method to prepare 3a
in a 1,4-type bond form to the naphthoquinone 1a, using initially, the conditions reported in the
literature (Figure 1) [19 23]. These conditions are shown as room temperature synthesis (RTS) and
–e derivatives, the corresponding amino acid 2a–c was added
,
reflux synthesis (RS) in Table 1. Base effect was studied using potassium carbonate (K2CO3) and
trimethylamine (TEA) in equimolar proportion. However, RTS and RS methodologies showed trace
or no production of the desired compounds when no base was used. Only for RS and base addition
were the yields increased to a range of 20–50%. A modified methodology was performed with
microwave (MAS) irradiation to determine the effect on the system. Interestingly, when the system
was subjected to microwave radiation, the yield of the derivatives was in the range of 28–30% without
base. Furthermore, the compound yields were increased when a base was used. The results indicate
that TEA and potassium hydroxide (KOH) were better bases, among others, to produce the derivative
compounds (Table 1). It was determined that the optimum pH for greater product formation was
9–10. At a lower pH, the reactions became very slow, and under higher pH, the presence of secondary

Molecules 2019, 24, 4285
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products was observed, as when the K2CO3 was used in an equimolar concentration (1 mmol) and not
in solution.
Figure 1. Preparation of 3a–e and 4a–e derivatives.
Table 1. Effect of reaction conditions of 3a–c derivatives in their yields.
Method
Compound Yield (%)
3b
Base
3a
3c
None
TEA
K2CO3
Nr
Tp
Tp
Nr
Tp
Tp
Nr
Tp
Tp
RTS
RS
None
TEA
K2CO3
Tp
32
23
Tp
50
50
Tp
30
20
None
TEA
K2CO3
AcONa
KOH
30
74
65
59
74
30
73
64
56
73
28
71
62
52
72
MAS
RTS: Room temperature synthesis (25 ◦C for 24–48 h); RS: Reflux synthesis (90 ◦C for 24 h); MAS: Microwave-assisted
synthesis (110 ◦C, 250 W, 25 min); Nr: No reaction; Tp: Trace product.
With these established conditions, we performed the synthesis of compounds 3a–e and 4a–e
with different bases and stoichiometric proportions between NQ and amino acids under MAS and
ultrasound (UAS) as an alternative method (Table 2). It was determined that the best yields were
obtained with KOH and TEA solutions. On the other hand, the yields were dependent of the proportion
of each amino acid in the solution; besides, reaction times were reduced significantly to 25 min under
MAS. In most cases, lower yields were obtained under UAS in comparison with MAS. Remarkably,
the yield was dependent on pH, stoichiometry, and base used for each amino acid incorporation
(Table 2).
In several investigations, the generation of compounds 3a
reported [19–29], and their boiling point, infrared, and H NMR spectra have been determined.
,
3b
,
3d
,
4a, and 4d has been
1
Compounds 4b
evaluations, but we did not find their complete characterization [33
been previously reported and, therefore, their complete characterization was carried out.
,
4c have been reported and used as intermediates for secondary reactions or biological
,
34], while 3c 3e, and 4e have not
,

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Table 2. Synthesis of 3a–e and 4a–e derivatives by MAS and UAS.
MAS a (%)
TEA KOH
80 86
UAS b (%)
TEA KOH
68 78
Compound Nq–aa
3a
3b
3c
1:2.0
1:1.2
1:1.5
1:1.5
1:1.5
1:2.0
1:1.2
1:1.5
1:1.5
1:1.5
85
82
81
79
91
95
78
91
78
91
87
85
80
86
87
89
82
85
75
71
67
77
86
85
80
75
75
81
65
78
82
80
70
68
85
85
3d
3e
4a
4b
4c
4d
4e
a MAS: 110 ◦C, 250 W, 25 min. b UAS: 25–40 ◦C, 1 h. Optimized conditions: Dioxane/water (4:1), TEA (1 mmol)/KOH
(3N) 5 mL. Nq–aa: Naphthoquinone–amino acid proportion.
2.2. Electrochemical Studies by Cyclic Voltammetry
The electrochemical reduction behavior of 1,4-naphthoquinone–amino acid (3a
–e) and
2,3-dichloro-1,4-naphhtoquinone derivatives (4a ) was investigated by cyclic voltammetry (CV)
–
e
in TBABF4 0.1M/DMSO as supporting electrolyte at a scan rate of 100 mV
cyclic voltammograms curves of compounds 3a–e and 4a–e, respectively.
·
s
−1. Figure 2A,B shows the
In Figure 2A,B, the CV profiles of the naphthoquinone derivative compounds mainly display one
quasi-reversible redox reaction process as compared to the two reduction peaks of typical quinone
derivatives in aprotic media [35
significant electroactive processes in the studied potential range. The irreversible reduction peak
observed near to 0.8 V for 3b and 0.9 V for 4a in Figure 2A,B, respectively, are related to the
oxidation reactions occurring by electron dislocations of the quinone derivatives at more positive
potentials, > 0.7 V or > 0.5 V for both 1,4-naphthoquinone and 2,3-dichloro-1,4-naphtoquinone–amino
,36]. Interestingly, compound 3a (Figure 2A) does not show any
−
–
e
−
–e
−
−
acid derivatives, respectively. Additionally, the irreversible reduction–oxidation peaks were not
observed when further cyclic voltammetry studies were performed at different scan rates in the
cathodic potential region for the evaluation of the redox reactions.
A
B
Figure 2. Cyclic Voltammetry curves of 5 mM naphthoquinone–amino acid derivatives (
and ( 4a
in TBABF4 0.1 M/DMSO at 100 mV s−1 on a glassy carbon disk working electrode at
room temperature.
A) 3a–e
B
)
–e

Molecules 2019, 24, 4285
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The main electrochemical parameters of the redox reaction of each naphthoquinone derivative
are reported in Table 3. Overall, the half-wave potentials (E1/2) for the redox reaction peak were near
−
1.24 V for 3b
–
e
and 1.12 V for 4a–e, respectively (Table 3). The redox reaction is a diffusion-controlled
−
process, since the cathodic peak current density (ipc) or the anodic peak current density (ipa) are
proportional to the square root of the scan rate (ipc vs. v1/2) [37,38].
In addition to this, the ratio of the cathodic peak current (ipc) to the anodic peak current (ipa) is
close to one for the naphthoquinone–amino acid derivatives 3b
systems (Table 3). However, the potential values of cathodic peak (Epc) to anodic peak (Epa) separation
Ep) are quite large and disagree with the theoretical value reported for a one-electron reversible
system, Ep > 60 mV, as shown in Table 3 [37]. Thus, the redox reaction process is considered
–e and 4a–e, as described for reversible
(
∆
∆
quasi-reversible. Furthermore, it is unclear if the reduction reaction of the quinone moiety occurs
via a single-step two-electron process as compared to the two successive one-electron transfer steps
producing semiquinone (Q−) and quinone dianion (Q2−), as typically observed for well-behaved
quinone derivatives (Q) in nonaqueous media [35,39–43].
Table 3. Electrochemical parameters of naphthoquinone derivatives with amino acid substituents a.
c
b
Epa
(V)
Epc
(V)
E1/2
(V)
-
ipa
ipc
∆Ep
(V)
-
|ipa/ipc
|
Compound
(mA cm−2
)
3a
3b
3c
3d
3e
4a
4b
4c
-
-
-
-
-
−1.18
−1.15
−1.17
−1.16
−1.04
−1.07
−1.04
−1.07
−1.05
−1.30
−1.30
−1.31
−1.32
−1.18
−1.19
−1.16
−1.20
−1.15
0.12
0.15
0.13
0.16
0.14
0.12
0.12
0.13
0.11
−1.24
−1.23
−1.24
−1.24
−1.11
−1.13
−1.10
−1.13
−1.10
0.28
0.16
0.28
0.05
0.36
0.37
0.27
0.28
0.26
−0.29
−0.17
−0.29
−0.06
−0.38
−0.41
−0.31
−0.26
−0.26
0.95
0.93
0.94
0.91
0.97
0.90
0.89
1.06
0.98
4d
4e
a
Determined by cyclic voltammetry in TBABF4 0.1 M/DMSO at 100 mV/s. The potentials are given with respect to
the Ag/Ag+ pseudo-reference electrode; b ∆Ep = Epc − Epa
;
E1/2 = (Epa + Epc)/2.
c
2.3. Effect of Naphthoquinone–Amino Acid Derivatives in Cell Proliferation of Cervical Cancer Cell Line SiHa
and Breast Cancer Cell Line MCF-7
SiHa cells were treated with 0.75 mM of naphthoquinone–amino acid derivatives 3a
–e and 4a–e;
however, the concentration was extremely toxic for the cell line (data not shown). Therefore, 0.1 mM
was used to register the proliferation effect on SiHa and MCF-7 cells. Compounds with glycine (3a
and asparagine (3e) substituents showed the most potent effect, inhibiting ~80% of proliferation in
SiHa cells, while in MCF-7, glycine (3a), chloride glycine (4a), and chloride asparagine (4e) showed a
proliferation inhibition near 90% (Figure 3A,B). Interestingly, the chloride substituent did not modify
the effect in SiHa cells, suggesting an effect directly mediated by the amino acids glycine and asparagine
(compare 3a and 3e versus 4a and 4e, Figure 3A), while in MCF-7, this substituent increased the
)
effect of naphthoquinone–amino acid compounds (compare 3a, 3b, and 3c versus 4a, 4b, and 4c). In
contrast, 4b increased 10% of inhibition compared with 3b 4b = 72.5% versus 3b = 61.3%; Figure 3A).
(
However, chloride incorporation did not always diminish proliferation, as it could be observed in 3c
versus 4c presenting 62.6% and 54.8% of proliferation inhibition in SiHa cells, respectively (Figure 3A).
Remarkably, in MCF-7 cells, chloride addition increased the proliferation inhibition effect of the
compounds (Figure 3B), suggesting a specific and particular effect for breast cell line. The 3d compound
with amino acid phenylalanine showed a bigger proliferation inhibition in MCF-7 cells (57%) over
SiHa cells (40%). Interestingly, for the chlorine compound 4d, a similar effect was observed for both
cell lines, presenting 69.8% of proliferation inhibition (Figure 3A,B).

Molecules 2019, 24, 4285
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Figure 3. Proliferation effect of naphthoquinone–amino acid derivatives was evaluated in cancer cell
lines derived from cervix and breast. ( ) SiHa cervical cancer cells were treated with 0.1 mM of
A
naphthoquinone–amino acid derivatives to assay proliferation rate at 72 h post-treatment. (B) MCF-7
breast cancer cells were treated with 0.1 mM of naphthoquinone–amino acid derivatives to assay
proliferation rate at 72 h post-treatment. Cells treated with 0.001% of DMSO were used as control.
3. Discussion
The naphthoquinone–amino acid derivatives have been previously reported; nonetheless, in
this investigation, we performed a very extensive analysis about the reactivity of the equivalents of
each amino acid used in the reactions. The results show a direct dependence of the pH on derivative
production since, under pH = 9, reactions became very slow and above pH = 10.5, several secondary
reactions appeared and reduced the yield, generating some different compounds that make difficult
the purification. We optimized reactions under ultrasonic and microwave irradiation, particularly
in the latter one, showing the highest efficiency obtaining the best yields 79–91% (3a–e) and 78–91%
(4a–e) compared with the literature reported data, and significantly reducing the reaction times from
days or hours to only a few minutes under the optimized conditions.
A full description of the electron-transfer mechanism of the naphthoquinones with amino acid
substituents is beyond the scope of the present paper. However, it is assumed that the quinone dianion
is possibly stabilized by an intramolecular hydrogen bond, since the dissociation of proton donor
groups, i.e., carboxyl group, in DMSO is not facile, thus, cannot protonate the dianion [44
such a case, the second redox process merges with the first (Figure 2), owing to the stabilization of
the dianion by a hydrogen bond, as reported elsewhere [35 46 48 49]. For instance, the E1/2 values
computed for compounds 3b (~1.24 V) are located at the mid-point of the half-wave potential values
determined for the semiquinone (Q−) and the dianion (Q2−) redox reaction peaks of the parental
1,4-naphtoquinone compound studied at same experimental conditions, 0.9 V or 1.6 V, respectively
(data not shown). Likewise, the redox reaction process peak of the naphthoquinone–amino acid and
–47]. In
,
, ,
–
e
−
−
chloride derivatives (4a
–
e
) is positioned at E1/2
~ 1.12 V, as shown in Table 3, while the E1/2 values for
−
the Q− and Q2– redox reaction steps of the 2,3-dichloro-1,4 naphthoquinone compound are
0.65 V
−
and −1.37 V, respectively (data not show).
Particularly, naphthoquinone derivatives with glycine and chloride substituents (4a) present a
noticeable electrochemical improvement regarding naphthoquinones with glycine (3a) (Figure 2). These
results are associated to the electron-accepting capacity of the chloride substituent, and possibly to
some conformational effects which modify the electronic configuration of the naphthoquinone–amino
acid derivatives, facilitating the reduction processes [41
compounds with amino acids and chloride substituents (4a
derivatives (3a ), as estimated by their E1/2 values, since reactive oxygen species (ROS) generation
,50]. It is then expected that naphthoquinone
–e) present higher biological activity than
–
e

Molecules 2019, 24, 4285
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is facilitated at compounds with more positive E1/2
[
39–
42
,49
,51]. However, additional factors such
as solvents, nature of supporting electrolyte, protonation–deprotonation equilibrium, and intra- and
intermolecular hydrogen bonding also play important roles in determining the half-wave potential of
the redox reaction processes, probably affecting cellular biological processes.
Recently, a proliferation inhibition effect of naphthoquinone–amino acid derivatives in different
cell lines was described. In the ileocecal adenocarcinoma cell line HCT-8, it was shown an 86, 84, and
59% of proliferation inhibition with phenylalanine, alanine, and proline naphthoquinone derivatives.
In the breast cancer cell line MDAMB-435, it was shown a 100, 100, and 36%, while in human multiforme
cell line SF-295 86, 83, and 59% of proliferation inhibition, respectively [23]. Glycine incorporation
in naphthoquinone structure is used to add methyl groups that would extend the molecules to
resemble other amino acids presenting different activities [17]. Naphthoquinones with amino acids
could probably interact with proteins and/or complexes, inhibiting several molecular processes like
replication, transcription, and translation. Moraes et al. [23] added three complete amino acids to
naphthoquinone structure, showing similar cytotoxicity to doxorubicin. The effect of glycine, alanine,
and phenylalanine derivatives synthesized by Moraes et al. was similar to our results. Marastoni et
al. [18] incorporated leucine, asparagine, phenylalanine, and serine into naphthoquinone with diamine
alkyl spacers, inhibiting three subunits of proteasome linked to proliferation inhibition of the breast
cancer cell line MDA and ovarian cancer cell line A2780 in the range of 100
µ
M, similar as in the
present study. However, in our study, we used a number of 1
×
105 cells, while they used 15
×
103 cells;
therefore, the molecules per cell that we used in our study was less than Marastoni et al.’s study.
The newly synthetized compounds with asparagine–naphthoquinones inhibited ~85% of SiHa
cell proliferation. Inhibition grade of naphthoquinone–amino acid derivatives 3a
ranked from 85 to 40% in SiHa cells. In contrast, MCF-7 cells presented an effect ranking from 90 to
30% of proliferation inhibition. In order from higher to lower, the compounds 4a 4e 4c, and 4d and
, 3e, 3b, 3c, and 3d
,
,
4b showed their effect. It could be theorized that the differential effects observed may be dependent
on hydropathy index and protein occurrence of the compounds. Glycine and asparagine present
the negative hydropathy index
−
0.4 and 4.5, respectively. Nevertheless, it should be noted that
−
asparagine’s hydropathy index is more negative that glycine’s hydropathy index, even though it is
more abundant in proteins than asparagine—7.2 and 5.1, respectively. This hypothesis needs several
experimental studies to be addressed. However, glycine– and asparagine–naphthoquinones, as well as
dichloride compounds, show selectively antitumorigenic properties in cervix and breast cancer cell
lines, respectively, positioning them as promising compounds for specific cancer treatment.
4. Materials and Methods
4.1. General
Commercially supplied 1,4-naphthoquinone, 2,3-dichloronaphthoquinone, alanine, glycine,
methionine, phenylalanine, and asparagine were used for the synthesis without further purification.
1H and 13C nucleus magnetic resonance (NMR) spectra were recorded on a Bruker Fourier 300 MHz
spectrometer (1H at 300, 13C at 75 MHz, Silberstreifen, Rheinstetten, Germany) and Bruker Avance III
400 MHz spectrometer (1H at 400, 13C at 101 MHz, Silberstreifen, Rheinstetten, Germany). The spectra
were acquired from solution in dimethylsulfoxide-d6 and methanol-d4 at room temperature, TMS as
internal reference, the chemical shifts (δ) are expressed in part per million (ppm) and the coupling
constants (J) in Hz. High-resolution mass spectra (HRMS) were measured with a Jeol JMS-AccuTOF
through DART (Direct Analysis in Real Time, Peabody, MA, USA) and by ESI (electrospray ionization) in
an Agilent 6200 Series TOF (Santa Clara, CA, USA) and 6500 Series Q-TOF LC/MS System (Santa Clara,
CA, USA). Infrared spectra were recorded on a Thermo Scientific NICOLET iS10 with ATR dispositive
(SMART iTR, Madison, WI, USA). Melting points were determined using a Bicote-Stuart SMO 10
apparatus (Stone, Staffordshire, UK) and were uncorrected. Microwave-assisted synthesis (MAS) was
performed on a CEM Mars 6 oven with a carrousel device (Matthews, NC, USA). Ultrasound-assisted

Molecules 2019, 24, 4285
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synthesis (UAS) was carried out in an Autoscience Ultrasonic cleaner-AS2060B with 60 Watts of
power (Lewisville, TX, USA). TLC was performed using silica gel 60 PF254 containing gypsum (Merck,
Darmstadt, Germany). The isolated reaction products were found to be >95% purity by NMR analysis
(See Supplementary Materials).
4.2. General Procedure for the Optimization of the Reaction Conditions for the Synthesis of Compounds 3a–c
A solution of the appropriate amino acid 2a–c (1 mmol) in 40 mL of EtOH/H2O (4:1) was basified
with several bases (pH = 9–10), and then a solution of 1 mmol of 1,4-naphthoquinone (1a) in ethanol
(10 mL) was added. The mixture was activated by different methods, such as room temperature (RTS),
reflux (RS), and microwave irradiation (MAS). Solvent and base effect were studied in the reaction
yields, using K2CO3 (3 N) and triethylamine (TEA) (Figure 1). The progress reaction was monitored
by TLC, using MeOH/CHCl3 (9:1) as eluent mixture. Finally, 20 mL of HCl (1 N) was added and
the precipitated product was filtered and purified by flash column chromatography, starting with
dichloromethane (DCM) and changing the polarity to finish with methanol.
4.3. Optimization of the Reaction Conditions for the Synthesis of Compounds 3a–c Under
Microwave Irradiation
In 15 mL of dioxane/H2O (4:1) or 30 mL of EtOH/H2O (4:1), 1.5 to 2 mmol of respective amino
acid was added, the mixture was basified with several bases (TEA, K2CO3 aq., AcONa, or KOH aq.)
to obtain a pH of 9–10, and activated under microwave irradiation with the following parameters:
◦
110 C, 250 W for a time of 10 min. Then, a solution of naphthoquinone (1a) in 5 mL or 20 mL of
the respective mixture was added; the reaction solution was irradiated with the same parameters by
30 min. After the time of reaction, 20 mL of HCl (1 N) was added, and the compounds were purified
with the methodology previously descripted.
4.4. Optimized Reaction Conditions Under Microwave Irradiation for the Synthesis of Compounds 3a–e
and 4a–e
A solution of respective amino acid (1.5–2.0 mmol) in dioxane/H2O (4:1, 15 mL for 3a
or 20 mL for 4a derivatives) was basified with TEA or KOH aq. (pH 9–10) and activated under
microwave irradiation (110 ◦C, 250 W for a time of 10 min). Then, a solution of naphthoquinone 5 mL
1a) or 2,3-dichloro-1,4-naphthoquinone (1b) 10 mL of dioxane/water mixture was added; the reaction
–e derivatives
–
e
(
mixture was irradiated with the same parameters by 20 min. After the time of reaction, 20 mL of
HCl (1N) was added, and the compounds were purified with the methodology previously descripted.
Under this methodology, compounds 3a, 3d and 3e, 4b, 4c, and 4e were purified as was previously
described, while 3b, 3c, 4a, and 4d were obtained by filtration without further purification.
4.5. General Procedure for the Synthesis of Compounds 3a–e and 4a–6 Under Ultrasonic Irradiation
For 3a–e derivatives, 15 mL of dioxane/H2O (4:1) was used, and 20 mL for 4a–e compounds. Then,
1.5–2.0 mmol of respective amino acid in solution dioxane/H2O was basified with TEA or KOH aq. to
pH 9–10, and activated by ultrasound at 25 ◦C and 15 min. Then, 5 mL a solution of naphthoquinone
(
1a) or 10 mL of 2,3-dichloro-1,4-naphthoquinone (same proportion dioxane/H2O) was added; the
reaction mixture was irradiated again for 45 min (maximum temperature of 40 ◦C). After the time of
reaction, 20 mL of HCl (1 N) was added, and the products were purified by column chromatography
with the previously described methodology.
4.6. Spectroscopic Characterization of Amino Acid–1,4-naphthoquinone Derivatives
2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)acetic acid (3a). Red-brown powder. Yield: 86%.
m. p.: 166–168 ◦C; IR (ATR): 3366, 1721, 1681, 1600, 1555, 1505, 1299, 1123 cm−1; 1H NMR (300 MHz,
DMSO-d6) δ: 12.96 (s, 1H), 7.99 (dd, J = 7.6/0.9 Hz, 1H), 7.93 (dd, J = 7.6/0.9 Hz, 1H), 7.83 (td, J =

Molecules 2019, 24, 4285
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7.5/1.4 Hz, 1H), 7.74 (td, J = 7.5/1.4 Hz, 1H), 7.46 (t, J = 6.2 Hz, 1H, NH), 5.63 (s, 1H), 3.99 (d, J = 6.2 Hz,
2H) ppm. (Characterization according to literature [23].)
2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino) propanoic acid (3b). Yellow powder. Yield: 91%.
m. p.: 145–147 ◦C; IR (ATR): 3357, 1725, 1685, 1604, 1550, 1492, 1366, 1310, 1298 cm−1; 1H NMR (300
MHz, DMSO-d6) δ: 13.08 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.84 (t, J = 7.5 Hz, 1H),
7.74 (t, J = 7.5 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H, NH), 5.66 (s, 1H), 4.21 (q, J = 7.3 Hz, 1H), 1.45 (d, J =
7.0 Hz, 3H) ppm. (Characterization according to literature [23].)
2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-4-(methylthio)butanoic acid (3c). Orange
powder. Yield: 87%. m. p.: 146–148 ◦C; IR (ATR): 3339, 1716, 1676, 1600, 1550 1505 1447, 1334, 1307,
1222 cm−1; 1H NMR (DMSO-d6)
δ
: 13.19 (s, 1H), 8.00 (td, J = 7.3/1.2 Hz, 1H), 7.94 (td, J = 7.4/1.2 Hz, 1H),
7.84 (td, J = 7.5/1.4 Hz, 1H), 7.75 (td, J = 7.5/1.5 Hz, 1H), 7.40 (d, J = 8.3 Hz, 1H, NH), 5.70 (s, 1H), 4.27
(td, J = 8.1/5.1 Hz, 1H), 2.64–2.41 (m, 2H), 2.28–2.08 (m, 2H), 2.05 (s, 3H) ppm; 13C NMR (DMSO-d6)
δ:
182.3, 181.7, 172.7, 148.5, 135.4, 133.2, 132.9, 130.8, 126.5, 125.8, 101.3, 54.1, 30.4, 30.1, 15.0 ppm; HRMS
(DART/Q-TOF) m/z: [M + H]+ for C15H16NO4S: 306.0755; Found: 306.0801.
2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenylpropanoic acid (3d). Red powder.
Yield: 85%. m. p.: 215–217 ◦C; IR (ATR): 3348, 1680, 1604, 1564, 1496, 1334, 1249, 1119 cm−1; 1H NMR
(300 MHz, DMSO-d6) δ: 7.93 (d, J = 8.0 Hz, 1H), 7.91 (d, J = 8.6 Hz, 1H), 7.80 (t, J = 7.4 Hz, 1H), 7.69 (t, J
= 7.4 Hz, 1H), 7.28 (d, J = 4.2 Hz, 1H, NH), 7.15 (m, 4H), 7.11 (s, 1H), 5.57 (s, 1H), 4.01 (q, J = 5.2 Hz,
1H), 3.14 (m, J = 7.2 Hz, 2H) ppm. (Characterization according to literature [23].)
4-amino-2-((1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-4-oxobutanoic acid (3e). Dark-red
powder. Yield 79%. M. p.: 147–149 ◦C; IR (ATR): 3438, 1734, 1682, 1591, 1528, 1379, 1276, 1231 cm−1
;
NMR 1H (300 MHz, DMSO-d6)
1H), 7.75 (s, 2H, NH), 7.69 (t, J = 7.2 Hz, 1H), 7.37 (d, J = 7.0 Hz, 1H, NH), 6.91 (s, 1H, NH), 5.67 (s, 1H),
δ: 7.96 (d, J = 7.9 Hz, 1H), 7.91 (d, J = 7.5 Hz, 1H), 7.81 (t, J = 7.0 Hz,
3.93 (q, J = 6.3/5.8 Hz, 1H), 2.64–2.41 (m, 1H) ppm; 13C NMR (DMSO-d6)
δ: 181.7, 181.4, 172.8, 172.7,
146.9, 134.9, 133.3, 132.2, 130.3, 125.9, 125.4, 99.8, 53.6, 38.3 ppm; HRMS (ESI/Q-TOF) m/z: [M + Na]+
for C14H12N2O5Na: 311.0644; Found: 311.0675.
4.7. Spectroscopic Characterization of Amino Acid–2,3-dichloronaphthoquinone Derivatives
2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)acetic acid (4a). Orange powder. Yield:
91%. M. p.: 168–170 ◦C; IR (ATR): 3339, 1718, 1671 1591, 1564, 1420, 1249, 1105, 714 cm−1; 1H NMR
(300 MHz, DMSO-d6) δ: 12.95 (s, 1H), 7.98 (dd, J = 7.4/1.4 Hz, 2H), 7.84 (td, J = 7.5/1.4 Hz, 1H), 7.75
(td, J = 7.5/1.4 Hz, 1H), 7.53 (t, J = 6.3 Hz, 1H, NH), 4.39 (d, J = 6.6 Hz, 2H) ppm. (Characterization
according to literature [24].)
2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)propanoic acid (4b). Orange–red
powder. Yield: 95%. m. p.: 227–229 ◦C; IR (ATR): 3339, 1716, 1676, 1600, 1550 1505 1447, 1334, 1307,
1222 cm−1; 1H NMR (400 MHz, Methanol-d4)
δ
: 7.96 (dd, J = 7.5/3.8 Hz, 2H), 7.72 (td, J = 7.5/1.3 Hz,
1H), 7.63 (td, J = 7.6/1.2 Hz, 1H), 7.21 (d, J = 8.3 Hz, 1H), 4.96 (q, J = 6.8 Hz, 1H), 1.54 (d, J = 6.8 Hz, 3H)
ppm; 13C NMR (101 MHz, Methanol-d4)
δ: 186.5, 179.8, 176.7, 149.5, 134.5, 132.3, 131.4, 129.9, 126.4,
125.9, 121.1, 53.4, 20.4 ppm; HRMS (DART/Q-TOF) m/z: [M + H]+ for C13H10ClNO4: 280.0332; Found:
280.0387.
2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-4-(methylthio)butanoic acid
(4c).
◦
Orange powder. Yield: 89%. m. p.: 185–188 C; IR (ATR): 3276, 1710, 1676, 1586, 1546, 1375,
1137, 1006, 723 cm−1; 1H NMR (400 MHz, DMSO-d6)
δ
8.23 (d, J = 7.3 Hz, 2H), 8.09 (t, J = 7.4 Hz, 1H),
8.01 (t, J = 7.5 Hz, 1H), 7.10 (d, J = 8.1 Hz, 1H, NH), 5.06 (q, J = 5.9 Hz, 1H), 2.87–2.70 (m, 2H), 2.49–2.40
(m, 1H), 2.27 (s, 3H), 1.51–1.46 (m, 1H) ppm; 13C NMR (101 MHz, DMSO-d6)
179.9, 175.4, 173.1, 143.8,
δ
135.1, 132.7, 132.11, 129.8, 126.6, 125.9, 119.7, 56.4, 33.8, 28.9, 14.8 ppm. HRMS (DART/Q-TOF) m/z: [M
+ H]+ for C15H14ClNO4S: 340.0366; Found: 340.0401.
2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-3-phenylpropanoic
acid
(4d).
Orange–red powder. Yield: 91%. m. p.: 175–177 ◦C; IR (ATR): 3303, 1743, 1676, 1600, 1555, 1406, 1294,
1
1262, 1226, 700 cm−1; H NMR (300 MHz, DMSO-d6)
δ
: 7.94 (dd, J = 9.0/2.9 Hz, 2H), 7.81 (td, J =

Molecules 2019, 24, 4285
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7.5/1.5 Hz, 1H), 7.73 (td, J = 7.5/1.5 Hz, 1H), 7.29–7.15 (m, 5H), 6.68 (d, J = 4.3 Hz, 1H), 5.32 (q, J =
6.2 Hz, 1H), 3.25 (dd, J = 5.7/2.8 Hz, 2H) ppm. (Characterization according to literature [22,29].)
4-amino-2-((3-chloro-1,4-dioxo-1,4-dihydronaphthalen-2-yl)amino)-4-oxobutanoic acid (4e). Red
powder. Yield: 85%. m. p.: 207 ◦C; IR (ATR): 3442, 3280, 3195, 1671, 1626, 1595, 1559 1393, 1298, 1276,
719 cm−1; 1H NMR (300 MHz, DMSO-d6)
δ: 7.94 (dd, J = 7.9/4.5 H, 2H), 7.81 (t, J = 6.1 Hz, 1H), 7.72 (t, J
= 7.4 Hz, 1H), 7.54 (d, J = 3.7 Hz, 1H), 7.04 (s, 1H), 5.00–4.81 (m, 1H), 2.70–2.54 (m, 2H).; 13C NMR
(DMSO-d6) δ: 181.7, 181.4, 172.8, 172.7, 146.9, 134.9, 133.3, 132.2, 130.3, 125.9, 125.4, 99.8, 53.6, 38.3 ppm;
HRMS (ESI/Q-TOF) m/z: [M + Na]+ for C14H11ClN2O5Na: 345.0254; Found: 345.0287.
4.8. Cyclic Voltammetry
Cyclic voltammetry (CV) was performed in a conventional three-electrode electrochemical cell
using a potentiostat/galvanostat (EG&G, PAR VersaStat 3). A polished glassy carbon disk electrode
(GC) (0.07 cm2, BASi USA) was employed as working electrode. Prior to each experiment, the GC
electrode was polished to mirror finishing with 0.3 mm and 0.05 mm alumina and washed with distilled
water, and sonicated in ethanol for 5 min. The counter electrode was a Pt wire (Alfa-Aesar, USA),
and a home-made Ag/Ag+ pseudo-reference electrode was used as a reference. The pseudo-reference
electrode consisted of a silver wire in a body-glass (PINE research, USA) filled with a solution of
0.01 M silver nitrate (AgNO3, BASi, USA) in tetrabutylammonium tetrafluoroborate (TBABF4, Sigma
Aldrich, USA) 0.1 M in dimethyl sulfoxide (DMSO, Sigma Aldrich, USA). The supporting electrolyte
consisted of TBABF4 0.1 M dissolved in DMSO. As received TBABF4 and DMSO were used without
further purification or drying processes. Furthermore, the testing solutions were prepared by adding
compounds 3a
cyclic voltammetry (CV) was carried out in a range from
100 mV s−1 in the cathodic direction to identify any electron transfer reaction. The redox reaction
–
e
or 4a
–
e
to the supporting electrolyte for a final concentration of 5
×
10−3 M. Then,
−
2.0 to 0.75 V vs. Ag/Ag+ at a scan rate of
processes associated to compounds 3a–e or 4a–e were further studied by CV at a scan rate of 10, 25, 50,
100, 200, and 300 mV·s−1
.
4.9. Cell Lines
The HPV-16 positive cervical tumor line SiHa and the breast tumor cell line MCF-7 were grown
in Dulbecco’s Modified Eagle’s Medium (DMEM) (Invitrogen Corporation, Carlsbad CA, United
States) enriched with 5% fetal bovine serum (FBS). Medium change and passage were performed
every 3 and 4 days, respectively. The SiHa cell line was kindly provided by Ph.D. Gariglio’s Lab from
CINVESTAV-IPN. The MCF-7 cell line was kindly provided by Ph.D. Victor Treviño from Tecnol
ógico
de Monterrey.
4.10. Cell Proliferation Analysis
Cell proliferation was quantified by violet crystal dye in 1 phosphate-buffered saline (PBS)
×
(2.7 mM KCl, 1.8 mM KH2PO4, 136 mM NaCl, 10 mM Na2HPO4 pH 7.4). The treated cells were
incubated in methanol for 15 min and washed two times with water. Cells were dyed with 0.1% crystal
violet and washed three times with water, and finally, violet crystal was recovered with 10% acid acetic
to be analyzed in microplate reader Multiskan GO Spectrophotometer (Thermo Scientific
Finland).
™, Ratastic,
5. Conclusions
As we previously mentioned, the synthesis of naphthoquinone–amino acid derivatives has been
reported; nonetheless, neither microwave- or ultrasonic-assisted synthesis has been reported. The
obtained results on the analysis of the synthetic methodology showed a direct dependence of the
stoichiometry of the reactants and the pH, due to the need of the amino acids for their deprotonation,
and the yield was increased with the microwave system. On the other hand, some products were
obtained directly by precipitation, only with the acidification of reaction mixture after a few hours of

Molecules 2019, 24, 4285
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rest without further purification. Since naphthoquinones possess redox properties, cyclic voltammetry
studies were done, concluding that the redox reaction is a diffusion-controlled quasi-reversible process.
The biological evaluation showed a potent proliferation inhibition in cervical and breast cancer cells
lines. Remarkably, naphthoquinone–amino acid derivatives, as well as dichloride compounds, showed
selectively antitumorigenic properties in cervix and breast cancer cell lines, respectively, positioning
them as promising compounds for specific cancer treatment.
Supplementary Materials: The following are available online: NMR and IR spectra of the characterization compounds.
Author Contributions: E.R.-Á. performed the synthesis experiment and characterization of the derivatives. D.d.L.
and L.L. designed the synthesis experiment, analyzed the data, and wrote the paper. J.G.A.-H. performed the
anticancer experiment, and J.A.L. designed the cancer experiment, analyzed the data, and wrote the paper of the
anticancer effect. I.L.E.-G., M.A.M.-S., and H.H. performed the electrochemical experiments, and analyzed and
described this section. All authors read and approved the manuscript.
Funding: This research was funded by the National Council for Science and Technology, Grant number
CB-2013-01-220844 and PhD Scholarship number 304251.
Acknowledgments: E.R.-
especially to Braulio Rodr
Huerta Salazar, Mar a del Carmen Garc
Á
í
., D.d.L. and L.L. acknowledge to the group of Instituto de Qu
guez Molina, to the technical support (Ma. de los ngeles Peña Gonz
a Gonz lez and Everardo Tapia Mendoza.), and to all the facilities to
í
mica of the UNAM,
Á
ález, Elizabeth
í
í
á
perform the characterization of the compounds and material used for the experiments.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds 3a–e and 4a–e are available from the authors.
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