Half-wave potentials and in vitro cytotoxic evaluation of 3-acylated 2,5-bis(phenylamino)-1,4-benzoquinones on cancer cells

Article abstract of DOI:10.3390/molecules24091780

A broad range of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones were synthesized and their voltammetric values, as well as in vitro cancer cell cytotoxicities, were assessed. The members of this series were prepared from acylbenzoquinones and phenylamines, in moderate to good yields (47-74%), through a procedure involving a sequence of two in situ regioselective oxidative amination reactions. The cyclic voltammograms of the aminoquinones exhibit two one-electron reduction waves to the corresponding radical-anion and dianion, and two quasi-reversible oxidation peaks. The first and second half-wave potential values (E1/2) of the members of the series were sensitive to the push-pull electronic effects of the substituents around the benzoquinone nucleus. The in vitro cytotoxic activities of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones against human cancer cells (bladder and prostate) and non-tumor human embryonic kidney cells were measured using the MTT colorimetric method. The substitution of both aniline groups, by either methoxy (electron donating effect) or fluorine (electron withdrawal effect), decreased the cytotoxicity in the aminoquinones. Among the members of the unsubstituted phenylamino series, two of the 18 compounds showed interesting anti-cancer activities. A preliminary assay, looking for changes in the expression of selected genes, was performed. In this context, the two compounds increased TNF gene expression, suggesting an association with an inflammatory-like response.

Full text of DOI:10.3390/molecules24091780

molecules
Article
Half-Wave Potentials and In Vitro Cytotoxic
Evaluation of 3-Acylated 2,5-Bis(phenylamino)-1,4-
benzoquinones on Cancer Cells
Julio Benites 1,2,
*
, Jaime A. Valderrama 1,2, Maryan Ramos 1, Maudy Valenzuela 1,
Angélica Guerrero-Castilla 1 , Giulio G. Muccioli 3 and Pedro Buc Calderon 1,4
1
Química y Farmacia, Facultad de Ciencias de la Salud, Universidad Arturo Prat, Casilla 121,
Iquique 1100000, Chile; jaimeadolfov@gmail.com (J.A.V.); maryan.ramos01@gmail.com (M.R.);
maudyta@gmail.com (M.V.); anguerrero@unap.cl (A.G.-C.); pedro.buccalderon@uclouvain.be (P.B.C.)
Instituto de Ciencias Exactas y Naturales, Universidad Arturo Prat, Casilla 121, Iquique 1100000, Chile
2
3
Bioanalysis and Pharmacology of Bioactive Lipids (BPBL), Louvain Drug Research Institute,
Université catholique de Louvain, 72 Avenue E. Mounier, BPBL 7201, 1200 Brussels, Belgium;
giulio.muccioli@uclouvain.be
Research Group in Metabolism and Nutrition, Louvain Drug Research Institute,
4
Université catholique de Louvain, 73 Avenue E. Mounier, 1200 Brussels, Belgium
*
Correspondence: juliob@unap.cl; Tel.: +56-57-2252-6275
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Jean Jacques Vanden Eynde
Received: 2 April 2019; Accepted: 16 April 2019; Published: 8 May 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A broad range of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones were synthesized and
their voltammetric values, as well as in vitro cancer cell cytotoxicities, were assessed. The members
of this series were prepared from acylbenzoquinones and phenylamines, in moderate to good yields
(47–74%), through a procedure involving a sequence of two in situ regioselective oxidative amination
reactions. The cyclic voltammograms of the aminoquinones exhibit two one-electron reduction
waves to the corresponding radical-anion and dianion, and two quasi-reversible oxidation peaks.
The first and second half-wave potential values (E1/2) of the members of the series were sensitive to
the push-pull electronic effects of the substituents around the benzoquinone nucleus. The in vitro
cytotoxic activities of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones against human cancer cells
(bladder and prostate) and non-tumor human embryonic kidney cells were measured using the MTT
colorimetric method. The substitution of both aniline groups, by either methoxy (electron donating
effect) or fluorine (electron withdrawal effect), decreased the cytotoxicity in the aminoquinones.
Among the members of the unsubstituted phenylamino series, two of the 18 compounds showed
interesting anti-cancer activities. A preliminary assay, looking for changes in the expression of selected
genes, was performed. In this context, the two compounds increased TNF gene expression, suggesting
an association with an inflammatory-like response.
Keywords: acylated 2,5-bis(phenylamino)-1,4-benzoquinones; oxidative amination; half-wave potential;
cancer cells; cytotoxicity
1. Introduction
The molecular frameworks of diverse naturally-occurring cytotoxic compounds, such as
smenospongine [
as their key structural component (Figure 1). In the field of synthetic cytotoxic aminoquinones,
those derived from 1,4-naphthoquinone [ ] and their heterocyclic analogues [ 17] have received
1], streptonigrin [2], and mansouramicyn C [3], contain the aminoquinoid moiety
4–8
9–
considerable attention. However, the synthesis and cytotoxic evaluation of aminobenzoquinones has
hitherto received little attention and was mainly focused on the 2,5-bis(arylamino)-1,4-benzoquinone
Molecules 2019, 24, 1780; doi:10.3390/molecules24091780
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1780
2 of 14
derivatives as compounds
A
and
B. The earlier findings of Verter and Rogers [18] reported that specific
2,5-bis(alkylamino)-3,6-dimethoxy-1,4-benzoquinones, prepared from polyporic acid, increased the
survival of L1210-bearing mice by 50%. In addition, Sarcoma 180 accumulated slower in treated
Swiss albino mice than in control animals [19]. Further studies, described by Mathew et al. [20], on
2,5-bis(phenylamino)benzoquinone
A and the corresponding chlorine analogue B, demonstrated a
good in vitro inhibitory activity against human colon adenocarcinoma proliferation. Compound
also found to be active against the leukemia L1210 screening in vitro.
B was
Figure 1. Examples of natural and synthetic antitumor active aminoquinones.
It is worth noting that synthetic 2,5-bis(arylamino)-1,4-benzoquinones containing acyl and
alkoxycarbonyl substituents at C-3 have been used as precursor of benz- and naphthoisoxazolequinones
[21–23]. Some of the members of these heterocycle series exhibit in vitro activity as radiosensitizers and
cytotoxic properties. Here, we wish to report the synthesis of a broad variety of 3-acyl-2,5-bis(arylamino)-
1,4-benzoquinones, in order to give information on their redox properties and in vitro cytotoxic activity
on cancer cells.
2. Results and Discussion
The members of the series of 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones (Scheme 1) were
prepared from 1,4-benzoquinone and a set of aldehydes and phenylamines. The acylhydroquinone
precursors 1a
aldehydes, according to our previously-reported procedure [24]. The preparation of 3-acyl-2,5-bis
(phenylamino)-1,4-benzoquinones 2a was accomplished through a sequence involving: (a) Oxidation
of the acylhydroquinones 1a , to the respective acylquinones, with silver (I) oxide (2.5 equiv.) in
–q were synthesized by the solar Friedel Crafts photoacylation of quinone 1 with
–q
–
q
dichloromethane (DCM) and (b) oxidative amination of the 2-acylquinones resulting in (b) with
phenylamines (2 equiv.) in ethanol under aerobic conditions. This procedure provides the substituted
benzoquinones 2a–q in the yield range 47–74% (Table 1), and is a modification of the procedure,
described by Shäfer and Aguado in [25], for the synthesis of compound 2a and some of their substituted
phenylamino analogs.

Molecules 2019, 24, 1780
3 of 14
Scheme 1. General access to 3-acyl-2,5-diphenylamino-1,4-benzoquinones 2a–q.
were established by 1H- and 13C-nuclear magnetic resonance
The structures of compounds 2a–q
(NMR), bi-dimensional nuclear magnetic resonance (2D-NMR), and high resolution mass spectroscopy
(HRMS). Heteronuclear multiple bond correlation (HMBC) experiments of the members of the series,
as those of the representative congeners 2b and 2p (Figure 2), allowed us to establish the location of
the substituents around the benzoquinone nuclei.
Figure 2. Heteronuclear multiple bond correlations (HMBCs) of compounds 2b and 2p.
It is worth mentioning that inspection of minimal energy conformation of compounds 2b and
2p, performed by MM2 calculation (ChemBio3D 11.0, PerkinElmer, MA, USA), shows a co-planar
orientation between the benzoquinone nucleus and the phenylamino substituent at C-5 (Supplementary
Materials). Furthermore, it was also observed that rotation of the substituents linked to the 2,3-quinone
double bond are strongly hindered (Figure 3).
Figure 3. Optimized 3D-structure of compounds 2b and 2p.

Molecules 2019, 24, 1780
4 of 14
The members of the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinone series 2a
–
q
were evaluated for
their half-wave potentials (EI 1/2 and EII 1/2). They were measured by cyclic voltammetry in acetonitrile
at room temperature, using a platinum electrode and 0.1 M tetraethylamoniumtetrafluoroborate as the
supporting electrolyte [26]. The voltammograms were run in the potential range from 0.0 to 2.0 V
−
versus non-aqueous Ag/Ag+. The cathodic peaks related to the reduction of quinone, and the anodic
one due to its re-oxidation, were observed for the compounds as well-defined quasi-reversible waves.
The EI
values for the first one-electron, which is related with the formation of the semiquinone
1/2
radical anion, fell within the range of
transfer, corresponding to the dianion formation [27
to 860 mV (Table 1). Taking into account the notable differences of the E1/2 values for each of the
one-electron transfer processes, it is evident that they are due to the push-pull electronic effects of the
substituents located in the 1,4-benzoquinone nucleus. Table 1, regarding the EI and EII
values of
−
850 to
−
500 mV. The EI 1/2 values for the second one-electron
28], were located within the range of 1170
,
−
−
1/2
1/2
the unsubstituted phenylamino members 2a
affects the second half-wave potential (
EII 1/2 = 300 mV) over the first one (
of the insertion of methoxy substituents into the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinone scaffold,
as in compounds 2i
2m, was related to a significant cathodic shift of the EI 1/2 when compared to EII
–h
, indicates that the nature of the acyl substituents mainly
∆
∆
EII 1/2 = 90 mV). The effect
–
1/2. This fact could be attributed to the donor effect of the 3,4,5-trimethoxyphenyl group, which is
transmitted to the quinonoid nucleus through the amino group.
Table 1. Yield and half-wave potential (EI,II 1 ) values of compounds 2a–q.
2
Product
Nº
−EI
−EII
1/2
(mV)
1/2
(mV)
R1
R2
R3
R4
Yield (%) a
ClogP
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2ñ
2o
2p
2q
CH3
C3H7
C5H11
H
H
H
H
H
H
H
H
MeO
MeO
MeO
MeO
MeO
H
H
H
H
H
H
H
H
H
H
H
H
H
MeO
MeO
MeO
MeO
MeO
F
F
F
F
F
H
H
H
H
H
H
H
H
MeO
MeO
MeO
MeO
MeO
H
H
H
H
H
64
56
57
59
55
58
56
60
74
57
63
67
64
47
62
52
49
55
0.14
1.21
2.05
2.88
1.69
1.57
0.56
1.93
1.19
1.06
0.67
−0.20
1.17
2.26
2.13
1.74
0.88
2.24
565
570
570
590
560
560
570
500
800
740
610
790
850
560
710
550
680
730
1170
1150
1090
910
1140
1120
870
1030
950
950
1010
930
1010
1040
860
C7H15
3,4-(OMe)2Ph
3,4,5-(OMe)3Ph
Furan-2-yl
Thiophen-2-yl
Ph
4-MeOPh
3-OMe-4-HOPh
Furan-2-yl
Thiophen-2-yl
Ph
4-MeOPh
3-MeO-4-HOPh
Furan-2-yl
Thiophen-2-yl
920
950
980
a
The products were isolated by column chromatography. Yields are based on acylhydroquinones 1a–q.
The effects on redox properties and lipophilia by either electron-donating or electron-withdrawing
groups in the 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinone scaffold (compounds 2i 2m versus 2n 2q
were also examined. Table 1 shows that the methoxy group (as in compounds 2i 2m) led to increased
0.2 to 1.19). Conversely, the fluorine
–
–
)
–
values of EI
group (as in compounds 2n
(
−
610 to
−
850 mV) but reduced values of ClogP (
2q) led to decreased values of EI
−
1/2
–
(
−
550 to 730 mV) but enhanced
−
1/2

Molecules 2019, 24, 1780
5 of 14
values of ClogP (0.88 to 2.26). As compared to previous series, it should be noted that the EI
values
1/2
of the unsubstituted phenylamino members 2a–h ranged between −500 and −590 mV.
The 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones 2a–q were evaluated for their in vitro cytotoxic
activities against normal human embryonic kidney cells (HEK-293 cells) and two human cancer cell lines
(T24 and DU-145 cells) in 72 h drug exposure assays. The cytotoxic activities of the new compounds
were measured using conventional microculture tetrazolium reduction assays [29]. The cytotoxic
activities are expressed in terms of IC50. Doxorubicin, a clinically used anti-cancer agent, was taken as
a positive control. The cytotoxic activity data are summarized in Table 2.
Table 2. IC50
±
SEM (
µM) values of 2a–q on T24 (bladder), DU-145 (prostate), and HEK-293 kidney cells.
Quinone
T24
DU-145
Mean Value
HEK-293
2a
2b
2c
2d
2e
>100
>100
96.0 ± 7.9
92.2 ± 8.7
45.2 ± 4.5
23.5 ± 2.1
47.7 ± 4.2
20.4 ± 2.1
39.9 ± 3.6
80.9 ± 9.7
92.9 ± 1.4
72.4 ± 2.6
45.9 ± 3.2
23.0 ± 2.3
48.6 ± 3.0
>100
98.0
96.1
30.7
28.7
52.9
36.1
40.0
78.2
96.4
78.8
72.9
35.0
55.9
87.2
100.0
100.0
95.0
100
>100
>100
>100
>100
>100
85.8 ± 0.9
61.7 ± 4.2
>100
16.3 ± 1.7
34.0 ± 3.7
58.2 ± 7.1
51.8 ± 6.2
40.2 ± 4.0
75.6 ± 9.1
>100
2f
2g
2h
2i
2j
2k
2l
2m
2n
2ñ
2o
2p
2q
DOX
>100
85.3 ± 1.9
56.9 ± 5.2
28.3 ± 1.5
33.0 ± 4.8
30.7 ± 3.1
>100
>100
47.1 ± 2.0
63.2 ± 0.6
74.5 ± 2.2
>100
>100
>100
>100
0.46 ± 0.08
>100
>100
>100
>100
90.0 ± 6.4
72.8 ± 7.0
>100
>100
0.93 ± 0.06
0.69
4.27 ± 0.34
Cells were seeded into 96-well plates at a density of 10,000 cells/well for 24 h and then incubated for 48 h, with or
without the quinone derivatives. At the end of the incubation, aliquots of cell suspensions were taken and the MTT
test was performed, as described in the Materials and Methods section. Results are expressed as mean values
SEM (µM) (n = 3). DOX = Doxorubicin.
±
Regarding the cytotoxicity of aminoquinones, it should be noted that the DU-145 cells were more
sensitive than T24 and HEK-293 cells. When comparing the IC50 values calculated for the most active
aminoquinones in both cancer cell lines, their activity ranged from 16.3 to 51.80
at least one order of magnitude higher than that obtained with doxorubicin (0.43 and 0.93
and DU-145 cells, respectively). The unsubstituted phenylamino members 2a 2h were largely more
active than the compounds 2i 2q, showing that the substitution of both aniline groups—by either
methoxy (electron donating effect) or fluorine (electron withdrawal effect)—decreased the cytotoxicity
of the aminoquinones. Among the series 2a 2h, the compounds 2c (IC50 values of 16.3 and 45.2 M in
T24 and DU-145 cells, respectively) and 2d (IC50 values of 34.0 and 23.5 M in T24 and DU-145 cells,
respectively) were the most active; displaying, in addition, a highly selective effect, as HEK-293 cells
were affected only at doses higher than 100 M. Moreover, within the same series, compounds 2f and 2g
µM. Such values were
µM in T24
–
–
–
µ
µ
µ
showed similar cytotoxic activities to previous compounds, but with a much lower selectivity. Outside
this series, compound 2l was also active, but without any selectivity as it affected both HEK-293 and
cancer cells. Thus, it may be concluded that, among all the tested aminoquinones, congeners 2c and 2d
displayed the best cytotoxic activities, exhibiting high selectivity and lipophilicity values. Therefore,
they represent lead-molecules for further investigations in exploring both intracellular targets as well
as their molecular mechanism of action.
In order to gain information about potential molecular targets, a preliminary assay for gene
expression was conducted. To this end, some genes were selected, regarding their key role in cell

Molecules 2019, 24, 1780
6 of 14
survival, for instance mTOR, TP53, TNF, and so on. Table 3 shows the relative expression levels of the
genes implicated in anti-cancer effects in T24 cells after treatment with 2c and 2d.
Table 3. Relative expression levels of genes implicated in anti-cancer effects in T24 cells after treatment
with compounds 2c and 2d.
Gene
Symbol
Relative Expression Levels in T24 Cells (µM)
Gene Name
Vehicle
2c
2d
Apoptosis regulator (BCL2), transcript variant alpha
Mechanistic target of rapamycin kinase
Glutathione S-transferase pi 1
Glutathione-disulfide reductase
Cell division cycle 25A
Tumor protein p53
v-Ha-ras Harvey rat sarcoma viral oncogene homolog
Histone deacetylase 3
BCL2
mTOR
GSTP1
GSR
CDC25A
TP53
HRAS
HDAC3
HDAC4
TNF
1.00 ± 0.05
1.00 ± 0.04
1.00 ± 0.06
1.00 ± 0.05
1.00 ± 0.06
1.00 ± 0.04
1.00 ± 0.06
1.00 ± 0.06
1.00 ± 0.05
1.00 ± 0.03
1.28 ± 0.15
0.99 ± 0.13
0.90 ± 0.08
0.96 ± 0.05
1.54 ± 0.13 ***
1.06 ± 0.11
1.07 ± 0.13
1.03 ± 0.15
1.08 ± 0.13
2.04 ± 0.11 ***
0.80 ± 0.04
0.95 ± 0.02
1.21 ± 0.04 *
1.07 ± 0.04
1.07 ± 0.03
1.01 ± 0.04
1.33 ± 0.06 *
1.02 ± 0.05
1.01 ± 0.02
1.44 ± 0.10 **
Histone deacetylase 4
Tumor necrosis factor
Cells were treated with 2c and 2d for 48 h. Total RNA was extracted for qPCR. The mRNA expression levels were
evaluated and further normalized to B2M level and the quantification performed according to the Delta–Delta Ct
method (2−∆∆Ct method) with respect to the vehicle-treated group (reference group). The values represent the mean
±
SEM, with n = 5 for each group. Significant differences, in comparison with the vehicle-treated group: * p < 0.05;
** p < 0.01; and *** p < 0.001.
Compound 2c (and, to a lesser extent, 2d) enhanced the expression of the TNF gene. Prior to
drawing a definitive conclusion, such an increase in gene expression should be further confirmed by
measuring its protein levels and activity. Nevertheless, it should be emphasized that a local increase in
TNF concentration is not only associated with an inflammatory response, but also with an immunogenic
response able to activate tumor-specific cytotoxic T lymphocytes, which can seek out and destroy
tumor cells and reduce tumor lesions [30]. In this context, the compounds 2c and 2d display a potential
application in cancer immunotherapy that deserves to be further investigated.
3. Materials and Methods
3.1. General Information
All the solvents and reagents were purchased from different companies, such as Aldrich (St. Louis,
MO, USA) and Merck (Darmstadt, Germany), and were used as supplied. Melting points (mp) were
determined on a Stuart Scientific SMP3 (Staffordshire, UK) apparatus and are un-corrected. The IR
spectra were recorded on an FT IR Bruker spectrophotometer, model Vector 22 (Bruker, Rheinstetten,
1
Germany), using KBr disks, and the wave numbers are given in cm−1. H- and 13C-NMR spectra were
recorded on a Bruker Avance-400 instrument (Bruker, Ettlingen, Germany) in CDCl3 or DMSO-d6 at 400
and 100 MHz, respectively. Chemical shifts are expressed in ppm downfield relative to tetramethylsilane,
and the coupling constants (J) are reported in Hertz. Data for the 1H-NMR spectra are reported as
follows: s = singlet, br s = broad singlet, d = doublet, m = multiplet, and the coupling constants (J)
are in Hz. Bi-dimensional NMR techniques and distortion-less enhancement by polarisation transfer
(DEPT) were used for the signal assignment. Chemical shifts are expressed in ppm downfield relative
to tetramethylsilane, and the coupling constants (J) are reported in Hertz. The HRMS data for all
final compounds were obtained using a LTQ-Orbitrap mass spectrometer (Thermo-Fisher Scientific,
Waltham, MA, USA) with the analysis performed using an atmospheric-pressure chemical ionization
(APCI) source, operated in positive mode. Silica gel Merck 60 (70–230 mesh, from Merck) was used for
preparative column chromatography and thin layer chromatography (TLC) aluminum foil 60F254 was
used for analytical thin layer chromatography. The acylbenzohydroquinones (1a–q) were prepared
according to a previously-reported procedure [24].

Molecules 2019, 24, 1780
7 of 14
3.2. Chemistry
Preparation of 3-Acyl-2,5-bis(phenylamino)-1,4-benzoquinones 2a–q, General Procedure.
Suspensions of the acylhydroquinones 1a
mg) in dichloromethane (30 mL) were left with stirring for 30 min at room temperature (rt). The mixtures
were filtered, the solids were washed with dichloromethane (3 25 mL), and the filtrates were evaporated
–q (1 equiv.), Ag2O (2.0 equiv.) and MgSO4 anhydrous (300
×
under reduced pressure. The residues were dissolved in ethanol, the phenylamines (2 equiv.) were
added to the solutions, and the mixtures were left with stirring at rt for 24 h. The solvents were removed
under reduced pressure and the residues were column-chromatographed over silica gel (petroleum
ether/EtOAc) to yield the corresponding pure 3-acyl-2,5-bis(phenylamino)- 1,4-benzoquinones 2a–q.
3-Acetyl-2,5-bis(phenylamino)-1,4-benzoquinone 2a (64%); red solid; mp: 200–201 ◦C. 1H-NMR (DMSO-d6):
δ
2.30 (s, 1H, Me), 5.80 (s, 1H, quinone), 7.22 (m, 4H, arom.), 7.36 (m, 4H, arom.), 7.45 (dd, 2H, J = 7.5,
7.7 Hz, arom.), 9.55 (s, 1H, 5-N Ph), 11.31 (br s, 1H, 2-N 31.9, 96.4, 109.1,
H
H
Ph). 13C-NMR (DMSO-d6):
δ
123.9 (3C), 124.2 (2C), 125.8, 126.2, 128.7 (2C), 129.3 (2C), 137.6, 139.2, 147.6, 178.3, 178.5, 199.3. HRMS
(APCI): [M + H]+ calcd. for C20H16N2O3: 332.11609; found 332.11359.
3-Butiryl-2,5-bis(phenylamino)-1,4-benzoquinone 2b (56%); red solid; mp: 115–116 ◦C. 1H-NMR (DMSO-d6):
δ
0.78 (t, 3H, J = 7.4 Hz, -CH2-CH2-CH3), 1.23 (m, 2H, -CH2-CH2-CH3), 2.63 (t, 2H, J = 7.4 Hz,
CH2-CH2-CH3), 5.81 (s, 1H, quinone), 7.15 (d, 2H, J = 7.4 Hz, H-arom.), 7.20 (t, 1H, J = 7.4 Hz, H-arom.),
7.25 (t, 1H, J = 7.3 Hz, H-arom.), 7.32 (t, 2H, J = 7.8 Hz, H-arom.), 7.38 (d, 2H, J = 7.4 Hz, H-arom.), 7.45
(t, 2H, J = 7.8 Hz, H-arom.), 9.51 (s, 1H, 5-N Ph), 10.85 (br s, 1H, 2-N
Ph). 13C-NMR (DMSO-d6):
-
H
H
δ
13.7, 16.6, 45.6, 96.0, 109.2, 123.9 (2C), 124.4 (2C), 125.8, 126.2, 128.7 (2C), 129.3 (3C), 137.6, 139.0, 147.5,
178.2, 178.8, 201.6. HRMS (APCI): [M + H]+ calcd. for C22H20N2O3: 360.14739; found 360.15469.
◦
1
3-Hexanoyl-2,5-bis(phenylamino)-1,4-benzoquinone 2c (57%); brown solid; mp: 140–141 C. H-NMR
(DMSO-d6): 0.83 (t, 3H, J = 7.1 Hz, -CH2-CH2-CH2-CH2-CH3), 1.17 (m, 6H, -CH2-CH2-CH2-CH2-CH3),
δ
2.63 (t, 2H, J = 7.0 Hz, -CH2-CH2-CH2-CH2-CH3), 5.80 (s, 1H, quinone), 7.14 (d, 2H, J = 7.4 Hz, H-arom.),
7.20 (t, 1H, J = 7.4 Hz, H-arom.), 7.25 (t, 1H, J = 7.3 Hz, H-arom.), 7.32 (t, 2H, J = 7.7 Hz, H-arom.), 7.38
(d, 2H, J = 7.4 Hz, H-arom.), 7.45 (t, 2H, J = 7.8 Hz, H-arom.), 9.51 (s, 1H, 5-NHPh), 10.79 (br s, 1H,
2-NH δ 13.8, 22.0, 22.8, 30.8, 43.5, 95.9, 109.3, 123.9 (2C), 124.4 (2C), 125.8,
Ph). 13C-NMR (DMSO-d6):
126.2, 128.8 (2C), 129.3 (3C), 137.6, 139.0, 147.5, 178.2, 178.8, 201.6. HRMS (APCI): [M + H]+ calcd. for
C24H24N2O3: 388.17869; found 388.18599.
◦
1
3-Octanoyl-2,5-bis(phenylamino)-1,4-benzoquinone 2d (59%); brown solid; mp: 91–92 C. H-NMR
(DMSO-d6): 0.86 (t, 3H, J = 7.0 Hz, -(CH2)6C 3, 1.23 (m, 10H, -CH2-(C 2)5CH3), 2.63 (t, 2H,
J = 6.5 Hz, -C 2(CH2)5CH3), 5.81 (s, 1H, quinone), 7.14 (d, 2H, J = 7.5 Hz, H-arom.), 7.19 (t, 1H,
J = 7.4 Hz, H-arom.), 7.25 (t, 1H, J = 7.2 Hz, H-arom.), 7.32 (t, 2H, J = 7.7 Hz, H-arom.), 7.38 (d, 2H,
J = 7.4 Hz, H-arom.), 7.45 (t, 2H, J = 7.8 Hz, H-arom.), 9.51 (s, 1H, 5-N Ph), 10.81 (br s, 1H, 2-N Ph).
δ
H
H
H
H
H
13C-NMR (DMSO-d6):
δ 13.9, 22.0, 23.1, 28.5, 28.6, 31.1, 43.6, 95.9, 109.3, 123.8 (2C), 124.4 (2C), 125.8,
126.1, 128.7 (2C), 129.3 (3C), 137.6, 139.0, 147.5, 178.2, 178.8, 201.6. HRMS (APCI): [M + H]+ calcd. for
C26H28N2O3: 416.20999; found 416.21710.
3-(3,4-Dimethoxybenzoyl)-2,5-bis(phenylamino)-1,4-benzoquinone 2e (55%); gray solid; mp: 230.5–231.5 ◦C.
1H-NMR (DMSO-d6):
δ
3.67 (s, 3H, OMe), 3.82 (s, 3H, OMe), 5.91 (s, 1H, quinone), 6.81 (t, 3H, J = 7.6 Hz
H-arom.), 6.94 (m, 4H, H-arom.), 7.26 (t, 2H, J = 7.2 Hz, H-arom.), 7.41 (d, 2H, J = 7.7 Hz, H-arom.), 7.46
(t, 2H, J = 7.4 Hz, H-arom.), 9.42 (s, 1H, 5-N Ph), 9.54 (br s, 1H, 2-N
Ph). 13C-NMR (DMSO-d6):
,
H
H
δ
55.3, 55.7, 95.3, 107.7, 109.9, 110.3, 123.8 (2C), 124.0, 125.7 (2C), 125.7, 126.0, 128.0 (2C), 129.3 (3C), 130.9,
137.7, 146.0, 147.3, 148.3, 152.8, 178.4, 179.3, 191.5. HRMS (APCI): [M + H]+ calcd. for C27H22N2O5:
454.15287; found 454.15964.
3-(3,4,5-Trimethoxybenzoyl)-2,5-bis(phenylamino)-1,4-benzoquinone 2f (58%); brown solid; mp: 216.5–217.5 ◦C
1H-NMR (CDCl3):
3.80 (s, 6H, OMe), 3.89 (s, 3H, OMe), 6.19 (s, 1H, quinone), 6.74 (s, 2H, H-arom.),
.
δ

Molecules 2019, 24, 1780
8 of 14
6.82 (m, 2H, H-arom.), 7.06 (m, 3H, H-arom.), 7.28 (m, 3H, H-arom.), 7.44 (t, 2H, J = 7.8 Hz, H-arom.),
8.23 (s, 1H, 5-N Ph), 8.48 (br s, 1H, 2-N 56.4 (2C), 61.0, 106.5 (2C), 123.1
Ph). 13C-NMR (CDCl3):
H
H
δ
(3C), 125.7 (2C), 126.6, 127.1, 129.0 (2C), 129.9 (3C), 133.1, 136.7, 136.9, 142.8, 146.0, 146.7, 152.9 (2C),
178.3, 179.3, 191.8. HRMS (APCI): [M + H]+ calcd. for C28H24N2O6: 484.16344; found 484.16364.
3-(Furan-2-carbonyl)-2,5-bis(phenylamino)-1,4-benzoquinone 2g (56%); brown solid; mp: 141.5–142.5 ◦C.
1H-NMR (DMSO-d6):
δ 5.89 (s, 1H, quinone), 6.88 (d, 2H, J = 7.4 Hz, H-arom.), 7.01 (m, 3H, H- arom),
7.09 (t, 1H, J = 4.3 Hz, H-arom.), 7.26 (t, 1H, J = 7.1 Hz, H-arom.), 7.41 (d, 2H, J = 7.7 Hz, H-arom.), 7.46
(t, 2H, J = 7.7 Hz, H-arom.), 7.58 (d, 1H, J= 3.5 Hz, H-arom.), 7.84 (d, 1H, J = 4.7 Hz, H-arom.), 9.45
(s, 1H, 5-NHPh), 9.65 (br s, 1H, 2-NH δ 95.3, 107.7, 123.9 (2C), 125.6 (2C),
Ph). 13C-NMR (DMSO-d6):
125.8, 126.2, 127.9, 128.0 (2C), 129.3 (2C), 133.9, 134.8, 137.5, 137.6, 145.2, 146.0, 147.4, 178.1, 179.0, 184.8.
HRMS (APCI): [M + H]+ calcd. for C23H16N2O4: 384.11101; found 384.11164.
3-(Thiophen-2-carbonyl)-2,5-bis(phenylamino)-1,4-benzoquinone 2h (60%); brown solid; mp: 245–246 ◦C.
1H-NMR (DMSO-d6):
δ 5.88 (s, 1H, quinone), 6.57 (d, 1H, J = 3.3 Hz, H-arom.), 6.90 (m, 2H, H- arom),
7.00 (d, 1H, J = 3.2 Hz, H-arom.), 7.04 (d, 3H, J = 5.1 Hz, H-arom.), 7.26 (t, 1H, J = 7.1 Hz, H-arom.),
7.40 (d, 2H, J = 7.9 Hz, H-arom.), 7.46 (t, 2H, J = 7.5 Hz, H-arom.), 7.81 (s, 1H, H-arom.), 9.46 (s, 1H,
Ph). 13C-NMR (DMSO-d6):
5-NHPh), 9.68 (br s, 1H, 2-NH δ 95.3, 107.2, 112.5, 118.2, 123.9 (2C), 125.2
(2C), 125.8, 126.3, 128.1 (2C), 129.3 (2C), 137.6, 137.7, 146.2, 146.8, 147.4, 153.1, 178.1, 179.0, 180.0. HRMS
(APCI): [M + H]+ calcd. for C23H16N2O3S: 400.08816; found 400.09555.
3-Benzoyl-2,5-bis-[(3,4,5-trimethoxyphenyl)amino]-1,4-benzoquinone 2i (74%); brown solid; mp: 224–225 ◦C.
1H-NMR (CDCl3)
δ
: 3.54 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.80 (s, 3H, OMe),
5.97 (s, 2H, H-arom.) 6.14 (s, 1H, quinone), 6.49 (s, 2H, H-arom.), 7.31 (m, 1H, H-arom.), 7.45 (m, 1H,
H-arom.), 7.56 (d, 2H, J = 7.3 Hz, H-arom.), 8.16 (s, 1H, 5-N Ph), 8.42 (br s, 1H, 2-N
Ph). 13C-NMR
(CDCl3) : 55.8 (2C), 56.4 (2C), 60.9, 61.1, 95.9, 101.0 (2C), 102.9 (2C), 107.0, 128.4 (2C), 128.8 (2C), 132.3,
δ
: 3.87 (s, 9H, 3
×
OMe),
H
H
δ
132.5, 133.0, 136.7, 136.9, 137.6, 145.9, 147.0, 153.2 (2C), 154.1 (2C), 178.2, 179.0, 192.9. HRMS (APCI):
[M + H]+ calcd. for C31H30N2O9: 574.19513; found 574.20187.
3-(4-Methoxybenzoyl)-2,5-bis-[(3,4,5-trimethoxyphenyl)amino]-1,4-benzoquinone 2j (57%); red solid; mp:
190–191 ◦C. 1H-NMR (CDCl3)
δ
: 3.57 (s, 6H, 2
OMe), 5.99 (s, 2H, H-arom.), 6.11 (s, 1H, quinone), 6.49 (s, 2H, H-arom.), 6.77 (d, 2H, J = 8.8 Hz,
H-arom.), 7.53 (d, 2H, J = 8.8 Hz, H-arom.), 8.16 (s, 1H, 5-N Ph), 8.37 (br s, 1H, 2-N
Ph). 13C-NMR
(CDCl3) : 55.6, 55.8 (2C), 56.4 (2C), 60.8, 61.1, 95.8, 101.0 (2C), 102.9 (2C), 107.3, 113.5 (2C), 131.0, 131.2
×
OMe), 3.77 (s, 3H, OMe), 3.82 (s, 3H, OMe), 3.87 (s, 9H,
3
×
H
H
δ
(2C), 132.4, 132.6, 136.7, 136.8, 145.6, 147.0, 153.0 (2C), 154.0 (2C), 163.5, 178.2, 179.2, 191.3. HRMS
(APCI): [M + H]+ calcd. for C32H32N2O10: 604.20570; found 604.21238.
3-(4-Hydroxy-3-methoxybenzoyl)-2,5-bis-[(3,4,5-trimethoxyphenyl)amino]-1,4-benzoquinone 2k (63%); brown
solid; mp: 152–152 ◦C. IR (KBr)
ν
cm–1: 3246 (N-H), 3245 (N-H), 1662 (C=O), 1649 (C=O), 1636
max
(C=O). 1H-NMR (CDCl3)
3.89 (s, 6H, 2 OMe), 5.99 (s, 2H, H-arom.), 6.13 (s, 2H, quinone + OH), 6.50 (s, 2H, H-arom.), 6.81 (d,
1H, J = 8.2 Hz, H-arom.), 6.95 (d, 1H, J = 1.8 Hz, arom), 7.25 (dd, 1H, J = 8.2, 1.9 Hz, arom), 8.15 (s,
1H, 5-N Ph), 8.34 (br s, 1H, 2-N : 55.9 (2C), 56.2, 56.5 (2C), 61.0, 61.2, 95.8,
Ph). 13C-NMR (CDCl3)
δ
: 3.61 (s, 6H, 2
×
OMe), 3.75(s, 3H, OMe), 3.85(s, 3H, OMe), 3.88(s, 3H, OMe),
×
H
H
δ
101.0 (2C), 103.1 (2C), 107.3, 109.3, 113.2, 125.2, 131.0, 132.4, 132.6, 136.7, 145.7, 146.8, 147.1, 150.6 (2C),
153.1 (2C), 154.1 (2C), 178.2, 179.2, 191.6. HRMS (APCI): [M + H]+ calcd. for C32H32N2O11: 620.20061;
found 620.20726.
3-(Furan-2-carbonyl)-2,5-bis-[(3,4,5-trimethoxyphenyl)amino]-1,4-benzoquinone 2l (67%); red solid; mp:
202–203 ◦C. 1H-NMR (CDCl3)
δ
: 3.66 (s, 6H, 2
OMe), 6.12 (s, 1H, quinone), 6.17 (s, 2H, H-arom.), 6.44 (dd, 1H, J = 3.6, 1.6 Hz, H-arom.), 6.49 (s,
2H, H-arom.), 6.88 (dd, 1H, J = 3.6, 0.8 Hz, H-arom.), 7.47 (d, 1H, J = 1.6, 0.8 Hz, H-arom.), 8.11 (s, 1H,
5-N Ph), 8.47 (s, 1H, 2-N : 56.0 (2C), 56.4 (2C), 61.0, 61.1, 96.0, 100.9 (2C),
Ph). 13C-NMR (CDCl3)
×
OMe), 3.78(s, 3H, OMe), 3.87 (s, 3H, OMe), 3.88 (s, 6H,
2
×
H
H
δ
102.2 (2C), 106.7, 112.9, 117.4, 132.4, 132.5, 136.7, 137.1, 145.7, 146.1, 147.1, 153.4 (2C), 153.5, 154.1 (2C),
177.8, 178.9, 180.2. HRMS (APCI): [M + H]+ calcd. for C29H28N2O10: 564.17440; found 564.18120.

Molecules 2019, 24, 1780
9 of 14
3-(Thiophen-2-carbonyl)-2,5-bis-[(3,4,5-trimethoxyphenyl)amino]-1,4-benzoquinone 2m (64%); red solid; mp:
174.5–175.5 ◦C. 1H-NMR (CDCl3)
: 3.62 (s, 6H, 2 OMe), 3.79 (s, 3H, OMe), 3.87 (s, 3H, OMe), 3.88 (s,
6H, 2 OMe), 6.09 (s, 2H, H-arom.), 6.12 (s, 1H, quinone), 6.49(s, 2H, H-arom.), 7.0 (dd, 1H, J = 4.9, 3.8
Hz, H-arom.), 7.43 (dd, 1H, J = 3.8, 1.2 Hz, H-arom.), 7.55 (dd, 1H, J = 4.9, 1.2 Hz, H-arom.), 8.15 (s, 1H,
5-N Ph), 8.44 (s, 1H, 2-N : 56.0 (2C), 56.4 (2C), 61.0, 61.2, 95.9, 101.0 (2C),
Ph). 13C-NMR (CDCl3)
δ
×
×
H
H
δ
103.0 (2C), 107.5, 127.6, 132.2, 132.5, 133.2, 134.6, 136.7, 137.1, 145.6, 145.7, 147.0, 153.3 (2C), 154.1 (2C),
177.8, 178.9, 184.5. HRMS (APCI): [M + H]+ calcd. for C29H28N2O9S: 580.15155; found 580.15880.
◦
3-Benzoyl-2,5-bis-[(4-fluorophenyl)amino]-1,4-benzoquinone 2n (47%); brown solid; mp: 250–251 C.
1H-NMR (CDCl3)
δ
: 6.02 (s, 1H, quinone), 6.68 (t, 2H, J = 8.5 Hz, H-arom.), 6.79 (dd, 2H, J = 8.7, 4.8 Hz,
H-arom.), 7.14 (m, 2H, H-arom.), 7.24 (m, 2H, H-arom.), 7.33 (t, 2H, J = 7.6 Hz, H-arom.), 7.48 (t, 1H,
J = 7.4 Hz, H-arom.), 7.54 (d, 1H, J = 1.4 Hz, H-arom.), 7.57 (m, 1H, H-arom.), 8.06 (s, 1H, 5-N Ph), 8.41
(br s, 1H, 2-N : 95.7, 107.5, 115.8, 116.1, 116.7 (2C), 117.1, 125.3, 125.4, 127.7,
Ph). 13C-NMR (CDCl3)
H
H
δ
127.8, 128.5 (2C), 128.8 (2C), 132.6, 132.7, 132.8, 132.9, 133.4, 137.6, 147.1, 178.4, 179.2, 193.1. HRMS
(APCI): [M + H]+ calcd. for C25H16F2N2O3: 430.11290; found 430.11942.
3-(4-Methoxybenzoyl)-2,5-bis-[(4-fluorophenyl)amino]-1,4-benzoquinone 2ñ (62%); brown solid; mp: 239–240 ◦C.
1H-NMR (CDCl3)
δ
: 3.86 (s, 3H, OMe), 6.00 (s, 1H, quinone), 6.70 (t, 2H, J = 8.5 Hz, H-arom.), 6.81 (dd,
4H, J = 8.8, 4.0 Hz, H-arom.), 7.13 (t, 2H, J = 8.5 Hz, H-arom.), 7.23 (m, 2H, H-arom.), 7.53 (d, 2H,
J = 8.9 Hz, H-arom.), 8.09 (s, 1H, 5-N Ph), 8.33 (br s, 1H, 2-N : 55.5, 95.5,
Ph). 13C-RMN (CDCl3)
H
H
δ
107.7, 113.5 (3C), 115.5, 116.6, 116.9, 125.2, 125.3, 127.6, 127.7, 130.8, 131.2 (2C), 132.4, 132.5, 132.7, 132.7,
145.8, 147.0, 163.7, 178.3, 179.2, 191.4. HRMS (APCI): [M + H]+ calcd. for C26H18F2N2O4: 460.12346;
found 460.13003.
3-(4-Hydroxy-3-methoxybenzoyl)-2,5-bis-[(4-fluorophenyl)amino]-1,4-benzoquinone 2o (52%); brown solid;
mp: 221–222 ◦C. 1H-NMR (CDCl3)
δ
: 3.85 (s, 3H, OMe), 6.01 (s, 1H, OH), 6.11 (s, 1H, quinone), 6.79
(m, 5H, H-arom.), 6.95 (m, 1H, H-arom.), 7.15 (m, 2H, H-arom.), 7.26 (m, 4H, H-arom.), 8.09 (s, 1H,
5-N Ph), 8.28 (s, 1H, 2-N : 56.1, 95.6, 107.8, 109.3, 113.6, 115.6, 115.9, 116.7,
Ph). 13C-NMR (CDCl3)
H
H
δ
117.0, 125.3, 125.4, 125.5, 128.1, 128.2, 130.9, 132.5, 132.6, 132.7, 132.8, 145.9, 146.8, 147.1, 150.8, 178.4,
179.3, 191.6. HRMS (APCI): [M + H]+ calcd. for C26H18F2N2O5: 476.11838; found 476.12468.
3-(Furan-2-carbonyl)-2,5-bis-[(4-fluorophenyl)amino]-1,4-benzoquinone 2p (49%); brown solid; mp:
271.5–272.5 ◦C. 1H-NMR (DMSO-d6)
δ
: 5.78(s, 1H, quinone), 6.59 (m, 1H, arom.), 6.90 (m, 4H, H-arom.),
7.05 (d, 1H, J = 3.4 Hz, H-arom.), 7.30 (t, 2H, J = 8.8 Hz, H-arom.), 7.43 (dd, 4H, J = 9.0, 5.0 Hz, H-arom.),
7.85 (m, 1H, H-arom.), 9.50(s, 1H, 5-N Ph), 9.67 (s, 1H, 2-N : 95.1, 107.2,
Ph). 13C-NMR (DMSO-d6)
H
H
δ
112.6, 114.5, 115.0, 116.0, 116.3, 129.2, 126.3 (2C), 127.7, 127.9, 133.9 (2C), 134.0, 134.1, 146.6, 147.1, 147.7,
153.0, 178.1, 178.9, 180.1. HRMS (APCI): [M + H]+ calcd. for C23H14F2N2O4: 420.09216; found 420.09920.
3-(Thiophen-2-carbonyl)-2,5-bis-[(4-fluorophenyl)amino]-1,4-benzoquinone 2q (55%); brown solid; mp:
278–279 ◦C. 1H-NMR (CDCl3)
δ: 5.78(s, 1H, quinone), 6.86 (m, 4H, H-arom.), 7.10 (s, 1H, H-arom.),
7.36 (m, 4H, H-arom.), 7.60 (s, 1H, H-arom.), 7.89 (s, 1H, H-arom.), 9.48 (s, 1H, 5-N Ph), 9.63 (s, 1H,
H
2-NH δ: 95.1, 107.7, 114.7, 115.0, 116.0, 116.3, 126.2, 126.3, 128.1 (2C), 128.2,
Ph). 13C-NMR (CDCl3)
133.7, 133.8, 133.9, 134.0, 134.3, 135.2, 145.1, 146.3, 147.7, 178.1, 178.9, 185.0. HRMS (APCI): [M + H]+
calcd. for C23H14F2N2O3S: 436.06932; found 436.07638.
3.3. Biological Assays
3.3.1. Cell Lines and Cell Cultures
Human cancer cell lines T24 (bladder) and DU-145 (prostate), and non-tumor HEK-293 cells were
obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cultures were
maintained at a density of 1
were cultured in high-glucose Dulbecco’s modified Eagle medium (Gibco, Grand Island, NY, USA)
supplemented with 10% fetal calf serum, penicillin (100 U/mL), and streptomycin (100 g/mL). All
cultures were kept at 37 ◦C in 95% air/5% CO2 at 100% humidity. Phosphate-buffered saline (PBS) was
×
105 cells/mL and the medium was changed at 48- to 72-h intervals. They
µ

Molecules 2019, 24, 1780
10 of 14
purchased from Gibco. Cells were incubated at the indicated times at 37 ◦C, with or without quinones
at various concentrations.
3.3.2. Cytotoxic Assays
The cytotoxicity of the quinones was assessed by following the reduction of MTT
(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan blue [31]. Cells were
seeded into 96-well plates at a density of 10,000 cells/well for 24 h and then incubated for 48 h, with
or without the quinone derivatives. Doxorubicin was used as the standard chemotherapeutic agent
(positive control). Cells were then washed twice with warm PBS and incubated with MTT (0.5 mg/mL)
for 2 hours at 37 ◦C. Blue formazan crystals were solubilized by adding 100
µL DMSO/well, and the
optical density of the coloured solutions was subsequently read at 550 nm. Results are expressed
as percentage of MTT reduction, compared to untreated control conditions. The IC50 values were
calculated using the GraphPad Prism software (San Diego, CA, USA).
3.3.3. Quantitative real-time PCR (qPCR) Assay
The T24 cells were cultured as previously mentioned. They were seeded into 6-well plates
(
2 × 105 cells/well) and, after 24 h of incubation, they were treated for 48 h with 2c and 2d (at 32 and
68 µM, respectively). Afterwards, they were washed with phosphate-buffered saline. The cellular
lysate was prepared with E.Z.N.A.
preserve and immediately stabilize the total RNA for the subsequent gene expression assays. The total
RNA isolated from the cells using the E.Z.N.A. HP Total RNA Isolation Kit (Omega Bio-tek) was
®RNA-Lock Reagent (Omega Bio-tek, Norcross, GA, USA) to
®
reverse-transcribed to cDNA using the AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies,
Santa Clara, CA, USA) and 1000 ng of the RNA sample.
The cDNA synthesized was employed for qPCR using Brilliant III Ultra-Fast SYBR
Master Mix (Agilent Technologies) in a Mx3000P qPCR System (Agilent Technologies), employing a
96-well plate with 20 L of PCR reaction per well and 10 pmol each of forward and reverse gene-specific
®Green QPCR
µ
primers. Ten genes were analyzed (see Table 4). The relative gene expressions were determined using
Beta-2-microglobulin (B2M) as housekeeping, and the delta-delta Ct method (2−∆∆Ct method) with
regard to the vehicle-treated group (i.e., the reference group). Five biological replicates were used
from each group (treated and reference group). The qPCR reactions were run by duplicates and
negative controls contained no cDNA, as previously reported [32,33]. The GraphPad Prism software
was used for statistical analyses of the relative gene expressions. The comparisons between means
were performed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons
test. All statistical analyses were performed with a significance level of p < 0.05.

Molecules 2019, 24, 1780
11 of 14
Table 4. Quantitative real-time (qPCR) Primer Sequences.
Amplicon
Size (pb)
Gene Name
Apoptosis
Gene Symbol
BCL2
Entrez Gene ID
Forward (50 → 30)
Reverse (50 → 30)
Apoptosis regulator (BCL2),
transcript variant alpha
NM_000633.2
NM_004958.3
ATGTGTGTGGAGAGCGTCAA
TCCGAGAGATGAGTCAAGAGG
GAGACAGCCAGGAGAAATCAA
CACCTTCCACTCCTATGAGGC
181
PI3 Kinases & Phosphatases
Mechanistic target of
rapamycin kinase
mTOR
141
Drug Metabolism/Oxidative Stress
Glutathione S-transferase pi 1
Glutathione-disulfide reductase
GSTP1
GSR
NM_000852.3
NM_000637.4
CATCTACACCAACTATGAGGCG
CACTTGCGTGAATGTTGGATG
AGCAGGGTCTCAAAAGGCTTC
TGGGATCACTCGTGAAGGCT
81
242
Cell Cycle
Cell division cycle 25A
Tumor protein p53
CDC25A
TP53
NM_001789.2
NM_000546.5
TGGGCCATTGGACAGTAAAG
ACAGCTTTGAGGTGCGTGTTT
TCCCAACAGCTTCTGAGGTA
CCCTTTCTTGCGGAGATTCTCT
76
77
Ras Signaling
V-Ha-Ras Harvey rat sarcoma
viral oncogene homolog
HRAS
NM_005343
GACGTGCCTGTTGGACATC
CTTCACCCGTTTGATCTGCTC
166
Histone Deacetylases
Histone deacetylase 3
Histone deacetylase 4
HDAC3
HDAC4
NM_003883.3
NM_006037.3
TCTGGCTTCTGCTATGTCAACG
AGCGTCCGTTGGATGTCAC
CCCGGTCAGTGAGGTAGAAAG
CCTTCTCGTGCCACAAGTCT
136
169
Inflammation
Tumor necrosis factor
Housekeeping
TNF
B2M
NM_000594.3
AGAACTCACTGGGGCCTACA
GCTCCGTGTCTCAAGGAAGT
GGCATCTTCAAACCTCCATG
177
Beta-2-microglobulin
NM_004048.2
ATGAGTATGCCTGCCGTGTGA
97

Molecules 2019, 24, 1780
12 of 14
4. Conclusions
In summary, we have synthesized of a number 3-acyl-2,5-bis(phenylamino)-1,4-benzoquinones
and assessed their voltammetric values and cytotoxicities of cancer cells in vitro. The members of the
series 2a–q were prepared from acylbenzoquinones and phenylamines, in moderate to good yields
(47–74%). The first and second half-wave potential values (E 1/2) of the members of the series were
sensitive to the push-pull electronic effects of the substituents around the benzoquinone nucleus, as
shown by the cyclic voltammograms of the aminoquinones. The preliminary results of the biological
evaluation of the compounds 2a–q showed interesting in vitro cytotoxic activity on cancer cells. In this
context, there were two more active compounds which increased TNF gene expression, suggesting an
association with an inflammatory-like response.
SupplementaryMaterials: The following NMR spectra for compounds 2b, 2g, 2h, 2j, 2m, 2ñ, 2p are online available.
Author Contributions: J.B. proposed the subject and designed the study. J.A.V. designed the experiments and
wrote the paper. M.R. and M.V. carried out the chemical experiments. A.G.-C. performed the biological evaluation.
G.G.M. carried out the HRMS analysis. P.B.C. designed the biological assays and wrote the paper. All the authors
read and approved the final manuscript.
Funding: This research was funded by Fondo Nacional de Ciencia y Tecnología (FONDECYT), grant number
1150030 and VRIIP0154-18-UNAP.
Acknowledgments: We thank Pilar Díaz from the Universidad Arturo Prat, Chile, for technical support.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Kong, D.; Yamori, T.; Kobayashi, M.; Duan, H. Antiproliferative and antiangiogenic activities of
Smenospongine, a marine sponge sesquiterpene aminoquinone. Mar. Drugs 2011, 9, 154–161. [CrossRef]
Gould, S.J.; Weimeb, S.M. Streptonigrin. Fortschr. Chem. Org. Naturst. 1982, 41, 77–111.
Hawas, U.W.; Shaaban, M.; Shaaban, K.A.; Speitling, M.; Maier, A.; Kelter, G.; Fiebig, H.H.; Meiners, M.;
Helmke, E.; Laatsch, H. Mansouramycins A–D, cytotoxic isoquinolinequinones from a marine streptomycete.
J. Nat. Prod. 2009, 72, 2120–2124. [CrossRef] [PubMed]
2.
3.
4.
5.
Valderrama, J.A.; Cabrera, M.; Benites, J.; Ríos, D.; Inostroza-Rivera, R.; Muccioli, G.G. Synthetic approaches
and in vitro cytotoxic evaluation of 2-acyl-3-(3,4,5-trimethoxyanilino)-1,4-naphthoquinones. RSC Adv. 2017
7, 24813–24821. [CrossRef]
,
Valderrama, J.A.; Garrido, J.; Delgado, V.; Benites, J.; Theoduloz, C. Access to New Cytotoxic Quinone-Amino
Acid Conjugates Linked through A Vinylic Spacer from 2-Acylnaphthoquinones and Methyl 3-Aminocrotonate.
Molecules 2017, 22, 2281. [CrossRef]
6.
7.
Benites, J.; Valderrama, J.A.; Bettega, K.; Curi Pedrosa, R.; Buc Calderon, P.; Verrax, J. Biological evaluation of
donor-aceptor aminonaphthoquinones as antitumor agents. Eur. J. Med. Chem. 2010, 45, 6052–6057. [CrossRef]
Sarma, M.D.; Ghosh, R.; Patra, A.; Hazra, B. Synthesis of novel aminoquinonoid analogues of diospyrin and
evaluation of their inhibitory activity against murine and human cancer cells. Eur. J. Med. Chem. 2008, 43,
1878–1888. [CrossRef] [PubMed]
8.
9.
Miguel del Corral, J.M.; Castro, M.A.; Gordaliza, M.; Martín, M.L.; Gamito, A.M.; Cuevas, C.; San Feliciano, A.
Synthesis and cytotoxicity of new heterocyclic terpenylnaphthoquinones. Bioorg. Med. Chem. 2006, 14, 2816–2827.
[CrossRef] [PubMed]
Ibacache, J.A.; Faundes, J.; Montoya, M.; Mejías, S.; Valderrama, J.A. Preparation of Novel Homodimers
Derived from Cytotoxic Isoquinolinequinones. A Twin Drug Approach. Molecules 2018, 23, 439. [CrossRef]
[PubMed]
10. Pathania, D.; Kuang, Y.; Sechi, M.; Neamati, N. Mechanisms underlying the cytotoxicity of a novel
quinazolinedione-based redox modulator, QD232, in pancreatic cancer cells. Br. J. Pharmacol. 2015, 172,
50–63. [CrossRef] [PubMed]
11. Bernardo, P.H.; Khanijou, J.K.; Lam, T.H.; Tong, C.T.; Chai, C.L.L. Synthesis and potent cytotoxic activity of
8-and 9-anilinophenanthridine-7,10-diones. Tetrahedron Lett. 2011, 52, 92–94. [CrossRef]

Molecules 2019, 24, 1780
13 of 14
12.
V
á
squez, D.; Rodríguez, J.A.; Theoduloz, C.; Buc Calderon, P.; Valderrama, J.A. Studies on quinones. Part
46. Synthesis and in vitro antitumor evaluation of Aminopyrimidoisoquinolinequinones. Eur J. Med. Chem.
2010, 45, 5234–5242.
13. Valderrama, J.A.; Ibacache, J.A.; Arancibia, V.; Rodriguez, J.; Theoduloz, T. Studies on quinones. Part 45: Novel
7-aminoisoquinoline-5,8-quinone derivatives with antitumor properties on cancer cell lines. Bioorg. Med.
Chem. 2009, 17, 2894–2901. [CrossRef] [PubMed]
14. Hsu, T.S.; Chen, C.; Lee, P.T.; Chiu, S.J.; Liu, H.F.; Tsai, C.C.; Chao, J.I. 7-Chloro-6-piperidin-1-yl-quinoline-5,8-
dione (PT-262), a novel synthetic compound induces lung carcinoma cell death associated with inhibiting
ERK and CDC2 phosphorylation via a p53-independent pathway. Cancer Chemother. Pharmacol. 2008, 62,
799–808. [CrossRef]
15. Lodygin, D.; Menssen, A.; Hermeking, H. Induction of the Cdk inhibitor p21 by LY83583 inhibits tumor cell
proliferation in a p53-independent manner. J. Clin. Invest. 2002, 110, 1717–1727. [CrossRef] [PubMed]
16. Ryu, C.K.; Jeong, H.J.; Lee, S.K.; You, H.J.; Choi, K.U.; Shim, J.Y.; Heo, Y.H.; Lee, C.O. Effects of
6-arylamino-5,8-quinolinediones and 6-chloro-7-arylamino-5,8-isoquinolinediones on NAD(P)H: Quinone
oxidoreductase (NQO1) activity and their cytotoxic potential. Arch. Pharm. Res. 2001, 24, 390–896. [CrossRef]
17. Lee, Y.S.; Wurster, R.D. MechanismofpotentiationofLY83583-inducedgrowthinhibitionbysodiumnitroprusside
in human brain tumor cells. Cancer Chemother. Pharmacol. 1995, 36, 341–344. [CrossRef] [PubMed]
18. Verter, H.S.; Rogers, J. Preparation of 2,5-Bis(alkylamino)-3,6-dimethoxy-p-benzoquinones. J. Org. Chem.
1966, 31, 987–988. [CrossRef]
19. Burton, J.F.; Cain, H.F. Antileukaemic activity of polyporic acid. Nature 1959, 184, 1326–1327. [CrossRef]
20. Mathew, A.E.; Zee-Cheng, R.K.Y.; Cheng, C.C. Amino-substituted p-benzoquinones. J. Med. Chem. 1986, 29,
1792–1795. [CrossRef]
21. Schafer, W.; Moore, H.W.; Aguado, A. Synthesen des 2,1-Benzoxazol-4,7-chinon-Systems. Synthesis 1974
,
30–32. [CrossRef]
22. Suto, M.J.; Stier, M.A.; Winters, R.T.; Turner, W.R.; Pinter, C.D.; Elliot, W.E.; Sebolt-Leopold, J.S. Synthesis and
evaluation of a series 3,5-disubstituted benzisoxazole-4,7-diones. Potent radiosensitizers in vitro. J. Med. Chem.
1991, 34, 3290–3294. [CrossRef]
23. Benites, J.; Valderrama, J.A.; Ramos, M.; Muccioli, G.G.; Buc Calderon, P. Targeting Akt as strategy to kill cancer
cells using 3-substituted 5-anilinobenzo[c]isoxazolequinones: A preliminary study. Biomed. Pharmacother.
2018, 97, 778–783. [CrossRef]
24. Benites, J.; Rios, D.; Díaz, P.; Valderrama, J.A. The solar-chemical photo-Friedel-Crafts heteroacylation of
1,4-quinones. Tetrahedron Lett. 2011, 52, 609–611. [CrossRef]
25. Schäfer, W.; Aguado, A. Oxidative Amination of Hydroquinones. Angew. Chem. Internat. Edit. 1971, 10, 405–406.
[CrossRef]
26. Ibacache, J.A.; Delgado, V.; Benites, J.; Theoduloz, C.; Arancibia, V.; Muccioli, G.; Valderrama, J.A. Synthesis,
half wave potentials and antiproliferative activity of 1-aryl-substituted Aminoisoquinolinequinones. Molecules
2014, 19, 726–739. [CrossRef]
27. De Abreu, F.C.; de Ferraz, P.A.; Goulart, M.O.F. Some applications of electrochemistry in biomedical chemistry.
Emphasis on the correlation of electrochemical and bioactive properties. J. Braz. Chem. Soc. 2002, 13, 19–35.
[CrossRef]
28. Aguilar-Martinez, M.; Cuevas, G.; Jimenez-Estrada, M.; González, I.; Lotina-Hennsen, B.; Macias-Ruvalcaba, N.
An experimental and theoretical study of the substituent effects on the redox properties of 2-[(R-phenyl)amine]-
1,4-naphthalenediones in acetonitrile. J. Org. Chem. 1999, 64, 3684–3694. [CrossRef] [PubMed]
29. Scudiero, D.A.; Shoemaker, R.H.; Paull, K.D.; Monks, A.; Tierney, S.; Nofziger, T.H.; Currens, M.J.; Seniff, D.;
Boyd, M.R. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture
using human and other tumor cell lines. Cancer Res. 1988, 48, 4827–4833.
30. Showalter, A.; Limaye, A.; Oyer, J.L.; Igarashi, R.; Kittipatarin, Ch.; Copik, A.J.; Khaled, A.R. Cytokines in
immunogenic cell death: Applications for cancer immunotherapy. Cytokine 2017, 97, 123–132. [CrossRef]
31. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and
cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [CrossRef]
32. Guerrero-Castilla, A.; Olivero-Verbel, J. Altered gene expression in HepG2 cells exposed to a methanolic coal
dust extract. Environ. Toxicol. Pharmacol. 2014, 38, 742–750. [CrossRef] [PubMed]

Molecules 2019, 24, 1780
14 of 14
33. Lima, L.; Gaiteiro, C.; Peixoto, A.; Soares, J.; Neves, M.; Santos, L.L.; Ferreira, J.A. Reference Genes for
Addressing Gene Expression of Bladder Cancer Cell Models under Hypoxia: A Step Towards Transcriptomic
Studies. PLoS ONE 2016, 11, e0166120. [CrossRef] [PubMed]
Sample Availability: Samples of the compounds 2b, 2g, 2h, 2j, 2m, 2ñ, 2p are available from the authors.
©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).