Norhierridin B, a new hierridin B-based hydroquinone with improved antiproliferative activity

Article abstract of DOI:10.3390/molecules25071578

Hierridin B (6), a methylated hydroquinone isolated from the marine picocyanobacterium Cyanobium sp. LEGE 06113, moderately inhibited the growth of colon adenocarcinoma HT-29 cells. Aiming to improve the potential antitumor activity of this natural produc

Full text of DOI:10.3390/molecules25071578

molecules
Article
Norhierridin B, a New Hierridin B-Based
Hydroquinone with Improved
Antiproliferative Activity
Pedro Brandão 1,† , Joana Moreira 1,2,†, Joana Almeida 3 , Nair Nazareth 3,
Ivo E. Sampaio-Dias 4 , Vitor Vasconcelos 2,5 , Rosário Martins 2,6 , Pedro Leão 2
,
Madalena Pinto 1,2 , Lucília Saraíva 3,
*
and Honorina Cidade 1,2,
*
1
Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences,
Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal;
pedrocgbrandao@gmail.com (P.B.); joana.m26@hotmail.com (J.M.); madalena@ff.up.pt (M.P.)
CIIMAR/CIMAR, Interdisciplinary Centre of Marine and Environmental Research, University of Porto,
Edifício do Terminal de Cruzeiros do Porto de Leixões, Avenida General Norton de Matos, S/N,
4450-208 Matosinhos, Portugal; vmvascon@fc.up.pt (V.V.); mrm@ess.ipp.pt (R.M.); pleao@ciimar.up.pt (P.L.)
LAQV/REQUIMTE, Laboratory of Microbiology, Department of Biological Sciences, Faculty of Pharmacy,
University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal;
2
3
JoanaAlmeida15@gmail.com (J.A.); naircampos@gmail.com (N.N.)
LAQV/REQUIMTE, Department of Chemistry and Biochemistry, Faculty of Sciences, University of Porto,
Rua do Campo Alegre 687, 4169-007 Porto, Portugal; idias@fc.up.pt
Department of Biology, Faculty of Sciences, University of Porto, Rua do Campo Alegre, Edifício FC4,
4169-007 Porto, Portugal
Health and Environment Research Centre, School of Health, Polytechnic Institute of Porto, Rua Dr. Ant
4
5
6
ónio
Bernardino de Almeida, 400, 4200-072 Porto, Portugal
*
Correspondence: lucilia.saraiva@ff.up.pt (L.S.); hcidade@ff.up.pt (H.C.);
Tel.: +351-220428584 (L.S.); +351-220428688 (H.C.)
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 10 March 2020; Accepted: 29 March 2020; Published: 30 March 2020
Abstract: Hierridin B (6), a methylated hydroquinone isolated from the marine picocyanobacterium
Cyanobium sp. LEGE 06113, moderately inhibited the growth of colon adenocarcinoma HT-29 cells.
Aiming to improve the potential antitumor activity of this natural product, the demethylated analogue,
norhierridin B (10), as well as its structurally-related quinone (9), were synthesized and evaluated for
their growth inhibitory effect on a panel of human tumor cell lines, including the triple-negative breast
cancer (TNBC) cells MDA-MB-231, SKBR3, and MDA-MB-468. Norhierridin B (10) showed a potent
growth inhibitory effect on all cancer cell lines. Moreover, the growth inhibitory effect of compound
10 on MDA-MB-231 cells was associated with cell cycle arrest and apoptosis. Norhierridin B (10
interfered with several p53 transcriptional targets, increasing p21, Bax, and MDM2, while decreasing
Bcl-2 protein levels, which suggested the potential activation of a p53 pathway. Altogether, these
results evidenced a great improvement of the antitumor activity of hydroquinone 10 when compared
)
to 6 and its structurally-related quinone (9). Notably, hydroquinone 10 displayed a prominent growth
inhibitory activity against TNBC cells, which are characterized by high therapeutic resistance.
Keywords: antitumor activity; hierridin B; hydroquinones; quinones
Molecules 2020, 25, 1578; doi:10.3390/molecules25071578
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1578
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1. Introduction
Quinones/hydroquinones constitute a family of metabolites, widespread in nature, with a
wide range of biological activities, including cytotoxicity to cancer cells [ ]. Particularly, alkyl
1,4-benzoquinones/hydroquinones with aliphatic side chains have been found attractive for their
in vitro growth inhibitory effect against several human cancer cell lines [ ] (Figure 1). Among these
secondary metabolites, a wide array of bioactive compounds has been isolated from nature, namely
from plant species and marine organisms. For instance, compounds (Figure 1), isolated from
1–3
3,4
1–3
Ardisia virens, exhibited a potent inhibitory effect against breast adenocarcinoma MCF-7, non-small
cell lung cancer NCI-H460, and glioblastoma SF-268 cell lines (concentration that caused cell growth
inhibition of 50%, GI50, of 2.21–11.32
µM) [4], with hydroquinone 3 being more potent than quinone
2
. Toluquinol ( ) (Figure 1), isolated from the marine fungus Penicillium sp. HL-85-ALS5-R004,
4
is another example of a natural product with a promising in vitro growth inhibitory effect in breast
adenocarcinoma MDA-MB-231 and glioblastoma U87-MG cells, as well as in promyelocytic leukemia
HL-60, fibrosarcoma HT-1080, and colorectal adenocarcinoma HT-29 cell lines [5]. Interestingly, when
comparing the in vitro growth inhibitory activity of quinones with structurally related hydroquinones,
it seems that the presence of a hydroquinone could be more favorable for activity. In fact, the IC50
values obtained for quinone
]. Moreover, toluquinone (
), showed lower in vitro growth inhibitory activity against a panel of cancer cell lines of breast
2
are slightly higher than those of the structurally related hydroquinone
3
[
4
5), a synthetic analogue of the marine hydroquinone toluquinol
(4
adenocarcinoma (MDA-MB-231, GI50 of 3.4
5.6 1.9 M), glioblastoma (U87-MG, GI50 of 8.8
and colorectal adenocarcinoma (HT-29, GI50 of 9.5
2.3 0.8 M, HL-60: GI50 of 1.7 0.5 M, U87-MG: GI50 of 5.6
and HT-29: GI50 of 4.1 0.4 M), further supporting that the presence of a hydroquinone is favorable
for the antitumor activity [5].
±
0.1
0.9
µ
M), promyelocytic leukemia (HL-60, GI50 of
M), fibrosarcoma (HT-1080, GI50 of 4.9 1.7 M),
M) than toluquinol ( ) (MDA-MB-231: GI50 of
1.5 M, HT-1080: GI50 of 1.4 0.6
±
µ
±
µ
±
µ
±
0.5
µ
5
±
µ
±
µ
±
µ
±
µM
±
µ
Figure 1. Structure of quinones and hydroquinones with cytotoxic activity.

Molecules 2020, 25, 1578
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Recently, following the search for new bioactive secondary metabolites from marine cyanobacteria,
we identified hierridin B ( ), a methylated hydroquinone derivative with a C15 aliphatic chain isolated
from the marine picocyanobacterium Cyanobium sp. LEGE 06,113 [ ]. Compound moderately inhibited
the growth of colon adenocarcinoma HT-29 cell line, with a GI50 of 100.2 M [ ]. In order to obtain new
6
6
6
µ
6
structurally-related quinone/hydroquinone with improved antiproliferative activity in cancer cell lines,
the demethylated hierridin B derivative, norhierridin B (10), as well as structurally-related quinone
9
and compound 6, were synthesized and evaluated for their growth inhibitory effect in a panel of
human tumor cell lines.
2. Results and Discussion
2.1. Synthesis
Norhierridin B (10) was synthesized according to the strategy illustrated on Scheme 1. In order to
explore the growth inhibitory effect of demethylated hierridin B (
6) derivative (10), as well as of its
1,4-benzoquinone analogue ( ), a synthetic pathway was planned in order to obtain this analogue as
9
an intermediate. Firstly, Grignard addition to o-vanilin using tetradecyl magnesium bromide in situ
prepared from Mg turnings and tetradecyl bromide in dry diethyl ether afforded the corresponding
carbinol
catalyzed by palladium on carbon (Pd/C) using sulfuric acid as hydrogen donor, as reported by
Gonz lez et al. [ ]. Nevertheless, as no reaction occurred, a similar reaction with Pd/C using formic acid
as hydrogen donor was conducted, affording the reduced derivative with 93% yield. The high yield
obtained reinforces the advantage of using palladium catalysts and formic acid in the catalytic transfer
hydrogenolysis of benzylic alcohols, as described previously [ 10]. Phenol was oxidized by streaming
oxygen into a solution of DMF containing salcomine as catalyst, affording the 1,4-benzoquinone with
using Na2S2O4 as a reducing agent afforded norhierridin
7 with 14% yield. The reduction of α-hydroxy group was firstly attempted by hydrogenolysis
á
7
8
8
–
8
9
good yield (82%). The reduction of quinone
9
B (10) with 87% yield.
Scheme 1. Synthesis of norhierridin B (10). (a) C14H29Br, Mg, Et2O, 35 ◦C, 2 h; (b) HCOOH, Pd/C,
EtOH/H2O, 80 ◦C, 4 h; (c) O2, salcomine, DMF, r. t., 24 h; (d) Na2S2O4, CHCl3/H2O, r.t., 10 min.
The structure elucidation of the new hydroquinone 10 was established by NMR and HRMS
(Figures S4 and S5). The newly synthesized quinone , as well as intermediates and 8, were
characterized by NMR techniques, and data were in accordance with that previously reported [11 12].
13C-NMR assignments were determined by 2D heteronuclear single quantum correlation (HSQC) and
9
7
,
heteronuclear multiple bond correlation (HMBC) experiments. Hierridin B (6) was synthesized and
characterized as described previously [13].

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2.2. Biological Activity
Tumor Cell Growth Inhibitory Activity Evaluation
The growth inhibitory effect of norhierridin B (10), as well as of its structurally related
1,4-benzoquinone ( ), intermediates and 8, and hierridin B ( ), was evaluated in a panel of
9
7
6
distinct human cancer cell lines of melanoma (A375), hepatocarcinoma (Huh-7), colon (HCT116),
and triple-negative breast cancer (TNBC) (MDA-MB-231, SKBR3, and MDA-MB-468), using the
sulforhodamine (SRB) assay. A dose-response curve was obtained for each compound in the distinct
cell lines and the concentration that induced half of growth inhibition (GI50) was determined after 48 h
treatment (Table 1).
Table 1. Effect of compounds 6–10 on the growth of human cancer cell lines.
GI50 (µM)
Cell line/Compound
6
7
8
9
10
Etoposide
MDA-MB-231
SKBR3
MDA-MB-468
9.65 ± 0.15
14.0 ± 0.0
7.85 ± 2.15
16.0 ± 1.7
27.5 ± 0.5
26.5 ± 0.5
5.83 ± 1.10
4.35 ± 0.15
6.65 ± 0.90
20.6 ± 1.9
2.95 ± 0.15
29.6 ± 0.5
0.61 ± 0.07
0.77 ± 0.06
0.68 ± 0.13
2.0 ± 0.4
>50
>50
>50
>50
>50
>50
28.8 ± 3.3
5.06 ± 1.24
4.77 ± 1.09
2.14 ± 0.36
0.9 ± 0.07
3.43 ± 0.85
0.67 ± 0.05
>50
31.00 ± 7.00
32.9 ± 4.2
>50
26.0 ± 3.2
A375
Huh-7
HCT116
0.61 ± 0.03
3.2 ± 0.6
GI50 was determined by sulforhodamine (SRB) assay after 48 h treatment (growth obtained with vehicle was set as
100%). Data are mean ± SEM of two to five independent experiments. Etoposide was used as positive control.
The obtained GI50 values revealed that all newly synthesized compounds (
had a much higher growth inhibitory activity than hierridin B ( ) in all cancer cell lines (Table 1).
Norhierridin B (10) was found to be the most effective against all cancer cell lines, exhibiting much
lower GI50 values than its methyl derivative hierridin B ( ). These results suggest that the presence of
7, 8, 9, and 10)
6
6
two p-hydroxy groups in the hydroquinone scaffold are important for the in vitro growth inhibitory
activity. In addition, norhierridin B (10) displayed higher activity than its structurally related quinone
(9), reinforcing that the presence of a hydroquinone scaffold is more favorable for the activity than
a quinone scaffold, as already reported by Chang et al. [
structurally related hydroquinones/quinones.
4] and Cheng-Sánchez et al. [5] for other
Interestingly, norhierridin B (10) exhibited a prominent growth inhibitory activity against TNBC
cells MDA-MB-231, SKBR3, and MDA-MB-468, which are well-known by their high therapeutic
resistance [14]. Moreover, an evident reduction of the norhierridin B (10) antiproliferative effect could
be observed by SRB assay in non-tumorigenic MCF10A breast epithelial cell lines (10.36
±
1.96 µM,
three independent experiments). In fact, supporting the marked antiproliferative effect of compound
10 on MDA-MB-231 cells, observed by SRB assay (Figure 2A), a pronounced inhibition of the growth
of these cancer cells was also observed at 2 µM by colony formation assay (Figure 2B,C).

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Figure 2. Norhierridin B (10) inhibits the growth of breast adenocarcinoma MDA-MB-231 cells.
) Dose-response curve for the growth of MDA-MB-231 cells treated with compound 10 for 48 h,
determined by SRB assay; data are mean SEM of four independent experiments; growth obtained
with vehicle was set as 100%. (B, C) Colony formation assay for MDA-MB-231 cells treated with
compound 10 for nine days: ( ) images correspond to a representative experiment of two; ( ) graph
represents mean SEM of two independent experiments; values significantly different from DMSO: ***
(
A
±
B
C
±
p < 0.001, unpaired Student’s t-test.
It was also verified that the growth inhibitory effect of compound 10 on MDA-MB-231 cells was
associated with the induction of apoptosis and cell cycle arrest. In fact, the 1 µM concentration of
compound 10 caused a significant G0/G1-phase cell cycle arrest (Figure 3A), which was consistent with
the visible increase of p21 protein expression levels observed by western blot analysis (Figure 3C).
Moreover, by cell viability assay, 1 and 2 µM of compound 10 significantly increased the percentage
of dead cells (Figure 3B), which was associated with an increase of the pro-apoptotic protein Bax
and a decrease of the anti-apoptotic protein Bcl-2 (Figure 3C). It is interesting to note that compound
10 interfered with the protein expression levels of several p53 transcriptional targets, such as p21,
Bax, Bcl-2, and MDM2 (Figure 3C), suggesting the activation of a p53 pathway in MDA-MB-231 cells.
Collectively, these results indicate that compound 10 is very effective against TNBC cells. The poor
prognosis and limited therapeutic options commonly observed with this type of cancer [14] render
compound 10 worthy of further studies to confirm its promising application as potential anticancer
agent, particularly against TNBC.

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Figure 3. Norhierridin B (10) induces cell cycle arrest and death and interferes with p53 transcriptional
targets. (A) Cell cycle progression was analyzed after 48 h treatment with compound 10 in
MDA-MB-231 cells; data are mean SEM of three independent experiments; values significantly
different from DMSO: *p < 0.05, unpaired Student’s t-test. (B) Percentage of death cells determined by
trypan blue assay for MDA-MB-231 cells treated with compound 10 for 48 h; data are mean SEM
±
±
of three independent experiments; values significantly different from DMSO: *p < 0.05, **p < 0.001,
unpaired Student’s t-test. (C) Protein expression levels of p53 targets in MDA-MB-231 cells was
analyzed by western blot after 48 h treatment with 2 µM compound 10. Immunoblots are representative
of three independent experiments; GAPDH was used as loading control.
3. Material and Methods
3.1. Synthesis
All reactions were monitored by thin-layer chromatography (TLC). Purifications of compounds
were carried out by flash chromatography using Macherey-Nagel silica gel 60 (0.04–0.063 mm).
Melting points were obtained in a Köfler microscope (Wagner and Munz, Munich, Germany) and are
1
uncorrected. H-NMR and 13C-NMR spectra were taken in CDCl3 at room temperature, on a Bruker
Avance 300 instrument (300.13 MHz for 1H and 75.47 MHz for 13C, Bruker Biosciences Corporation,
Billerica, MA, USA). Chemical shifts are expressed in ppm values relative to tetramethylsilane (TMS)
used as an internal reference; 13C-NMR assignments were made by 2D (HSQC and HMBC) NMR
experiments (long-range 13C-1H coupling constants were optimized to 7 Hz). HRMS mass spectrum
was recorded as ESI (electrospray ionization) mode on a MicrOTOF spectrometer (Bruker Corporation,
Billerica, MA, USA) at C.A.C.T.I.-University of Vigo, Spain. The commercially available reagents,
and etoposide, were purchased from Sigma Aldrich Co. (Sintra, Portugal). Reagents and solvents were
purified and dried according to the usual procedures described elsewhere [15]. Hierridin B (6) was
synthesized as described previously [13]. The following materials were synthesized and purified by
the described procedures.

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3.1.1. Synthesis of 2-(1-hydroxypentadecyl)-6-methoxyphenol (7)
Tetradecyl magnesium bromide was prepared from Mg turnings (2.01 g, 0.62 eq.) and tetradecyl
bromide (37 g, 0.13 mol) in dry diethyl ether (200 mL) under a nitrogen atmosphere. o-Vanilin (10 g,
0.066 mol) was gradually added, and the mixture was then refluxed for 2 h, cooled, and poured into a
saturated solution of NH4Cl (150 mL). The layers were separated, and the aqueous phase thoroughly
extracted with diethyl ether (3
anhydrous sodium sulfate, and evaporated under reduced pressure. The residue was purified by
×
200 mL). The organic layer was washed with water, dried over
flash column chromatography (silica gel, n-hexane: ethyl acetate 8:2), affording
7 (3.23 g, 14%) as a
white solid.
2-(1-hydroxypentadecyl)-6-methoxyphenol (
H-3,-4,-5), 6.38 (s, 1H, 1-OH), 4.87 (t, J = 6.0 Hz, 1H, H-10), 3.88 (s, 3H, 2-OCH3), 1.84–1.80 (m, 2H, H-20),
7 δ = 6.83–6.78 (m, 3H,
): 1H-NMR (CDCl3, 300.13 MHz):
1.30–1.24 (m, 24 H, H-30-140), 0.87 (t, J = 6.5 Hz, 3H, H-150); 13C-NMR (CDCl3, 75.47 MHz)
δ: 146.8
(C-2), 143.1 (C-1), 129.8 (C-6), 119.6 (C-4), 119.2 (C-5), 109.7 (C-3), 72.0 (C-10), 56.0 (2-OCH3), 37.2 (C-20),
31.9–22.7 (C-20-140), 14.1 (C-150).
3.1.2. Synthesis of 2-methoxy-6-pentadecylphenol (8)
In a 250 mL two neck round-bottomed flask equipped with a water-cooled condenser and a
magnetic stirrer, ethanol (99 mL), water (20 mL), compound
7 (3.17 g, 9 mmol), formic acid (0.68 mL),
and Pd/C 10% (w/w) (1.259 g) were added. The reactor was placed in a preheated oil bath and
maintained at 80 ◦C under atmospheric pressure for 4 h. The reactor was allowed to cool down to r.t.,
and the ethanol was evaporated under reduced pressure. Water was added (20 mL), and the resulting
solution was extracted with diethyl ether (3
sodium sulfate and evaporated under reduced pressure affording a solid, which was crystallized from
diethyl ether: methanol affording the title compound (8) (2,81 g, 93%) as a white solid.
×
100 mL). The organic layer was dried over anhydrous
1
2-methoxy-6-pentadecylphenol (
8
): H-NMR (CDCl3, 300.13 MHz):
δ
= 6.80–6.70 (m, 3H, H-3,-4,-5), 5.66
(s, 1H, 1-OH), 3.87 (s, 3H, 2-OCH3), 2.62 (t, J = 7.6 Hz, 1H, H-10), 1.65–1.57 (m, 2H, H-20), 1.27–1.25
(m, 24 H, H-30-140), 0.87 (t, J = 6.5 Hz, 3H, H-150); 13C-NMR (CDCl3, 75.47 MHz)
δ: 146.3 (C-2), 143.4
(C-1), 128.8 (C-6), 122.3 (C-5), 119.1 (C-4), 108.1 (C-3), 55.9 (2-OCH3), 31.9 (C-20), 30.9 (C-10), 37.2 (C-20),
29.8–22.7 (C-20-140), 14.1 (C-150).
3.1.3. Synthesis of 2-methoxy-6-pentadecyl-1,4-benzoquinone (9)
A solution of
8 (2.17 g, 6 mmol) in DMF was bubbled with O2 for 5 min and, after the O2
atmosphere, was kept over the reaction vessel. Salcomine was added (380 mg, 1.17 mmol), and the
reaction was stirred at r.t. for 24 h. After, diethyl ether (20 mL) was added, and the organic layer
was washed with 0.1 M HCl (2
dried over anhydrous sodium sulfate, and the solvent evaporated. The obtained solid was purified
by crystallization from petroleum ether 40–60 ◦C, affording the title compound (
9) (1.85 g, 82%) as a
×
10 mL), water (10 mL) and brine (10 mL). The organic layer was
yellow solid.
1
2-methoxy-6-pentadecyl-1,4-benzoquinone (
9
): H-NMR (CDCl3, 300.13 MHz):
δ
= 6.49–6.47 (m, 1H, H-5),
5.87 (d, J = 2.4 Hz, 1H, H-3), 3.82 (s, 3H, 2-OCH3), 2.44 (ddd, J = 7.5 Hz, 1.4, 2H, H-10), 1.54–1.45 (m, 2H,
H-20), 1.30–1.25 (m, 24 H, H-30-140), 0.88 (t, J= 6.5, 3H, H-150); 13C-NMR (CDCl3, 75.47 MHz)
δ: 187.7
(4-CO), 182.2 (1-CO), 158.9 (C-2), 147.6 (C-6), 132.9 (C-5), 107.1 (C-3), 55.3 (2-OCH3), 28.7 (C-10), 27.7
(C-20), 29.8–22.7 (C-20-140), 14.1 (C-150).
3.1.4. Synthesis of 2-methoxy-6-pentadecylbenzene-1,4-diol (10).
Compound
9 (1.86 g, 0.005 mol) was dissolved in chloroform and added to a 10% solution of
Na2S2O4 in hot water. The mixture was shaken for 10 min, and, after the aqueous layer was drawn
off, the chloroform was shaken with brine, dried over anhydrous sodium sulfate, and the solvent

Molecules 2020, 25, 1578
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evaporated under reduced pressure. The residue was purified by crystallization from n-hexane,
affording the title compound (10) (1.31 g, 87%) as a white solid.
2-methoxy-6-pentadecylbenzene-1,4-diol (10): m.p. (n-hexane) = 92–94 ◦C; 1H-NMR (CDCl3, 300.13 MHz):
1H-NMR (CDCl3, 300.13 MHz):
δ
6.31 (d, J = 2.8 Hz, 1H, H-3), 6.20 (d, J = 2.8 Hz, 1H, H-5), 3.84 (s, 3H,
2-OCH3), 2.56 (t, J = 7.5 Hz, 2H, H-10), 1.60–1.52 (m, 2H, H-20), 1.30–1.25 (m, 24H, H-30-140), 0.87 (t, J =
6.5 Hz, 3H, H-150); 13C-NMR (CDCl3, 75.47 MHz):
97.0 (C-3), 56.3 (2-OCH3), 31.9 (C-20), 30.9 (C-10), 29.8–22.7 (C-20-140), 14.1 (C-150); ESI-TOF-HRMS (+)
m/z: Anal. Calc. for C24H37O3 (M+H+): 349.27320; found: 349.27372.
δ 148.3 (C-2), 147.6 (C-4), 137.3 (C-1), 107.9 (C-5),
3.2. Biological Activity
3.2.1. Human Cell Lines and Growth Conditions
Human breast adenocarcinoma MDA-MB-231, SK-BR-3, MDA-MB-468, melanoma A375,
hepatocellular carcinoma Huh-7, colorectal carcinoma HCT116, and non-tumorigenic breast epithelial
MCF10A cell lines were purchased from ATCC (Rockville, MD, USA). Cancer cells were cultured in
RPMI-1640 medium with UltraGlutamine (Biowest, VWR, Carnaxide, Portugal) with 10% fetal bovine
serum (FBS; Gibco, Alfagene, Lisboa, Portugal). MCF10a cells were cultured in DMEM:F12 with 10% FBS
or MEGM SingleQuot Kit Suppl. & Growth Factors, respectively (Lonza, VWR, Carnaxide, Portugal).
Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2. Mycoplasma detection was
routinely performed using the MycoAlert™ PLUS kit (Lonza).
3.2.2. Sulforhodamine B (SRB) Assay
Human cell lines were incubated in 96-well plates at a final density of 5.0
A375, Huh-7 and HCT116 cells), 1.0
104 cells/well (for MDA-MB-231 and MCF10A) and 7.5
cells/well (for SKBR3 and MDA-MB-468 cells) for 24 h. Cells were then treated with serial dilutions
(0.313 M to 50 M) of compounds for 48 h. The effect of these compounds on in vitro growth was
×
103 cells/well (for
103
×
×
µ
µ
assessed using the SRB assay. Cells were fixated with 12.5% TCA for 1 h at 4 ◦C. Plates were then
stained with 0.25% SRB (Sigma-Aldrich, Sintra, Portugal) and washed with 1% acetic acid. The bound
dye was solubilized in 0.12% Tris Base and the absorbance was measured at 510 nm in a microplate
reader (Biotek Instruments Inc., Synergy, MX, USA). The maximum concentration of solvent used in
this assay (1% DMSO) was included as control. The GI50 was then determined.
3.2.3. Colony Formation Assay
MDA-MB-231 cells were seeded in 6-well plates at a density of 5.0
×
102 cells/well, followed by
incubation with hydroquinone 10 for 9 days. Formed colonies were fixed with 10% MeOH and 10%
AcOH for 10 min, and then they were stained with 0.5% crystal violet (Sigma-Aldrich, Sintra, Portugal)
in 1:1 MeOH/H2O for 15 min. Colonies containing more than 20 cells were counted.
3.2.4. Cell Viability Assay
The percentage of viable cells was determined using the Trypan Blue Assay.
Briefly, MDA-MB-231 cells were seeded in 24-well plates at 6
×
104 cells/well for 24 h, followed
by treatment with hydroquinone 10 for 24 h. Cells were then harvested and resuspended in 0.4%
Trypan Blue (Sigma-Aldrich, Sintra, Portugal), and the number of viable/dead cells were counted using
a Leica light optical microscope (Wetzlar). Vehicle (0.25% DMSO) was included as a control.
3.2.5. Cell Cycle Analysis
Cell cycle and apoptosis were analyzed as described [16]. Briefly, MDA-MB-231 cells were
incubated in 6-well plates at a density of 2.25
hydroquinone 10 or DMSO only for 48 h. For cell cycle analysis, cells were stained with propidium
×
105 cells/well for 24 h. Cells were then treated with

Molecules 2020, 25, 1578
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iodide (PI; Sigma-Aldrich, Sintra, Portugal); cell cycle scattering was analyzed by flow cytometry and
cell cycle phases were quantified using FlowJoX v10.0.7 (Treestar, Woodburn, OR, USA).
3.2.6. Western Blot Analysis
MDA-MB-231 cells were seeded in 6-well plates at a density of 2.25
×
105 cells/well for 24 h,
followed by treatment with 2 M hydroquinone 10. Protein extracts were quantified using the Bradford
µ
reagent (Sigma-Aldrich, Sintra, Portugal). Proteins were run in SDS-PAGE and transferred to a
Whatman nitrocellulose membrane from Protan (VWR, Carnaxide, Portugal). After blocking, proteins
were identified using a mouse monoclonal anti-anti-MDM2 (D-12), anti-Bax (2D2), anti-Bcl-2 (C-2),
or a rabbit polyclonal anti-p21 (C-19), followed by an anti-mouse or anti-rabbit horseradish-peroxidase
(HRP)-conjugated secondary antibody. For the loading control, a mouse monoclonal anti-GAPDH
(6C5) was used. The signal was detected with the ECL Amersham kit from GE Healthcare (VWR,
Carnaxide, Portugal). The ChemiDoc™ XRS Imaging System from Bio-Rad Laboratories (Amadora,
Portugal) for Microsoft Windows 10 was used for detection.
3.2.7. Statistical analysis
Data were statistically analyzed using GraphPad Prism version 7.0. Different statistical tests were
used accordingly to the dataset; p values <0.05 or < 0.001 were considered statistically significant.
4. Conclusions
In this work, the demethylated derivative of hierridin B (
6) and norhieridin B (10), as well as
its structurally related quinone ( ), were synthesized for the first time. The study of their effect on
9
the in vitro cell growth inhibition of human tumor cell lines revealed that norhierridin B (10) was the
most effective in all cancer cell lines, exhibiting much lower IC50 values than its structurally related
quinone (9) and hierridin B (6). Particularly, hydroquinone 10 exhibited a noticeable growth inhibitory
activity against the TNBC cells MDA-MB-231, SKBR3, and MDA-MB-468, with these being effects
associated with cell cycle arrest; increase of p21 and Bax; and decrease of Bcl-2 protein expression levels.
The interference of hydroquinone 10 in MDA-MB-231 cells with the protein expression levels of the
p53 transcriptional targets p21, Bax, Bcl-2, and MDM2 suggests the activation of a p53 pathway, which
should be further explored in the future. Altogether, these results indicate that hydroquinone 10 could
be an effective cytotoxic agent against TNBC cells, which should be further explored in the future.
Supplementary Materials: The following are available online, Figure S1: 1H and 13C-NMR of compound
Figure S2: 1H and 13C-NMR of compound , Figure S3: 1H and 13C-NMR of compound , Figure S4: 1H and
13C-NMR of compound 10, Figure S5: HRMS of compound 10.
7,
8
9
Author Contributions: V.V., R.M., P.L., M.P., and H.C. conceptualized the study. H.C. planned all aspects of
the study. J.M. and P.B. performed the synthesis, purification, and structure elucidation of compounds 10
I.E.S.-D. performed the synthesized of compound . J.M. wrote the manuscript. N.N. and J.A. performed the
7– .
6
tumor cell growth assays. H.C. and M.P. designed the experimental work concerning the synthesis. L.S. designed
the experimental work concerning the antitumor activity. V.V., R.M, P.L., L.S., and H.C. revised the manuscript.
All authors agreed to the final version of the manuscript.
Funding: This research was supported by national funds through FCT—Foundation for Science and Technology
within the scope of UIDB/04423/2020, UIDP/04423/2020 and UIDB/50006/2020. Joana Moreira acknowledges her
grant (SFRH/BD/135852/2018).
Acknowledgments: The authors thank Sara Cravo for all the technical and scientific support.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.

Molecules 2020, 25, 1578
10 of 10
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Sample Availability: Samples of the compounds 7, 9 and 10 are available from the authors.
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2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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(CC BY) license (http://creativecommons.org/licenses/by/4.0/).