Synthesis and antitumor activity evaluation of compounds based on toluquinol

Article abstract of DOI:10.3390/md17090492

Encouraged by the promising antitumoral, antiangiogenic, and antilymphangiogenic properties of toluquinol, a set of analogues of this natural product of marine origin was synthesized to explore and evaluate the effects of structural modifications on their cytotoxic activity. We decided to investigate the effects of the substitution of the methyl group by other groups, the introduction of a second substituent, the relative position of the substituents, and the oxidation state. A set of analogues of 2-substituted, 2,3-disubstituted, and 2,6-disubstituted derived from hydroquinone were synthesized. The results revealed that the cytotoxic activity of this family of compounds could rely on the hydroquinone/benzoquinone part of the molecule, whereas the substituents might modulate the interaction of the molecule with their targets, changing either its activity or its selectivity. The methyl group is relevant for the cytotoxicity of toluquinol, since its replacement by other groups resulted in a significant loss of activity, and in general the introduction of a second substituent, preferentially in the para position with respect to the methyl group, was well tolerated. These findings provide guidance for the design of new toluquinol analogues with potentially better pharmacological properties.

Full text of DOI:10.3390/md17090492

marine drugs
Article
Synthesis and Antitumor Activity Evaluation of
Compounds Based on Toluquinol
Iván Cheng-Sánchez 1
,†
, José A. Torres-Vargas
2,3,†
, Beatriz Martínez-Poveda 2,3
,
1
, Miguel Ángel Medina 2,3,4 , Francisco Sarabia * and
1,
Guillermo A. Guerrero-Vásquez
Ana R. Quesada 2
,3,4,
*
1
Department of Organic Chemistry, Faculty of Sciences, University of Málaga, Campus de Teatinos s/n,
9071 Málaga, Spain
2
2
Department of Molecular Biology and Biochemistry, Faculty of Sciences, University of M
álaga, Campus de
Teatinos s/n, 29071 Málaga, Spain
IBIMA (Biomedical Research Institute of Málaga), 29071 Málaga, Spain
CIBER of Rare Diseases; Group U741 (CB06/07/0046), 29071 Málaga, Spain
3
4
*
†
Correspondence: frsarabia@uma.es (F.S.); quesada@uma.es (A.R.Q.); Tel.: +34-952-134-258 (F.S.)
Iván Cheng-Sánchez and José A. Torres-Vargas contributed equally to the execution of this work.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 28 July 2019; Accepted: 18 August 2019; Published: 23 August 2019
Abstract: Encouraged by the promising antitumoral, antiangiogenic, and antilymphangiogenic
properties of toluquinol, a set of analogues of this natural product of marine origin was synthesized
to explore and evaluate the effects of structural modifications on their cytotoxic activity. We decided
to investigate the effects of the substitution of the methyl group by other groups, the introduction
of a second substituent, the relative position of the substituents, and the oxidation state. A set of
analogues of 2-substituted, 2,3-disubstituted, and 2,6-disubstituted derived from hydroquinone were
synthesized. The results revealed that the cytotoxic activity of this family of compounds could rely on
the hydroquinone/benzoquinone part of the molecule, whereas the substituents might modulate the
interaction of the molecule with their targets, changing either its activity or its selectivity. The methyl
group is relevant for the cytotoxicity of toluquinol, since its replacement by other groups resulted in a
significant loss of activity, and in general the introduction of a second substituent, preferentially in the
para position with respect to the methyl group, was well tolerated. These findings provide guidance
for the design of new toluquinol analogues with potentially better pharmacological properties.
Keywords: toluquinol; thymoquinone; marine hydroquinone; antitumor; natural compound analogues
1
. Introduction
Hydroquinones and quinones are ubiquitous in nature, playing essential roles in oxidative
metabolism, including in electron transport chains and photosynthesis, and are produced as primary
or secondary metabolites by many organisms. A myriad of hydroquinones and quinones have been
isolated from marine sources, including microbes, algae, and marine invertebrates, such as ascidians,
sponges, cnidarians, and mollusks [
including cytotoxicity to cancer cells, probably related to their involvement in redox cycling and/or
Michael-1,4-addition reactions [ ]. In many cases, the molecules responsible for the biological activities
detected in some marine macroorganisms derive from the complex secondary metabolite relationship
between the marine microorganisms and their hosts [ ]. Marine fungi are a rich source of structurally
diverse compounds with a high pharmaceutical potential [ ]. In the course of our ongoing research
program aimed at the search and characterization of new drug candidates of a marine origin, toluquinol
1,2]. Some of them display a wide range of biological activities,
3
4
5
(2-methylhydroquinone, compound
1) was isolated from the culture broth of the marine fungus
Mar. Drugs 2019, 17, 492; doi:10.3390/md17090492
www.mdpi.com/journal/marinedrugs

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Penicillium sp. HL-85-ALS5-R004 [
6
]. The antitumoral, antiangiogenic, and antilymphangiogenic
activities of toluquinol [6,7] make this compound a good example of the potential of marine-derived
compounds in the treatment of cancer. Encouraged by these results, we decided to explore the
effects of structural modifications of toluquinol on its antitumoral properties in order to establish a
structure-activity relationship as a guide to identify and develop either more potent or more specific
analogues based on this natural product. These structural modifications included the substitution of
the methyl group of toluquinol by different substituents (derivatives
substituent (derivatives 11), the effect of the position of this second substituent (derivatives 12
and, finally the oxidation state of the hydroquinone, such as the case of toluquinol ( ) and toluquinone
2–
7), the introduction of a second
8–
–13),
1
(14), and thymoquinol (15) and thymoquinone (16) (Figure 1).
Figure 1. Structures of toluquinol (1) and programmed set of analogues 2–16.
. Results and Discussion
2
2
.1. Synthesis of Compounds 2–15
Forthesynthesisofanalogues
to which the trifluoromethyl derivative
2
and
3
2
, weusedtheproceduresdescribedintheliterature[
and the prenyl derivative were prepared by direct treatment
of 1,4-hydroquinone (17) with 5-(trifluoromethyl)-dibenzothiophenium trifluoromethanesulfonate
Umemoto’s reagent) and prenol, respectively, under acidic conditions (Scheme 1). Whereas the
preparation of proceeded without further issues, the synthesis of was more complicated due to
the low yield of the coveted prenyl derivative , obtained at a poor 15% yield, and the formation
of sub-products that could be identified as the disubstituted derivative 18 and the benzopyrane 19
8,9], according
3
(
2
3
3
,
obtained in 3% and 2.7% yields, respectively. The three compounds obtained from this reaction could be
separated by flash column chromatography and sub-products 18 and 19 were considered for inclusion
in the biological studies (Scheme 1).

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Scheme 1. Synthesis of analogues 2 and 3.
For the synthesis of analogues , we considered a Suzuki coupling reaction as a straightforward
4–7
and powerful method [10] to install a vinyl-type substituent via direct coupling of the 2-bromo-1,4-
hydroquinone 20, prepared by bromination of 17 [11], with the corresponding pinacol boronic ester.
The optimization of this coupling reaction led us to establish Pd[PPh ] and K CO as the most
3
4
2
3
suitable catalyst and base, respectively. On the other hand, the solvent, temperature, and reaction
time depended on the boronic ester employed. Thus, when the reactions were undertaken using the
◦
solvent system 1,4-dioxane/H O at 80 C, which are the usual reaction conditions for Suzuki couplings
2
(Conditions A), we observed an important degree of degradation of the reaction mixture, detecting
only small amounts of the corresponding coupling products. In light of these discouraging results,
we found that reactions undertaken in THF instead of 1,4-dioxane produced better results in terms
of the resulting yields of the coupling products. With this new solvent system, products
were obtained in 92 and 32% yields, requiring a reaction time for completion of 48 h (Conditions B),
and products and were obtained in 79 and 56% yields, respectively, after 15 h for the completion of
the coupling reactions (Conditions C) (Scheme 2).
4 and 5
6
7
Scheme 2. Synthesis of analogues 4–7 via Suzuki couplings (* Conditions A: Pd[PPh ] , solid K CO ,
3
4
2
3
◦
◦
dioxane, H O, 80 C, 15 h; conditions B: Pd[PPh ] , 2 M aq. K CO , THF, 80 C, 48 h; conditions C:
2
3 4
2
3
◦
Pd[PPh ] , 2 M aq. K CO , THF, 80 C, 15 h).
3
4
2
3

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For the cases of the 2,6-disubstituted 1,4-hydroquinone derivatives
8–
11, we devised their syntheses
based again on a Suzuki coupling reaction as the most suitable method for rapid access to the targeted
molecules. To this end, the bromo derivative of toluquinol 22 was prepared according to the procedures
described in the literature [12] from o-cresol (21), and the corresponding Suzuki couplings were
achieved following the modified conditions previously established for the synthesis of derivatives
4–7. However, on this occasion we found that, for some cases (compounds 8 and 11), the solvent
system 1,4-dioxane/H O provided better results compared with the use of THF as solvent, whereas for
2
compounds 9 and 10, THF was the solvent of choice for the Suzuki coupling (Scheme 3).
Scheme 3. Synthesis of disubstituted analogues 8–11 via Suzuki couplings (* Conditions A: Pd[PPh ] ,
3 4
◦
◦
solid K CO , dioxane, H O, 80 C, 15 h; conditions B: Pd[PPh ] , 2 M aq. K CO , THF, 80 C, 48 h).
2
3
2
3 4
2
3
As a continuation of the synthetic strategy based on a Suzuki reaction for the installation of
new substituents, the synthesis of the corresponding 2,5-disubstituted analogues requires the bromo
derivative 24 as the key starting material. The synthesis of this bromo derivative was achieved
according to the procedure described in the literature [13], starting from toluquinol (
1) and proceeding
through bromo toluquinone 23, prepared in three steps from . The reduction of this bromo quinone by
1
the action of sodium thiosulfate provided the bromo precursor 24 at a 44% yield. The Suzuki coupling
reaction of 24 with the corresponding pinacol boronic ester afforded the desired 2,5-disubstituted 12 at
a reasonable 50% yield (Scheme 4).
Scheme 4. Synthesis of the p-disubstituted analogue 12 via a Suzuki reaction.

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Finally, compounds 2,3-dimethyl-1,4-hydroquinone (13) and toluquinone (14) were commercially
available and, in the case of thymoquinol (15), its preparation was achieved from commercial
thymoquinone (16) by reduction with sodium thiosulfate [14].
2
.2. Antitumoral Properties of the Compounds
The synthesized 1,4-hydroquinone derivatives 2–12 and 15 and the commercially available
compounds 13, 14, and 16 were subjected to biological evaluation with regard to their effect on cell
growth against a panel of various human cancer cell lines. They included MDA-MB-231 (human breast
adenocarcinoma), HL-60 (human promyelocytic leukemia), U87-MG (human glioblastoma), HT-1080
(
human fibrosarcoma), and HT-29 (human colorectal adenocarcinoma). In these studies, toluquinol (
1)
was used as a control, for comparison purposes.
To elucidate the effect of the toluquinol analogs on the growth of tumors, logarithmically
proliferating cells were treated with serial dilutions of the compounds for 72 h. Then, the number
of viable cells was evaluated by the addition of MTT, and results were expressed as a percentage of
viable cells versus the untreated controls. Representative dose-response curves are presented in the
Supplementary Materials. In those figures each point represents the mean of quadruplicates, and SD
values were typically lower than 10% of the mean values and were omitted for clarity. The half-maximal
inhibitory concentration (IC ) value was calculated from each dose-response curve as the concentration
50
of compound yielding 50% of the control cells’ survival. Table 1, containing the IC50 values (means
SD of three independent experiments with quadruplicate samples each one), summarizes the results of
these biological evaluations.
±
Table 1. In vitro antitumor activities of toluquinol and analogues (IC , µM) [a].
5
0
Tumor Cell Line
U87-MG [d]
Structure
Compound
MDA-MB-231 [b]
HL-60 [c]
HT-1080 [e]
HT-29 [f]
Toluquinol (1)
2.3 ± 0.8
1.7 ± 0.5
5.6 ± 1.5
1.4 ± 0.6
4.1 ± 0.4
Compound 2
14.5 ± 1.8
13.9 ± 1.1
31.7 ± 5.6
41.2 ± 11.4
29.7 ± 7.1
Compound 3
Compound 18
Compound 4
>100
>100
50.0 ± 3.4
7.0 ± 1.2
3.0 ± 0.7
>100
>100
>100
>100
>100
>100
4.1 ± 1.1
10.4 ± 3.6
3.7 ± 1.8
7.9 ± 0.1
Compound 5
Compound 6
3.9 ± 1.5
6.7 ± 1.8
5.3 ± 2.0
6.3 ± 1.3
29.7 ± 4.9
15.9 ± 4.4
8.4 ± 1.4
6.1 ± 2.5
15.5 ± 4.5
12.3 ± 2.5

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Table 1. Cont.
Tumor Cell Line
U87-MG [d]
Structure
Compound
MDA-MB-231 [b]
HL-60 [c]
HT-1080 [e]
HT-29 [f]
Compound 7
4.2 ± 1.8
6.2 ± 1.7
8.8 ± 0.8
3.2 ± 1.9
9.8 ± 3.2
Compound 8
Compound 9
Compound 10
Compound 11
2.2 ± 0.3
2.6 ± 1.3
3.2 ± 1.2
2.4 ± 0.3
3.1 ± 1.7
3.6 ± 1.5
4.7 ± 0.8
3.1 ± 1.3
10.0 ± 2.1
15.8 ± 2.3
16.0 ± 1.3
9.6 ± 1.3
3.3 ± 0.1
5.5 ± 1.9
5.0 ± 1.5
2.9 ± 1.0
8.1 ± 0.6
14.2 ± 4.3
15.6 ± 1.2
7.7 ± 1.6
Compound 12
1.6 ± 0.5
2.0 ± 0.3
2.9 ± 1.4
3.2 ± 1.6
1.6 ± 0.5
2.0 ± 0.9
5.6 ± 1.9
5.2 ± 1.2
5.1 ± 0.4
10.2 ± 2.0
15.1 ± 3.2
8.8 ± 0.9
1.1 ± 0.2
1.6 ± 0.3
2.0 ± 1.1
4.0 ± 0.9
7.9 ± 2.0
6.5 ± 2.6
8.2 ± 1.4
12.9 ± 1.6
Compound 13
Thymoquinol
1
1
3
.0 ± 0.1
.0 ± 0.1
(
15)
Thymoquinone
16)
(
Toluquinone
14)
.4 ± 0.1
>100
4.9 ± 1.7
>100
9.5 ± 0.5
>100
(
Compound 19
82.0 ± 5.9
>100
[a]
Half-maximal inhibitory concentration (IC50) values calculated from dose-response curves as the concentration
of compound yielding 50% of control cell survival. They are expressed as means
+
SD of three independent
[b]
[c]
experiments with quadruplicate samples each.
MDA-MB-231: Human breast adenocarcinoma. HL-60: Human
promyelocytic leukemia. [ U87-MG: Human glioblastoma.
d]
[e]
HT-1080: Human fibrosarcoma. HT-29: Human
[f]
colorectal adenocarcinoma.
2
.2.1. Antitumor Properties with Structure-Activity Relationship (SAR) of 2-Substituted
Hydroquinones 2–7
Relevant conclusions can be derived from the results presented in Table 1. At first sight, they clearly
indicate a relevant role of the methyl group of toluquinol in the cytotoxic activity of this compound,
since compounds lacking this methyl group (
2–7) exhibited significantly increased IC50 values for all
the tested cell lines. In this regard, substitution of the methyl group of toluquinol by a trifluoromethyl

Mar. Drugs 2019, 17, 492
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group (compound
2) caused a 6- to 30-fold increase in the IC50 values for all the cell types, approaching
the recently reported IC50 values for 1,4-hydroquinone in tumor cell lines [15]. These results are also in
agreement with our previous results showing that substitution of the methyl group of toluquinol with
either a carboxylic acid (as in gentisic acid) or a sulfonic acid (as in calcium dobesilate) provoked a
notable decrease in the growth inhibitory activity of the compounds, as deduced from the higher IC50
values obtained for them in endothelial and tumor cells [6].
Many bioactive quinones and hydroquinones derived from marine organisms have one or more
prenyl groups in their molecules [1–3]. Our results show that the substitution of a prenyl group
(
compound ) for a toluquinol methyl group practically abrogated the generic antitumor activity of the
3
compound that only exerted a moderate cytotoxic effect against the HL60 human leukemia cell line.
This activity is consistent with that already described in P388 murine leukemia cells for this natural
marine compound, first isolated from the tunicate Aplidium californicum [16,17]. Disubstituted prenyl
hydroquinones are also frequently found in marine organisms. Although a correlation between their
substitution pattern and the producing organism has been proposed, there is some evidence that the
pharmacophore responsible for the cytotoxicity of these naturally occurring secondary metabolites
could be the quinone, hydroquinone, or naphthoquinone nucleus [
the inclusion of a second isoprenyl group in the molecule (compound 18, obtained as a sub-product in
the synthesis of compound , as shown in Scheme 1) caused a 7-fold increase in the antileukemic activity
1,18]. As shown in Table 1,
3
of the new compound, which remained noncytotoxic for the other tumor cell lines. This suggests that
the structural modification of the natural isoprenyl hydroquinone can effectively increase its activity,
maintaining the interesting putative specificity of this compound to leukemia cells.
It is worth noting that, when substituted vinyl groups were introduced instead of the methyl group,
their corresponding derivatives (compounds
being the most active in this series and compound
effect of the steric hindrance when either a bulky substituent is present, such as the tert-butyl group
compound ), or an increased number of methyl groups are included in the aliphatic chain (compound
). In any case, all of them showed significantly lower cytotoxicity than toluquinol, indicating that
4
–7
) retained some antitumor activities, with compound
7
6
the least active, as an indication of the detrimental
(
6
5
these structural changes are not well tolerated with regard to their antiproliferative properties and
again suggesting the important role of the methyl group for the cytotoxicity of this compound.
2
.2.2. Antitumor Properties with SAR of 2,6-Disubstituted Hydroquinones 8–11
Our results show that good tolerance was displayed after the inclusion of a second substituent in
the biological properties of the resulting disubstituted 1,4-hydroquinones (compounds
toluquinol derivatives presented antitumoral activities in the same low M range as the original
molecule. The biological evaluation of the subset comprised by the 2,6-disubstituted-1,4-hydroquinones
compounds 11) revealed a number of intriguing findings. Although the resulting cytotoxicity values
8–13). All those
µ
(
8–
showed that the introduction of a second substituent in the toluquinol molecule was well tolerated,
a significant decrease in the cytotoxic activity was only observed as the steric volume of the substituent
increased (compounds
9 and 10). This is in agreement with the abovementioned results obtained with
the monosubstituted vinyl derivatives. We analyzed the effect of the position of the second substituent
in the 1,4-hydroquinone.
2
.2.3. Antitumor Properties with SAR of 2,3-Disubstituted Hydroquinone 13 and 2,5-Disubstituted
Hydroquinones 12 and 15
A comparison of the IC50 values obtained for 2,3-dimethyl-1,4-hydroquinone (13), the 2,5-
disubstituted derivatives (compounds 12 and 15), and the 2, 6-disubstituted derivatives (compounds
8
and 11) indicates that, although no major differences were observed when non-bulky groups were
included in any of those positions, in general, 2,5-disubstituted compounds seemed to exhibit higher
cytotoxic activity. In this regard, compound 12 proved to be one of the most potent analogues,
exhibiting cytotoxic activity higher than that of the 2,6-disubstituted counterpart (compound 11).

Mar. Drugs 2019, 17, 492
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Special mention should be made of thymoquinol (compound 15), which in our assays demonstrated
very similar activity to that of toluquinol. Several thymoquinol glycosides have been isolated from plants
used in folk medicine to treat infections [19,20]. The main reason that we included thymoquinol in this
study is that it is the reduced form of thymoquinone (compound 16), the major active ingredient of Nigella
sativa, known as black seed or black cumin, which has been used as a traditional remedy to treat a variety
of diseases for more than 2000 years [21]. Known as “the miracle herb of the century,” its impressive
biological activities include antibacterial, antifungal, antiviral, antihelminthic, anti-inflammatory,
immunomodulatory, and anticancer, as well as curative properties against cardiovascular diseases,
diabetes, asthma, and oral or kidney diseases [22]. Thymoquinone, also found in many other
medicinal plants, presents a wide range of biological activities of pharmacological value, including an
interesting antitumor activity derived from its antiproliferative, anti-oxidant, cytotoxic, apoptogenic,
and antimetastatic effects [21,23]. Thymoquinone has been recently identified as a potent anticancer
compound capable of inducing apoptotic cell death by upregulating the expression of apoptotic
genes p53 and caspase 9 and downregulating the expression of anti-apoptotic genes such as Bcl2 [24].
The antiangiogenic activity of thymoquinone may also contribute to enhancing the pharmacological
potential of this compound in oncology [25,26].
2
.2.4. Antitumor Properties with SAR of Benzoquinones 14, 16, and Benzopyrane 19
To shed light on the effect of the oxidation state of the toluquinol derivatives on their biological
properties, we compared the activities of toluquinol (1) and thymoquinol (15) with those of their
respective quinones, toluquinone (14) and thymoquinone (16). Our results show that the reduced
compounds have better activities than those of their corresponding quinones, although these differences
were not very significant. Although this contradicts the previous statement that the keto form of
thymoquinone could be responsible for the pharmacological properties of the compound [27], it is
in agreement with recent reported data showing that the reduction of thymoquinone, toluquinone,
and other quinones to their respective hydroquinones increased their cytotoxic potency and selectivity
on four cell lines of ovarian cancer [28]. In any case, it cannot be ruled out that both species might
play a role in the toxicity of the compounds, which could generate a redox cycle in the tumor cell,
responsible for the ROS production leading to cell death, in a similar mechanism to that proposed to
explain the cytotoxic activities of marine terpenoquinones [3]. Supporting this hypothesis, the cytotoxic
activity of thymoquinone has been related to the induction of oxidative damage in cancer cells,
resulting in apoptosis [29]. Thymoquinone has a dual effect, with antioxidant properties at low
concentration, but pro-oxidant ones at higher concentrations, derived from ROS generation [30].
Acting as a redox-cycler, the quinone can be metabolized by enzymatic or non-enzymatic reactions
to hydroquinone or semiquinone, generating superoxide anion radicals [31]. The relevance of the
cyclic interconversion of quinones and hydroquinones in the activity of these compounds might also
explain the negligible cytotoxic activity of compound 19 (obtained as a subproduct in the synthesis of
compound
3
, as shown in Scheme 1), in which this interconversion between the two forms is prevented.
3
. Experimental Section
3
.1. General Techniques
All reactions were carried out under an argon atmosphere with dry, freshly distilled solvents
under anhydrous conditions, unless using aqueous reagents or otherwise noted. All solvents used in
reactions were dried and distilled using standard procedures. Tetrahydrofuran (THF) was distilled
from sodium benzophenone, and methylene chloride (CH Cl ) from calcium hydride. Yields refer
2
2
1
to chromatographically and spectroscopically ( H NMR) homogeneous materials, unless otherwise
stated. All solutions used in workup procedures were saturated unless otherwise noted. All reagents
were purchased at highest commercial quality and used without further purification unless otherwise
stated. All reactions were monitored by thin-layer chromatography (TLC) using 0.25-mm silica

Mar. Drugs 2019, 17, 492
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gel plates (60F-254), using UV light (254 nm) as the visualizing agent and acidic ceric ammonium
molybdate/phosphomolybdic acid or potassium permanganate solutions and heat as the developing
agents. Flash column chromatography (FCC) was performed using silica gel (60 Å, particle size
2
30–400 mesh) under air pressure. All solvents used for chromatographic purifications were distilled
1
13
prior to use. H and C NMR spectra were recorded on a Bruker DPX-400 MHz instrument (Fällanden,
Switzerland) and calibrated using residual undeuterated solvent as an internal reference. Chemical
shifts are reported in ppm, with the resonance resulting from incomplete deuteration of the solvent as
the internal standard ( CDCl : 7.26 ppm, s and 77.0 ppm, t; CD OD: 4.87 ppm, s, 3.31 ppm, quin
and 49.1 ppm, sep; 13C D OS: 2.49 ppm, quin and 39.52 ppm, sep). Data are reported as follows:
13
13
3
3
2
6
1
chemical shift
δ/ppm (multiplicity, coupling constants J (Hz) and integration ( H only)). The following
abbreviations were used to explain the multiplicities: s = singlet; d = doublet; t = triplet; q = quartet;
13
quin = quintet; b = broad; m = multiplet or combination thereof. C signals are singles, unless
otherwise stated. High-resolution mass spectrometry (HRMS) was performed on a Thermo Fisher
Scientific H-ESI and APCI mass spectrometer (Waltham (MA), USA) in positive mode and using an ion
trap (Orbitrap) as the mass analyzer type. HRMS signals are reported to four decimal places and are
within
±
5 ppm of theoretical values. Melting points were collected using a Gallenkamp melting point
◦
apparatus (London, UK), using a gradient of 0.5 C per min.
3
.2. Biological Material and Methods
All the cancer cell lines used in this study were obtained from the American Type Culture Collection
(ATCC, Manassas, Virginia, USA). Human fibrosarcoma HT-1080 and human glioblastoma U87-MG
cells were maintained in Eagle’s Minimum Essential Medium (EMEM), supplemented with 10% FBS.
Human colon adenocarcinoma HT-29 cells were maintained in DMEM containing glucose (4.5 g/L)
supplemented with 10% FBS. Human breast cancer carcinoma MDA-MB-231 cells were maintained
in a RPMI1640 medium supplemented with 10% FBS. Acute promyelocytic leukemia HL-60 cells
were maintained in a RPMI1640 medium supplemented with 20% FBS. All culture media contained
glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50
and all cell lines were grown at 37 C in a humidified 5% CO atmosphere.
µ
g/mL), and amphotericin (1.25
µg/mL),
◦
2
3
.3. Synthesis
3
.3.1. 2-(Trifluoromethyl)benzene-1,4-diol (2)
Hydroquinone 17 (50 mg, 0.45 mmol, 1.0 equiv.), 5-(Trifluoromethyl) dibenzothiophenium
trifluoromethanesulfonate (362 mg, 0.9 mmol, 2.0 equiv.) and pyridine (0.04 mL, 0.45 mmol, 1.0 equiv.)
◦
were dissolved in DMF (2 mL) and the reaction mixture was stirred for 72 h at 25 C. After this time,
the reaction mixture was diluted with water and the aqueous phase was extracted with Et O twice.
2
The combined organic phases were washed sequentially with a saturated aqueous CuSO solution
4
and brine, dried over MgSO , filtered, and the solvent removed under reduced pressure. The crude
4
mixture was purified by flash column chromatography (silica gel, 5% EtOAc in hexanes
→
20% EtOAc
in hexanes) to obtain
2
(39 mg, 49%) as a yellow oil [
8
]: R = 0.23 (silica gel, 30% EtOAc in hexanes);
f
1
H NMR (400 MHz, CD OD) δ 6.88 (d, J = 2.9 Hz, 1 H), 6.81 (ddd, J = 8.8, 2.9, 0.6 Hz, 1 H), 6.76 (dd,
J = 4.9, 4.4 Hz, 1 H); 19F NMR (400 MHz, CD OD) δ −63.78; HRMS (H-ESI) m/z: [M + H] calculated
3
+
3
for C H F O 179.0320; found 179.0318.
7
6
3
2
3
.3.2. 2-(3-Methylbut-2-en-1-yl)benzene-1,4-diol (3), 2,5-bis-(3-Methylbut-2-en-1-yl)benzene-1,4-diol
(
18) and 2,2-dimethyl-6-chromanol (19): Reaction of 17 with Prenol
Hydroquinone 17 (300 mg, 2.72 mmol, 1.0 equiv.) and 3-methyl-2-buten-1-ol (0.55 mL, 5.45 mmol,
2
0
.0 equiv.) were dissolved in a 1:1 mixture of Et O:CH Cl (10 mL). Then, BF
.2 equiv.) was added at 0 C and the reaction mixture was stirred for 48 h at 25 C. After this time,
•
OEt (0.1 mL, 0.54 mmol,
2
2
2
3
2
◦
◦
cold water was added and the aqueous phase was extracted with EtOAc. The organic phase was

Mar. Drugs 2019, 17, 492
10 of 16
washed with brine, dried over MgSO , filtered, and the solvent removed under reduced pressure.
4
The residue was purified by flash column chromatography (silica gel, 15% EtOAc in hexanes
EtOAc in hexanes) to obtain (72 mg, 15%), together with side products 18 (21 mg, 3%) and 19 (13 mg,
.7%) [ ]. [ ]: white solid, R = 0.30 (silica gel, 50% EtOAc in hexanes); m.p. = 101–103 C; H NMR
400 MHz, CDCl3) δ 6.68 (d, J = 8.3 Hz, 1 H), 6.63–6.54 (m, 2 H), 5.29 (t, J = 7.3 Hz, 1 H), 3.29 (d,
→
25%
3
◦
1
2
9
3
f
(
J = 7.0 Hz, 2 H), 1.77 (s, 3 H), 1.76 (s, 3 H); 13C NMR (100 MHz, CDCl )
δ
149.7, 147.5, 131.6, 128.8, 122.6,
3
+
1
15.7, 115.1, 112.4, 27.8, 24.5, 16.4; HRMS (H-ESI) m/z: [M + H] calculated for C H O 179.1072;
11
15
2
1
found 179.1064. [18]: white solid; H NMR (400 MHz, CDCl )
H), 3.31–3.24 (m, 1 H), 2.71 (dd, J = 13.0, 6.4 Hz, 1 H), 1.82–1.69 (m, 12 H); C NMR (100 MHz, CDCl )
δ 6.70–6.45 (m, 4 H), 5.29 (t, J = 7.9 Hz, 1
3
13
3
+
δ
147.2, 131.4, 125.8, 122.9, 115.6, 27.5, 24.6, 16.5; HRMS (H-ESI) m/z: [M + H] calculated for C H O
16
23
2
1
2
47.1698; found 247.1708. [19]: red oil; H NMR (400 MHz, CDCl ) δ 6.65 (d, J = 8.6 Hz, 1 H), 6.62–6.53
3
1
3
(m, 2 H), 2.72 (t, J = 6.8 Hz, 2 H), 1.77 (t, J = 6.8 Hz, 2 H), 1.31 (s, 6 H); C NMR (100 MHz, CDCl )
δ
3
+
1
49.8, 146.9, 121.3, 117.1, 114.8, 113.9, 73.2, 32.5, 25.6, 22.2; HRMS (H-ESI) m/z: [M + H] calculated for
C H O 179.1072; found 179.1071.
11
15
2
3
.3.3. Synthesis of Compounds 20 and 22
-Bromobenzene-1,4-diol (20). Bromine (0.56 mL, 10.90 mmol, 1.2 equiv.) was added dropwise to
2
t
◦
a solution of 1,4-hydroquinone (17) (1.0 g, 9.08 mmol, 1.0 equiv.) in BuOMe (20 mL) at -15 C and
the reaction mixture was stirred for 2 h. After this time, the reaction mixture was quenched by the
addition of 10% aqueous sodium thiosulfate solution and extracted with Et O twice. The combined
2
organic phases were washed with brine, dried over MgSO , filtered, and the solvent removed under
4
reduced pressure. The residue was purified by flash column chromatography (silica gel, 10% EtOAc in
hexanes) to obtain 20 (1.3 g, 78%) as a colorless solid [11]: R = 0.35 (silica gel, 30% EtOAc in hexanes);
f
1
H NMR (400 MHz, CD OD)
δ
6.89 (d, J = 2.8 Hz, 1 H), 6.72 (d, J = 8.7 Hz, 1 H), 6.60 (dd, J = 8.7, 2.9 Hz,
H); C NMR (100 MHz, CD OD) δ 152.0, 148.2, 120.3, 117.7, 116.4, 110.7.
3
13
1
3
2-Bromo-6-methylbenzene-1,4-diol (22). Bromine (2.8 mL, 55.48 mmol, 2.0 equiv.) was added
dropwise over 15 min to a solution of o-cresol (21) (2.9 mL, 27.74 mmol, 1.0 equiv.) in AcOH (30 mL) at
◦
◦
0
C and the reaction mixture was stirred at 25 C for 1 h. After this time, cold water was added and the
resulting white precipitate was filtered and washed with cold water to obtain 2,4-dibromo-6-methyl
phenol (S1) (5.8 g, 80%) as a colorless solid that did not require further purification [12]: R = 0.60
f
◦
1
(
1
1
20% EtOAc in hexanes); m.p. = 56–57 C; H NMR (400 MHz, CDCl )
δ
7.43 (dd, J = 2.3, 0.6 Hz,
H), 7.21–7.19 (m, 1 H), 2.27 (t, J = 0.7 Hz, 3 H); 13C NMR (100 MHz, CDCl3)
149.8, 133.1, 131.3,
27.7, 112.0, 110.4, 16.5. A solution of CrO (1.5 g, 15.31 mmol, 1.1 equiv.) in H O (4 mL) was added
3
δ
3
2
to a solution of 2,4-dibromo-6-methyl phenol (3.7 g, 13.91 mmol, 1.0 equiv.) in Ac O (7 mL)/CH CN
2
3
◦
(
3.5 mL) and the resulting mixture was heated at 60 C for 1.5 h. After this time, the reaction mixture
was cooled to 25 C and diluted with water. Then, the aqueous phase was extracted with CH Cl three
◦
2
2
times, and the combined organic phases were washed with brine, dried over MgSO , filtered, and the
4
solvent removed under reduced pressure to obtain 2-bromo-6-methyl-p-benzoquinone (S2) (2.3 g, 85%)
as an orange solid that did not require further purification: R = 0.45 (silica gel, 20% EtOAc in hexanes);
f
◦
1
m.p. = 92–93 C; H NMR (400 MHz, CDCl )
2
δ
7.23 (d, J = 2.4 Hz, 1 H), 6.65 (dq, J = 2.4, 1.6 Hz, 1 H),
3
.14 (d, J = 1.6 Hz, 3 H); 13C NMR (100 MHz, CDCl3)
δ
184.8, 179.9, 145.7, 138.1, 137.3, 133.4, 16.8.
The resulting p-benzoquinone derivative (1.0 g, 4.97 mmol, 1.0 equiv.) was dissolved in a mixture
◦
of EtOH (10 mL)/H O (2 mL) and the mixture was heated at 50 C. Then, Na S O (1 g, 5.97 mmol,
1
2
2
2
4
◦
.2 equiv.) was added and the reaction mixture was heated at 50 C for 1.5 h. After this time, EtOH was
removed under reduced pressure, the crude mixture was diluted with water, and the aqueous phase
was extracted with EtOAc. The organic phase was washed with brine, dried over MgSO , filtered,
4
and the solvent removed under reduced pressure to obtain a solid that was washed with cold CH Cl
2
2
to obtain 22 (585 mg, 57%) as a pale yellow solid that did not require further purification: R = 0.25
f
1
(
silica gel, 20% EtOAc in hexanes); H NMR (400 MHz, CDCl )
δ
6.82 (dd, J = 2.9, 0.5 Hz, 1 H), 6.60 (dd,
3

Mar. Drugs 2019, 17, 492
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J = 2.9, 0.7 Hz, 1 H), 2.25 (t, J = 0.5 Hz, 3 H); 13C NMR (100 MHz, CDCl3)
δ
148.8, 144.8, 126.5, 117.7,
+
1
15.7, 109.6, 16.9; HRMS (H-ESI) m/z: [M + H] calculated for C H BrO 202.9708; found 202.9726.
7
8
2
3
.3.4. General Procedures for Suzuki Couplings: Synthesis of the 2-Substituted and 2,6-Disubstituted
Hydroquinones 4–11
Condition A: To a solution of boronic acid pinacol ester derivative (2.0 equiv.), bromo derivative
(
1.0 equiv.), Pd[PPh ] (0.2 equiv.), and K CO (3.6 equiv.) in 1,4-dioxane was added a drop of water
3 4 2 3
◦
and the reaction mixture was heated at 80 C for 15 h. After this time, a saturated aqueous NH Cl
4
solution was added and the aqueous phase was extracted with EtOAc three times. The combined
organic phases were washed with brine, dried over MgSO , filtered, and the solvent removed under
4
reduced pressure. The residue was purified by flash column chromatography (silica gel) to obtain
the corresponding product. Condition B: Boronic acid pinacol ester derivative (1.1 equiv.), bromo
derivative (1.0 equiv.), Pd[PPh ] (0.05 equiv.), and K CO (2.0 M aqueous solution, 6.6 equiv.) were
3
4
2
3
◦
dissolved in THF and the reaction mixture was heated at 80 C for 48 h. After this time, a saturated
aqueous NH Cl solution was added and the aqueous phase was extracted with EtOAc three times.
4
The combined organic phases were washed with brine, dried over MgSO , filtered, and the solvent
4
removed under reduced pressure. The residue was purified by flash column chromatography (silica gel)
to obtain the corresponding product. Conditions C: Boronic acid pinacol ester derivative (1.1 equiv.),
bromo derivative (1.0 equiv.), Pd[PPh ] (0.05 equiv.), and K CO (2.0 M aqueous solution, 6.6 equiv.)
3
4
2
3
◦
were dissolved in THF and the reaction mixture was heated at 80 C for 15 h. After this time, a saturated
aqueous NH Cl solution was added and the aqueous phase was extracted with EtOAc three times.
4
The combined organic phases were washed with brine, dried over MgSO , filtered, and the solvent
4
removed under reduced pressure. The residue was purified by flash column chromatography (silica
gel) to obtain the corresponding product.
2
-(2-Methylprop-1-en-1-yl)benzene-1,4-diol (
(50 mg, 0.29 mmol, 1.0 equiv.) by treatment with 2-methyl-1-propenylboronic acid pinacol ester
0.06 mL, 0.29 mmol, 1.1 equiv.), Pd[PPh ] (15 mg, 0.01 mmol, 0.05 equiv.), and K CO (0.9 mL, 2.0 M
4). Compound 4 was prepared from bromohydroquinone
2
(
0
3
4
2
3
aqueous solution, 1.72 mmol, 6.6 equiv.) in THF (2 mL), according to the general procedure for Suzuki
couplings (conditions B), as described above. Purification by flash column chromatography (silica gel,
2
0% EtOAc in hexanes) afforded
4
(39 mg, 92%) as a pale yellow oil: R = 0.48 (silica gel, 50% EtOAc in
f
1
hexanes); H NMR (400 MHz, MeOD)
δ
6.60 (d, J = 8.6 Hz, 1 H), 6.56 (d, J = 3.0 Hz, 1 H), 6.49 (ddd,
J = 8.6, 3.0, 0.4 Hz, 1 H), 6.21–6.14 (m, 1 H), 1.88 (d, J = 1.4 Hz, 3 H), 1.76 (d, J = 1.1 Hz, 3 H); 13C NMR
100 MHz, MeOD) 150.7, 148.7, 136.3, 127.6, 121.9, 117.7, 116.8, 114.9, 26.5, 19.6; HRMS (H-ESI) m/z:
M + H] calculated for C H O 165.0916; found 165.0938.
(
[
δ
+
10
13
2
2-(3-Methylbut-2-en-2-yl)benzene-1,4-diol (5). Compound 5 was prepared from bromohydroquinone
2
0 (50 mg, 0.26 mmol, 1.0 equiv.) by treatment with 3-Methyl-2-buten-2-ylboronic acid pinacol ester
(
0.06 mL, 0.29 mmol, 1.1 equiv.), Pd[PPh ] (15 mg, 0.01 mmol, 0.05 equiv.), and K CO (0.9 mL, 2.0 M
3 4 2 3
aqueous solution, 1.72 mmol, 6.6 equiv.) in THF (2 mL), according to the general procedure for Suzuki
coupling (conditions B). Purification by flash column chromatography (silica gel, 5% EtOAc in hexanes
→
15% EtOAc in hexanes) gave
in hexanes); H NMR (400 MHz, CD OD)
5
(15 mg, 32%) as a pale yellow oil: R = 0.40 (silica gel, 50% EtOAc
f
1
δ 6.61 (d, J = 8.7 Hz, 1 H), 6.50 (dd, J = 8.6, 3.0 Hz, 1 H),
3
6
3
.37 (d, J = 2.9 Hz, 1 H), 1.86 (dd, J = 1.4, 1.0 Hz, 3 H), 1.79 (d, J = 0.5 Hz, 3 H), 1.50 (dd, J = 2.7, 1.3 Hz,
13
H); C NMR (100 MHz, CD OD)
δ 149.7, 146.1, 132.4, 128.1, 126.2, 115.7, 115.6, 113.4, 20.7, 18.7, 18.6;
3
+
HRMS (H-ESI) m/z: [M + H] calculated for C H O 179.1072; found 179.1065.
11
15
2
(
E)-2-(3,3-Dimethylbut-1-en-1-yl)benzene-1,4-diol (
bromohydroquinone 12 (100 mg, 0.53 mmol, 1.0 equiv.) by treatment with 2-t-Butyl-E-vinylboronic acid
pinacol ester (0.14 mL, 0.58 mmol, 1.1 equiv.), Pd[PPh ] (31 mg, 0.03 mmol, 0.05 equiv.), and K CO
6). Compound 6 was prepared from
3
4
2
3
(
1.75 mL, 2.0 M aqueous solution, 3.49 mmol, 6.6 equiv.) in THF (4 mL), according to the general
procedure for Suzuki coupling (conditions C). Purification by flash column chromatography (silica gel,
5% EtOAc in hexanes) afforded (80 mg, 79%) as a pale yellow oil: R = 0.33 (silica gel, 50% EtOAc
1
6
f

Mar. Drugs 2019, 17, 492
12 of 16
1
in hexanes); H NMR (400 MHz, CD OD)
6
1
δ
6.81 (t, J = 3.0 Hz, 1 H), 6.58 (dd, J = 12.4, 3.9 Hz, 2 H),
3
.47 (dd, J = 8.6, 2.9 Hz, 1 H), 6.14 (d, J = 16.3 Hz, 1 H), 1.11 (s, 9 H); 13C NMR (100 MHz, CD OD)
51.2, 148.6, 141.9, 127.2, 120.8, 117.3, 115.5, 112.9, 34.3, 30.1; HRMS (H-ESI) m/z: [M + H] calculated
δ
3
+
for C H O 193.1229; found 193.1238.
12
17
2
(
E)-2-(3-Methoxyprop-1-en-1-yl) benzene-1,4-diol (
bromohydroquinone 20 (50 mg, 0.26 mmol, 1.0 equiv.) by treatment with trans-3- Methoxy-1-
propenylboronic acid pinacol ester (0.06 mL, 0.29 mmol, 1.1 equiv.), Pd[PPh ] (15 mg, 0.01 mmol,
7). Compound 7 was prepared from
3
4
0
.05 equiv.), and K CO (0.90 mL, 2.0 M aqueous solution, 1.72 mmol, 6.6 equiv.) in THF (2 mL),
2 3
according to the general procedure for Suzuki coupling (conditions C). Purification by flash column
chromatography (silica gel, 25% EtOAc in hexanes) gave
7 (26 mg, 56%) as a pale yellow oil: R = 0.30
f
1
(
silica gel, 50% EtOAc in hexanes); H NMR (400 MHz, CD OD)
δ
6.88–6.82 (m, 2 H), 6.62 (d, J = 8.5 Hz,
3
1
(
H), 6.54 (dd, J = 8.6, 2.9 Hz, 1 H), 6.22 (dt, J = 16.0, 6.3 Hz, 1 H), 4.07 (dd, J = 6.3, 1.4 Hz, 2 H), 3.36
151.2, 149.2, 129.4, 126.1, 125.6, 117.5, 116.7, 113.6, 74.7, 57.9;
HRMS (H-ESI) m/z: [M + H] calculated for C H O 181.0865; found 181.0884.
s, 3 H); 13C NMR (100 MHz, CD OD)
δ
3
+
10
13
3
2
-Methyl-6-(2-methylprop-1-en-1-yl)benzene-1,4-diol (
derivative 22 (58 mg, 0.29 mmol, 1.0 equiv.) by treatment with 2-methyl-1-propenylboronic acid
pinacol ester (0.12 mL, 0.58 mmol, 2.0 equiv.), Pd[PPh ] (66 mg, 0.06 mmol, 0.2 equiv.), and K CO
8). Compound 8 was prepared from bromo
3
4
2
3
(
142 mg, 1.03 mmol, 3.6 equiv.) in 1,4-dioxane (1.5 mL), according to the general procedure for Suzuki
coupling (conditions A). Purification by flash column chromatography (silica gel, 20% EtOAc in
1
hexanes) afforded
8
(29 mg, 58%) as a yellow oil: R = 0.50 (silica gel, 30% EtOAc in hexanes); H NMR
f
(
400 MHz, CD OD)
δ
6.44 (dd, J = 3.0, 0.5 Hz, 1 H), 6.38–6.36 (m, 1 H), 6.17 (s, 1 H), 2.14 (s, 3 H), 1.89 (d,
3
13
J = 1.2 Hz, 3 H), 1.73 (d, J = 1.0 Hz, 3 H); C NMR (100 MHz, CD OD)
1
δ
149.1, 145.0, 135.9, 126.4, 125.7,
3
+
20.5, 115.3, 113.6, 24.9, 18.2, 15.4; HRMS (H-ESI) m/z: [M + H] calculated for C H O 179.1072;
11
15
2
found 179.1081.
-Methyl-6-(3-methylbut-2-en-2-yl)benzene-1,4-diol (
2
9). Compound 9 was prepared from bromo
derivative 22 (50 mg, 0.24 mmol, 1.0 equiv.) by treatment with 3-methyl-2-buten-2-ylboronic acid
pinacol ester (0.06 mL, 0.26 mmol, 1.1 equiv.), Pd[PPh ] (14 mg, 0.01 mmol, 0.05 equiv.) and K CO
3
4
2
3
(0.8 mL, 2.0 M aqueous solution, 1.72 mmol, 6.6 equiv.) in THF (2 mL), according to the general
procedure for Suzuki coupling (conditions B). Purification by flash column chromatography (silica gel,
1
0% EtOAc in hexanes) gave
9
(16 mg, 35%) as a pale yellow oil: R = 0.60 (silica gel, 30% EtOAc in
f
1
hexanes); H NMR (400 MHz, CDCl )
δ
6.52 (dd, J = 3.0, 0.7 Hz, 1 H), 6.33 (dd, J = 3.1, 0.5 Hz, 1 H),
3
2
.21 (t, J = 0.6 Hz, 3 H), 1.89 (dd, J = 1.5, 1.0 Hz, 3 H), 1.85 (d, J = 0.7 Hz, 3 H), 1.55 (q, J = 1.4 Hz, 3 H);
13
C NMR (100 MHz, CDCl3) δ 148.4, 143.8, 132.1, 130.7, 124.7, 124.6, 116.1, 112.2, 21.7, 20.1, 20.0, 16.3;
+
HRMS (H-ESI) m/z: [M + H] calculated for C H O 193.1229; found 193.1234.
12
17
2
(
E)-2-(3,3-Dimethylbut-1-en-1-yl)-6-methylbenzene-1,4-diol (10). Compound 10 was prepared
from bromo derivative 22 (100 mg, 0.53 mmol, 1.0 equiv.), by treatment with 2-t-Butyl-E-vinylboronic
acid pinacol ester (0.14 mL, 0.58 mmol, 1.1 equiv.), Pd[PPh ] (31 mg, 0.03 mmol, 0.05 equiv.), and K CO
3
4
2
3
(1.75 mL, 2.0 M aqueous solution, 3.50 mmol, 6.6 equiv.) in THF (4 mL), according to the general
procedure for Suzuki coupling (conditions B). Purification by flash column chromatography (silica
gel, 15% EtOAc in hexanes) gave 10 (40 mg, 79%) as a pale yellow oil: R = 0.40 (silica gel, 30% EtOAc
f
1
in hexanes); H NMR (400 MHz, CDCl )
δ 6.67–6.65 (m, 1 H), 6.52 (dd, J = 3.0, 0.6 Hz, 1 H), 6.43 (d,
J = 16.2 Hz, 1 H), 6.16 (d, J = 16.2 Hz, 1 H), 2.20 (t, J = 0.6 Hz, 3 H), 1.12 (s, 9 H); C NMR (100 MHz,
3
13
CDCl )
δ
148.8, 144.9, 144.9, 125.6, 125.2, 118.8, 116.3, 110.9, 33.7, 29.5, 16.1; HRMS (H-ESI) m/z: [M +
3
+
H] calculated for C H O 207.1385; found 207.1367.
13
19
2
(
E)-2-(3-Methoxyprop-1-en-1-yl)-6-methylbenzene-1,4-diol (11). Compound 10 was prepared
from bromo derivative 22 (50 mg, 0.25 mmol, 1.0 equiv.) by treatment with trans-3-methoxy-1-
propenylboronic acid pinacol ester (0.10 mL, 0.49 mmol, 2.0 equiv.), Pd[PPh ] (57 mg, 0.05 mmol,
3
4
0
.2 equiv.), and K CO (122 mg, 0.89 mmol, 3.6 equiv.) in 1,4-dioxane (1.5 mL), according to the general
2 3
procedure for Suzuki coupling (conditions A). Purification by flash column chromatography (silica gel,
5% EtOAc in hexanes) afforded 11 (24 mg, 51%) as a pale yellow oil: R = 0.30 (silica gel, 50% EtOAc
2
f

Mar. Drugs 2019, 17, 492
13 of 16
1
in hexanes); H NMR (400 MHz, CD OD)
0
δ
6.97–6.90 (m, 1 H), 6.71 (d, J = 3.0 Hz, 1 H), 6.50 (dd, J = 3.0,
3
13
.7 Hz, 1 H), 6.16 (dt, J = 15.9, 6.3 Hz, 1 H), 4.08 (dd, J = 6.3, 1.4 Hz, 2 H), 3.37 (s, 3 H), 2.15 (s, 3 H);
C
NMR (100 MHz, CD OD)
δ
150.1, 145.2, 128.2, 127.1, 125.6, 124.9, 116.9, 109.3, 73.2, 56.6, 15.5; HRMS
3
+
(H-ESI) m/z: [M + H] calculated for C H O 195.1021; found 195.1023.
11
15
3
3
.3.5. Synthesis of Compounds 23, 24, and 2,5-Disubstituted Hydroquinones 12 and 15
-Bromo-5-methylcyclohexa-2,5-diene-1,4-dione (23). To a solution of toluquinol ( ) (2.5 g, 20.14 mmol,
2
1
1
.0 equiv.) in acetone (15 mL) was added K CO (14 g, 100.70 mmol, 5.0 equiv.) and Me SO (5.7 mL,
2
3
2
4
60.41 mmol, 3.0 equiv.) and the reaction mixture was stirred for 3 h. After this time, the reaction
mixture was diluted with water and the aqueous phase was extracted with Et O. The organic phase
2
was washed with brine, dried over MgSO , filtered, and the solvent removed under reduced pressure
4
to obtain the corresponding dimethoxy derivative (~20 mmol), which was used in the next steps
without purification. To a solution of the dimethoxy derivative obtained above (~20 mmol) and NaOAc
(3.3 g, 40.28 mmol, 2.0 equiv.) in AcOH (20 mL) was added bromine (1.2 mL, 2.15 mmol, 1.1 equiv.)
over 25 min and, after the addition, the reaction mixture was stirred for 1 h. Then, the reaction mixture
◦
was quenched by a slow addition of a saturated aqueous NaHCO solution at 0 C. The aqueous
3
phase was then extracted with EtOAc and the organic phase washed with brine, dried over MgSO4,
filtered, and the solvent removed under reduced pressure to obtain the corresponding bromo derivative
(~20 mmol), which was used in the next step without purification. The bromo derivative obtained
above (~20 mmol) was dissolved in CH CN (35 mL). Then, CAN (28 g, 50.34 mmol, 2.5 equiv.) and H O
3
2
◦
(
20 mL) were added and the reaction mixture was stirred for 1 h at 25 C. After this time, the reaction
mixture was diluted with water and the aqueous phase was extracted with Et O twice. The combined
2
organic phases were washed with brine, dried over MgSO , filtered, and the solvent removed under
4
reduced pressure. The residue was purified by flash column chromatography (silica gel, 1% EtOAc in
hexanes) to obtain compound 23 (1.5 g, 37% over 3 steps) as an orange solid [13]: R = 0.45 (silica gel,
f
1
2
3
0% EtOAc in hexanes); H NMR (400 MHz, CDCl )
δ
7.29 (s, 1 H), 7.26 (s, 2 H), 2.08 (d, J = 1.6 Hz,
3
13
H); C NMR (100 MHz, CDCl ) δ 185.1, 179.5, 146.5, 138.1, 137.5, 132.6, 15.7.
3
2
-Bromo-5-methylbenzene-1,4-diol (24). Quinone 23 (264 mg, 1.31 mmol, 1.0 equiv.) was dissolved
◦
in a mixture of EtOH/H O (3/0.5 mL) and heated to 50 C. Then, Na S O (264 mg, 1.57 mmol, 1.2 equiv.)
was added and the reaction mixture was stirred at 50 C for 1.5 h. After this time, the reaction mixture
was cooled to 25 C and the EtOH was removed under reduced pressure. The resulting residue was
2
2
2
4
◦
◦
diluted with water and extracted with EtOAc twice and the combined organic phases washed with
brine, dried over MgSO , filtered, and the solvent removed under reduced pressure to obtain a yellow
4
solid. This solid was washed with cold CH Cl to afford pure compound 24 (118 mg, 44%) as a pale
2
2
yellow solid that did not require further purification: R = 0.52 (silica gel, 50% EtOAc in hexanes);
f
1
13
H NMR (400 MHz, CD OD)
δ
6.83 (s, 1 H), 6.64 (d, J = 0.7 Hz, 1 H), 2.08 (s, 3 H); C NMR (100 MHz,
148.7, 146.4, 124.9, 117.9, 117.8, 105.6, 14.6; HRMS (H-ESI) m/z: [M + H] calculated for
3
+
CD OD)
δ
3
C H BrO 202.9708; found 202.9715.
7
8
2
(
E)-2-(3-Methoxyprop-1-en-1-yl)-5-methylbenzene-1,4-diol (12). Compound 12 was prepared
from bromo derivative 24 (113 mg, 0.56 mmol, 1.0 equiv.) by treatment with trans-3-Methoxy-1-
propenylboronic acid pinacol ester (0.24 mL, 1.11 mmol, 2.0 equiv.), Pd[PPh ] (129 mg, 0.11 mmol,
3
4
0
.2 equiv.), and K CO (277 mg, 2.00 mmol, 3.6 equiv.) in 1,4-dioxane (3 mL), according to the general
2 3
procedure for Suzuki coupling (conditions A). Purification by flash column chromatography (silica gel,
5
% EtOAc in hexanes
→
20% EtOAc in hexanes) gave compound 12 (54 mg, 50%) as a pale yellow oil:
1
R = 0.50 (silica gel, 50% EtOAc in hexanes); H NMR (400 MHz, CD OD)
δ
6.85–6.78 (m, 2 H), 6.52
f
3
(
3
d, J = 0.6 Hz, 1 H), 6.15 (dt, J = 15.9, 6.5 Hz, 1 H), 4.06 (dd, J = 6.4, 1.3 Hz, 2 H), 3.35 (s, 3 H), 2.11 (s,
13
H); C NMR (100 MHz, CD OD)
δ
147.9, 147.5, 128.2, 125.4, 123.4, 121.4, 117.6, 111.7, 73.5, 56.5, 14.8;
3
+
HRMS (H-ESI) m/z: [M + H] calculated for C H O 195.1021; found 195.1017.
11
15
3
Thymoquinol 15. To a solution of thymoquinone (16) (100 mg, 0.61 mmol, 1.0 equiv.) in MeOH
2 mL) was added a solution of Na S O (225 mg, 1.29 mmol, 2.1 equiv.) in H O (2 mL). The reaction
(
2
2
4
2

Mar. Drugs 2019, 17, 492
14 of 16
◦
mixture was stirred for 2 h at 25 C and, after this time, the organic solvent was removed under reduced
pressure. Then, the crude mixture was diluted with water and the aqueous phase was extracted
with EtOAc. The organic phase was washed with brine, dried over MgSO , filtered, and the solvent
4
removed under reduced pressure. The residue was purified by flash column chromatography (silica
gel, 20% EtOAc in hexanes) to obtain 15 (50 mg, 49%) as a pale brown solid [14]: R = 0.50 (silica gel,
f
1
3
6
1
0% EtOAc in hexanes); H NMR (400 MHz, CD OD)
δ
6.56 (s, 1 H), 6.48 (s, 1 H), 3.17 (dt, J = 13.8,
δ 147.8, 146.6, 132.8, 121.6, 116.9,
12.1, 26.3, 21.8, 14.5; HRMS (H-ESI) m/z: [M + H] calculated for C H O 167.1072; found 167.1058.
3
.9 Hz, 1 H), 1.17 (s, 3 H), 1.15 (s, 3 H); 13C NMR (100 MHz, CD OD)
3
+
10
15
2
3
.4. Cell Growth Assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT dye reduction assay
in 96-well microplates was used [32]. This assay is dependent on the reduction of MTT by
mitochondrial dehydrogenases of a viable cell to a blue formazan product, which can be measured
3
3
spectrophotometrically. 3
a total volume of 100
×
10 HL-60, and 2
×
10 HT-1080, HT-29, MDA-MB-231, and U87MG cells in
µ
L of their respective growth medium were incubated with serial dilutions 1:1 of
the tested compounds. After three days of incubation (37 C and 5% CO in a humid atmosphere), 10
◦
µL
2
of MTT (5 mg/mL in phosphate-buffered saline) were added to each well, and the plate was incubated
◦
for a further 4 h at 37 C. The resulting formazan was dissolved in 150
µL of 0.04 N HCl/2-propanol
and read at 550 nm. IC50 values were calculated from semi-logarithmic dose-response plots as those
concentrations of the compound yielding 50% cell survival, taking the values obtained for the control
to be 100%. IC50 results are expressed as means ± S.D. of at least three independent experiments.
4
. Conclusions and Future Perspectives
Bioactive marine compounds can serve as the basis for the synthesis of derivatives with improved
pharmacological properties. The synthesis of a set of analogues of the natural product toluquinol has
been described and their in vitro cytotoxicity against a panel of cancer cell lines was compared.
Our results indicate that the cytotoxic activity of this family of compounds could rely on the
hydroquinone part of the molecule, probably due to its capability to undergo redox cycling. Although
the hydroquinones seemed to be more cytotoxic than their quinone counterparts, the differences
between them were not very significant. Substituents in the ring may modulate the interaction of
the molecule with their targets, changing either the activity or the selectivity. Tolerance for biological
activity was much more restricted for the methyl group of the molecule, whose replacement by other
groups resulted in a significant loss of cytotoxic activity. An increased selectivity was obtained when
prenyl groups were used as substituents. The inclusion of a second substituent group in the toluquinol
molecule was well tolerated and originated a series of derivatives with cytotoxic activities that were
similar to or even higher than that of the natural product. These findings provide guidance for the
design of new toluquinol analogues with potentially better pharmacological properties. Two of the
compounds studied here (toluquinol and thymoquinone) have been reported to inhibit angiogenesis
in vitro and in vivo. Further optimization and development of the new compounds and biochemical
studies to determine the mechanism of the biological actions of this family of compounds, with an
emphasis on their putative antiangiogenic activity and their undesired toxic effects, are currently
in progress.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/17/9/492/s1.
Author Contributions: Conceptualization, A.R.Q., F.S., M.
Validation and Analysis, A.R.Q., F.S., J.A.T.-V., I.C.-S., M.
A.R.Q., F.S.; Writing—Review and Editing, A.R.Q., F.S., M.
F.S., B.M.-P.
Á
Á
Á
.M., B.M.-P.; Methodology, J.A.T.-V., I.C.-S., G.A.G.-V.;
.M., B.M.-P.; Writing—Original Draft Preparation,
.M., B.M.-P.; Funding Acquisition, A.R.Q., M. .M.,
Á

Mar. Drugs 2019, 17, 492
15 of 16
Funding: Supported by grants BIO2014-56092-R, CTQ2014-60223-R (MINECO and FEDER), P12-CTS-1507,
and UMA18-FEDERJA-220 (Andalusian Government and FEDER) and funds from group BIO-267 (Andalusian
Government). The “CIBER de enfermedades raras” is an initiative of the ISCIII (Spain). The funders had no role
in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Acknowledgments: J.A.T.-V. and I.C.-S. thank the Ministerio de Educación, Cultura y Deporte for their predoctoral
fellowships (FPU programme).
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.
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
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