Flavonoid glycosides with a triazole moiety for marine antifouling applications: Synthesis and biological activity evaluation

Article abstract of DOI:10.3390/md19010005

Over the last decades, antifouling coatings containing biocidal compounds as active ingre-dients were used to prevent biofouling, and eco-friendly alternatives are needed. Previous research from our group showed that polymethoxylated chalcones and glycosy

Full text of DOI:10.3390/md19010005

marine drugs
Article
Flavonoid Glycosides with a Triazole Moiety for Marine
Antifouling Applications: Synthesis and Biological
Activity Evaluation
Daniela Pereira 1,2,† , Catarina Gonçalves 2,†, Beatriz T. Martins 1, Andreia Palmeira 1,2 , Vitor Vasconcelos 2,3
,
Madalena Pinto 1,2 , Joana R. Almeida 2,
*
, Marta Correia-da-Silva 1,2,
*
and Honorina Cidade 1,2
1
Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de
Farmácia, Universidade do Porto, R. Jorge de Viterbo Ferreira 228, 4050-313 Porto, Portugal;
dmpereira@ff.up.pt (D.P.); beatriz.martins@itqb.unl.pt (B.T.M.); apalmeira@ff.up.pt (A.P.);
madalena@ff.up.pt (M.P.); hcidade@ff.up.pt (H.C.)
CIIMAR—Centro Interdisciplinar de Investigação Marinha e Ambiental, Avenida General Norton de Matos,
S/N, 4450-208 Matosinhos, Portugal; catarina.goncalves@ciimar.up.pt (C.G.); vmvascon@fc.up.pt (V.V.)
Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre S/N,
4169-007 Porto, Portugal
2
3
*
†
Correspondence: jalmeida@ciimar.up.pt (J.R.A.); m_correiadasilva@ff.up.pt (M.C.-d.-S.)
These authors contributed equally to this work.
Abstract: Over the last decades, antifouling coatings containing biocidal compounds as active ingre-
dients were used to prevent biofouling, and eco-friendly alternatives are needed. Previous research
from our group showed that polymethoxylated chalcones and glycosylated flavones obtained by
synthesis displayed antifouling activity with low toxicity. In this work, ten new polymethoxylated
flavones and chalcones were synthesized for the first time, including eight with a triazole moiety.
Eight known flavones and chalcones were also synthesized and tested in order to construct a quanti-
tative structure-activity relationship (QSAR) model for these compounds. Three different antifouling
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Pereira, D.; Gonçalves, C.;
profiles were found: three compounds (1b
a macrofouling species (Mytilus galloprovincialis), two compounds (6a and 6b) exhibited inhibitory
activity against the biofilm-forming marine bacteria Roseobacter litoralis and one compound (7b
exhibited activity against both mussel larvae and microalgae Navicula sp. Hydrogen bonding accep-
tor ability of the molecule was the most significant descriptor contributing positively to the mussel
larvae anti-settlement activity and, in fact, the triazolyl glycosylated chalcone 7b was the most potent
compound against this species. The most promising compounds were not toxic to Artemia salina,
highlighting the importance of pursuing the development of new synthetic antifouling agents as an
ecofriendly and sustainable alternative for the marine industry.
, 11a and 11b) exhibited anti-settlement activity against
Martins,
B.T.;
Palmeira,
Pinto,
A.;
Vasconcelos,
V.;
M.;
Almeida, J.R.; Correia-da-Silva, M.;
Cidade, H. Flavonoid Glycosides
with a Triazole Moiety for Marine
Antifouling Applications: Synthesis
and Biological Activity Evaluation.
Mar. Drugs 2021, 19, 5. https://
dx.doi.org/10.3390/md19010005
)
Received: 5 December 2020
Accepted: 21 December 2020
Published: 24 December 2020
Keywords: flavonoids; synthesis; click chemistry; biofouling; antifouling; eco-friendly alternatives
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
1. Introduction
Marine biofouling, resulting from the accumulation of marine micro and macroorganisms
on submerged surfaces, has been a huge problem for maritime industries, causing several
technical and economic problems, including corrosion of materials and the increase in fuel
consumption. Moreover, marine biofouling is associated with environmental and health
problems, due to an increase in gas emissions and the spread of invasive species [1,2].
Biocidal paints containing organotin compounds, namely tributyltin (TBT), were widely
used for decades in the maritime industry to prevent biofouling. However, due to their neg-
ative effect on the environment and on live organisms, these substances were completely
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
banned in 2008 by the international maritime organization [3]. Since then, some booster
biocides, such as Irgarol 1051 or Sea-nine 211, in combination with copper, have been
used; nevertheless, even these compounds have demonstrated toxicity on living organisms.
Mar. Drugs 2021, 19, 5. https://dx.doi.org/10.3390/md19010005
https://www.mdpi.com/journal/marinedrugs

Mar. Drugs 2021, 19, 5
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Therefore, it is imperative to find new antifouling (AF) compounds with environmentally
safe characteristics [4–6]. Several non-toxic marine natural products with AF activity have
been reported; among them, some flavonoids presented potential AF activity and low
toxicity, suggesting their potential as new lead compounds for the development of new AF
agents [7].
Previous works from our group reported some glycosylated flavones [
cones [ ] with potential AF activity. Interestingly, when comparing the anti-settlement
activity against Mytilus galloprovincialis of previously described chalcones, it seemed
that the presence of a polymethoxylated B-ring could be important for this activity [ ].
8] and chal-
9
9
Moreover, the introduction of a triazole moiety is associated with an increase in AF ac-
tivity [10]. In fact, over the last decade, there has been a great interest in the synthesis
of 1,2,3-triazoles due to the fact of these moieties behaved as more than passive linkers.
They carried favorable physicochemical properties, showing importance to biological ac-
tivity [11
,12]. This approach has been used to generate a vast array of compounds with
biological potential [13
–
16], namely with AF activity [10 17 18]. Moreover, some antimi-
, ,
crobial agents are based on nitrogen heterocycles, including the triazole-based biocides
fluconazole and itraconazole, which suggest their potential to act as AF agents [10].
Based on this, the present work aims to synthesize new potential AF polymethoxylated
chalcone and flavone derivatives with glycosyl groups incorporating a 1,2,3-triazole moiety
using a click chemistry approach. The potential of synthesized compounds as benign
AF agents was assessed against the adhesive larvae of the macrofouling mussel Mytilus
galloprovincialis and the biofilm-forming marine bacteria Cobetia marina, Vibrio harveyi,
Pseudoalteromonas atlantica, Halomonas aquamarina and Roseobacter litoralis. The most promis-
ing compounds were submitted to complementary assays to evaluate their viability as AF
agents, including the evaluation of possible mechanisms of action related with adhesion
and neurotransmission pathways. These compounds were also tested for anti-microalgal
activity towards Navicula sp. and general ecotoxicity using nauplii of the marine shrimp
Artemia salina.
2. Results and Discussion
2.1. Synthesis and Structure Elucidation
A series of four glycosylated flavones and four glycosylated chalcones bearing a 1,2,3-
triazole moiety was synthesized. To prepare glycosylated flavones (Scheme 1), flavones 1a
and 1b, used as building blocks, were synthesized by the Mentzer synthesis, through direct
thermal cyclocondensation of phloroglucinol and β-ketoesters, with good yields, as described
by Seijas et al. [19]. However, instead of a microwave (MW) irradiation, the synthesis
of flavones 1a and 1b was performed in a muffle furnace. After, the propargylation of
flavones 1a and 1b was achieved with propargyl bromide, giving rise to flavones 2a and
2b with 66% and 55% yield, respectively. Copper(I)-catalysed azide alkyne cycloaddition
(CuAAC), commonly referred as click chemistry, was developed by the Sharpless and
Meldal groups in 2002, and is the most useful reaction for the regioselective synthesis of
1,4-disubstituted-1,2,3-triazole ring [20
and an aliphatic azide using copper (I) as a catalyst in low-time and mild conditions,
with high yields and few by-products [20 22]. Therefore, the incorporation of the triazole
,21]. This involves a reaction of a terminal alkyne
–
linked glycosidic moiety in flavones 2a and 2b was accomplished by CuAAC under MW
irradiation, giving rise to flavones 3a, 3b, 4a and 4b with 49–82% yield (Scheme 1).

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Scheme 1. Synthesis of flavones 1a–1b, 2a–2b, 3a–3b and 4a–4b.
(i) 240 ◦C, 60–80 min, 74–77%; (ii) Cs2CO3,
tetrabutylammonium bromide (TBAB), acetone, 60 ◦C, 6 h, 55–66%; (iii) Sodium ascorbate, CuSO4.5H2O,
tetrahydrofuran (THF):water, microwave (MW), 30 min, 49–82%; (iv) NaN3, acetone:water, r.t., 3 h, 72%.
The first step in the synthetic process to obtain glycosylated chalcones (Scheme 2)
was the propargylation of 2,4-dihydroxyacetophenone with propargyl bromide. As for
the synthesis of flavones 2a
bromide, in the presence of anhydrous Cs2CO3 and tetrabutylammonium bromide (TBAB).
Nevertheless, in addition to the desired 4-O-monosubstituted acetophenone ( ),
–2b, firstly this reaction was accomplished with propargyl
5
the 2,4-disubstituted acetophenone was obtained. Therefore, this reaction was performed in
the presence of anhydrous K2CO3, as described by Zhao et al. [23], with slight modifications,
and the 4-O-monosubstituted acetophenone (5) was successfully obtained as expected,
with a 76% yield. Afterwards, the base-catalysed aldol reaction of this propargylated
acetophenone with benzaldehydes afforded chalcones 6a and 6b with moderate yields,
which were subsequently submitted to MW assisted CuAAC with azide sugar derivatives,
affording triazole linked glycosylated chalcones 7a, 7b, 8a and 8b with 45–65% yield.
In order to perform structure–activity relationship studies, structure related non-
glycosylated chalcones were also synthesized (Scheme 3). Firstly 2,4-dihydroxyacetophenone
was protected with methoxymethyl chloride affording
and 10b were prepared by base-catalyzed aldol reaction of
trimethoxybenzaldehyde with 33% and 47% yield, respectively, as described before [24
9
with 84% yield. Chalcones 10a
and 3,4-dimethoxy- and 3,4,5-
25],
9
,
with slight modifications. Chalcones 11a and 11b were obtained with moderate yields
by deprotection of methoxymethyl group at C-40 of intermediate chalcones 10a and 10b
,
as described by Loureiro et al. [26].
The 2,3,4,6-tetra-O-acetyl-
for the synthesis of glycosylated derivatives 3a
2,3,4,6-tetra-O-acetyl- -D-glucopyranosyl bromide and sodium azide, as described by
Adesoye et al. [27], with 72% yield.
β-D-glucopyranosyl azide (12), used as a building block
,
3b 7a and 7b, was synthesized from
,
α

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◦
Scheme 2. Synthesis of chalcones 6a–6b, 7a–7b and 8a–8b. (i) K2CO3, acetone, 60 C, 1 h, 76%; (ii) 40% NaOH, methanol,
microwave (MW), 3 h, 41–43%; (iii) Sodium ascorbate, CuSO4.5H2O, tetrahydrofuran (THF):water, microwave (MW), 1 h,
45–65%; (iv) NaN3, acetone:water, r.t., 3 h, 72%.
Scheme 3. Synthesis of chalcones 10a
microwave (MW), 4 h, 33–47%; (iii) p-Toluenesulfonic acid (PTSA), methanol, 50 C, 5 h, 24–31%.
–
10b and 11a
–
11b. (i) K2CO3, acetone, 60◦◦C, 1 h, 84%; (ii) 40% NaOH, methanol,
The newly synthesized compounds, 2a, 2b, 3a, 3b, 4a, 4b, 7a, 7b, 8a and 8b were
characterized by high resolution mass spectrometry (HRMS) and nuclear magnetic reso-
nance (NMR). The coupling constants of the vinylic system (JHα-Hβ = 15.5–15.3 Hz) confirm
the (E)-configuration for all synthesized chalcones. The NMR spectra of the newly syn-
thesized compounds 3a
flavone scaffold and chalcone precursors. Additionally, signals of a triazole ring (
8.61–7.73 s,
C200 144.1–141.6 and C300 125.2–121.5) and a glycosyl moiety were observed.
The position of the triazole ring on these compounds was evidenced by the correlation
,
3b
,
4a,
4b
,
7a
,
7b
,
8a and 8b showed characteristic signals for the
δ
H-300
δ
δ

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found in the heteronuclear multiple bond correlation (HMBC) spectra between the proton
signals of H-100 and the carbon signals of C-200 and C-300.
2.2. Mussel (Mytilus galloprovincialis) Larvae Anti-Settlement Activity
Mussels are one of the main macrofouling organisms present on ships and submerged
maritime structures worldwide; thus, they are a target species used in settlement inhibition
bioassays [28,29]. Due to the presence of a muscular sensory foot, mussel plantigrade
larvae are highly specialized in adhesion to the submerged surfaces and the fixation
is made through the production of byssal threads [30], which constitutes the endpoint
of this bioassay. Therefore, for the evaluation of the AF activity of the compounds to-
wards macrofouling species, the ability of the synthetized flavonoids to inhibit the settle-
ment of Mytilus galloprovincialis larvae at 50
in addition to glycosylated flavones 3a 3b
non-glycosylated flavones 1a and 2a
tested in order to perform SAR studies. Results showed that among 18 tested flavonoids
(10 chalcones and 8 flavones), seven chalcones (6a 6b 7b 8a 8b 11a and 11b) and only
three flavones (1b 4a and 4b) presented a percentage of settlement 40%,
µ
M was assessed. In this screening bioassay,
4a and 4b and chalcones 7a 7b 8a and 8b
and chalcones 6a 10a and 11a were
,
–
,
,
,
,
–
b
b
–
b,
–
b
–b
,
,
,
,
,
,
≤
suggesting that chalcone scaffold seems to be more promising for anti-settlement activity.
These 10 compounds were further selected for dose–response studies in order to determine
LC50/EC50 values.
Among these, three chalcones (7b
,
11a
, 11b) and one flavone (1b) revealed effective
anti-settlement activity (EC50 < 25 µg/mL), with triazolyl glycosylated chalcone 7b being
the most potent (EC50 = 3.28
(> 60.98) (Table 1).
µM; 2.43
µg
·
mL−1), showing the highest therapeutic ratio
Table 1. Antifouling (AF) effectiveness and toxicity parameters of flavones 1b
chalcones 6a, 6b, 7b, 8a, 8b, 11a and 11b towards mussel plantigrade larvae.
,
4a and 4b and
−1
Compound
EC50 (µM)
EC50 (µg·mL
2.87
)
LC50 (µM)
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
> 200
LC50/EC50
> 23.98
> 4.70
8.34 (95% CI:
4.2–13.36)
1b
4a
42.55 (95% CI:
34.90–52.80)
48.22 (95% CI:
30.57–85.40)
84.52 (95% CI:
45.07–267.02)
85.56 (95% CI:
44.84–291.41)
3.28 (95% CI:
1.97–4.74)
35.83 (95% CI:
19.22–74.74)
53.90 (95% CI:
29.98–126.88)
18.10 (95% CI:
13.95–23.44)
9.64 (95% CI:
3.85–17.22)
32.75
38.56
28.60
31.52
2.43
4b
6a
> 4.15
> 2.37
6b
7b
8a
> 2.34
> 60.98
> 5.58
27.07
42.35
5.44
8b
11a
11b
> 3.71
> 11.05
> 20.75
3.18
EC50: minimum concentration that inhibited 50% of larval settlement; LC50: median lethal dose;
LC50/EC50: therapeutic ratio; CI: confidence interval. EC50 are recommend to be less than 25
and therapeutic ratio higher than 15 for effective AF agents [31].
µg/mL
2.3. Quantitative Structure—Activity Relationship
Quantitative structure–activity relationship (QSAR) studies have been used for several
years to point out small molecules’ properties that are relevant for activity, and to forecast
the activity of new compounds [32]. Therefore, a QSAR model was built to highlight the

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descriptors that are being relevant for anti-settlement activity against M. galloprovincialis
plantigrades of the tested flavonoids. In this work, a 2D-QSAR model was elaborated using
the Comprehensive Descriptors for Structural and Statistical Analysis (CODESSA 2.7.2)
software package, which calculates approximately 500 descriptors. The heuristic method
performs a pre-selection of descriptors by eliminating descriptors that are not available
for each structure, that have a small variation in magnitude, that are correlated pairwise,
and that have no statistical significance. The heuristic method is a very useful method for
searching the best set of descriptors, without restrictions on the data set size [33].
The correlation coefficient (R2), squared standard error (S2), and Fisher’s value (F)
were used to evaluate the validity of regression equation [34]. As the rules of QSAR estab-
lish that there must be one descriptor for each five molecules used to build the model [34],
three descriptors were used to build the QSAR equation. The multilinear regression
analysis using Heuristic method for 15 compounds in the three-descriptor model is
shown in Figure 1. The compounds are uniformly distributed around the regression line
(Figure 1), which suggests that the obtained model has satisfactory predictive ability.
Figure 1. Quantitative structure-activity relationship (QSAR) model obtained with the heuristic method for 15 molecules
with the CODESSA software (R2 = 0.7945, F = 14.18, S2 = 0.0243). X,
X and t-test are the regression coefficient of the linear
∆
model, standard errors of the regression coefficient, and the t significance coefficient of the determination, respectively.
AF = antifouling activity.
The best QSAR equation had a R2 of 0.7945, Fisher value of 14.18, and S2 of 0.0243,
which reveals that the proposed model has statistical validity [35]. The R2 is higher than 0.6,
which is an indicator of a good fit to the regression line [36], representing close to 80% of the
total variance in AF activity shown by the test compounds. The QSAR model is significant
at a 95% level, as shown by the Fisher F-test (F = 14.18), which is higher than the tabulated
value (3.59), as desired for a statistically significant model [35]. The squared standard
deviation S2 is small and close to zero (s2 = 0.0243), proving that the model is significant
and has low variation about the regression line [37]. The reliability of the resulting QSAR
model was explored using two different types of validation criteria: external validation
by using a test set and internal validation by leave-one-out (LOO) cross-validation [38].

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The model was able to predict the activity of an external test set with an average difference
of 0.19 from the experimental value [39]. Moreover, the cross-validated R2 (Q2 = 0.5953)
from the LOO internal validation process is higher than 0.5 and smaller than the overall R2,
as expected, and the difference between R2 and Q2 is lower than 0.3, which indicates that
the model does not suffer from overfitting [40].
By interpreting the molecular descriptors in the regression model (Figure 1), it is
possible to have some insight into structural characteristics that are likely to be responsible
for AF activity of the studied compounds. There are three descriptors included in the
regression model, which proved to be important features and provide statistically signifi-
cant contributions to the QSAR equation.
As indicated by the higher t-test value, hydrogen bonding acceptor ability of the
molecule (HACA1) is a charged partial surface area (CPSA) descriptor that appeared as the
most significant descriptor for the obtained QSAR model, contributing positively to the AF
activity [41]. HACA1 is determined by the equation:
HACA1 =
SA A ∈ XH−acceptor
(1)
∑
A
where SA stands for solvent-accessible surface area of H-bonding acceptor atoms, selected
by threshold charge. This descriptor proves the importance of the hydrogen bonding
acceptor properties for the activity of the test compounds [42].
The topological descriptor average complementary information content of order 2
(CIC2) descriptor is predicted as being negatively implied in the AF activity of the test
compounds [41]. The CIC2 descriptor represents the difference between the maximum
possible complexity of a molecule and its real topological information. It belongs to
the multi-graph information content indices and it describes neighborhood symmetry of
second order [43]. The constitutional descriptor number of triple bonds is also responsible
for a decrease in activity.
The molecular descriptors used in the QSAR model demonstrate that the mechanism
underlying the AF activity of flavonoids is mainly related to their HACA1, and it may
be prejudiced by topological CIC2 and by the presence of triple bonds. Interestingly,
the triazolyl glycosylated chalcone 7b, with the most promising anti-settlement activity,
is one of the compounds with more hydrogen-bonding acceptors. In contrast,
propargylated flavones 2a and 2b had a percentage of settlement higher than 40% at
50 µM, and therefore were not selected for dose–response studies and for the determina-
tion of the LC50/EC50 values. Moreover, propargylated chalcones 6a and 6b showed the
lowest activity. Overall, the examination of the molecular descriptors reported in this work
can lead to a better understanding of the relation between the structure and AF activity
of flavonoids.
2.4. Biofilm-Forming Marine Bacteria Growth Inhibitory Activity
Although the macrofouling species represent the most problematic component of
fouling in terms of biomass and negative repercussions, the first micro-colonizers are
also of extreme importance, since they represent the basis of the fouling community,
and ultimately, they may modulate the colonization of further species by inducing or
inhibiting species adhesion via biochemical cues [44]. Thus, synthesized flavonoids were
further evaluated for their ability to inhibit the growth of five marine biofilm-forming
bacteria, Vibrio harveyi, Cobetia marina, Halomonas aquamarina, Pseudoalteromonas atlantica
and Roseobacter litoralis.
Results showed that only the bacterial growth of Roseobacter litoralis was meaningfully
compromised by tested compounds, with significant inhibitory activity for propargylated
chalcones 6a and 6b (Figure 2). These compounds were selected for concentration–response
analysis (Figure 3).

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Figure 2. Bacterial growth inhibition screening of flavonoid derivatives (15
µM) towards five biofilm-forming ma-
rine bacteria: Vibrio harveyi, Cobetia marina, Halomonas aquamarina, Pseudoalteromonas atlantica and Roseobacter litoralis.
B: Negative control with 1% dimethyl sulfoxide (DMSO); C: positive control with penicillin–streptomycin–neomycin-
stabilized solution. * indicates significant differences at p < 0.05 (Dunnett test), against the negative control (B).
Figure 3. Concentration–response growth inhibition activity of compounds 6a and 6b towards Roseobacter litoralis.
B: DMSO negative control; C: positive control with penicillin–streptomycin–neomycin-stabilized solution.
Compounds 6a and 6b presented low anti-bacterial activity towards R. litoralis with
EC30 values of 135 and 83.5 µM, respectively.
2.5. Biofilm—Forming Marine Diatoms Growth Inhibitory Activity
The most promising compounds regarding anti-settlement activity (1b
, 7b, 11a, 11b)
were further evaluated for their ability to inhibit the growth of the biofilm-forming

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microalgae Navicula sp. This marine diatom is a major biofouling species that very effec-
tively colonizes submerged surfaces by secreting adhesive extracellular polymer substances
(EPS), and thus is a good representative of fouling microalgae.
Only triazolyl glycosylated chalcone 7b showed significant inhibitory activity with
the concentration–response analyses revealing an EC50 value of 41.76
suggesting the ability of this compound to act also as a promising AF agent against
microfouling species.
µ
M; 30.94
µg ,
mL−1
·
2.6. In Vitro Acetylcholinesterase (AChE) and Tyrosinase (Tyr) Activities
The identification of the mechanism of action associated with AF activity remains
a challenge for the scientific community. According to Qian et al. (2013) antifoulants
appear to affect settlement through distinct pathways, which can be classified roughly into
several categories such as inhibitors of ion channel function, inhibitors of quorum sensing,
blockers of neurotransmission or inhibitors of adhesive production or release [45].
Moreover, some specific target molecules in fouling organisms have been determined,
such as AChE, which seems to be involved in cholinergic neural signaling during the
settlement [46]. It is known that the commercial booster biocide Sea-Nine 211 acts by
this mechanism [47
isms, territrem A and pulmonarin [49
ing compounds to modulate the activity of AChE was evaluated (1b
,
48], as well as two natural compounds isolated from marine organ-
50]. For this reason, the ability of the most promis-
7b 11a and 11b).
,
,
,
AChE activity was significantly induced for chalcones 7b and 11b (Figure 4). Induced AChE
activity has been described as an exposure effect that is in some cases associated with apop-
tosis [51], and thus the specific target behind these compounds’ bioactivity should be
further explored in future work.
Figure 4. AChE activity of the most promising compounds 1b 7b
, 11a and 11b. B: Dimethyl sulfoxide
(DMSO) (1% in water). C: Eserine (200 M, water). * indicates significant differences at p < 0.05
µ
(Dunnett test), against the negative control (B).
A well-known pathway in the production of biological adhesives of mussels is the
3,4-dihydroxyphenyl-L-alanine (L-DOPA) metabolism that functions in the production of
DOPA-containing mussel byssal plaques by the action of Tyr that catalyses the conversion
of DOPA precursor into DOPA residues [46,52]. Considering this, the most promising
compounds in the inhibition of mussel adhesion were tested for their ability to inhibit Tyr
(Figure 5). Results show that flavone 1b is able to significantly decrease Tyr activity at all
the concentrations tested, reaching 23.5% of inhibition at 100 µM. Therefore, the inhibition
of this enzyme, with a crucial effect in the formation of mussel adhesive, could be one of
the mechanisms involved in the inhibition of the mussel settlement. This also highlights
a specific AF mode of action related with mussel adhesion and explains the absence of
activity against bacteria and diatoms.

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Figure 5. Tyr activity of the most promising compounds 1b, 7b
,
11a and 11b. B: Dimethyl sulfoxide
(DMSO) (1% in water). C: Kojic acid (1.4 mM, water). * indicates significant differences at p < 0.05
(Dunnett test), against the negative control (B).
2.7. Environmental Fate Parameters: Artemia Salina Ecotoxicity Bioassay
Ecotoxicity assays carried out on non-target organisms aim to understand how tested
compounds can affect sensitive non-target organisms and influence the health status of
the surrounding ecosystem [53]. Artemia salina is a species of small crustaceans that live in
salty marine environments and are used as test organisms because of their easy culture,
short generation time, cosmopolitan distribution and commercial availability of their eggs
in latent form [54].
Ecotoxicity results showed that the most promising compounds 1b
are non-toxic to Artemia salina (less than 10% mortality) at both concentrations tested
(25 and 50
M) (Figure 6), in contrast to the commercial AF agent ECONEA® which was
previously shown by our group to cause 100% lethality at the same concentrations and
conditions [ ]. These results suggest that any of the tested compounds could be a good
, 7b, 11a and 11b
µ
9
alternative, being more environmentally compatible antifoulants.
Figure 6. Mortality rate of Artemia salina nauplii after 48 h of exposure to compounds 1b
11b, B: DMSO (1% in filtered seawater). C: K2Cr2O7 (13.6 µM, filtered seawater).
, 7b, 11a and

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3. Materials and Methods
3.1. Synthesis and Structure Elucidation of Chalcones and Flavones
MW reactions were performed using a glassware setup for atmospheric pressure
reactions and a 100 mL Teflon reactor (internal reaction temperature measurements with a
fiber-optic probe sensor) and were carried out using an Ethos MicroSYNTH 1600 Microwave
Labstation from Milestone (Thermo Unicam, Oeiras, Portugal). The reactions were moni-
tored by analytical thin-layer chromatography (TLC) Macherey-Nagel Silica gel 60 F254
(Macherey-Nagel, Dueren, Germany), Purifications of compounds were carried out by
flash chromatography using Macherey-Nagel silica gel 60 (0.04–0.063 mm) (Macherey-
Nagel, Dueren, Germany), preparative TLC using Macherey-Nagel silica gel 60 (GF254)
(Macherey-Nagel, Dueren, Germany) plates and crystallization. Melting points were ob-
tained in a Köfler microscope (Wagner and Munz, Munich, Germany) and are uncor-
1
rected. H and 13C NMR spectra were taken in CDCl3 or DMSO-d6 at room temperature,
on Bruker Avance 300 and 500 instruments (Bruker Biosciences Corporation, Billerica, MA,
USA) (300.13 MHz or 500 MHz for 1H and 75.47 or 120 MHz for 13C). Chemical shifts are
expressed in δ (ppm) values relative to tetramethylsilane (TMS) 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 spectra of com-
pounds 2a, 2b, 3a, 3b, 4a, 4b, 7a, 7b and 8b were performed on an APEXQe FT-ICR
MS (Bruker Daltonics, Billerica, MA) equipped with a 7T actively shielded magnet, at
C.A.C.T.I.—University of Vigo, Spain. Ions were generated using a Combi MALDI-
electrospray ionization (ESI) source. HRMS mass spectrometry of compound 8a was
performed on an LTQ OrbitrapTM XL hybrid mass spectrometer (Thermo Fischer Scientific,
Bremen, Germany) controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0. at CEMUP—
University of Porto, Portugal. Phloroglucinol, ethyl 3,4-dimethoxybenzoylacetate,
ethyl 3,4,5-trimethoxybenzoylacetate, 2,4-dihydroxyacetophenone and 2,3,4,6-tetra-O-acetyl-
α
-D-glucopyranosyl bromide were purchased from Sigma Aldrich (St. Louis, MO, USA).
3,4-Dimethoxybenzaldehyde and 3,4,5-trimethoxybenzaldehyde were purchased from
Acros Organics (Janssen Pharmaceuticalaan, Geel, Belgium). 2-Azidoethyl-2,3,4,6-tetra-O-
acetyl-β-D-glucopyranoside was purchased from Synthose (Concord, ON, Canada).
3.1.1. Synthesis of Flavones 1a and 1b
A mixture of phloroglucinol (0.175 g, 1.39 mmol) and ethyl 3,4-dimethoxybenzoylacetate
(0.700 g, 2.78 mmol) or ethyl 3,4,5-trimethoxybenzoylacetate (0.739 g, 2.78 mmol) was
heated at 240 ◦C in muffle furnace (Thermo Fisher Scientific, Oeiras, Portugal) for 60–100
min. Afterwards, the crude mixture was dissolved in 10% NaOH (20 mL) and washed
with diethyl ether (2
×
20 mL), and the product was precipitated by adding 37% HCl.
The solid was filtered and washed with water, and the flavones 1a and 1b were obtained
with 74% and 77% yields, respectively. The structure elucidation of compounds 1a and
2b was established by H and 13C NMR techniques and data were in accordance with
1
previously reported results [19].
3.1.2. Synthesis of 7-O-Propargylflavones 2a and 2b
To a solution of 1a (0.200 g, 0.64 mmol) or 1b (0.200 g, 0.58 mmol), cesium carbonate
(0.207 g, 0.64 mmol or 0.189 g, 0.58 mmol), tetrabutylammonium bromide (TBAB) (0.205 g,
0.64 mmol or 0.187 g, 0.58 mmol) in anhydrous acetone (20 mL), and propargyl bromide
solution, 80 wt.% in toluene (0.071 mL, 0.64 mmol or 0.065 mL, 0.58 mmol), were added.
The mixture was refluxed at 60 ◦C during 6 h and filtered. The filtrate was evaporated
under reduced pressure and purification was carried out by flash column chromatography
(SiO2; n-hexane: ethyl acetate), 8:2) followed by crystallization in acetone.
2-(3,4-dimethoxyphenyl)-5-hydroxy-7-(prop-2-yn-1-yloxy)-4H-chromen-4-one (2a). Light yel-
◦
1
low solid; Yield: 66%; m.p.: 222–224 C (acetone); H NMR (DMSO-d6, 500 MHz), δ:
12.93 (1H, s, OH-5), 7.71 (1H, dd, J = 8.5 and 2.2 Hz, H-60), 7.57 (1H, d, J = 2.2 Hz, H-20),
7.14 (1H, d, J = 8.7 Hz, H-50), 7.03 (1H, s, H-3), 6.87 (1H, d, J = 2.2 Hz, H-8), 6.44 (1H, d,

Mar. Drugs 2021, 19, 5
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J = 2.3 Hz, H-6), 4.94 (2H, d, J = 2.5 Hz, H-100), 3.88 (3H, s, 30-OCH3), 3.85 (3H, s, 40-
OCH3), 3.66 (1H, t, J = 2.4 Hz, H-300). 13C NMR (DMSO-d6, 120 MHz)
δ: 182.3 (C4), 164.0
(C2), 163.2 (C7), 161.3 (C5), 157.3 (C8a), 152.5 (C40), 149.2 (C30), 122.9 (C10), 120.5 (C60),
111.9 (C50), 109.6 (C20), 105.3 (C4a), 104.3 (C3), 98.8 (C6), 94.0 (C8), 79.2 (C300), 78.6 (C200),
56.4 (C100), 56.1, 56.0 (30-OCH3 and 40-OCH3). HRMS (ESI+) m/z calcd for C20H17O6
[M + H+] 353.10196, found 353.10174.
5-hydroxy-7-(prop-2-yn-1-yloxy)-2-(3,4,5-trimethoxyphenyl)-4H-chromen-4-one (2b).
Light yellow solid; Yield: 55%; m.p.: 211–213 C (acetone); 1H NMR (DMSO-d6, 300.13 MHz),
◦
δ
: 12.87 (1H, s, OH-5), 7.36 (2H, s, H-20 and H-60), 7.16 (1H, s, H-3), 6.91 (1H, d,
J = 2.3 Hz, H-8), 6.46 (1H, d, J = 2.3 Hz, H-6), 4.96 (2H, d, J = 2.4 Hz, H-100), 3.91 (6H, s, 30-
OCH3 and 50-OCH3), 3.76 (3H, s, 40- OCH3), 3.70 (1H, t, J = 2.4 Hz, H-300).
13C NMR (DMSO-d6, 75.47 MHz)
δ: 182.2 (C4), 163.4 (C2), 163.1 (C7), 161.2 (C5), 157.1 (C8a),
153.3 (C30 and C50), 140.9 (C40), 125.8 (C10), 105.3 (C3), 105.2 (C4a), 104.2 (C20 and C60),
00
00
0
00
0
0
98.7 (C6), 93.9 (C8), 79.1 (C2 ), 78.4 (C3 ), 60.3 (4 -OCH3), 56.4 (C1 , 3 -OCH3 and 5 -OCH3).
HRMS (ESI+) m/z calcd for C21H19O7 [M + H+] 383.11253, found 383.11233.
3.1.3. Synthesis of Flavone-Triazolyl-Glycosides 3a and 3b
To a solution of 2a (0.100 g, 0.28 mmol) or 2b (0.100 g, 0.27 mmol) and 2,3,4,6-
tetra-O-acetyl-β-D-glucopyranosyl azide (0.106 g, 0.28 mmol or 0.102 g, 0.27 mmol)
in THF/water solvent mixture (2:1; 30 mL), sodium ascorbate (0.225 g, 1.14 mmol or 0.216 g,
1.09 mmol) and copper(II) sulphate pentahydrate (0.142 g, 0.57 mmol or 0.136 g, 0.54 mmol)
were added. The reaction vessel was sealed and the mixture was kept stirring and heated
◦
for 30 min at 70 C under MW irradiation of 500 W. After cooling, the reaction mixture was
filtered and concentrated under reduced pressure. The water suspension was extracted
with ethyl acetate (2
×
20 mL), and the combined organic layers were dried over anhydrous
sodium sulphate, evaporated under reduced pressure, and then purified by crystallization
in acetone.
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((2-(3,4-dimethoxyphenyl)-5-hydroxy-4-oxo-4H-
chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)tetrah◦ydro-2H-pyran-3,4,5-triyl triacetate
1
(
3a). Light yellow solid; Yield: 82%; m.p.: 143–145 C (acetone); H NMR (DMSO-d6,
500 MHz), δ
: 12.93 (1H, s, OH-5), 8.61 (1H, s, H-300), 7.71 (1H, dd, J = 8.5 and 2.1 Hz, H-60),
7.60 (1H, d, J = 2.2 Hz, H-20), 7.15 (1H, d, J = 8.7 Hz, H-50), 7.05 (1H, s, H-0300), 6.94 (1H, d,
J = 2.2 Hz, H-8), 6.47 (1H, d, J = 2.2 Hz, H-6), 6.39 (1H, d, J = 9.2 Hz, H-1 ), 5.68 (1H, t,
J = 9.4 Hz), 5.56 (1H, t, J = 9.5 Hz), 5.19 (1H, t, J = 9.8 Hz) (H-2000, H-3000, H-4000), 5.32 (2H, s,
H-100), 4.39–4.36 (1H, m, H-5000), 4.15–4.07 (2H, m, H-6000), 3.86 (3H, s, 40-OCH3), 3.83 (3H,
s, 30-OCH3), 2.03, 1.99, 1.96, 1.77 (12H, s, 2000, 3000, 4000, -COCH3). 13C NMR (DMSO-d6,
120 MHz) δ COCH3), 163.7 (C2 and
: 182.1 (C4), 170.1, 169.6, 169.4, 168.5 (2000, 3000, 4000, 6000-
C7), 161.2 (C5), 158.0 (C8a), 152.3 (C40), 149.1 (C30), 142.7 (C200), 124.0 (C300), 122.7 (C10),
120.2 (C60), 111.7 (C50), 109.5 (C20), 105.0 (C3), 104.1 (C4a), 98.7 (C6), 93.7 (C8), 83.9 (C1000),
73.3 (C5000), 72.1, 70.1, 67.5 (C2000, C3000, C4000), 61.8 (C6000), 61.7 (C100), 55.9, 55.8 (30-OCH3
and 40-OCH3), 20.5, 20.4, 20.3, 19.9 (2000, 3000, 4000, 6000-CO
C34H36N3O15 [M + H+] 726.21409, found 726.21380.
C
H3). HRMS (ESI+) m/z calcd for
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-(((5-hydroxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-
chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-◦1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
(
3b). Yellow solid; Yield: 79%; m.p.: 169–171 C (acetone); 1H NMR (DMSO-d6, 300.13 MHz),
δ
: 12.85 (1H, s, OH-5), 8.61 (1H, s, H-300), 7.38 (2H, s, H-20 and H-60), 7.17 (1H, s, H-3),
7.00 (1H, d, J = 2.2 Hz, H-8), 6.49 (1H, d, J = 2.2 Hz, H-6), 6.39 (1H, d, J = 9.1 Hz, H-1000),
5.68 (1H, t, J = 9.3 Hz), 5.56 (1H, t, J = 9.4 Hz), 5.19 (1H, t, J = 9.7 Hz) (H-2000, H-3000, H-4000),
5.33 (2H, s, H-100), 4.40–4.36 (1H, m, H-5000), 4.17–4.06 (2H, m, H-6000), 3.91 (6H, s, 30-OCH3
and 50-OCH3), 3.76 (3H, s, 40-OCH3), 2.03, 2.00, 1.96, 1.77 (12H, s, 2000, 3000, 4000, 6000-COCH3).
13C NMR (DMSO-d6, 75.47 MHz)
δ
: 182.2 (C4), 170.1, 169.6, 169.4, 168.5 (2000, 3000, 4000,
000
0
0
00
6 -
COCH3), 163.8 (C7), 163.4 (C2), 161.2 (C5), 157.3 (C8a), 153.3 (C3 and C5 ), 142.7 (C2 ),
140.9 (C40), 125.8 (C10), 123.9 (C300), 105.1 (C3 and C4a), 104.2 (C20 and C60), 98.7 (C6),

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93.9 (C8), 83.9 (C1000),073.3 (C5000), 702.1, 70.1, 67.5 (C2000, C3000, C4000), 61.8 (C100 and C6000),
60.3 (4 -OCH3), 56.3 (3 -OCH3 and 5 -OCH3), 20.5, 20.4, 20.3, 19.9 (2 , 3 , 4 , 6 -COCH3).
0
000 000 000 000
HRMS (ESI+) m/z calcd for C35H38N3O16 [M + H+] 756.22466, found 756.22445.
3.1.4. Synthesis of Flavone-Triazolyl-Glycosides 4a and 4b
To a solution of 2a (0.090 g, 0.26 mmol) or 2b (0.100 g, 0.26 mmol) and 2-azidoethyl-
2,3,4,6-tetra-O-acetyl- -D-glucopyranoside (0.109 g, 0.26 mmol) in tetrahydrofuran/water
β
solvent mixture (2:1; 30 mL), sodium ascorbate (0.207 g, 1.05 mmol) and copper(II) sulphate
pentahydrate (0.131 g, 0.52 mmol) were added. The reaction vessel was sealed and the
mixture was kept stirring and heated for 30 min at 70 ◦C under MW irradiation of 500 W.
After cooling, the reaction mixture was filtered and concentrated under reduced pressure.
The water suspension was extracted with ethyl acetate (2
×
20 mL), and the combined
organic layers were dried over anhydrous sodium sulphate, evaporated under reduced
pressure, and then purified by crystallization in ethyl acetate/n-hexane (4a) or by flash
column chromatography (SiO2; n-hexane: ethyl acetate, 3:7) (4b).
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(4-(((2-(3,4-dimethoxyphenyl)-5-hydroxy-4-oxo-
4H-chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl
triacetate (4a). Light brown solid; Yield: 60%; m.p.: 99–100 ◦C (n-hexane: ethyl acetate);
1H NMR (DMSO-d6, 300.13 MHz),
δ
: 12.94 (1H, s, OH-5), 8.15 (1H, s, H-300),07.72 (1H, dd,
J = 8.5, 1.8 Hz, H-60), 7.59 (1H, d, J = 1.9 Hz, H-20), 7.14 (1H, d, J = 8.6 Hz, H-500), 7.05 (1H, s,
H-3), 6.96 (1H, d, J = 2.1 Hz, H-8), 6.47 (1H, d, J = 2.1 Hz, H-6), 5.28 (2H, s, H-1 ), 5.23 (1H, t,
J = 9.6 Hz), 4.90 (1H, t, J = 9.7 Hz), 4.77–4.71 (1H, m) (H-20000, H-30000, H-40000), 4.72 (1H, d,
J = 8.1 Hz, H-10000), 4.60–4.57 (2H, m, H-60000), 4.19–4.01 (5H, m, H-1000, H-2000, H-50000),
3.89 (3H, s, 30-OCH3), 3.86 (3H, s, 40-OCH3), 2.02, 1.98, 1.92, 1.89 (12H, s, 20000, 30000
,
40000, 60000-COCH3). 13C NMR (DMSO-d6, 75.47 MHz)
δ: 182.1 (C4), 170.1, 169.6, 169.3,
169.0 (20000, 30000, 40000, 60000
-COCH3), 163.9 (C7), 163.7 (C2), 161.2 (C5), 157.2 (C8a), 152.3
(C40), 149.0 (C30), 141.7 (C200), 125.2 (C300), 122.8 (C10), 120.2 (C60), 111.7 (C50), 109.5 (C20),
105.0 (C4a), 104.1 (C3), 99.2 (C10000), 98.6 (C6), 93.5 (C8), 071.9, 70.7, 68.1 (C20000, C30000, C40000),
000
000
0000
00
0
0000
70.6, 67.4, 61.7 (C1 , C2 , C5 ), 61.9 (C1 ), 55.9, 55.8 (3 -OCH3 and 4 -OCH3), 49.4 (C6 ),
20.5, 20.4, 20.3, 20.3 (20000, 30000, 40000, 60000-CO
C
H3). HRMS (ESI+) m/z calcd for C36H40N3O16
[M + H+] 770.24031, found 770. 23792.
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(4-(((5-hydroxy-4-oxo-2-(3,4,5-trimethoxyphenyl)-4H-
chromen-7-yl)oxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl tri-
◦
acetate (4b). Light yellow solid; Yield: 49%; m.p.: 96–98 C (n-hexane: ethyl acetate);
1H NMR (DMSO-d6, 300.13 MHz),
δ
: 12.86 (1H, s, OH-5), 8.15 (1H, s, H-300), 7.38 (2H, s,
H-20 and H-60), 7.17 (1H, s, H-3), 7.02 (1H, d, J = 2.2 Hz, H-8), 6.49 (1H, d, J = 2.2 Hz, H-6),
5.29 (2H, s, H-100), 5.23 (1H, t, J = 9.5 Hz), 4.90 (1H, t, J = 9.7 Hz), 4.74 (1H, t, J = 8.8 Hz)
(H-20000, H-30000, H-40000), 4.84 (1H, d, J = 8.0, H-10000), 4.60–4.56 (2H, m, H-60000), 4.21–4.01 (5H,
m, H-50000, H-1000 and H-2000), 3.91 (6H, s, 30-OCH3 and 50-OCH3), 3.76 (3H, s, 40-OCH3),
2.02, 1.98, 1.92, 1.89 (12H, s, 20000, 30000, 40000, 60000-COCH3). 13C NMR (DMSO-d6, 75.47 MHz)
δ
: 182.2 (C4), 170.1, 169.6, 169.3, 169.0 (20000, 30000, 400000,0 60000
-COCH3), 164.0 (C7), 163.4 (C2),
0
0
0
0
00
161.1 (C5), 157.3 (C8a), 153.3 (C3 and C5 ), 141.6 (C2 ), 140.9 (C4 ), 125.8 (C1 ), 125.2 (C3 ),
105.3 (C4000a0), 105.1 (C0300)0, 104.2 (C20 and C60), 99.2 (C1000000)0, 98.7 (C6), 93.7 (C80), 71.9, 70.7, 70.6,
0000
0000
000
00
0
68.1 (C2 , C3 , C4 and C5 ), 67.4, 61.7 (C1 , C2 ), 62.0 (C1 ), 60.3 (4 -OCH3), 56.4 (3
and 50-OCH3), 49.4 (C60000), 20.5, 20.4, 20.3, 20.3 (20000, 30000, 40000, 60000-CO H3). HRMS (ESI+)
C
m/z calcd for C37H42N3O17 [M + H+] 800.25087, found 800.24829.
3.1.5. Synthesis of Propargyloxyacetophenone 5
To a solution of 2,4-dihydroxyacetophenone (1.00 g, 6.57 mmol) and potassium carbon-
ate (0.91 g, 6.57 mmol) in anhydrous acetone (20 mL), propargyl bromide solution, 80 wt.%
in toluene (0.73 mL, 6.57 mmol) was added. The mixture was refluxed at 60 ◦C during 3 h.
Then, the reaction mixture was filtered, evaporated under reduced pressure and purified
by flash column chromatography (SiO2; n-hexane: ethyl acetate), 9:1), giving rise to
5 with

Mar. Drugs 2021, 19, 5
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76% yield. The structure elucidation of compound
5
was established by 1H and 13C NMR
techniques and data were in accordance with previously reported results [23].
3.1.6. Synthesis of Propargyloxychalcones 6a and 6b
To a solution of 5 (0.350 g, 1.84 mmol) in methanol (20 mL) was added a solution of 40%
NaOH in methanol, until pH 14, under stirring. Afterwards, 3,4-dimethoxybenzaldehyde
(0.612 g, 3.68 mmol) or 3,4,5-trimethoxybenzaldehyde (0.772 g, 3.68 mmol) was slowly
added to the reaction mixture. The reaction was submitted to MW irradiation at 180 W at
70 ◦C for 4 h. After, a solution of 10% HCl was added until pH 5, and the obtained solid
was filtered, washed with water, and purified by crystallization with methanol, giving rise
to chalcone 6a and 6b with 41% and 43% yield, respectively. The structure elucidation of
compounds 6a and 6b was established by 1H and 13C NMR techniques and data were in
accordance with previously reported results [23].
3.1.7. Synthesis of Chalcone-Triazolyl-Glycosides 7a and 7b
To a solution of 6a (0.140 g, 0.41 mmol) or 6b (0.200 g, 0.54 mmol) and 2,3,4,6-tetra-
O-acetyl-β-D-glucopyranosyl azide (0.309 g, 0.82 mmol or 0.405 g, 1.09 mmol) in tetrahy-
drofuran (THF)/water solvent mixture (2:1; 30 mL), sodium ascorbate (0.328 g, 1.66 mmol
or 0.430 g, 2.17 mmol) and copper(II) sulphate pentahydrate (0.207 g, 0.83 mmol or 0.271
g, 1.09 mmol) were added. The reaction mixture was heated for 1 h at 70 ◦C under MW
irradiation of 250 W with agitation. After cooling, the reaction mixture was filtered and
concentrated under reduced pressure. The water suspension was extracted with ethyl
acetate (2
×
20 mL) and the combined organic layers were washed with water (1
×
20 mL),
dried over anhydrous sodium sulphate, evaporated under reduced pressure, and then
purified by crystallization with methanol.
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((4-(€-3-(3,4-dimethoxyphenyl)acryloyl)-3- hydrox-
yphenoxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7a).
◦
1
Yellow solid; yield: 51%; m.p.: 169–171 C (methanol); H NMR (CDCl3, 300.13 MHz),
: 13.49 (1H, s, OH-20), 7.89 (1H, s, H-300), 7.86 (1H, d, J = 9.3 Hz, H-60), 7.84 (1H, d,
δ
J = 15.5 Hz, H-β), 7.43 (1H, d, J = 15.5 Hz, H-α), 7.26–7.22 (1H, m, H-6), 7.16 (1H, d,
J= 1.9 Hz, H-2), 6.90 (1H, d, J= 8.3 Hz, H-5), 6.58–6.55 (2H, m, H-30 and H-50), 5.90 (1H,
d, J= 9.2 Hz, H-1000), 5.48–5.38 (2H, m), 5.27–5.18 (1H, m) (H-2000, H-3000,H-4000), 5.24 (2H, s,
H-100), 4.33–4.11 (2H, m, H-6000), 4.04–3.98 (1H, m, H-5000), 3.96 (3H, s, 3-OCH3), 3.93 (3H, s,
4-OCH3), 2.07, 2.06, 2.02, 1.86 (12H, s, 2000, 3000, 4000, 6000-COCH3). 13C NMR (CDCl3,
75.47 MHz) δ COCH3), 166.6
: 192.0 (CO), 170.6, 170.0, 169.5, 169.1 (2000, 3000, 4000, 6000-
(C20), 164.5 (C40), 10501.8 (C3), 149.4 (C4) 145.0 (C
β
), 144.1 (C200), 131.4 (C600), 127.8 (C1),
0
0
123.6 (C6), 121.5 (C3 ), 118.0 (C
α
), 114.7 (C1 ), 111.3 (C5), 110.3 (C2), 107.9 (C5 ), 102.3 (C3 ),
000
000
000
000
000
00
000
85.9 (C1 ), 75.3 (C5 ), 72.7, 70.4, 67.8 (C2 , C3 , C4 ), 62.0 (C1 ), 61.6 (C6 ), 56.2 (3-OCH3),
56.1 (4-OCH3), 20.8, 20.7, 20.6, 20.3 (2000, 3000, 4000, 6000-CO
C
H3). HRMS (ESI+) m/z calcd for
C34H38N3O14 [M + H+] 712.23483, found 712.23337.
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(4-((3-hydroxy-4-((E)-3-(3,4,5-trimethoxyphenyl)
acryloyl)phenoxy)methyl)-1H-1,2,3-triazol-◦1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate
(
δ
7b). Yellow solid; yield: 57%; m.p.: 127–128 C (quamarnol); 1H NMR (CDCl3, 300.13 MHz),
: 13.41 (1H, s, OH-20), 7.89 (1H, s, H-300), 7.87 (1H, d, J = 8.8 Hz, H-60), 7.81 (1H, d,
J = 15.4 Hz, H-
β
), 7.45 (1H, d, J = 15.4 Hz, H-
α
), 6.87 (2H, s, H-2 and H-6), 6.60–6.56 (2H, m,
0
0
000
000
000
H-3 and H-5 ), 5.91–5.88 (1H, m, H-1 ), 5.48–5.39 (2H, m), 5.27–5.21 (1H, m) (H-2 , H-3 ,
H-4000), 5.25 (2H, s, H-100), 4.34–4.13 (2H, m, H-6000), 4.03–4.00 (1H, m, H-5000), 3.93 (6H, s,
3-OCH3 and 5-OCH3), 3.91 (3H, s, 4-OCH3), 2.08, 2.07, 2.02, 1.87 (12H, s, 2000, 3000000, 4000000, 6000000-
COCH3). 13C NMR (CDCl3, 75.47 MHz)
δ: 191.9 (CO), 170.6, 170.0, 169.5, 169.1 (2 , 3 , 4 ,
6000-
131.5 (C60), 130.3 (C1), 121.5 (C300), 119.5 (C
102.3 (C30), 86.0 (C1000), 75.4 (C5000), 72.7, 70.4, 67.8 (C2000, C3000, C4000), 62.1 (C100), 61.6 (C6000),
C
OCH3), 166.6 (C20), 164.6 (C40), 153.7 (C3 and C5), 145.0 (C
β
), 144.1 (C200), 140.8 (C4),
α
), 114.7 (C10), 108.0 (C50), 105.9 (C2 and C6),

Mar. Drugs 2021, 19, 5
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61.2 (4-OCH3), 56.4 (3-OCH3 and 5-OCH3), 20.8, 20.7, 20.6, 20.3 (2000, 3000, 4000, 6000-CO
CH3).
HRMS (ESI+) m/z calcd for C35H40N3O15 [M + H+] 742.24539, found 742.24366.
3.1.8. Synthesis of Chalcone-Triazolyl-Glycosides 8a and 8b
To a solution of 6a (0.050 g, 0.15 mmol) or 6b (0.2500 g, 0.68 mmol) and 2-azidoethyl-
2,3,4,6-tetra-O-acetyl- -D-glucopyranoside (0.123 g, 0.30 mmol or 0.566 g, 1.36 mmol) in
β
THF/water solvent mixture (2:1; 30 mL), sodium ascorbate (0.117 g, 0.59 mmol or 0.538 g,
2.71 mmol) and copper(II) sulphate pentahydrate (0.074 g, 0.30 mmol or 0.339 g, 1.36 mmol)
were added. The reaction mixture was heated for 1 h at 70 ◦C under MW irradiation of
250 W with agitation. After cooling, the reaction mixture was filtered and the THF in the
filtrate was evaporated under reduced pressure. Then, the water suspension was extracted
with ethyl acetate (2
×
20 mL) and the combined organic layers were washed with water
(1 × 20 mL), dried over anhydrous sodium sulphate, concentrated under reduced pressure,
and then purified by preparative TLC (SiO2; n-hexane: ethyl acetate, 2:8) (8a) or flash
column chromatography (SiO2; n-hexane: ethyl acetate, 5:5) (8b).
(2R,3R,4S,5R,6R)-2-(acetoxymethyl)-6-(2-(4-((4-((E)-3-(3,4-dimethoxyphenyl)acryloyl)-3-
hydroxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl tri-
acetate (8a). Yellow solid; yield: 65%; m.p.: 77–79 ◦C (n-hexane: ethyl acetate); 1H NMR
(CDCl3, 300.13 MHz),
δ
: 13.49 (1H, s, OH-20), 7.87 (1H, d, J = 9.7 Hz, H-60), 7.85 (1H, d,
J = 15.4 Hz, H- α), 7.25 (1H, dd, J = 8.1,
β
), 7.73 (1H, s, H-300), 7.44 (1H, d, J = 15.4 Hz, H-
1.8, H-6), 7.16 (1H, d, J = 1.8, H-2), 6.91 (1H, d, J = 8.4, H-5), 6.60–6.56 (2H, m, H-30 and
H-50), 5.23 (2H, s, H-100), 5.18 (1H, t, J = 9.4), 5.06 (1H, t, J = 9.6), 5.03–4.97 (1H, m) (H-20000
H-30000, H-40000), 4.68–4.52 (2H, m, H-60000), 4.48 (1H, d, J = 7.9, H-10000), 4.27–4.21 (2H, m),
4.15–4.10 (1H, m), 3.90–3.81 (1H, m) (H-1000, H-2000), 3.96 (3H, s, 3-OCH3), 3.94 (3H, s,
,
4-OCH3), 3.72–3.66 (1H, m, H-50000), 2.08, 2.01, 1.99, 1.95 (12H, s, 20000, 30000, 40000
,
60000-COCH3). 13C NMR (CDCl3, 75.47 MHz)
δ
: 192.0 (CO), 170.7, 170.3, 169.6, 169.5
), 143.1
), 114.7 (C10), 111.3 (C5),
(20000, 30000, 40000, 60000 OCH3), 166.6 (C20), 164.6 (C40), 151.8 (C4), 149.4 (C3) 144.9 (C
-C β
(C200), 131.5 (C60), 127.9 (C1), 124.6 (C300), 010020 3.6 (C6), 118.1 (C
α
0
0
0000
0000
0000
110.3 (C2), 107.9 (C5 ), 102.3 (C3 ), 100.6 (C1 ), 72.1 (C5 ), 72.6, 71.1, 68.3 (C2 , C3 and
C40000), 67.8, 61.8 (C1000 and C2000), 62.1 (C100), 56.2 (30-OCH3), 56.1 (40-OCH3), 50.3 (C60000),
20.9, 20.7, 20.7, 20.7 (20000, 30000, 40000, 60000-CO
C
H3). HRMS (ESI+) m/z calcd for C36H42N3O15
[M + H+] 756.261044, found 756.26373.
(2R,3R,4S,5S,6S)-2-(acetoxymethyl)-6-(2-(4-((3-hydroxy-4-((E)-3-(3,4,5-trimethoxyphenyl)
acryloyl)phenoxy)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)tetrahydro-2H-pyran-3,4,5-triyl tri-
acetate (8b). Yellow solid; yield: 45%; m.p.: 78–81 ◦C (n-hexane: ethyl acetate); 1H NMR
(CDCl3, 300.13 MHz),
J = 15.4 Hz, H-
δ
: 13.41 (1H, s, OH-20), 7.87 (1H, d, J = 9.8 Hz, H-60), 7.81 (1H, d,
β
), 7.74 (1H, s, H-300), 7.46 (1H, d, J = 15.4 Hz, H-
α), 6.87 (2H, s, H-2 and
H-6), 6.61–6.57 (2H, m, H-30 and H-50), 5.24 (2H, s, H-100), 5.18 (1H, t, J= 9.4), 5.07 (1H, t,
J = 9.6), 4.99 (1H, t, J = 8.7) (H-20000, H-30000, H-40000), 4.68–4.52 (2H, m, H-60000), 4.48 (1H, d,
0000
000
000
J = 7.9, H-1 ), 4.28–4.21 (2H, m), 4.13–4.08 (2H, m) (H-1 and H-2 ), 3.93 (3H, s, 4-OCH3),
30.09001 (600H00 , s0,0003-O00C00 H3 and 5-OCH3), 3.72–3.66 (1H, m, H-50000), 2.04, 2.02, 1.99, 1.95 (12H, s,
2
, 3 , 4 , 6 -COCH3). 13C NMR (CDCl3, 75.47 MHz)
δ: 191.9 (CO), 170.7, 170.3, 169.6,
169.5 (2000000, 30000, 40000, 60000
-
C
OCH3), 166.7 (C20), 164.8 (0C0 40), 153.6 (C3 and C50), 144.9 (C
β
0),
0
143.0 (C2 ), 140.7 (C4), 131.5 (C6 ), 130.4 (C1), 124.7 (C3 ), 119.6 (C
α
), 114.6 (C1 ), 108.0 (C5 ),
105.9 (C2 and C6), 102.3 (C30), 100.6 (C10000), 72.1 (C50000), 72.6, 71.1, 68.3 (C20000, C30000 and
C40000), 67.8, 61.9 (C1000 and C2000), 62.1 (C100), 60.5 (4-OCH3), 56.4 (3-OCH3 and 5-OCH3),
50.3 (C60000), 21.2, 20.9, 20.7, 20.7 (20000, 30000, 40000, 60000-CO
C37H44N3O16 [M + H+] 786.27161, found 786.26915.
C
H3). HRMS (ESI+) m/z calcd for
3.1.9. Synthesis of Acetophenone 9
To a solution of 2,4-dihydroxyacetophenone (1.00 g, 6.57 mmol) and potassium car-
bonate (2.73 g, 19.72 mmol) in anhydrous acetone (20 mL), chloromethyl methyl ether
◦
(0.749 mL, 9.86 mmol) was added and the mixture refluxed for 1 h at 60 C. Then, the reaction

Mar. Drugs 2021, 19, 5
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mixture was filtered, evaporated under reduced pressure and purified by flash column chro-
matography (SiO2; n-hexane: ethyl acetate, 9:1), giving rise to
9 with 84% yield. The structure
elucidation of compound
9
was established by 1H and 13C NMR techniques and data were
in accordance with previously reported results [55].
3.1.10. Synthesis of Chalcones 10a and 10b
To a solution of
9 (0.500 g, 2.55 mmol) in methanol (20 mL) a solution of 40%
NaOH in methanol was added until pH 14, under stirring. Then, a solution of 3,4-
dimethoxybenzaldehyde (0.847 g, 5.10 mmol) or 3,4,5-trimethoxybenzaldehyde (1.00 g,
5.10 mmol) in methanol was slowly added to the reaction mixture. The reaction was
submitted to MW irradiation at 180 W at 70 ◦C for 4 h. Then, a solution of 10% HCl was
added until pH 5, and the obtained solid was filtered and washed with water and purified
by crystallization with methanol, giving rise to 10a and 10b with 33% and 47% yield,
respectively. The structure elucidation of both compounds was established by 1H and 13
C
NMR techniques and data of 10a were in accordance with previously reported results [24].
Although the synthesis of compound 10b has been previously reported [25], the NMR
data are described here for the first tim
(3,4,5-trimethoxyphenyl)prop-2-en-1-one (10b). Yellow solid; yield: 47%; m.p.: 132–134 ◦C
(methanol); 1H NMR (CDCl3, 500 MHz), : 13.29 (1H, s, OH-20), 7.85 (1H, d, J = 9.1 Hz,
€(E)-1-(2-hydroxy-4-(methoxymethoxy)phenyl)-3-
δ
H-60), 7.81 (1H, d, J = 15.4 Hz, H-
β), 7.45 (1H, d, J = 15.4 Hz, H- α) 6.87 (2H, s, H-2 and
H-6), 6.65 (1H, d, J = 2.4 Hz, H-30), 6.60 (1H, dd, J = 9.0,2.5 Hz, H-50), 5.23 (2H, s, H-100),
3.93 (6H, s, 3-OCH3 and 5-OCH3), 3.91 (3H, s, 4-OCH3), 3.49 (3H, s, H-200). 13C NMR
(CDCl3, 120 MHz) δ β),
: 192.0 (CO), 166.4 (C20), 163.8 (C40), 153.6 (C3 and C5), 144.9 (C
140.7 (C4), 131.4 (C60), 130.4 (C1), 119.5 (C ), 115.0 (C10), 108.4 (C50), 105.9 (C2 and C6),
α
104.1 (C30), 94.2 (C100), 61.2 (4-OCH3), 56.6 (C200), 56.4 (3-OCH3 and 5-OCH3).
3.1.11. Synthesis of Chalcones 11a and 11b
To a solution of 10a (0.200 g, 0.58 mmol) or 10b (0.250 g, 0.67 mmol) in methanol
(10 mL), p-toluenesulfonic acid monohydrate (0.110 g, 0.58 mmol or 0.127 g, 0.67 mmol)
was added. The reaction was submitted to conventional heating at 50 ◦C for 5 h. After the
addiction of 10 mL of water, methanol was evaporated, and the aqueous solution was
extracted with ethyl acetate (2
×
20 mL). The organic phase was washed with water
(1 20 mL), dried over anhydrous sodium sulphate and concentrated under reduced
×
pressure, giving rise to an orange solid. The crude product was purified by flash column
chromatography (n-hexane: ethyl acetate, 8:2) (11a) or crystallization with chloroform (11b),
giving rise to chalcone 11a and 11b with 24% and 31% yield, respectively. The structure
elucidation of compounds 11a and 11b was established by 1H and 13C NMR techniques
and data were in accordance with previously reported results [56,57].
3.1.12. Synthesis of 2,3,4,6-Tetra-O-Acetyl-β-D-Glucopyranosyl Azide (12)
To a solution of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1.00 g, 2.43 mmol)
in acetone: water (9 mL, 2:1), sodium azide (0.197 g, 3.04 mmol) was added and the reaction
mixture stirred at room temperature for 3 h. After, the acetone was evaporated under
reduced pressure and a white solid was filtered. Crystallization of the solid with ethanol
afforded 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl azide 12 with 72% yield. The structure
elucidation was established by 1H and 13C NMR techniques and data were in accordance
with previously reported results [27].
3.2. Mussel (Mytilus galloprovincialis) Larvae Anti-Settlement Activity
Mussel (Mytilus galloprovincialis) plantigrades were collected in 0juvenile ◦aggregates
◦
00
0
00
during low neap tides at Memória beach, Matosinhos, Portugal (41 13 59 N; 8 43 28 W).
In laboratory, mussel plantigrade larvae (0.5–2 mm) were isolated in a binocular magnifier
(Olympus SZX2-ILLT, Tokyo, Japan) to a petri dish with filtered seawater, and those
with functional foot and competent exploring behaviour were selected for the bioassays.

Mar. Drugs 2021, 19, 5
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The flavonoids were screened at 50
condition and 5 larvae per well, for 15 h, in the darkness at 18
µ
M in 24-well microplates with 4 well replicates per
±
1 ◦C, following Almeida
et al. (2015) [58]. Test solutions were obtained by dilution of the compounds stock solutions
(50 mM) in DMSO and prepared with filtered seawater. All bioassays included a negative
control with DMSO and a positive control with CuSO4, a potent AF agent. After the
exposure period, the anti-settlement activity was determined by the presence/absence of
attached byssal threads produced by each individual larvae.
All compounds that caused more than 60% of settlement inhibition ( 40% of settlement)
≤
in the screening bioassay were considered active and selected for the determination of
the semi-maximum response concentration that inhibited 50% of larval settlement (EC50),
at compounds concentrations of 3.12, 6.25, 12.5, 25, 50, 100, 200 µM.
3.3. Quantitative Structure–Activity Relationship
The eighteen flavonoid derivatives (1a
,
1b
,
2a, 2b, 3a, 3b, 4a, 4b from Scheme 1; 6a,
6b
,
7a 7b 8a 8b from Scheme 2; and 10a 10b
,
,
,
,
,
11a
,
11b from Scheme 3) were used to build
a QSAR model using the experimental data obtained from the mussel (Mytilus galloprovin-
cialis) larvae anti-settlement activity in vivo bioassay (AF activity = log(100/%settlement).
AF activity was selected as a dependent variable in the QSAR analysis. The 18 molecules
were randomly distributed into a training set (15 molecules) and a test set (3 molecules).
CODESSA software (version 2.7.10, University of Florida, Gainesville, FL, USA) was
used to calculate more than 500 constitutional, topological, geometrical, electrostatic,
quantum-chemical and thermodynamical molecular descriptors [59]. The heuristic multilin-
ear regression methodology was chosen to perform a complete search for the best multilinear
correlations with a multitude of descriptors of the training set [60]. The 2D-QSAR model
with the best square of the correlation coefficient (R2), F-test (F), and squared standard error
(S2) was selected. The final model was further validated using the test set and leave-one-out
(LOO) internal validation.
3.4. Inhibitory Activity against Biofilm-Forming Marine Bacteria Growth
For anti-bacterial screening, five strains of marine biofilm-forming bacteria from the
Spanish Type Culture Collection (CECT): Cobetia marina CECT 4278, Vibrio harveyi CECT 525,
Halomonas aquamarina CECT 5000, Pseudoalteromonas atlantica CECT 570, and Roseobacter
litoralis CECT 5395 were used. Bacteria were inoculated and incubated for 24 h at 26 ◦C in
marine broth (Difco) at an initial density of 0.1 (OD600) in 96 well flat-bottom microtiter
plates and exposed to the test compounds at 15 µM. Test solutions were obtained by
dilution of the compounds stock solutions (50 mM) in DMSO. Bacterial growth inhibition
in the presence of the compounds was determined in quadruplicate at 600 nm using a
microplate reader (Biotek Synergy HT, Vermont, USA). Negative and positive controls
used were a solution of marine broth with DMSO, and a solution of marine broth with
penicillin–streptomycin–neomycin, respectively. Compounds exerting a significant anti-
bacterial activity (Dunnet test, p < 0.05) in the screening bioassays were selected for the
determination of the effective inhibitory concentration (EC50).
3.5. Inhibitory Activity against Biofilm-Forming Marine Diatom Growth
The anti-microalgal activity of the most promising compounds was also evaluated
against a benthic marine diatom, Navicula sp., purchased from the (Telde, Gran Canaria)
Spanish Collection of Algae (BEA). Diatom cells were inoculated in f/2 medium (Sigma) at
an initial concentration of 2–4
plates for 10 days at 20 ◦C. Navicula growth inhibition in the presence of each compound
at 15 M was determined in quadruplicate and quantified based on the difference in
cell densities among the treatments, and cells were counted using a Neubauer counting
chamber. A positive control with cycloheximide (3.55 M) and a negative control with
f/2 medium 0.1% DMSO were included. Compounds that showed significant inhibitory
×
106 cells mL−1 and grown in 96-well flat-bottom microtiter
µ
µ

Mar. Drugs 2021, 19, 5
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activity in the screening assay (Dunnet test, p < 0.05) were selected for further determination
of their effective inhibitory concentrations (EC50).
3.6. In Vitro Acetylcholinesterase (AChE) and Tyrosinase (Tyr) Activities
The ability of the most promising compounds to inhibit AChE and Tyr was tested
to assess their potential mode of action related with neurotransmission disruption or
impairment of adhesive metabolism pathways, respectively.
AChE activity was evaluated using Electrophorus electric AChE Type V-S (SIGMA
C2888, E.C. 3.1.1.7), according to Ellman et al. (1961) [61] with some modifications [58,62].
Reaction solution containing 1 M phosphate buffer pH 7.2, 10 mM dithiobisnitrobenzoate
(DTNB) (acid dithiobisnitrobenzoate and sodium hydrogen carbonate in phosphate buffer)
and 0.075 M acetylcholine iodide was added to pure AChE enzyme (0.25 U/mL) and each
test compound (final concentration of 25, 50 and 100
All tests included a positive control with eserine (200
µ
M, 1% DMSO) in quadruplicate.
µM, water) and a negative control
with 1% DMSO in water. The optical density was measured at 412 nm in a microplate
reader (Biotek Synergy HT, Winooski, Vermont, USA) during 5 min at 25 ◦C.
Tyr inhibition assay was performed using Agaricus bisporus Tyr (EC1.14.18.1) according
to Adhikari et al. (2008) [63] with some modifications [8]. The enzymatic reaction follows
the catalytic conversion of L-Dopa to dopaquinone and the formation of dopachrome by
measuring the absorbance at 475 nm. Briefly, Tyr (25 U/mL) was added to 50 mM phosphate
buffer pH 6.5 and the tested compounds at 25, 50 and 100 µM (final concentrations,
1% DMSO). The enzymatic activity was triggered by the addition of L-dopa (25 mM).
Kojic acid (1.4 mM, water) was included as positive control and 1% DMSO in water as
negative control.
3.7. Environmental Fate Parameters: Artemia Salina Ecotoxicity Bioassay
The brine shrimp (Artemia salina) nauplii lethality test was used to determine
the toxicity of promising AF compounds to non-target organisms [64]. Artemia salina
eggs were allowed to hatch in seawater for 48 h at 25 ◦C. Bioassays were performed in
96-wells microplates with 15–20 nauplii per well and 200
µL of the compounds test solution.
Compounds were tested at final concentrations of 25 and 50
µM (filtered seawater with
1% DMSO). All tests included K2Cr2O7 (13.6 µM) as positive control and DMSO (1%) as
negative control. Bioassays were run in the dark at 25 ◦C, and the percentage of mortality
was determined after 48 h of exposure.
3.8. Statistical Analysis
Datasets from anti-settlement, antibacterial and anti-microalgal bioassay, and determi-
nation of AChE and Tyr activities, were analysed by one-way analysis of variance (ANOVA)
followed by a multi-comparisons Dunnett’s test against negative control (p < 0.05). For the
AF bioassays, the half maximum response concentration (EC50) values for each compound,
when applicable, were calculated using Probit regression analysis. Significance was consid-
ered at p < 0.01, and 95% lower and upper confidence limits (95%LCL; UCL). The software
IBM SPSS Statistics 26 (Armonk, New York, USA) was used for statistical analysis.
4. Conclusions
In this study, eight new triazole-flavonoid hybrids were synthesized using the click
chemistry approach. From the series of synthesized compounds, flavone 1b and chalcones
7b, 11a and 11b showed significant anti-settlement activity towards the macrofouling
species Mytilus galloprovincialis adhesive larvae. Regarding the compounds’ structures,
HACA1 was the most significant descriptor for the obtained QSAR model, contributing
positively to the AF activity. Particularly, triazolyl glycosylated chalcone 7b, with a high
number of hydrogen-bonding acceptors, showed the most effective anti-settlement activity
(EC50 = 3.28
µ
M; 2.43
µ
g
·
mL−1) with the highest therapeutic ratio (LC50/EC50 > 60.98),
exhibiting also a significant inhibitory activity against the marine diatom Navicula sp.

Mar. Drugs 2021, 19, 5
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(EC50 = 41.76
µ
M; 30.94
µ
g
·
mL−1), suggesting potential in the suppression of biofouling
colonization succession. Flavone 1b, which was effective against the settlement of mussel
larvae, also showed capacity to inhibit the activity of Tyr, which might explain the specific
AF activity against mussel larvae. Ecotoxicity studies on the non-target species Artemia
salina revealed that the flavonoids 1b
nauplii of this sensitive crustacean, even at 50
,
7b
,
11a and 11b did not show ecotoxicity to the
M, a concentration much higher than
µ
their EC50. These results disclosed synthetic flavonoids, particularly a new chalcone
incorporating a 1,2,3- triazole ring (7b), with potential to be a good environmentally
compatible alternative to the majority of the antifoulants in use. Flavonoids are ubiquitous
in Nature and, therefore, they come with the advantage that they have been selected during
evolution to have high specificity, high efficiency and some might be potential nontoxic
inhibitors of fouling. Natural compounds are usually biodegradable, not leaving residue
in the environment, and are thus considered one of the most promising alternatives to
the biocides in use. However, the yields of natural compounds from marine organisms
are generally poor, hindering their development as AF agents. Moreover, optimizing a
micro-organism for enhanced production of antifoulant is generally laborious and time
consuming. Synthesis of nature-like antifoulants seems to be a more sustainable way to
create an opportunity to produce commercial supplies for the antifouling industry.
Author Contributions: Conceptualization, H.C.; methodology, J.R.A.; M.C.-d.-S.; and H.C.;
compounds synthesis, D.P.; B.T.M.; compounds structure elucidation, D.P.; quantitative structure-
activity relationship studies, A.P.; in vivo AF bioassays, enzymatic determinations and ecotoxico-
logical bioassays, C.G.; J.R.A.; resources, M.P.; V.V.; writing—original draft preparation, D.P.; C.G.;
writing—review and editing, D.P.; C.G.; J.R.A.; M.C.-d.-S.; H.C.; M.P.; V.V.; supervision, J.R.A.;
M.C.-d.-S.; H.C.; project administration, J.R.A. and M.C.-d.-S.; funding acquisition, V.V., J.R.A. and
M.C.-d.-S. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by national funds through the FCT—Foundation for Science
and Technology within the scope of UIDB/04423/2020 and UIDP/04423/2020 and under the
projects: NASCEM-PTDC/BTA-BTA/31422/2017 (POCI-01-0145-FEDER-031422), and PTDC/AAG-
TEC/0739/2014 (POCI-01-0145-FEDER-016793; Project 9471-RIDTI) co-financed by FCT,
PORTUGAL2020 COMPETE 2020, through the European Regional Development Fund (ERDF);
and by the structured program of R&D&I ATLANTIDA (reference NORTE-01-0145-FEDER-000040),
supported by the North Portugal Regional Operational Programme (NORTE2020), through the ERDF.
D. P. and C. G. acknowledge the grants they received (SFRH/BD/147207/2019 and NASCEM/BI-
Lic/2019-53, respectively).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article.
Acknowledgments: The authors thank Sara Cravo for all the technical and scientific support.
Conflicts of Interest: The authors declare no conflict of interest.
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