Potential of synthetic chalcone derivatives to prevent marine biofouling

Article abstract of DOI:10.1016/j.scitotenv.2018.06.169

Biofouling represents a major economic, environmental and health concern for which new eco-friendly solutions are needed. International legislation has restricted the use of biocidal-based antifouling coatings, and increasing efforts have been applied in the search for environmentally friendly antifouling agents. This research work deals with the assessment of the interest of a series of synthetic chalcone derivatives for antifouling applications. Sixteen chalcone derivatives were synthesized with moderate yields (38–85%). Antifouling bioactivity of these compounds was assessed at different levels of biological organization using both anti-macrofouling and anti-microfouling bioassays, namely an anti-settlement assay using mussel (Mytilus galloprovincialis) larvae, as well as marine bacteria and microalgal biofilms growth inhibition bioassays. Results showed that three compounds (11, 12, and 16) were particularly active against the settlement of mussel larvae (EC₅₀ 7.24–34.63 μM), being compounds 12 and 16 also able to inhibit the growth of microfouling species (EC₅₀ 4.09–20.31 μM). Moreover, the most potent compounds 12 and 16 were found to be non-toxic to the non-target species Artemia salina (<10% mortality at 25 μM). A quantitative structure-activity relationship model predicted that descriptors describing the ability of molecules to form hydrogen bonds and encoding the shape, branching ratio and constitutional diversity of the molecule were implied in the antifouling activity against the settlement of mussel larvae. This work elucidates for the first time the relevance of synthesizing chalcone derivatives to generate new non-toxic products to prevent marine biofouling.

Full text of DOI:10.1016/j.scitotenv.2018.06.169

Science of the Total Environment 643 (2018) 98–106
Contents lists available at ScienceDirect
Science of the Total Environment
journal homepage: www.elsevier.com/locate/scitotenv
Potential of synthetic chalcone derivatives to prevent marine biofouling
J.R. Almeida a, J. Moreira b, D. Pereira b, S. Pereira a, J. Antunes a,c, A. Palmeira a,b, V. Vasconcelos a,c, M. Pinto a,b
,
a,b
M. Correia-da-Silva a,b, , H. Cidade
⁎
a
Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), Universidade do Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos,
Portugal
b
Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira 228, 4050-313 Porto, Portugal
Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre, P 4069-007 Porto, Portugal
c
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
• Marine biofouling impacts atmosphere,
hydrosphere, and biosphere negatively.
• Antifouling paints in use release toxic
and persistent organic pollutants.
• Synthesis of new nontoxic antifoulants
was achieved in this work.
• These compounds are able to be ob-
tained in short time and in suitable
amounts.
a r t i c l e i n f o
a b s t r a c t
Article history:
Biofouling represents a major economic, environmental and health concern for which new eco-friendly solutions
are needed. International legislation has restricted the use of biocidal-based antifouling coatings, and increasing
efforts have been applied in the search for environmentally friendly antifouling agents. This research work deals
with the assessment of the interest of a series of synthetic chalcone derivatives for antifouling applications. Six-
teen chalcone derivatives were synthesized with moderate yields (38–85%). Antifouling bioactivity of these com-
pounds was assessed at different levels of biological organization using both anti-macrofouling and anti-
microfouling bioassays, namely an anti-settlement assay using mussel (Mytilus galloprovincialis) larvae, as well
as marine bacteria and microalgal biofilms growth inhibition bioassays. Results showed that three compounds
(11, 12, and 16) were particularly active against the settlement of mussel larvae (EC50 7.24–34.63 μM), being
compounds 12 and 16 also able to inhibit the growth of microfouling species (EC50 4.09–20.31 μM). Moreover,
the most potent compounds 12 and 16 were found to be non-toxic to the non-target species Artemia salina
(b10% mortality at 25 μM). A quantitative structure-activity relationship model predicted that descriptors de-
scribing the ability of molecules to form hydrogen bonds and encoding the shape, branching ratio and constitu-
tional diversity of the molecule were implied in the antifouling activity against the settlement of mussel larvae.
This work elucidates for the first time the relevance of synthesizing chalcone derivatives to generate new non-
toxic products to prevent marine biofouling.
Received 15 April 2018
Received in revised form 6 June 2018
Accepted 13 June 2018
Available online xxxx
Keywords:
Antifouling
Chalcones
Synthesis
Settlement
QSAR
Ecotoxicity
© 2018 Published by Elsevier B.V.
⁎
Corresponding author at: Faculdade de Farmácia, Universidade do Porto, Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Rua Jorge Viterbo
Ferreira n° 228 (E2), 4050-313 Porto, Portugal.
E-mail address: m_correiadasilva@ff.up.pt (M. Correia-da-Silva).
https://doi.org/10.1016/j.scitotenv.2018.06.169
0048-9697/© 2018 Published by Elsevier B.V.

J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
99
1. Introduction
2. Material and methods
Both macro and microorganisms are part of the community respon-
sible for the natural process known as biofouling, which occurs when
marine species attach to natural or artificial underwater surfaces
(Callow and Callow, 2002). Marine biofouling causes not only material
and economic loss for the marine sector operations, but also creates a
series of environmental and health problems affecting atmosphere, hy-
drosphere, biosphere due to over-consumption of fuel and carbon diox-
ide emissions, and also the spread of pathogenic bacteria and
indigenous species which contributes to health problems and biodiver-
sity reduction (Qian et al., 2010; Schultz et al., 2011).
Antifouling (AF) paints have been and remain the primary strategy
for biofouling control. Biocidal paints based on tributyltin (TBT) have
been effective AF agents. Nevertheless, these coatings were banned in
several countries since 2008 (IMO, 2008), given its detrimental effect
to non-target organisms and whole marine environment (Antizar-
Ladislao, 2008). In fact, environmental studies demonstrated that
organotin compounds do not readily degrade in the environment, af-
fecting marine organisms and possibly biomagnifying through the
food chains (Konstantinou and Albanis, 2004). Booster biocides based
on copper, zinc, and several organic compounds were more recently in-
troduced as AF agents, but have also been found to be harmful to many
non-target organisms (Thomas and Brooks, 2010). Therefore, there is a
high demand for environmentally benign, non-toxic AF agents as an al-
ternative to the biocide-based coatings currently in use (Almeida and
Vasconcelos, 2015).
A wide range of diverse natural AF compounds have been identified
lately (Qian et al., 2015; Satheesh et al., 2016; Wang et al., 2017). These
compounds have been reported as acting against micro and
macrofouling species with low toxicity, and were recently considered
as models to the synthesis of nature-inspired AF agents namely,
synoxazolidinone A (Trepos et al., 2014), 2,5-diketopiperazine (Liao
et al., 2015), zosteric acid (Almeida et al., 2017; Catto et al., 2015),
batatasin III (Moodie et al., 2018; Moodie et al., 2017a), polygodial
(Moodie et al., 2017b).
Chalcones represent one of the major subclasses of flavonoids and
have long been recognised for their myriad of biological activities
(Singh et al., 2014). Regarding AF properties, the evaluation of their ef-
fect is limited to studies using marine bacterial biofilms such as Vibrio
natriegens, Bacillus flexus, and Pseudomonas fluorescens (Sivakumar
et al., 2010a; Sivakumar et al., 2010b). Moreover, chalcones have been
used as anticorrosive agents (Bouklah et al., 2006), alone and combined
with iodide ions to synergize the activity of the latter in acid-mediated
corrosion of steel (Bouklah et al., 2003; Elayyoubi et al., 2002). Consid-
ering the antibacterial and slimicidal activities together with anticorro-
sive properties, chalcones were proposed as ideal candidates to be used
as AF agents in anticorrosive coatings (Sivakumar et al., 2010a). At the
best of our knowledge, no studies concerning their effects on other foul-
ing organisms have been conducted.
2.1. Synthesis and structure elucidation
Microwave (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 in an Ethos MicroSYNTH 1600 Microwave
Labstation from Milestone. The reactions were monitored by thin-layer
chromatography (TLC). Compounds purification was performed by
flash column chromatography using Macherey-Nagel silica gel 60
(0.04–0.063 mm), and preparative thin-layer chromatography (TLC)
using Macherey-Nagel silica gel 60 (GF254) plates. Melting points
were obtained in a Köfler microscope and are uncorrected. 1H and 13
C
NMR spectra were taken in CDCl3 at room temperature, on Bruker
Avance 300 instrument (300.13 MHz for 1H and 75.47 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 C,
H coupling constants were optimized to 7 Hz). HRMS mass spectra
were recorded at C.A.C.T.I.-University of Vigo, Spain. Experiments were
performed on an APEXQe FT-ICR MS (Bruker Daltonics, Billerica, MA),
equipped with a 7 T actively shielded magnet. Ions were generated
using a Combi MALDI-electrospray ionization (ESI) source. Ionization
was achieved by electrospray, using a voltage of 4500 V applied to the
needle, and a counter voltage of 300 V applied to the capillary. Samples
were prepared by adding a spray solution of 70:29.9:0.1 (v/v/v) CH3OH/
water/formic acid or 70:29.9:0.1 (v/v/v) CH3CN/water/formic acid to a
solution of the sample at a v/v ratio of 1 to 5% to give the best signal-
to-noise ratio. Data acquisition was performed using the ApexControl
software version 3.0.0, and data processing was performed using the
DataAnalysis software, version 4.0 both from Bruker Daltonics. 2-
Hydroxy-4,6-dimethoxyacetophenone and benzaldehydes were pur-
chased from Sigma Aldrich. Chalcone derivatives 1–6 (33–42%)
(Pereira et al., 2016), 8 (46%) (Detsi et al., 2009), 9 (60%) (Kadival
et al., 1962), 10 (47%) (Boeck et al., 2006), 11 (40%) (Boeck et al.,
2006), 12 (77%) (Thieury et al., 2017), 13 (39%) (Detsi et al., 2009), 14
(38%) (Alvim et al., 2010), 15 (71%) (Mateeva et al., 2002), and 16
(73%) (Neves et al., 2012) were synthesized as described elsewhere.
The NMR data of compound 9 was described for the first time, as indi-
cated below. The new chalcone derivative 7 was synthesized and puri-
fied by the following procedures.
2.1.1. Synthesis of chalcone 7
2-Hydroxy-4-methoxy-3-propylacetophenone was synthesized
(quantitative yield) and characterized according to a previous described
procedure (Pereira et al., 2016). Then, to a solution of 2-hydroxy-4-
methoxy-3-propylacetophenone (1.000 mmol, 0.208 g) in methanol
was added an aqueous solution of 40% sodium hydroxide until
pH 13–14. Then, a solution of 2 mmol of 4-chlorobenzaldehyde in meth-
anol was slowly added to the reaction mixture. The reaction was sub-
mitted to successive 15 min periods of MW irradiation at 180 W. Total
irradiation time was 30 min and the final temperature was 75 °C. The
solution was extracted with chloroform (3 × 50 mL). The combined or-
ganic layers were rinsed with brine and water, dried over anhydrous so-
dium sulfate and evaporated under reduced pressure. The obtained
residue was purified by flash column chromatography (SiO2; n-
hexane/ethyl acetate 8:2) affording compound 7 as orange crystals in
57% yield.
(E)-1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-(4-Clorophenyl)
prop-2-en-1-one (7). mp (ethyl acetate): 109–112 °C; 1H NMR (CDCl3,
300.13 MHz): δ 13.30 (1H, s, 2′-OH), 7.82 (1H, d, J = 15.0 Hz, H-β),
7.78 (1H, d, J = 9.2 Hz, H-6′), 7.58 (1H, d, J = 15.0 Hz, H-α), 7.57 (2H,
d, J = 8.5 Hz, H-2,6), 7.40 (2H, d, J = 8.5 Hz, H-3,5), 6.50 (1H, d, J =
9.2 Hz, H-5′), 3.90 (3H, s, 4′-OCH3), 2.66 (2H, t, J = 7.7 Hz, H-1″),
1.59–1.51 (2H, m, H-2″), 0.96 (3H, t, J = 7.4 Hz, H-3″); 13C NMR (CDCl3,
In this research work, a series of chalcone derivatives was synthe-
sized (Fig. 1, 1–16) and evaluated for their AF activity against both
micro and macrofouling species, namely five biofilm-forming marine
bacteria (Cobetia marina, Vibrio harveyi, Pseudoalteromonas atlantica,
Halomonas aquamarina and Roseobacter litoralis), four marine diatom
strains (Cylindrotheca sp., Halamphora sp., Nitzschia sp. and Navicula
sp.) and the adhesive larvae of the macrofouling mussel Mytilus
galloprovincialis. A quantitative structure-activity relationship (QSAR)
model to predict the AF activity against larvae of Mytilus galloprovincialis
was also developed.
Compounds showing promising AF bioactivity were submitted to
complementary assays to evaluate the viability of the selected com-
pounds as AF agents, including the assessment of general ecotoxicity
using Artemia salina standard ecotoxicity assay, and the evaluation of
possible mechanisms of action related with adhesion and neurotrans-
mission pathways.

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J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
Fig. 1. Structure of chalcone derivatives 1–16.
75.47 MHz): δ 191.7 (CO), 163.5 (C-4′), 163.1 (C-2′), 142.3 (C-β), 136.1
(C-4), 133.1 (C-1), 129.3 (C-2,-6), 129.0 (C-3,-5), 128.8 (C-6′), 120.8 (C-
α), 118.4 (C-3′), 114.1 (C-1′), 101.8 (C-5′), 55.5 (4′-OCH3), 24.2 (C-1″),
21.6 (C-2″), 14.0 (C-3″); ESI-TOF-HRMS (+) m/z: Anal. Calc. for
16 were prepared in filtered seawater and obtained by dilution of the
compounds stock solutions (50 mM) in DMSO. A negative control
(0.01% DMSO) and a positive control (5 μM CuSO4) were included in
all bioassays. The anti-settlement response of ECONEA® (50 μM), a
commercial AF agent, was also included for comparative purposes.
After exposure, the anti-settlement activity was determined by the
presence/absence of efficiently attached byssal threads produced by
each individual for all the tested conditions.
Compounds causing significant settlement inhibition (p b 0.01) at 50
μM in the screening bioassay compared to negative control were consid-
ered bioactive. The bioactive compounds were selected for further test-
ing and serial dilutions of compounds stock solutions (200, 100, 50, 25,
12.5, 6.25, and 3.12 μM) were performed to determine the semi-
maximum response concentrations that inhibited 50% larval settlement
(EC50) and the median lethal dose (LC50), if applicable.
C
19H20ClO3 (M + H+): 331.10955; found: 331.10862.
(E)-3-(2,4-dichlorophenyl)-1-(2-hydroxy-4,6-dimethoxyphenyl)
prop-2-en-1-one (9): mp (ethanol): 182–183 °C; 1H NMR (CDCl3,
300.13 MHz): δ 14.15 (s, 2′-OH), 8.05 (d, J = 15.6 Hz, H-β), 7.85 (d, J
= 15.6 Hz, H-α), 7.62 (d, J = 8.7 Hz, H-6), 7.45 (d, J = 2.1 Hz, H-2),
7.28 (dd, J = 8.7, 2.1 Hz, H-5), 6.12 (d, J = 2.3 Hz, H-5′), 5.96 (d, J =
2.3 Hz, H-3′), 3.90 (s, 3H, 4′-OCH3), 3.84 (s, 3H, 6′-OCH3); 13C NMR
(CDCl3, 75.47 MHz) δ: 192.0 (CO), 168.5 (C-4′), 166.5 (C-6′), 162.4 (C-
2′), 136.6 (C-β), 135.9 (C-4), 132.5 (C-1), 130.4 (C-2), 130.1 (C-3),
130.0 (C-α), 128.5 (C-6), 127.5 (C-5), 106.2 (C-1′), 93.8 (C-3′), 91.3
(C-5′), 55.9 (4′-OCH3), 55.6 (6′-OCH3); ESI-TOF-HRMS (+) m/z: Anal.
Calc. for C17H15Cl2O4 (M + H+): 353.03419; found: 353.03380.
2.4. QSAR model construction
2.2. Mussel adhesive larvae sampling and processing
The 3D structures of the 16 chalcones and positive control Tralopyril
were drawn using Hyperchem 7.5 (Hypercube, FL, USA) (Froimowitz,
1993), being minimized by the AM1 semi-empirical method with the
Polak-Ribiere conjugate gradient algorithm (RMS b 0.1 kcal·Å−1·-
mol−1) (Zhang et al., 2006). Twelve molecules were used to construct
QSAR models using the biological data obtained in vitro studies for AF
activity (AF = −log(incrustration / Maximal_incrustration)). AF activ-
ity was adopted as a dependent variable in the QSAR analysis. The 17
molecules were randomly distributed into a training set (12 molecules)
and a test set (5 molecules). CODESSA software (version 2.7.10, Univer-
sity of Florida, USA) was used to calculate N500 constitutional,
topological, geometrical, electrostatic, quantum-chemical, and thermo-
dynamical molecular descriptors (Katritsky et al., 2004). The heuristic
multilinear regression procedures available in the framework of the
CODESSA program was used to perform a complete search for the best
Mussel (Mytilus galloprovincialis) juvenile aggregates were sampled
during low neap tides in intertidal pools, at Memória beach, Matosinhos,
Portugal (41° 13′ 59″ N; 8° 43′ 28″ W). In the laboratory, mussel planti-
grade adhesive larvae (0.5–2 mm) were screened among the mussel ag-
gregates samples in a binocular magnifier (Olympus SZX2-ILLT),
isolated in a petri dish with filtered seawater and gently washed to re-
move adhered organic particles immediately before the bioassays.
2.3. Mussel larvae anti-settlement bioassays
M. galloprovincialis plantigrade larvae showing exploring behaviour
were selected and exposed to the series of chalcone derivatives 1–16
at 50 μM in 24-well microplates with 4 replicates for 15 h, at 18
1
°C, in the darkness (Almeida et al., 2017). Solutions of compounds 1–

J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
101
multilinear correlations with a multitude of descriptors of the training
2.7. Artemia salina ecotoxicity bioassay
set (Katritsky et al., 2004). The 2D-QSAR model with the best correlation
coefficient (R2), F-test (F), standard error (s), as cross-validation (Q2)
was selected. The final model was further validated using the test set.
Bioactive chalcones 11, 12, and 16 were tested for general
ecotoxicity using the brine shrimp (Artemia salina) nauplii lethality
test (Almeida et al., 2017; Meyer et al., 1982). A. salina eggs were
allowed to hatch in nutrient-enriched seawater for approximately
48 h at 25 °C. Test solutions of the selected compounds were pre-
pared in filtered seawater. Test concentrations were 50 and 25 μM
and a concentration correspondent to the anti-settlement EC50
value for each compound, when applicable. Toxicity tests were per-
formed in the darkness in 96-wells microplates with eight replicates
per condition and 15–20 nauplii per well. 13.6 μM K2Cr2O7 was in-
cluded as positive control and 1% DMSO as negative control. Percent-
age of mortality was assessed as endpoint at the end of the exposure
period.
2.5. In vitro evaluation of acetylcholinesterase and tyrosinase activities
Acetylcholinesterase (AChE) and tyrosinase (Tyr) inhibitory activity
were determined as potential mechanisms of action of AF compounds
11, 12, and 16, as described previously (Almeida et al., 2017). The poten-
tial of compounds 11, 12, and 16 to inhibit AChE activity was evaluated
using Electrophorus electric AChE Type V-S (SIGMA C2888, E.C. 3.1.1.7),
according to Ellamn et al. (Ellman et al., 1961) with some modifications
(Almeida et al., 2015). Briefly, to a reaction solution containing 10 mM
of 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in 1 M of phosphate
buffer pH 7.2 (with sodium hydrogen carbonate) and 75 mM of
acetyltiocholine iodide, pure AChE enzyme (0.25 U/mL) and each tested
compound (final concentration of 50 μM) was added. The optical den-
sity was measured at 412 nm in a microplate reader during 5 min at
25 °C. A negative control (B) with 1% DMSO and a positive control
with 200 μM eserine was also included.
Tyr inhibition assays were conducted using Agaricus bisporus Tyr (EC
1.14.18.1) according to Adhikari et al. (2008) with slight modifications
(Almeida et al., 2017). The enzymatic reaction follows the catalytic con-
version of L-Dopa to dopaquinone and the formation of dopachrome by
measuring the absorvance at 475 nm. Briefly, Tyr (25 U/mL) is added to
50 mM fosfate buffer pH 6.5 and the tested compound at 50 μM. The en-
zymatic activity was triggered by the addition of L-dopa (25 mM). Kojic
acid (1.4 mM) was included as positive control and 1% DMSO as nega-
tive control.
2.8. Data analysis
One-way analysis of variance (ANOVA) followed by a Dunnett test
against the DMSO control (p b 0.01) was used to analyse anti-
settlement, antibacterial and anti-microalgal screening data as well as
Artemia salina ecotoxicity data. Semi-maximum response concentration
that inhibited 50% mussel larval settlement (EC50), response concentra-
tion that inhibited bacterial growth (EC) and the median lethal dose
(LC50) for each bioactive compound were assessed using Probit regres-
sion analysis, when applicable. Pearson Goodness-of-fit (Chi-Square)
significance was considered at p b 0.01 for this analysis, and 95% lower
and upper confidence limits (95% LCL; UCL) were presented. Therapeu-
tic ratio (LC50/EC50) was used as a measure of effectiveness vs toxicity of
compounds (Almeida and Vasconcelos, 2015; Qian et al., 2010;
Rittschof, 1999). The software IBM SPSS Statistics 25 was used for statis-
tical analysis.
2.6. Antibacterial and anti-diatom bioassays
For antibacterial activity screening, five strains of marine biofilm-
forming bacteria Cobetia marina CECT 4278, Vibrio harveyi CECT 525,
Halomonas aquamarina CECT 5000, Pseudoalteromonas atlantica CECT
570 and Roseobacter litoralis CECT 5395 from the Spanish Type Culture
Collection (CECT) 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 compounds 11, 12, and
3. Results and discussion
3.1. Synthesis and structure elucidation
A series of structure-related chalcone derivatives with different sub-
stitution patterns in A and B rings were synthesized (Fig. 1, 1–16) and
evaluated for their AF activity. The chemical diversity was selected in
order to allow structure-activity relationship (SAR) studies. Chalcones
1–15 were prepared by base-catalysed aldol reaction of appropriately
substituted acetophenones with benzaldehydes with moderate yields.
Chalcone 16 was synthesized by the O-alkylation of the non prenylated
chalcone 15 with prenyl bromide in presence of tetrabutylammonium
hydroxide at room temperature leading to prenyloxychalcone 16 (73%
yield) as previously reported (Neves et al., 2012). The synthetic approach
followed for the synthesis of the new chalcone 7 is described in Scheme 1.
Firstly, 2-hydroxy-4-methoxy-3-propylacetophenone was prepared
by the methylation of 2,4-dihydroxy-3-propylacetophenone with
dimethylsulfate in presence of anhydrous potassium carbonate and anhy-
16 at 9.7, 9.7 and 6.9 μM, respectively (corresponding to 3 μg·mL−1
)
test concentration. Bacterial growth inhibition in the presence of the
tested compounds was determined in quadruplicate at 600 nm using
a microplate reader (Biotek Synergy HT, Vermont, USA). Marine broth
with 0.1% DMSO and 1:100 penicilin-streptomycin-neomycin stabilized
solution (Sigma P4083) were used as negative (B) and positive
(C) controls, respectively.
For the anti-microalgal screening, four strains of benthic marine di-
atoms, Cylindrotheca sp., Halamphora sp., Nitzschia sp. and Navicula sp.
were purchased from the Spanish Collection of Algae and inoculated
inf/2medium(Sigma)ataninitialconcentrationof2–4×106 cellsmL−1
,
and grown in 96 well flat-bottom microtiter plates for 10 days at 20 °C.
Diatoms growth inhibition in the presence of chalcones 11, 12 and 16 at
9.7, 9.7 and 6.9 μM, respectively, was determined in quadruplicate, and
quantified by the difference in cell densities among treatments, counted
using a Neubauer counting chamber. f/2 medium with 0.1% DMSO and
cycloheximide (3.55 μM) were used as negative and positive controls,
respectively. The anti-bacterial and anti-microalgal activity of the
comercial AF agent ECONEA® was also included in the same bioassay
conditions (8.7 μM) as reference. Compounds producing b25% growth
inhibition were considered not active. Compounds that showed signifi-
cant inhibitory activity at screening assays were selected for further de-
termination of inhibitory effect concentrations (EC). A serial dilution of
the stock solution was used to obtain final concentrations from 27.6 μM
to 1.7 μM.
drous acetone with quantitative yield. After, compound
7 was
prepared by base-catalysed aldol reaction of 2-hydroxy-4-methoxy-3-
propylacetophenone with 4-chlorobenzaldehyde by MW assisted organic
synthesis, according to the strategy illustrated on Scheme 1.
The structure elucidation of all compounds was established on the
basis of NMR and HRMS techniques. The HRMS allowed to confirm the
molecular formula and the purity of compounds. The characterization
of compound 9 was performed for the first time by 1H, 13C NMR and
HRMS. 13C NMR assignments were determined by 2D heteronuclear sin-
gle quantum correlation (HSQC) and heteronuclear multiple bond cor-
relation (HMBC) experiments. The coupling constants of the vinylic
system (JHα-Hβ = 16.0–15.0 Hz) confirm the (E)-configuration for all
synthesized chalcones.

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J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
Scheme 1. Synthesis of chalcone 7.
3.2. Anti-settlement activity
a prenyl side chain also in A ring which has revealed to inhibit the set-
tlement of cyprids of B. albicostatus, with low toxicity (Feng et al., 2009).
For compounds 11, 12, and 16 the anti-settlement activity was eval-
uated at lower and higher concentrations to determine the EC50 values
(Fig. 3). Chalcone 12 (EC50 = 7.24 μM) showed to be the most effective
larval settlement inhibitor, followed by compounds 16 (EC50 = 16.48
μM) and 11 (EC50 = 34.63 μM) (Table 1). Considering toxicity, none of
these compounds caused mortality to the target species
M. galloprovincialis plantigrades at the maximum tested concentration
(200 μM), while the commercial AF agent ECONEA® was found to
exert some toxicity (LC50 = 107.89 μM) (Table 1) (Almeida et al.,
2017). Among these chalcones, the lower levels of toxicity and higher
effectiveness to mussel larvae was observed for 12 with LC50 levels
higher than 200 μM and LC50/EC50 higher than 27.6, respectively.
These results indicate that 12 had the best performance as promising
AF agent against this target species, given the higher AF effectiveness,
no toxicity until 200 μM, and therapeutic ratios higher than 25 (Fig. 3;
Table 1). Noteworthy, although 12 presented lower levels of effective-
ness than ECONEA® (Table 1), this compound has evidenced more po-
tential as ecofriendly AF agent considering the non-toxic effects found.
Mytilus spp. are among the most common macrofouling species with
worldwide representatives and studies focusing on anti-settlement bio-
assays with Mytilus galloprovincialis adhesive plantigrade larvae have
produced consistent results (Almeida et al., 2017; Almeida and
Vasconcelos, 2015; Bers and Wahl, 2004; Carl et al., 2011; Dobretsov,
1999; Rajagopal et al., 2003). Therefore, the anti-settlement of
M. galloprovincialis larvae was selected as the first screening assay for
the evaluation of the AF activity of the synthesized compounds.
From the series of chalcone derivatives tested, polymethoxylated
chalcones 11, 12, and 16 presented highly significant anti-settlement re-
sponses (N80% inhibition) at 50 μM (p b 0.01) against the settlement of
mussel M. galloprovincialis larvae when compared to the negative con-
trol (Fig. 2). When comparing the results of chalcones 1–7 with the
same A ring substitution pattern, the results revealed that the presence
of three methoxy groups on B ring at positions 3,4,5 (compound 1) and
2,4,5 (compound 5) is associated with a more favourable anti-
settlement activity than the presence of a lower number of methoxy
groups or a chlorine atom. In contrast, for chalcones 8–16 the best activ-
ity was obtained for compounds with only one methoxy group at B ring
(compounds 11 and 12). The presence of a 3,4,5-trimethoxyphenyl B
ring is only associated with an enhancement of activity for the 2′-
prenyloxychalcone 16. In fact, when comparing the results of
prenylated derivative 16 (55% inhibition at 50 μM) with the non
prenylated chalcone 15 (10% induction at 50 μM) it seems that the pres-
ence of a prenyloxy group at C-2′ is associated with a significant (p b
0.01) enhancement of the anti-settlement activity. Together, these re-
sults may suggest the importance of the presence of a trimethoxyphenyl
B ring associated with a lipophilic side chain in A ring, such as a propyl
or a prenyl group, to induce bioactivity. It is noteworthy to point out that
compound 16 is structure-related with 2′-methoxykurarinone, isolated
from the Chinese medicinal plant Sophora flavescens, a AF flavonoid with
3.3. QSAR model
QSAR have been successfully established to predict different types of
biological activities and/or properties (Dudek et al., 2006). Accordingly,
with the overall anti-settlement activity against M. galloprovincialis lar-
vae results, a QSAR model was built to establish a relationship between
the chalcones structures and their AF properties, which will which will
allow the design of new active compounds. In this work, a 2D-QSAR
model was built using Comprehensive Descriptors for Structural and
Statistical Analysis (CODESSA) software package (CODESSA software
version 2.7.2, University of Florida, USA). A large number of molecular
descriptors divided into five categories (constitutional, topological, geo-
metrical, electrostatic and quantum-chemical) were generated. The
heuristic method proceeds with a preselection of descriptors by elimi-
nating those descriptors that are not available for each structure, de-
scriptors having a small variation in magnitude for all structures,
descriptors found to be correlated pairwise, and descriptors found to
be of no statistical significance. The heuristic method is a very useful
tool for searching the best pool of descriptors. It is a quick method and
Fig. 2. Anti-settlement activity of compounds 1–16 at 50 μM towards plantigrade larvae of
the mussel Mytilus galloprovincialis. * means significant differences at p b 0.01 (Dunnett
test); B: DMSO control (0.01%); C: 5 μM CuSO4 as positive control; ECONEA® (50 μM)
was used for comparative purposes.
Fig. 3. Dose-response activity regarding anti-settlement of the promising compounds 11,
12, and 16. B: DMSO control (0.01%); C: 5 μM CuSO4 as positive control.

J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
103
Table 1
the variation of the residuals or the variation about the regression line.
Thus, standard deviation is an absolute measure of quality of fit and
Antifouling effectiveness and toxicity parameters of compounds 11, 12, and 16 towards
mussel plantigrades.
should have
a low value for the regression to be significant
(Gramatica, 2013). The cross-validated R2 (Q2) process repeats the re-
gression many times on subsets of data and R is computed using the
predicted values of the missing molecules. Q2 (0.8020) is smaller than
the overall R2 (0.8595), as expected, but still the difference between
R2 and Q2 is lower than 0.3, which indicates that the model has good
predictive power (Gramatica, 2013). External (test set) predictivity
was used as validation criteria, and the model was able to predict the
AF activity with an average difference of 0.28 from the experimental
value (Golbraikh et al., 2003). From all the above, it can be concluded
that the QSAR model is applicable for AF activity, which suggests that
the model may have predictive capacity for more AF hits.
Compound
EC50 (μM)
LC50 (μM)
LC50/EC50
11
12
16
34.63 (95% CI: 20.47–60.48)
7.24 (95% CI: 3.75–11.24)
16.48 (95% CI: 11.10–23.53)
4.012 (95% CI: 0.38–9.54)
N200
N200
N200
107.78
N5.78
N27.61
N12.14
26.86
ECONEA®
EC50, minimum concentration that inhibited 50% of larval settlement; LC50, the median le-
thal dose; LC50/EC50, therapeutic ratio; CI, confidence interval. Note: a therapeutic ratio
(LC50/EC50) higher than 15 is recommended (Wang et al., 2017).
presents no restrictions on the size of the data set (Dunn and Hopfinger,
2002).
The correlation coefficient (R2) (a statistical measure of how close
the data are to the fitted regression line), standard error (s) (which con-
sists of an absolute measure of the quality of fit), Fisher's value
(F) (which represents the F-ratio between the variance of actual and
predicted activity), and cross-validation (Q2) (which measures the
goodness-of-prediction) were employed to judge the validity of regres-
sion equation (Kubinyi, 1993). A major point in developing QSAR model
is the number of descriptors used to elaborate the equation. Laws of
QSAR establish that it should be one descriptor for each five molecules
(Kubinyi, 1993); therefore, as the training set was composed of 12 mol-
ecules, 2 descriptors were used to build the QSAR models. The
multilinear regression analysis using Heuristic method for 12 com-
pounds in the 2-parameter model is represented by:
Descriptors selected as influential are those describing the ability of
molecules to form hydrogen bonds (Area-weighted surface charge of
hydrogen bonding acceptor atom - HASA2) and encoding the shape,
branching ratio and constitutional diversity of the molecule (Average
Information content (order 2) – AIC2).
Geometric descriptors affecting the shape of chalcones as well as rel-
ative charged surface area descriptors have already been described as
important for antibiofouling activity (Sivakumar et al., 2010b). More-
over, an appropriate balance between hydrophobicity and hydrogen
bond forming capacity has also been described as an influential physico-
chemical factor for the settlement inhibition (Moodie et al., 2017a).
Very low logP values originate weak antifouling activity (compound
15 has the lowest logP value and the lowest antifouling activity)
(Moodie et al., 2017a).
AF ¼ −0:14079 HASA2–1:0969 AIC2 þ 6:0319
ꢀ
ꢁ
R2 ¼ 0:8595; s2 ¼ 0:0118; F ¼ 27:52; Q2 ¼ 0:8020
ð1Þ
3.4. Acetylcholinesterase and tyrosinase activities
where HASA2 is the area-weighted surface charge of hydrogen bonding
acceptor atom and AIC2 is Average Information content (order
2) (Fig. 4).
Molecular mechanisms of the AF compounds are largely source of
speculation in the literature (Qian et al., 2013). Given the lack of precise
information about the genomic and proteomic background of major
fouling organisms, identifying molecular targets and mapping the
mechanisms associated with AF activity remains a challenge. AF com-
pounds described in literature with known molecular targets mainly
act on ion channels, enzymes, adhesive production and neurotransmis-
sion blocking (Qian et al., 2013). The mechanisms of action of the most
promising non-toxic AF compounds was investigated, concerning neu-
rotransmission and adhesive metabolism, namely inhibition of AChE
and Tyr activities. These functions modulation is known to have effects
on organism settlement and thus are described as a mode of action of
some AF bioactive compounds (Chen and Qian, 2017). In fact, it has
been reported that AChE may be involved in the settlement of marine
organisms, and that Tyr is a phenoloxidase enzyme with an important
role in the formation of the DOPA-containing adhesive plaques of mus-
sels (Faimali et al., 2003; Hellio et al., 2000; Mansueto et al., 2012).
The best training model had a quality (R2) of 0.8595, Fisher value
(F) of 27.52, s2 = 0.0118, and cross-validation (Q2) of 0.8020, which
demonstrate that the proposed model has statistical stability and valid-
ity (Bewick et al., 2003). The squared correlation coefficient R2 is a rela-
tive measure of quality of fit by regression equation. Correspondingly, it
represents N85% of the total variance (R2 = 0.8595) in AF activity exhib-
ited by the test molecules. Its value is close to 1.0 which represents the
better fit to the regression line (Alexander et al., 2015). The F-test re-
flects the ratio of the variance explained by the model and the variance
due to the error in the regression. High value of the F-test indicates that
the model is statistically significant. The QSAR model is significant at
95% level as shown by their Fischer ratio values which exceed the tabu-
lated values (4.25) as desired for a meaningful correlation (Liu and
Long, 2009). Standard deviation s2 is 0.0118 and this value expresses
Fig. 4. Plot of experimental versus calculated AF activity according to QSAR model.

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J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
Fig. 7. Antibacterial activity of compounds 11, 12, and 16 at 9.7, 9.7 and 6.9 μM,
respectively towards five biofilm-forming marine bacteria Cobetia marina, Vibrio harveyi,
Pseudoalteromonas atlantica, Halomonas aquamarina and Roseobacter litoralis. *indicates
significant diferences (p b 0.01, Dunnett test) against the negative control (B: 0.1%
DMSO); 1:100 penicilin-streptomycin-neomycin stabilized solution (Sigma P4083) was
used as positive control (C); ECONEA® was used for comparative purposes.
Fig. 5. Acetylcholinesterase activity of the most promising AF chalcones 12, and 16. B: 1%
DMSO. C: Eserine (200 μM).
However, AChE (Fig. 5) and Tyr (Fig. 6) inhibition was not found for any
of the tested compounds, indicating that the observed AF activity seems
to be not related with these metabolic functions.
inhibited the growth of the biofilm-forming marine diatom Navicula
sp. (EC50 = 6.75 μM), showing also some inhibitory effect against the
growth of other tested diatoms (EC50 7.04–20.31 μM) (Fig. 8, Table 2).
Chalcone 12 displayed a low growth inhibition towards Cylindrotheca
sp., Nitzschia sp. and Navicula sp. (b25% inhibition), and any effect on
Halamphora sp. (Fig. 8, Table 2).
3.5. Antibacterial and anti-microalgal activities
Marine biofilms are known to modulate in some degree the succes-
sion of colonization of macrofouling species by producing specific
chemical cues that attract or repulse the successive fouling species
(Hadfield, 2011; Hadfield and Paul, 2001). For that reason, efforts on
AF screenings are often based only on anti-biofilm properties (Ding
et al., 2018; Salta et al., 2013). However, the AF response at different
levels of biological organization, including both macrofoulers and
biofilm-forming species is ideal to draw the full picture of promising
AF agents (Martin-Rodriguez et al., 2015). In this study, marine
biofilm-forming bacterial and microalgae (diatoms) were used to
broad the AF effectiveness assessment and better understand the selec-
tivity of the promising compounds towards microfouling species.
Among the three compounds selected from the initial screening as
potential AF compounds (11, 12, and 16), compound 11 was not able
to prevent microfouling (biofilms), causing an induction of growth of
all the strains tested in both antibacterial and antidiatom screenings
(Figs. 7 and 8). In contrast, compounds 12 and 16 showed inhibitory ac-
tivity against bacterial growth of H. aquamarina (EC50 = 18.67 and
18.78 μM, respectively), in higher levels than observed for ECONEA
(EC30 = 15.31 μM) and also against Roseobacter litoralis (EC50 = 4.09
and 12.34 μM, respectively) (Fig. 7, Table 2), being however this
last strain less sensitive to compounds 12 and 16 than to ECONEA®
(EC50 = 0.22 μM). Compounds 12 and 16 showed lower bioactivity
against all the other bacteria tested (b25%).
Concerning the anti-microalgal bioassays, the observed bioactivity
for chalcones is below the levels of efficacy obtained for ECONEA®
(~50% growth inhibition at the same concentration). Thus, these two
compounds seem to affect in some way the marine biofilm community,
however being not as potent as ECONEA® concerning anti-microalgal
properties. Nevertheless, compound 12 and 16 were able to inhibit the
growth of microfouling biofilms (EC50 4.09–20.31 μM) allied with the
previously mentioned Mytillus larval anti-settlement activity, being
these characteristics together very promising to take these compounds
in consideration as AF agents. The broad AF responses of compounds 12
and 16 towards different levels of biological organization (micro and
macro species) highlights that the target of these compounds is most
probably acting on the biofouling colonization succession, rather than
on a specific mechanism related with invertebrate adhesion. This is
also confirmed by the negative responses towards the modulation of
the studied enzymes AChE and Tyr.
3.6. Ecotoxicity assessment
For the most promising AF compounds (11, 12, and 16) the potential
ecotoxicity was assed using the nauplii of the marine crustacean Artemia
salina at concentrations ranging from 50 μM and the concentration
Compounds 12 and 16 also showed a promising inhibitory effect of
microalgal growth. Particularly, compound 16 significantly (p b 0.01)
Fig. 8. Anti-microalgal activity of compounds 11, 12, and 16 at 9.7, 9.7 and 6.9 μM,
respectively, towards four biofilm-forming marine diatoms Cylindrotheca sp.,
Halamphora sp., Nitzschia sp. and Navicula sp. *indicates significant diferences (p b 0.01,
Dunnett test) against the negative control (B: 0.1% DMSO); Cycloheximide (3.55 μM)
was used as positive control (C); ECONEA® was used for comparative purposes (8.7 μM).
Fig. 6. Tyrosinase activity of the most promising AF chalcones 12, and 16. B: 1% DMSO. C:
Kojic acid (1.4 mM).

J.R. Almeida et al. / Science of the Total Environment 643 (2018) 98–106
105
Table 2
Bacteria and microalgal growth inhibition of compounds 12 and 16 vs ECONEA®.
ECONEA®
12
16
EC30 (μM)
EC50 (μM)
EC50 (μM)
EC50 (μM)
Bacteria
Diatoms
Halomonas aquamarina
Roseobacter litoralis
Cylindrotheca sp.
Halamphora sp.
Nitzschia sp.
15.31 (95% CI: 7.3–70.4)
–
–
18.67 (95% CI: 8.3–98.6)
4.09 (95% CI: 0.9–8.2)
18.78 (95% CI: 13.4–31.1)
12.34 (95% CI: 9.8–16.4)
7.04 (95% CI: 6.1–8.2)
14.65 (95% CI:9.7–60.9)
20.31 (95% CI:13.4–60.6)
6.75 (95% CI: 5.3–8.7)
0.22 (95% CI: 0.004–0.517)
5.22 (95% CI: 2.9–10.8)
6.04 (95% CI: 5.1–7.2)
4.97 (95% CI: 2.6–11.0)
5.65 (95% CI: 2.8–15.9)
Navicula sp.
EC30 and EC50, minimum concentration that inhibited 30 and 50% of bacterial and diatoms growth, respectively; CI, confidence interval.
correspondent to the anti-settlement EC50 value for each compound. For
compounds 12 and 16 no significant differences were found at the con-
centrations tested against the negative control (p b 0.01), however re-
sults showed slightly N10% mortality at the concentration of 50 μM for
both compounds (13.92% and 14.03%, respectively) (Fig. 9). The ob-
served lethality is not significantly different (p b 0.01) from the negative
control (filtered seawater) and thus these compounds are considered
non-toxic to this non-target species. Compound 11 showed significant
toxicity even with a concentration (25 μM) below the EC50 value for
mussel larvae anti-settlement activity (34.63 μM) (Fig. 9). Overall re-
sults suggest that compounds 12 and 16 are more suitable as AF agents
considering the lower toxicity to the environment.
consideration as promising candidates to a new generation of non-
toxic AF agents, and contributing to minimize the economic, environ-
mental, and health problems associated with biofouling.
Acknowledgements
Support for this work was provided by the Strategic Funding UID/
Multi/04423/2013 through national funds provided by FCT – Founda-
tion for Science and Technology and European Regional Development
Fund (ERDF), in the framework of the programme PT2020 and by the
project PTDC/AAG-TEC/0739/2014 - COMPETE (POCI-01-0145-FEDER-
016793) - RIDTI (Project 9471) and by the project INNOVMAR
(reference NORTE-01-0145-FEDER-000035, within Research Line
NOVELMAR), supported by North Portugal Regional Operational Pro-
gramme (NORTE 2020), under the PORTUGAL 2020 Partnership Agree-
ment, through the ERDF).
4. Conclusions
The present scenario of marine antifoulants calls for more environ-
mental compatible alternatives than the active principles now in use.
Natural products are one of the most promising sources of nontoxic
AF agents. Nevertheless, the development of suitable AF agents requires
the compounds produced in high amounts, and this is often not possible
to reach by isolation from natural sources. Alternatively, new agents ob-
tained from chemical synthesis can mimic promising non-toxic natural
products with the advantage of being produced in large scale, in a short
period of time, avoiding the exhaustion of the natural resources.
In this study synthetic chalcone derivatives were obtained with good
yields and their potential for AF application was disclosed, particularly
for polymethoxylated chalcones 11, 12, and 16. Chalcones 12 and 16
showed high anti-settlement potential (EC50 b 25 μM−1) towards larvae
of Mytilus galloprovincialis, and some inhibitory effect on bacteria and
diatoms growth, suggesting their potential in the suppression of bio-
fouling colonization succession. The ecotoxicity study on both target
and non-target species revealed that these compounds did not show
toxicity at environmentally relevant concentrations. This research de-
scribes for the first time the potential application of the synthesized
chalcone derivatives to prevent biofouling, giving inputs to their
JM, SP, JA and JRA also acknowledge FCT for the grants PTCD/DTP-
FTO/1981/2014-BI-2016-054, PTDC/AAG-TEC/0739/2014-BI-2017-004,
PhD scholarship SFRH/BD/99003/2013 and postdoctoral scholarship
SFRH/BPD/87416/2012, respectively.
Author contributions
MCS planned all the aspects of this study. Synthesis procedures were
performed by JM and DP. HC and MP designed the experimental work
concerning the synthesis. QSAR model was constructed by AP. In vivo
AF bioassays were performed by JRA and SP and antibacterial and
anti-microalgal experiments were performed by JA and JRA. Artemia sa-
lina bioassays were performed by JRA and SP. In vitro enzymatic assays
were executed by JRA. JRA and VV designed the experimental work
concerning the biological activity. JRA and HC wrote this manuscript.
MCS, VV, and MP had an important contribution in the revision of the
manuscript.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.scitotenv.2018.06.169.
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