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Article

Flavonoid Glycosides with a Triazole Moiety for Marine Antifouling Applications: Synthesis and Biological Activity Evaluation

by
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
2
CIIMAR—Centro Interdisciplinar de Investigação Marinha e Ambiental, Avenida General Norton de Matos, S/N, 4450-208 Matosinhos, Portugal
3
Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua do Campo Alegre S/N, 4169-007 Porto, Portugal
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Mar. Drugs 2021, 19(1), 5; https://doi.org/10.3390/md19010005
Submission received: 5 December 2020 / Revised: 19 December 2020 / Accepted: 21 December 2020 / Published: 24 December 2020
(This article belongs to the Special Issue Marine Natural Products with Antifouling Activity)
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Abstract

:
Over the last decades, antifouling coatings containing biocidal compounds as active ingredients 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 quantitative structure-activity relationship (QSAR) model for these compounds. Three different antifouling profiles were found: three compounds (1b, 11a and 11b) exhibited anti-settlement activity against 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 acceptor 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.

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 negative effect on the environment and on live organisms, these substances were completely 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. Therefore, it is imperative to find new antifouling (AF) compounds with environmentally safe characteristics [4,5,6]. 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 [8] and chalcones [9] 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 [9]. Moreover, the introduction of a triazole moiety is associated with an increase in AF activity [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 activity [11,12]. This approach has been used to generate a vast array of compounds with biological potential [13,14,15,16], namely with AF activity [10,17,18]. Moreover, some antimicrobial 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 promising 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 [20,21]. This involves a reaction of a terminal alkyne and an aliphatic azide using copper (I) as a catalyst in low-time and mild conditions, with high yields and few by-products [20,21,22]. Therefore, the incorporation of the triazole 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).
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–2b, firstly this reaction was accomplished with propargyl bromide, in the presence of anhydrous Cs2CO3 and tetrabutylammonium bromide (TBAB). Nevertheless, in addition to the desired 4-O-monosubstituted acetophenone (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 9 with 84% yield. Chalcones 10a and 10b were prepared by base-catalyzed aldol reaction of 9 and 3,4-dimethoxy- and 3,4,5-trimethoxybenzaldehyde with 33% and 47% yield, respectively, as described before [24,25], with slight modifications. Chalcones 11a and 11b were obtained with moderate yields by deprotection of methoxymethyl group at C-4′ of intermediate chalcones 10a and 10b, as described by Loureiro et al. [26].
The 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl azide (12), used as a building block for the synthesis of glycosylated derivatives 3a, 3b, 7a and 7b, was synthesized from 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide and sodium azide, as described by Adesoye et al. [27], with 72% yield.
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 resonance (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 synthesized compounds 3a, 3b, 4a, 4b, 7a, 7b, 8a and 8b showed characteristic signals for the flavone scaffold and chalcone precursors. Additionally, signals of a triazole ring (δH-3″ 8.61–7.73 s, δC2″ 144.1–141.6 and δC3″ 125.2–121.5) and a glycosyl moiety were observed. The position of the triazole ring on these compounds was evidenced by the correlation found in the heteronuclear multiple bond correlation (HMBC) spectra between the proton signals of H-1″ and the carbon signals of C-2″ and C-3″.

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 towards macrofouling species, the ability of the synthetized flavonoids to inhibit the settlement of Mytilus galloprovincialis larvae at 50 μM was assessed. In this screening bioassay, in addition to glycosylated flavones 3a, 3b, 4a and 4b and chalcones 7a, 7b, 8a and 8b, non-glycosylated flavones 1a–b and 2a–b and chalcones 6a–b, 10a–b and 11a–b were 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%, 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 µM; 2.43 μg·mL−1), showing the highest therapeutic ratio (> 60.98) (Table 1).

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 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 establish 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.
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]. 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 significant 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:
HACA 1 =   ∑ A S A     A ∈ X H − acceptor
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 determination 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).
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 microalgae Navicula sp. This marine diatom is a major biofouling species that very effectively 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 µM; 30.94 µg·mL−1, suggesting the ability of this compound to act also as a promising AF agent against microfouling species.

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 [47,48], as well as two natural compounds isolated from marine organisms, territrem A and pulmonarin [49,50]. For this reason, the ability of the most promising compounds to modulate the activity of AChE was evaluated (1b, 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 apoptosis [51], and thus the specific target behind these compounds’ bioactivity should be further explored in future work.
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.

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, 7b, 11a and 11b are non-toxic to Artemia salina (less than 10% mortality) at both concentrations tested (25 and 50 μM) (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 [9]. These results suggest that any of the tested compounds could be a good alternative, being more environmentally compatible antifoulants.

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 monitored 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 obtained in a Köfler microscope (Wagner and Munz, Munich, Germany) and are uncorrected. 1H 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 compounds 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 1H and 13C NMR techniques and data were in accordance with 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 yellow solid; Yield: 66%; m.p.: 222–224 °C (acetone); 1H NMR (DMSO-d6, 500 MHz), δ: 12.93 (1H, s, OH-5), 7.71 (1H, dd, J = 8.5 and 2.2 Hz, H-6′), 7.57 (1H, d, J = 2.2 Hz, H-2′), 7.14 (1H, d, J = 8.7 Hz, H-5′), 7.03 (1H, s, H-3), 6.87 (1H, d, J = 2.2 Hz, H-8), 6.44 (1H, d, J = 2.3 Hz, H-6), 4.94 (2H, d, J = 2.5 Hz, H-1″), 3.88 (3H, s, 3′-OCH3), 3.85 (3H, s, 4′-OCH3), 3.66 (1H, t, J = 2.4 Hz, H-3″). 13C NMR (DMSO-d6, 120 MHz) δ: 182.3 (C4), 164.0 (C2), 163.2 (C7), 161.3 (C5), 157.3 (C8a), 152.5 (C4′), 149.2 (C3′), 122.9 (C1′), 120.5 (C6′), 111.9 (C5′), 109.6 (C2′), 105.3 (C4a), 104.3 (C3), 98.8 (C6), 94.0 (C8), 79.2 (C3″), 78.6 (C2″), 56.4 (C1″), 56.1, 56.0 (3′-OCH3 and 4′-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-2′ and H-6′), 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-1″), 3.91 (6H, s, 3′-OCH3 and 5′-OCH3), 3.76 (3H, s, 4′- OCH3), 3.70 (1H, t, J = 2.4 Hz, H-3″). 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 (C3′ and C5′), 140.9 (C4′), 125.8 (C1′), 105.3 (C3), 105.2 (C4a), 104.2 (C2′ and C6′), 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)tetrahydro-2H-pyran-3,4,5-triyl triacetate (3a). Light yellow solid; Yield: 82%; m.p.: 143–145 °C (acetone); 1H NMR (DMSO-d6, 500 MHz), δ: 12.93 (1H, s, OH-5), 8.61 (1H, s, H-3″), 7.71 (1H, dd, J = 8.5 and 2.1 Hz, H-6′), 7.60 (1H, d, J = 2.2 Hz, H-2′), 7.15 (1H, d, J = 8.7 Hz, H-5′), 7.05 (1H, s, H-3), 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-2‴, H-3‴, H-4‴), 5.32 (2H, s, H-1″), 4.39–4.36 (1H, m, H-5‴), 4.15–4.07 (2H, m, H-6‴), 3.86 (3H, s, 4′-OCH3), 3.83 (3H, s, 3′-OCH3), 2.03, 1.99, 1.96, 1.77 (12H, s, 2‴, 3‴, 4‴, -COCH3). 13C NMR (DMSO-d6, 120 MHz) δ: 182.1 (C4), 170.1, 169.6, 169.4, 168.5 (2‴, 3‴, 4‴, 6‴-COCH3), 163.7 (C2 and C7), 161.2 (C5), 158.0 (C8a), 152.3 (C4′), 149.1 (C3′), 142.7 (C2″), 124.0 (C3″), 122.7 (C1′), 120.2 (C6′), 111.7 (C5′), 109.5 (C2′), 105.0 (C3), 104.1 (C4a), 98.7 (C6), 93.7 (C8), 83.9 (C1‴), 73.3 (C5‴), 72.1, 70.1, 67.5 (C2‴, C3‴, C4‴), 61.8 (C6‴), 61.7 (C1″), 55.9, 55.8 (3′-OCH3 and 4′-OCH3), 20.5, 20.4, 20.3, 19.9 (2‴, 3‴, 4‴, 6‴-COCH3). HRMS (ESI+) m/z calcd for C34H36N3O15 [M + H+] 726.21409, found 726.21380.
(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-3″), 7.38 (2H, s, H-2′ and H-6′), 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-1‴), 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-2‴, H-3‴, H-4‴), 5.33 (2H, s, H-1″), 4.40–4.36 (1H, m, H-5‴), 4.17–4.06 (2H, m, H-6‴), 3.91 (6H, s, 3′-OCH3 and 5′-OCH3), 3.76 (3H, s, 4′-OCH3), 2.03, 2.00, 1.96, 1.77 (12H, s, 2‴, 3‴, 4‴, 6‴-COCH3). 13C NMR (DMSO-d6, 75.47 MHz) δ: 182.2 (C4), 170.1, 169.6, 169.4, 168.5 (2‴, 3‴, 4‴, 6‴-COCH3), 163.8 (C7), 163.4 (C2), 161.2 (C5), 157.3 (C8a), 153.3 (C3′ and C5′), 142.7 (C2″), 140.9 (C4′), 125.8 (C1′), 123.9 (C3″), 105.1 (C3 and C4a), 104.2 (C2′ and C6′), 98.7 (C6), 93.9 (C8), 83.9 (C1‴), 73.3 (C5‴), 72.1, 70.1, 67.5 (C2‴, C3‴, C4‴), 61.8 (C1″ and C6‴), 60.3 (4′-OCH3), 56.3 (3′-OCH3 and 5′-OCH3), 20.5, 20.4, 20.3, 19.9 (2‴, 3‴, 4‴, 6‴-COCH3). 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-β-D-glucopyranoside (0.109 g, 0.26 mmol) in tetrahydrofuran/water 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-3″), 7.72 (1H, dd, J = 8.5, 1.8 Hz, H-6′), 7.59 (1H, d, J = 1.9 Hz, H-2′), 7.14 (1H, d, J = 8.6 Hz, H-5′), 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-2⁗, H-3⁗, H-4⁗), 4.72 (1H, d, J = 8.1 Hz, H-1⁗), 4.60–4.57 (2H, m, H-6⁗), 4.19–4.01 (5H, m, H-1‴, H-2‴, H-5⁗), 3.89 (3H, s, 3′-OCH3), 3.86 (3H, s, 4′-OCH3), 2.02, 1.98, 1.92, 1.89 (12H, s, 2⁗, 3⁗, 4⁗, 6⁗-COCH3). 13C NMR (DMSO-d6, 75.47 MHz) δ: 182.1 (C4), 170.1, 169.6, 169.3, 169.0 (2⁗, 3⁗, 4⁗, 6⁗-COCH3), 163.9 (C7), 163.7 (C2), 161.2 (C5), 157.2 (C8a), 152.3 (C4′), 149.0 (C3′), 141.7 (C2″), 125.2 (C3″), 122.8 (C1′), 120.2 (C6′), 111.7 (C5′), 109.5 (C2′), 105.0 (C4a), 104.1 (C3), 99.2 (C1⁗), 98.6 (C6), 93.5 (C8), 71.9, 70.7, 68.1 (C2⁗, C3⁗, C4⁗), 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 (2⁗, 3⁗, 4⁗, 6⁗-COCH3). 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 triacetate (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-3″), 7.38 (2H, s, H-2′ and H-6′), 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-1″), 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-2⁗, H-3⁗, H-4⁗), 4.84 (1H, d, J = 8.0, H-1⁗), 4.60–4.56 (2H, m, H-6⁗), 4.21–4.01 (5H, m, H-5⁗, H-1‴ and H-2‴), 3.91 (6H, s, 3′-OCH3 and 5′-OCH3), 3.76 (3H, s, 4′-OCH3), 2.02, 1.98, 1.92, 1.89 (12H, s, 2⁗, 3⁗, 4⁗, 6⁗-COCH3). 13C NMR (DMSO-d6, 75.47 MHz) δ: 182.2 (C4), 170.1, 169.6, 169.3, 169.0 (2⁗, 3⁗, 4⁗, 6⁗-COCH3), 164.0 (C7), 163.4 (C2), 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 (C4a), 105.1 (C3), 104.2 (C2′ and C6′), 99.2 (C1⁗), 98.7 (C6), 93.7 (C8), 71.9, 70.7, 70.6, 68.1 (C2⁗, C3⁗, C4⁗ and C5⁗), 67.4, 61.7 (C1‴, C2‴), 62.0 (C1″), 60.3 (4′-OCH3), 56.4 (3′ and 5′-OCH3), 49.4 (C6⁗), 20.5, 20.4, 20.3, 20.3 (2⁗, 3⁗, 4⁗, 6⁗-COCH3). HRMS (ESI+) 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 carbonate (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 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 tetrahydrofuran (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-hydroxyphenoxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate (7a). Yellow solid; yield: 51%; m.p.: 169–171 °C (methanol); 1H NMR (CDCl3, 300.13 MHz), δ: 13.49 (1H, s, OH-2′), 7.89 (1H, s, H-3″), 7.86 (1H, d, J = 9.3 Hz, H-6′), 7.84 (1H, d, 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-3′ and H-5′), 5.90 (1H, d, J= 9.2 Hz, H-1‴), 5.48–5.38 (2H, m), 5.27–5.18 (1H, m) (H-2‴, H-3‴,H-4‴), 5.24 (2H, s, H-1″), 4.33–4.11 (2H, m, H-6‴), 4.04–3.98 (1H, m, H-5‴), 3.96 (3H, s, 3-OCH3), 3.93 (3H, s, 4-OCH3), 2.07, 2.06, 2.02, 1.86 (12H, s, 2‴, 3‴, 4‴, 6‴-COCH3). 13C NMR (CDCl3, 75.47 MHz) δ: 192.0 (CO), 170.6, 170.0, 169.5, 169.1 (2‴, 3‴, 4‴, 6‴-COCH3), 166.6 (C2′), 164.5 (C4′), 151.8 (C3), 149.4 (C4) 145.0 (Cβ), 144.1 (C2″), 131.4 (C6′), 127.8 (C1), 123.6 (C6), 121.5 (C3″), 118.0 (Cα), 114.7 (C1′), 111.3 (C5), 110.3 (C2), 107.9 (C5′), 102.3 (C3′), 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 (2‴, 3‴, 4‴, 6‴-COCH3). 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 (7b). Yellow solid; yield: 57%; m.p.: 127–128 °C (quamarnol); 1H NMR (CDCl3, 300.13 MHz), δ: 13.41 (1H, s, OH-2′), 7.89 (1H, s, H-3″), 7.87 (1H, d, J = 8.8 Hz, H-6′), 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, 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-4‴), 5.25 (2H, s, H-1″), 4.34–4.13 (2H, m, H-6‴), 4.03–4.00 (1H, m, H-5‴), 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, 2‴, 3‴, 4‴, 6‴-COCH3). 13C NMR (CDCl3, 75.47 MHz) δ: 191.9 (CO), 170.6, 170.0, 169.5, 169.1 (2‴, 3‴, 4‴, 6‴-COCH3), 166.6 (C2′), 164.6 (C4′), 153.7 (C3 and C5), 145.0 (Cβ), 144.1 (C2″), 140.8 (C4), 131.5 (C6′), 130.3 (C1), 121.5 (C3″), 119.5 (Cα), 114.7 (C1′), 108.0 (C5′), 105.9 (C2 and C6), 102.3 (C3′), 86.0 (C1‴), 75.4 (C5‴), 72.7, 70.4, 67.8 (C2‴, C3‴, C4‴), 62.1 (C1″), 61.6 (C6‴), 61.2 (4-OCH3), 56.4 (3-OCH3 and 5-OCH3), 20.8, 20.7, 20.6, 20.3 (2‴, 3‴, 4‴, 6‴-COCH3). 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-β-D-glucopyranoside (0.123 g, 0.30 mmol or 0.566 g, 1.36 mmol) in 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 triacetate (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-2′), 7.87 (1H, d, J = 9.7 Hz, H-6′), 7.85 (1H, d, J = 15.4 Hz, H-β), 7.73 (1H, s, H-3″), 7.44 (1H, d, J = 15.4 Hz, H-α), 7.25 (1H, dd, J = 8.1, 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-3′ and H-5′), 5.23 (2H, s, H-1″), 5.18 (1H, t, J = 9.4), 5.06 (1H, t, J = 9.6), 5.03–4.97 (1H, m) (H-2⁗, H-3⁗, H-4⁗), 4.68–4.52 (2H, m, H-6⁗), 4.48 (1H, d, J = 7.9, H-1⁗), 4.27–4.21 (2H, m), 4.15–4.10 (1H, m), 3.90–3.81 (1H, m) (H-1‴, H-2‴), 3.96 (3H, s, 3-OCH3), 3.94 (3H, s, 4-OCH3), 3.72–3.66 (1H, m, H-5⁗), 2.08, 2.01, 1.99, 1.95 (12H, s, 2⁗, 3⁗, 4⁗, 6⁗-COCH3). 13C NMR (CDCl3, 75.47 MHz) δ: 192.0 (CO), 170.7, 170.3, 169.6, 169.5 (2⁗, 3⁗, 4⁗, 6⁗-COCH3), 166.6 (C2′), 164.6 (C4′), 151.8 (C4), 149.4 (C3) 144.9 (Cβ), 143.1 (C2″), 131.5 (C6′), 127.9 (C1), 124.6 (C3″), 123.6 (C6), 118.1 (Cα), 114.7 (C1′), 111.3 (C5), 110.3 (C2), 107.9 (C5′), 102.3 (C3′), 100.6 (C1⁗), 72.1 (C5⁗), 72.6, 71.1, 68.3 (C2⁗, C3⁗and C4⁗), 67.8, 61.8 (C1‴ and C2‴), 62.1 (C1″), 56.2 (3′-OCH3), 56.1 (4′-OCH3), 50.3 (C6⁗), 20.9, 20.7, 20.7, 20.7 (2⁗, 3⁗, 4⁗, 6⁗-COCH3). 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 triacetate (8b). Yellow solid; yield: 45%; m.p.: 78–81 °C (n-hexane: ethyl acetate); 1H NMR (CDCl3, 300.13 MHz), δ: 13.41 (1H, s, OH-2′), 7.87 (1H, d, J = 9.8 Hz, H-6′), 7.81 (1H, d, J = 15.4 Hz, H-β), 7.74 (1H, s, H-3″), 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-3′ and H-5′), 5.24 (2H, s, H-1″), 5.18 (1H, t, J= 9.4), 5.07 (1H, t, J = 9.6), 4.99 (1H, t, J = 8.7) (H-2⁗, H-3⁗, H-4⁗), 4.68–4.52 (2H, m, H-6⁗), 4.48 (1H, d, 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), 3.91 (6H, s, 3-OCH3 and 5-OCH3), 3.72–3.66 (1H, m, H-5⁗), 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 (2⁗, 3⁗, 4⁗, 6⁗-COCH3), 166.7 (C2′), 164.8 (C4′), 153.6 (C3 and C5), 144.9 (Cβ), 143.0 (C2″), 140.7 (C4), 131.5 (C6′), 130.4 (C1), 124.7 (C3″), 119.6 (Cα), 114.6 (C1′), 108.0 (C5′), 105.9 (C2 and C6), 102.3 (C3′), 100.6 (C1⁗), 72.1 (C5⁗), 72.6, 71.1, 68.3 (C2⁗, C3⁗ and C4⁗), 67.8, 61.9 (C1‴ and C2‴), 62.1 (C1″), 60.5 (4-OCH3), 56.4 (3-OCH3 and 5-OCH3), 50.3 (C6⁗), 21.2, 20.9, 20.7, 20.7 (2⁗, 3⁗, 4⁗, 6⁗-COCH3). HRMS (ESI+) m/z calcd for C37H44N3O16 [M + H+] 786.27161, found 786.26915.

3.1.9. Synthesis of Acetophenone 9

To a solution of 2,4-dihydroxyacetophenone (1.00 g, 6.57 mmol) and potassium carbonate (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 mixture was filtered, evaporated under reduced pressure and purified by flash column chromatography (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 13C 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€(E)-1-(2-hydroxy-4-(methoxymethoxy)phenyl)-3-(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), δ: 13.29 (1H, s, OH-2′), 7.85 (1H, d, J = 9.1 Hz, H-6′), 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-3′), 6.60 (1H, dd, J = 9.0,2.5 Hz, H-5′), 5.23 (2H, s, H-1″), 3.93 (6H, s, 3-OCH3 and 5-OCH3), 3.91 (3H, s, 4-OCH3), 3.49 (3H, s, H-2″). 13C NMR (CDCl3, 120 MHz) δ: 192.0 (CO), 166.4 (C2′), 163.8 (C4′), 153.6 (C3 and C5), 144.9 (Cβ), 140.7 (C4), 131.4 (C6′), 130.4 (C1), 119.5 (Cα), 115.0 (C1′), 108.4 (C5′), 105.9 (C2 and C6), 104.1 (C3′), 94.2 (C1″), 61.2 (4-OCH3), 56.6 (C2″), 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 juvenile aggregates 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. The flavonoids were screened at 50 µM in 24-well microplates with 4 well replicates per condition and 5 larvae per well, for 15 h, in the darkness at 18 ± 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 galloprovincialis) 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 multilinear 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 × 106 cells mL−1 and grown in 96-well flat-bottom microtiter plates for 10 days at 20 °C. Navicula growth inhibition in the presence of each compound at 15 μM 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 μM) and a negative control with f/2 medium 0.1% DMSO were included. Compounds that showed significant inhibitory 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 μM, 1% DMSO) in quadruplicate. All tests included a positive control with eserine (200 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 determination 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 considered 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 µM; 2.43 µg·mL−1) with the highest therapeutic ratio (LC50/EC50 > 60.98), exhibiting also a significant inhibitory activity against the marine diatom Navicula sp. (EC50 = 41.76 µM; 30.94 µg·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, 7b, 11a and 11b did not show ecotoxicity to the nauplii of this sensitive crustacean, even at 50 µM, a concentration much higher than 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 ecotoxicological 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.

References

  1. Amara, I.; Miled, W.; Slama, R.B.; Ladhari, N. Antifouling processes and toxicity effects of antifouling paints on marine environment. A review. Environ. Toxicol. Pharmacol. 2018, 57, 115–130. [Google Scholar] [CrossRef] [PubMed]
  2. Wang, K.-L.; Wu, Z.-H.; Wang, Y.; Wang, C.-Y.; Xu, Y. Mini-Review: Antifouling Natural Products from Marine Microorganisms and Their Synthetic Analogs. Mar. Drugs 2017, 15, 266. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Dubalska, K.; Rutkowska, M.; Bajger-Nowak, G.; Konieczka, P.; Namieśnik, J. Organotin Compounds: Environmental Fate and Analytics. Crit. Rev. Anal. Chem. 2013, 43, 35–54. [Google Scholar] [CrossRef]
  4. Bao, V.W.W.; Leung, K.M.Y.; Lui, G.C.S.; Lam, M.H.W. Acute and chronic toxicities of Irgarol alone and in combination with copper to the marine copepod Tigriopus japonicus. Chemosphere 2013, 90, 1140–1148. [Google Scholar] [CrossRef]
  5. Kyei, S.K.; Darko, G.; Akaranta, O. Chemistry and application of emerging ecofriendly antifouling paints: A review. J. Coat. Technol. Res. 2020, 17, 315–332. [Google Scholar] [CrossRef]
  6. Rossini, P.; Napolano, L.; Matteucci, G. Biotoxicity and life cycle assessment of two commercial antifouling coatings in marine systems. Chemosphere 2019, 237, 124475. [Google Scholar] [CrossRef]
  7. Martins, B.T.; Correia da Silva, M.; Pinto, M.; Cidade, H.; Kijjoa, A. Marine natural flavonoids: Chemistry and biological activities. Nat. Prod. Res. 2019, 33, 3260–3272. [Google Scholar] [CrossRef]
  8. Almeida, J.R.; Correia-da-Silva, M.; Sousa, E.; Antunes, J.; Pinto, M.; Vasconcelos, V.; Cunha, I. Antifouling potential of Nature-inspired sulfated compounds. Sci. Rep. 2017, 7, 42424. [Google Scholar] [CrossRef] [Green Version]
  9. Almeida, J.R.; Moreira, J.; Pereira, D.; Pereira, S.; Antunes, J.; Palmeira, A.; Vasconcelos, V.; Pinto, M.; Correia-da-Silva, M.; Cidade, H. Potential of synthetic chalcone derivatives to prevent marine biofouling. Sci. Total Environ. 2018, 643, 98–106. [Google Scholar] [CrossRef]
  10. Trojer, M.A.; Movahedi, A.; Blanck, H.; Nydén, M. Imidazole and Triazole Coordination Chemistry for Antifouling Coatings. J. Chem. 2013, 2013, 23. [Google Scholar]
  11. Hou, J.; Liu, X.; Shen, J.; Zhao, G.; Wang, P.G. The impact of click chemistry in medicinal chemistry. Expert Opin. Drug Discov. 2012, 7, 489–501. [Google Scholar] [CrossRef] [PubMed]
  12. Totobenazara, J.; Burke, A.J. New click-chemistry methods for 1,2,3-triazoles synthesis: Recent advances and applications. Tetrahedron Lett. 2015, 56, 2853–2859. [Google Scholar] [CrossRef]
  13. Agalave, S.G.; Maujan, S.R.; Pore, V.S. Click chemistry: 1,2,3-triazoles as pharmacophores. Chem Asian J. 2011, 6, 2696–2718. [Google Scholar] [CrossRef] [PubMed]
  14. Kolb, H.C.; Sharpless, K.B. The growing impact of click chemistry on drug discovery. Drug Discov. Today 2003, 8, 1128–1137. [Google Scholar] [CrossRef]
  15. Ma, N.; Wang, Y.; Zhao, B.X.; Ye, W.C.; Jiang, S. The application of click chemistry in the synthesis of agents with anticancer activity. Drug Des. Devel Ther. 2015, 9, 1585–1599. [Google Scholar]
  16. Tron, G.C.; Pirali, T.; Billington, R.A.; Canonico, P.L.; Sorba, G.; Genazzani, A.A. Click chemistry reactions in medicinal chemistry: Applications of the 1,3-dipolar cycloaddition between azides and alkynes. Med. Res. Rev. 2008, 28, 278–308. [Google Scholar] [CrossRef]
  17. Andjouh, S.; Blache, Y. Screening of bromotyramine analogues as antifouling compounds against marine bacteria. Biofouling 2016, 32, 871–881. [Google Scholar] [CrossRef]
  18. Kantheti, S.; Narayan, R.; Raju, K.V.S.N. The impact of 1,2,3-triazoles in the design of functional coatings. RSC Adv. 2015, 5, 3687–3708. [Google Scholar] [CrossRef]
  19. Seijas, J.A.; Vazquez-Tato, M.P.; Carballido-Reboredo, R. Solvent-free synthesis of functionalized flavones under microwave irradiation. J. Org. Chem 2005, 70, 2855–2858. [Google Scholar] [CrossRef]
  20. Rostovtsev, V.V.; Green, L.G.; Fokin, V.V.; Sharpless, K.B. A stepwise huisgen cycloaddition process: Copper(I)-catalyzed regioselective "ligation" of azides and terminal alkynes. Angew. Chem. Int. Ed. 2002, 41, 2596–2599. [Google Scholar] [CrossRef]
  21. Tornøe, C.W.; Christensen, C.; Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-Triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 2002, 67, 3057–3064. [Google Scholar] [CrossRef] [PubMed]
  22. Liang, L.; Astruc, D. The copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) “click” reaction and its applications. An overview. Coord Chem Rev. 2011, 255, 2933–2945. [Google Scholar] [CrossRef]
  23. Zhao, L.; Mao, L.; Hong, G.; Yang, X.; Liu, T. Design, synthesis and anticancer activity of matrine-1H-1,2,3-triazole-chalcone conjugates. Bioorg. Med. Chem. Lett. 2015, 25, 2540–2544. [Google Scholar] [CrossRef] [PubMed]
  24. Park, J.; Khloya, P.; Seo, Y.; Kumar, S.; Lee, H.K.; Jeon, D.-K.; Jo, S.; Sharma, P.K.; Namkung, W. Potentiation of ΔF508- and G551D-CFTR-Mediated Cl- Current by Novel Hydroxypyrazolines. PLoS ONE 2016, 11, e0149131. [Google Scholar] [CrossRef] [Green Version]
  25. Zhang, J.; Fu, X.L.; Yang, N.; Wang, Q.A. Synthesis and cytotoxicity of chalcones and 5-deoxyflavonoids. Sci. World J. 2013, 2013. [Google Scholar] [CrossRef] [Green Version]
  26. Loureiro, D.R.P.; Magalhães, Á.F.; Soares, J.X.; Pinto, J.; Azevedo, C.M.G.; Vieira, S.; Henriques, A.; Ferreira, H.; Neves, N.; Bousbaa, H.; et al. Yicathins B and C and Analogues: Total Synthesis, Lipophilicity and Biological Activities. ChemMedChem 2020, 15, 749–755. [Google Scholar] [CrossRef]
  27. Adesoye, O.G.; Mills, I.N.; Temelkoff, D.P.; Jackson, J.A.; Norris, P. Synthesis of a d-Glucopyranosyl Azide: Spectroscopic Evidence for Stereochemical Inversion in the SN2 Reaction. J. Chem. Educ. 2012, 89, 943–945. [Google Scholar] [CrossRef]
  28. Briand, J.F. Marine antifouling laboratory bioassays: An overview of their diversity. Biofouling 2009, 25, 297–311. [Google Scholar] [CrossRef]
  29. Kojima, R.; Kobayashi, S.; Satuito, C.G.P.; Katsuyama, I.; Ando, H.; Seki, Y.; Senda, T. A Method for Evaluating the Efficacy of Antifouling Paints Using Mytilus galloprovincialis in the Laboratory in a Flow-Through System. PLoS ONE 2016, 11, e0168172. [Google Scholar] [CrossRef] [Green Version]
  30. Aldred, N.; Ista, L.K.; Callow, M.E.; Callow, J.A.; Lopez, G.P.; Clare, A.S. Mussel (Mytilus edulis) byssus deposition in response to variations in surface wettability. J. R Soc. Interface 2006, 3, 37–43. [Google Scholar] [CrossRef]
  31. Qian, P.Y.; Xu, Y.; Fusetani, N. Natural products as antifouling compounds: Recent progress and future perspectives. Biofouling 2010, 26, 223–234. [Google Scholar] [CrossRef] [PubMed]
  32. Dudek, A.Z.; Arodz, T.; Gálvez, J. Computational methods in developing quantitative structure-activity relationships (QSAR): A review. Comb. Chem. High. Throughput Screen. 2006, 9, 213–228. [Google Scholar] [CrossRef] [PubMed]
  33. Dunn, W.J.; Hopfinger, A.J. 3D QSAR of Flexible Molecules Using Tensor Representation; Kluwer Academic Publishers: NewYork, NY, USA, 2002; Volume 3. [Google Scholar]
  34. Kubinyi, H. QSAR: Hansch Analysis and Related Approaches; Wiley-VCH: Weinhem, Germany, 1993; Volume 1. [Google Scholar]
  35. Veerasamy, R.; Rajak, H.; Jain, A.; Sivadasan, S.; Christapher, P.V.; Agrawal, R. Validation of QSAR Models-Strategies and Importance. Int. J. Drug Design Discov. 2011, 2, 511–519. [Google Scholar]
  36. Alexander, D.L.J.; Tropsha, A.; Winkler, D.A. Beware of R(2): Simple, Unambiguous Assessment of the Prediction Accuracy of QSAR and QSPR Models. J. Chem. Inf. Model. 2015, 55, 1316–1322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Liu, P.; Long, W. Current Mathematical Methods Used in QSAR/QSPR Studies. Int. J. Mol. Sci. 2009, 10, 1978–1998. [Google Scholar] [CrossRef] [PubMed]
  38. Gramatica, P. On the Development and Validation of QSAR Models. In Computational Toxicology: Volume II; Reisfeld, B., Mayeno, A.N., Eds.; Humana Press: Totowa, NJ, USA, 2013; pp. 499–526. [Google Scholar]
  39. Golbraikh, A.; Shen, M.; Xiao, Z.; Xiao, Y.-D.; Lee, K.-H.; Tropsha, A. Rational selection of training and test sets for the development of validated QSAR models. J. Comput.-Aided Mol. Des. 2003, 17, 241–253. [Google Scholar] [CrossRef]
  40. OECD. Guidance Document on the Validation of (Quantitative) Structure-Activity Relationships [(Q)SAR] Models; OECD: Paris, France, 2007. [Google Scholar]
  41. Todeschini, R.; Consonni, V. Molecular Descriptors for Chemoinformatics; Wiley-VCH: Weinheim, Germany, 2009; Volume I. [Google Scholar]
  42. Dunn, W.J. Handbook of Molecular Descriptors. Methods and Principles in Medicinal Chemistry Series. Volume 11 By Roberto Todeschini and Viviana Consonni (Universita degli Studi di Milano-Bicocca). Edited by R. Mannold, H. Kubinyi, and H. Timmerman. Wiley-VCH:  Weinheim and New York. 2000. xxi + 668 pp. 498 DM. ISBN 3-527-29913-O. J. Am. Chem. Soc. 2001, 123, 7198. [Google Scholar]
  43. Roy, K.; Kar, S.; Das, R.N. Understanding the Basics of QSAR for Applications in Pharmaceutical Sciences and Risk Assessment; Academic Press: Boston, MA, USA, 2015. [Google Scholar]
  44. Almeida, J.R.; Vasconcelos, V. Natural antifouling compounds: Effectiveness in preventing invertebrate settlement and adhesion. Biotechnol. Adv. 2015, 33, 343–357. [Google Scholar] [CrossRef]
  45. Qian, P.Y.; Chen, L.; Xu, Y. Mini-review: Molecular mechanisms of antifouling compounds. Biofouling 2013, 29, 381–400. [Google Scholar] [CrossRef]
  46. Chen, L.; Qian, P.Y. Review on Molecular Mechanisms of Antifouling Compounds: An Update since 2012. Mar. Drugs 2017, 15, 264. [Google Scholar] [CrossRef] [Green Version]
  47. Chen, L.; Au, D.W.; Hu, C.; Peterson, D.R.; Zhou, B.; Qian, P.Y. Identification of Molecular Targets for 4,5-Dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT) in Teleosts: New Insight into Mechanism of Toxicity. Environ. Sci. Technol. 2017, 51, 1840–1847. [Google Scholar] [CrossRef] [PubMed]
  48. Chen, L.; Zhang, H.; Sun, J.; Wong, Y.H.; Han, Z.; Au, D.W.; Bajic, V.B.; Qian, P.Y. Proteomic changes in brain tissues of marine medaka (Oryzias melastigma) after chronic exposure to two antifouling compounds: Butenolide and 4,5-dichloro-2-n-octyl-4-isothiazolin-3-one (DCOIT). Aquat. Toxicol. (Amst. Neth.) 2014, 157, 47–56. [Google Scholar] [CrossRef] [PubMed]
  49. Nong, X.H.; Wang, Y.F.; Zhang, X.Y.; Zhou, M.P.; Xu, X.Y.; Qi, S.H. Territrem and butyrolactone derivatives from a marine-derived fungus Aspergillus terreus. Mar. Drugs 2014, 12, 6113–6124. [Google Scholar] [CrossRef] [PubMed]
  50. Tadesse, M.; Svenson, J.; Sepcic, K.; Trembleau, L.; Engqvist, M.; Andersen, J.H.; Jaspars, M.; Stensvag, K.; Haug, T. Isolation and synthesis of pulmonarins A and B, acetylcholinesterase inhibitors from the colonial ascidian Synoicum pulmonaria. J. Nat. Prod. 2014, 77, 364–369. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Jiang, W.; Duysen, E.G.; Lockridge, O. Induction of plasma acetylcholinesterase activity and apoptosis in mice treated with the organophosphorus toxicant, tri-o-cresyl phosphate. Toxicol. Res. 2012, 1, 55–61. [Google Scholar] [CrossRef]
  52. Martin-Rodriguez, A.J.; Babarro, J.M.; Lahoz, F.; Sanson, M.; Martin, V.S.; Norte, M.; Fernandez, J.J. From broad-spectrum biocides to quorum sensing disruptors and mussel repellents: Antifouling profile of alkyl triphenylphosphonium salts. PLoS ONE 2015, 10, e0123652. [Google Scholar] [CrossRef] [Green Version]
  53. Katranitsas, A.; Castritsi-Catharios, J.; Persoone, G. The effects of a copper-based antifouling paint on mortality and enzymatic activity of a non-target marine organism. Mar. Pollut. Bull. 2003, 46, 1491–1494. [Google Scholar] [CrossRef]
  54. Koutsaftis, A.; Aoyama, I. Toxicity of four antifouling biocides and their mixtures on the brine shrimp Artemia salina. Sci. Total Environ. 2007, 387, 166–174. [Google Scholar] [CrossRef]
  55. Sugamoto, K.; Matsusita, Y.-i.; Matsui, K.; Kurogi, C.; Matsui, T. Synthesis and antibacterial activity of chalcones bearing prenyl or geranyl groups from Angelica keiskei. Tetrahedron 2011, 67, 5346–5359. [Google Scholar] [CrossRef]
  56. Lim, S.S.; Jung, S.H.; Ji, J.; Shin, K.H.; Keum, S.R. Synthesis of flavonoids and their effects on aldose reductase and sorbitol accumulation in streptozotocin-induced diabetic rat tissues. J. Pharm. Pharmacol. 2001, 53, 653–668. [Google Scholar] [CrossRef]
  57. Neves, M.P.; Cravo, S.; Lima, R.T.; Vasconcelos, M.H.; Nascimento, M.S.J.; Silva, A.M.S.; Pinto, M.; Cidade, H.; Corrêa, A.G. Solid-phase synthesis of 2′-hydroxychalcones. Effects on cell growth inhibition, cell cycle and apoptosis of human tumor cell lines. Bioorg. Med. Chem. 2012, 20, 25–33. [Google Scholar] [CrossRef] [PubMed]
  58. Almeida, J.R.; Freitas, M.; Cruz, S.; Leão, P.N.; Vasconcelos, V.; Cunha, I. Acetylcholinesterase in Biofouling Species: Characterization and Mode of Action of Cyanobacteria-Derived Antifouling Agents. Toxins 2015, 7, 2739–2756. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  59. Katritsky, A.; Karelson, M.; Lobanov, V.S.; Dennington, R.; Keith, T. CODESSA 2.7.10; Semichem, Inc.: Shawnee, KS, USA, 2004. [Google Scholar]
  60. Ćwik, J.; Koronacki, J. A Heuristic Method of Model Choice for Nonlinear Regression. In Proceedings of the Rough Sets and Current Trends in Computing, Berlin, Heidelberg, 22–26 June 1998; pp. 68–74. [Google Scholar]
  61. Ellman, G.L.; Courtney, K.D.; Andres, V.; Featherstone, R.M. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 1961, 7, 88–95. [Google Scholar] [CrossRef]
  62. Cunha, I.; Garcia, L.M.; Guilhermino, L. Sea-urchin (Paracentrotus lividus) glutathione S-transferases and cholinesterase activities as biomarkers of environmental contamination. J. Environ. Monit 2005, 7, 288–294. [Google Scholar] [CrossRef]
  63. Adhikari, A.; Devkota, H.P.; Takano, A.; Masuda, K.; Nakane, T.; Basnet, P.; Skalko-Basnet, N. Screening of Nepalese crude drugs traditionally used to treat hyperpigmentation: In vitro tyrosinase inhibition. Int. J. Cosmet. Sci. 2008, 30, 353–360. [Google Scholar] [CrossRef]
  64. Meyer, B.N.; Ferrigni, N.R.; Putnam, J.E.; Jacobsen, L.B.; Nichols, D.E.; McLaughlin, J.L. Brine shrimp: A convenient general bioassay for active plant constituents. Planta Med. 1982, 45, 31–34. [Google Scholar] [CrossRef]
Marinedrugs 19 00005 sch001 550
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%.
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%.
Marinedrugs 19 00005 sch001
Marinedrugs 19 00005 sch002 550
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 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%.
Marinedrugs 19 00005 sch002
Marinedrugs 19 00005 sch003 550
Scheme 3. Synthesis of chalcones 10a–10b and 11a–11b. (i) K2CO3, acetone, 60 °C, 1 h, 84%; (ii) 40% NaOH, methanol, microwave (MW), 4 h, 33–47%; (iii) p-Toluenesulfonic acid (PTSA), methanol, 50 °C, 5 h, 24–31%.
Scheme 3. Synthesis of chalcones 10a–10b and 11a–11b. (i) K2CO3, acetone, 60 °C, 1 h, 84%; (ii) 40% NaOH, methanol, microwave (MW), 4 h, 33–47%; (iii) p-Toluenesulfonic acid (PTSA), methanol, 50 °C, 5 h, 24–31%.
Marinedrugs 19 00005 sch003
Marinedrugs 19 00005 g001 550
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, ΔX 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.
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, ΔX 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.
Marinedrugs 19 00005 g001
Marinedrugs 19 00005 g002 550
Figure 2. Bacterial growth inhibition screening of flavonoid derivatives (15 µM) towards five biofilm-forming marine 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 2. Bacterial growth inhibition screening of flavonoid derivatives (15 µM) towards five biofilm-forming marine 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).
Marinedrugs 19 00005 g002
Marinedrugs 19 00005 g003 550
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.
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.
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Marinedrugs 19 00005 g004 550
Figure 4. AChE activity of the most promising compounds 1b 7b, 11a and 11b. B: Dimethyl sulfoxide (DMSO) (1% in water). C: Eserine (200 μM, water). * indicates significant differences at p < 0.05 (Dunnett test), against the negative control (B).
Figure 4. AChE activity of the most promising compounds 1b 7b, 11a and 11b. B: Dimethyl sulfoxide (DMSO) (1% in water). C: Eserine (200 μM, water). * indicates significant differences at p < 0.05 (Dunnett test), against the negative control (B).
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Marinedrugs 19 00005 g005 550
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).
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).
Marinedrugs 19 00005 g005
Marinedrugs 19 00005 g006 550
Figure 6. Mortality rate of Artemia salina nauplii after 48 h of exposure to compounds 1b, 7b, 11a and 11b, B: DMSO (1% in filtered seawater). C: K2Cr2O7 (13.6 μM, filtered seawater).
Figure 6. Mortality rate of Artemia salina nauplii after 48 h of exposure to compounds 1b, 7b, 11a and 11b, B: DMSO (1% in filtered seawater). C: K2Cr2O7 (13.6 μM, filtered seawater).
Marinedrugs 19 00005 g006
Table
Table 1. Antifouling (AF) effectiveness and toxicity parameters of flavones 1b, 4a and 4b and chalcones 6a, 6b, 7b, 8a, 8b, 11a and 11b towards mussel plantigrade larvae.
Table 1. Antifouling (AF) effectiveness and toxicity parameters of flavones 1b, 4a and 4b and chalcones 6a, 6b, 7b, 8a, 8b, 11a and 11b towards mussel plantigrade larvae.
CompoundEC50 (μM)EC50 (μg·mL−1)LC50 (µM)LC50/EC50
1b8.34 (95% CI: 4.2–13.36)2.87> 200> 23.98
4a42.55 (95% CI: 34.90–52.80)32.75> 200> 4.70
4b48.22 (95% CI: 30.57–85.40)38.56> 200> 4.15
6a84.52 (95% CI: 45.07–267.02)28.60> 200> 2.37
6b85.56 (95% CI: 44.84–291.41)31.52> 200> 2.34
7b3.28 (95% CI: 1.97–4.74)2.43> 200> 60.98
8a35.83 (95% CI: 19.22–74.74)27.07> 200> 5.58
8b53.90 (95% CI: 29.98–126.88)42.35> 200> 3.71
11a18.10 (95% CI: 13.95–23.44)5.44> 200> 11.05
11b9.64 (95% CI: 3.85–17.22)3.18> 200> 20.75
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 µg/mL and therapeutic ratio higher than 15 for effective AF agents [31].
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Pereira, D.; Gonçalves, C.; Martins, B.T.; Palmeira, A.; Vasconcelos, V.; Pinto, 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://doi.org/10.3390/md19010005

AMA Style

Pereira D, Gonçalves C, Martins BT, Palmeira A, Vasconcelos V, Pinto M, Almeida JR, Correia-da-Silva M, Cidade H. Flavonoid Glycosides with a Triazole Moiety for Marine Antifouling Applications: Synthesis and Biological Activity Evaluation. Marine Drugs. 2021; 19(1):5. https://doi.org/10.3390/md19010005

Chicago/Turabian Style

Pereira, Daniela, Catarina Gonçalves, Beatriz T. Martins, Andreia Palmeira, Vitor Vasconcelos, Madalena Pinto, Joana R. Almeida, Marta Correia-da-Silva, and Honorina Cidade. 2021. "Flavonoid Glycosides with a Triazole Moiety for Marine Antifouling Applications: Synthesis and Biological Activity Evaluation" Marine Drugs 19, no. 1: 5. https://doi.org/10.3390/md19010005

APA Style

Pereira, D., Gonçalves, C., Martins, B. T., Palmeira, A., Vasconcelos, V., Pinto, M., Almeida, J. R., Correia-da-Silva, M., & Cidade, H. (2021). Flavonoid Glycosides with a Triazole Moiety for Marine Antifouling Applications: Synthesis and Biological Activity Evaluation. Marine Drugs, 19(1), 5. https://doi.org/10.3390/md19010005

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