Synthesis of Eugenol Derivatives and Evaluation of their

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Current Pharmaceutical Design, 2020, 26, 1-11 1 RESEARCH ARTICLE

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Synthesis of Eugenol Derivatives and Evaluation of their Antifungal Activity Against Fusarium solani f. sp. piperis

Sarah Canal Maximinoa#, Jessyca Aparecida Paes Dutraa#, Ricardo Pereira Rodriguesa, Rita de Cássia Ribeiro Gonçalvesa, Pedro Alves Bezerra de Moraisb, José Aires Venturac, Ricardo Pinto Schuencka, Valdemar La-cerda Júniord, Rodrigo Rezende Kitagawaa# and Warley de Souza Borgesa,d#*

aGraduate Program of Pharmaceutical Sciences, Health Sciences Center, Federal University of Espírito Santo, Avenida Marechal Campos 1468, Maruípe, 29047-105,Vitória, ES, Brazil. bDepartment of Chemistry and Physics, Exact, Natural and Health Sciences Center, Federal University of Espírito Santo, Alto Universitário, s/n, Guararema, Alegre, ES, Brazil. cCapixaba Institute for Re-search, Technical Assistance and Rural Extension, Rua Afonso Sarlo 160, Bento Ferreira, 29052-010, Vitória, ES, Brazil. dDepartment of Chemistry, Exact Sciences Center, Federal University of Espírito Santo, Avenida Fernando Ferrari 514, Goiabeiras, 29075-910, Vitória, ES, Brazil

A R T I C L E H I S T O R Y

Received: October 31, 2019 Accepted: March 13, 2020 DOI:

10.2174/1381612826666200403120448

Abstract: Background: Fusarium solani f. sp. piperis is a phytopathogen that causes one of the most destructive diseases in black pepper crops, resulting in significant economic and crop production losses. Consequently, the control of this fungal disease is a matter of current and relevant interest in agriculture. Objective: The objective was to synthesize eugenol derivatives with antifungal activity. Methods: In this study, using bimolecular nucleophilic substitution and click chemistry approaches, four new and three known eugenol derivatives were obtained. The eugenol derivatives were characterized and their antifungal and cytotoxic effects were evaluated. Results: Eugenol derivative 4 (2-(4-allyl-2-methoxyphenoxy)-3-chloronaphthalene-1,4-dione) was the most ac-tive against F. solani f. sp. piperis and showed acceptable cytotoxicity. Compound 4 was two-fold more effective than tebuconazole in an antifungal assay and presented similar cytotoxicity in macrophages. The in silico study of β-glucosidase suggests a potential interaction of 4 with amino acid residues by a cation-π interaction with residue Arg177 followed by a hydrogen bond with Glu596, indicating an important role in the interactions with 4, justifying the antifungal action of this compound. In addition, the cytotoxicity after metabolism was evaluated as a mimic assay with the S9 fraction in HepG2 cells. Compound 4 demonstrated maintenance of cytotoxicity, showing IC50 values of 11.18 ± 0.5 and 9.04 ± 0.2 µg mL-1 without and with the S9 fraction, respectively. In contrast, eugenol (257.9 ± 0.4 and 133.5 ± 0.8 µg mL-1), tebuconazole (34.94 ± 0.2 and 26.76 ± 0.17 µg mL-1) and especially car-bendazim (251.0 ± 0.30 and 34.7 ± 0.10 µg mL-1) showed greater cytotoxicity after hepatic biotransformation. Conclusion: The results suggest that 4 is a potential candidate for use in the design of new and effective com-pounds that could control this pathogen.

Keywords: Antifungal, eugenol derivatives, cytotoxicity, Piper nigrum, Fusarium solani f. sp. piperis.

1. INTRODUCTION The black pepper (Piper nigrum L.) is the most-consumed spice species in the world. It is mainly cultivated in Asian countries such as India, Indonesia, Thailand, Vietnam and China, as well as in South America, in Brazil [1-2]. Brazil is the third-largest producer of pepper in the world and the fifth largest exporter of this com-modity [3]. The Brazilian national harvest reached almost 79 thou-sand tons in 2017 [4]. However, the phytopathogen Fusarium solani f. sp. piperis has been responsible for a decline in productivity and an increase in production costs for Brazilian black pepper crops [5-7]. The infec-tion causes vessel obstruction leading to root rot, or Fusarium dis-ease (fusariosis). The first symptoms start at the roots or branches and promote yellowing and falling of leaves, stem blight, root rot, and plant death. This fungus produces secondary metabolites, such as naphthoquinones and fusaric acid, which have toxigenic proper-ties capable of inducing root rot and rust [5-6, 8-10]. *Address correspondence to this author at the Graduate Program of Pharma-ceutical Sciences, Health Sciences Center, Federal University of Espírito Santo, Avenida Marechal Campos 1468, Maruípe, 29047-105,Vitória, ES, Brazil; E-mail:

Control measures have been taken to increase black pepper resistance to fusariosis, including micrografting, micropropagation, and in-vitro plant regeneration. However, the narrow genetic vari-ability and the homogeneous plantations of this crop have led to the spread of the fungus [11-12]. Strategies for F. solani f. sp. piperis control are limited because there is no officially approved fungicide in Brazil and information on resistant cultivars is scarce [8]. Nevertheless, synthetic fungi-cides, such as carbendazim and tebuconazole, have been widely used for control of this fungal disease. Their indiscriminate use has led to the appearance of resistant isolates that increase production costs [10,13]. Furthermore, the World Health Organization classifies some fungicides, including carbendazim and tebuconazole, as hazardous chemicals and as possible human carcinogens and teratogens, re-spectively. In addition, with respect to carbendazim, its repeated applications lead to accumulation in and contamination of various ecosystems [14-15]. These undesirable effects have induced a search for new and effective fungicide agents, mainly from natural resources [7,16-18]. Recently, plant-derived natural antifungals have been reported as alternatives to chemical fungicides. The application of essential oils has attracted much attention as a method of controlling fungal

2 Current Pharmaceutical Design, 2020, Vol. 26, No. 00 Maximino et al.

infections due to their antimicrobial properties and low toxicity [5,19]. Eugenol (4-allyl-2-methoxyphenol) is the major constituent of essential oil from Syzygium aromaticum (clove). This molecule exhibits several applications in the pharmaceutical, cosmetic, food, and agricultural industries [20]. It has a wide range of biological activities, including antifungal characteristics [21]. Furthermore, eugenol has structural standards that enable the semi-synthesis of compounds with applicability in the pharmaceutical and agro-chemical industries [22-23]. Various studies of the antifungal activ-ity of eugenol and analogs against phytopathogenic fungi have been reported [16,19]. However, there are few reports on the antifungal activity of eugenol against F. solani f. sp. piperis. Thus, we report here the synthesis of a series of eugenol deriva-tives and their antifungal activity and cytotoxic effects.

2. MATERIALS AND METHODS

2.1 General Experimental Procedures Solvents with analytical grades and purity higher than 99.5% were purchased from Synth (São Paulo, SP, Brazil). Dimethylfor-mamide, commercial azides, commercial halides, potassium car-bonate, sodium ascorbate, copper sulfate, and deuterated solvents (CDCl3 and DMSO-D6) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received. Microwave reactions were conducted using a CEM Discover Synthesis Unit (CEM Corp., Matthews, USA). The machine consists of a continuous focused microwave power delivery system with operator-selectable power output from 0 to 300 W. Reactions were performed in glass vessels (capacity 10 mL) sealed with a septum. For the open layer chroma-tography, a 2.5 × 25 cm glass column filled with silica gel was used as the stationary phase with a particle size of 0.04-0.063 mm, packed in hexane:ethyl acetate(7:3) as the mobile phase. Nuclear magnetic resonance (NMR) spectra were recovered on a Varian 400 MHz instrument (Palo Alto, USA) using deuterated chloroform (CDCl3) and dimethylsulfoxide (DMSO-D6) as the solvent and tetramethylsilane (TMS) as the internal standard. The chemical shifts (δ) are reported in ppm and J values in Hertz (Hz). High-resolution mass spectra were obtained with a quadrupole time-of-flight instrument (UltrOTOF-Q, Bruker Daltonics, Billerica, MA, USA) equipped with an ESI ion source by direct injection of the compound dissolved in methanol (MeOH). The high-resolution mass spectrometry (HR-ESI-MS) of all compounds was acquired using a capillary voltage of 3,900 V, dry gas flow at 4 L hr-1, and nitrogen as the nebulizer gas. The infrared (IR) spectrum was re-corded on a Bruker Tensor 27 FT-IR Spectrometer (Bremen, Ger-many) scanning from 4000 to 500 cm-1. 2.2. Synthesis of eugenol derivatives Eugenol was isolated from clove essential oil according to the standard procedure [24], using hydrodistillation. The synthetic pro-cedures used to obtain compounds 1-7 followed the methodology described by Brito et al. [25] with modifications. Synthesis of eugenol derivatives is shown in Scheme 1. In a 25-mL round bottom flask containing eugenol (1 equiva-lent) in acetone (5 mL), under stirring and ice bath, potassium car-bonate (1 equivalent) was added. The system was left 30 min under this condition. Commercial halides (1.5 equiv.) were then added and the system was kept at room temperature under stirring for over 12 h. The reaction solutions were concentrated under reduced pres-sure and the final products were purified by column chromatogra-phy using silica gel as the stationary phase and hexane:acetate (7:3, v/v) as the mobile phase to obtain compounds 1-5. In a microwave tube (10 mL capacity) containing compound 5 (1 equivalent) in 1.0 mL DMF, 4 equivalents of commercial azides, 0.1 equivalents of sodium ascorbate and 0.03 equivalents of a 0.1 mol L-1 solution of CuSO4 were added. Subsequently, the tube was

sealed and microwave irradiated at 150 W and 70 °C for 20 min. After this process, the reaction solutions were concentrated under reduced pressure and the final products were purified by column chromatography using silica gel as the stationary phase and hex-ane:acetate (7:3, v/v) as mobile phase to obtain compounds 6 and 7. Scheme 1. (i) potassium carbonate, acetone, and commercial halide, R.T.; (ii) sodium ascorbate, dimethylformamide, copper sulfate and commercial azides, MW; 70 °C.

Compound 1 (4-allyl-2-methoxy-1-((4-nitrobenzyl)oxy) ben-zene) was obtained as a yellowish oil with a yield of 16.4% (0.015 g, 0.05 mmol). TLC: Rf = 0.33 (hexane:ethyl ether, 4:1, v/v); FT-IR (Attenuated total reflection - ATR) vmax / cm-1 2920, 1604, 1509, 1229. 1H NMR (400 MHz, CDCl3) δ 8.22 (d, 2H, J 8.6 Hz, CH), 7.62 (d, 2H, J 8.6 Hz, CH), 6.77 (d, 1H, J 8.2 Hz, CH), 6.76 (brs, 1H, CH), 6.67 (dd, 1H, J 1.9; 8.2 Hz, CH), 5.95 (m, 1H, CH), 5.21 (s, 2H, CH2), 5.08 (m, 1H, CH2), 5.06 (m, 1H, CH2), 3.89 (s, 3H, CH3), 3.34 (d, 2H, J 6.6 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ 149.7 (CO), 147.5 (CN), 145.8 (CO), 145.0 (C), 137.4 (CH), 134.3 (C), 127.5 (2C), 123.8 (2H), 120.4 (CH), 115.8 (CH2), 114.6 (CH), 112.5 (CH), 70.2 (CH2), 56.0 (OCH3), 39.8 (CH2). These data are consistent with the literature [26]. HR-ESI-MS [M + H]+ Found: 300.1230, Calc. for C17H18NO4

+ 300.1230; [M + Na]+ Found: 322.1051, Calc. for C17H17NNaO4

+ 322.1050; [2M + Na]+ Found: 621.2221, Calc. for C34H34N2NaO8

+ 621. 2207. Compound 2 (2-(4-allyl-2-methoxyphenoxy)acetonitrile) was also obtained as a yellowish oil, with a yield of 62.6% (0.770 g, 3.7 mmol). TLC: Rf = 0.25 (hexane:ethyl ether, 9:1, v/v); FT-IR (ATR) vmax / cm-1 2984, 2360, 1740, 1243, 1047. 1H NMR (400 MHz, CDCl3) δ 6.97 (d, 1H, J 7.8 Hz, CH), 6.75 (m, 2H, CH), 5.93 (m, 1H, CH), 5.10 (d, 1H, J 6.2 Hz, CH2), 5.07 (s, 1H, CH2), 3.85 (s, 3H, CH3), 3.33 (d, 2H, J 5.7 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ 150.4 (CO), 144.0 (CO), 137.1 (C), 137.0 (CH), 120.8 (CH), 117.9 (CH), 116.1 (CH2), 115.4 (CN), 112.8 (CH), 55.8 (OCH2), 39.9 (CH2). HR-ESI-MS [M + H]+ Found: 204.1019, Calc. for C12H14NO2

+ 204.1019; [M + Na]+ Found: 226.0837, Calc. for C12H13NNaO2

+ 226.0838; [M + K]+ Found: 243.0991, Calc. for C12H13KNO2

+ 242.0578. Compound 3 (4-allyl-2-methoxy-1-((2-((phenylsulfonyl) methyl)benzyl)oxy)benzene) was obtained as a white powder with a yield of 31.4% (0.039 g, 0.95 mmol). TLC: Rf = 0.65 (hexane:ethyl acetate, 4:1, v/v); FT-IR (ATR) vmax / cm-1 2918, 1519, 1235, 1035, 688. 1H NMR (400 MHz, CDCl3) δ 7.65 (d, 2H, J 7.8 Hz, CH), 7.60 (t, 1H, J 7.8 Hz, CH), 7.44 (t, 2H, J 7.8 Hz, CH), 7.40 (d, 1H, J 7.4 Hz, CH), 7.32 (t, 1H, J 7.4 Hz, CH), 7.22 (t, 1H, J 7.4 Hz, CH), 7.07 (d, 1H, J 7.4 Hz, CH), 6.82 (d, 1H, J 8.2 Hz, CH), 6.73

O

OH i

O

OR

R=NO2

CNS

O O Cl

O

O

1 2 3 54

O

O ii

O

O

5

NNN

R'

R'= NO2SOO

6 7

NN

NR'

+

Synthesis of Eugenol Derivatives and Evaluation of their Antifungal Activity Against Current Pharmaceutical Design, 2020, Vol. 26, No. 00 3

(brs, 1H, CH), 6.69 (brd, 1H, J 8.2 Hz, CH), 5.97 (m, 1H, CH), 5.09 (m, 2H, CH2), 4.95 (s, 2H, CH2), 4.68 (s, 2H, CH2), 3.83 (s, 3H, CH3), 3.35 (d, 2H, J 6.6 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ 150.1 (CO), 146.1 (CO), 138.4 (CS), 137.5 (CH), 137.2 (C), 134.3 (C), 133.7 (CH), 132.6 (CH), 130.1 (CH), 129.1 (CH), 129.0 (2CH), 128.6 (2CH), 128.3 (CH), 127.2 (C), 120.5 (CH), 115.9 (CH), 115.7 (CH2), 112.4 (CH), 70.5 (OCH2), 59.4 (SCH2), 55.7 (OCH3), 39.9 (CH2). HR-ESI-MS [M + H]+ Found: 409.1468, Calc. for C24H25O4S+ 409.1468; [M + Na]+ Found: 431.1285, Calc. for C24H24NaO4S+ 431.1288; [M + K]+ Found: 447.1024, Calc. for C24H24KO4S+ 447.1027; [2M + Na]+ Found: 839.2661, Calc. for C48H48NaO8S2

+ 839.2683. Compound 4 (2-(4-allyl-2-methoxyphenoxy)-3-chloronaph-thalene-1,4-dione) was obtained as a red powder with a yield of 57.7% (0.124 g, 0.35 mmol). TLC: Rf = 0.24 (hexane:ethyl ether, 4:1, v/v); FT-IR (ATR) vmax / cm-1 2923, 1673, 1666, 1264, 712. 1H NMR (400 MHz, CDCl3) δ 8.18 (dd, 1H, J 1.5; 7.4 Hz, CH), 8.01 (dd, 1H, J 1.5; 7.0 Hz, CH), 7.76 (ddd, 1H, J 1.5; 7.0; 7.4 Hz, CH), 7.72 (dt, 1H, J 1.5; 7.4 Hz, CH), 7.00 (d, 1H, J 8.6 Hz, CH), 6.76 (m, 1H, CH), 6.76 (m, 1H, CH), 5.95 (m, 1H, CH), 5.11 (m, 1H, CH2), 5.07 (t, 1H, J 1.5 Hz, CH2), 3.73 (s, 3H, CH3), 3.37 (d, 2H, J 7.0 Hz CH2). 13C NMR (100 MHz, CDCl3) δ 178.6 (CO), 177.5 (CO), 155.1 (C), 148.7 (CO), 144.0 (CO), 137.1 (CH), 137.0 (C), 134.3 (CH), 134.0 (CH), 131.2 (C), 130.5 (C), 128.3 (CCl), 127.1 (CH), 126.9 (CH), 121.1 (CH), 118.3 (CH), 116.1 (CH2), 113.2 (CH), 56.2 (OCH3), 39.9 (CH2). HR-ESI-MS [M + H]+ Found: 355.0730, Calc. for C20H16ClO4

+ 355.0732; [M + Na]+ Found: 377.0545, Calc. for C20H15ClNaO4

+ 377.0551; [2M + Na]+ Found: 731.1199, Calc. for C40H30Cl2NaO8

+ 731.1210. Compound 6 (4-((4-allyl-2-methoxyphenoxy)methyl)-1-(4-nitrobenzyl)-1H-1,2,3-triazole) was obtained as a yellowish oil with a yield of 71.3% (0.067 g, 0.17 mmol). TLC: Rf = 0.37 (hex-ane:ethyl acetate, 1:1, v/v); FT-IR (ATR) vmax / cm-1 3065, 1517, 1232, 837. 1H NMR (400 MHz, DMSO-D6) δ 8.31 (s, 1H, CH), 8.24 (d, 2H, J 8.6 Hz, CH), 7.52 (d, 2H, J 8.6 Hz, CH), 7.01 (d, 1H, J 8.2 Hz, CH), 6.79 (brs, 1H, CH), 6.67 (brd, 1H, J 8.2 Hz, CH), 5.93 (m, 1H, CH), 5.80 (s, 2H, CH2), 5.09 (s, 2H, CH2), 5.04 (m, 2H, CH2), 3.29 (d, 2H, J 6.6 Hz, CH2). 13C NMR (100 MHz, DMSO-D6) δ 149.1 (CO), 147.2 (CN), 145.6 (CO), 143.4 (C), 143.3 (CN), 137.9 (CH), 133.0 (C), 129.0 (2CH), 125.1 (CN), 123.9 (2CH), 120.1 (CH), 115.5 (CH2), 114.3 (CH), 112.5 (CH), 61.9 (OCH2), 55.4 (OCH3), 51.9 (NCH2), 39.1 (CH2). These data are consistent with the literature [27]. HR-ESI-MS [M + H]+ Found: 381.1557, Calc. for C20H21N4O4

+ 381.1557; [2M + Na]+ Found: 783.2847, Calc. for C40H40N8NaO8

+ 783.2861. Compound 7 (4-((4-allyl-2-methoxyphenoxy)methyl)-1-(2-((phenylsulfonyl)methyl)benzyl)-1H-1,2,3-triazole) was obtained as a yellowish oil in quantitative yield. TLC: Rf = 0.75 (hexane:ethyl ether, 7:3, v/v); FT-IR (ATR) vmax / cm-1 2933, 1516, 1303, 1124, 691. 1H NMR (400 MHz, CDCl3) δ 8.15 (s, 1H, CH), 7.82 (d, 2H, J 7.4, CH), 7.76 (t, 1H, J 7.4 Hz, CH), 7.64 (t, 2H, J 7.4, CH), 7.32 (t, 1H, J 7.4 Hz, CH), 7.24 (t, 1H, J 7.4 Hz, CH), 7.10 (d, 1H, J 7.4 Hz, CH), 7.04 (d, 1H, J 7.4 Hz, CH), 6.97 (d, 1H, J 7.8 Hz, CH), 6.76 (brs, 1H, CH), 6.65 (brd, 1H, J 7.8 Hz, CH), 5.91 (m, 1H, CH), 5.67 (s, 2H, NCH2), 5.04 (s, 2H, OCH2), 5.03 (m, 2H, CH2), 4.91 (s, 2H, SCH2), 3.83 (s, 3H, OCH3), 3.26 (d, 2H, J 6.6 Hz, CH2). 13C NMR (100 MHz, CDCl3) δ 149.1 (CO), 145.6 (CO), 143.4 (CN), 138.4 (CS), 137.9 (CH), 136.4 (C), 134.1 (CH), 133.1 (CH), 133.0 (C), 129.3 (CH), 129.2 (2CH), 128.7 (CH), 128.1 (2CH), 128.1 (CH), 124.8 (NCH), 120.1 (CH), 115.5 (CH2), 114.3 (CH), 112.5 (CH), 61.9 (OCH2), 57.7 (SCH2), 55.4 (OCH3), 50.0 (OCH2), 38.9 (CH2). HR-ESI-MS [M + H]+ Found: 490.1796, Calc. for C27H28N3O4S+ 490.1795; [M + Na]+ Found: 512.1620, Calc. for C27H27N3NaO4S+ 512.1614; [M + K]+ Found: 528.1359, Calc. for C27H27N3KO4S+ 528.1354.

2.3. Antifungal Assays

2.3.1. Fungal Strain Fusarium solani f. sp. Piperis strain (E-053) was obtained from the Incaper Phytopathology Laboratory fungi collection, Brazil. The fungus was cultivated on potato dextrose agar (PDA) and incubated at 25 °C for 7 days with a 12 h photoperiod.

2.3.2. Inoculum Preparation For the preparation of the inoculum, the mycelium was used. The fungus cultured on PDA was covered with 1 mL of 0.85% sterile saline solution, obtaining a homogeneous suspension. For the broth microdilution method, the conidial suspension, with an opti-cal density of 0.15–0.17, was prepared in saline solution (0.85%) by turbidimetry and diluted 1:50 to obtain a standardized inoculum (0.4x104 to 5x104 conidia mL-1).

2.3.3. Broth Microdilution Method The minimum inhibitory concentration (MIC) was determined by the broth microdilution method according to standardized proto-col M38-A2 of the Clinical Laboratory Standards Institute [28]. For this analysis, the eugenol derivatives were tested at different con-centrations. The plates were then incubated at 28 °C and analyzed after 48 hours by visual inspection of the turbidity. The MIC was defined as the lowest concentration of the substance at which the tested micro-organism did not show visible growth. The experi-ments were performed in triplicate. Tebuconazole and carbendazim were used as positive controls.

2.3.4. Determination of Minimum Fungicidal Concentration The minimum fungicidal concentrations (MFCs) were deter-mined according to the methodology previously described by Xie et al. [29], with some adaptations. The MFCs were determined as the lowest concentrations resulting in no growth in the culture after 2 days.

2.4. In silico Methods

2.4.1. Homology Modeling The search for potential models for Fusarium solani f. sp. piperis was performed with the SWISS-MODEL server (SMTL, last update: 2019-05-15, latest version of PDB included: 2019-05-10). The target sequence was searched with the BLAST server [30]. The geometry of the resulting model was normalized by force field calculation [31].

2.4.2. Homology Model Validation The quality of the model was evaluated using the parameterized QMEAN score function for the SWISS-MODEL server [32].

2.4.3. Docking Calculations

Molecular docking calculations were performed with the mod-eled protein-ligand complex of β-glucosidase from Fusarium solani sp. based on the PDB code: 5JU6 in GOLD software v.5 (Cam-bridge Crystallographic Data Center (CCDC)) [33-34], selecting the score function Goldscore. The grid region for docking was set over the glucose binding site, comprising an 11 Å radius sphere. The docking runs from the genetic algorithm were set to 10 runs per ligand. Docking poses were ranked using the Goldscore score func-tion, and the docking parameters were validated according to the redocking results.

2.5. Cytotoxicity Assay

2.5.1. Cell Lineages Murine macrophages RAW 264.7 (ATCC® TIB-71™) and Human hepatoma cells (HepG2) (ATCC® CRL-10741™) were


provided by the Banco de Células do Rio de Janeiro, Brazil (BCRJ). Macrophages were maintained in Dulbecco's Modified Eagle Me-dium (DMEM) and HepG2 in DMEM/F-12, which were supple-mented with 10% fetal bovine serum and incubated at 37 °C with a 5% CO2 atmosphere until reaching the confluence about 70-90%. Macrophages were dissociated using a cell scraper, and HepG2 cells were trypsinized and counted in a Neubauer chamber to obtain the required concentration for the cytotoxicity assay.

2.5.2. Basal Cytotoxicity Aliquots (0.2 mL) of the medium containing the RAW cell suspension were seeded into 96-well tissue-culture plates at 105 cells mL-1. Then, the plates were incubated at 37 oC under 5% CO2 for 24 h for adherence. After this period, the medium was removed, and the cells were treated with medium modified with various con-centrations of eugenol and eugenol derivatives (50-400 µgmL-1) or unmodified medium (control) and incubated for 24 h under the same conditions. Afterward, the medium was removed, and the plates were prepared for the MTT-tetrazolium assay [35]. Tebu-conazole and carbendazim were used as controls to compare their cytotoxic effect. The assays were performed in triplicate and re-peated at least three times.

2.5.3. Cytotoxicity in Metabolic Activation System (S9 fraction) Aliquots (0.2 mL) of the medium containing HepG2 cells at the concentration 3x105 cells mL-1 were seeded into 96-well tissue-culture plates and incubated at 37 oC under 5% CO2 for 24 h for adherence. After this time, the medium was removed, and the cells were treated with a modified medium with various concentrations of eugenol and its’ derivatives, with or without the S9 system. The system consisted of 10% S9 fraction, 1% 0.4 M MgCl2, 1% 1.65 M KCl, 0.5%, 1 M d-glucose-6-phosphate disodium, 4% 0.1 M β-nicotinamide adenine dinucleotide, 54% 0.2 M phosphate buffer, and 29.5% sterile distilled water. After incubation for 24 hours at 37 oC under 5% CO2, the medium was removed and the basal cyto-toxicity and metabolism-mediated cytotoxicity were evaluated us-ing an MTT-tetrazolium assay. Tebuconazole and carbendazim were used as benchmarks to evaluate the cytotoxicity, and cyclo-phosphamide was used as a positive metabolism control. The con-centrations ranged from 12.5 to 1600 µg mL-1 for cyclophos-phamide and carbendazim, 6.25 to 400 µg mL-1 for eugenol, 3.12 to 200 µg mL-1 for compound 4 and 6.25 to 200 µg mL-1 for tebucona-zole. The assay was performed in triplicates and repeated at least two times [36-37].

2.6. Statistical Analysis Linear regression analysis with 95% confidence limits was used to define a dose-response curve and to compute the inhibitory con-centration, that is, the concentration needed to reduce absorbance of the system by 50% (IC50), the so-called cytotoxic midpoint, in both cytotoxic assays. All results were obtained from three independent experiments and expressed as mean ± SD (n=3). Statistically sig-nificant differences were determined by two-way analysis of vari-ance with post-hoc Bonferroni test (using the GraphPad Prism 5.0 statistics package). Means were considered statistically significant at p  <  0.05.

3. RESULTS AND DISCUSSION Natural products have been considered as a source for obtaining agricultural fungicides; essential clove oil and eugenol have shown excellent antifungal activity against filamentous fungi, including Fusarium species [16,18-19]. Structural modifications were carried out on the phenol group of eugenol using bimolecular nucleophilic substitution and click chemistry approaches, which resulted in seven derivatives. The synthesis and spectroscopic characterization of compounds 1, 5, and 6 have been described previously [26-27]. Compounds 2-4 and

7 are new and are chemically described here for the first time. Their chemical structures were determined by spectrometric and spectro-scopic methods, such as uni and bi-dimensional 1H and 13C NMR, FT-IR, and HR-ESI-MS. For all obtained compounds, the signals observed in the NMR spectra (δ in ppm) related to eugenol moiety were similar. Compounds 2-4 were obtained through bimolecular nucleo-philic substitution reaction. For compound 2, apart from the signals related to eugenol moi-ety, a hydrogen singlet (2H) was observed at δ 4.75 ppm bound to carbon at δ 55.8 ppm, indicating etherification with the methylene-cyano group. The carbon related to the cyano group was observed at δ 115.4 ppm. The structure was confirmed by HRESIMS Found: 204.1019, which is consistent with the formula C12H13NO2. For compound 3, the data obtained with HRESIMS established the molecular formula to be C24H24O4S. Hydrogen singlets were observed at 4.68 ppm (2H) and 4.95 ppm (2H), related to benziloxy and methylenesulfoxide groups, respectively. In the 13C NMR spec-trum, two methylene groups were observed at δ 59.4 ppm and 70.5 ppm. Four aromatic hydrogens were also observed at δ 7.07 ppm (d, 7.4 Hz), 7.22 ppm (t, 7.4 Hz), 7.32 ppm (t, 7.4 Hz) and 7.40 ppm (d, 7.4 Hz), confirming the presence of a 1,2-disubstitute ring in the structure. Three additional aromatic hydrogen signals were ob-served at δ 7.44 ppm (t, 2H, 7.8 Hz), 7.60 ppm (t, 7.8 Hz), and 7.65 ppm (d, 2H, 7.8 Hz), indicating a monosubstituted ring in the struc-ture and confirming the final structure of 3. With respect to compound 4, the 13C NMR spectrum showed 10 signals related to naphtoquinone moiety. Signals observed at δ 178.6 ppm and 177.5 ppm were related to ketone groups. Four addi-tional aromatic hydrogens were observed at δ 7.72 ppm (dt, 1.5; 7.4 Hz), 7.76 ppm (ddd, 1.5; 7.0; 7.4 Hz), 8.01 ppm (dd, 1.5; 7.0 Hz) and 8.18 ppm (dd, 1.5; 7.4 Hz), confirming a 1.2-disubstitute aro-matic ring in the structure. The molecular formula C20H15ClO4 was confirmed by HRESIMS. Compounds 6-7 were obtained through the click chemistry reaction. The hydrogen related to the triazole ring was observed at δ 8.31 ppm (s) in compound 6 and at δ 8.15 ppm (s) in compound 7. The carbons of the triazole ring were observed at δ 125.1 ppm and 143.3 ppm and at δ 124.9 ppm and 143.4 ppm for compounds 6 and 7, respectively. The benzyl group was observed at δ 5.80 ppm (s, 2H) and 5.67 ppm (s, 2H) in compounds 6 and 7, respectively. A meth-ylenesulfoxide group was observed at δ 4.91 (s, 2H) and δ 57.7 ppm in compound 7. For compound 6, four aromatic hydrogens were observed at δ 7.52 ppm (d, 2H, 8.6 Hz) and δ 8.24 ppm (d, 2H, 8.6 Hz). The mo-lecular formula C20H20N4O4 was confirmed by HRESIMS. For compound 7, four aromatic hydrogens were present at δ 7.04 ppm (d, 7.4 Hz), 7.10 ppm (d, 7.4 Hz), 7.24 ppm (t, 7.4 Hz) and 7.32 ppm (t, 7.4 Hz), confirming the presence of a 1,2-disubstituted ring in the structure. Three additional aromatic hydro-gen signals were observed at δ 7.64 ppm (t, 2H, 7.4 Hz), 7.76 ppm (t, 7.4 Hz), and 7.82 ppm (d, 2H, 7.4 Hz), indicating a monosubsti-tuted ring in the structure and confirming the final structure of 7. The molecular formula C27H27N3O4S was confirmed by HRESIMS.

3.1. Antifungal Activity The Fusarium genus is one of the main economic problems in agricultural and food production, causing serious damage to the production and reducing product quality [16]. In black pepper (Piper nigrum L.) crops affected by fusariosis (caused by Fusarium solani f. sp. piperis), there is an annual reduction of 3% in produc-tion. This severe disease can reduce a pepper crop’s productive cycle to 5 to 6 years, whereas a healthy pepper crop has a produc-tive cycle of 12 years on average [10].


The antifungal activities of eugenol and its derivatives were evaluated using Fusarium solani f. sp. piperis native isolate. Anti-fungal activity was evaluated based on minimum inhibitory concen-tration (MIC) and minimum fungicidal concentration (MFC). The fungicides tebuconazole and carbendazim, which are used in agri-culture, were included as positive controls. The results, shown in Table 1, indicate that compound 4 was the most active eugenol derivative to affect fungus viability. Compounds with MICs above 400 µg mL-1 were not evaluated for fungicidal activity. According to the results, eugenol exhibited fungistatic activity at 400 µg mL-1. In the literature, there are several reports of antifungal activity of eugenol or essential clove oil against filamentous fungi of Fusarium spp. [18,22,38-39] but only a few reports for F. solani f. sp. piperis. Eugenol’s antifungal activity might be due to its high lipophil-icity and the presence of functional groups. These characteristics allow this compound to move through the plasma membrane, caus-ing alteration of lipids in the fungal membrane, increasing perme-ability and consequently, causing cell content extravasation [38-40]. Compound 4 exhibited antifungal activity superior to that of tebuconazole, an important yieldsince Fusarium solani f. sp. piperis has shown resistance to tebuconazole compared to other Fusarium species, such as Fusarium oxysporum [16]. The promising antifungal activity of compound 4 is probably due to the eugenol scaffold and its association with the naphthoqui-none moiety in the structure. In the development of agrochemicals, naphthoquinones are extremely useful compounds due to their rela-tively non-toxic nature and their redox and acid–base properties, which can be synthetically modulated [41-42]. These compounds are recognized by their antimicrobial activity, including activity against filamentous fungi. The addition of halogen groups increases antifungal activity due to the ability to remove electrons and form free radicals [43-44]. Table 1. Antifungal activity of eugenol derivatives against F.

solani f. sp. piperis.

Fusarium solani f. sp. piperis Compounds

MIC (µg mL-1) MFC (µg mL-1)

Eugenol 400 >400

1 >400 Nt

2 >400 Nt

3 >400 Nt

5 25 50

6 >400 Nt

7 >400 Nt

Tebuconazole 50 100

Carbendazim 1.56 1.56

Nt (no tested)

3.2. Docking Studies on Eugenol Derivatives Many advances have been made in the study of phytopatho-genic fungi at the molecular and cellular levels, enabling the under-standing of the mechanisms of interaction between new phyto-chemicals and phytopathogenic species, such as Fusarium solani f. sp. piperis [5,45-46]. From this perspective, β-glucosidase proteins from fungi are attractive and promising targets for the design of new antifungal agents to control Fusarium disease in plants [5].

β-glucosidase enzyme plays an important role in various bio-logical processes. It occurs in most living organisms and catalyses the hydrolysis of O-glucosidic bonds, releasing glucose units [47]. In the binding site, the glucose chemical structure is in its β-anomeric state, adopting a C1 chair conformation. The glucose molecule is stabilized in a region between the N-terminal domain and a cleft comprised of several loops. The chemical structure of glucose is well stabilized by several hydrogen bonds to the amino acids Asp171, Arg235, His269, Asp354, and Glu596 with in a short dis-tance of 2.6–2.9 Å (Fig. 1). Fig. (1). Major amino acids involved in the β-glucosidase binding site of F. Solani: Asp171, Arg235, His269, Asp354, and Glu596. (A higher resolution / colour version of this figure is available in the electronic copy of the arti-cle). The docking accuracy was evaluated by redocking, i.e., extract-ing the co-crystal ligand (glucose) from the protein-ligand complex and performing its placement in the binding site through docking calculations. The resulting docking poses were then compared to the protein-ligand complex by measuring the Root Mean Square Deviation (RMSD), obtaining a value of 0.37 Å for the top docked pose and an average RMSD of 1.168 Å for the other docking poses from this calculation (Fig. 2). Fig. (2). Redocking of glucose in the generated model of β-glucosidase from F. solani. RMSD deviation at 0.37 Å and average RMSD = 1.168 Å. (A higher resolution / colour version of this figure is available in the electronic copy of the article).


Table 2. Docking results of selected compounds.

Compound Docking Score

Eugenol 56.820

1 69.005

2 49.892

3 85.884

4 67.812

6 78.652

7 91.353

Fig. (3). Docking results of the evaluated compounds: a) eugenol; b) Compound 1; c) Compound 2; d) Compound 3; e) Compound 4; f) Compound 6; g) Com-pound 7. (A higher resolution / colour version of this figure is available in the electronic copy of the article).


The evaluated compounds placed properly at the β-glucosidase active site, extending its docking from the catalytic core to a chan-nel comprised of aromatic residues, conferring an array of π-π in-teractions (Fig. 4). The docking score also indicated a potential for binding with score values ranging from 56 to 91 (Table 2), whereas the glucose top-ranked docking poses range from 57 to 62. The docking results indicated the potential for eugenol to form hydrogen bonds with the amino acids Lys268 (3.3 Å), Ser540 (3.0 Å), and Asp171 (2.6 Å). Part of its backbone is also stabilized by a π-interaction with Trp355 (Fig. 4a). Compound 1 is well stabilized by two additional π-stacking interactions with the amino acids Tyr598 (3.2 Å) and Trp380 (3.4 Å). Compounds 2, 3, and 6 presented an allyl-metoxy benzene ring system stabilized by π-stacking interac-tions, mainly with the amino acids Tyr598 and Trp380, ranging from 3.2 to 5.9 Å. The polar groups are stabilized by hydrogen bonds with nearby histidine and arginine residues (Fig. 3). The analysis of the docking poses at the β-glucosidase binding site of compound 4 indicates that its naphtoquinone ring system could be stabilized by a cation-π interaction with Arg177 residue followed by a hydrogen bond with Glu596. Compound 4 is also stabilized by π-stacking interactions in the allyl-metoxy benzene moiety with Trp380 and Tyr598 residues. These interactions could result in enhanced stability compared to the other evaluated com-pounds (Fig. 4f). The docking results suggest a potential for the evaluated compounds to bind to the β-glucosidase binding site, highlighting that the activity of compound 4 could be enhanced by the additional interactions it can produce at the active site.

3.3. Cytotoxic Effects of Eugenol Derivatives The use of agrochemicals as fungicides may result in exposure of the environment and humans to these chemicals. Humans could be exposed during production or application, or through the consumption of food products contaminated with residues of these chemicals [48]. The toxic profile of tebuconazole has been investigated by the European Food Safety Authority. Studies have shown effects on the endocrine organs, including hypertrophy and malformations of the adrenal glands, leading it to be classified as a developmental toxin [49]. Regarding carbendazim, this fungicide has been banned in the USA, most of the European Union and Australia due to its severe toxicity and persistent nature. Repeated applications of it lead to accumulation and contamination of various ecosystems, with long-lasting impacts on soil sustainability and human and animal health. However, developing countries, such as China, Brazil, and India, are still permitting its production and use [14]. Thus, in vitro models have been used to study the cytotoxicity pattern of new drugs or other foreign compounds [48]. In this study, the basal cytotoxicity of compounds that pre-sented antifungal activity was evaluated on a macrophage lineage to investigate their safety for use. The IC50 values, i.e., the compound concentrations that inhibit 50% of cell growth, are shown in Table 3. Values means ± standard deviation of two distinct experiments performed in triplicate. IC50 (concentration of tested compounds that reduce cell viability by 50%); Raw 264.7 (lineage of murine macrophages). Significantly different at level of p < 0.05 according to Bonferroni’s test.

Eugenol showed cytotoxicity to macrophages at the highest concentrations (200-400 µg mL-1) however, it was less cytotoxic than tebuconazole at these concentrations (p<0.05). These results confirm that the cytotoxicity and antifungal activity of eugenol are related to the same structural patterns, mainly due to the presence of a double bond in the allyl chain [38].

Table 3. Inhibitory concentration (IC50) values in µg mL-1 of

eugenol compounds and standards after treatment.

Compounds Raw 264.7

IC50

Eugenol 100.8 ± 0.79***

4 21.61 ± 0.23*

Tebuconazol 23.83 ± 0.72*

Carbendazim 25.98 ± 0.07*

Compound 4 showed a cytotoxicity profile similar to that of tebuconazole. Most likely, this is due to the presence of electro-philic carbons and the quinone nucleus from the naphthalene ring, which are capable of inducing the formation of deleterious radicals through redox cycles [50-52]. The variation in substitution of the eugenol molecule and the change in structural patterns could explain the observed differences in cytotoxicity results, as observed by Bar et al. [53] and Xie et al. [16]. The xenobiotic-metabolizing capacity is a central point for in vitro/in vivo comparisons in toxicity evaluation. The bioactivation is an important consideration for in vitro cytotoxicity assays, since the compound test may be biotransformed in vivo [54]. Thus, in vitro methods using the S9 system have been used as an important tool to study the biotransformation patterns of chemi-cals [55-56]. The use of the S9 fraction from liver is the most ap-propriate screening system compared to hepatocytes and micro-somes. The S9 fraction includes the phase I and II metabolic en-zymes, and it is relatively inexpensive, easy to use, and amenable to automation. Microsomes report phase I metabolism only, whereas hepatocytes are not easily automated [56]. In this study, we evaluated the cytotoxicity of antifungal active compounds on hepatic cells (HepG2) in the presence and absence of the S9 metabolic system. Dose-response curves for compounds and standards are shown in Fig. 4. The cyclophosphamide results confirm the assay efficiency in the cytotoxic evaluation post-metabolization. The dose-response curves obtained before and after the metabolic activation of tebu-conazole were similar, while carbendazim was bioactivated in the presence of the S9 system, resulting in an IC50 value 7 times lower than that of carbendazim in the absence of S9. Eugenol presented changes in dose-response curves before and after metabolization, with an increase in cytotoxicity of about 2 times (Fig. 5). The IC50 values of compound 4 in the presence and absence of S9 system were not statistically significantly different (ρ<0.05). Table 4 shows the IC50 values of the compounds in the absence and pres-ence of the metabolizing system. The results obtained with eugenol exposure to the S9 hepatic fraction were similar to those found by other authors. They reported increased eugenol cytotoxicity in the presence of the hepatic S9 fraction of rats or hamsters in hepatoma cells [57-58]. Babich et al. [57] reported that eugenol’s cytotoxicity after S9 exposure is related to depletion of the intracellular glutathione level. Maralhas et al. [59] also showed that eugenol, in the presence of S9, exhibited increases in cytotoxicity and genotoxicity. Compound 4 metabolization may be associated with the detoxi-fication of naphthoquinones by quinine oxido reductase enzyme (QR). According to Cuendet et al. [60], the reduction of electro-philic quinones by the QR enzymes is an important detoxification


Fig. (4). Dose-response curves for cyclophosphamide (A), tebuconazole (B), carbendazim, (C), eugenol (D), and compound 4 (E) in HepG2 cells in the pres-ence and absence of the metabolic activation system (S9 hepatic fraction). The data represent the mean ± SD of a distinct experiment performed in triplicate. (A higher resolution / colour version of this figure is available in the electronic copy of the article). route, converting quinines to hydroquinones. However, no signifi-cant difference in IC50 value was observed in compound 4 cytotox-icity in the presence and absence of the S9 system, indicating that either this compound was not metabolized or it was metabolized into another compound with the same cytotoxic profile. In recent years, tebuconazole toxicity has been reported as hav-ing hepatotoxic effects, causing long-term hepatocellular tumors, liver weight increase, and centrilobular hypertrophy in rats and mice [49,61-62]. Carbendazim toxicity has also been reported. It is known to manifest embryotoxicity, germ cell apoptosis, teratogene-sis, infertility, and developmental toxicity in different mammalian species [14].

In contrast with the toxicity reported for tebuconazole and car-bendazim, demonstrating hazards to human health primarily due to hepatotoxic effects [14,49,61,63], compound 4 is a promising can-didate for further studies and the development of a new agricultural fungicide against Fusarium solani f. sp. piperis. Future investigations aimed at mechanisms of action and geno-toxic activity are necessary for a better understanding of the cyto-toxic effects and for the determination of the potential of the com-pounds reported in this work as future agricultural fungicides. Con-sidering that other heteroaromatic ring-containing compounds dis-played biomedical potential due to their ability to interact with nu-cleic acids [64-66], also eugenol derivatives are interesting candi-dates as therapeutics that deserve future exploration of their DNA and RNA-binding ability.


Table 4. Inhibitory concentration (IC50) values of samples on human hepatocarcinoma cell line (HepG2) in the pres-ence and absence of the S9 hepatic system.

HepG2

IC50 (µg mL-1) Compounds

- S9 + S9

Eugenol 257.9 ± 0.4* 133.5 ± 0.8*†ab

Compound 4 11.8 ± 0.5* 9.04 ± 0.2*‡ab

Tebuconazole 34.94 ± 0.20* 26.76 ± 0.17*†

Carbendazim 251.0 ± 0.30* 34.71 ± 0.10*†

Cyclophosphamide 412.7 ± 0.17 65.40 ± 0.12†

Values expressed as mean ± SD of up to two separate experiments per-formed in triplicate. *p<0.05 (ANOVA) indicates significantly different results between cyclophosphamide (+ S9) and the samples. † p<0.05 (ANOVA) indicates significantly different results between the samples in the presence of the metabolizing system (+ S9) and in the absence (-S9). ‡ p<0.05 (ANOVA) indicates significantly different results between eugenol (prototype) and compounds in the presence of the metabolizing system (+ S9). ap<0.05 (ANOVA) indicates significantly different results between carbendazim and eugenol derivative in the presence of the metabolizing system (+ S9). bp<0.05 (ANOVA) indicates significantly different results between tebuconazole and eugenol derivative in the presence of the metabo-lizing system (+ S9).

CONCLUSION It was possible to demonstrate that structural modifications of the eugenol molecule resulted in one effective compound. Com-pound 4 showed promising fungicidal activity against Fusarium solani f. sp. piperis. In addition, compound 4’s cytotoxic activity was similar to that of tebuconazole and was more stable than that of carbendazim after metabolization. Thus, this compound is the most promising for further optimization due to its antifungal potency being greater than that of eugenol and tebuconazole and its accept-able cytotoxicity profile.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE Not applicable.

HUMAN AND ANIMAL RIGHTS No Animals/Humans were used for studies that are the basis of this research.

CONSENT FOR PUBLICATION Not applicable.

AVAILABILITY OF DATA AND MATERIALS Not applicable.

FUNDING None.

CONFLICT OF INTEREST The authors declare no conflict of interest, financial or other-wise.

ACKNOWLEDGEMENTS This study was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001, by the National Council of Scientific and Technological Develop-

ment - CNPq and the Foundation of Support to Research and Inno-vation of Espírito Santo (FAPES PPE-Agro no 76418880/16 and 76419363/16). We would also like to acknowledge INCTBioNat (CNPq 465637/2014-0), NCQP-UFES for additional support. RPR also thanks CAPES for the scholarship granted. SUPPORTIVE/SUPPLEMENTARY MATERIAL Supplementary information (IR, NMR, and HRMS) is available as a PDF file.

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Synthesis of Eugenol Derivatives and Evaluation of their