Eugenol derivatives: synthesis, characterization, and evaluation of antibacterial and antioxidant activities

Eugenol is the major component of clove essential oil and has demonstrated relevant biological potential with well-known antimicrobial and antioxidant action. Therefore, this work carried out the synthesis, purification, characterization, and evaluation of the antioxidant and antibacterial potential of 19 eugenol derivatives. The derivatives were produced by esterification reactions in the hydroxyl group (−OH) of eugenol with different carboxylic acids and also by addition reactions in the double bond of the allyl group. The derivatives had a promising antibacterial potential, including a lower minimum inhibitory concentration of 500 μg/mL than eugenol (1000 μg/mL). In addition, the derivatives were active against bacterial strains (Escherichia coli, Staphylococcus aureus) that eugenol itself showed no activity, thus increasing the spectrum of antibacterial action. As for the antioxidant activity, it was observed that the derivatives that involved esterification reactions in the hydroxyl group (−OH) of the eugenol molecule’s phenol resulted in a significant reduction of the antioxidant action (IC50 > 100 μg/mL) when compared with the eugenol precursor molecule (IC50 = 4.38 μg/mL). On the other hand, the structural changes located in the double bond affected much more smoothly the capacity of capturing radicals than the starting molecule, also being obtained derivatives with proximal antioxidant capacity (IC50 = 19.30 μg/mL) to commercial standards such as Trolox (IC50 = 16.00 μg/mL). Electronic supplementary material The online version of this article (10.1186/s13065-018-0407-4) contains supplementary material, which is available to authorized users.


Introduction
Molecular modification in structures of biologically active substances that occur naturally is one of the main strategies to enhance healthy biological effects, as well as to reduce eventual side effects [1][2][3]. In 1998, it was estimated that 60% of the antitumor and anti-infective drugs that entered the market or under clinical trial originated from natural products [4] via structural modifications. More recent data (December 2014) show that of the 237 drugs used as anti-infectious agents (antibacterial, antifungal, parasitic, and antiviral), excluding vaccines, recognized by public health agencies in the world, 138 (approximately 58.30%) are products of natural origin or derived from natural products. Thus, it is clear that this is a line of research with great potential for obtaining new drugs [5].
Eugenol, a natural substance used as a target molecule for the manufacture of bioactive compounds, was first isolated in 1929 and its commercial production began in 1940 in the United States. It can be produced synthetically; however, it is mainly extracted from Ocimum tenuiflorum, Cassia fistula, Zieria smithii, and Pimenta racemosa. Classified as a phenylpropanoid of the allyl-phenol type, eugenol is a pale yellow oil with clove odor and spicy taste. With numerous applications in the pharmaceutical, food, agricultural, and cosmetics industries [6,7], it showed promising antimicrobial and antioxidant effects [8][9][10][11][12], when evaluated against the fungi Cladosporium spp. [13]. Other activities are reported in literature, such as antiviral [14,15], anti-inflammatory [16], and inhibitor of platelet aggregation [17]. In addition, its anti-Leishmania activity together with its low cytotoxicity qualifies it as a promising source of new leishmanicidal [18].

Open Access
*Correspondence: fmsilva1986@yahoo.com.br; felipe.maia@ifrn.edu.br 1 Instituto Federal de Educação, Ciência e Tecnologia do Rio Grande do Norte (IFRN), RN 233, Km 02 N°999, Chapada do Apodi, Apodi, RN 59700-000, Brazil Full list of author information is available at the end of the article This broad spectrum of biological activity makes eugenol a target molecule for structural modifications in order to produce substances with therapeutic properties. Currently, among the various clinical pathologies, bacterial infections and cellular oxidation stand out, both with serious implications for public health. Due to the acquisition of resistance and the mutational capacity of microorganisms, commercial antibiotics are in many cases incapable of fighting bacterial proliferation, resulting in failures in the treatment associated with multiresistant bacteria. Consequently, bacterial resistance has become a global concern regarding public health [19][20][21].
Aerobic organisms have the ability to produce free radicals that, in excess, can initiate chain reactions that damage cells or cause death to the latter. As a consequence, several diseases arise, especially cardiovascular and neurodegenerative diseases. The fight against the effects resulting from the production of free radicals has been the dietary use of antioxidant substances with a significant effect in the prevention of these diseases [22][23][24][25][26][27][28]. Eugenol, according to reports in literature, combats oxidative stress with beneficial effects on health.
Thus, in view of the broad spectrum of biological activities of eugenol, the present study aimed to obtain its derivatives by means of esterification and addition reactions. All were submitted to the evaluation of the antibacterial and antioxidant potential with very interesting results.

Results and discussion
Structural modifications from eugenol, carried out on the hydroxyl group and the olefinic bond, were represented in reaction Schemes 1 and 2, respectively.
Evaluation of the antibacterial activity of eugenol derivatives using the inhibition zone technique, measured in millimeter (mm), demonstrates the potential for inhibition of microbial growth by a given substance. According to literature, substances with inhibition halos less than 7 mm, greater than 7 and less than 16 mm, and greater than 16 mm are considered inactive, moderately active, and antibacterial potential, respectively [32,33]. The results presented in Table 1 show the inhibition zones (halos) presented by the eugenol derivatives against six bacterial strains.
According to the results ( Table 1), acetylation of eugenol did not result in any benefit, since esters 1, 2, and 3 showed no antibacterial action against any of the strains. Also, esterification with benzoic acid and its p-substituted derivatives yielded derivatives 4-9 which exhibited random but still insufficient activities relative to eugenol itself.
Regarding derivatives 10-18 resulting from double bond addition, 10-14, 17, and 18 showed random and insufficient activities relative to eugenol. However, according to the classification shown above, derivative 16 showed a strong antibiotic effect against Bacillus cereus and a moderate effect against Staphylococcus aureus, Streptococcus, and Klebsiella pneumoniae, but was inactive in cases of Pseudomonas aeruginosa and Escherichia coli. It was of interest to observe that compound 16, except for the bacterium P. aeruginosa, showed greater Scheme 1 Eugenol derivatives (1-11) through reactions in the hydroxyl groups activity than eugenol itself. In the case of derivative 15, a triacetyl derivative, it is of particular interest to highlight the high antibacterial activity (inhibition halo 12) against E. coli relative to eugenol (inhibition halo 0). By contrast, 15 was inactive (inhibition halo 0) against P. aeruginosa, whereas eugenol was active (inhibition halo 12).
A recent work [34] revealed antimicrobial activity for eugenol against strains E. coli and S. aureus, exhibiting inhibition halos with diameters of 9.25 and 7.75 mm, respectively. Although the results in the present study did not show activity for these strains, it is worth mentioning that the amounts (3 mg) applied in the first one were 13 times higher than those (0.2 mg) used in the present study. Of the derivatives priorly mentioned, those with inhibition halos greater than 6 mm were subjected to microdilution tests to determine the minimum inhibitory concentration (MIC) which prevents visible growth of the bacteria. Table 2 shows the results for derivatives 4-18 expressed in μg/mL. Among the compounds tested, 8 and 16 showed the highest activity in inhibiting the strains. Compound 16 had the highest activity of all the derivatives involved in this study, and regarding K. pneumoniae and B. cereus strains, it was two times more active than eugenol. In contrast, compound 8 exhibited, in comparative terms, strong antibiotic activity against the E. coli strain, where the eugenol itself is inactive. Whereas epoxide 16 from eugenol showed strong relative activity, the corresponding acetate 17 showed a marginal effect.
In previous work [35], an MIC of 1200 μg/mL was recorded for eugenol against S. aureus bacteria, consistent with an MIC of 1000 μg/mL determined in the present study. These comparative data show that, like eugenol, several of its derivatives have a promising antimicrobial potential.

Pseudomonas aeruginosa Escherichia coli Staphylococcus aureus Streptococcus Klebsiella pneumoniae Bacillus cereus
In the specific case of eugenol, the relationship between the hydroxyl group and the antioxidant action was observed in a previous study [26] through derivatives 2, 4, 5, 6, and 9, in which all presented IC 50 is lower than eugenol.
On the other hand, the chemical modification in the double bond, in the case of the derivatives 12, 14, 16, 18, and 19, also caused reduction in the antioxidant capacity against the radical DPPH, however, much lower than that caused by the esterification of the hydroxyl group. Thus, derivatives 16 (IC 50 19.3 μg/mL) and 18 (IC 50 32 μg/ mL), for example, showed antioxidant action close to the Trolox standard (IC 50 16 μg/mL).
Derivatives 12, 14, 16, 18, and 19, with higher antioxidant action than the others, have a structural characteristic capable of enhancing this action. Although with IC 50 values higher than eugenol, the results reflect the behavior of the substances in vitro; however, in living biological systems, the antioxidant activity varies, among others, with factors such as the reduction potential in the medium, the displacement capacity of the radical structure formed, the ability to complex transition metals involved in the oxidative process, access to the site of action according to hydrophilicity or lipophilicity, and its partition coefficient [39,40]. The partition coefficient is closely related to the hydrophilic (or hydrophobic) character of the molecule. In the case of derivatives 12, 14, 16, 18, and 19, although less active than eugenol, the hydrophilicity is substantially different, especially for 12 and 14, which have additional hydroxyl groups allowing a higher degree of hydration and, consequently, greater interaction in aqueous media.

Conclusions
It was possible to demonstrate that structural modifications in the eugenol molecule resulted in some potentially antibacterial substances (e. g., 8, 15, 16). In addition, various derivatives (9, 10, 12, 13, 14, 15, 16, 17, and 18) have greater power in inhibiting the growth of certain strains regarding eugenol, as in the case of Streptococcus bacteria.
Regarding the antioxidant capacity of the derivatives, the study contributed to make an empirical evaluation of the structure-activity relationship, being observed that the hydroxyl group is decisive in inhibiting the propagation of free radicals. On the other hand, changes in the olefinic bond, although resulting in a slight reduction in the capacity to capture DPPH radicals, and the increase in the hydrophilic character can compensate and contribute as a differential in the antioxidant action.

General methods
GC-MS analyses were performed using a Shimadzu QP2010SE Plus instrument equipped with a Rtx ® -5MS (5% phenyl)-dimethylpolysiloxane capillary column (30 m × 0.25 mm) with a film thickness of 0.1 µm using He as carrier gas (1.0 mL/min) in split mode; the injector and detector temperatures were 240 and 280 °C, respectively; column temperature was programmed at 5 °C/ min from 60 to 80 °C (3 min), then at 30 °C/min to 280 °C  [43]. 14: Eugenol (820 mg, 5 mmol) in CH 2 Cl 2 (5 mL) was added dropwise to m-chloroperbenzoic acid (1.30 g) in CH 2 Cl 2 (15 mL) at 25 °C. After stirring for 24 h, 10% aq. Na 2 SO 3 (10 mL) was added to the mixture and the solution was washed two times with 5% NaHCO 3 (25 mL). The CH 2 Cl 2 layer was dried (Na 2 SO 4 ) and concentrated [44]. The reaction product (360 mg, 2 mmol) in 20% NaOH (10 mL) was heated at 80 °C for 2 h. The reaction mixture was cooled (28 °C), diluted with water, and neutralized with 10% HCl to pH 7.0. The water was removed under reduced pressure and the resultant mass was extracted with anhydrous EtOH (5 × 10 mL . Each suspension was further diluted to a final concentration of 1 × 10 6 NTU in NaCl solution (0.85%) with 10% MHB. A volume of 100 μL of each suspension was distributed into the wells of the microplates resulting in a final inoculum concentration of 5 × 10 5 NTU. The initial solution of the eugenol derivatives was made using 10 mg of each dissolved in 1 mL of DMSO/water (1:1). From this concentration (10 mg/mL), several dilutions were made in distilled water in order to obtain a stock solution of 2000 µg/mL. Further serial dilutions were performed in microplates by addition of MHB (100 µL) to reach a final concentration in the range of 7.8-1000 μg/mL. All the experiments were performed in triplicate and the microdilution trays were incubated in bacteriological oven at 35 °C for 24 h. After this period, the antibacterial activity was detected using a colorimetric method by adding 25 µL of the resazurin staining (0.01%) aqueous solution in each well of the microplate. The minimum inhibitory concentration (MIC) was defined as the lowest extract concentration that can inhibit bacterial growth, as indicated by resazurin staining (dead bacterial cells are not able to change the staining color by visual observation-blue to red).

Free radical scavenging activity (DPPH Assay)
The free radical scavenging activity was determined by the DPPH assay [46,47]. 2 mL of various concentrations (10,20,30,50,70, 100 µg/mL) of the compounds in methanol was added to 2 mL of a methanol solution of 6.6 × 10 −2 mM DPPH. The decrease in absorbance was determined at 517 nm at room temperature at 0 min, 1 min, and every 5 min for 1 h. For each antioxidant concentration tested, the reaction kinetics was plotted and from these graphs, the absorbance was read after 30 min. Inhibition of the DPPH radical in percent was calculated according to Eq. 1: Equation 1: Inhibition of the DPPH radical.
where Ablank is the absorbance of the control and Asample is the absorbance of the sample. Sample concentration providing 50% inhibition (IC 50 ) was calculated from the graph plotting inhibition percentage against sample concentration. Tests were carried out in triplicate, and Trolox and gallic acid were used as positive controls.