Synthesis. Phenoxyacetic acid was synthesized in 65% yield, a result similar to that reported by Yan et al. (2014), who achieved a yield of 67%. Initially, this acid was characterized by analyzing its mass spectrum. The appearance of the ion peak at m/z = 152 corresponds to the molecular mass of this compound.
The structural confirmation of phenoxyacetic acid was possible by analyzing the 1H NMR spectrum. The appearance of the signal at δH: 4.62 (s, 2H) corresponds to the hydrogens of the CH2-COOH group. Additionally, other signals observed in the hydrogen linked to the carbon of the aromatic ring region also contributed to the identification of the acid.
The structure of phenoxyacetic acid is part of the chemical backbone of the herbicides 2,4-D (2,4-dichloroacetic acid), 2,4-DB (2,4-diclophenoxybutyric acid), and 2,4,5-T (trichlorophenoxyacetic acid). The herbicide 2,4-D was developed by the British during the Second World War (1939–1945) due to the need to increase food production. Together with 2,4,5-T, this compound was used as Agent Orange in the Vietnam War (1954–1975)45.
Phenoxyacid class herbicides are used in agriculture in the form of quaternary ammonium salts (2,4-D dimethylammonium salt) or esters, such as 2,4-D-isooctyl ester (trade name), which can be more easily absorbed by plants. Herbicides that contain esters derived from 2,4-D have the trade name “2,4-D Ester”. Generally, 2,4-D derivatives are salts or esters containing a long-chain alkyl group, designed to reducing volatility and minimizing environmental damage46.
Structural modifications in molecules can lead to compounds with greater biological activity47. Considering this fact, phenoxyacetic acid was used in the synthesis of seven compounds containing the phenyl 2-phenoxyacetate nucleus through Steglich esterification, using DCC and DMAP in the presence of phenolic compounds (thymol, vanillin, eugenol, carvacrol, guaiacol, p-cresol, and β-naphthol), resulting in the formation of their respective esters: 2-isopropyl-5-methylphenyl 2-phenoxyacetate (1), 5-formyl-2-methoxyphenyl 2-phenoxyacetate (2), 4-allyl-2-methoxyphenyl 2-phenoxyacetate (3), 5-isopropyl-2-methylphenyl 2-phenoxyacetate (4), 2-methoxyphenyl 2-phenoxyacetate (5), p-tolyl 2-phenoxyacetate (6), and naphthalen-2-yl 2-phenoxyacetate (7).
In Scheme 2, the proposed mechanism for the Steglich Esterification Reaction was presented. Firstly, DCC, together with the carboxylic acid, forms a reactive intermediate that facilitates the nucleophilic attack of the hydroxyl on the electron-deficient carbon of the formed amide, thus generating the corresponding ester. DMAP is a strong nucleophile that is responsible for two functions in solution: firstly, it acts as an acyl transfer reagent, suppressing side reactions responsible for the formation of unwanted byproducts in the Steglich reaction. The second function is to form an amide intermediate that is much more reactive than the intermediate formed by the added DCC, facilitating the nucleophilic attack of the hydroxyl on the carbonyl. To obtain a good yield in this step, it is important to keep the system without the presence of water, as water would compete in the nucleophilic attack with the hydroxyl, causing a shift in the chemical equilibrium towards the formation of the starting reagent48,49.
Scheme 2. General mechanism for Steglich esterification reaction48,49.
All synthesized compounds were characterized by hydrogen-1 and carbon-13 nuclear magnetic resonance (1H and 13C NMR) spectroscopy, as well as mass spectrometry. These spectra are available in the ‘Supplementary Material’.
In addition to phenoxyacetic acid being part of the chemical structure of the herbicides 2,4-D, 2,4-DB, and 2,4,5-T, some of the phenols used in the Steglich esterification reactions were chosen because they have previously been reported to exhibit phytotoxic potential by our research group6,8,30, such as carvacrol, thymol, eugenol, and guaiacol. There are citations reporting the biological activities associated with phenols used as starting material. For instance, vanillin exhibits antitumor activity36, p-cresol demonstrates antibacterial activity37, and cytotoxic activity50. However, there are no reports in the literature regarding the phytotoxic activity of the phenolic compounds vanillin, p-cresol, and β-naphthol. However, several studies have investigated phenolic compounds as a type of autotoxins that exert harmful effects on plant growth. These compounds not only affect soil microbial communities but are also considered potential autotoxins capable of altering soil microbial conditions13. Considering these noted activities, phytotoxicity tests were conducted for these phenolic compounds.
Phytotoxicity of compounds 1–7. Esters 1–7 were subjected to evaluation for their phytotoxic potential on parameters such as germination (GP), germination speed index (GSI), root growth (RG), and aerial growth (AG) in L. sativa and S. bicolor. Among the esters analyzed, only those derived from eugenol and guaiacol, designated as esters 3 and 5, demonstrated a significant influence on the germination of L. sativa (eudicotyledonous). Notably, the concentration of 3 mmol L⁻¹ of the ester 3 showed an effect comparable to the positive control represented by the herbicide 2,4-D. The ester 5, at concentrations of 3 mmol L⁻¹ and 1.5 mmol L⁻¹, exhibited statistical similarity in relation to the positive control 2,4-D. Additionally, the concentration of 0.75 mmol L⁻¹ of the ester 5 also caused a significant reduction in the germination rate, as illustrated in Fig. 2.
Ester 3 exhibited a significant influence on the germination rate of S. bicolor, manifesting itself in a similar way to the herbicide 2,4-D when present in its highest concentration of 3 mmol L⁻¹. At concentrations of 3 mmol L⁻¹, 1.5 mmol L⁻¹, and 0.75 mmol L⁻¹ of ester 5, a similar reducing effect was observed, with concentrations of 3 mmol L⁻¹ and 1.5 mmol L⁻¹ showed an impact comparable to the 2,4-D herbicide. These results are represented in Fig. 3.
The GSI of L. sativa was affected by the ester 3, at 3 mmol L− 1, providing a strong inhibitory effect, being similar to the positive control 2,4-D. This same inhibitory effect was observed in seeds treated with ester 5, at concentrations of 1.5 and 3 mmol L− 1, resembling the positive control provided by the herbicide 2,4-D.
On the other hand, stimulatory effects on this variable were observed in seeds treated with ester 2, derived from vanillin, at concentrations of 0.75 mmol L− 1 and 0.1875 mmol L− 1 in relation to the solvent (Fig. 4) .
In the context of sorghum, esters 3 and 5 exerted adverse influences on the Germination Speed Index (GSI). When applied to seeds, the ester 3 at a concentration of 3 mmol L⁻¹ showed an effect similar to the herbicide 2,4-D. However, concentrations of 3 mmol L⁻¹ and 1.5 mmol L⁻¹ of the ester 5 reduced the sorghum GSI, resembling the positive control provided by the 2,4-D herbicide.
Regarding root growth of L. sativa, esters 3 and 5 showed an inhibitory effect compared to the negative control. Specifically, the presence of the ester 3 at a concentration of 3 mmol L⁻¹ inhibited this variable, showing statistical similarity with the herbicide 2,4-D. On the other hand, the ester 5 at concentrations of 3 mmol L⁻¹ and 1.5 mmol L⁻¹ exhibited an inhibitory effect, statistically comparable to the 2,4-D herbicide. In contrast, esters 1, 2, 4, 6, and 7 promoted stimulation of lettuce root growth, as shown in Fig. 7.
In relation to S. bicolor, root growth was inhibited by esters 1, 4, and 5 at a concentration of 3 mmol L− 1, being similar to 2,4-D. However, the ester 3 had a stimulating effect on this same variable (Fig. 7).
The aerial growth of L. sativa was affected by the ester 2 at a concentration of 3 mmol L− 1, promoting the reduction of this variable, equal to the positive control 2,4-D. In relation to the ester 5, concentrations of 0.75 and 1.5 mmol L− 1 caused a reduction in AG, being similar to the 2,4-D herbicide (Fig. 8).
The ester 3 inhibited the aerial growth of S. bicolor, with the greatest reduction in the length of the aerial part of the seedlings observed in treatments with this ester. All concentrations of the ester 3 resulted in a reduction in growth, with the concentration of 3 mmol L− 1 having a similar effect to the 2,4-D herbicide (Fig. 9).
Esters 3 and 5 are the most promising among those tested, as they presented the most significant results in the initial development of both L. sativa and S. bicolor. Ester 3, derived from eugenol, led to reduced germination, GSI, and RG of L. sativa, while in S. bicolor, it resulted in reduced germination, GSI, and AG. Vitalini et al51when working with eugenol at a concentration of 2 µL, observed the inhibition of the germination of Echinochloa oryzoides seeds. This same dose was able to significantly reduce the germination and GSI of Sinapis alba and Lolium multiflorum, as well as root and shoot swelling.
The ester 5, derived from guaiacol, reduced the germination of L. sativa and S. bicolor, the GSI of L. sativa and S. bicolor, the AG of L. sativa, and the RG of L. sativa and S. bicolor. Alves et al. (2021) observed similar effects when analyzing the impacts of guaiacol and guaiacoxyacetic acid, noting reductions in germination, GSI, RG, and AG of both L. sativa and S. bicolor. As observed for guaiacoxyacetic acid, guaiacol ester affected the development of model plants even at lower concentrations.
Kasugai et al.53 observed the breakdown of the cell wall of bacteria treated with guaiacol. Meanwhile, Alves et al.30found the death of meristematic cells in L. sativa roots exposed to guaiacol. The authors suggested that the possible action of guaiacol on cell walls may have led to the loss of essential molecules, compromising nutrient transport and cellular respiration in the organism.
The root growth of S. bicolor was affected by the treatment containing esters 1, 4, and 5. Studies involving thymol and its derivative, thymoxyacetic acid, reported the ability of these compounds to affect the germination percentage of L. sativa and S. bicolor at a concentration of 3 mmol L− 1 (ALVES et al.6). Inhibitory effects were also observed on the GSI of seeds of L. sativa, S. bicolor, Cucumis sativus L., and Amaranthus viridis L7. Studies report the ability of thymol to inhibit the growth of monocotyledonous and eudicot plants through the production of reactive oxygen species (ROS)52, corroborating the results obtained in this work.
Phytotoxic effects on seed germination and root growth of Hypericum perforatum exposed to Thymus pulegioides oil of the carvacrol chemotype and pure carvacrol were also observed54. Carvacrol also showed a strong phytotoxic effect on Amaranthus retroflexus, Avena fatua, Portulaca oleracea, and Erigeron bonariensis in post-emergence trials. These weeds were controlled for a period of 24 hours to 30 days, with higher doses showing greater and faster phytotoxic effects. These effects were related to the possible oxidative damage of this compound, which may have caused the reduction in growth and loss of photosynthetic pigments found55.
Cytogenotoxicity. The cytogenotoxicity of treatments with esters 1–7 was investigated in relation to changes in the mitotic index (MI), chromosomal alterarions (CA), and nuclear alterations (NA) in L. sativa root cells. Regarding the MI, the ester 3 demonstrated a significant increase in all concentrations compared to the negative control. Cells exposed to esters 4, 5, and 6 also showed an increase in the MI.
In the case of the ester 4, concentrations of 1.5 mmol L− 1 and 3 mmol L− 1 were responsible for this increase. For the ester 5, all concentrations induced an increase in MI, except the concentration of 3 mmol L− 1, which resulted in a reduction in MI comparable to the effect of the 2,4-D herbicide. The ester 6, at concentrations of 0.187 mmol L− 1 and 0.375 mmol L− 1, promoted an increase in MI (Fig. 10).
At a concentration of 3 mmol L− 1, the ester 5 completely inhibited the lettuce MI. Using concentrations of 3 mmol L− 1 and 1.5 mmol L− 1 of the ester 7, a reduction in the MI was observed, with the effect of the concentration of 1.5 mmol L− 1 being similar to that of 2,4-D (Fig. 10). No changes in the MI were identified in cells treated with esters 1, 2, and 3 (Fig. 10).
As for CA, an increase in this variable was observed in all treatments, with the exception of treatments containing esters 6 and 7. With these molecules, a reduction in the rate of this variable was observed, except at a concentration of 0.375 mmol L− 1 of the ester 6, which did not differ from the negative control. The ester 1 provided an increase in the CA rate at a concentration of 0.375 mmol L− 1. An increase was also recorded at concentrations of 3 mmol L− 1 and 0.75 mmol L− 1 of the ester 2 (Fig. 11).
At all concentrations tested, the ester 3 increased CA. In cells treated with the ester 4, there was a significant increase in CA according to increasing concentrations. In relation to the ester 5, at a concentration of 1.5 mmol L− 1, there was an increase in the rate of chromosomal alterations, while at a concentration of 0.375 mmol L− 1, it was reduced. Regarding the rate of AN, micronucleus, and condensed nucleus, no significant differences were found in relation to the negative control (Fig. 11).
Considering individual chromosomal alterations, late chromosomes, chromosomal breakage, bridge in anaphase and telophase, c-metaphase, adherent chromosome, and lost chromosome were observed (Fig. 12).
For chromosomal adhesion, an increase was recorded in cells treated with esters 1, 2, 3, and 4 in relation to the negative control. At concentrations of 0.75 mmol L− 1 and 0.375 mmol L− 1 of the ester 1, there was an increase in the rate of adherent chromosomes. For treatment with the ester 2, the increase was observed at concentrations of 3 mmol L− 1, 0.75 mmol L− 1, and 0.375 mmol L− 1. Only at a concentration of 1.5 mmol L− 1 did the ester 3 have a different effect than the negative control (Fig. 12). The ester 4, on the other hand, was responsible for the increase in the rate of adherent chromosomes only at concentrations of 0.375 mmol L− 1 and 0.1875 mmol L− 1 (Fig. 12).
Considering the c-metaphase rate, an increase was recorded in cells treated with the ester 3 at a concentration of 3 mmol L− 1, when compared to the negative control (Fig. 13).
There was an induction of an increase in the rate of late chromosomes (Fig. 13) by ester 4 at all concentrations except 0.75 mmol L− 1 and 0.187 mmol L− 1, and by ester 5 at a concentration of 0.75 mmol L− 1 (Fig. 13).
Cell cycle assessment can explain the negative effects found on early seedling development. In this study, cytotoxicity was estimated by the effect of esters 1–7 on the mitotic index.
MI is a variable used to indicate the cytotoxicity of a substance through its increase or reduction56. The increase in the MI is related to the disordered proliferation of unhealthy cells with a high frequency of chromosomal alterations. Although the rate of cell division is higher, the presence of these chromosomal alterations triggers cell cycle block8. This relationship was observed in this study, as the esters 3, 4, and 5, derived from eugenol, carvacrol, and guaiacol, respectively, showed an increase in both the MI and the CA rate. The increase in the MI was also observed in L. sativa treated with eugenoxyacetic acid30.
The reduction in MI is related to the action of chemical agents in the mitotic cycle, preventing cell proliferation and thus tissue growth56. The reduction in root growth (L. sativa and S. bicolor) and aerial growth (only S. bicolor) of plants exposed to a higher concentration of ester 5, derived from guaiacol, can be explained by the mitodepressant effect of this molecule. Alves et al. (2021) also observed a decrease in MI in L. sativa meristematic cells treated with guaiacol and guaiacoxyacetic acid.
The increased frequency of CA observed in this work indicates a genotoxic action on mitotic division, resulting in alterations in the structure and/or number of chromosomes. According to these alterations, these effects can be classified as clastogenic and aneugenic56. The clastogenic effect occurs when the agent interacts with the DNA molecule, inducing chromosomal breaks56. This alteration is due to chromosomal fragments derived from breaks in the genetic material57, and this effect was not statistically significant in this work.
Aneugenic agents act on cytoplasmic structures, such as the mitotic spindle, causing errors in chromosome segregation. These agents can also result in the elimination of genetic material and the formation of polyploid cells. Some examples are the formation of bridges, losses, late chromosomes, chromosomal adhesions, and c-metaphases56.
Esters 1, 2, 3, and 4, derived, respectively, from thymol, vanillin, eugenol, and carvacrol, induced an increase in the number of adherent chromosomes. Chromosomal adhesion is characterized by the loss of normal DNA condensation characteristics. The action of toxic agents on histones or other proteins responsible for chromatin organization leads to heterochromatinization of the nucleus, activating cell death mechanisms.
Cell death may explain the reduction in growth of roots and aerial part of L. sativa and S. bicolor seedlings exposed to esters 1, 3, and 558. Alves et al. 30 found a 5-fold increase in the rate of adherent chromosomes in cells treated with eugenol and eugenoxyacetic acid.
The ester 3, derived from eugenol, also induced an increase in cells with c-metaphase. C-metaphase occurs due to the inactivation of microtubules, preventing the formation of the mitotic spindle, which in its absence, leads to the random orientation of chromosomes on the equatorial plate, paralyzing the cycle in metaphase56.
All molecules, with the exception of the ester 2 derived from vanillin, caused the appearance of late chromosomes. These occur when the chromosomes are not connected with the spindle fiber and can move to either pole of the cell, leading to the formation of daughter cells with an unequal number of chromosomes 59.
The chemical structure of esters may have facilitated the interaction of thymol, eugenol, carvacrol, and guaiacol with cells, due to changes in the membrane structure. Its effects can be associated with the production of ROS, generating oxidative stress.
This stress can alter several physiological processes, such as mitochondrial respiration, and the distribution and organization of microtubules60. This is reflected in the aneugenic mode of action of these molecules. Thus, we can speculate that oxidative stress caused damage to the mitotic spindle of exposed cells, leading to the appearance of adherent chromosomes, c-metaphases, and lagging chromosomes.