*2.1. Laccase-Mediated Synthesis of 2,6-Dimethoxy-4-(phenylimino)cyclohexa-2,5-dienone Derivatives*

In this work, laccase catalyzed reactions using **1** and **2a–j** were carried out. It has been previously reported that using laccases from different fungal sources (*Trametes* sp. and *Rhizoctonia praticola*), catalyze reactions between phenolic compounds and anilines, heterodimeric compounds are formed [25,26,31,32], similarly found in this work (Scheme 1).

**2j/3j** Cl H H H **Scheme 1.** Reaction scheme for laccase-mediated synthesis between **1** and **2a–j**.

**2f/3f** H Cl Cl H **2g/3g** H H CF3 H **2h/3h** H Cl H Cl **2i/3i** Cl H H Cl

To determine the reaction yields in the formation of the products, different substrate ratios were analyzed. Excluding reactions 1, 7 and 10, most reactions reached higher yields using ratio 1:1 (syringic acid:aniline) (Table 1). Moreover, when aniline was used as substrate, the same result was reported [27], indicating that the increase of the concentration of one of them decrease the yield of the obtained compounds.

Highest yields were obtained using 3-chloroaniline and 3,5-dichloroaniline as substrates (**2b** and **2h**) (Table 1). This high yield could be explained because the oxidation by laccase (from *Trametes versicolor*) of 3-chloroaniline does not occur [33]. On the other hand, using methoxyanilines (**2c** and **2d**) low yields were obtained, due to a high amount of side products (data not shown).

On the other hand, yield did not increase when the enzyme concentration was increased (data not shown). Bollag et al. [31] showed that the prolonged incubation or higher enzyme amounts caused further polymerization reaction decreasing cross-coupling formation. Furthermore, Itoh et al. [34] concluded that reactivity of laccase mediated reaction between phenolic acids and chlorophenols is due to the substrate specificity of the laccase rather than the chemical property of the substrates, which could explain the lack of relations among electron donating and withdrawing groups and yield of the reactions.


**Table 1.** Percentage yields of products at different reactant ratios.

<sup>1</sup> ND: Not determined.

The ten synthetized compounds were purified using semipreparative chromatography and were identified (Figure 1) using 1H-NMR and 13C-NMR spectra and mass spectrometry (Figure S1–S30, Supplementary Materials). Compound **3b** showed two aliphatic proton signals (δ 3.670 (s, 3H, H8) and δ 3.874 (s, 3H, H7)) and two aliphatic carbon signals (δ 56.212 and δ 56.321) that determined the presence of two methoxy moieties. Two olefin hydrogen signals at higher fields (δ 6.010 (d, 1H, H3 *J* = 1.9 Hz) and δ 6.368 (d, 1H, H5 *J* = 1.9 Hz)), the olefin carbon signals (δ 98.583 (C3) and δ 111.717 (C5)) and one carbon signal at δ 176.633 (C1) indicated the quinonoid character of the products. Table 2 presents the NMR data (chemical shift assignments for short and long-range heteronuclear coupling) of compound **3b**.

The only difference in spectra signals between compounds **3a** and **3b** (Figures S2, S3, S5 and S6) was in the aromatic region of the spectra. Compound **3b** showed four aromatic proton signals δ 6.737 (d, 1H, H6', *J* = 8.0 Hz), δ 6.888 (s, 1H, H2'), δ 7.159 (d, 1H, H4', *J* = 8.0 Hz), and δ 7.316 (t, 1H, H5', *J* = 8.0 Hz) that indicated the existence of a *m*-substituted aromatic fragment. The assignation of the entire molecule was achieved by using two-dimensional NMR analysis. Therefore, identifying this compound as 4-(3'-chlorophenylimino)-2,6-dimethoxycyclohexa-2,5-dienone (Figure 1).

**Figure 1.** Structure of synthetized compounds **3a–j** 2,6-dimethoxy-4-(phenylimino)cyclohexa-2,5-dienone derivatives.

**Table 2.** 1H- and 13C- chemical shifts assignments for compound **3b**. Information from HSQC and HMBC experiments are also provided.


The spectra of compounds **3c–j** (Figures S8, S9, S11, S12, S14, S15, S17, S18, S20, S21, S23, S24, S26, S27, S29 and S30) only showed differences in the aromatic region; the assignment of the 1H and 13C-NMR spectra can be found in the spectroscopic data section (Section 3.4). Figure 1 shows the structures of the ten synthetized compounds in this work. To our knowledge compounds **3a** and **3f** were previously synthesized [31], 1H-NMR spectra for compounds **3a** and **3f** (spectroscopic data Section 3.4) have the same number of signals and comparable chemical shifts and coupling constants like those found by Bollag et al. [31]; furthermore, the mass spectra of **3a** and **3f** showed a base peak with *m*/*z* 277 and 311, respectively, corresponding to the molecular ions, equivalent to the previously described data [31]. Therefore, the other eight compounds (**3b**, **3c**, **3d**, **3e**, **3g**, **3h**, **3i** and **3j**) have not been previously reported.

Interesting, compound **3a** was also obtained using syringaldehyde instead of syringic acid in the reaction with 4-chloroaniline. This could be explained with an extra step when using syringaldehyde, an oxidation of the aldehyde to a carboxylic acid (syringic acid), similar oxidations has been previously described using several aromatic aldehydes with laccase, yielding carboxylic acids [35]. Hence, syringaldehyde is oxidized to syringic acid and then the same product (compound **3a**) could be found in both reactions, starting with syringaldehyde or with syringic acid. However, this synthesis had a very low yield (data not shown).

#### *2.2. Antifungal Activity*

Antifungal activity of compounds **1** and **2a–j** and compounds **3a–j** against *B. cinerea* was measured on mycelial growth in solid media and the EC50 were calculated using the mycelial growth (Tables 3 and 4). The most active compound was the 3,5-dichloro-substituted product (compound **3h**), while compound **3c** had the lowest activity. It has been reported that the substituent affects the antifungal activity of a molecule [36], for instance, the position of the chlorine atom in the aromatic ring is important for the antifungal activity against *B. cinerea* since para-substituted compound (**3a**) and ortho-substituted compound (**3j**) were more active than the meta-substituted compound and unsubstituted compound (EC50 = 0.14 ± 0.02) [27], while activity of meta-substituted compound (compound **3b**) and unsubstituted compound are similar [27]. The number of chlorine atoms in the aromatic ring is also important, both dichlorinated compounds **3f** and **3h** showed higher antifungal activity than mono chlorinated compounds **3a**, **3b**, and **3j**, however, dichlorinated compound **3i** showed an antifungal activity comparable to the monochlorinated compounds, therefore, the number and position of chlorine atoms in the aromatic ring seems to be important for the antifungal activity of these compounds.

**Table 3.** Effect of compounds **3a–j** on the mycelial growth of *B. cinerea* in solid medium.


\* No activity.

**Table 4.** Effect of the substrates on the mycelial growth of *B. cinerea* in solid medium.


Furthermore, the methoxy derivative compounds (**3c** and **3d**) were less active against the fungus than the other compounds, even the nonsubstituted compound **3** [27], the same effect was observed for aspirin derivatives, where the methoxy para-substituted derivative showed almost 30% less antifungal activity against *B. cinerea* than the chlorinated para-substituted compound [37]. Similar behavior was previously reported for oxadiazole derivatives when tested the activity of the methoxy meta-substituted oxadiazole derivative against *B. cinerea*, and its activity was less than half compared to the nonsubstituted compound [38]. Usually, the chloro-substituted compounds have higher antifungal activity in commercial fungicides, for example, chlorine compounds like boscalid, chlorothalonil, and iprodione have been used to control *B. cinerea* [6]. The antifungal activity of iprodione has been tested against this strain of *B. cinerea*, showing an EC50 of 0.015 ± 0.003 mM [27], this antifungal activity is in the same order of magnitude than the most active compound obtained in this work (**3h**). Additionally, *p*-nitro and *p*-trifluoromethyl compounds (**3e** and **3g**) were tested against this fungus, **3e** showed no antifungal activity, probably because of the low solubility of this molecule, for this reason **3e** was not used in further assays. Compound **3g** only showed an intermediate antifungal activity compared to the rest of the synthetized molecules in this work. Lastly, most of the substrates used in the reactions (i.e., **1** and **2a–j**) showed lower antifungal activity than the products (Table 4), only **2f** was more active than **3f**.
