In Vitro Antifungal Activity of New and Known Geranylated Phenols against Phytophthora cinnamomi Rands

A series of new and known geranylated phenol/methoxyphenol derivatives has been tested in vitro as inhibitor agents of mycelial growth of Phytophthora cinnamomi. The activity of tested compounds is correlated with the nature, number, and position of the substituent group on the aromatic ring. Results indicate that the most active geranylated derivatives are those having two hydroxyl groups (or one –OH and one –OCH3) attached to the aromatic ring. Interestingly, these derivatives are as active as Metalaxil®, a commonly used commercial fungicide. Thus, our results suggest that some of these compounds might be of agricultural interest due to their potential use as fungicides against P. cinnamomi. The effect of structure on fungicide activity is discussed in terms of electronic distribution on both the aromatic ring and side geranyl chain. All tested compounds have been synthesized by direct coupling of geraniol and the respective phenol. Interestingly, new digeranylated derivatives were obtained by increasing the reaction time.

Compound 17: In the 1 H NMR spectrum of compound 17 two doublet aromatic signals at δH = 6.79 (d, J = 8.5 Hz, 1H) and 6.67 (d, J = 8.5 Hz, 1H) ppm were observed, suggesting a tetrasubstituted aromatic pattern. Additionally, two signals of methoxyl groups at δH = 3.90 (s, 3H) and 3.84 (s, 3H) ppm, and a signal at δH = 5.88 ppm (s, 1H), assigned to the OH group, were observed. However, this information is not sufficient to locate the position of the geranyl chain in the aromatic ring. However, from the 1D NOESY spectrum the signal of the OH group (s, δH = 5.88 ppm) showed a spatial correlation with the signal at δH = 3.30 ppm (d, J = 7.2 Hz, 2H), which was assigned to the H-1' and showed a spatial correlation with the signals at δH = 6.79 ppm (d, J = 8.5 Hz, 1H) and δH = 1.71 ppm (s, 3H) assigned to ArH-5 and CH3-C3', respectively (Figure S.1.2) This information was consistent with the position of the geranyl chain being on the C-6 carbon of the aromatic ring. In agreement with the information observed from the 2D HMBC spectrum, the signal of H-1' showed 3 JH-C heteronuclear correlations with C-1, C-5, and C-3' at δC = 147.2, 123.5, and 136.2 ppm, respectively, and 2 JH-C correlations with C-6 and C-2' at δC = 120.7 and 122.2 ppm, respectively (Figure S.1.2). Figure S2. Mayor spatial NOE correlations (blue arrows) and main 1 H-13 C HMBC correlations (red arrows) observed for compound 17.
Compound 18: for the structural determination of regioisomer 18 an analysis similar to that made for 17 was carried out. In the 1 H NMR spectrum of compound 18 two doublet aromatic signals at δH = 6.78 (d, J = 8.4 Hz, 1H) and 6.65 (d, J = 8.4 Hz, 1H) ppm were observed, suggesting a tetrasubstituted aromatic pattern and an ortho-coupling system between these two hydrogens. Additionally, two signals of methoxyl groups at δH = 3.92 (s, 3H) and 3.83 (s, 3H) ppm, and a signal at δH = 5.61 ppm (s, 1H), assigned to the OH group, were observed. However, from the 1D NOESY spectrum, one of the methoxy groups (s, δH = 3.83 ppm) showed a spatial correlation with the signal at δH = 3.27 ppm (d, J = 7.2 Hz, 2H), which was assigned to the H-1'. This information suggests that the methoxy group is attached to the C-3 of the aromatic ring and that the geranyl chain is in an ortho position with respect to the methoxy group. Additionally, H-1' showed spatial coupling with an aromatic hydrogen at δH = 6.78 ppm (d, J = 8.4 Hz, 1H), assigned to H-5. H-1' also showed spatial coupling with the signal at δH = 1.71 ppm (s, 3H), assigned to CH3-C3' of the geranyl chain ( Figure  S.1.3). The data obtained from the 2D HMBC spectrum confirmed the proposed structure for regioisomer 18. In this way, H-1' showed 3 JH-C heteronuclear correlations with C-3 and C-5 of aromatic ring at δC = 139.7 and 124.2 ppm, respectively, and with C-3' at δC = 135.8 ppm of geranyl chain, while H-1' showed 2 JH-C correlation with C-4 at δC = 126.9 ppm and with C-2' (geranyl chain) at δC = 123.1 ppm ( Figure S3). Figure S3. Mayor spatial NOE correlations (blue arrows) and main 1 H-13 C HMBC correlations (red arrows) observed for compound 18.
Compound 21: From the 1 H NMR spectrum, the presence of a single aromatic signal at δH = 6.32 ppm (s, 1H) suggests that the aromatic system is penta-substituted and that only one geranyl chain was incorporated in a single position of the aromatic ring. Therefore, the structural determination for compound 21 does not require further analysis of spectroscopic data. However, Figure S4 shows the major spatial 1D NOESY and heteronuclear 2D HMBC correlations observed in both spectra. Figure S4. Mayor spatial NOE correlations (blue arrows) and main 1 H-13 C HMBC correlations (red arrows) observed for compound 21.

2D HMBC
Compound 29: In the 1 H NMR spectrum of compound 29 two singlet aromatic signals at δH = 6.74 and 6.67 ppm were observed, suggesting a tetrasubstituted aromatic pattern. This implies unequivocally that two geranyl chains were incorporated in ortho positions between these, generating an asymmetrical aromatic structure ( Figure S8). These signals were assigned to H-6 and H-3, respectively. In addition, the signals observed at δH = 5.39 and 3.84 ppm were assigned to the hydroxyl in C-1 (s, 1H) and the methoxyl group (s, 3H) in C-2 of the aromatic ring. From the 1D NOESY spectrum the H-6 signal (δH = 6.74 ppm) showed spatial coupling with a signal at δH = 3.26 ppm (d, J = 6.9 Hz, 2H), which was assigned to the H-1'' of geranyl chain, while the H-3 signal (δH = 6.67 ppm) showed spatial coupling with a signal at δH = 3.23 ppm (d, J = 7.0 Hz, 2H), which was assigned to the H-1' of another geranyl chain ( Figure S8). In this way the patterns of aromatic substitution of the geranyl chains in positions C-4 and C-5 are clearly defined. In addition, the structure of compound 29 and the positions of the geranyl chains were confirmed from the 2D HMBC spectrum. Thus, the H-6 signal showed 3 JH-C heteronuclear correlations with C-1'' and C-2 at δC = 31.2 and 144.5 ppm, respectively, and 2 JH-C correlation with C-6 at δC = 124.3 ppm. The H-3 signal showed 3 JH-C heteronuclear correlations with C-1' and C-1 at δC = 30.8 and 143.5 ppm respectively, and 2 JH-C correlation with C-4 at δC = 132.5 ppm ( Figure S8). Figure S8. Mayor spatial NOE correlations (blue arrows) and main 1 H-13 C HMBC correlations (red arrows) observed for compound 29.
Compound 32: A similar analysis to compound 21 was carried out for compound 32. From the 1 H NMR spectrum the presence of aromatic signals was not observed, suggesting that the aromatic system is fully substituted and that two geranyl chain were incorporated in the aromatic ring. Therefore, only one possible structure can be suggested for compound 32 and no further analysis of spectroscopic data is required. However, in Figure S9 the major spatial 1D NOESY and heteronuclear 2D HMBC correlations observed in both spectra are shown. Figure S9. Mayor spatial NOE correlations (blue arrows) and main 1 H-13 C HMBC correlations (red arrows) observed for compound 32. Table S1. Comparison of the effect of linear geranylated phenols/methoxyphenols derivatives on in vitro mycelial growth of P. cinnamomi and B. cinerea measured as a percentage of inhibition. three independent experiments ± standard deviation.