Synthesis of New Hydrated Geranylphenols and in Vitro Antifungal Activity against Botrytis cinerea

Geranylated hydroquinones and other geranylated compounds isolated from Aplydium species have shown interesting biological activities. This fact has prompted a number of studies where geranylated phenol derivatives have been synthesized in order to assay their bioactivities. In this work, we report the synthesis of a series of new hydrated geranylphenols using two different synthetic approaches and their inhibitory effects on the mycelial growth of Botrytis cinerea. Five new hydrated geranylphenols were obtained by direct coupling reaction between geraniol and phenol in dioxane/water and using BF3·Et2O as the catalyst or by the reaction of a geranylated phenol with BF3·Et2O. Two new geranylated quinones were also obtained. The synthesis and structural elucidation of all new compounds is presented. All hydrated geranylphenols efficiently inhibit the mycelial growth of B. cinerea. Their activity is higher than that observed for non-hydrated compounds. These results indicate that structural modification on the geranyl chain brings about an enhancement of the inhibition effect of geranylated phenol derivatives.

On the other hand, the anticancer properties, both in vitro and in vivo, of a group of prenylated quinones, i.e., 3-demethylubiquinone Q2 (9) and its synthetic analogs, were studied as a function of their molecular structure [20,21]. The results indicate that 9 and its derivatives are able to inhibit the Thus, the interesting biological activity shown by 1 and other prenyl derivatives [17] (see Figure 1) has prompted us to undertake the synthesis of a significant number of linear geranylated phenols, including Compounds 1-3 and some geranylated methoxyphenyl/acetate analogs [26][27][28][29][30][31][32], in order to evaluate the in vitro cytotoxic activity on some cancer lines and the inhibitory effects on the mycelial growth of plant pathogen Botrytis cinerea [29][30][31]. The latter is a facultative phytopathogenic fungus that attacks the flowers, fruits, leaves and stems of more than 200 plant species [33]. In Chile, there is a high incidence of this fungus, and its control by commercial fungicides (dicarboximides and benzimidazoles) is becoming more ineffective due to the appearance of highly resistant strains [34,35]. Thus, an increasing number of metabolites isolated from plants, hemisynthetic and synthetic products have been studied as an alternative to chemical fungicide [29,[36][37][38]. Previous work has shown that the anti-fungal activity of geranylated phenols is mainly determined by the presence of the geranyl chain and substitution on the aromatic ring [29,31]. However, there are no data regarding the fungicide activity of compounds carrying a modified geranyl chain, e.g., hydrated geranylphenols. Therefore, in this research, a study of the inhibitory effects on the mycelial growth of plant pathogen B. cinerea of geranylated phenols (Compounds 14-18), geranylated quinones (Compounds Thus, the interesting biological activity shown by 1 and other prenyl derivatives [17] (see Figure 1) has prompted us to undertake the synthesis of a significant number of linear geranylated phenols, including Compounds 1-3 and some geranylated methoxyphenyl/acetate analogs [26][27][28][29][30][31][32], in order to evaluate the in vitro cytotoxic activity on some cancer lines and the inhibitory effects on the mycelial growth of plant pathogen Botrytis cinerea [29][30][31]. The latter is a facultative phytopathogenic fungus that attacks the flowers, fruits, leaves and stems of more than 200 plant species [33]. In Chile, there is a high incidence of this fungus, and its control by commercial fungicides (dicarboximides and benzimidazoles) is becoming more ineffective due to the appearance of highly resistant strains [34,35]. Thus, an increasing number of metabolites isolated from plants, hemisynthetic and synthetic products have been studied as an alternative to chemical fungicide [29,[36][37][38]. Previous work has shown that the anti-fungal activity of geranylated phenols is mainly determined by the presence of the geranyl chain and substitution on the aromatic ring [29,31]. However, there are no data regarding the fungicide activity of compounds carrying a modified geranyl chain, e.g., hydrated geranylphenols. Therefore, in this research, a study of the inhibitory effects on the mycelial growth of plant pathogen B. cinerea of geranylated phenols (Compounds 14-18), geranylated quinones (Compounds Figure 2) is reported. The synthesis and structural elucidation of the new compounds (14-18, 20, 22-26) is also presented.  Figure 2) is reported. The synthesis and structural elucidation of the new compounds (14-18, 20, 22-26) is also presented.

Synthesis
Linear geranylated phenols/methoxyphenols have been synthesized by direct coupling of geraniol with the respective phenol or methoxyphenols. This reaction has been studied for many authors, because it is directly related to the synthesis of biologically-active phenolic terpenoids [11,14,20,[26][27][28][29][30]. The coupling is commonly carried out in strong mineral acids or aprotic solvents with Lewis acids, e.g., BF3·Et2O, in dioxane for the synthesis of tocopherols and geranyl and farnesyl analogs of the ubiquinones, p-toluenesulfonic acid in CH2CI2 for the synthesis of cannabigerol and related marihuana constituents [39]. Alternatively, BF3·Et2O/AgNO3 has been used as a catalyst and acetonitrile as a solvent [29,30]. In this work, Compounds 1, 3, 14, 15, 17 and 19 were synthesized through this reaction, using dioxane as the solvent, BF3·Et2O as the catalyst and in the presence or absence of a nitrogen atmosphere.
Direct coupling between o-cresol and p-cresol with geraniol under a nitrogen atmosphere leads to Compounds 14 and 15 with 3.1% and 12% yields, respectively (Scheme 1).

Synthesis
Linear geranylated phenols/methoxyphenols have been synthesized by direct coupling of geraniol with the respective phenol or methoxyphenols. This reaction has been studied for many authors, because it is directly related to the synthesis of biologically-active phenolic terpenoids [11,14,20,[26][27][28][29][30]. The coupling is commonly carried out in strong mineral acids or aprotic solvents with Lewis acids, e.g., BF 3¨E t 2 O, in dioxane for the synthesis of tocopherols and geranyl and farnesyl analogs of the ubiquinones, p-toluenesulfonic acid in CH 2 CI 2 for the synthesis of cannabigerol and related marihuana constituents [39]. Alternatively, BF 3¨E t 2 O/AgNO 3 has been used as a catalyst and acetonitrile as a solvent [29,30]. In this work, Compounds 1, 3, 14, 15, 17 and 19 were synthesized through this reaction, using dioxane as the solvent, BF 3¨E t 2 O as the catalyst and in the presence or absence of a nitrogen atmosphere.
Direct coupling between o-cresol and p-cresol with geraniol under a nitrogen atmosphere leads to Compounds 14 and 15 with 3.1% and 12% yields, respectively (Scheme 1).
In the search for a synthetic pathway to obtain hydrated geranylphenols, we have attempted two different approaches. In the first one, we explore the possibility of obtaining both geranylated quinones and hydrated geranylphenols in a one-pot synthesis. It has been reported that some hydrated geranylorcinols have been obtained as minor products in the coupling reaction of orcinol and geraniol in the presence of 1% aqueous oxalic acid at 80 °C [40]. Therefore, in this approach, the coupling reaction is carried out under air using dioxane as the solvent, BF3·Et2O as the catalyst and small amounts of added water. Interestingly, the obtained products depend on the chemical nature of the reacting phenol. Thus, coupling between geraniol and 1,4-hydroquinone leads to monogeranylated hydroquinone 1 and quinone 2, as well as digeranylated quinone 19 (Scheme 3); whereas, coupling between 2-metoxyhydroquinone and geraniol gives the disubstituted quinone 20 as the only product (Scheme 4). Finally, hydrated geranylphenols 22 and 23 were obtained in the coupling reaction between o-cresol and geraniol (Scheme 5). Following the same synthetic procedure, Compound 17 is obtained as a unique product by coupling between 2-metoxyhydroquinone and geraniol with 5.9% yield (Scheme 2). Following the same synthetic procedure, Compound 17 is obtained as a unique product by coupling between 2-metoxyhydroquinone and geraniol with 5.9% yield (Scheme 2). Standard acetylation (Ac2O/CH2Cl2/DMAP) of 15 and 17 gives the acetylated derivatives 16 and 18 with 94.8% and 98% yields, respectively.
In the search for a synthetic pathway to obtain hydrated geranylphenols, we have attempted two different approaches. In the first one, we explore the possibility of obtaining both geranylated quinones and hydrated geranylphenols in a one-pot synthesis. It has been reported that some hydrated geranylorcinols have been obtained as minor products in the coupling reaction of orcinol and geraniol in the presence of 1% aqueous oxalic acid at 80 °C [40]. Therefore, in this approach, the coupling reaction is carried out under air using dioxane as the solvent, BF3·Et2O as the catalyst and small amounts of added water. Interestingly, the obtained products depend on the chemical nature of the reacting phenol. Thus, coupling between geraniol and 1,4-hydroquinone leads to monogeranylated hydroquinone 1 and quinone 2, as well as digeranylated quinone 19 (Scheme 3); whereas, coupling between 2-metoxyhydroquinone and geraniol gives the disubstituted quinone 20 as the only product (Scheme 4). Finally, hydrated geranylphenols 22 and 23 were obtained in the coupling reaction between o-cresol and geraniol (Scheme 5). In the search for a synthetic pathway to obtain hydrated geranylphenols, we have attempted two different approaches. In the first one, we explore the possibility of obtaining both geranylated quinones and hydrated geranylphenols in a one-pot synthesis. It has been reported that some hydrated geranylorcinols have been obtained as minor products in the coupling reaction of orcinol and geraniol in the presence of 1% aqueous oxalic acid at 80˝C [40]. Therefore, in this approach, the coupling reaction is carried out under air using dioxane as the solvent, BF 3¨E t 2 O as the catalyst and small amounts of added water. Interestingly, the obtained products depend on the chemical nature of the reacting phenol. Thus, coupling between geraniol and 1,4-hydroquinone leads to monogeranylated hydroquinone 1 and quinone 2, as well as digeranylated quinone 19 (Scheme 3); whereas, coupling between 2-metoxyhydroquinone and geraniol gives the disubstituted quinone 20 as the only product (Scheme 4). Finally, hydrated geranylphenols 22 and 23 were obtained in the coupling reaction between o-cresol and geraniol (Scheme 5). In this reaction, Compounds 1, 3 and 19 were obtained with 28.0%, 7.6% and 1.9% yields, respectively. When this coupling is carried out in the presence of a nitrogen atmosphere and with no added water, Compound 1 is obtained as the exclusive product [26]. Recently, this reaction has been performed at higher temperatures using aluminum phenoxide as the catalyst, and a completely different pattern of products has been reported. Compound 1 and a mixture of digeranylated quinones (19 and di-ortho-geranylated quinone) were obtained with 40% and 27% yields, respectively, but Compound 3 was not identified [41].
On the other hand, a methoxy substitution in the hydroquinone induces a complete change in the product distribution, i.e., Compound 20 is obtained with 4.1% yield. It is worth mentioning that geranylated quinones (3,19,20) are formed only by coupling geraniol with 1,4-dihydroxybenzene systems. Probably, the oxidation to 1,4-quinone is enhanced by the redox properties of hydroquinone compounds.
Finally, in the coupling of geraniol with o-cresol, hydrated Compounds 22 and 23 were obtained with 9.0% and 10.5% yields, respectively.   In this reaction, Compounds 1, 3 and 19 were obtained with 28.0%, 7.6% and 1.9% yields, respectively. When this coupling is carried out in the presence of a nitrogen atmosphere and with no added water, Compound 1 is obtained as the exclusive product [26]. Recently, this reaction has been performed at higher temperatures using aluminum phenoxide as the catalyst, and a completely different pattern of products has been reported. Compound 1 and a mixture of digeranylated quinones (19 and di-ortho-geranylated quinone) were obtained with 40% and 27% yields, respectively, but Compound 3 was not identified [41].
On the other hand, a methoxy substitution in the hydroquinone induces a complete change in the product distribution, i.e., Compound 20 is obtained with 4.1% yield.
Finally, in the coupling of geraniol with o-cresol, hydrated Compounds 22 and 23 were obtained with 9.0% and 10.5% yields, respectively.
The formation of Compounds 22 and 23 may be explained by the proposed mechanism depicted in Scheme 6. In this reaction, Compounds 1, 3 and 19 were obtained with 28.0%, 7.6% and 1.9% yields, respectively. When this coupling is carried out in the presence of a nitrogen atmosphere and with no added water, Compound 1 is obtained as the exclusive product [26]. Recently, this reaction has been performed at higher temperatures using aluminum phenoxide as the catalyst, and a completely different pattern of products has been reported. Compound 1 and a mixture of digeranylated quinones (19 and di-ortho-geranylated quinone) were obtained with 40% and 27% yields, respectively, but Compound 3 was not identified [41].
On the other hand, a methoxy substitution in the hydroquinone induces a complete change in the product distribution, i.e., Compound 20 is obtained with 4.1% yield.
Finally, in the coupling of geraniol with o-cresol, hydrated Compounds 22 and 23 were obtained with 9.0% and 10.5% yields, respectively. In this reaction, Compounds 1, 3 and 19 were obtained with 28.0%, 7.6% and 1.9% yields, respectively. When this coupling is carried out in the presence of a nitrogen atmosphere and with no added water, Compound 1 is obtained as the exclusive product [26]. Recently, this reaction has been performed at higher temperatures using aluminum phenoxide as the catalyst, and a completely different pattern of products has been reported. Compound 1 and a mixture of digeranylated quinones (19 and di-ortho-geranylated quinone) were obtained with 40% and 27% yields, respectively, but Compound 3 was not identified [41].
On the other hand, a methoxy substitution in the hydroquinone induces a complete change in the product distribution, i.e., Compound 20 is obtained with 4.1% yield.
It is worth mentioning that geranylated quinones (3,19,20) are formed only by coupling geraniol with 1,4-dihydroxybenzene systems. Probably, the oxidation to 1,4-quinone is enhanced by the redox properties of hydroquinone compounds.
Finally, in the coupling of geraniol with o-cresol, hydrated Compounds 22 and 23 were obtained with 9.0% and 10.5% yields, respectively.
The formation of Compounds 22 and 23 may be explained by the proposed mechanism depicted in Scheme 6.
In the first step, an allylic carbocation is formed by the reaction of BF 3¨E t 2 O with geraniol, which is then coupled with phenol via Electrophilic Aromatic Substitution (EArS) (Step 2). In presence of water, the adduct BF 3¨H2 O is presumably formed by nucleophilic displacement of an ether molecule by H 2 O (Step 3). Subsequently, this adduct reacts with the geranyl chain by a Markovnikov-type addition, forming a stable tertiary carbocation, which is then hydrated by reaction with a water molecule (Steps 3 and 4). Finally, the remaining olefinic bond is hydrated by BF 3¨H2 O, and a completely hydrated geranyl chain is obtained (Step 5). It is worth mentioning that water is added 24 h after the coupling reaction has been started. This means that Step 3 begins when most of the geranylphenol has already been formed. In the first step, an allylic carbocation is formed by the reaction of BF3·Et2O with geraniol, which is then coupled with phenol via Electrophilic Aromatic Substitution (EArS) (Step 2). In presence of water, the adduct BF3·H2O is presumably formed by nucleophilic displacement of an ether molecule by H2O (Step 3). Subsequently, this adduct reacts with the geranyl chain by a Markovnikov-type addition, forming a stable tertiary carbocation, which is then hydrated by reaction with a water molecule (Steps 3 and 4). Finally, the remaining olefinic bond is hydrated by BF3·H2O, and a completely hydrated geranyl chain is obtained (Step 5). It is worth mentioning that water is added 24 h after the coupling reaction has been started. This means that Step 3 begins when most of the geranylphenol has already been formed.
Based on this result, our second approach consists of the direct hydration of the side chain by the reaction of a geranylated phenol with a Lewis acid (BF3·Et2O) in dioxane and in the presence of water. Compound 17 was submitted to this reaction, and compounds 21, 24-26 were obtained with 11.1%, 10.7%, 24% and 18.9% yields, respectively (Scheme 7). Based on this result, our second approach consists of the direct hydration of the side chain by the reaction of a geranylated phenol with a Lewis acid (BF 3¨E t 2 O) in dioxane and in the presence of water. Compound 17 was submitted to this reaction, and compounds 21, 24-26 were obtained with 11.1%, 10.7%, 24% and 18.9% yields, respectively (Scheme 7).
Compounds 24 and 26 may be formed through Steps 3-5 of the mechanism proposed in Scheme 6. However, in this reaction, Compound 25 is formed by cyclization of the tertiary carbocation formed in Step 3, the formation of tertiary carbocation in the C-7 1 position of geranyl chain, 6-endo-trig cyclization from the C2 1 -C3 1 double bond and hydration by the subsequent nucleophilic attack of water on the tertiary carbocation in the C-3 1 position (Scheme 8).
The carbocation intermediates appearing in Schemes 6 and 8 have been proposed for coupling of phenol with geraniol and various reactions of geraniol in acidic aqueous solution [39,40]. Compounds 24 and 26 may be formed through Steps 3-5 of the mechanism proposed in Scheme 6. However, in this reaction, Compound 25 is formed by cyclization of the tertiary carbocation formed in Step 3, the formation of tertiary carbocation in the C-7′ position of geranyl chain, 6-endo-trig cyclization from the C2′-C3′ double bond and hydration by the subsequent nucleophilic attack of water on the tertiary carbocation in the C-3′ position (Scheme 8) Scheme 8. Proposed mechanism for formation of Compound 25. Reaction of Lewis acid (BF3·Et2O) with water, addition Markovnikov of H by BF3H2O adduct on C-6′ position of geranyl chain and carbocation formation in C-7′, 6-endo-trig cyclization and later geranyl chain hydration.
The carbocation intermediates appearing in Schemes 6 and 8 have been proposed for coupling of phenol with geraniol and various reactions of geraniol in acidic aqueous solution [39,40].
Compounds 14-18, 20, 22-26 are new, and their structural characterization is described in the next section.

Structure Determination
The chemical structures of all compounds synthesized in this work were mainly established by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy techniques. All NMR spectra are given in Figure S1. In this section, the NMR data used to determine the chemical structure of new compounds, geranylated phenol derivatives (14)(15)(16)(17)(18), geranylated quinone (20) and hydrated geranylphenols (22)(23)(24)(25)(26), are discussed in detail. Compound 19 has been already reported, but it has been included in this section because the NMR assignation given in literature is not right [41]. Compounds 24 and 26 may be formed through Steps 3-5 of the mechanism proposed in Scheme 6. However, in this reaction, Compound 25 is formed by cyclization of the tertiary carbocation formed in Step 3, the formation of tertiary carbocation in the C-7′ position of geranyl chain, 6-endo-trig cyclization from the C2′-C3′ double bond and hydration by the subsequent nucleophilic attack of water on the tertiary carbocation in the C-3′ position (Scheme 8) The carbocation intermediates appearing in Schemes 6 and 8 have been proposed for coupling of phenol with geraniol and various reactions of geraniol in acidic aqueous solution [39,40].
Compounds 14-18, 20, 22-26 are new, and their structural characterization is described in the next section.

Structure Determination
The chemical structures of all compounds synthesized in this work were mainly established by 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy techniques. All NMR spectra are given in Figure S1. In this section, the NMR data used to determine the chemical structure of new compounds, geranylated phenol derivatives (14)(15)(16)(17)(18), geranylated quinone (20) and hydrated geranylphenols (22)(23)(24)(25)(26), are discussed in detail. Compound 19 has been already reported, but it has been included in this section because the NMR assignation given in literature is not right [41].
Compound 19: The symmetrical molecular structure was confirmed by the substitution pattern in the olefin zone and by the intensity of integrated signals of hydrogen atoms in quinone and olefinic portion. For instance, the signal at δH = 6.70 ppm (s, 2H, H-3 and H-6) indicates the presence of two In the 1 H-NMR spectrum of the acetylated derivative 16, a singlet at δ H = 2.29 ppm (3H, CH 3 CO) was observed. Additionally, in the 13 C NMR spectrum, the signals appearing at δ C = 169.6 (C=O) and 20.8 (CH 3 ) ppm confirmed the presence of monoacetylated derivative 16.
Compound 19: The symmetrical molecular structure was confirmed by the substitution pattern in the olefin zone and by the intensity of integrated signals of hydrogen atoms in quinone and olefinic portion. For instance, the signal at δH = 6.70 ppm (s, 2H, H-3 and H-6) indicates the presence of two In the 1 H-NMR spectrum of the acetylated derivative 18, two singlet signals at δ H = 2.29 and 2.28 ppm (each 3H, CH 3 CO) were observed. Additionally, in the 13 C NMR spectrum, the signals appearing at δ = 169.2 (COCH 3 -C4), 168.9 (COCH 3 -C1) ppm and δ = 20.8 (CH 3 COO-C1) and 20.6 (CH 3 COO-C4) ppm, confirmed the presence of diacetylated derivative 18.
Compound 19: The symmetrical molecular structure was confirmed by the substitution pattern in the olefin zone and by the intensity of integrated signals of hydrogen atoms in quinone and olefinic portion. For instance, the signal at δ H = 6.70 ppm (s, 2H, H-3 and H-6) indicates the presence of two identical H. In a previous report, two different signals were found and assigned to these H (δ H = 6.48 ppm, 1H, s, H-13 and δ H = 6.52 ppm, 1H, s, H-16). A detailed assignment of 1 H-NMR signals is given in the experimental part, and the corresponding spectrum is shown in the Supplementary Material. Additionally, spatial correlations (NOE) were observed for the signals at δ H = 6.70 ppm and at δ H = 3.21 ppm (4H, d, J = 6.8, H-1 1 ) and for the latter and the signal at δ H = 1.73 ppm, assigned to Int. J. Mol. Sci. 2016, 17, 840 9 of 18 CH 3 -C3 1 (Figure 5a). Finally, in the 13 C NMR spectrum, only one signal at δ C = 187.6 ppm (C-1 and C-4) of the carbonyl group was observed, confirming the symmetrical structure of Compound 19. identical H. In a previous report, two different signals were found and assigned to these H (δH = 6.48 ppm, 1H, s, H-13 and δH = 6.52 ppm, 1H, s, H-16). A detailed assignment of 1 H-NMR signals is given in the experimental part, and the corresponding spectrum is shown in the Supplementary Material. Additionally, spatial correlations (NOE) were observed for the signals at δH = 6.70 ppm and at δH = 3.21 ppm (4H, d, J = 6.8, H-1′) and for the latter and the signal at δH = 1.73 ppm, assigned to CH3-C3′ (Figure 5a). Finally, in the 13 C NMR spectrum, only one signal at δC = 187.6 ppm (C-1 and C-4) of the carbonyl group was observed, confirming the symmetrical structure of Compound 19. Compound 20: The presence of double geranyl chain substitution on the quinone nucleus was confirmed by the observation of two doublet signals in the 1 H-NMR spectrum at δH = 3.15 ppm (2H, J = 7.4 Hz) and 3.12 ppm (2H, J = 7.0 Hz), which were assigned to the hydrogens H-1′ and H-2′′, respectively. Additionally, the presence of only one hydrogen at δH = 6.32 ppm (1H, s, H-6) demonstrates the degree of tetra-substitution on the quinone moiety. Differentiation between geranyl chains was established by the HMBC correlations observed for H-1′ at 3 JH-C with C-4 (δC = 187.9 ppm; C=O) and H-1′ at 3 JH-C with C-2 (δC = 155.1 ppm; C-OCH3) and at 2 JH-C with C-3 (δC = 131.9 ppm) (Figure 5b). Similarly, the signal of H-1′′ showed 3 JH-C correlations with C-4 (δC = 187.9 ppm; C=O) and C-6 (δC = 130.5 ppm) and 2 JH-C with C-5 (δC = 148.3 ppm) (Figure 5b). In this spectrum, a 2 JH-C coupling of H-8 (δH = 2.78-2.74, m, 2H) with C-1′ (δC = 120.4 ppm) and a 3 JH-C coupling between the signals of C-2′and C-6′ at δC = 151.9 and 126.9 ppm, respectively, were observed. In addition, correlations at 3 JH-C between the CH3-Ar group (δH = 2.16 ppm) with C-2′ and C-4′ at δC = 151.9 and 128.4 ppm, respectively, and a 2 JH-C with C-3′ (δC = 126.2) were observed (Figure 6a). The presence of two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary carbinolic signals at δC = 75.8 and 71.0 ppm in the 13 C NMR spectrum. These were assigned to carbons C-6 and C-2, respectively, by two-dimensional (2D) HMBC correlations. Thus, H-8 showed correlation at 3 JH-C with carbinolic carbon at C-6 (δC = 75.8 ppm), whereas the methyl groups at δC = 29.2 ppm (CH3-1 and CH3-C2) showed correlations at 2 JH-C with C-2 (δC = 71.0 ppm) (Figure 6a).
Because Compound 24 was obtained from 17 by the hydration reaction of it, the aromatic substitution pattern was maintained for Compounds 24 and 25. Thus, in Compound 24, the presence of two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary carbinolic signals at δ C = 75.8 and 70.9 ppm in the 13 C NMR spectrum. These signals were assigned to carbons C-3 1 and C-7 1 , respectively, by two-dimensional (2D) HMBC correlations. Thus, the CH 3 -C7 1 and CH 3 -8 1 groups showed correlation at 2 J H-C with carbinolic carbon at C-7 1 (δ C = 70.9 ppm), and therefore, the signal at δ C = 75.8 ppm was unequivocally assigned to C-3 1 (Figure 7a); while for Compound 25, the methylene group (at δ C = 22.7 ppm, assigned as C-7 1 ) showed a correlation at 2 J H-C with C-2 (δ C = 114.2 ppm) and C-1 1 (δ C = 48.3 ppm) and 3 J H-C with a tertiary carbinolic carbon at δ C = 76.7 ppm assigned to C-2 1 . Additionally, the CH 3 -C2 1 group at δ H = 1.20 ppm (3H, s) showed coupling at 2 J H-C with C-2 1 and 3 J H-C with tertiary C-1 1 (δ C = 48.3 ppm) (Figure 7b) Thus, the cyclohexane structure is confirmed for geranyl chain. Finally, mono-hydroxylation in the geranyl chain for Compound 26 was mainly established by 13 C NMR data and 2D HMBC correlations. Only one signal of carbinolic carbon at δ C = 70.9 ppm in the 13 C NMR spectrum was observed, and the methyl groups at δ H = 1.22 (6H, s, CH 3 -C7 1 and H-8 1 ) showed 2 J H-C correlations with this carbon (C-7 1 , δ C = 70.9 ppm) (Figure 7c). In addition, these methyl groups showed 3 J H-C correlations with C-6 1 (δ C = 43.3 ppm) (Figure 7c). presence of two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary carbinolic signals at δC = 72.9 and 71.3 ppm in the 13 C NMR spectrum. These signals were assigned to carbons C-6 and C-2, respectively, by two-dimensional (2D) HMBC correlations. Thus, the CH3-C6 group showed correlation at 2 JH-C with carbinolic carbon at C-6 (δC = 72.9 ppm), while the methyl groups at δC = 31.2 and 29.9 ppm (CH3-1 and CH3-C2, respectively) showed correlations at 2 JH-C with C-2 (δC = 71.3 ppm) (Figure 6b). Because Compound 24 was obtained from 17 by the hydration reaction of it, the aromatic substitution pattern was maintained for Compounds 24 and 25. Thus, in Compound 24, the presence of two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary carbinolic signals at δC = 75.8 and 70.9 ppm in the 13 C NMR spectrum. These signals were assigned to carbons C-3′ and C-7′, respectively, by two-dimensional (2D) HMBC correlations. Thus, the CH3-C7′ and CH3-8′ groups showed correlation at 2 JH-C with carbinolic carbon at C-7′ (δC = 70.9 ppm), and therefore, the signal at δC = 75.8 ppm was unequivocally assigned to C-3′ ( Figure 7a); while for Compound 25, the methylene group (at δC = 22.7 ppm, assigned as C-7′) showed a correlation at 2 JH-C with C-2 (δC = 114.2 ppm) and C-1′ (δC = 48.3 ppm) and 3 JH-C with a tertiary carbinolic carbon at δC = 76.7 ppm assigned to C-2′. Additionally, the CH3-C2′ group at δH = 1.20 ppm (3H, s) showed coupling at 2 JH-C with C-2′ and 3 JH-C with tertiary C-1′ (δC = 48.3 ppm) (Figure 7b) Thus, the cyclohexane structure is confirmed for geranyl chain. Finally, mono-hydroxylation in the geranyl chain for Compound 26 was mainly established by 13 C NMR data and 2D HMBC correlations. Only one signal of carbinolic carbon at δC = 70.9 ppm in the 13 C NMR spectrum was observed, and the methyl groups at δH = 1.22 (6H, s, CH3-C7′ and H-8′) showed 2 JH-C correlations with this carbon (C-7′, δC = 70.9 ppm) (Figure 7c). In addition, these methyl groups showed 3 JH-C correlations with C-6′ (δC = 43.3 ppm) (Figure 7c).

In Vitro Antifungal Activity against B. cinerea.
All studied compounds (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) were tested for in vitro antifungal activity on the mycelial growth of B. cinerea strain GM7 using the agar radial assay with Potato Dextrose Agar (PDA). Figure 8 shows an assay where the B. cinerea mycelium grows in medium containing only PDA and 1% ethanol (Figure 8a All studied compounds (14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) were tested for in vitro antifungal activity on the mycelial growth of B. cinerea strain GM7 using the agar radial assay with Potato Dextrose Agar (PDA). Figure 8 shows an assay where the B. cinerea mycelium grows in medium containing only PDA and 1% ethanol (Figure 8a The inhibition of mycelial growth is evaluated by measuring colony diameters in the presence and absence of the tested compounds. The results, expressed as the percentage of inhibition, are summarized in Table 1. The data indicate that geranylated derivatives of o-and p-cresol (14-16) have no effect on the mycelial growth of B. cinerea. However, the methoxy derivatives of geranylated p-cresol (17 and 18) exhibit a significative increase in the inhibitory activity. This result is in line with previous work where a family of methoxy geranylated derivatives was studied [30].
On the other hand, geranylated quinones (19)(20)(21) show an important activity (greater than 50% at the higher tested concentrations) that is independent of the number of geranyl chains. In the case of geranylated phenols, it was found that antifungal activity decreases with the increasing number of prenyl chains [29,30].  The inhibition of mycelial growth is evaluated by measuring colony diameters in the presence and absence of the tested compounds. The results, expressed as the percentage of inhibition, are summarized in Table 1. The percentage of inhibition of mycelial growth is based on colony diameter measurements after 72 h of incubation. Each point represents the mean of at least three independent experiments˘the standard deviation. 1 C´refers to the negative control; and 2 C+ refers to the positive control (Captan).
The data indicate that geranylated derivatives of oand p-cresol (14-16) have no effect on the mycelial growth of B. cinerea. However, the methoxy derivatives of geranylated p-cresol (17 and 18) exhibit a significative increase in the inhibitory activity. This result is in line with previous work where a family of methoxy geranylated derivatives was studied [30].
On the other hand, geranylated quinones (19)(20)(21) show an important activity (greater than 50% at the higher tested concentrations) that is independent of the number of geranyl chains. In the case of geranylated phenols, it was found that antifungal activity decreases with the increasing number of prenyl chains [29,30].
All hydrated geranylphenols exhibit activities on mycelial growth inhibition that are in the range 30%-95% at 250 ppm. A comparison of the percentages of inhibition measured for geranylated phenol and their respective hydrated compound, i.e., 14 with 23, 17 with 24 and 26, shows that the latter are more active than the parent compound. This effect is larger for compounds carrying only one hydroxyl group in the side chain (25 and 26) than for completely hydrated compounds (22)(23)(24). In other words, incorporation of hydroxyl groups in the side chain, by hydration of the geranyl chain, brings about an enhancing effect on the antifungal activity. Previous work was focused on the effect of substitution in the phenol ring, and the results suggested that the inhibition effect depends mainly on the presence of the prenyl chain [29,31]. In this context, these results are important because, as far as we know, this is the first report of the effect of the side chain structure on the fungicide activity of geranylated compounds.
Finally, it is interesting to stress that Compound 26 stands out as being as active as Captan, a fungicide that is currently used for infection control in some crops.

General
Chemicals were obtained from Merck (Darmstadt, Germany) or Aldrich (St. Louis, MO, USA) and were used without further purification. A detailed description of conditions used to register Fourier transform infrared (FT-IR) spectra, high resolution mass spectra and 1 H, 13 C, 13 C DEPT-135, selective gradients 1D 1 H NOESY, gs-2D Heteronuclear Single Quantum Coherence (HSQC) and gs-2D HMBC spectra has been given elsewhere [30]. Silica gel (Merck 200-300 mesh) was used for Column Chromatography (C.C.) and silica gel plates HF 254 for thin layer chromatography (TLC). TLC spots were detected by heating after spraying with 25% H 2 SO 4 in H 2 O.

Coupling Reaction in Presence of Nitrogen
The coupling of geraniol and phenols was carried out using boric trifluoride etherate BF 3¨E t 2 O as the catalyst and dioxane as the solvent. Experimental details for a typical reaction have been given elsewhere [30].