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Synthesis of New Hydrated Geranylphenols and in Vitro Antifungal Activity against Botrytis cinerea Mauricio Soto 1 , Luis Espinoza 1 , María I. Chávez 1 , Katy Díaz 1 , Andrés F. Olea 2 and Lautaro Taborga 1, * 1

2

*

Departamento de Química, Universidad Técnica Federico Santa María, Valparaíso 2340000, Chile; [email protected] (M.S.); [email protected] (L.E.); [email protected] (M.I.C.); [email protected] (K.D.) Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile, Santiago 8910339, Chile; [email protected] Correspondence: [email protected]; Tel.: +56-32-265-4225

Academic Editor: Zdenek Wimmer Received: 18 April 2016; Accepted: 25 May 2016; Published: 3 June 2016

Abstract: 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 ¨ Et2 O as the catalyst or by the reaction of a geranylated phenol with BF3 ¨ Et2 O. 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. Keywords: geranylated phenol derivatives; hydrated geranyl; synthesis; structural elucidation; growth inhibition effect; Botrytis cinerea; fungicide

1. Introduction The subgroups of linear geranylated quinones, geranylated hydroquinones and meroterpenes are represented by an important number of metabolites isolated from ascidians belonging to the genus Aplydium [1,2]. The first biologically active tunicate metabolites were 2-geranylhydroquinone (1) and 2-geranyl hydroquinone diacetate (2) (Figure 1), isolated from Aplydium sp. and Phacelia crenulata [3,4] and Pyrola japonica [5], respectively, and later found in many others Aplydium species. It has been shown that these compounds exhibit antitumoral activity [6]. Additionally, several linear quinones/hydroquinones carrying a geranyl type side chain (Compounds 1, 3–11; Figure 1) have been obtained from diverse Aplydium species [7–12]. Compound 1 and 2-geranylbenzoquinone (3) exhibit interesting and various biological activities [3,8,13–17], whereas Compounds 5 and 6 show antioxidant activities [8], and Compound 4 shows cytotoxicity activity against P-388 mouse lymphoma suspension culture [18]. Additionally, linear geranylmethoxyphenol/acetates derivatives isolated from Phacelia ixodes [19] are cytotoxic, allergenic and insecticidal. 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

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growth of human cancer cell lines and of the solid Ehrlich carcinoma in mice by inducing apoptosis in cancer Thecancer anticancer activity of the thissolid structural-related family compounds depends on the growth ofcells. human cell lines and of Ehrlich carcinoma inof mice by inducing apoptosis in length of the The polyprenyl chain and on of the methoxyl groups in the quinone parton of the the cancer cells. anticancer activity ofthe thisposition structural-related family of compounds depends molecule [20].polyprenyl The most effective compounds were of those a side chain thequinone geranylpart typeof(two length of the chain and on the position the having methoxyl groups inofthe the isoprene units) andmost one methoxyl group at thewere para-position to the geranyl molecule [20]. The effective compounds those having a side chainchain. of the geranyl type (two Cyclodiprenyl hydroquinones, such as methoxyconidiol (12) and conitriol isoprene units) and one methoxyl group at the para-position to the geranyl chain. (13), have been isolated from Aplidium aff. densum [22] and Aplidium conicum [9], respectively. Methoxyconidiol and Cyclodiprenyl hydroquinones, such as methoxyconidiol (12) and conitriol (13), have been isolated its methoxy derivative their[9], biological activities on human cancer lines from Aplidium aff. densumwere [22] synthesized, and Aplidium and conicum respectively. Methoxyconidiol and its cell methoxy and sea urchin embryos were assessed [23]. A detailed description of the isolation and biological derivative were synthesized, and their biological activities on human cancer cell lines and sea urchin activitieswere of these and other structures of natural prenylquinones, hydroquinones and meroterpenes embryos assessed [23]. A detailed description of the isolation and biological activities of these and can bestructures found in [24,25]. other of natural prenylquinones, hydroquinones and meroterpenes can be found in [24,25].

Figure 1. naturally-occurring linear geranylated hydroquinones/quinones 1–11,1–11, and 1. Structure Structureofofsome some naturally-occurring linear geranylated hydroquinones/quinones cyclodiprenyl meroterpenes 12–13. and cyclodiprenyl meroterpenes 12–13.

Thus, the theinteresting interestingbiological biological activity shown and prenyl other prenyl derivatives (see Thus, activity shown by 1 by and1other derivatives [17] (see [17] Figure 1) Figure 1) has prompted us to undertake the synthesis of a significant number of linear geranylated has prompted us to undertake the synthesis of a significant number of linear geranylated phenols, phenols, including Compounds 1–3 and some geranylated methoxyphenyl/acetate analogs [26–32], including Compounds 1–3 and some geranylated methoxyphenyl/acetate analogs [26–32], in order to in order to the in vitro cytotoxic activity on some and the inhibitory on evaluate theevaluate in vitro cytotoxic activity on some cancer lines cancer and thelines inhibitory effects on theeffects mycelial the mycelial of Botrytis plant pathogen Botrytis Thephytopathogenic latter is a facultative growth of plantgrowth pathogen cinerea [29–31]. The cinerea latter is [29–31]. a facultative fungus phytopathogenic fungus that attacks the flowers, fruits, leaves and stems of 200 plant that attacks the flowers, fruits, leaves and stems of more than 200 plant speciesmore [33]. than In Chile, there species [33]. In Chile, there is a high incidence of this fungus, and its control by commercial fungicides is a high incidence of this fungus, and its control by commercial fungicides (dicarboximides and (dicarboximides is and benzimidazoles) is becoming ineffective of due to theresistant appearance of [34,35]. highly benzimidazoles) becoming more ineffective due tomore the appearance highly strains resistant strains [34,35]. Thus, an increasing number of metabolites isolated from plants, Thus, an increasing number of metabolites isolated from plants, hemisynthetic and synthetic products hemisynthetic andas an synthetic products havefungicide been studied as Previous an alternative chemical have been studied alternative to chemical [29,36–38]. work hastoshown that fungicide [29,36–38]. Previous work has shown that the anti-fungal activity of geranylated phenols is the anti-fungal activity of geranylated phenols is mainly determined by the presence of the geranyl mainly the aromatic presencering of [29,31]. the geranyl chain and on the aromatic chain anddetermined substitutionby on the However, there are substitution no data regarding the fungicide ring [29,31]. However, there are no data regarding the fungicide activity of compounds carrying a activity of compounds carrying a modified geranyl chain, e.g., hydrated geranylphenols. modified geranyl chain, e.g., hydrated geranylphenols. Therefore, in this research, a study of the inhibitory effects on the mycelial growth of plant Therefore, in this research, a phenols study of(Compounds the inhibitory effects on the mycelial growth of plant pathogen B. cinerea of geranylated 14–18), geranylated quinones (Compounds pathogen B. cinerea of geranylated phenols (Compounds 14–18), geranylated quinones (Compounds

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19–21) and and hydrated hydrated geranylphenols geranylphenols derivatives (Compounds 22–26) (see Figure 2) is reported. reported. The synthesis and structural elucidation of the new compounds (14–18, 20, 22–26) is also presented. of the new compounds (14–18, 20, 22–26) is also presented.

Figure 2. 2. Chemical geranylated quinones quinones (19–21) (19–21) and and Figure Chemical structures structures of of geranylated geranylated phenols phenols (14–18) (14–18) geranylated derivatives (22–26) (22–26) that that have have been been studied studied in in this this work. work. hydrated geranylphenols derivatives

2. Results 2. Resultsand andDiscussion Discussion 2.1. Synthesis

phenols/methoxyphenols have Linear geranylated phenols/methoxyphenols have been been synthesized synthesized by by direct coupling of methoxyphenols. ThisThis reaction has been for many geraniol with with the therespective respectivephenol phenoloror methoxyphenols. reaction has studied been studied for authors, because it is directly related to the synthesis of biologically-active phenolic many authors, because it is directly related to the synthesis of biologically-active phenolic terpenoids [11,14,20,26–30]. The coupling is commonly carried out in strong mineral acids or aprotic with Lewis Lewis acids, acids, e.g., e.g., BF BF33¨·Et solvents with Et22O, O,in indioxane dioxane for for the the synthesis synthesis of of tocopherols tocopherols and geranyl and farnesyl analogs of the ubiquinones, p-toluenesulfonic acid in CH 2CI 2 for the of farnesyl analogs of the ubiquinones, p-toluenesulfonic acid in CH2 CI2 for the synthesis of synthesis cannabigerol cannabigerol and related marihuana[39]. constituents [39]. Alternatively, BF33·Et 2O/AgNO 3 has used and related marihuana constituents Alternatively, BF3 ¨ Et2 O/AgNO has been used asbeen a catalyst as a catalyst andas acetonitrile as a solvent [29,30]. this work, Compounds 3, 14, 17 and 19 were and acetonitrile a solvent [29,30]. In this work,In Compounds 1, 3, 14, 15, 171,and 1915, were synthesized synthesized reaction, using dioxane as BF the3 ¨ solvent, BF3·Et 2O as the and in the through this through reaction,this using dioxane as the solvent, Et2 O as the catalyst andcatalyst in the presence or presenceofora absence a nitrogen atmosphere. absence nitrogenofatmosphere. Direct coupling coupling between betweeno-cresol o-cresoland andp-cresol p-cresolwith withgeraniol geraniolunder under a nitrogen atmosphere leads Direct a nitrogen atmosphere leads to to Compounds and with 3.1% 12% yields, respectively (Scheme Compounds 14 14 and 15 15 with 3.1% andand 12% yields, respectively (Scheme 1). 1).

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Scheme Scheme 1. 1. Synthesis Synthesis of of Compounds Compounds 14, 14, 15 15 and and 16. 16.

Following the same synthetic procedure, Compound 17 is obtained as a unique product by 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). coupling between 2-metoxyhydroquinone and geraniol with 5.9% yield (Scheme 2).

Synthesis of Compounds 17 and 18. Scheme 2. Synthesis

Standard acetylation (Ac (Ac222O/CH O/CH222Cl Standard acetylation Cl222/DMAP) /DMAP)ofof1515and and17 17gives givesthe theacetylated acetylatedderivatives derivatives 16 16 and and 18 with with 94.8% 94.8% and and 98% 98% yields, yields, respectively. respectively. 18 In search for for aa synthetic synthetic pathway pathway to to obtain obtain hydrated hydrated geranylphenols, geranylphenols, we have attempted attempted In the the search we have two explore the the possibility possibility of of obtaining obtaining both both geranylated geranylated two different different approaches. approaches. In In the the first first one, one, we we explore quinones andhydrated hydrated geranylphenols a one-pot synthesis. hasreported been reported some quinones and geranylphenols in ainone-pot synthesis. It has It been that somethat hydrated hydrated geranylorcinols have been obtained as minor products in the coupling reaction of orcinol geranylorcinols have been obtained as minor products in the coupling reaction of orcinol and geraniol and geraniol in the presence of 1% aqueous oxalic acid at 80 °C [40]. Therefore, in this approach, the in the presence of 1% aqueous oxalic acid at 80 ˝ C [40]. Therefore, in this approach, the coupling 3 2 coupling reaction is carried out under air using dioxane as the solvent, BF 3 ·Et 2 O as the catalyst and reaction is carried out under air using dioxane as the solvent, BF3 ¨ Et2 O as the catalyst and small small amounts of added Interestingly, the obtained products depend the chemical amounts of added water.water. Interestingly, the obtained products depend on theon chemical naturenature of the of the reacting phenol. Thus,between coupling between geraniol and 1,4-hydroquinone leads to reacting phenol. Thus, coupling geraniol and 1,4-hydroquinone leads to monogeranylated monogeranylated hydroquinone 1 well and quinone 2, as wellquinone as digeranylated quinone 19 (Scheme 3); hydroquinone 1 and quinone 2, as as digeranylated 19 (Scheme 3); whereas, coupling whereas, coupling between 2-metoxyhydroquinone and geraniol gives the disubstituted quinone 20 between 2-metoxyhydroquinone and geraniol gives the disubstituted quinone 20 as the only product as the only producthydrated (Schemegeranylphenols 4). Finally, hydrated geranylphenols andcoupling 23 werereaction obtained in the (Scheme 4). Finally, 22 and 23 were obtained22 in the between coupling reaction between o-cresol and geraniol (Scheme 5). o-cresol and geraniol (Scheme 5).

Scheme 3. Synthesis of Compounds 1, 3 and 19.

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 Int. J. Mol. Sci. 2016, 17, 840 5 ofno 18 Int. J. Mol. Sci. 2016, 17, 840 5 of 18 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 Scheme 3. Synthesis of Compounds 1, 3 and 19. different pattern of products has been reported. Compound 1 and a mixture of digeranylated quinones and di-ortho-geranylated quinone) obtained with 40% In this(19reaction, Compounds 1, 3 and 19 were were obtained with 28.0%, 7.6% and and 27% 1.9% yields, yields, respectively, but Compound 3 was not identified [41]. respectively. When this coupling is carried out in the presence of a nitrogen atmosphere and with no Onwater, the other hand, a 1methoxy substitution in the hydroquinone induces this a complete in added Compound is obtained as the exclusive product [26]. Recently, reactionchange has been the product distribution, i.e., Compound 20 is obtained with 4.1% yield. 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 3. of 1, 19. the product distribution, i.e.,Scheme Compound 20 is obtained with 4.1% yield. Scheme 3. Synthesis Synthesis of Compounds Compounds 1, 33 and and 19. Scheme 4. Synthesis of Compound 20. 28.0%, 7.6% and 1.9% yields, In this reaction, Compounds 1, 3 and 19 were obtained with respectively. When this coupling is carried out in the presence of a nitrogen atmosphere and with no It is worthCompound mentioning1 that geranylated 19, 20) are formed onlythis by coupling geraniol added water, is obtained as thequinones exclusive(3,product [26]. Recently, reaction has been with 1,4-dihydroxybenzene systems. Probably, the oxidation to 1,4-quinone is enhanced by the redox performed at higher temperatures using aluminum phenoxide as the catalyst, and a completely properties of hydroquinone compounds. different pattern of products has been reported. Compound 1 and a mixture of digeranylated Finally, in the coupling of geraniol withquinone) o-cresol, hydrated Compounds andand 23 were quinones (19 and di-ortho-geranylated obtained with 22 40% 27%obtained yields, Scheme 4. 4. Synthesis Synthesis of of were Compound 20. Scheme Compound 20. with 9.0% andbut 10.5% yields, respectively. respectively, Compound 3 was not identified [41]. On other hand, a methoxy substitution in the(3,hydroquinone induces complete change in It is the worth mentioning that geranylated quinones 19, 20) are formed onlyaby coupling geraniol the product distribution, i.e., Compound 20 is obtained with 4.1% yield. 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.

Scheme 5. 5. Synthesis Synthesis of of Compounds Compounds 22 22 and 23 23 by by coupling coupling of of o-cresol o-cresol and and geraniol, geraniol, in in the the presence presence of of Scheme Schemeand 4. Synthesis of Compound 20. atmosphere. water and the absence of a nitrogen water and the absence of a nitrogen atmosphere.

It is worth mentioning that geranylated quinones (3, 19, 20) are formed only by coupling geraniol The formation of Compounds 22 and 23 may be explained by the proposed mechanism depicted this reaction, Compounds 3 and 19the were obtained with 28.0%,is 7.6% and by 1.9% withIn 1,4-dihydroxybenzene systems.1,Probably, oxidation to 1,4-quinone enhanced theyields, redox in Scheme 6. respectively. this coupling is carried out in the presence of a nitrogen atmosphere and with properties of When hydroquinone compounds. no added water, Compound is obtained the exclusive [26]. Recently, this reaction Finally, inSynthesis the coupling of1 geraniol o-cresol, hydrated Compounds 22 and 23 were obtained Scheme 5. of Compounds 22with andas23 by coupling ofproduct o-cresol and geraniol, in the presence of has been performed at higher temperatures using aluminum phenoxide as the catalyst, and a completely atmosphere. water and the absence of a nitrogen with 9.0% and 10.5% yields, respectively. different pattern of products has been reported. Compound 1 and a mixture of digeranylated quinones Thedi-ortho-geranylated formation of Compounds 22 and 23 may be explained by and the proposed mechanism depicted (19 and quinone) were obtained with 40% 27% yields, respectively, but in Scheme 6. 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, the coupling of geraniol o-cresol, hydrated Compounds 22 and 23presence were obtained Scheme in 5. Synthesis of Compounds 22with and 23 by coupling of o-cresol and geraniol, in the of with water 9.0% and 10.5% yields, and the absence of arespectively. nitrogen atmosphere. The formation of Compounds 22 and 23 may be explained by the proposed mechanism depicted The formation of Compounds 22 and 23 may be explained by the proposed mechanism depicted in Scheme 6. in Scheme In the 6. first step, an allylic carbocation is formed by the reaction of BF3 ¨ Et2 O with geraniol, which is then coupled with phenol via Electrophilic Aromatic Substitution (EArS) (Step 2). In presence of water, the adduct BF3 ¨ H2 O is presumably formed by nucleophilic displacement of an ether molecule by H2 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

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(Steps 3 and 4). Finally, the remaining olefinic bond is hydrated by BF3 ¨ 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 Int. J. Mol.been Sci. 2016, 17, 840 6 of 18 already formed.

Scheme 6. 6. Proposed Proposed mechanism mechanism for for the the formation formation of Scheme of Compounds Compounds 22 22 and and 23. 23.

In the first step, an allylic carbocation is formed byofthe of BF3·Et2O geraniol, Based on this result, our second approach consists thereaction direct hydration of with the side chain which by the is then coupled with phenol via Electrophilic Aromatic Substitution (EArS) (Step 2). In presence of reaction of a geranylated phenol with a Lewis acid (BF3 ¨ Et2 O) in dioxane and in the presence of water. water, the adduct 3·H2O is presumably formed nucleophilic of an ether molecule Compound 17 wasBF submitted to this reaction, andbycompounds 21,displacement 24–26 were obtained with 11.1%, by H 2 O (Step 3). Subsequently, this adduct reacts with the geranyl chain by a Markovnikov-type 10.7%, 24% and 18.9% yields, respectively (Scheme 7). addition, forming24a and stable tertiary carbocation, which then hydrated by reaction a water Compounds 26 may be formed through Stepsis3–5 of the mechanism proposedwith in Scheme 6. molecule (Steps 3 and 4). Finally, the remaining olefinic bond is hydrated by BF 3 ·H 2 O, and However, in this reaction, Compound 25 is formed by cyclization of the tertiary carbocation formed ina completely hydratedof geranyl is obtained (Step It is worth mentioning that water iscyclization added 24 Step 3, the formation tertiarychain carbocation in the C-715). position of geranyl chain, 6-endo-trig 1 1 h after reaction started. Thissubsequent means thatnucleophilic Step 3 begins when most on of the the from thethe C2coupling -C3 double bond has and been hydration by the attack of water 1 geranylphenol has already been formed. tertiary carbocation in the C-3 position (Scheme 8). Based on this result, our second approach the direct of thefor side chain by The carbocation intermediates appearing in consists Schemesof 6 and 8 havehydration been proposed coupling of the reaction a geranylated phenol with aofLewis acidin(BF 3·Et2O) in dioxane and[39,40]. in the presence of phenol with of geraniol and various reactions geraniol acidic aqueous solution water. Compound14–18, 17 was20, submitted this and reaction, compounds 21, 24–26 were obtainedinwith Compounds 22–26 aretonew, their and structural characterization is described the 11.1%, 10.7%, 24% and 18.9% yields, respectively (Scheme 7). next section.

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Scheme 7. Synthesis of Compounds 21 and 24–26.

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 Scheme Synthesis of Compounds Compounds 21and and 24–26. Scheme 7.7. Synthesis of 21 24–26. 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) 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. 8. Proposed Proposed mechanism mechanism for for formation formation of ofCompound Compound25. 25.Reaction Reactionof ofLewis Lewisacid acid(BF (BF¨3·Et 2O) Scheme 3 Et2 O) with water, addition Markovnikov of H by BF 3H2O adduct on C-6′1 position of geranyl chain and with water, addition Markovnikov of H by BF3 H2 O adduct on C-6 position of geranyl chain and 1 , 6-endo-trig carbocation formation formation in in C-7 C-7′, 6-endo-trig cyclization cyclization and and later later geranyl geranyl chain chain hydration. hydration. carbocation

The carbocation intermediates appearing in Schemes 6 and 8 have been proposed for coupling 2.2. Structure Determination Scheme 8. geraniol Proposed and mechanism formation Compound 25. Reaction ofsolution Lewis acid (BF3·Et2O) of phenol with variousfor reactions of of geraniol in acidic aqueous [39,40]. The chemical structures of all compounds synthesized in this work were mainly established by 1D with water, addition Markovnikov of H by BF 3H2O adduct on C-6′ position of geranyl chain and Compounds 14–18, 20, 22–26 are new, and their structural characterization is described in the carbocation C-7′, 6-endo-trig cyclization and later geranyl chain hydration. and Nuclear formation Magneticin Resonance (NMR) spectroscopy techniques. All NMR spectra are given in next2D section. Figure S1. In this section, the NMR data used to determine the chemical structure of new compounds, The carbocation intermediates appearing in Schemes and 8 have beengeranylphenols proposed for coupling geranylated phenol derivatives (14–18), geranylated quinone 6(20) and hydrated (22–26), 2.2. Structure Determination of phenol with geraniol and various reactions of geraniol in acidic aqueous solution [39,40]. are discussed in detail. Compound 19 has been already reported, but it has been included in this section The chemical structures of all compounds in this work were mainly established by Compounds 20, 22–26 andsynthesized their is described in the because the NMR 14–18, assignation givenare in new, literature is notstructural right [41].characterization Compounds 14–18: The 1 H-NMR 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy techniques. All NMR spectra are given next section. spectrum of Compound 14 shows a pattern characteristic of aromatic tri-substitution, i.e., singlet signal in Figure S1. In this section, the NMR data used to determine the chemical structure of new at 6.93 ppm (1H, H-3, meta-coupling of H-3 with H-5 was not detected); doublet at 6.88 ppm (1H, compounds, geranylated phenol derivatives (14–18), geranylated quinone (20) and hydrated Structure Determination J2.2. = 8.0, H-5); and doublet at 6.69 ppm (1H, J = 8.0, H-6). The position of the geranyl chain on the geranylphenols (22–26), are discussed in detail. Compound 19 has been already reported, but it has aromatic has been established two-dimensional (2D) Heteronuclear Multiple Bond Correlation Thering chemical structures of allby compounds synthesized in this work were mainly established by been included in this section because the assignation given in literature is not right [41].1 2 J NMR 1 with C-4 (HMBC) correlations. In this spectrum, a coupling of H-1 (δ = 133.9 ppm) and C-2 H-C spectroscopy techniques. All C NMR spectra are given 1D and 2D Nuclear Magnetic Resonance (NMR) Compounds 14–18: The3 1H-NMR spectrum of Compound 14 shows a pattern characteristic of (δ = 123.6 S1. ppm) JH-C coupling between the signals of C-1, C-3, C-31 and C-5 at δC =of130.9, inC Figure In and this asection, the NMR data used to determine the chemical structure new aromatic tri-substitution, i.e., singlet signal at 6.93 ppm (1H, H-3, meta-coupling of H-3 with H-5 was 3J 135.7 and 126.7 ppm, respectively, were observed. Further correlations at between the CH -Ar H-C 3 compounds, geranylated phenol derivatives (14–18), geranylated quinone (20) and hydrated not detected); doublet at 6.88 ppm (1H, J = 8.0, H-5); and doublet at 6.69 ppm (1H, J = 8.0, H-6). The group (δ = 2.23 ppm) with C-1 and C-3 at δ = 151.8 and 130.9 ppm, respectively, were observed H C Compound 19 has been already reported, but it has geranylphenols (22–26), are discussed in detail. position of the geranyl chain on the1 aromatic ring has been established by two-dimensional (2D) (Figure 3a). On in thethis other hand,because the H-NMR spectrum of Compound shows a is singlet signal at been included section the NMR assignation given in15literature not right [41]. Heteronuclear Multiple Bond Correlation (HMBC) correlations. In this spectrum, a 2JH-C coupling of 1 6.93 ppm (1H, H-3, meta-coupling of H-3 with H-5 was not detected) and two doublet signals at Compounds 14–18: The H-NMR spectrum of Compound 14 shows a pattern characteristic of 6.92 ppm tri-substitution, (1H, J = 8.7 Hz,i.e., H-5) and 6.73 (1H, J = 8.7 Hz, H-6). a of signal at aromatic singlet signal at 6.93 ppm (1H, H-3,Additionally, meta-coupling H-3 appearing with H-5 was 5.07 (s, 1H)doublet was assigned the OH aromatic substitution shows not ppm detected); at 6.88to ppm (1H,group. J = 8.0,The H-5); and doublet at 6.69pattern ppm (1H, J =unequivocally 8.0, H-6). The position of the geranyl chain on the aromatic ring has been established by two-dimensional (2D) Heteronuclear Multiple Bond Correlation (HMBC) correlations. In this spectrum, a 2JH-C coupling of

C-1, C-3, C-3′ and C-5 at δCC = 130.9, 135.7 and 126.7 ppm, respectively, were observed. Further correlations at 33JH-C H-C between the CH33-Ar group (δH H = 2.23 ppm) with C-1 and C-3 at δC C = 151.8 and 1 1 130.9 ppm, respectively, were observed (Figure 3a). On the other hand, the H-NMR spectrum of Compound 15 shows a singlet signal at 6.93 ppm (1H, H-3, meta-coupling of H-3 with H-5 was not detected) and two signals at 6.92 ppm (1H, J = 8.7 Hz, H-5) and 6.73 (1H, J = 8.7 Hz, 8H-6). Int. J. Mol. Sci. 2016, 17, doublet 840 of 18 Additionally, a signal appearing at 5.07 ppm (s, 1H) was assigned to the OH group. The aromatic substitution pattern shows unequivocally that the geranyl chain is attached to the ortho position at that the geranyl chain attachedoftothe thegeranyl ortho position at the hydroxyl groups. position of the geranyl hydroxyl groups. Theisposition chain on aromatic ring The has been confirmed by 2D chain on the aromatic ring has been confirmed by 2D HMBC correlations. In this spectrum, the signal HMBC correlations. In this spectrum, the signal at δHH = 3.35 ppm assigned to H-1′ (2H d, J = 7.2 Hz) 1 3 3 2 at δH =3 3.35 ppm assigned to H-1 d, JC-3 = 7.2 shows with C-1and (δC2 J=H–C 152.1), C-3 shows JH-C H-C coupling with C-1 (δCC = (2H 152.1), (δCCHz) = 127.8) andJH-C C-3′coupling (δCC = 138.1 ppm) H–C coupling 1 (δ = 138.1 ppm) and 2 J 1 (δ = 126.6 and 121.8 ppm, (δ = 127.8) and C-3 coupling with C-2 and C-2 with C-2 and C-2′ (δ C = 126.6 and 121.8 ppm, respectively; Figure 3b) Additionally, the signal at δ H = C H–C C C C H 1 ) showed spatial correlations respectively; Figure 3b) Additionally, the signal at δ = 3.35 ppm (H-1 3.35 ppm (H-1′) showed spatial correlations with theHsignals at δHH = 6.93, 5.07 and 1.79 ppm, assigned with theOH signals δH33-C3′, = 6.93, 5.07 and 1.79 ppm, assigned toδH-3, OH ppm and CH -C3331-Ar) , respectively; while to H-3, andat CH respectively; while the signal at H = 2.23 (s, 3CH showed spatial H the signal at δ = 2.23 ppm (s, CH -Ar) showed spatial correlations with H-3 and H-5 (Figure 3c). H H-3 and H-5 (Figure 3 correlations with 3c).

Figure 3. 3. Main Figure Main observed observed correlations: correlations: 2D 2D Heteronuclear Heteronuclear Multiple Multiple Bond Bond Correlation Correlation (HMBC), (HMBC), Compound 14 (a) (a) and and Compound Compound 15 15 (b); (b); 1D 1D Nuclear Nuclear Overhauser Overhauser Effect Effect Spectroscopy Spectroscopy (NOESY) Compound Compound 15 (c). 3 In the the 111H-NMR of the the acetylated acetylated derivative derivative 16, 16, aa singlet singlet at at δδHH = = 2.29 In H-NMR spectrum spectrum of 2.29 ppm ppm (3H, (3H, CH CH33CO) CO) H 13 13 C = 169.6 (C=O) and C NMR spectrum, the signals appearing at δ C was observed. Additionally, in the 13 was observed. Additionally, in the C NMR spectrum, the signals appearing at δC = 169.6 (C=O) and 20.8 (CH3) ppm confirmed the presence of monoacetylated derivative 16. 20.8 (CH33 ) ppm confirmed the presence of monoacetylated derivative 16. 11 Compound 17: Compound 17: In In the the 1H-NMR H-NMR spectrum, spectrum, aa pattern pattern characteristic characteristic of of aromatic aromatic tetra-substitution, tetra-substitution, i.e., two singlet signals at 6.67 (1H, H-3) and 6.43 (1H, H-6), was observed. i.e., two singlet signals at 6.67 (1H, H-3) and 6.43 (1H, H-6), was observed. The The position position of of the the geranyl geranyl chain on the aromatic ring was established by two-dimensional (2D) HMBC correlations. In this chain on the aromatic ring was established by two-dimensional (2D) HMBC correlations. In this 3 H–C H–C coupling of H-1′1 with C-2 (δC C = 118.5 ppm) and C-2′ spectrum, aa 222JJH–C 1 (δCC = 121.8 ppm) and a3 3JH–C spectrum, H–C coupling of H-1 with C-2 (δC = 118.5 ppm) and C-2 (δC = 121.8 ppm) and a JH–C coupling between the signals of C-1, C-3 and C-3′ at δCC = 147.6, 115.3 and 139.2 ppm, respectively, coupling between the signals of C-1, C-3 and C-31 at δC = 147.6, 115.3 and 139.2 ppm, respectively, H–C between the CH33O group (δH H were observed. observed. In In addition, addition, aa correlation correlationatat333JJH–C C-5 were between the CH3 O group (δH == 3.83 3.83 ppm) ppm) and and C-5 H–C C = 145.4 ppm) and correlations between 222JH–C H–C and 33J3 H–C of the OH-C4 group (δH H = 5.15 ppm) with C(δ C H–C (δC = 145.4 ppm) and correlations between JH–C and JH–C of the OH-C4 group (δH = 5.15 ppm) with 3 (δC = 115.3 ppm) were also observed (Figure 4). C-3 C(δC = 115.3 ppm) were also observed (Figure 4).

Figure 4. 4. Major 2D HMBC HMBC observed observed correlations correlations for Figure for Compound Compound 17. 17.

In the the 111H-NMR the acetylated acetylated derivative derivative 18, 18, two two singlet singlet signals signals at at δδHHH = = 2.29 2.29 and and In H-NMR spectrum spectrum of of the 13C NMR spectrum, the signals 13 13 3 CO) were observed. Additionally, in the 2.28 ppm (each 3H, CH 2.28 ppm (each 3H, CH33CO) were observed. Additionally, in the C NMR spectrum, the signals appearing at at δδ == 169.2 169.2 (COCH (COCH333-C4), -C4), 168.9 168.9 (COCH (COCH333-C1) 20.8 (CH (CH333COO-C1) COO-C1) and and appearing -C1) ppm ppm and and δδ == 20.8 3COO-C4) ppm, confirmed the presence of diacetylated derivative 18. 20.6 (CH 3 20.6 (CH3 COO-C4) ppm, confirmed the presence of diacetylated derivative 18. Compound 19: 19: The The symmetrical symmetricalmolecular molecularstructure structurewas wasconfirmed confirmed substitution pattern Compound byby thethe substitution pattern in in the olefin zone and by the intensity of integrated signals of hydrogen atoms in quinone and olefinic the olefin zone and by the intensity of integrated signals of hydrogen atoms in quinone and olefinic portion. For ppm (s, 2H, H-3H-3 andand H-6)H-6) indicates the presence of two portion. Forinstance, instance,the thesignal signalatatδHHδ = 6.70 = 6.70 ppm (s, 2H, indicates the presence of H

two 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-11 ) and for the latter and the signal at δH = 1.73 ppm, assigned to

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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 1H-NMR signals is given in the experimental part, and the corresponding spectrum is shown in the Supplementary Int. J. Mol. Sci. 2016, 17, 840 9 of 18 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 CCNMR one signal atat δCδ=C 187.6 ppm (C-1 andand CCH33-C3′ -C31 (Figure (Figure5a). 5a).Finally, Finally,in inthe the1313 NMRspectrum, spectrum,only only one signal = 187.6 ppm (C-1 4) of of thethe carbonyl group was observed, confirming thethe symmetrical structure ofof Compound 19.19. C-4) carbonyl group was observed, confirming symmetrical structure Compound

Figure 5. Main observed correlations: 1D NOE Compound 19 (a), 2D HMBC Compound 20 (b).

Compound 20: double geranyl geranyl chain chain substitution substitution on the quinone quinone nucleus nucleus was was Compound 20: The The presence presence of of double on the 1H-NMR 1 spectrum at δ H = 3.15 ppm (2H, confirmed by the observation of two doublet signals in the confirmed by the observation of two doublet signals in the H-NMR spectrum at δH = 3.15 ppm 1 and J = 7.4 (2H, J(2H, = 7.0J = Hz), were were assigned to thetohydrogens H-1′ and (2H, J =Hz) 7.4and Hz) 3.12 and ppm 3.12 ppm 7.0 which Hz), which assigned the hydrogens H-1H-2′′, 11 , respectively. respectively. Additionally, the presence of only one one hydrogen at at δH δ= = 6.32 H-2 Additionally, the presence of only hydrogen 6.32ppm ppm(1H, (1H,s,s, H-6) H-6) H demonstrates the degree of tetra-substitution on the quinone moiety. Differentiation between demonstrates the degree of tetra-substitution on the quinone moiety. Differentiation between geranyl geranyl 3JH–C with C-4 chains was wasestablished established byHMBC the HMBC correlations observed at (δ chains by the correlations observed for H-11 at 3for JH–C H-1′ with C-4 C = 187.9 ppm; 3 2 1 3 2 (δ C = 187.9 ppm; C=O) and H-1′ at J H–C with C-2 (δC = 155.1 ppm; C-OCH 3) and at JH–C with C-3 (δC = C=O) and H-1 at JH–C with C-2 (δC = 155.1 ppm; C-OCH3 ) and at JH–C with C-3 (δC = 131.9 ppm) 3JH–C correlations with C-4 (δC = 187.9 11 showed 3 J showed 131.9 ppm) 5b). of H-1′′ (Figure 5b). (Figure Similarly, theSimilarly, signal of the H-1signal H–C correlations with C-4 (δC = 187.9 ppm; C=O) 2 ppm;C-6 C=O) C-6 ppm) (δC = 130.5 and C-5 JH–C(δwith C-5 (δC = 148.3 ppm) (Figure 5b). and (δC and = 130.5 and 2ppm) JH–C with C = 148.3 ppm) (Figure 5b). 1H-NMR spectrum shows a pattern characteristic of aromatic tri1 Compound 22: The Compound 22: The H-NMR spectrum shows a pattern characteristic of aromatic tri-substitution, 1 ) and = 7.3 Hz, 1H, δH-4′) and δH = 6.90 (J = 7.4 Hz, 11H, substitution, i.e., doublet δH =(J6.95 i.e., doublet signals at δHsignals = 6.95 at ppm = 7.3ppm Hz,(J1H, H-4 H = 6.90 (J = 7.4 Hz, 1H, H-6 ), a H-6′), a doublet double doublet at δ(J H = 6.70 (J = 7.3 and 7.4 Hz,1 1H, H-5′). The position of the geranyl double signal atsignal δH = 6.70 = 7.3 and 7.4 Hz, 1H, H-5 ). The position of the geranyl chain on chain on the aromatic ring was established by two-dimensional HMBC correlations. In this the aromatic ring was established by two-dimensional (2D) HMBC(2D) correlations. In this spectrum, a 2 3 2spectrum, 1 (δ 3 J and a JH-C H-8 (δH = m, 2.78–2.74, m,C-1 2H) with C-1′ (δ C = 120.4 ppm) a JH-C coupling JH-C coupling ofcoupling H-8 (δH =of2.78–2.74, 2H) with = 120.4 ppm) and a coupling between H-C C 1 1 at δ C-6′ between theof signals C-2′and at δC = 151.9 and 126.9 ppm, respectively, were observed. the C-2 Int. J.signals Mol. Sci. 2016, 17,and 840ofC-6 10 of 18 C = 151.9 and 126.9 ppm, respectively, were observed. In addition, 3J 1 and 1 atand In addition, at correlations at 3JH-C between the CH3(δ -Ar=group (δH = with 2.16 ppm) with C-2′ C-4′ at δ C = correlations between the CH -Ar group 2.16 ppm) C-2 C-4 δ = 151.9 and H-C 3 H C 2 1 (δwith 151.9 and of 128.4 respectively, aC-3 JH-C C-3′ (δ C = 126.2) were(Figure observed (Figure 6a). presence twoppm, hydroxyl in the geranyl was confirmed by the 6a). observation of The two 128.4 ppm, respectively, andgroups a 2 JH-C and with 126.2) were observed The presence of C = chain 13 presence of two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary carbinolic signals δC = 72.9 andwas 71.3 ppm in the C observation NMR spectrum. signals were two hydroxyl groups in theatgeranyl chain confirmed by the of twoThese tertiary carbinolic tertiary carbinolic at ppm δC-2, C = 75.8 and ppm in the 13C NMR spectrum. These were assigned 13 C71.0 assigned to C-671.0 and byspectrum. two-dimensional (2D)assigned HMBC correlations. signals at δCcarbons = 75.8signals and inrespectively, the NMR These were to carbons C-6Thus, and 2JH-C with carbinolic to carbons C-6 and C-2, respectively, by two-dimensional (2D) HMBC correlations. Thus, H-8 showed 3 the CH 3 -C6 group showed correlation at carbon at C-6 (δ C = 72.9 ppm), while the C-2, respectively, by two-dimensional (2D) HMBC correlations. Thus, H-8 showed correlation at JH-C 3JH-C with carbinolic carbon at C-6 (δC = 75.8 ppm), whereas the methyl groups at δ2C = correlation at methyl groups at δC = 31.2 and(δ29.9 (CH3-1whereas and CH3the -C2,methyl respectively) at 3J-1 Hwith carbinolic carbon at C-6 75.8 ppm), groupsshowed at δC = correlations 29.2 ppm (CH C = ppm 2JH-C with C-2 (δC = 71.0 ppm) (Figure 6a). 29.2 ppm (CH 3= -171.3 andppm) CH 3-C2) showed at 2 J correlations C with C-2 (δ Cshowed (Figure 6b). and CH -C2) correlations at with C-2 (δ = 71.0 ppm) (Figure 6a). 3 H-C C Compound 23: A similar analysis was conducted to elucidate the structure of this compound. The 1H-NMR spectrum shows a pattern characteristic of aromatic tri-substitution, i.e., a singlet signal at δH = 6.95 ppm (1H, H-2′, meta-coupling of H-2′ with H-6′ was not detected), a doublet signal at δH = 6.90 (J = 8.1 Hz, 1H, H-6′) and a doublet signal at δH = 6.68 (J = 8.1 and 1H, H-5′). The position of the geranyl chain on the aromatic ring was established by two-dimensional (2D) HMBC correlations. In this spectrum, a 2JH-C coupling of H-8 (δH = 2.65–2.52, m, 2H) with C-1′ (δC = 135.5 ppm) and a 3JH-C coupling between signals of C-2′and C-6′ at δC = 130.9 126.7 ppm,22; respectively, were Figure 6. the 2D HMBC observed correlations for:and (a) Compound (b) Compound 23. observed. Main 3 In addition, correlations at JH-C between the CH3-Ar group (δH = 2.22 ppm) with C-2′ and C-4′ at δC = 130.9Because and 151.6 ppm, respectively, and a 2JH-C with17C-3′ (δ C = hydration 123.4) were observed (Figure 6b). The Compound 24 was obtained from byto the reaction of aromatic Compound 23: A similar analysis was conducted elucidate the structure of it, thisthe compound. 1 H-NMRpattern substitution wasshows maintained for characteristic Compounds 24 25. Thus, in Compound the presence The spectrum a pattern of and aromatic tri-substitution, i.e.,24, a singlet signal 1 1 1 of two hydroxyl groups in the geranyl chain was confirmed by the observation of twosignal tertiary at δH = 6.95 ppm (1H, H-2 , meta-coupling of H-2 with H-6 was not detected), a doublet at 13C NMR spectrum. These signals1 were assigned to 1and signals at δ C = 75.8 70.9 ppm in the δcarbinolic = 6.90 (J = 8.1 Hz, 1H, H-6 ) and a doublet signal at δ = 6.68 (J = 8.1 and 1H, H-5 ). The position of H H carbons C-3′ and C-7′, respectively, by two-dimensional (2D) HMBC correlations. Thus, the CH3-C7′ and CH3-8′ groups showed correlation at 2JH-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 2JH-C with C-2 (δC = 114.2 ppm) and C-1′ (δC = 48.3 ppm) and 3JH-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

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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 13C NMR spectrum. These signals were Int. J. Mol. Sci. 17, 840 of 18 assigned to 2016, carbons C-6 and C-2, respectively, by two-dimensional (2D) HMBC correlations. 10 Thus, the CH3-C6 group showed correlation at 2JH-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 2JHthe geranyl chain on the aromatic ring was established by two-dimensional (2D) HMBC correlations. C with C-2 (δC = 71.3 ppm) (Figure 6b). In this spectrum, a 2 JH-C coupling of H-8 (δH = 2.65–2.52, m, 2H) with C-11 (δC = 135.5 ppm) and a 3 JH-C coupling between the signals of C-21 and C-61 at δC = 130.9 and 126.7 ppm, respectively, were observed. In addition, correlations at 3 JH-C between the CH3 -Ar group (δH = 2.22 ppm) with C-21 and C-41 at δC = 130.9 and 151.6 ppm, respectively, and a 2 JH-C with C-31 (δC = 123.4) were observed (Figure 6b). The 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 Figureshowed 6. Main 2D HMBC observed for: (a) carbon Compound 22; (δ (b) Compound 23. CH3 -C6 group correlation at 2 JH-Ccorrelations with carbinolic at C-6 C = 72.9 ppm), while the methyl groups at δC = 31.2 and 29.9 ppm (CH3 -1 and CH3 -C2, respectively) showed correlations at Because Compound 24 was obtained from 17 by the hydration reaction of it, the aromatic 2J H-C with C-2 (δC = 71.3 ppm) (Figure 6b). substitution pattern was maintained for Compounds 24 and 25. Thus, in Compound 24, the presence Because Compound 24 was obtained from 17 by the hydration reaction of it, the aromatic of two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary substitution pattern was maintained for Compounds 24 and 25. Thus, in Compound 24, the presence of carbinolic signals at δC = 75.8 and 70.9 ppm in the 13C NMR spectrum. These signals were assigned to two hydroxyl groups in the geranyl chain was confirmed by the observation of two tertiary carbinolic carbons C-3′ and C-7′, respectively, by two-dimensional (2D) HMBC correlations. Thus, the CH3-C7′ signals at δC = 75.8 and 70.9 ppm in the 13 C NMR spectrum. These signals were assigned to carbons 2JH-C with carbinolic carbon at C-7′ (δC = 70.9 ppm), and and1 CH3-8′ groups showed correlation at C-3 and C-71 , respectively, by two-dimensional (2D) HMBC correlations. Thus, the CH3 -C71 and therefore, the signal at δC = 75.8 ppm was unequivocally assigned to C-3′ (Figure 7a); while for CH3 -81 groups showed correlation at 2 JH-C with carbinolic carbon at C-71 (δC = 70.9 ppm), and therefore, Compound 25, the methylene group (at δC = 22.7 ppm, assigned as C-7′) showed a correlation at 2JH-C the signal at δC = 75.8 ppm was unequivocally assigned to C-31 (Figure 7a); while for Compound carbon at with C-2 (δC = 114.2 ppm) and C-1′ (δC = 48.3 ppm) and 31JH-C with a tertiary carbinolic 25, the methylene group (at δC = 22.7 ppm, assigned as C-7 ) showed a correlation at 2 JH-C with C-2 δC = 76.7 ppm assigned to C-2′. Additionally, the CH3-C2′ group at δH = 1.20 ppm (3H, s) showed (δC = 114.2 ppm) and C-11 (δC = 48.3 ppm) and 3 JH-C with a tertiary carbinolic carbon at δC = 76.7 ppm coupling at 2JH-C1 with C-2′ and 3JH-C with tertiary C-1′ (δC = 48.3 ppm) (Figure 7b) Thus, the cyclohexane assigned to C-2 . Additionally, the CH3 -C21 group at δH = 1.20 ppm (3H, s) showed coupling at 2 JH-C structure1 is confirmed for geranyl chain. Finally, mono-hydroxylation in the geranyl chain for with C-2 and 3 JH-C with tertiary C-11 (δC = 48.3 ppm) (Figure 7b) Thus, the cyclohexane structure is Compound 26 was mainly established by 13C NMR data and 2D HMBC correlations. Only one signal confirmed for geranyl chain. Finally, mono-hydroxylation in the geranyl chain for Compound 26 was of carbinolic carbon at13 δC = 70.9 ppm in the 13C NMR spectrum was observed, and the methyl groups mainly established by C NMR data and 2D HMBC correlations. Only one signal of carbinolic carbon at δH = 1.22 (6H, s, CH3-C7′ and H-8′) showed 2JH-C correlations with this carbon (C-7′, δC = 70.9 ppm) at δC = 70.9 ppm in the 13 C NMR spectrum was observed, and the methyl groups at δH = 1.22 (6H, s, (Figure 7c). In addition, these methyl groups showed 3JH-C correlations with C-6′ (δC = 43.3 ppm) CH3 -C71 and H-81 ) showed 2 JH-C correlations with this carbon (C-71 , δC = 70.9 ppm) (Figure 7c). In (Figure 7c). addition, these methyl groups showed 3 JH-C correlations with C-61 (δC = 43.3 ppm) (Figure 7c).

Figure 7. 7. Major Major 2D 2D HMBC HMBC observed observed correlations correlations for for Compounds Compounds 24 24 (a), (a), 25 25 (b) (b) and and 26 26 (c). (c). Figure

2.3. In Vitro Antifungal Activity against B. cinerea. All studied compounds (14–26) 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, negative control), Captan at 250 ppm (Figure 8b, used in this study as a positive control) and two different concentrations of Compound 26 (Figure 8c, 150 ppm; Figure 8d, 250 ppm).

All studied compounds (14–26) 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, negative control), Captan at 250 ppm (Figure 8b, used in this study as a positive control) Int. J. Mol. Sci. 2016, 17, 840 11 of 18 and two different concentrations of Compound 26 (Figure 8c, 150 ppm; Figure 8d, 250 ppm).

(a)

(b)

(c)

(d)

Figure Effectof of hydrated geranylphenol the mycelial in vitro growth mycelialof growth cinerea. Figure 8.8.Effect hydrated geranylphenol 26 on26 theon in vitro B. cinerea.of(a)B.Negative (a) Negative control; the medium contains only Potato Dextrose Agar (PDA) and 1% ethanol. (b) control; the medium contains only Potato Dextrose Agar (PDA) and 1% ethanol. (b) Positive control; Positive control; Captan at 250 ppm.26(c) 26Compound at 150 ppm.26(d) Captan at 250 ppm. (c) Compound atCompound 150 ppm. (d) atCompound 250 ppm. 26 at 250 ppm.

The inhibition of mycelial growth is evaluated by measuring colony diameters in the presence and The inhibition of mycelial growth is evaluated by measuring colony diameters in the presence absence of the tested compounds. The results, expressed as the percentage of inhibition, are and absence of the tested compounds. The results, expressed as the percentage of inhibition, are summarized in Table 1. summarized in Table 1. The data indicate that geranylated derivatives of o- and p-cresol (14–16) have no effect on the mycelial of B. cinerea. However, the methoxy derivatives of geranylated p-cresol (17and and 18) Tablegrowth 1. Percentage of inhibition of geranylated phenols (14–18), geranylated quinones (19–21) exhibit a significative increase in the inhibitory activity.of This result is in line work hydrated geranylphenols (22–26) on the mycelial growth B. cinerea strain GM7 at with 72 h inprevious vitro. where a family of methoxy geranylated derivatives was studied [30]. On the other hand, geranylated (19–21) show anGrowth important (greater than 50% Percentagequinones of Inhibition on Mycelial of B.activity cinerea in Vitro (%) Compounds at the higher tested concentrations) of the number of geranyl 50 that mg/Lis independent 150 mg/L 250 chains. mg/L In the case of geranylated phenols, it was found that antifungal activity decreases with the increasing number 14 0˘0 0˘0 0˘0 of prenyl chains15[29,30]. 9˘4 6˘3 8˘5 16 0˘0 0˘0 9˘0 17 2 56 ˘ 2 geranylated quinones 56 ˘ 0(19–21) and Table 1. Percentage of inhibition 49 of ˘ geranylated phenols (14–18), 18 3 mycelial growth of48B.˘cinerea 3 2 vitro. hydrated geranylphenols (22–26) 36 on˘ the strain GM7 at 52 72 ˘ h in 19 30 ˘ 2 51 ˘ 1 69 ˘ 1 20 43 Percentage ˘8 58 ˘ 73 ˘ 8 of Inhibition on8Mycelial Growth of 21 36 ˘ 0 64 ˘ 0 75 ˘ 0 B. cinerea in Vitro (%) Compounds 22 0˘0 30 ˘ 2 53 ˘ 3 500mg/L 1500mg/L 250 mg/L 23 0˘ ˘0 28 ˘ 7 24 36 ˘ 0 66 ˘ 4 67 ˘5 14 0±0 0±0 0±0 25 50 ˘ 6 81 ˘ 5 90 ˘ 1 15 691± ˘ 3 0 8 ± 5 94 ˘ 0 26 81 ˘90± 4 0 ˘ 0 0 ˘ 0 C´ 1 16 ±0 0±0 9±0 0˘0 94 ˘ 5 94 ˘ 0 99 ˘ 0 C+ 2 17 49 ± 2 56 ± 2 56 ± 0 The percentage of inhibition of mycelial growth is based on colony diameter measurements after 72 h of 18 36 ± 3 48 ± 3 52 ± 2 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). 19 30 ± 2 51 ± 1 69 ± 1

20

43 ± 8

58 ± 8

73 ± 8

The data indicate21that geranylated of o-64and 36 derivatives ±0 ± 0 p-cresol (14–16) 75 ± 0have no effect on the mycelial growth of B. 22 cinerea. However,0 ±the methoxy derivatives of geranylated 0 30 ± 2 53 ± 3 p-cresol (17 and 18) exhibit a significative increase in the inhibitory activity. This result is in line with previous work where 23 0±0 0±0 28 ± 7 a family of methoxy geranylated derivatives was studied [30]. 24 36 ± 0 66 ± 4 67 ± 5 On the other hand, geranylated quinones (19–21) show an important activity (greater than 50% at 25 50 ± 6 81 ± 5 90 ± 1 the higher tested concentrations) that is independent of the number of geranyl chains. In the case of ±0 91 ±decreases 0 0 geranylated phenols, 26 it was found that81antifungal activity with 94 the± increasing number of 1 0±0 0±0 0±0 prenyl chains [29,30].C− 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 (22–24). In

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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. 3. Experimental Section 3.1. 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 HF254 for thin layer chromatography (TLC). TLC spots were detected by heating after spraying with 25% H2 SO4 in H2 O. 3.2. Synthesis 3.2.1. Coupling Reaction in Presence of Nitrogen The coupling of geraniol and phenols was carried out using boric trifluoride etherate BF3 ¨ Et2 O as the catalyst and dioxane as the solvent. Experimental details for a typical reaction have been given elsewhere [30]. (E)-4-(3,7-dimethylocta-2,6-dienyl)-2-methylphenol (14): Coupling of o-cresol (1.0 g, 9.3 mmol) and geraniol (1.5 g, 9.7 mmol) was carried out in dioxane (20 mL) with BF3 ¨ Et2 O (0.9 mL, 7.2 mmol) as the catalyst. Two fractions were obtained from the C.C. Fraction I: Compound 14 (64 mg, 3.1% yield) obtained as a yellow viscous oil; Fraction II: unreacted o-cresol (922 mg) that was recovered. Compound 14: IR (cm´1 ) 3385, 2966, 2923, 2854, 1668, 1611, 1508, 1453, 1377, 1262, 1205, 1115, 772; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.93 (1H, s, H-3), 6.88 (1H, d, J = 8.0, H-5), 6.69 (1H, d, J = 8.0, H-6), 5.32–5.29 (1H, m, H-21 ), 5.11–5.09 (1H, m, H-61 ), 4.55 (1H, s, OH), 3.25 (2H, d, J = 7.2, H-11 ), 2.23 (3H, s, CH3 -C2), 2.12–2.08 (2H, m, H-51 ), 2.06–2.04 (2H, m, H-41 ), 1.69 (3H, s, CH3 -C31 ), 1.68 (3H, s, H-81 ), 1.60 (3H, s, CH3 -C71 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 151.8 (C-1), 135.7 (C-31 ), 133.9 (C-4), 131.4 (C-71 ), 130.9 (C-3), 126.7 (C-5), 124.3 (C-61 ), 123.6 (C-21 ), 123.4 (C-2), 114.8 (C-6), 39.7 (C-41 ), 33.3 (C-11 ), 26.6 (C-51 ), 25.7 (C-81 ), 17.7 (CH3 -C71 ), 16.1 (CH3 -C31 ); 15.8 (CH3- C4); MS m/z (%) M+ 244 (48.3), 201 (16.7), 175 (100), 160 (36.7), 147 (31.7), 133 (35.0), 121 (68.3), 106 (13.3), 91 (20.0), 69 (28.3), 41 (31.7). (E)-2-(3,7-dimethylocta-2,6-dienyl)-4-methylphenol (15): Coupling of p-cresol (1.02 g, 9.4 mmol) and geraniol (1.46 g, 9.4 mmol) was carried out in dioxane (20 mL) with BF3 ¨ Et2 O (1.17 mL, 9.5 mmol) as the catalyst. Two fractions were obtained from the C.C. Fraction I: Compound 15 (264 mg, 12% yield) obtained as a yellow viscous oil; Fraction II: unreacted p-cresol (728 mg) that was recovered. Compound 15: IR (cm´1 ) 3446, 2966, 2919, 2857, 1611, 1506, 1446, 1376, 1260, 1197, 1105, 1040, 924, 810; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.93 (1H, s, H-3), 6.92 (1H, d, J = 8.7, H-5), 6.73 (1H, d, J = 8.7, H-6), 5.36–5.32 (1H, m, H-21 ), 5.12–5.09 (1H, m, H-61 ), 5.07 (1H, s, OH), 3.35 (2H, d, J = 7.2, H-11 ), 2.28 (3H, s, CH3 -C4), 2.16–2.14 (2H, m, H-51 ), 2.11–2.09 (2H, m, H-41 ), 1.79 (3H, s, CH3 -C31 ), 1.71 (3H, s, CH3 -C71 ), 1.62 (3H, s, H-81 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 152.1 (C-1), 138.1 (C-31 ), 131.9 (C-71 ), 130.5 (C-5), 129.8 (C-4), 127.8 (C-3), 126.6 (C-2), 123.8 (C-71 ), 121.8 (C-21 ), 115.6

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(C-6), 39.7 (C-41 ), 29.7 (C-11 ), 26.4 (C-51 ), 25.7 (C-81 ), 20.5 (CH3- C4); 17.7 (CH3 -C71 ), 16.0 (CH3 -C31 ); MS m/z (%) M+ 244 (70), 201 (33.3), 175 (88.3), 159 (65), 147 (40), 133 (35), 121 (100: M+ -123 (C9 H15 )), 105 (30), 91 (36.7), 69 (40), 41 (45). (E)-2-(3,7-dimethylocta-2,6-dienyl)-5-methoxybenzene-1,4-diol (17): Coupling of 2-methoxyhydroquinone (2.02 g, 14.4 mmol) and geraniol (2.36 mL, 13.2 mmol) was carried out in dioxane (20 mL) with BF3 ¨ Et2 O (1.62 mL, 12.9 mmol) as the catalyst. Two fractions were obtained from the C.C. Fraction I: Compound 17 (233 mg, 5.9% yield) was obtained as a brown viscous oil; Fraction II: unreacted 2-methoxyhydroquinone (1.76 g) that was recovered. Compound 17: IR (cm´1 ) 3420, 2965, 2924, 2852, 1603, 1520, 1446, 1196, 1105, 835; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.67 (1H, s, H-3), 6.43 (1H, s, H-6), 5.28 (1H, t, J = 7.2 Hz, H-21 ), 5,15 (1H, bs, OH-C4), 5.06 (1H, t, J = 5.5 Hz, H-61 ), 4.87 (1H, bs, OH-C1), 3.83 (3H, s, CH3 O), 3.26 (2H, d, J = 7.2 Hz, H-11 ), 2.12–2.10 (2H, m, H-51 ), 2.08–2.05 (2H, m, H-41 ), 1.76 (3H, s, CH3 -C31 ), 1.69 (3H, s, H-81 ), 1.60 (3H, s, CH3 -C71 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 147.6 (C-1), 145.4 (C-5), 139.2 (C-31 ), 138.6 (C-4), 132.1 (C-71 ), 123.8 (C-61 ), 121.8 (C-21 ), 118.5 (C-2), 115.3 (C-3), 100.4 (C-6), 56.1 (CH3 O-C5), 39.7 (C-41 ), 29.4 (C-11 ), 26.4 (C-51 ), 25.7 (C-81 ), 17.7 (CH3 -C71 ), 16.2 (CH3 -C31 ); MS m/z (%) 276 (39.5: M+ ), 191 (21), 175 (9.9), 153 (100: M+ -123 (C9 H15 )), 91 (4.9), 69 (16), 41 (16). 3.2.2. Coupling Reaction in the Absence of Nitrogen and with Added Water The main difference in the experimental procedure of this reaction is that, after the addition and stirring were completed, 5 mL of H2 O were added, and the stirring was continued for another 24 h. The crude was chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity (19.8:0.2 Ñ 8.0:12.0). 2-Geranylhydroquinone (1), 2-geranylquinone (3) and 2,5-bisgeranylquinone (19): Coupling of 1,4-hydroquinone (1.01 g, 9.2 mmol) and geraniol (1.35 g, 5.5 mmol) was carried out in dioxane (30 mL) with BF3 ¨ Et2 O (0.46 g, 3.2 mmol) as the catalyst. Three fractions were obtained from the C.C. Fraction I: Compound 19 (42 mg, 1.9% yield) obtained as a brown viscous oil. Compound 19: 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.70 (2H, s, H-3 and H-6), 5.03 (2H, t, J = 6.8 Hz, H-21 ), 4.94 (2H, t, J = 6.3 Hz, H-61 ), 3.21 (4H, d, J = 6.8, H-11 ), 2.07–2.02 (4H, m, H-51 ), 1.98–1.95 (4H, m, H-41 ), 1.73 (6H, s, CH3 -C31 ), 1.68 (6H, s, H-81 ), 1.57 (6H, s, CH3 -C71 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 187.6 (C-1 and C-4), 143.6 (C-2 and C-5), 137.5 (C-31 and C-311 ), 136.2 (C-3 and C-6), 131.5 (C-71 and C-711 ), 123.9 (C-61 and C-611 ), 119.5 (C-21 and C-211 ), 39.7 (C-41 and C-411 ), 26.5 (C-11 and C-111 ), 25.7 (C-51 and C-511 ), 25.3 (C-81 and C-811 ), 17.7 (CH3 -C71 and CH3 -C711 ), 16.4 (CH3 -C31 and CH3 -C311 ). Fraction II: Compound 3 (166 mg, 7.6% yield) obtained as a brown viscous oil. Fraction III: Compound 1 (616 mg, 28% yield) obtained as a brown viscous oil. The spectroscopic data (IR, MS and NMR) for 1 and 3 were consistent with those previously reported [26]. 3,5-Bis((E)-3,7-dimethylocta-2,6-dienyl)-2-methoxycyclohexa-2,5-diene-1,4-dione (20): Coupling of 2-methoxyhydroquinone (500 mg, 3.6 mmol) and geraniol (0.65 mL, 3.6 mmol) was carried out in dioxane (20 mL) with BF3 ¨ Et2 O (0.46 g, 3.2 mmol) as the catalyst. Two fractions were obtained from the C.C. Fraction I: Compound 20 (60 mg, 4.1% yield) was obtained as a brown viscous oil; Fraction II: unreacted 2-methoxyhydroquinone (419 mg) that was recovered. Compound 20: IR (cm´1 ) 2966, 2924, 2854, 1649, 1602, 1446, 1376, 1323, 1207, 1161, 1121, 954, 887; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.32 (1H, s, H-6), 5.15–5.13 (1H, m, H-21 ), 5.11–5.08 (1H, m, H-61 ), 5.07–5.04 (1H, m, H-211 ), 5.03–5.01 (1H, m, H-611 ), 3.99 (3H, s, CH3 O-C2), 3.15 (2H, d, J = 7.4 Hz, H-11 ), 3.12 (2H, d, J = 7.0 Hz, H-111 ), 2.11–2.09* (2H, m, H-51 ), 2.07–2.05 (2H, m, H-411 ), 2.04–2.02* (2H, m, H-511 ), 1.97–1.95 (2H, m, H-41 ), 1.73 (3H, s, CH3 -C311 ), 1.69** (3H, s, H-81 ), 1.67** (3H, s, H-811 ), 1.64*** (3H, s, CH3 -C71 ), 1.63 (3H, s, CH3 -C31 ), 1.60*** (3H, s, CH3 -C711 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 187.9 (C-4), 184.3 (C-1), 155.1 (C-2), 148.3 (C-5), 139.7 (C-311 ), 136.9 (C-31 ), 131.9 (C-3), 131.8* (C-71 ), 131.4* (C-711 ), 130.5 (C-6), 124.1

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(C-611 ), 123.9 (C-61 ), 120.1 (C-21 ), 118.0 (C-211 ), 60.9 (CH3 O-C2), 39.7 (C41 ), 39.6 (C411 ), 27.3 (C-111 ), 26.6** (C-51 ), 26.4** (C-511 ), 25.7*** (C-81 ), 25.6*** (C-811 ), 22.5 (C-11 ), 17.7 ¤ (CH3 -C-71 ), 17.6 ¤ (CH3 -C-711 ), 16.1 ¤¤ (CH -C-31 ), 16.1 ¤¤ (CH -C-311 ); MS m/z (%)M+ 410 (8.8), 327 (100: M+ -83 (C H )), 243 (3.5), 227 3 3 6 11 (8.8), 207 (3.5), 189 (5.3), 91 (3.5), 69 (12.3), 41 (10.5). * ** *** ¤ ¤¤ : interchangeable signals. 8-(2-Hydroxy-3-methylphenyl)-2,6-dimethyloctane-2,6-diol (22) and 8-(4-hydroxy-3-methylphenyl)2,6-dimethyloctane-2,6-diol (23): Coupling of o-cresol (1.0 g, 9.3 mmol) and geraniol (1.55 g; 10.0 mmol) was carried out in dioxane (20 mL) with BF3 ¨ Et2 O (1.2 mL, 10.0 mmol) as the catalyst. Three fractions were obtained from the C.C. Fraction I: unreacted o-cresol (473 mg) that was recovered. Fraction II: Compound 22 (225 mg, 9.0% yield) obtained as a brown viscous oil. IR (cm´1 ) 3375, 2968, 2866, 1595, 1467, 1376, 1264, 1220, 1152, 1112, 935, 763; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.95 (1H, d, J = 7,3 Hz, H-41 ), 6.90 (1H, d, J = 7,4 Hz, H-61 ), 6.70 (1H, dd, J = 7,4 and 7.3 Hz, H-51 ), 2.78–2.74 (2H, m, H-8), 2.16 (3H, s, CH3 -C31 ), 1.85–1.73 (2H, m, H-7), 1.68–1.62 (2H, m, H-5), 1.56–1.52 (2H, m, H-4), 1.51–1.47 (2H, m, H-3), 1.29 (3H, s, CH3 -C6), 1.23 (6H, s, H-1 and CH3 -C2); 13 C NMR (CDCl3 , 100.6 MHz) δ 151.9 (C-21 ), 128.2 (C-41 ), 126.9 (C-61 ), 126.2 (C-31 ), 120.4 (C-11 ), 118.8 (C-51 ), 75.8 (C-6), 71.0 (C-2), 44.3 (C-3), 40.5 (C-5), 31.3 (C-7), 29.3 and 29.2 (C-1 and CH3 -C2), 24.2 (CH3 -C6), 22.3 (C-8), 18.4 (C-4), 16.0 (CH3 -C31 ). MS m/z (%) M+ 280 (< 1%), 262 (55.4), 244 (12.5), 229 (14.3), 201 (19.6), 188 (10.7), 173 (64.3), 161 (48.2), 145 (10.7), 121 (100: M+ -159 (C9 H19 O2 )); 109 (16.1), 91 (28.6), 77 (12.5), 59 (14.3), 43 (14.3). Fraction III: Compound 23 (263 mg, 10.5% yield) obtained as a brown viscous oil. IR (cm´1 ) 3358, 2970, 2933, 2866, 1509, 1457, 1376, 1263, 1222, 1117, 1001, 980, 816; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.95 (1H, s, H-21 ), 6.90 (1H, d, J = 8,1 Hz, H-61 ), 6.68 (1H, d, J = 8,1 Hz, H-51 ), 2.65–2.52 (2H, m, H-8), 2.22 (3H, s, CH3 -C31 ), 1.83–1.73 (1H, m, H-7), 1.71–1.65 (2H, m, H-3), 1.64–1.61 (1H, m, H-7), 1.55–1.49 (1H, m, H-5), 1.48–1.42 (2H, m, H-4), 1.41–1.36 (1H, m, H-5), 1.24 (9H, s, CH3 -C6, CH3 -C2 and H-1); 13 C NMR (CDCl3 , 100.6 MHz) δ 151.6 (C-41 ), 135.5 (C-11 ), 130.9 (C-21 ), 126.7 (C-61 ), 123.4 (C-31 ), 114.7 (C-51 ), 72.9 (C-6), 71.3 (C-2), 45.7 (C-7), 36.9 (C-4), 34.8 (C-5), 31.2 (C-1), 29.9 (CH3 -C2), 29.3 (C-8), 27.7 (CH3- C6), 16.5 (C-3), 15.7 (CH3 -C31 ); MS m/z (%) 281 (2.0: M + 1), 262 (23.5), 244 (41.2), 229 (11.8), 201 (15.7), 187 (15.7), 173 (37.3), 161 (39.2), 121 (100: M+ -159 (C9 H19 O2 )), 109 (64.7), 91 (15.7), 69 (19.6), 43 (19.6). 3.2.3. Acetylation of Geranylated Phenols Geranylated phenols were acetylated by following a described acetylation method [30]. (E)-2-(3,7-dimethylocta-2,6-dienyl)-4-methylphenyl acetate (16): Acetylation of Compound 15 (100 mg, 0.4 mmol) with Ac2 O (0.54 g, 5.3 mmol), DMAP (2.0 mg) and pyridine (1.0 mL) in dichloromethane (20 mL) gives Compound 16 as a viscous yellow oil (111.2 mg, 94.8% yield). Compound 16: IR (cm´1 ) 2966, 2923, 1763, 1447, 1496, 1367, 1213, 1191, 1105, 1010, 901, 824; 1 H-NMR (CDCl3 , 400.1 MHz) δ 7.03 (1H, s, H-3), 7.02 (1H, d, J = 7.9 Hz, H-5), 6.90 (1H, d, J = 7.9 Hz, H-6), 5.26–5.22 (1H, m, H-21 ), 5.13–5.09 (1H, m, H-61 ), 3.22 (2H, d, J = 7.2 Hz, H-11 ), 2.32 (3H, s, CH3 -C4), 2.29 (3H, s, CH3 CO), 2.13–2.09 (2H, m, H-51 ), 2.07–2.02 (2H, m, H-41 ), 1.70 (3H, s, CH3 -C31 ), 1.69 (3H, s, H-81 ), 1.61 (3H, s, CH3 -C71 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 169.6 (CO), 146.6 (C-1), 136.5 (C-31 ), 135.6 (C-4), 132.9 (C-2), 131.4 (C-71 ), 130.6 (C-3), 127.5 (C-5), 124.1 (C-61 ), 121.7 (C-21 ), 121.6 (C-6), 39.6 (C-41 ), 28.6 (C-11 ), 26.5 (C-51 ), 25.6 (C-81 ), 20.9 and 20.8 (CH3- C4 and CH3 CO), 17.7 (CH3 -C71 ), 16.1 (CH3 -C31 ). MS m/z (%) M+ 286 (8.9), 243 (42.9), 201 (21.4), 187 (10.4), 175 (100: M+ -111 (C7 H11 O)), 159 (53.6), 123 (69.6), 91 (17.9), 69 (28.6), 43 (26.8). (E)-2-(3,7-dimethylocta-2,6-dienyl)-5-methoxy-1,4-phenylene diacetate (18): Reaction of Compound 17 (65 mg, 0.18 mmol) with Ac2 O (0.54 g, 5.3 mmol), DMAP (2.0 mg) and pyridine (1.0 mL) in dichloromethane (20 mL) gives Compound 18 as a viscous yellow oil (83 mg, 98% yield). Compound 18: IR (cm´1 ) 2965, 2919, 2854, 1766, 1509, 1445, 1368, 1201, 1181, 1157, 1012, 906; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.87 (1H, s, H-3), 6.65 (1H, s, H-6), 5.20 (1H, t, J = 7.2 Hz, H-21 ),

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5.09 (1H, t, J = 5.5 Hz, H-61 ), 3.78 (3H, s, CH3 O), 3.15 (2H, d, J = 7.16, H-11 ), 2.29 (3H, s, COCH3 ), 2.28 (3H, s, COCH3 ), 2.10–2.07 (2H, m, H-51 ), 2.05–2.02 (2H, m, H-41 ), 1.68 (3H, s, CH3 -C31 ), 1.65 (3H, s, H-81 ), 1.60 (3H, s, CH3 -C71 ), 13 C NMR (CDCl3 , 100.6 MHz) δ 169.2 (COCH3 -C4), 168.9 (COCH3 -C1), 149.5 (C-5), 146.6 (C-1), 137.4 (C-4), 137.1 (C-31 ), 131.5 (C-71 ), 125.5 (C-2), 124.1 (C-61 ), 123.3 (C-3), 121.1 (C-21 ), 106.9 (C-6), 56.1 (CH3 O), 39.6 (C-41 ), 27.7 (C-11 ), 26.4 (C-51 ), 25.6 (C-81 ), 20.8 (CH3 COO-C1), 20.6 (CH3 COO-C4), 17.7 (CH3 -C71 ), 16.1 (CH3 -C31 ); MS m/z (%) M+ 360 (22.2), 318 (16.7), 276 (76.7), 207 (22.2), 191 (44.4), 175 (16.7), 153 (100: M+ -207 (C13 H19 O2 )), 123 (21.0), 91 (4.9), 69 (24.4), 43 (33.3). 3.2.4. Reaction of Geranylated Phenols with BF3 ¨ Et2 O (E)-2-(3,7-dimethylocta-2,6-dien-1-yl)-5-methoxycyclohexa-2,5-diene-1,4-dione (21), 2-(3,7-dihydroxy-3, 7-dimethyloctyl)-5-methoxybenzene-1,4-diol (24), 2-((2-hydroxy-2,6,6-trimethylcyclohexyl)methyl)-5methoxybenzene-1,4-diol (25) and (E)-2-(7-hydroxy-3,7-dimethyloct-2-en-1-yl)-5-methoxybenzene-1, 4-diol (26): To a solution of Compound 17 (100 mg; 0.36 mmol) in dioxane (30 mL) was slowly added dropwise BF3 ¨ Et2 O (0.5 mL, 4.2 mmol) and H2 O (0.5 mL, 27.8 mmol) with stirring at room temperature and without a N2 atmosphere. The crude of the reaction was washed, extracted and chromatographed on silica gel [30]. Five fractions were obtained. Fraction I: unreacted Compound 17 (23 mg) that was recovered. Fraction II: Compound 24 (11 mg, 11.1% yield) obtained as a brown viscous oil. IR (cm´1 ) 2927, 2853, 1674, 1648, 1603, 1454, 1375, 1207, 1174, 987; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.46 (1H, s, H-3), 5.94 (1H, s, H-6), 5.17–5.13 (1H, m, H-21 ), 5.09–5.06 (1H, m, H-61 ), 3.82 (3H, s, CH3 O-C5), 3.14 (2H, d, J = 7.0 Hz, H-11 ), 2.12–2.08 (2H, m, H-41 ), 2.07–2.04 (2H, m, H-51 ), 1.69 (3H, s, H-81 ), 1.61 (3H, s, CH3 -C31 ), 1.60 (3H, s, CH3 -C71 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 187.6 (C-1), 182.4 (C-4), 158.7 (C-5), 149.6 (C-2), 140.1 (C-31 ), 131.9 (C-71 ), 130.3 (C-3), 123.9 (C-61 ), 117.8 (C-21 ), 107.7 (C-6), 56.2 (CH3 O-C5), 39.6 (C-51 ), 27.3 (C-11 ), 26.4 (C-41 ), 25.7 (C-81 ), 17.7 (CH3 -C71 ), 16.1 (CH3 -C31 ); MS m/z (%) 274 (8.5: M+ ), 259 (5.1), 191 (100: M+ -83 (C6 H11 )), 176 (10.2), 148 (5.1), 91 (3.4), 69 (5.1), 41 (5.1). Fraction III: Compound 25 (25 mg, 24% yield) obtained as a brown viscous oil. IR (cm´1 ) 355, 3455, 2959, 2927, 2854, 1629, 1509, 1446, 1376, 1278, 1198, 1154, 1121, 1042, 951, 864; 1 H-NMR (CDCl3 , 400.1 MHz) δ 6.61 (1H, s, H-3), 6.33 (1H, s, H-6), 5.12 (1H, bs, HO-C4), 3.81 (3H, s, CH3 O), 2.65–2.45 (2H, m, CH2 -C2), 1.93–1.91 (1H, m, H-31 ), 1.65–1.64 (1H, m, H-41 ), 1.65–1.63 (1H, m, H-11 ), 1.60–1.56 (1H, m, H-31 ), 1.60–1.56 (1H, m, H-41 ), 1.49–1.46 (1H, m, H-51 ), 1.32–1.30 (1H, m, H-51 ), 1.20 (3H, s, CH3 -C21 ), 0.98 (3H, s, CH3 -C61 ), 0.89 (3H, s, CH3 -C61 ); 13 C NMR (CDCl3 , 100.6 MHz) δ 146.2 (C-1), 145.3 (C-5), 138.9 (C-4), 114.3 (C-3), 114.2 (C-2), 100.3 (C-6), 76.7 (C-21 ), 55.9 (CH3 O-C-5), 48.3 (C-11 ), 41.5 (C-51 ), 40.0 (C31 ), 33.4 (C-61 ), 32.1 (CH3 -C61 ), 22.7 (CH2 -C-2), 20.7 (CH3 -C61 ), 19.8 (C-41 ), 19.6 (CH3 -C21 ); MS m/z (%) 276 (54.9: M+ -H2 O), 191 (18.2), 153 (100), 69 (8.5), 41 (7.3). Fraction IV: Compound 26 (20 mg, 18.9% yield) obtained as a brown viscous oil. IR (cm´1 ) 3396, 2968, 2937, 1646, 1603, 1521, 1446, 1374, 1274, 1196, 1018, 867; 1 H-NMR (CDCl , 400.1 MHz) δ 6.67 (1H, s, H-3), 6.42 (1H, s, H-6), 5.29 (1H, m, H21 ), 3.82 (3H, s, 3 CH3 O), 3.26 (2H, d, J = 7.1 Hz H-11 ), 2.07–2.03 (2H, m, H-41 ), 1.76 (3H, s, CH3 -C31 ), 1.52–1.48 (2H, m, H-61 ), 1.46–1.41 (2H, m, H-51 ), 1.22 (6H, s, CH3 -C71 and H-81 ); 12 C NMR (CDCl3 , 100.6 MHz) δ 147.4 (C-1), 145.4 (C-5), 139.3 (C-4), 138.3 (C-31 ), 122.0 (C-21 ), 118.5 (C-2), 115.3 (C-3), 100.4 (C-6), 70.9 (C-71 ), 56.1 (CH3 O-C5), 43.3 (C-61 ), 39.9 (C-41 ), 29.2 (CH3 -C71 and C81 ), 28.9 (C-11 ), 22.5 (C-51 ), 16.1 (CH3 -C31 ); MS m/z (%) 292 (8.9: M+ -H2 ), 277 (3.6), 219 (1.8), 203 (8.9), 191 (100: M+ -H2 -101 (C6 H13 O)), 176 (8.9), 148 (3.6), 91 (1.8), 69 (1.8), 43 (1.8). Fraction V: Compound 21 (12 mg, 10.7% yield) obtained as brown viscous oil. The spectroscopic data (IR, MS and NMR) were consistent with those previously reported [27]. 3.3. In Vitro Effect of the Compounds on the Mycelial Growth of B. cinerea The antifungal activities of all tested compounds were evaluated using the radial growth test at final concentrations of 50, 150 and 250 mg/L in PDA medium [37]. Captan was used as the positive control, whereas PDA medium containing 1% ethanol was considered as the negative control. The

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percentages of inhibitions were determined following a standard method [42]. Experimental conditions have been detailed elsewhere [30]. 4. Conclusions Hydrated geranylphenols were synthesized by following two different synthetic pathways: direct coupling of geraniol with o-cresol in dioxane with added water and using BF3 ¨ Et2 O as the catalyst; or by the reaction of a geranylated phenol with BF3 ¨ Et2 O in dioxane and added water. Interestingly, the coupling of geraniol to hydroquinones gives completely different products. On the other hand, the mycelial growth inhibition of hydrated geranylphenols is in the range of 30%–95% at 250 ppm. The percentages of inhibition induced by hydrated compounds (23 and 26) are higher than those produced by the respective geranylated phenol (14 and 17). The enhancement of the antifungal activity is larger for hydrated compounds carrying only one hydroxyl group in the side chain (25 and 26) than for completely hydrated compounds (22–24). It is worth stressing that the new Compound 26 exhibits antifungal activity similar to Captan, a common fungicide used to control B. cinerea. Finally, as far as we know, this is the first study relating the structure of the geranyl chain with the antifungal activity of geranylated phenols. Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/17/6/ 840/s1. Acknowledgments: The authors thank FONDECYT and Dirección General de Investigación, Innovación y Postgrado of Universidad Técnica Federico Santa María (DGIIP-USM) for financial support through Grant No. 1120996 and Línea No. 116.13.12., respectively. Mauricio Soto thanks DGIIP-USM for the “Programa de Incentivo a la Iniciación Científica” (PIIC-2014) fellowship. Author Contributions: Luis Espinoza supervised the whole study; María I. Chávez and Mauricio Soto performed the synthesis of all compounds; Lautaro Taborga collaborated in the synthesis and structural determination of geranylphenols by spectroscopic methods; Katy Díaz carried out the study of the mycelial growth of B. cinerea; Luis Espinoza, Andres F. Olea, Lautaro Taborga and Katy Díaz collaborated in the discussion and interpretation of results; Andres F. Olea and Luis Espinoza wrote the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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