Antifungal toxicity of linear geranylphenol. Influence of oxigenate substituents

Article abstract of DOI:10.1016/j.fct.2017.05.027

Twenty four linear geranylphenols were evaluated for their antifungal properties against ATCC and clinical isolates of Candida albicans and Cryptococcus neoformans. For the analysis of their antifungal behavior the compounds were grouped into two series:

Full text of DOI:10.1016/j.fct.2017.05.027

Accepted Manuscript
Antifungal toxicity of linear geranylphenol. Influence of oxigenate substituents
Lautaro Taborga, Maximiliano Sortino, Carrasco Héctor, Estefanía Butassi, Susana
Zacchino, Luis Espinoza
PII:
S0278-6915(17)30255-7
10.1016/j.fct.2017.05.027
FCT 9062
DOI:
Reference:
To appear in: Food and Chemical Toxicology
Received Date: 30 March 2017
Revised Date: 12 May 2017
Accepted Date: 13 May 2017
Please cite this article as: Taborga, L., Sortino, M., Héctor, C., Butassi, Estefaní., Zacchino, S.,
Espinoza, L., Antifungal toxicity of linear geranylphenol. Influence of oxigenate substituents, Food and
Chemical Toxicology (2017), doi: 10.1016/j.fct.2017.05.027.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
“Antifungal toxicity of Linear Geranylphenol. Influence of Oxigenate
Substituents”
Authors:
Lautaro Taborga1*, Maximiliano Sortino2, Héctor Carrasco3, Estefanía Butassi2, Susana Zacchino2
and Luis Espinoza1.
1Departamento de Química, Universidad Técnica Federico Santa María, Av. España N° 1680,
Valparaíso, Chile; E-Mails: lautaro.taborga@usm.cl (L.T.); luis.espinozac@usm.cl (L.E.)
2Área Farmacognosia, Facultad de Cs. Bioquímicas y Farmacéuticas, Universidad Nacional de
Rosario, Suipacha 531, (2000) Rosario, Argentina; E-mails: szaabgil@citynet.net.ar (S.Z.);
msortino@fbioyf.unr.edu.ar (M.S.); fefabutassi@hotmail.com (E.B.)
3Instituto de Ciencias Químicas Aplicadas, Facultad de Ingeniería, Universidad Autónoma de Chile,
El Llano Subercaseaux 2801, San Miguel, Santiago 8900000, Chile; E-Mails:
hector.carrasco@uautonoma.cl (H.C.)
*Correspondence: lautaro.taborga@usm.cl; Tel.: +56-032-265-4225; Fax: +56-032-265-4782.
Abstract:
Twenty four linear geranylphenols were evaluated for their antifungal properties against ATCC and
clinical isolates of Candida albicans and Cryptococcus neoformans. For the analysis of their
antifungal behavior the compounds were grouped into two series: (i) compounds with only one
geranyl substituent on the benzene ring and (ii) compounds with two geranyl moieties on the
benzene ring. Results showed that compounds of series (i) present better antifungal activities than
those of series (ii). In addition, within group (i) all compounds showed better activities against C.
neoformans than against C. albicans which can be easily verified by comparing MIC100 or MIC50 of
each compound against both fungi. Di- (10 and 11) and tri- hydroxy (3 and 4) compounds showed
significant anti-cryptoccocal activity, being 3, 10 and 11 highly active with MIC100 or MIC50 = 3.9
µg/mL similar to the standard drug amphotericin B. Moreover, when evaluating the toxicity of
compounds 6, 10 and 11 on the HDF cell line (human dermal fibroblasts), results were obtained
with IC50 values > 100 µΜ, considered as non-toxic for the cell. This indicates that the toxicity of
the analyzed compounds is selective towards fungi, which makes them a very attractive family for
the development of future drugs.
Keywords:
linear geranylphenols; antifungal activity; Candida albicans; Cryptococcus neoformans.

ACCEPTED MANUSCRIPT
1. Introduction:
In the last years, fungi have emerged as major cause of human infections especially among
immunocompromised hosts having an enormous impact on morbidity and mortality (Mathew and
Nath, 2009). Although there are several available drugs for the treatment of systemic and
superficial mycoses, any of them is completely effective (Brown and Wright, 2005; Chen et al.,
2010) and therefore quickly develops resistance due to the large-scale use (Mukherjee et al., 2003)
and all possess a certain degree of toxicity (Kontoyiannis, 2012). There is, therefore, an urgent
need for new antifungal chemical structures, alternatives to the existing ones (Vicente et al.,
2003).
On the other hand, linear geranylphenols are an interesting sub-class of secondary metabolites
whose terpene portion may have a length of up to eight isoprene units (De Rosa et al., 1995). They
were found mainly in marine organisms such as brown algae (Capon et al., 1981; Faulkner, 1986;
Gerwick and Fenical, 1981; Ochi et al., 1979), sponges (Bifulco et al., 1995; De Rosa et al., 1995),
alcyonaceas (Bowden and Coll, 1981), gorgonians (Ravi and Wells, 1982) and ascidians (Faulkner,
1993; Fu et al., 1997, 1994; Guella et al., 1987; Howard et al., 1979; Targett and Keeran, 1984).
Among them, geranylphenols with mono-, di- and sesquiterpene (Capon et al., 1981; Faulkner,
1986; Gerwick and Fenical, 1981; Ochi et al., 1979) chains were isolated from brown algae while
structures with longer linear chains were isolated from sponges (Bifulco et al., 1995; De Rosa et al.,
1995).
Potent biological activities including anti-inflammatory (Bauer et al., 2011; Quang et al., 2006),
antifungal (Danelutte et al., 2003) anti-HIV (Manfredi et al., 2001), antioxidant (Yamaguchi et al.,
2006) as well as antineoplastic (Han et al., 2007; Liu et al., 2008) have been reported for this family
of compounds. Some particular compounds such as 2-geranylbenzoquinone (1) (isolated from the
ascindian Synoicum castellatum Kott.) (Carroll et al., 1993), 2-geranylhydroquinone (2) isolated
from Cordia alliodora (ex Ruiz & Pav.) Oken tree (Manners and Jurd, 1977), Phacelia crenulata
Torr. ex S. Watson (Reynolds and Rodriguez, 1979), Aplidium antillense Gravier (Benslimane et al.,
1988) and the tunicate Amaroucium multiplicatum Sluiter (Sato and Shindo, 1989), have shown
antimicrobial, anticancer, protective and antioxidant effects, among others (Figure 1) (Benslimane
et al., 1988; De Rosa et al., 1994; Reynolds and Rodriguez, 1979; Rudali and Menetrier, 1967;
Rudall, 1966; Sato and Shindo, 1989).
Figure 1. Structure of geranylphenols with biological activity

ACCEPTED MANUSCRIPT
Based on the data mentioned above, our research group has been devoted in the last years to the
synthesis of linear geranylphenols, which were obtained by direct geranylation reactions between
.
geraniol and the corresponding phenol derivatives, using BF3 OEt2 as the first catalyst and AgNO3 as
the second one (Espinoza et al., 2014). Eight of them have been previously evaluated for their in
vitro inhibitory effect of the mycelial growth of Botrytis cinerea, a fungus that affects important
agricultural crops (Espinoza et al., 2014). Analysis of the results suggested that compounds having
hydroxyl groups attached to the aromatic nucleus (compounds 2, 3 and 4, Figure 2) exhibited a
higher percentage of inhibitory activity of the growth of B. cinerea mycelium. In addition the
biological activity, decreased or was gone when the compounds were acetylated (Espinoza et al.,
2014).
Figure 2. Geranylphenols that showed inhibitory effect over B. cinerea.
In order to deepen the knowledge on the antifungal behavior of geranylphenols, we report here
the antifungal activity of a series of 24 geranylphenols, two of them new ones (7 and 9), against
standardized as well as clinical isolates of clinically important yeasts belonging to Candida and
Cryptococcus genera in order to get structure-antifungal activity relationships, and to know the
potential usefulness of these compounds as hits and heads of series for their further development.
2. Materials and Methods:
2.1 Chemistry
2.1.1 Synthesis
Unless otherwise stated, all chemical reagents purchased (Merck, Darmstadt, Germany or Aldrich,
St. Louis, MO, USA) were of the highest commercially available purity and were used without
previous purification. IR spectra were recorded as thin films in a FT-IR Nicolet 6700
spectrophotometer (Thermo Scientific, San Jose, CA, USA) and frequencies are reported in cm−1.
Low resolution mass spectra were recorded on an Agilent 5973 spectrometer (Agilent
Technologies, Santa Clara, CA, USA) at 70 eV ionizing voltage, in a GC 6890N DB-5 m, 30 m × 0.25
mm × 0.25 μm column, and data are given as m/z (% rel. int.). High resolution mass spectra were
recorded on an LTQ Orbitrap XL spectrometer (Thermo Scientific, San Jose, CA, USA) by applying a
voltage of 1.8 kV in the positive and 1.9 kV in the negative, ionization mode. The spectra were
recorded using full scan mode, covering a mass range from m/z 100–1,300. The resolution was set
to 50,000 and maximum loading time for the ICR cell was set to 250 ms. 1H, 13C, 13C DEPT-135, sel.
1
gs-1D H NOESY, gs-2D HSQC and gs-2D HMBC spectra were recorded in CDCl3 solutions and are
1
referenced to the residual peaks of CHCl3 at δ = 7.26 ppm and δ = 77.0 ppm for H and 13C,

ACCEPTED MANUSCRIPT
respectively, on a Bruker Avance 400 Digital NMR spectrometer (Bruker, Rheinstetten, Germany),
1
operating at 400.1 MHz for H and 100.6 MHz for 13C. Chemical shifts are reported in δ ppm and
coupling constants (J) are given in Hz. Silica gel (Merck 200–300 mesh) was used for C.C. and silica
gel plates HF254 for TLC. TLC spots were detected by heating after spraying with 25% H2SO4 in H2O.
(E)-3-(3,7-dimethylocta-2,6-dienyl)benzene-1,2-diol
(5),
(E)-4-(3,7-dimethylocta-2,6-
dienyl)benzene-1,2-diol (7).
To a solution of catechol (1.0 g, 9.1 mmol) and geraniol (1.4 g, 9.1 mmol) in acetonitrile (25 mL),
.
saturated with AgNO3, was slowly added BF3 OEt2 (0.46 g, 3.2 mmol) dropwise with stirring at
room temperature and under a N2 atmosphere. After the addition was completed, the stirring was
continued for 48 h. The end of the reaction was verified by TLC, and then the mixture was poured
onto crushed ice (30 g) and extracted with EtOAc (3 × 20 mL). The organic layer was washed with
5% NaHCO3 (15 mL, 5%) and water (2 × 15 mL), dried over Na2SO4, and filtered. The solvent was
evaporated under reduced pressure. The crude was re-dissolved in CH2Cl2 (5 mL) and
chromatographed on silica gel with petroleum ether/EtOAc mixtures of increasing polarity
(19.8:0.2→0.2:19.8). Two fracꢀons were obtained. Fracꢀon I: 108 mg of viscous reddish oil, 4.8%
yield, identified as compound 5; Fraction II: 239 mg of viscous reddish oil, 10.75% yield, identified
as compound 7.
Compound 5: IR (cm−1): 3418; 2967; 2922; 1620; 1590; 1475; 1375; 1278. 1H-NMR: 6.78 (d, J = 8.4
Hz, 1H, H-6); 6.75 (dd, J = 8.4 and 6.8 Hz, 1H, H-5); 6.66 (d, J = 6.8 Hz, 1H, H-4); 5.38 (s, 1H, OH-C2);
5.33-5.31 (m, 2H, H-2’ and OH-C-1); 5.05-5.04 (m, 1H, H-6’); 3.37 (d, J = 7.1 Hz, 2H, H-1’); 2.14-2.12
(m, 2H, H-5’); 2.11-2.10 (m, 2H, H-4’); 1.78 (s, 3H, CH3-C3’); 1.69 (s, 3H, H-8’); 1.60 (s, 3H, CH3-C7’).
13C-NMR: 144.3 (C-1); 141.9 (C-2); 138.9 (C-3’); 132.2 (C-7’); 127.4 (C-3); 123.7 (C-6’); 121.7 (C-2’);
121.4 (C-4); 120.7 (C-5); 113.2 (C-6); 39.6 (C-4’); 29.9 (C-1’); 26.3 (C-5’); 25.7 (C-8’); 17.7 (CH3-C7’);
16,1 (CH3-C3’). Spectroscopic data of compound 5 were consistent with the literature. (Taborga et
al., 2013)
Compound 7: IR (cm−1): 3402; 2973; 2922; 1605; 1442; 1375; 1278. 1H-NMR: 6.77 (d, J = 7.6 Hz, 1H,
H-6); 6.70 (s, 1H, H-3); 6.61 (d, J = 7.6 Hz, 1H, H-5); 5.29 (t, J = 6.7 Hz, 1H, H-2’); 5.10 (t, J = 6.4 Hz,
1H, H-6’); 3.24 (d, J = 7.2 Hz, 2H, H-1’); 2.11-2.08 (m, 2H, H-5’); 2.05-2.02 (m, 2H, H-4’); 1.68 (s, 6H,
CH3-C3’ and H-8’); 1.60 (s, 3H, CH3-C7’). 13C-NMR: 143.4 (C-2); 141.3 (C-1); 136.1 (C-3’); 135.0 (C-4);
131.5 (C-7’); 124.3 (C-6’); 123.1 (C-2’); 120.7 (C-5); 115.4 (C-3); 115.3 (C-6); 39.7 (C-4’); 33.4 (C-1’);
26.6 (C-5’); 25.7 (C-8’); 17.7 (CH3-C7’); 16.1 (CH3-C3’). HRMS: (M + 1) calcd. for C16H22O2: 247.1620,
found: 247.1628.
(E)-4-(3,7-dimethylocta-2,6-dienyl)-1,3-phenylene diacetate (9).
Ac2O (1.08 g, 10.6 mmol) was added to a solution of compound 6 (70 mg, 0.284 mmol), DMAP (3.0
mg) and pyridine (1.0 mL) in dichloromethane (30 mL). The end of the reaction was verified by TLC
(1 h), and the mixture was extracted with EtOAc (2 × 10 mL). The organic layer was washed with
5% KHSO4 (2 × 10 mL) and water (2 × 10 mL), dried over Na2SO4, and filtered. The solvent was
evaporated under reduced pressure. Compound 9 was obtained as dark yellow oil (92.2 mg, 98.9%
yield). Compound 9: IR (cm−1): 2964; 2917; 2849; 1770; 1640; 1612; 1495; 1450; 1369; 1198; 1140;
1014; 973; 913. 1H-NMR: 7.22 (d, J = 8.4 Hz, 1H, H-5); 6.92 (dd, J = 2.1 and 8.4 Hz, 1H, H-6); 6.85 (d,
J = 2.1 Hz, 1H, H-2); 5.22 (t, J = 6.8 Hz, 1H, H-2’); 5.10 (t, J = 6.0 Hz, 1H, H-6’); 3.22 (d, J = 7.1 Hz, 2H,

ACCEPTED MANUSCRIPT
H-1’); 2.29 (s, 3H, COCH3); 2.27 (s, 3H, COCH3); 2.11-2.08 (m, 2H, H-5’); 2.06-2.05 (m, 2H, H-
4’); 1.68 (s, 3H, H-8’); 1.68 (s, 3H, CH3-C3’); 1.60 (s, 3H, CH3-C7’). 13C-NMR: 169.1 (COCH3); 168.9
(COCH3); 148.9 (C-3); 148.9 (C-1); 137.1 (C-3’); 131.5 (C-7’); 130.9 (C-4); 130.1 (C-5); 124.1 (C-6’);
121.1 (C-2’); 119.0 (C-6); 155.7 (C-2); 39.6 (C-4’); 28.0 (C-1’); 26.5 (C-6’); 25.6 (C-8’); 21.0 (COCH3);
20.8 (COCH3); 17.6 (CH3-C7’); 16.0 (CH3 -C3’).
2.2 Biology
2.2.1 Microorganisms and media
For the antifungal evaluation, standardized strains from the American Type Culture Collection
[(ATCC), Rockville, MD, USA] C. albicans ATCC 10231 and C. neoformans ATCC 32264 as well as
clinical isolates of the Reference Center in Mycology (CEREMIC, CCC, Rosario, Argentina) and
Malbrán Institute [(IM), Av. Velez Sarsfield 563, Buenos Aires] were used. The isolates included six
strains of C. albicans, four non-albicans Candida strains (C. krusei, C. tropicalis, C. glabrata and C.
parapsilopsis) and six strains of C. neoformans whose voucher specimens are presented in Table 2.
Strains were grown on Sabouraud-chloramphenicol agar slants for 48 h at 30 °C, maintained on
slopes of Sabouraud-dextrose agar (SDA, Oxoid) and sub-cultured every 15 days to prevent
pleomorphic transformations. Inocula were obtained according to reported procedures(CLSI
(Clinical and Laboratory Standars Institute), 2008) and adjusted to 1-5 x103 cells with colony
forming units (CFU)/mL.
2.2.2 Cell line
HDF (human dermal fibroblasts) were obtained from ATCC.
2.2.3 Cell culture
HDF cell line was maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and
Ham's F12 medium, containing 10% heat-inactivated fetal bovine serum (FBS), amphotericin (2.5
mg/mL), penicillin (100 U/mL) and streptomycin (100 mg/mL).
2.2.4 Fungal growth inhibition percentage determination.
Microdilution techniques were performed in 96-well microplates according to the guidelines M-
27A3 of Clinical and Laboratory Standards Institute (CLSI) for yeasts (CLSI (Clinical and Laboratory
Standars Institute), 2008). For the assay, compound test-wells (CTWs) were prepared with stock
solutions of each compound in DMSO (maximum concentration ≤ 1%), diluted with RPMI-1640, to
final concentrations of 250-3.9 μg/mL. An inoculum suspension (100 μL) was added to each well
(final volume in the well = 200 μL). A growth control-well (GCW) (containing medium, inoculum,
and the same amount of DMSO used in a CTW, but compound-free) and a sterility control-well
(SCW) (sample, medium, and sterile water instead of inoculum) were included for each tested
fungus. Microtiter trays were incubated in a moist, dark chamber at 30 °C for 48 h for both yeasts.
Microplates were read in a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA,
USA). Tests were performed in triplicate. Reduction of growth for each compound concentration
was calculated as follows: % of inhibition = 100 - (OD 405 CTW − OD 405 SCW)/(OD 405 GCW − OD
405 SCW). The means ± SEM were used for constructing the dose-response curves with SigmaPlot
11.0 software that represent % inhibition vs concentration of each compound. Two endpoints

ACCEPTED MANUSCRIPT
were recorded from this assay and the dose-response curves: the Minimum Inhibitory
Concentration that results in total fungal growth inhibition (MIC100) and the minimum
concentration that inhibits 50 % of the fungal growth (MIC50). Amphotericin B (Sigma-Aldrich, St
Louis, Mo, USA) was used as the standard positive drug.
3. Results and Discussion:
3.1. Chemistry
3.1.1. Synthesis
The syntheses of most compounds, as well as their chemical characterization, have been
previously reported by our research group. Compounds 5, 6 and 13 were reported in Taborga et al
(2013); 2, 3, 4, 8, 15, 16, 19, 21, in Espinoza et al. (2014); 10-12, 14, 18, 20, 23 and 24, in Taborga
et al., 2015 (Taborga et al., 2015) and 17, 22 and 25, in Taborga et al., 2016 (Taborga et al., 2016).
Structures 7 and 9 are new compounds and their syntheses and characterization are reported here
for the first time.
Of the whole series, compounds 5 and 6 have been previously synthesized by the traditional
method used for another geranyl methoxy-phenols (Fedorov et al., 2006), i.e. direct coupling
.
between geraniol and pyrocatechol or resorcinol in dioxane solution catalyzed with BF3 Et2O. The
yields of this reaction were 4.8% and 11.9% for compounds 5 and 6, respectively (Taborga et al.,
2013). However, when the geranylation of these hydroquinones was carried out following the
modified Electrophilic Aromatic Substitutions (EAS) reaction (Scheme 1), compound 5 was
obtained with identical yield, whereas compound 6 was obtained with 15.0 % yield that is slightly
higher than the obtained with the traditional method. Additionally, in the reaction of
pyrocathecol, the new compound 7 was obtained with 10.7 % yield. The chemical structure of 7
was mainly determined by NMR spectroscopic techniques and the results are detailed in section
1.2. The spectroscopic data of compounds 5 and 6 were consistent with those previously reported
(Taborga et al., 2013).
Scheme 1. Synthesis of compounds 5 and 7.
The new compound 9 was obtained from 6 by standard acetylation (Ac2O and DMAP in CH2Cl2)
with 98.9% yield (see Scheme 2). The presence of a diacetylated derivative was confirmed by NMR
1
spectroscopy: in the H-NMR spectrum two singlets at δH = 2.29 and 2.27 ppm (each 3H, CH3CO)
were observed, whereas in the 13C-NMR spectrum signals at δC = 21.0 (COCH3), 20.8 (COCH3),
169.1 and 168.9 ppm (C=O) were found.

ACCEPTED MANUSCRIPT
Scheme 2. Synthesis of compound 9.
3.1.2 Structure determination
The chemical structure of the new compound 7 was mainly established by NMR spectroscopy.
Thus, in this section the NMR data used to determine the chemical structure of geranylphenol 7 is
discussed in detail, whereas those corresponding to the novel acetylated derivative 9 have been
given in the previous paragraph.
1
The H NMR spectrum of compound 7 shows aromatic signals at δH = 6.77 (d, J = 7.6 Hz, 1H); δH =
6.70 (s, 1H) and δH = 6.61 (d, J = 7.6 Hz, 1H), which were assigned to hydrogen atoms H-6, H-3 and
H-5, respectively. These results confirm the aromatic mono-substitution at 4-position.
Additionally, in the HMBC spectrum, the signal at δH = 3.24 ppm assigned to H-1’ (d, J = 7.2 Hz, 2H,
H-1’) shows 3JH-C coupling with C-3 (δC = 115.4), C-5 (δC = 120.7 ppm) and C-3’ (δC = 136.1 ppm), and
2JH-C coupling with C-4 and C-2’ (δC = 135.0 and 123.1 ppm, respectively). These HMBC correlations
are shown in Figure 3. In order to establish the E geometry of the C-2’-C-3’ double bond of the
geranyl chain, selective 1D NOESY NMR experiments were recorded for compound 7. These
correlations are shown in Figure 3, where the most important ones correspond to those observed
between the H-1’ and the methyl group in the C-3’ position.
Figure 3. Most important 2D 1H-13C HMBC correlations (blue) and selective 1D NOESY correlations
(red) observed for compound 7.
3.2 Biology
3.2.1 Antifungal activity
The antifungal properties of the twenty four geranylphenols were investigated against clinical
important fungal species. Standardized strains of Cryptococcus neoformans and Candida albicans
of the American Type Culture Collection (ATCC) and also clinical isolates of C. neoformans and C.
albicans and non-albicans Candida strains were used as targets for the antifungal assays.
Results were expressed as the percentages of inhibition of each fungus in the range 250-3.9 µg/mL
by using the standardized microbroth dilution method CLSI M-27A3 (CLSI, 2008) which assures
reproducible results.
The selection of C. neoformans was due to the fact that this opportunistic yeast is the main cause
of cryptococcal meningoencephalitis which has a high mortality rate among patients with
profoundly impaired infections (Trpković et al., 2012). Even though new antifungal drugs have

ACCEPTED MANUSCRIPT
been developed in recent years (Roemer and Krysan, 2014), the availability of antifungal agents
with anti-cryptococcal activity is still limited and sometimes the strains develop quickly resistance
(Perkins et al., 2005). This scenario has motivated the search of new compounds with anti-C.
neoformans properties (Cordeiro et al., 2012).
In turn, C. albicans is the fourth leading cause of nosocomial bloodstream infection (BSI) in
intensive care units, causing fatal invasive candidiasis in a high percentage of patients (Pfaller and
Diekema, 2007). As a consequence, new chemical structures with anticandidal activities are highly
welcome. In addition although C. albicans was in the past the usual species associated with
invasive and BSI infections, at present non-albicans Candida spp. such as C. tropicalis, C. glabrata,
C. parapsilopsis, C. krusei and C. lusitaneae, constitute more than a half of isolates in human
candidiasis (Pfaller and Diekema, 2007).
For a more comprehensive analysis of the antifungal results, the compounds were grouped into
two series: (i) compounds with only one geranyl substituent (mono-geranyl) and (ii) compounds
with two geranyl moieties (bi-geranyl) on the aromatic ring. Compounds of series (i) were sub-
divided in two sub-groups: (i.1) includes compounds with two oxygenated substituents (2, 5, 6, 7,
8 and 9); (i.2) includes compounds with three extra substituents (3, 4, 10, 11, 12, 13, 14, 15, 16
and 17). All compounds were tested at first against the standardized strains C. albicans ATCC
10231 and C. neoformans ATCC 32264. Results are showed in Table 1.
Table 1. Antifungal activity, part a
250
125
62.5
31.25
15.65
7.8
3.9
MIC100
MIC50
group
Structure
Name pathogen
µg/mL µg/mL
µg/mL µg/mL
µg/mL µg/mL
µg/mL µg/mL
µg/mL
100
100
100
100
0.0
0.0
0.0
31.25
31.25
Cn
2
90
100
45.2±1.1 13.4±0.3
7.6±0.6
59.6±3.4
6.3±0.8
100
5.7±0.5
0.0
4.2±1.0
0.0
1.1±0.1
0.0
>250
62.5
>250
7.8
125
31.25
>250
3.9
Ca
100
8.5±0.6
100
100
6.0±1.3
100
Cn
5
33.1±2.1
100
3.4±1.0
100
1.6±0.1
2.0±0.9
Ca
95.5±2.7 54.0±1.8
Cn
I
i.1
6
100
97.8±0.8 57.4±3.1 13.9±1.0
3.7±0.6
59
1.8±0.4
27
0.0
0
125
62.5
15.6
15.6
125
Ca
100
100
100
100
100
31.2
62.5
>250
>250
Cn
7
100
96.0±2.3 88.2±1.4 46.5±1.9 13.5±0.8
6.2±0.5
Ca
68.3±0.9 54.9±0.5 38.3±1.2 34.3±1.3 32.6±0.5 29.7±0.6 24.8±1.0
29.5±1.3 23.7±1.0 12.2±0.1 10.2±0.7 9.6±0.5 5.8±0.7
Cn
8
0
>250
Ca

ACCEPTED MANUSCRIPT
31.2±15. 15.5±1.1
9
0.0
9.8±0.9
100
0.0
4.8±2.6
100
0.0
1.8±0.3
100
0.0
2.6
>250
>250
>250
>250
Cn
9
25.2±0.2 13.4±0.5
0.6±0.1
0.4±0.1
Ca
Cn
Ca
Cn
100
100
100
100
100
100
3.9
1.9
3
77.9±0.1 66.5±1.8 11.8±0.4
4.5±1.8
2.3±0.7
125
31.2
100
100
100
100
100
100
100
100
86.3±5.4
4.0±0.3
100
6.6±1.8
2.4±0.2
100
1.7±1.1
0.1±0.2
100
0.0
0.0
62.5
>250
3.9
31.2
250
4
66.2±3.1 10.0±0.8
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
Ca
100
100
100
100
100
100
100
100
100
3.9
10
11
12
13
14
15
16
17
96.8±0.7 72.1±2.5 13.6±0.6
2.1±0.1
100
31.2
3.9
15.6
3.9
100
100
100
6.3±1.8
0.0
92.0±2.1 29.4±1.6
2.5±0.3
0.0
31.2
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
31.2
125
65.7±3.8 52.1±3.3 42.6±1.6 32.7±2.0
0.0
1.4±0.1
0.0
58.9±0.1 28.8±1.3 14.2±1.2
66.4±2.7 25.5±0.6 15.1±1.3
6.4±0.7
0.0
0.0
0.0
250
i.2
0.0
0.0
250
31.0±2.1 19.8±2.3 18.1±1.3 17.6±1.9 13.1±1.8
5.3±0.2
0.0
2.6±1.4
0.0
>250
>250
>250
250
15.8±2.3
16.0±1.3
68.3±3.9
20.0±2.7
29.6±1.5
20.0±2.7
0.0
0.0
6.2±0.3
0.0
0.0
4.8±1.2
0.0
0.0
4.4±1.4
0.0
8.4±0.8
2.3±0.4
8.9±0.8
7.1±0.1
8.9±0.8
4.1±1.1
0.0
0.0
0.0
3.7±0.3
0.0
3.2±1.0
0.0
1.5±0.5
0.0
1.4±0.7
0.0
0.5 ±0.0
0.0
>250
>250
>250
250
3.7±0.3
0.0
3.2±1.0
0.0
1.5±0.5
0.0
1.4±0.7
0.0
0.5±0.0
0.0
63.7±1.7 12.2±1.5
9.5±2.1 5.4±0.6
4.1±0.2
3.0±0.6
2.9±0.0
2.1±0.5
2.0±0.4
>250
Table 1 Antifungal activity, part b
38.3±2.8
34.2±1.8
33.6±1.5
15.0±1.3
33.2±1.5
26.2±6.1
41.7±12.6
14.5±1.1
40.4±0.2 16.5±1.8
0.0
1.8±0.8
0.0
0.0
0.0
0.0
0.0
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
Cn
18
13.0±1.1
18.0±5.5
7.3±1.8
30.8±2.2
9.2±2.4
0.0
5.6±1.4
0.0
1.8±0.4 0.6 ±0.0
>250
>250
>250
>250
>250
>250
>250
>250
>250
>250
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
Ca
Cn
0.0
1.3±1.2
0.0
0.0
0.0
0.0
19
20
21
3.4±0.9
0.0
2.3±0.3
0.0
0.0
0.0
0.0
4.5±0.9
0.0
2.8±0.2
0.0
1.1±0.0
0.0
1.2±0.0
0.0
1.0±0.4
0.0
ii
5.2±1.6
3.6±0.9
0.0
0.0
0.0
0.0
0.0
26.0±10.7 24.7±11.9
0.0
0.0
0.0
0.0
22
23
9.5±2.1
5.4±0.6
4.1±0.2
0.0
3.0±0.6
0.0
2.9±0.0
0.0
2.1±0.5
0.0
2.0±0.4
0.0
47.6±1.4
29.1±0.4

ACCEPTED MANUSCRIPT
31.6±0.7
56.0±4.6
18.2±0.0
62.8±1.8
43.5±1.1
13.3±2.3
10.5±2.8
11.1±0.5
11.8±1.4
6.9±1.2
0.0
4.1±1.4
0.0
1.4±0.8
0.0
0.4±0.1
0.0
0.0
0.0
0.0
0.0
0.0
>250
>250
>250
>250
>250
>250
250
Ca
Cn
Ca
Cn
Ca
24
25
6.0±0.2
0.0
4.5±0.0
0.0
4.4±0.8
0.0
2.1±0.1
0.0
>250
250
OCH3
G
24.8±0.1 14.5±1.2 8.4±0.3
6.0±0.5
5.2±1.4
>250
H3C
OCH3
G
100
100
100
100
100
100
100
100
100
100
100
100
100
100
0.25
0.50
Cn
Ca
Amphotericin B
Table 1. Inhibition percentages of the mono- (group i) and bi- geranyl (group ii) phenols and
derivatives at different two-fold concentrations (250-3.9 µg/mL) against Candida albicans ATCC
10231 (Ca) and Cryptococcus neoformans ATCC 32264 (Cn). MIC100 and MIC50 are included too
From Table 1, it is clear that compounds containing two geranyl substituents (group ii) (no matter
the other substituents on the aromatic ring) are very weak inhibitors of C. neoformans (all MIC100
and almost all MIC50 are higher than 250 µg/mL) and of C. albicans (MIC100 and MIC50
> 250
µg/mL).
Instead, group (i) with one geranyl substituent, showed better activities against C. neoformans
than against C. albicans which can be easily verified by comparing MIC100 or MIC50 of each
compound against both fungi. As a mere example, from sub-group i.1, 5 possesses MIC100 = 62.5
µg/mL against C. neoformans and MIC
> 250 µg/mL against C. albicans and from sub-group i.2, 3
shows MIC100 = 3.9 µg/mL against C. neoformans and 125 µg/mL against C. albicans.
From the results obtained with C. neoformans, the following structure-activity relationships can be
drawn: (a) within sub-group (i.1) the phenolic compounds (2, 5, 6 and 7) clearly show better
activities than non-phenolic structures (8 and 9); (b) among compounds of sub-group i.2, only di-
(10 and 11) and tri- hydroxylated (3 and 4) compounds showed significant anti-cryptoccocal
activity (100% inhibition at concentrations ≤ 250 µg/mL), being 3, 10 and 11 highly active with
MIC100 or MIC50 = 3.9 µg/mL, similar to the standard drug amphotericin B (Figures 4a-c).
Series i.1
120
5
6
2
100
7
8
80
60
40
20
0
9
0
50
100
150
200
250
300
Concentrations (µg/mL)
Series i.2

ACCEPTED MANUSCRIPT
Series ii
Figure 4. Comparative antifungal activities of compounds of series (i) [(i.1) (i.2)] and (ii) against C.
neoformans. Amphotericin B inhibits 100 % growth at 0.25 µg/mL against C. neoformans (not
included).
3.2.2 Second order studies with clinical isolates
In order to gain insight into the potential of the most active compounds against not only
standardized strains but on clinical isolates of the tested fungal spp, the most promising
compounds were tested against an extended panel of C. neoformans strains that were isolated
from immunocompromised patients suffering fungal infections. They were also tested against
clinical isolates of C. albicans and non-albicans Candida strains. Results are recorded in Table 2.
Table 2. Minimum Inhibitory Concentrations (MIC100 and MIC50) and Minimum Fungicidal activity
(MFC) of selected mono-geranyl compounds 6, 7, 3, 10 and 11 against six C. neoformans, six C.
albicans and four non-albicans Candida isolated strains. For the sake of comparison, MIC100 and
MIC50 of all compounds against the ATCC standardized strain C. albicans ATCC 10231 and C.
neoformans ATCC 32264 were included.
3
6
7
10
11
Amph. B
MIC100
Voucher
Strain
MIC100 MIC50 MFC MIC100 MIC50 MFC MIC100 MIC50 MFC MIC100 MIC50 MFC MIC100 MIC50 MFC
specimen
C. neoformans
C. neoformans
C. neoformans
C. neoformans
C. neoformans
ATCC 32264 3.9
1.9
3.9
7.8
3.9 15.6 31.25 15.6 62.5
7.8 15.6 125 >250 >250 3.9
7.8 31.2 250 >250 >250 3.9
250 >250 >250 7.8
15.6 15.6 15.6 >250 >250 >250 3.9
3.9
1.9
1.9
1.9
1.9
3.9
3.9
7.8
3.9
3.9
3.9
3.9
3.9
3.9
7.8
7.8
7.8
0.25
0.12
0.12
0.25
0.12
IM 983040
IM 972724
IM 042074
IM 983036
31.2 15.6 15.6
31.2 15.6 15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
15.6
7.8 31.2
7.8 15.6
15.6 15.6 31.2
15.6 15.6

ACCEPTED MANUSCRIPT
7.8
7.8
C. neoformans
C. neoformans
IM 003190
IM 003230
15.6
15.6
3.9
15.6 31.2 15.6 31.2 >250 >250 >250 7.8
3.9
3.9
15.6
15.6
7.8
7.8
15.6
31.2
0.25
0.50
7.8 31.2
15.6
7.8 15.6
250 >250 >250 7.8
125
62.5 31.2 125
250
>250
62.5
125
125
125
125
125
125
125
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
C. albicans
ATCC 10231 125 31.2
125
62.5
125
62.5
31.2 15.6
31.2 31.2
0.25
0.75
0.50
0.75
0.56
0.75
0.50
CCC 125
CCC 126
CCC 127
CCC 128
CCC 129
CCC 130
125 62.5 250 >250 >250 >250 31.2 15.6 62.5 62.5 31.2
62.5 62.5 62.5 125 62.5 250 >250 >250 >250 31.2 15.6 62.5 62.5 31.2
31.2 31.2 62.5 125 62.5 250 >250 >250 >250 31.2 15.6 62.5 62.5 31.2
62.5 62.5 125
125 62.5 125 62.5 62.5 62.5 >250 >250 >250 31.2 15.6 125 31.2 31.2
31.2 31.2 31.2 62.5 62.5 62.5 >250 >250 >250 31.2 15.6 62.5 62.5 31.2
125 62.5 250 >250 >250 >250 31.2 31.2 31.2 31.2 31.2
C. glabrata
CCC 115
62.5 31.2 62.5 125
125 250 >250 >250 >250 62.5 31.2 125 62.5 31.2
125 62.5 250 >250 >250 >250 62.5 15.6 125 62.5 31.2
125 125 250 >250 >250 >250 62.5 31.2 125 125 62.5
62.5
125
125
125
1.50
0.75
0.50
0.50
C. parapsilopsis CCC 124
62.5 31.2 125
125 62.5 125
C. krusei
CCC 117
CCC 131
C. tropicalis
62.5 15.6 62.5 125 62.5 250 >250 >250 >250 62.5 15.6 125 62.5 31.2
MIC100 and MIC50: concentrations of each compound that produced 100 % or 50 % reduction of the
growth control respectively. ATCC= American Type Culture Collection (Illinois, USA); CCC= Center
of Mycological Reference (Rosario, Argentina), IM = Malbrán Institute (Buenos Aires, Argentina).
C. albicans = Candida albicans; C. glabrata = Candida glabrata; C. parapsilopsis = Candida
parapsilopsis; C. krusei = Candida krusei; C. tropicalis = Candida tropicalis; C. neoformans =
Cryptococcus neoformans. Amph B = Amphotericin B.
As it can be observed in Table 2, all clinical strains of C. neoformans were more susceptible to
geranylphenols than all clinical strains of C. albicans and non-albicans Candida strains similarly to
the behavior showed against the standardized strains in Table 1. The only exception was
compound 7 that was almost inactive in all the isolated strains with MICs ≥ 250 µg/mL.
It is also clear from Table 2 that compounds 3, 6, 10 and 11 are compounds that deserve attention
as hits for the development of a new antifungal drug against C. neoformans. They inhibit 100% of
standardized as well as clinical isolates of C. neoformans at very low concentrations (3.9-31.2
µg/mL), and inhibit 50% of their growth at concentrations between 1.9-15.6 µg/mL. In addition,
they all are fungicide (kill the fungi rather than inhibit them), thus avoiding recurrence.
3.2.3 In vitro human dermal fibroblasts growth inhibition assay.
The cytotoxic activity of the most active compounds was evaluated following the procedure
previously described by our group (Taborga et al., 2016). The IC50 obtained from these assays are
shown in Table 3.
Table 3. Inhibitory Concentrations (IC50) of selected mono-geranyl compounds 6, 7, 3, 10 and 11
on human dermal fibroblast (HDF)
The cytotoxicity values on human dermal fibroblast (HDF)
Compound number
IC50
3
6
7
10
11
ND
ND
>100µM
>100µM
>100µM
ND = Not Determined

ACCEPTED MANUSCRIPT
The cytotoxicity assay performed on compounds 6, 10 and 11 on human dermal fibroblast (HDF),
showed values of IC50 greater than 100 µM, indicating that the toxicity of these compounds is
negligible. This is in agreement with the results previously reported by our group that showed that
the toxic activity of this type of compounds depends strongly on the substitution of geranyl chains.
Compounds substituted with two geranyl chains on the aromatic ring are more toxic than
compounds substituted with only one geranyl chain. The results indicate that the compounds
mono-attached by geranyl do not present significant toxic activity in the tests carried out in vitro in
the non-tumor cell lines (HDF) nor in the tumor cell lines (Taborga et al., 2016).
4. Conclusions:
From the values of inhibition percentages of fungal growth reported in Table 1, we conclude that
the compounds having a geranyl substituent (series i) were more active than bi-geranyl
compounds (series ii) against C. neoformans strains. Additionally, geranylphenols 2, 3-7, 10, and 11
were more active than their acetylated or methoxylated derivatives. This effect can be clearly
observed by comparing, within sub-series i.2, compounds 4, 13, and 16 and 11, 12 and 17.
Dramatic decrease in biological activity is observed when comparing data between pairs of
compounds 2 with 8; 6 with 9 or 3 with 15. Therefore the decreasing order of biological activities
in this series of compounds could be established as: Ar-OH >> Ar-OCH3 ≈ Ar-OAc against C.
neoformans strains. Although the biological activity of these compounds against C. albicans
decreases, the trend observed is similar. We believe that this effect presumably is caused by the
change of polarity in the structures of the compounds from more polar hydroxyl functions to less
polar methoxy or acetoxy functions. This effect is also in accordance to the incorporation of two
geranyl chains on the aromatic ring (bi-geranyl, series ii). In addition, from the values reported
against clinical isolates (Table 2) the compounds 3, 6, 10 and 11 (series i) showed interesting
MIC100 and MIC50 values. However, compound 7 showed no activity in the other strains. These
results are considered significant because this series of compounds (3, 6, 10 and 11) can act
against to several strains of Candida and C. neoformans.
Based on the reported data in Table 1 the observed decreasing order in the growth inhibition
effect on C. neoformans and C. albicans assay was 10 ≥ 11 > 3 > 7 ≈ 4, while a similar effect was
observed in clinical isolates for compounds 10 and 11 (Table 2).
Finally, it was observed that the activity of the compounds analyzed is selective towards fungi,
considering that the compounds showed growth inhibition of pathogenic fungi, and a negligible
cytotoxicity on human non-tumor cell lines. This results making them a very attractive family for
the development of future drugs for the treatment of mycotic infections.
Acknowledgments:
The authors thank to FONDECYT (grant No. 11160319) and the Dirección General de Investigación
y Postgrado (DGIP-USM grant No. 116.13.12) of Universidad Técnica Federico Santa María. SZ and
MS acknowledges to ANPCyT, PICT 2014-1170 and National University of Rosario, project BIO381;

ACCEPTED MANUSCRIPT
MS is a member of the research career of CONICET. EB acknowledges CONICET for the doctoral
fellowship
Author Contributions:
LE, LT and SZ designed research; SZ, LE, LT, MS, EB and HC performed research and analyzed the
data; LE, LT, HC and SZ wrote the paper. All authors read and approved the final manuscript.
Conflicts of Interest:
“The authors declare no conflict of interest."
References:
Bauer, J., Koeberle, A., Dehm, F., Pollastro, F., Appendino, G., Northoff, H., Rossi, A., Sautebin, L.,
Werz, O., 2011. Arzanol, a prenylated heterodimeric phloroglucinyl pyrone, inhibits
eicosanoid biosynthesis and exhibits anti-inflammatory efficacy in vivo. Biochem. Pharmacol.
81, 259–268.
Benslimane, A.F., Pouchus, Y.F., Le Boterff, J., Verbist, J.F., 1988. Substances cytoxiques et
antibacteriennes de L´ascidie Aplidium antillense. J. Nat. Prod. 51, 582–583.
Bifulco, G., Bruno, I., Minale, L., Riccio, R., Debitus, C., Bourdy, G., Vassas, A., Lavayre, J., 1995.
Bioactive prenylhydroquinone sulfates and a novel C31 furanoterpene alcohol sulfate from
the marine sponge, Ircinia sp. J. Nat. Prod. 58, 1444–1449.
Bowden, B.F., Coll, J.C., 1981. Studies of Australian soft corals. XXVI. Tetraprenylbenzoquinone
derivatives from a Nephthea species of soft coral (Octocorallia, Alcyonacea). Aust. J. Chem.
34, 2677–2681.
Brown, E.D., Wright, G.D., 2005. New targets and screening approaches in antimicrobial drug
discovery. Chem. Rev. 105, 759–774.
Capon, R.J., Ghisalberti, E.L., Jefferies, P.R., 1981. Isoprenoid dihydroquinones from a brown alga,
Cystophora sp. Phytochemistry 20, 2598–2600.
Carroll, A.R., Bowden, B.F., Coll, J.C., 1993. Studies of Australian ascidians .3. A new
tetrahydrocannabinol derivative from the Ascidian Synoicum-castellatum. Aust. J. Chem. 46,
1079–1083.
Chen, S.C., Playford, E.G., Sorrell, T.C., 2010. Antifungal therapy in invasive fungal infections. Curr.
Opin. Pharmacol. 10, 522–530.
CLSI (Clinical and Laboratory Standars Institute), 2008. No Title, in: Wayne (Ed.), Reference Method
for Broth Dilution Antifungal Susceptibility Testing of Yeasts M27A3. Wayne (PA), pp. 1–25.
Cordeiro, R.D.A., Nogueira, G.C., Brilhante, R.S.N., Teixeira, C.E.C., Mourão, C.I., Castelo-Branco,
D.D.S.C.M., Paiva, M.D.A.N., Ribeiro, J.F., Monteiro, A.J., Sidrim, J.J.C., Rocha, M.F.G., 2012.
Farnesol inhibits in vitro growth of the Cryptococcus neoformans species complex with no
significant changes in virulence-related exoenzymes. Vet. Microbiol. 159, 375–380.

ACCEPTED MANUSCRIPT
Danelutte, A.P., Lago, J.H.G., Young, M.C.M., Kato, M.J., 2003. Antifungal flavanones and
prenylated hydroquinones from Piper crassinervium Kunth. Phytochemistry 64, 555–559.
De Rosa, S., Crispino, A., De Giulio, A., 1995. Sulfated polyprenylhydroquinones from the sponge
Ircinia spinosula. J. Nat. Prod. 58, 1450–1454.
De Rosa, S., De Giulio, A., Iodice, C., 1994. Biological effects of prenylated hydroquinones:
structure-activity relationship studies in antimicrobial, brine shrimp, and fish lethality assays.
J. Nat. Prod. 57, 1711–1716.
Espinoza, L., Taborga, L., Díaz, K., Olea, A.F., Peña-Cortés, H., 2014. Synthesis of linear
geranylphenols and their effect on mycelial growth of plant pathogen Botrytis cinerea.
Molecules 19, 1512–26.
Faulkner, D.J., 1993. Marine natural products. Nat. Prod. Rep. 10, 497–539.
Faulkner, D.J., 1986. Marine natural products. Nat. Prod. Rep. 3, 1–33.
Fedorov, S., Radchenko, O., Shubina, L., 2006. Evaluation of cancer preventive activity and
structure-activity relationships of 3-demethylubiquinone Q2 , isolated from the ascidian
aplidium glabrum, and its synthetic analogues. Pharm. Res. 23, 70–81.
Fu, X., Hossain, M.B., Helm, D. Van Der, Schmitz, F.J., van der Helm, D., 1994. Longithorone A:
Unprecedented Dimeric Prenylated Quinone from the Tunicate Aplydium longithorax. J. Am.
Chem. Soc. 116, 12125–12126.
Fu, X., Hossain, M.B., Schmitz, F.J., Helm, D. Van Der, 1997. Type quinones from the tunicate
aplidium longithorax. J. Org. Chem. 62, 3810–3819.
Gerwick, W.H., Fenical, W., 1981. Ichthyotoxic and cytotoxic metabolites of the tropical brown alga
Stypopodium zonale (Lamouroux) Papenfuss. J. Org. Chem. 46, 22–27.
Guella, G., Mancini, I., Pietra, F., 1987. Verapliquinones: Novel Diprenylquinones from an Aplidium
sp. (Ascidiacea) of Ile-Verte Waters, Brittany. Helv. Chim. Acta 70, 621–626.
Han, Q.-B., Qiao, C.-F., Song, J.-Z., Yang, N.-Y., Cao, X.-W., Peng, Y., Yang, D.-J., Chen, S.-L., Xu, H.-
X., 2007. Cytotoxic prenylated phenolic compounds from the twig bark of Garcinia
xanthochymus. Chem. Biodivers. 4, 940–946.
Howard, B.M., Clarkson, K., Bernstein, R.L., 1979. Simple prenylated hydroquinone derivatives
from the marine urochordate aplidium californicum. Natural anticancer and antimutagenic
agents. Tetrahedron Lett. 20, 4449–4452.
Kontoyiannis, D.P., 2012. Invasive mycoses: strategies for effective management. Am. J. Med. 125,
S25–S38.
Liu, Q., Shu, X., Wang, L., Sun, A., Liu, J., Cao, X., 2008. Albaconol, a plant-derived small molecule,
inhibits macrophage function by suppressing NF-kappaB activation and enhancing SOCS1
expression. Cell. Mol. Immunol. 5, 271–278.
Manfredi, K.P., Vallurupalli, V., Demidova, M., Kindscher, K., Pannell, L.K., 2001. Isolation of an
anti-HIV diprenylated bibenzyl from Glycyrrhiza lepidota. Phytochemistry 58, 153–157.
Manners, G.D., Jurd, L., 1977. The hydroquinone terpenoids of Cordia alliodora. J. Chem. Soc.

ACCEPTED MANUSCRIPT
Perkin Trans. 1 405–410.
Mathew, B.P., Nath, M., 2009. Recent approaches to antifungal therapy for invasive mycoses.
ChemMedChem 4, 310–323.
Mukherjee, P.K., Leidich, S.D., Isham, N., Leitner, I., Ryder, N.S., Ghannoum, M.A., 2003. Clinical
Trichophyton rubrum strain exhibiting primary resistance to terbinafine. Antimicrob. Agents
Chemother. 47, 82–86.
Ochi, M., Kotsuki, H., Inoue, S., Taniguchi, M., Tokoroyama, T., 1979. Isolation of 2-(3, 7, 11-
trimethyl-2, 6, 10-dodecatrienyl) hydroquinone from the brown seaweed Dictiopteris
undulata. Chem. Lett. 831–832.
Perkins, A., Gomez-Lopez, A., Mellado, E., Rodriguez-Tudela, J.L., Cuenca-Estrella, M., 2005. Rates
of antifungal resistance among Spanish clinical isolates of Cryptococcus neoformans var.
neoformans. J. Antimicrob. Chemother. 56, 1144–1147.
Pfaller, M. a., Diekema, D.J., 2007. Epidemiology of invasive candidiasis: a persistent public health
problem. Clin. Microbiol. Rev. 20, 133–163.
Quang, D.N., Hashimoto, T., Arakawa, Y., Kohchi, C., Nishizawa, T., Soma, G.-I., Asakawa, Y., 2006.
Grifolin derivatives from Albatrellus caeruleoporus, new inhibitors of nitric oxide production
in RAW 264.7 cells. Bioorg. Med. Chem. 14, 164–168.
Ravi, B.N., Wells, R.J., 1982. Lipid and terpenoid metabolites of the Gorgonian Plexura flava. Aust.
J. Chem. 35, 105–112.
Reynolds, G., Rodriguez, E., 1979. Geranylhydroquinone: A contact allergen from trichomes of
Phacelia crenulata. Phytochemistry 18, 1567–1568.
Roemer, T., Krysan, D.J., 2014. Antifungal drug development: challenges, unmet clinical needs, and
new approaches. Cold Spring Harb. Perspect. Med. 4, 1–15.
Rudali, G., Menetrier, L., 1967. Research on the radioprotective action of geranyl-hydroquinone.
Therapie 22, 895–904.
Rudall, G., 1966. Research on the radioprotective action of geranyl-hydroquinone. C. R. Seances
Soc. Biol. Fil. 160, 1365–9.
Sato, A., Shindo, T., 1989. Antioxidant metabolites from the tunicate Amaroucium multiplicatum. J.
Nat. Prod. 52, 975–981.
Taborga, L., Díaz, K., Olea, A.F., Reyes-Bravo, P., Flores, M.E., Peña-Cortés, H., Espinoza, L., 2015.
Effect of Polymer Micelles on Antifungal Activity of Geranylorcinol Compounds against
Botrytis cinerea. J. Agric. Food Chem. 63, 6890–6896.
Taborga, L., Espinoza, L., Moller, A., Carrasco, H., Cuellar, M., Villena, J., 2016. Antiproliferative
effect and apoptotic activity of linear geranylphenol derivatives from phloroglucinol and
orcinol. Chem. Biol. Interact. 247, 22–29.
Taborga, L., Vergara, A., Fernández, M.J., Osorio, M., Carvajal, M., Madrid, A., Marilaf, F., Carrasco,
H., Espinoza, L., 2013. Synthesis and NMR structure determination of new linear
geranylphenols by direct geranylation of activated phenols. J. Chil. Chem. Soc. 58, 1790–
1796.

ACCEPTED MANUSCRIPT
Targett, N.M., Keeran, W.S., 1984. A terpenehydroquinone from the marine Ascidian Aplidium
constellatum. J. Nat. Prod. 4, 1975–1976.
Trpković, a., Pekmezović, M., Barać, A., Crnčević Radović, L., Arsić Arsenijević, V., 2012. In vitro
antifungal activities of amphotericin B, 5-fluorocytosine, fluconazole and itraconazole against
Cryptococcus neoformans isolated from cerebrospinal fluid and blood from patients in Serbia.
J. Mycol. Med. 22, 243–248.
Vicente, M.F., Basilio, A., Cabello, A., Pelaez, F., Valca, J., 2003. Microbial natural products as a
source of antifungals. Clin. Microbiol. Infect. 9, 15–32.
Yamaguchi, L.F., Lago, J.H.G., Tanizaki, T.M., Mascio, P. Di, Kato, M.J., 2006. Antioxidant activity of
prenylated hydroquinone and benzoic acid derivatives from Piper crassinervium Kunth.
Phytochemistry 67, 1838–1843.

ACCEPTED MANUSCRIPT
Highlights


Geranylphenols have antifungal activity against Candida albicans and Cryptococcus
neoformans.
The bioactivity of geranylphenols is related to the amount and type of substitution in the
aromatic ring.


Some geranylphenols studied showed similar activity to standard drug Amphotericin B.
Compound analyzed is selective, showed growth inhibition of fungi, and a negligible
cytotoxicity on human cell lines.

The geranylphenols is a very attractive family for the development of future drugs for the
treatment of mycotic infections.