Structural requirements for the antifungal activities of natural drimane sesquiterpenes and analogues, supported by conformational and electronic studies

Article abstract of DOI:10.3390/molecules18022029

Seventeen drimanes including polygodial (1), isopolygodial (2), drimenol (3) and confertifolin (4) obtained from natural sources and the semi-synthetic derivatives 5-17 obtained from 1-3, were evaluated in vitro for antifungal properties against a unique panel of fungi with standardized procedures by using two end-points, MIC₁₀₀ and MIC₅₀. A SAR analysis of the whole series, supported by conformational and electronic studies, allowed us to show that the Δ^7,8 -double bond would be one of the key structural features related to the antifungal activity. The MEPs obtained for active compounds exhibit a clear negative minimum value (deep red zone) in the vicinity of the Δ^7,8 -double bond, which is not present in the inactive ones. Apart of this negative zone, a positive region (deep blue) appears in 1, which is not observed either in its epimer 2 nor in the rest of the active compounds. The LogP of active compounds varies between 2.33 and 3.84, but differences in MICs are not correlated with concomitant variations in LogP values.

Full text of DOI:10.3390/molecules18022029

Molecules 2013, 18, 2029-2051; doi:10.3390/molecules18022029
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Structural Requirements for the Antifungal Activities of
Natural Drimane Sesquiterpenes and Analogues,
Supported by Conformational and Electronic Studies
Marcos Derita 1, Iván Montenegro 2, Francisco Garibotto 3,4, Ricardo D. Enriz 3,4
,
Mauricio Cuellar Fritis 2,† and Susana A. Zacchino 1,†,
*
1
Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas,
Universidad Nacional de Rosario, Suipacha 531, 2000 Rosario, Argentina
Laboratorio de Ciencias Químicas y Recursos naturales, Facultad de Farmacia,
2
3
Universidad de Valparaíso, Avenida Gran Bretaña 1111, Playa Ancha, 2340000 Valparaíso, Chile
Departamento de Química, Facultad de Química, Bioquímica y Farmacia,
Universidad Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina
IMIBIO-SL (CONICET), Chacabuco 915, 5700 San Luis, Argentina
4
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: szaabgil@citynet.net.ar;
Tel.: +54-341-4375315.
Received: 18 December 2012; in revised form: 22 January 2013 / Accepted: 31 January 2013 /
Published: 5 February 2013
Abstract: Seventeen drimanes including polygodial (1), isopolygodial (2), drimenol (3)
and confertifolin (4) obtained from natural sources and the semi-synthetic derivatives 5–17
obtained from 1–3, were evaluated in vitro for antifungal properties against a unique panel
of fungi with standardized procedures by using two end-points, MIC100 and MIC50. A SAR
analysis of the whole series, supported by conformational and electronic studies, allowed
us to show that the Δ7,8 -double bond would be one of the key structural features related to
the antifungal activity. The MEPs obtained for active compounds exhibit a clear negative
minimum value (deep red zone) in the vicinity of the Δ7,8 -double bond, which is not
present in the inactive ones. Apart of this negative zone, a positive region (deep blue)
appears in 1, which is not observed either in its epimer 2 nor in the rest of the active
compounds. The LogP of active compounds varies between 2.33 and 3.84, but differences
in MICs are not correlated with concomitant variations in LogP values.

Molecules 2013, 18
Keywords: antifungal agents; drimanes; structure-activity relationships; stereo-electronic
2030
studies
1. Introduction
In the course of our ongoing search for antifungal compounds from natural sources, we recently
reported that the aerial parts of Polygonum acuminatum Kunth., used in the Argentinean traditional
medicine for ailments related to fungal infections [1], possessed antifungal properties against yeasts
and dermatophytes, these results supporting their ethnopharmacological use [2]. Polygodial (1, Figure 1)
was isolated from the most active extract as the main compound responsible for the activity, although
other drimane derivatives such as isopolygodial (2) drimenol (3) and confertifolin (4) (Figure 1)
showed antifungal properties too [2].
Figure 1. Drimanes isolated from Polygonum acuminatum.
Although the claims that 1 possesses antifungal activity were consistent in all previous reports [3–6],
contradictory reports were found in the literature regarding the antifungal activities of its natural
analogue 2, which was variously reported to be either active or inactive, and based on this information,
conflicting opinions about the main structural features required for drimanes to display antifungal
activities were put forth. They can be summarized as follows: Fujita and Kubo stated that the
- orientation of the C-9 carbonyl group was indispensable for activity, since 1 (with an aldehyde in
the C-9 orientation) but not 2 (with a C-9 aldehyde) possessed antifungal activity [7]. Conversely,
Anke and Sterner [8] found that 2 did possess antifungal activity, although lower than that of 1, which
was corroborated by us in the paper cited above [2] and in a subsequent one [9]. Another structural
feature which was assigned a role in the antifungal activity was the ,-unsaturated C-8 aldehyde
moiety which, according to Taniguchi et al., was essential for activity [10]. However, this appears to
be a contradiction with their own previous report in which 2 was devoid of antifungal activity,
although it possess the ,-unsaturated C-8 aldehyde moiety [7,10]. Fujita and Kubo also reported that
1 lost its antifungal activity against Saccharomyces cerevisiae in certain media, because of formation
of a pyrrole between its aldehydes and the amines present in the medium [7]. Considering that, with
the formation of a pyrrole, both aldehydes disappear, this report opens a new question: if one or both
aldehydes are necessary for the antifungal activity.
In order to clarify the main features required for drimanes to display antifungal activities, we tested
herein a series of 17 compounds 1–17 (most of them not previously assayed for antifungal activities)
for antifungal properties against a unique panel of nine fungal strains, with a standardized
methodology, in the same laboratory. To guarantee confident and reproducible results, the whole series

Molecules 2013, 18
2031
was tested following the Clinical and Laboratory Standards Institute (CLSI) [11,12] standardized
procedures. CLSI has established consensus procedures to facilitate the agreement among laboratories
in measuring the susceptibility of yeasts (updated in 2008 as M-27 A3) and of filamentous fungi
(updated in 2008 as M-38 A2) to antifungal agents, with broth dilution methods. The standardized
parameters detailed in both documents include protocols for the preparation of antifungal stock
solutions, dilutions for testing, inocula, culture medium, temperature and incubation time, endpoint
definitions and reference MIC ranges for microdilution testing of both, the established and newly
introduced antifungal agents.
Among the tested analogues of 1, compounds 2–4 were isolated from P. acuminatum leaves and the
remaining compounds 5–17 were prepared by isomerization, reduction, oxidation and methylenation
reactions using the natural compounds 1, 2 and 3 as starting materials. They were tested against three
yeasts: Candida albicans, S. cerevisiae and Cryptococcus neoformans; three Aspergillus spp.
(A. flavus, A. fumigatus and A. niger) and three dermatophytes: Microsporum gypseum, Trichophyton
rubrum and Trichophyton mentagrophytes by using two end-points MIC100 and MIC50. A SAR
analysis of the whole series, supported by conformational and electronic studies on some selected
compounds is presented herein, with the aim of determining the stereo-electronic characteristics related
to the antifungal behavior and thus contribute to the understanding of the structural properties of
antifungal drimanes.
2. Results and Discussion
2.1. Chemistry
Sesquiterpenes 1–4 were obtained from P. acuminatum leaves as described previously [2].
Compounds 1–3 were used as starting materials for the preparation of compounds 5–17.
2.1.1. Compounds Obtained from 1
Scheme 1 shows the modifications performed on compound 1 that led to compounds 5–8.
Reduction of 1 with excess of NaBH4 in MeOH at rt produced (−)-(5S,10S)-(9R)-7-drimene-11,12-diol
(5) and (−)-(5aS,9aS,9bR)-6,6,9a-trimethyl-1,3,5,5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan (6).
Isodrimeninol (7) was obtained by reduction of 1 with 0.5 mmol of NaBH4 for each mmol of drimane
and compound 8 was a methylenation product obtained by treating 1 with one equivalent of the bulky
Tebbe´s reagent, following previously reported procedures [13]. The identities of 5, 7 and 8 were
established by comparison with previously reported data [13–16]. Compound 6, which was only
previously reported once [16], is described in this work.
2.1.2. Compounds Obtained from 2
Scheme 2 shows the modifications performed on compound 2. Reduction of 2 with excess NaBH4
in MeOH at r.t. produced (+)-(5S,10S)-(9S)-7-drimene-11,12 diol (isodrimendiol, 9) [17] which, by
catalytic hydrogenolysis using Pd/C (10% Pd), was converted into isodrimenol 10 [15,18].
Epoxidation of 9 with m-chloroperbenzoic acid (MCPBA) produced a single epoxide 11 in 75% yield.
The 7-H signal in the 1H-NMR spectrum (δ 3.3, brdd, J = 5.2 Hz, 5.2 Hz) [19] and the signal for C-5,

Molecules 2013, 18
2032
appearing at a δ 15.2 ppm lower than that corresponding to 9 in the 13C-NMR spectrum, confirmed the
α configuration of 11, as reported for similar compounds [20]. The epoxide 11 was refluxed with
LiAlH4 in THF to afford the triol 12 in 85% yield. Oxidative degradation of 12 with sodium periodate
gave the ketol 13 in 75% yield [21].
Scheme 1. Modifications performed on 1 to obtain compounds 5–8.
CHO
8
H
1.a
2
1
11
CH2OH
O
CHO
12
9
6
9b
12
CH2OH
3
H
CHO
8
7
3a
4
9a
9
10
5
1.b
5a
+
7
5
H
H
H
6
33%
11
10
5
62%
1
1.c
HO
O
7
H
Conditions and reagents: (1.a) Tebbe’s reagent (C5H5)2TiCH2ClAl(CH3)2; (1.b) NaBH4 (excess),
MeOH rt; (1.c) NaBH4 (0.5 mmol per mmol of 1), MeOH rt.
Scheme 2. Modifications performed on 2 to obtain compounds 9–13.
11
CH2OH
CHO
CH2OH
12
CHO
CH2OH
CH3
9
7
2.a
2.b
2
9
10
H
H
H
CH2OH
CH2OH
CH2OH
CH2OH
O
CH2OH
OH
O
2.d
2.e
H
H
12
11
H
13
Conditions and reagents: (2.a) NaBH4 (excess), MeOH rt; (2.b) H2, Pd/C (10%); (2.c) MCPBA,
DCM; (2.d) LiAlH4, THF; (2.e) NaIO4, THF/H2O.
2.1.3. Compounds Obtained from 3
Scheme 3 shows the modifications performed on 3. Oxidation of 3 with Jones reagent afforded
drimenal 14 and the unsaturated ketone 15. On the other hand, the unsaturated ketone 16 was obtained
by treating 3 with the oxidation reagent pyridinium chlorochromate (PCC) [14]. Their structures were
determined by comparison with the previously reported data [14,22,23]. In addition, catalytic

Molecules 2013, 18
2033
hydrogenation of 3 using Pd/C (10% Pd), led to the saturated drimanol 17 whose spectroscopic data
were in accordance to the literature [24].
Scheme 3. Modifications performed on 3 to obtain compounds 14–17.
Conditions and reagents: (3.a): Jones reagent; (3.b): Pyridinium chlorochromate (PCC); (3.c) H2,
Pd/C (10%).
2.2. Antifungal Activity
To carry out the antifungal evaluation, concentrations of naturally occurring drimanes 1–4 and
semi-synthetic derivatives 5–17 up to 250 µg/mL were incorporated to growth media according to
CLSI guidelines [11]. Table 1 summarizes the concentration of drimanes that completely inhibited
(MIC100) the growth of nine opportunistic pathogenic fungi including yeasts (C. albicans,
C. neoformans, and S. cerevisiae), hialohyphomycetes (Aspergillus spp.) as well as dermatophytes
(Microsporum and Trichophyton spp). In addition to MIC determinations, the Minimum Fungicidal
Concentration (MFC) of each active compound against this panel was accomplished by sub-culturing a
sample of media from MIC tubes showing no growth, onto drug free agar plates (Table 1).
Table 1. Minimum Inhibitory Concentration (MIC100) and Minimum Fungicidal
Concentration (MFC) of naturally occurring (1–4) and semi-synthetic (5–17) drimanes,
against human opportunistic pathogenic fungi.
Compounds
Structure
Yeasts
Sc
Aspergillus spp
Dermatophytes
Nº
1
Ca
Cn
Afum
Afl
An
Mg
Tr
Tm
CHO
CHO
MIC100
MFC
3.9
15.6
7.8
I
I
I
62.5
125
62.5
62.5
125
7.8
31.2
7.8
-
-
-
125
H
CHO
CHO
2
3
MIC100
MFC
62.5
125
62.5
125
62.5
125
I
-
I
-
I
-
62.5
125
62.5
125
62.5
125
H
CH2OH
MIC100
MFC
I
-
I
-
125
I
I
-
I
-
I
-
62.5
125
62.5
125
62.5
125
H

Molecules 2013, 18
2034
Table 1. Cont.
O
O
4
MIC100
MFC
I
-
250
I
-
I
-
I
-
I
-
125
250
62.5
125
62.5
H
-
125
CH2OH
CH2OH
5
6
7
8
MIC100
MFC
125
-
250
-
250
-
I
-
I
-
I
-
250
250
250
250
250
250
H
O
MIC100
MFC
250
I
250
125
250
I
-
I
-
I
-
62.5
125
62.5
125
62.5
125
H
I
HO
1
O
MIC100
MFC
250
I
250
125
250
I
-
I
-
I
-
31.2
125
31.2
125
31.2
125
H
I
CHO
MIC100
MFC
250
I
250
125
250
I
-
I
-
I
-
62.5
125
62.5
125
62.5
125
H
I
CH2OH
CH2OH
MIC100
MFC
250
I
250
125
250
I
-
I
-
I
-
125
125
125
250
250
250
9
H
I
CH2OH
MIC100
MFC
125
250
125
125
31.2
62.5
I
-
I
-
I
-
125
250
62.5
125
250
250
10
11
H
CH2OH
CH2OH
O
MIC100
MFC
I
-
I
I
-
I
-
I
-
I
-
I
-
I
-
I
-
H
-
I
CH2OH
CH2OH
OH
MIC100
I
I
I
I
I
I
I
I
H
12
13
MFC
-
I
-
-
I
-
-
I
-
-
I
-
-
I
-
-
I
-
-
250
I
-
250
250
-
250
250
CH2OH
MIC100
MFC
O
H
CHO
MIC100
MFC
125
125
125
125
31.2
31.2
I
-
I
-
I
-
62.5
125
62.5
125
125
250
14
15
MIC100
MFC
I
I
250
-
I
-
I
-
I
-
250
250
250
250
250
250
-
-

Molecules 2013, 18
2035
Table 1. Cont.
MIC100
MFC
250
I
250
250
250
I
-
I
-
I
-
250
250
250
250
250
250
16
17
I
MIC100
MFC
I
I
-
I
I
-
I
-
I
-
I
I
I
-
-
-
-
-
St drugs
Amph B
MIC
MIC
MIC
0.78
1.56
0.5
0.50
3.12
0.5
0.25
0.39
0.25
0.50
0.78
0.12
0.50
0.78
0.5
0.50
1.56
0.25
0.12
0.04
0.05
0.075 0.075
0.01 0.025
0.025 0.025
Terbinafine
Ketoconazole
Ca: Candida albicans ATCC 10231, Sc: Saccharomyces cerevisiae ATCC 9763, Cn: Cryptococcus
MIC50 (µg/mL) neoformans ATCC 32264; An: Aspergillus niger ATCC 9029, Afu: Aspergillus fumigatus
ATCC 26934; Afl: Aspergillus flavus ATCC 9170, Mg: Microsporum gypseum C 115; Tr: Trichophyton
rubrum C113, Tm: Trichophyton mentagrophytes ATCC 9972; Amph B: Amphotericin B. I: inactive
(MIC > 250 µg/mL).
Regarding the sensitivity of the different fungi to drimanes, it can be stated that: (i) all compounds,
but not 11, 12 and 17, exhibited moderate activity (MICs  250 µg/mL) against dermatophytes and
yeasts; (ii) no member of the series was active against Aspergillus spp; (iii) all active compounds
showed fungicidal rather than fungistatic properties against most fungi, since they not only inhibit but
also kill them at Minimum Fungicidal Concentrations (MFC)  250 µg/mL.
2.3. Structure-Activity Relationships (SAR)
Since no differences in MICs among the members were observed against dermatophytes
(one-dilution-step MIC changes are within the range of experimental error) [25], the SAR studies were
performed on the activity of the series against yeasts (C. albicans, S. cerevisiae and C. neoformans)
which showed striking differences.
For a better analysis of the differential behavior of these compounds against yeasts, we selected the
clinically important fungi C. neoformans and C. albicans as targets. C. neoformans is a fungus that
remains an important life-threatening complication for immunocompromised hosts, particularly for
patients who have undergone transplantation of solid organs [26]. Therefore, the activity of compounds
against this fungus has a true clinical relevance. In turn, C. albicans is one of the leading causes of
nosocomial blood stream infections worldwide [27].
Thus, compounds 1–10, 14–16 that showed activity against at least one yeast were re-tested against
the standardized strains C. albicans ATCC 10231 and C. neoformans ATCC 32264 determining, this
time, MIC50 (the minimum concentration of compounds that inhibit 50% of growth), a less stringent
end-point highly used [27] and recommended by CLSI [10]. MIC50 consistently represent the in vitro
activity of compounds and many times provide a better correlation with other measurements of
antifungal activity [27]. Results are shown in Table 2.

Molecules 2013, 18
Table 2. MIC50 values of representative members of the drimane series against C. albicans
2036
ATCC 10231 (Ca) and C. neoformans ATCC 32264 (Cn).
MIC50 (µg/mL)
Ca
1.9
Cn
1.9
LogP
2.33
2.33
3.83
3.35
2.76
3.40
3.24
3.67
2.77
3.83
3.53
3.84
3.37
4.30
1
2
31.2
62.5
62.5
250
62.5
62.5
31.2
250
62.5
62.5
250
31.2
I
31.2
31.2
62.5
250
31.2
31.2
31.2
31.2
15.6
62.5
I
3
4
5
6
7
8
9
10
14
15
16
17
62.5
I
Amphotericin B
0.78
0.25
From the analysis of the structures and the activities displayed by all compounds against
C. albicans and C. neoformans, some structure-activity relationships can be extracted. They were
grouped according to the influence of each structural feature such as 7,8-double bond in the drimane
skeleton, configuration of C-9, presence of an aldehyde at C-9 (or at C-8) and presence of an
α,β-unsaturated aldehyde moiety at C-8.
2.3.1. Role of the 7,8-Double Bond in the Drimane Skeleton Alone and in α,β Position Respective to
the Aldehyde on C-8
The 7,8-double bond in the drimane scheleton appears to be one of the necessary structural features
for antifungal activity since inactive compounds 11, 12, 13 and 17 all lack this 7,8-double bond. The
comparison of the activities of 3 with its saturated derivative 17 (devoid of antifungal activity), clearly
reinforces this observation. A computer-assisted analysis supporting this observation is presented
below, in Section 2.4.
In addition, it can be observed that the 7,8-double bond in active structures can be found either
alone as in compounds 3, 5–10 and 14, or as being part of the C-8 α,β-unsaturated aldehyde moiety
(constituting a Michael acceptor group) as in compounds 1 and 2. The fact that both drimane structural
types do display antifungal activity suggests that antifungal drimanes could not act via a Michael
addition. This finding differs from the antifungal structural requirements of some other sesquiterpenes
such as the sesquiterpenelactones (+)-costunolide and (−)-dehydrocostunolide isolated from Centaurea
plants [28] and the synthetic tuberiferin and dehydrobrachylaenolide [29], whose antifungal activities
were reported to be enhanced by a Michael acceptor group. Nevertheless, our findings are not in any
way strange since many other diverse structural compounds not containing a Michael acceptor group,
were found to display strong antifungal properties [30,31].

Molecules 2013, 18
2037
2.3.2. Role of the Configuration of C-9
The comparison of activities of 1 and 2 shows that, in contrast to Fujita and Kubo’s previous
report [7], both compounds do possess antifungal activity. However, the 9-derivative 2 displays 16
and eight times lower activity than its epimer 1, against C. albicans and C. neoformans, respectively.
This result is consistent with Anke and Sterner’s [8] and Derita et al.’s [2] previous studies, who both
found that 2 did display antifungal activities, but lower than 1. The different antifungal activities
displayed by compounds 1 and 2 are supported by their different electronic distributions which are
presented in Section 2.4.
2.3.3. Role of an Aldehyde at C-9 and of a CH2OH at C-8
The presence of an aldehyde group at C-9 appears not to be indispensable for activity, as can be
observed in compounds 3–7, 9, 10, 15 and 16. Otherwise, the comparison of diol 9 with the
monoalcohol 10, suggests that a CH2OH at C-8 does not contribute to the activity either.
2.4. Conformational and Electronic Studies
To gain a better understanding of the above experimental results, computer-assisted conformational
and electronic studies were performed on all compounds of the series. The purpose was to obtain
precise information about the antifungal drimanes’ stereoelectronic characteristics that give support to
the relationships between structure and activity.
2.4.1. Conformational Analysis
For the conformational analysis, each ring of the drimane scheleton was named A and B (Figure 2).
The essential conformational problem of drimane sesquiterpenes, involves two aspects:
(a) The conformational behavior of torsional angles 1-5 which determines the spatial ordering of
ring A.
(b) The orientation of torsional angles 6-9 which determines the overall shape of ring B. In this
ring, they are also important torsional angles 10 and 11, which are related to CH2OH
substituents as R2 or R1.
Our B3LYP/6311G(d.p) calculations indicated that a chair, and not a boat form, is the energetically
preferred spatial ordering for ring A in compound 1. The energy gap obtained between both
conformers is 14.7 Kcal/mol, indicating that this compound is fairly rigid and possesses a restricted
conformational flexibility. Similar results were obtained for all compounds possessing this structural
feature like compounds 1–3, 5, 8–10 and 14. Considering that, in these compounds, ring B is a
cyclohexene, each of the three possible conformations: sofa (also called half-boat), half-chair and boat,
were evaluated.

Molecules 2013, 18
Figure 2. General representation of drimanes reported here, showing the torsional angles
2038
that determine the spatial ordering of rings A and B.
R2
OH
OH








R1



H2
C





H
Our theoretical results indicated that the so-called “half-boat” conformation is the highly preferred
low-energy form Figure 3a which is in agreement with experimental data obtained for structurally
related compounds [32–34]. Considering that all these compounds adopt similar low flexible
conformations, it is clear that this would not be the cause of the different activities displayed against
fungi. The same can be said for compounds 4, 6 and 7, for which our theoretical calculations indicate
that the conformational flexibility is even more restricted than the observed for the above compounds
as a consequence of the presence of a third fused ring. The energetically preferred conformation
obtained for compound 6 is shown in Figure 3b. Closely similar conformations were obtained for
compounds 4 and 7.
Figure 3. Spatial view of the lowest energy conformations obtained for compounds 1 (a)
and 6 (b) from DFT (B3LYP/6-311G(d,p) optimizations. The chair and half-boat forms
adopted by rings A and B respectively, may be appreciated in this figure.
Thus, the knowledge of the stereo-electronic properties of these drimanes will surely help to
elucidate the structural requirements for the antifungal activity. For this analysis, Molecular
Electrostatic Potentials (MEPs) are particularly valuable, because they allow the visualization and
assessment of the capacity of a molecule to interact electrostatically with a putative-binding site [35–37].
Figure 4 gives the MEPs obtained for compounds 1, 2 and 5, which showed high, moderate and low
activities respectively. The MEP obtained for 1 (Figure 4a) exhibits three characteristic regions: a clear
negative minimum value (deep red zone with V(r)min ≈ −0.1139 el/au3) in the vicinity of Δ7,8 double
bond; a positive region (deep blue zone with V(r)max ≈ 0.1136 el/au3) located near the CH3 group at
C10; and an extended hydrophobic zone (deep and light green zone with an almost neutral potential

Molecules 2013, 18
2039
V(r)med ≈ −0.0011 el/au3) in the rest of the molecule. Considering that 1 is the most active of the series,
this MEP should account for the characteristics of the electronic general pattern for all the molecules
possessing antifungal effect. Interestingly, the MEP obtained for its epimer 2 (Figure 4b), showed a
marked electronegative zone near the double bond Δ7,8 similar to the showed by 1 but a little smaller.
In turn, the positive zone is not observed in 2 which is a clear difference with 1. Regarding compound
5, concomitantly with its lower activity, it showed a red negative zone but considerably smaller than
that of 2 (Figure 4c).
Figure 4. Electrostatic potential-encoded electron density surfaces of compounds 1 (a), 2
(b) and 5 (c). The surfaces were generated using B3LYP/6-311G++(d,p) single point
calculations. The coloring represents electrostatic potential with red and blue indicating the
electronegative or the electropositive areas respectively. The color-coded is shown at the
left, where red is representing the most negative and the blue, the most positive values,
respectively. Intermediate negative and positive values are represented by colours that
downgrades from orange to light blue.
For the inactive compounds 13 and 17, the electronegative zone is totally missing (Figure 5). This is
an expected result considering that these compounds do not possess a double bond in such portion of
their structures.
Finally, we calculated logP (the logarithm of the partition coefficient in a biphasic system, e.g.,
n-octanol/water) for all drimanes which are shown in Table 2. It is known that LogP describes the
macroscopic hydrophobicity of a molecule, which is a factor related to its ability to penetrate
membranes of fungal cells and to reach the interacting sites, thus influencing the antifungal activity of
compounds [38]. LogP values for the different active drimanes of the series varied from 2.33 to 3.84,
which fall within the range of values observed for other antifungal compounds [31,39,40]. However,
although these values indicate that they all are lipophilic compounds [39,41], LogP have similar values
for drimanes with clear different activity, suggesting that this parameter would not have a direct

Molecules 2013, 18
2040
correlation with the antifungal activity of the compounds of Table 2 when acting against C. albicans
and C. neoformans.
Figure 5. Molecular Electrostatic Potential (MEPs) obtained for compounds 13 (a) and 17 (b).
Thus, the most active drimane 1 and its epimer 2, both possess the same LogP value = 2.33 yet their
activity is quite different. The same can be observed for compound 14 which has a LogP = 3.53 and
displays lower activities. This behavior of drimanes was dissimilar to that of sesquiterpene lactones,
for which a clear inverse relationship between polarity and antifungal activity was observed [28,29].
However, we must take into account that the sesquiterpene lactone skeletons are quite different from
those of the drimane sesquiterpenes. In addition, different fungal species were used as the test species
in each work; here, the yeasts C. albicans and C. neoformans were used, while in the previous reports
the filamentous fungus Cunninghamella echinulata [28] and Phycomyces blakesleeanus were used [29].
It has been often observed completely different behaviors for certain compounds towards different type
of fungi [42]. As the authors stated in [28], there is not much information about the chemical structure-
antifungal activity relationships for sesquiterpenoids and deeper studies should be necessary. In this
work, we add information that could be useful for a better knowledge of drimane sesquiterpenes
behavior against human opportunistic and pathogenic fungi.
3. Experimental
3.1. Chemistry
1
Melting points were determined in a Büchi melting point apparatus and are uncorrected. The H-
and 13C-NMR spectra were run on a Bruker DPX 300 spectrometer operating at 300 and 75 MHz,
respectively, using chloroform-d as solvent and tetramethylsilane as internal standard. The mass
spectra were scanned on a Hewlett Packard HP Engine-5989 spectrometer (equipped with a direct inlet
probe) operating at 70 eV.
3.1.1. Isolation of Natural Compounds 1–4
Compounds 1–4 were isolated from DCM extracts of leaves of P. acuminatum Kunth.
(Polygonaceae) collected on the bank of Coronda River at Puerto Gaboto, Santa Fe Province,

Molecules 2013, 18
2041
Argentina, in March 2010. The plant was identified by Prof. Susana Gattuso and a voucher specimen
was deposited at the Herbarium of the Vegetal Biology Area of UNR, Suipacha 531, Rosario,
Argentina (UNR # 2359). The extraction methodology and isolation of pure compounds was
performed according to reported procedures [1]. Compounds 1–4 were identified by micromelting
point, optical rotation and spectroscopic data, including 1H- and 13C-NMR [43–47] and were compared
with authentic samples obtained previously in our laboratory [1,48,49] for polygodial (1) and
isopolygodial (2) or with literature data [50,51], for drimenol (3) and for confertifolin (4) [44,52].
3.1.2. General Procedure for Synthesis of 5 and 6
To a magnetically well-stirred solution of 1 (2.1 mmol) in MeOH (25 mL) acidified with 5 drops of
1 M HCl, NaBH4 (8.4 mmol) was added carefully in small portions. The mixture was refluxed at
50 °C for 4 h and most of the solvent was evaporated in vacuo. Water (20 mL) was added cautiously
and extractions with EtOAc were performed. The organic phase was dried with MgSO4, evaporated
and submitted to column chromatography (CC) to give compounds 5 (1.3 mmol) and 6 (0.7 mmol) in
yields of 62% and 33%, respectively.
(−)-(5S,10S)-(9R)-7-Drimene-11,12diol (drimendiol) (5): White solid; mp: 74 °C; [α]D25 −8° (CHCl3, c
1
= 1.0). IR (film) max 3286, 2924 cm−1. H-NMR:  5.78 (1H, m, H-7); 4.33 (1H, d, J = 11.9 Hz,
H-12B); 4.00–3.90 (4H, m, H-11B, 12A and 2OH); 3.64 (1H, dd, J = 8.7 and 10.4 Hz, H-11A);
2.15–1.90 (4H, m, H-1β, 6α, 6β and 9); 1.58–1.40 (3H, m, H-2α, 2β and 3β); 1.24–1.10 (3H, m, H-1α,
13
3α and 5); 0.88 and 0.86 (6H, 2s, 13-Me and 14-Me); 0.75 (3H, s, 15-Me). C-NMR:  136.9 (C-8);
127.1 (C-7); 67.3 (C-12); 61.2 (C-11); 54.4 (C-5); 49.4 (C-9); 42.0 (C-3); 39.3 (C-1); 35.6 (C-10);
33.2 (C-13); 32.9 (C-4); 23.5 (C-6); 21.9 (C-14); 18.8 (C-2); 14.5 (C-15). MS m/z (EI): 238 [M+].
All these data are coincident with the literature [13,16].
25
(−)-(5aS,9aS,9bR)-6,6,9a-Trimethyl-1,3,5,5a,6,7,8,9,9a,9b-decahydronaphtho[1,2-c]furan (6): [α]D
−17° (CHCl3, c = 1.0), IR (film) max 2980, 1660, 1460, 1380, 1365 cm−1. H-NMR:  5.80 (1H, m,
1
H-4); 4.35 (1H, dd, J = 1.1 and 12.0 Hz, H-3β); 3.99 (1H, d, J = 12.0 Hz, H-3α); 3.91 (1H, dd, J = 2.1
and 10.9 Hz, H-1α); 3.68 (1H, dd, J = 8.3, 10.9 Hz, H-1β); 2.15 (1H, m, H-9b); 2.20–1.20 (9H, m,
H-9α and β, H-8α and β, H-7α and β, H-5α and β, H-5a); 0.90 and 0.88 (6H, 2s, 12-Me and 11);
0.77 (3H, s, 10-Me). 13C-NMR:  136.9 (C-3a), 127.4 (C-4); 67.4 (C-3); 61.4 (C-1); 54.4 (C-9b);
49.4 (C-5a); 42.0 (C-7); 39.3 (C-9); 35.6 (C-9a); 33.2 (C-11); 32.9 (C-6); 23.6 (C-5); 21.9 (C-10); 18.8
(C-8); 14.5 (C-12). MS m/z (EI): 220 [M+]. This is the first time that this compound is described in detail.
3.1.3. Synthesis of (−)-(1R,5aS,9aS,9bR)-6,6,9a-Trimethyl-1,3,5,5a,6,7,8,9,9a,9b-decahydro
naphto[2,1c]furan-1-ol (isodrimeninol, 7)
Compound 1 (0.55 mmol) was dissolved in MeOH (10 mL) acidified with 3 drops of 1 M HCl and
NaBH4 (0.27 mmol) was added carefully in small portions. The mixture was stirred during 30 min at
0 °C and then for 3 h at rt. Water (10 mL) was added cautiously and extractions with EtOAc were
performed. The organic phase was dried with MgSO4, evaporated and submitted to CC to give
25
compound 7 (0.39 mmol) in 71% yield. Colorless oil; [α]D −12° (CHCl3, c = 1.0); IR (film) max

Molecules 2013, 18
2042
1
3400, 1630, 1390, 1370, cm−1. H-NMR:  5.52 (1H, m, H-4); 5.29 (1H, brd, J = 2.2 Hz, H-1); 4.50
(1H, brd, J = 11.3 Hz, H-3β); 4.19 (1H, brd, J = 11.3 Hz, H-3α); 3.04 (1H, brs, -OH); 2.21 (1H, brd,
J = 2.2, H-9b); 2.13 (1H, m, H-5α); 1.92 (1H, m, H-5β); 1.80 (1H, ddd, J = 2.7, 4.7 and 13.2 Hz,
H-9β); 1.61 (1H, m, H-8β); 1.49 (2H, m, H-8α and 7β); 1.31 (3H, m, H-5a, 9α and 7α); 0.93; 0.90 (6H,
2s, 10-Me and 11); 0.83 (3H, s, 12-Me). 13C-NMR:  136.5 (C-3a), 117.1 (C-4), 99.4 (C-1),
68.9 (C-3), 61.6 (C-9b), 49.8 (C-5a), 42.4 (C-7), 39.8 (C-9), 33.4 (C-9a), 33.1 (C-11), 32.9 (C-6),
23.6 (C-5), 21.5 (C-10), 18.5 (C-8), 14.0 (C-12). MS m/z (EI): 236 [M+]. All these data agree with the
literature values [14].
3.1.4. Synthesis of (−)-(1R,4aS,8aS)-5,5,8a-Trimethyl-2-vinyl-1,4,4a,5,6,7,8,8a-octahydro naphtalene-
1-carbaldehyde (8) [11]
Compound 1 (2.14 mmol) was dissolved in anhydrous THF (5 mL), the solution was stirred at 0 °C
under nitrogen, and Tebbe´s reagent [(C5H5)2TiCH2ClAl(CH3)2] (4 mL of a 0.5M solution in toluene,
2.0 mmol) was added dropwise. The dark brown solution was allowed to warm to room temperature
for 10 min, 0.1 M NaOH solution was added (10 drops) and the slurry was filtered through a short pad
of silica gel. The filtrate was evaporated and purified by CC to give compound 8 as a colorless oil,
yield 25%; [α]D25 −17° (CHCl3, c = 1.0); IR (film) max 3090, 2720, 1719, 1643, 1607 cm−1. 1H-NMR:
 9.48 (1H, d, J = 5.1 Hz, CHO); 6.35 (1H, dd, J = 11.2 and 17.9 Hz, Hc); 6.14–6.11 (1H, m, H-3);
4.93 (1H, d, J = 11.2 Hz, Ha); 4.82 (1H, d, J = 17.9 Hz, Hb); 2.82 (1H, brs, H-1); 2.29–2.19 (2H, m,
H-4α and β); 1.87–1.81 (1H, m, H-8β); 1.54–1.15 (6H, m, H-8α, H-7α and β, H-6α and β, H-4a); 1.03
(3H, s, 8a-Me); 0.95 and 0.91 (6H, 2s, 5-Meα and β). 13C-NMR:  206.7 (CHO); 138.5 (=CH);
132.8 (C-3); 131.5 (C-2); 112.4 (=CH2); 62.6 (C-1); 48.9 (C-4a); 41.9 (C-6); 40.2 (C-8); 37.3 (C-8a);
33.3 (5-Me); 33.1 (C-5); 24.1 (C-4); 22.3 (5-Me); 18.1 (C-7); 15.5 (8a-Me). MS m/z (EI): 232 [M+].
All these data are coincident with the literature [13].
3.1.5. Synthesis of (+)-(5S,10S)-(9S)-7-Drimene-11,12diol (isodrimendiol, 9)
To a magnetically well-stirred solution of 2 (1.06 mmol) in MeOH (20 mL) acidified with 5 drops
of 1 M HCl, NaBH4 (4.2 mmol) was added carefully in small portions. The mixture was refluxed at
50 °C for 4 h and most of the solvent was evaporated in vacuo. Water (20 mL) was added cautiously
and extractions with EtOAc were performed. The organic phase was dried with MgSO4, evaporated
25
and submitted to CC to give compound 9 (0.83 mmol) as white solid, yield 78%, mp: 129 °C; [α]D
+10° (CHCl3, c = 1.0). IR (film) max 3286, 2924, 1640, 1045 cm−1. 1H-NMR:  5.83 (1H, t, J = 3.5 Hz,
H-7); 4.13 (1H, d, J = 12.0 Hz, H-12B); 4.00 (1H, d, J = 12.0 Hz, H-12A); 3.92 (1H, dd, J = 4.5 and
11.0 Hz, H-11B); 3.64 (1H, dd, J = 5.4 and 11.0 Hz, H-11A); 2.60 (2H, brs, 2OH); 2.20–1.90 (3H, m,
H-6α and β, H-9β); 1.67–1.20 (7H, m, H-1α and β, H-2α and β, H-3α and β, H-5); 0.92 (6H, 2s, 15-Me
13
and 14-Me); 0.88 (3H, s, 13-Me). C-NMR:  137.2 (C-8); 127.5 (C-7); 67.7 (C-12); 63.1 (C-11);
54.1 (C-5); 43.4 (C-9); 42.7 (C-3); 36.5 (C-1); 35.9 (C-10); 33.1 (C-4); 33.0 (C-15); 24.3 (C-6); 22.0
(C-14); 21.7 (C-13); 18.8 (C-2). MS m/z (EI): 238 [M+]. All these data are in agreement with the
literature values [16].

Molecules 2013, 18
2043
3.1.6. Synthesis of (+) [(1R,4aS,8aS)-2,5,5,8a-Tetramethyl-1,4,4a,5,6,7,8,8a-octahydro naphthalen-1-
yl]methanol (isodrimenol, 10)
Diol 9 (0.34 mmol) was dissolved in DCM (15 mL) and catalytic quantities of Pd/C (10% Pd) were
added. The mixture was left under a hydrogen atmosphere during 4 h and the reaction product was
filtered, concentrated and purified by CC. Compound 10 (0.29 mmol) was thus obtained in 85% yield;
25
1
[α]D +10° (CHCl3, c = 1.0); IR (film) max 3405, 2922, 1620 cm-1; H-NMR:  5.57 (1H, m, H-7);
3.74 (2H, brd, J = 3.0, H-11A and B); 1.74 (3H, brs, 12-Me); 2.10–1.20 (10H, m, H-1α and β, H-2α
and β, H-3α and β, H-5, H-6α and β, H-9); 0.93 (3H, s, 15-Me); 0.91 and 0.87 (6H, 2s, 13-Me and
14-Me). 13C-NMR:  131.1 (C-8); 124.6 (C-7); 61.3 (C-11); 57.6 (C-5); 43.4 (C-9); 42.7 (C-3);
36.8 (C-1); 36.1 (C-10); 33.2 (C-13); 33.0 (C-4); 24.0 (C-6); 23.0 (C-14); 22.2 (C-12); 21.7 (C-2);
18.8 (C-15). MS m/z (EI): 222 [M+]. All these data are in agreement with the literature [14,17].
3.1.7. Synthesis of (−)-7α,8-Epoxy-(9S)-drimane-11,12-diol (11) [20]
To a solution of diol 9 (4.2 mmols) in CH2Cl2 (30 mL) MCPBA acid (5.4 mmol) was added in
small portions at rt during 10 min. Stirring was continued at rt for 35 min. The reaction mixture was
washed with NaHCO3 and water, dried and concentrated. The residue was chromatographed over silica
gel to afford compound 11 (75% yield); mp: 105 °C; [α]D25 −69° (CHCl3, c = 1.0); IR (film) max 3280,
1
3120, 3050 cm−1. H-NMR:  3.95–3.45 (4H, m, H-11A and B, H-12A and B); 3.36 (1H, H-7);
2.1 (1H, dd, J = 4.5 and 15.3 Hz, H-9); 1.74–1.64 (2H, m, H-6α and β); 1.61–1.10 (7H, m H-1α and β,
H-2α and β, H-3α and β, H-5); 0.96 (3H, s, 15-Me); 0.89 and 0.86 (6H, 2s, 13-Me and 14-Me).
13C-NMR:  66.6 (C-12); 62.0 (C-8); 60.6 (C-11); 56.8 (C-7); 48.3 (C-9); 42.4 (C-3); 39.0 (C-5);
36.2 (C-1); 34.9 (C-10); 32.9 (C-15); 32.8 (C-4); 22.8 (C-6); 22.7 (C-14); 22.2 (C-13); 18.3 (C-2). MS
m/z (EI): 254 [M+]. All these data are in agreement with the literature [20].
3.1.8. Synthesis of (−)-(9S)-Drimane-8,11,12-triol (12) [20]
To a stirred mixture of LiAlH4 (16 mmol) in dry THF (50 mL), a solution of the epoxide 11
(4.5 mmol) in dry THF (30 mL) was added and the mixture was heated at reflux temperature under
nitrogen for 4 h. The excess of reagent was decomposed by the addition of EtOAc and an aqueous
solution of HCl (10%). The mixture was extracted with EtOAc and the organic phase was washed with
NaHCO3 and water, dried and concentrated to give compound 12 (85% yield); mp: 118 °C; [α]D25 −41°
1
(CHCl3, c = 1.0); IR (film) max 3400, 3280, 3120 cm−1. H-NMR:  4.0 (2H, m, H-12A and B);
3.83–3.60 (3H, m, H-11A and B and 1OH); 3.33 (1H, brs, 1OH); 2.58 (1H, brs, 1OH); 1.89 (1H, m,
H-9); 1.80–1.10 (11H, m, H-1α and β, H-2α and β, H-3α and β, H-5, H-6α and β, H-7α and β);
1.05 (3H, s, 15-Me); 0.87 and 0.80 (6H, 2s, 13-Me and 14-Me). 13C-NMR:  76.4 (C-8); 69.5 (C-12);
60.9 (C-11); 56.9 (C-5); 48.6 (C-9); 42.1 (C-3); 37.6 (C-10); 37.4 (C-1); 33.4 (C-4); 33.1 (C-7);
33.0 (C-15); 23.7 (C-14); 21.8 (C-13); 20.0 (C-6); 18.8 (C-2). MS m/z (EI): 256 [M+]. All these data
are in agreement with the literature [20].

Molecules 2013, 18
2044
3.1.9. Synthesis of (−)-11-Hydroxy-(9R)-12-nordriman-8-one (13) [20]
To a stirred solution of NaIO4 (3.53 mmol) in water (15 mL), a solution of the triol 12 (2.54 mmol)
in MeOH (10 mL) was added at rt during 1.5 h. The reaction solution was extracted with EtOAc and
the organic phase was washed with NaHCO3 and water, dried and concentrated to give compound 13
25
1
(90% yield); mp: 112 °C; [α]D −22° (CHCl3, c= 1.0); IR (film) max 3280, 1720 cm−1. H-NMR: 
3.98 (1H, brd, J = 10 Hz, H-11A); 3.91 (1H, brd, J = 10 Hz, H-11B); 2.36–2.09 (2H, m, 2H-7); 2.37
(1H, dd, J = 4.3 and 1.8 Hz, H-9β); 2.11 (1H, ddd, J = 1.6, 6.2 and 9.8 Hz, H-7); 1.95 (1H, m, H-7);
1.70–1.16 (9H, m, H-1α and β, H-2α and β, H-3α and β, H-5, H-6α and β); 0.94 (3H, s, 15-Me); 0.92
13
and 0.84 (6H, 2s, 13-Me and 14-Me). C-NMR:  214.8 (C-8); 67.0 (C-9); 60.1 (C-11); 46.0 (C-5);
42.2 (C-3); 40.0 (C-10); 39.3 (C-1); 36.8 (C-7); 33.5 (C-15); 33.4 (C-4); 23.2 (C-6); 22.2 (C-13); 22.0
(C-14); 18.5 (C-2). All these data agree with the literature values [20].
3.1.10. General Procedure for Synthesis of 14 and 15 [13,16]
A magnetically stirred and cold (0 °C) solution of the alcohol substrate 3 (10 mmol) in acetone
(50 mL) was titrated with Jones reagent, until the orange-brown color of the reagent persisted for 30
seconds. The reaction mixture was stirred for an additional 1 h at rt, and then isopropanol (1 mL) was
added dropwise to destroy any excess of reagent. Water (100 mL) was added, and the suspension was
extracted with AcOEt, dried with MgSO4, concentrated, and purified by column chromatography to
give compounds 14 (0.68 mmol) and 15 (0.85 mmol) with yields of 30 and 38%, respectively.
(−)-(1S,8aS)-2,5,5,8a-Tetramethyl-1,4,4a,5,6,7,8,8a-octahydronaphthalene-1-carbaldehyde (drimenal,
25
14): colorless oil, [α]D −20° (CHCl3, c = 1.0); IR (film) max 2923, 2851, 1714 cm−1.
1H-NMR:  9.69 (1H, d, J = 5.1 Hz, H-11); 5.70 (1H, brs, H-7); 2.59 (1H, m, H-9); 2.05 (2H, m, H-6α
and β); 1.70–1.10 (7H, m, H-1α and β, H-2α and β, H-3α and β, H-5); 1.62 (3H, s, 12-Me); 1.05 (3H, s,
15-Me); 0.90 and 0.85 (6H, 2s, 13-Me and 14-Me). 13C-NMR:  206.7 (C-11); 127.8 (C-8); 125.5
(C-7); 67.6 (C-9); 49.1 (C-5); 42.0 (C-3); 40.4 (C-1); 37.0 (C-10); 33.3 (C-14); 33.0 (C-4); 23.7 (C-6);
22.1 (C-13); 21.6 (C-12); 18.3 (C-2); 15.7 (C-15). MS m/z (EI): 220 [M+]. All these data are in
agreement with the literature [16,21].
(−)-(4aS,8aS)-2,5,5,8a-Tetramethyl-4a,5,6,7,8,8a-hexahydro-4H-naphtalen-1-one (15): white solid;
25
1
mp: 81 °C; [α]D −74° (CHCl3, c= 1.0); IR (film) max 2923, 1663 cm−1. H-NMR:  6.69–6.67 (1H,
m, H-3); 2.30–2.25 (2H, m, H-4α and β); 1.89 (1H, brd, J = 13.4 Hz, H-8β); 1.75 (3H, d, J = 2.0 Hz,
Me-2); 1.70–1.10 (6H, m, H-8α, H-7α and β, H-6α and β, H-4a); 1.04 (3H, s, Me-8a); 0.99 and 0.91
13
(6H, 2s, Me-5α and β). C-NMR:  205.9 (C-1); 143.4 (C-3); 132.9 (C-2); 49.4 (C-4a); 45.1 (C-8a);
41.6 (C-6); 33.6 (C-5); 33.2 (C-8); 32.3 (Me-5); 24.4 (C-4); 22.3 (Me-5); 18.2 (C-7); 17.1 (Me-8a);
16.4 (Me-2). MS m/z (EI): 206 [M+]. All these data are in agreement with the literature [13,16,22].
3.1.11. Synthesis of (−)-(4aS,8aS)-3,4a,8,8-Tetramethyl-4a,5,6,7,8,8a-hexahydro-1H-naphtalen-2-one
(16) [14,17]
To a magnetically stirred solution of the alcohol substrate 3 (10 mmol) in DCM (50 mL),
pyridinium chlorochromate (10 mmol) was added. The brown suspension was stirred overnight, until

Molecules 2013, 18
2045
thin layer chromatography analysis showed the disappearance of the starting material. Celite (500 mg)
and EtOAc (50 mL) were added to the reaction mixture, and the slurry was filtered through a short pad
of silica gel, washing copiously with EtOAc. The filtrate was dried, evaporated, and purified by
column chromatography to give compound 16 (1.2 mmol) with 60% yield as a colorless oil; [α]D25 −7°
1
(CHCl3, c = 1.0); IR (film) max 2956, 1704, 1673 cm−1. H-NMR:  6,39 (1H, d, J = 1.3 Hz, H-4);
2.49 (1H, dd, J = 3.9 and 17.4 Hz, H-1α); 2.34 (1H, dd, J = 14.0 and 17.4 Hz, H-1β); 1.72 (3H, d,
J = 1.3 Hz, Me-3); 1.72 (1H, brdd, J = 13.9 and 3.9 Hz, H-5β); 1.70–1.10 (6H, m, H-5α, H-6α and β,
13
H-7α and β, H-8a); 1.07 (3H, s, Me-4a); 0.91 (3H, s, Me-8); 0.88 (3H, s, Me-8). C-NMR:  201.6
(C-2); 158.3 (C-4); 131.2 (C-3); 50.6 (C-8a); 41.2 (C-7); 38.4 (C-5); 36.9 (C-4a); 35.4 (C-1);
32.8 (C-8); 32.2 (Me-8); 20.9 (Me-8); 18.6 (C-6); 18.5 (Me-4a); 15.5 (Me-3). MS m/z (EI): 206 [M+].
All these data are in agreement with the literature [13,16,22].
3.1.12. Synthesis of (+) [(1S,2S,8aS)-2,5,5,8a-Tetramethyldecahydronaphthalen-1-yl]methanol
(drimanol, 17)
Drimenol (3, 0.36 mmol) was dissolved in DCM (10 mL) and catalytic quantities of Pd/C (10% Pd)
were added. The mixture was left under a hydrogen atmosphere during 4 h and the reaction product
was filtered, concentrated and purified by CC to obtain compound 17 (75% yield); [α]D25 +15° (CHCl3,
1
c = 1.0); IR (film) max 3620, 2950, 1380 cm−1. H-NMR:  3.86 (1H, dd, J = 4.5 and 10.7 Hz,
H-11B); 3.61 (1H, dd, J = 4.5 and 10.7 Hz, H-11A); 2.17 (1H, m, H-8); 1.65–1.10 (12H, m, H-1α and
β, H-2α and β, H-3α and β, H-5, H-6α and β, H-7α and β, H-9); 0.98 (3H, d, J = 7.5 Hz, 12-Me); 0.88
13
and 0.83 (9H, 3s, 13-Me and 15-Me). C-NMR:  61.0 (C-11); 56.5 (C-5); 55.7 (C-9); 41.9 (C-3);
39.9 (C-1); 37.6 (C-10); 34.5 (C-7); 33.6 (C-14); 33.2 (C-4); 28.5 (C-8); 21.6 (C-13); 18.4 (C-6);
17.5 (C-2); 17.1 (C-15), 15.6 (C-12). MS m/z (EI): 224 [M+]. All these data are in agreement with the
literature [23].
3.2. Antifungal Evaluation
3.2.1. Microorganisms and Media
For the antifungal evaluation, standardized strains from the American Type Culture Collection
(ATCC), Rockville, MD, USA, and CEREMIC (CCC), Centro de Referencia en Micología, Facultad
de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531 (2000)-Rosario, Argentina, were used in a
first instance screening: C. albicans ATCC 10231, S. cerevisiae ATCC 9763, C. neoformans ATCC
32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029, T. rubrum CCC 110,
T. mentagrophytes ATCC 9972, and M. gypseum CCC 115.
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 of cells or spore suspensions were obtained according to reported
procedures and adjusted to 1–5 × 103 cells/spores with colony forming units (CFU)/mL [11,12].

Molecules 2013, 18
2046
3.2.2. Antifungal Susceptibility Testing
Minimum inhibitory concentration (MIC) of each compound was determined by using broth
microdilution techniques according to the guidelines of the CLSI for yeasts (M27-A3) and for
filamentous fungi (M 38 A2) [11,12]. MIC values were determined in RPMI-1640 (Sigma, St. Louis,
MO, USA) buffered to pH 7.0 with MOPS. Microtiter trays were incubated at 35 °C for yeasts and
hialohyphomycetes and at 28–30 °C for dermatophytes strains in a moist, dark chamber, and MICs
were visually recorded at 48 h for yeasts, and at a time according to the control fungus growth, for the
rest of fungi.
For the assay, stock solutions of pure compounds were two-fold diluted with RPMI from 250 to
0.98 µg/mL (final volume = 100 µL) and a final DMSO concentration ≤1%. A volume of 100 µL of
inoculum suspension was added to each well with the exception of the sterility control where sterile
water was added to the well instead. Ketoconazole, terbinafine and amphotericin B were used as
positive controls.
Endpoints were defined as the lowest concentration of drug resulting in total inhibition (MIC100) of
visual growth compared to the growth in the control wells containing no antifungal. MIC50 was defined
as the lowest concentration of a compound that inhibited 50% of the growth control, respectively
(culture media with the microorganism but without the addition of any compound), and was
determined spectrophotometrically with the aid of a VERSA Max microplate reader (Molecular
Devices, Sunnyvale, CA, USA).
The Minimum Fungicidal Concentration (MFC) of each compound against each strain was also
determined as follows: After determining the MIC100, an aliquot of 5 µL sample was withdrawn from
each clear well of the microtiter tray and plated onto a 150-mm RPMI-1640 agar plate buffered with
MOPS (Remel, Lenexa, KS, USA.). Inoculated plates were incubated at 30 °C, and MFCs were
recorded after 48 h. The MFC was defined as the lowest concentration of each compound that resulted
in total inhibition of visible growth.
3.3. Calculations Methods
The DFT calculations, full geometry optimization and calculation of the harmonic vibrational
frequencies were performed using the GAUSSIAN 03 program [53]. Density Functional Theory (DFT)
calculations were employed in order to properly account for the electron correlation effects. The
widely employed hybrid method denoted by B3LYP was used, along with the double-zeta split valence
basis set 6-311G(d,p). This method includes a mixture of HF and DFT exchange terms and the
gradient corrected correlation functional of Lee, Yang and Parr [54,55], as proposed and parameterized
by Becke [56,57].
A preliminary conformational search was carried out using the GASCOS algorithm [58–60]
combined with PM6 an RHF/3-21G calculations. All the conformations obtained using this calculations
were confirmed from B3LYP/6311G(d, p) computations. With any conformational search, it is very
important to examine the structures obtained to make sure that they are true minima and not transition
structures or other structures with very low or zero forces on the atoms (stationary points). Thus, minima
were characterized through harmonic frequency analysis employing B3LYP/6311G(d,p) calculations.

Molecules 2013, 18
2047
The electronic studies were carried out using molecular electrostatic potentials (MEPs) [61]. The
low-energy conformations were obtained from our conformational search. Subsequently, single point
calculations were carried out. Thus, these MEPs were calculated using B3LYP/6-311G++(d,p) wave
functions and MEPs graphical presentations were created using the MOLEKEL program [62].
4. Conclusions
Results obtained here were aimed at contributing to clear up the main structural features necessary
for the antifungal activity of drimanes (that have been the subject of contradictory statements in the
literature) by testing them against a unique panel of fungal strains, with a standardized methodology, in
the same laboratory.
Regarding the absolute configuration of C-9, here we found that compounds with an aldehyde in
any of the two possible configurations did possess antifungal activity against C. albicans and
C. neoformans, although the 9-derivative 2 displays 16 and eight times lower activity than the 9-one
1. This result is in contrast with Fujita and Kubo [7], but consistent with Anke and Sterner’s [8] and
Derita et al.’s [2] previous studies, who both found that 2 did display antifungal activities but lower
than those of compound 1.
Another result demonstrated that aldehydes at C-9 or C-8 or a CH2OH at C-8 would not be a
requisite for activity. In addition, this work showed that the 7,8-double bond in the drimane scheleton
should be considered one of the necessary structural features for antifungal activity, either alone as in
compounds 3, 5–10 and 14, or as part of a C-8 α,β-unsaturated aldehyde moiety (constituting a
Michael acceptor group) as in compounds 1 and 2. The fact that both drimane structural types do
display antifungal activity clearly suggested that antifungal drimanes would not act via a Michael
addition. This finding differs from Taniguchi’s report [10]. However, as explained in the Introduction,
the statement of Taniguchi et al. in [10] contradicted their own previous report [7] in which 2
was shown to be devoid of antifungal activity, although it possessed the ,-unsaturated C-8 aldehyde
moiety. In addition, results obtained here showed that the electronic characteristics of drimanes play an
important role in their antifungal behavior. Thus, we found that the electronic distribution in the
vicinity of the 7,8 is important for activity and also we found that the most active compound 1
possessed a differential big positive zone in the MEP which could contribute to its high antifungal
activity. Regarding the influence of the differential hydrophobocity of compounds in the antifungal
behavior, results showed that variations in LogP values were not reflected in a concomitant variation
of activity.
Acknowledgements
SZ acknowledges ANPCyT PICT-2010 0608 and UNR BIO 258. RDE acknowledges ANPCyT,
PICT 2010-1832. Grants from Universidad Nacional de San Luis (UNSL) partially supported this
work. RDE and FG are members of the Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET-Argentina) staff.

Molecules 2013, 18
2048
References
1. Del Vitto, L.; Petenatti, E.; Petenatti, M. Recursos herbolarios de San Luis (Argentina) 2a parte:
Plantas exóticas cultivadas, adventicias y/o naturalizadas. Multequina 1998, 7, 29–48.
2. Derita, M.; Leiva, M.; Zacchino, S. Influence of plant part, season of collection and content of the
main active constituent, on the antifungal properties of Polygonum acuminatum Kunth.
J. Ethnopharmacol. 2009, 124, 377–383.
3. Mc Callion, R.; Cole, A.; Walker, J.; Blunt, J.; Munro, M. Antibiotic substances from New
Zealand plants. Planta Med. 1982, 44, 134–138.
4. De Almeida Alves, T.; Ribeiro, F.; Kloos, H.; Zani, C. Polygodial, the fungitoxic component from
the Brazilian medicinal plant Polygonum punctatum. Mem. Inst. Oswaldo Cruz. 2001, 96, 831–833.
5. Taniguchi, M.; Adachi, T.; Oi, S.; Kimura, A.; Katsumura, S.; Isoe, S.; Kubo, I. Structure-activity
relationship of the Warburgia sesquiterpene dialdehydes. Agric. Biol. Chem. 1984, 48, 73–78.
6. Lee, S.; Lee, J.; Lunde, C.; Kubo, I. In vitro antifungal susceptibilities of Candida albicans and
other fungal pathogens to polygodial, a sesquiterpene dialdehyde. Planta Med. 1998, 65, 204–208.
7. Fujita, K.; Kubo, I. Multifunctional action of antifungal polygodial against Saccharomyces
cerevisiae: Involvement of pyrrole formation on cell surface in antifungal action. Bioorg. Med. Chem.
2005, 13, 6742–6747.
8. Anke, H.; Sterner, O. Comparison of the antimicrobial and cytotoxic activities of twenty
unsaturated sesquiterpene dialdehydes from plants and mushrooms. Planta Med. 1991, 57, 344–347.
9. Derita, M.; Zacchino, S. Validation of the ethnopharmacological use of Polygonum persicaria for
its antifungal properties. Nat. Prod. Commun. 2011, 6, 931–933.
10. Taniguchi, M.; Yano, Y.; Tada, E.; Ikenishi, K.; Oi, S.; Haraguchi, H.; Hashimoti, K.; Kubo, I.
Mode of action of polygodial, an antifungal sesquiterpene dialdehyde. Agric. Biol. Chem. 1988,
52, 1409–1414.
11. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution
Antifungal Susceptibility Testing for Yeasts (M27 A3), 3rd ed.; CLSI: Wayne, PA, USA, 2008;
Volume 28, No. 14, pp. 1–25.
12. Clinical and Laboratory Standards Institute (CLSI). Reference Method for Broth Dilution
Antifungal Susceptibility Testing for and for Filamentous Fungi (M38 A2), 2nd ed.; CLSI: Wayne,
PA, USA, 2008; Volume 28, No. 16, pp. 1–35.
13. Cuellar, M.; Salas, C.; Cortés, M.; Morello, A.; Maya, J.; Preite, M. Synthesis and in vitro
trypanocide activity of several drimane-quinone derivatives. Bioorg. Med. Chem. 2003, 11,
2489–2497.
14. Cuellar, M.; Moreno, L.; Preite, M. Regioselective oxidative fragmentation of drimanic terpene
alcohols: a short, easy and efficient access to natural and synthetic 11-nordrimane terpene
derivatives. ARKIVOC 2003, 10, 169–177.
15. Sakio, Y.; Hirano, Y.; Hayashi, M.; Komiyama, K.; Ishibashi, M. Dendocarbins A-N, new
drimane sesquiterpenes from the nudibranch Dendrodoris carbunculosa. J. Nat. Prod. 2001, 64,
726–731.

Molecules 2013, 18
2049
16. Matsuda, H.; Pongpiriyadacha, Y.; Morikawa, T.; Kashima, Y.; Nakano, K.; Yoshikawa, M.
Protective effects of polygodial and related compounds on ethanol-induced gastric mucosal
lesions in rats: Structural requirements and mode of action. Bioorg. Med. Chem. Lett. 2002, 12,
477–482.
17. Cuellar Fritis, M. Synthesis of sesquiterpenquinones and aza-anthraquinones derivatives using
polygodial as chiral synthon. Doctoral Thesis, Pontificia Universidad Católica de Chile, Santiago,
Chile, 2002; p. 155.
18. Feng, J.; Yang, C.; Zhang, D.; Wang, J.; Fu, H.; Chen, H.; Li, X. Catalytic transfer hydrogenolysis
of α-methylbenzyl alcohol using palladium catalysts and formic acid. Appl. Catal. A Gen. 2009,
354, 38–43.
19. Donelly, D.; O´Reilly, J.; Chiaroni, A.; Polonsky, J. Crystal structure and absolute configuration
of a new sesquiterpenoid metabolite of Fomes annosus, 7α,8β,11-trihydroxydrimane. Chem. Soc.
Perkin Trans. 1 1980, 2196–2199.
20. González Sierra, M.; Colombo, M.; Zudenigo, M.; Rúveda, E. 13C NMR spectral analysis of
grindelane diterpenoid acids. Phytochemistry 1984, 23, 1685–1689.
21. Cortés, M.; Moreno, L.; López, J. Partial Synthesis of (−)-11,12-dinordriman-8-one and the (−)-
enantiomer of polywood. J. Chem. Res. (S) 1998, 36–37.
22. Scher, J.; Speakman, J.; Zapp, J.; Becker, H. Bioactivity guided isolation of antifungal compounds
from the liverwort Bazzania trilobata (L.) S. F. Gray. Phytochemistry 2004, 65, 2583–2588.
23. Moreno-Osorio, L.; Espinoza, L.; Cuellar, M.; Preite, M. LTA-mediated synthesis and complete
1
assignment of H and 13C NMR data of two natural 11-nordrimanes: Isonordrimenone and
polygonone. Magn. Reson. Chem. 2007, 45, 993–996.
24. Hellou, J.; Andersen, R. Terpenoids from the dorid nudibranch Cadlina luteomarginata.
Tetrahedron 1982, 38, 1875–1879.
25. Zhang, L.; Yan, K.; Zhang, Y.; Huang, R.; Bian, J.; Zheng, Ch.; Sun, H.; Chen, Z.; Sun, N.;
An, R.; et al. High-throughput synergy screening identifies microbial metabolites as combination
agents for the treatment of fungal infections. Proc. Natl. Acad. Sci. USA 2007, 104, 4606–4011.
26. Singh, N. Impact of current transplantation practices on the changing epidemiology of infections
in transplant recipients. Lancet Infect. Dis. 2003, 3, 156–161.
27. Pfaller, M.; Diekema, D. Epidemiology of invasive candidiasis: A persistent public health
problem. Clin. Microbiol. Rev. 2007, 20, 133–163.
28. Barrero, A.; Oltra, J.E.; Alvarez, M.; Raslan, D.; Saúde, D.; Akssira, M. New sources and
antifungal activity of sesquiterpene lactones. Fitoterapia 2000, 71, 60–64.
29. Justicia, J.; Álvarez de Cienfuegos, L.; Estévez, R.; Paradas, M.; Lasanta, A.; Oller, J.; Rosales,
A.; Cuerva, J.; Oltra, E. Ti-catalyzed transannular cyclization of epoxygermacrolides. Synthesis of
antifungal (+)-tuberiferine and (+)-dehydrobrachylaenolide. Tetrahedron 2008, 64, 11938–11943.
30. Vargas L.; Castelli, M.V.; Kouznetsov, V.; Urbina J.; López, S. Sortino, M.; Enriz, R.; Ribas,
J.C.; Zacchino, S. In vitro antifungal activity of new series of homoallylamines and related
compounds with inhibitory properties of the synthesis of fungal cell wall polymers.
Bioorg. Med. Chem. 2003, 11, 1531–1550.
31. Musiol, R.; Jampilek, J.; Buchta, V.; Silva, L.; Niedbala, H.; Podeszwa, B.; Palka, A.;
Majerz-Maniecka, K.; Oleksyn, B.; Polanski, J. Antifungal properties of new series of quinoline
derivatives. Bioorg. Med. Chem. 2006, 14, 3592–3598.

Molecules 2013, 18
2050
32. Setiadi, D.; Chass, G.; Torday, L.; Varro, A.; Papp, J. Vitamin E models. Conformational analysis
and stereochemistry of tetralin, chroman, thiochroman and selenochroman. J. Mol. Struct.
(Theochem.) 2002, 594, 161–172.
33. Suvire, F.; Andreu, I.; Bermejo, A.; Zamora, M.; Cortés, D.; Enriz, R. Conformational study of
N-alkyl-benzyltetrahydroisoquinolines alkaloid. J. Mol. Struct. (Theochem.) 2003, 666, 109–116.
34. Villagra, S.; Bernini, M.; Rodrıguez, A.; Zacchino, S.; Kouznetsov, V.; Enriz, R. Conformational
and electronic study of homoallylamines with inhibitory properties against polymers of fungal cell
wall. J. Mol. Struct. (Theochem.) 2003, 666, 587–598.
35. Politzer, P.; Truhlar, D. Chemical Applications of Atomic and Molecular Electrostatic Potentials;
Plenum Publishing: New York, NY, USA, 1981.
36. Carrupt, P.; El Tayar, N.; Karlén, A.; Festa, B. Molecular electrostatic potentials for
characterizing drug-biosystem interactions. Methods Enzymol. 1991, 202, 638–650.
37. Greeling, P.; Langenaeker, W.; De Proft, F.; Baeten, A. Molecular Electrostatic Potentials:
Concepts and Applications. Theoretical and Computational Chemistry; Elsevier Science B.V.:
Amsterdam, The Netherlands, 1996; p. 587.
38. Voda, K.; Boh, B.; Vrtacnik, M. A quantitative structure–antifungal activity relationship study of
oxygenated aromatic essential oil compounds using data structuring and PLS regression analysis
J. Mol. Model. 2004, 10, 76–84.
39. Leal, P.; Mascarello, A.; Derita, M.; Zuljan, F.; Nunes, R.; Zacchino, S.; Yunes, R. Relation
between lipophylicity of alkyl gallates and antifungal activity against yeasts and filamentous
fungi. Bioorg. Med. Chem. Lett. 2009, 19, 1793–1796.
40. Meléndez Gómez, C.; Kouznetsov, V.; Sortino, M.; Alvarez, S.; Zacchino, S. In vitro antifungal
activity of polyfunctionalized 2-(hetero)arylquinolines prepared through imino Diels Alder
reactions. Bioorg. Med. Chem. 2008, 16, 7908–8920.
41. Barfknecht, C.; Nichols, D. Correlation of psychotomimetic activity of phenetylamines and
amphetamines with 1-octanol-water partition coefficients. J. Med. Chem. 1975, 18, 208–210.
42. López, S.; Castelli, M.; Zacchino, S.; Domínguez, J.; Lobo, G.; Charris-Charris, J.; Cortés, J.;
Ribas, J.; Devia, C.; Rodríguez, A.; Enriz, R. In vitro antifungal evaluation and structure–activity
relationships of a new series of chalcone derivatives and synthetic analogues, with inhibitory
properties against polymers of the fungal cell wall. Bioorg. Med. Chem. 2001, 9, 1999–2013.
43. Jansen, B.; de Groot, A. Occurrence, biological activity and synthesis of drimane
sesquiterpenoids. Nat. Prod. Rep. 2004, 21, 449–477.
44. Appel, H.; Bond, R.; Overton, K. The constitution and stereochemistry of valdiviolide, fuegin,
winterin and futronolide. Tetrahedron 1963, 19, 635–641.
45. Ying, B.; Peiser, G.; Ji, Y.; Mathias, K.; Tutko, D.; Hwang, Y. Phytotoxic sesquiterpenoids from
Canella winterana. Phytochemistry 1995, 38, 909–915.
46. Barnes, C.; Loder, J. The structure of polygodial: A new sesquiterpene dialdehyde from
Polygonum hydropiper L. Austr. J. Chem. 1962, 15, 322–327.
47. Guillerm, D.; Delarue, M.; Jalali–Naini, M.; Lemaître, P.; Lallemand, J-Y. Synthesis of all
possible stereoisomers of polygodial. Tetrahedron Lett. 1984, 25, 1043–1048.

Molecules 2013, 18
2051
48. Castelli, V.; Lodeyro, A.; Malheiros, A.; Zacchino, S.; Roveri, O. Inhibition of the mitochondrial
ATP synthesis by polygodial, a naturally occurring dialdehyde unsaturated sesquiterpene.
Biochem. Pharmacol. 2005, 70, 82–89.
49. Malheiros, A.; Cechinel Filho, V.; Schmitt, C.; Yunes, R.; Escalante, A.; Svetaz, L.; Zacchino, S.;
Delle Monache, F. Antifungal activity of drimane sesquiterpenes from Drimys brasiliensis using
bioassay-guided fractionation. J. Pharm. Pharm. Sci. 2005, 8, 335–339.
50. Rodriguez, B.; Zapata, N.; Medina, P.; Viñuela, E. A complete 1H and 13C NMR data assignment
for four drimane sesquiterpenoids isolated from Drimys winterii. Magn. Reson. Chem. 2005, 43,
82–84.
51. Urban, S.; Capon, R. Absolute stereochemistry of puupehenone and related metabolites.
J. Nat. Prod. 1996, 59, 900–901.
52. Hueso-Rodriguez, J.; Rodríguez, B. A new and efficient route to optically active drimanes.
Synthesis of (+)-winterin, (+)-confertifolin, (+)-isodrimenin and (+)-bicyclofarnesol. Tetrahedron
1989, 45, 1567–1576.
53. Frisch, M.; Trucks, G.; Schlegel, H.; Scuseria, G.; Robb, M.; Cheeseman, J.; Montgomery, J., Jr.;
Vreven, T.; Kudin, K.; Burant, J.; et al. Gaussian 03, Revision B.05; Gaussian, Inc.: Pittsburgh,
PA, USA, 2003.
54. Lee, C.; Yang, W.; Parr, R. Development of the Colle-Salvetti correlation-energy formula into a
functional of the electron density. Phys. Rev. B 1988, 37, 785–789.
55. Miehlich, B.; Savin, A.; Stoll, H.; Preuss, M. Results obtained with the correlation energy density
functionals of Becke and Lee, Yang and Parr. Chem. Phys. Lett. 1989, 157, 200–207.
56. Becke, A. Density-functional exchange-energy approximation with correct asymptotic behavior.
Phys. Rev. A 1988, 38, 3098–3100.
57. Becke, A. Density-functional thermochemistry. III. The role of exact exchange. J. Chem. Phys.
1993, 98, 5648–5652.
58. Santagata, L.; Suvire, F.; Enriz, R.; Torday, L.; Csizmadia, I. A geometrical algorithm to search
the conformational space of flexible molecules. J. Mol. Struct. (Theochem.) 1999, 465, 33–67.
59. Santagata, L.; Suvire, F.; Enriz, R. An analytic ring closure condition for geometrical algorithm to
search the conformational space. J. Mol. Struct. (Theochem.) 2000, 507, 89–95.
60. Santagata, L.; Suvire, F.; Enriz, R. Partially relaxed ring closure conditions for geometrical
algorithm to search the conformational space for minimum energy conformations. J. Mol. Struct.
(Theochem.) 2001, 536, 173–188.
61. Politzer, P.; Daiker, K. The Force Concept in Chemistry; Deb, M.B., Ed.; van Nostrand Reinhold:
New York, NY, USA, 1981.
62. Flükiger, P.; Lüthi, H.; Portmann, S.; Weber, J. MOLEKEL 4.0; Swiss Center for Scientific
Computing: Manno, Switzerland, 2000.
Sample Availability: Samples of the compounds 1–4 are available from the authors.
© 2013 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).