(+)-epi-Epoformin, a Phytotoxic Fungal Cyclohexenepoxide: Structure Activity Relationships

Article abstract of DOI:10.3390/molecules23071529

(+)-epi-Epoformin (1), is a fungal cyclohexene epoxide isolated together with diplopimarane and sphaeropsidins A and C, a nor-ent-pimarane and two pimaranes, from the culture filtrates of Diplodia quercivora, a fungal pathogen for cork oak in Sardinia, Italy. Compound 1 possesses a plethora of biological activities, including antifungal, zootoxic and phytotoxic activity. The last activity and the peculiar structural feature of 1 suggested to carry out a structure activity relationship study, preparing eight key hemisynthetic derivatives and the phytotoxicity was assayed. The complete spectroscopic characterization and the activity in the etiolated wheat coleoptile bioassay of all the compounds is reported. Most of the compounds inhibited growth and some of them had comparable or higher activity than the natural product and the reference herbicide Logran. As regards the structure-activity relationship, the carbonyl proved to be essential for their activity of 1, as well as the conjugated double bond, while the epoxide could be altered with no significant loss.

Full text of DOI:10.3390/molecules23071529

molecules
Article
(+)-epi-Epoformin, a Phytotoxic Fungal
Cyclohexenepoxide: Structure Activity Relationships
Antonio Cala 1,† ID , Marco Masi 2,† ID , Alessio Cimmino 2 ID , José M. G. Molinillo 1,
Francisco A. Macias 1,* and Antonio Evidente 2
1
Allelopathy Group, Department of Organic Chemistry, School of Science, Institute of Biomolecules (INBIO),
University of Cádiz, C/República Saharaui 7, 11510 Puerto Real, Cádiz, Spain; antonio.cala@uca.es (A.C.);
chema.gonzalez@uca.es (J.M.G.M.)
Department of Chemical Sciences, University of Naples “Federico II”, Complesso Universitario Monte S.
2
Angelo, Via Cintia 4, 80126 Napoli, Italy; marco.masi@unina.it (M.M.); alessio.cimmino@unina.it (A.C.);
evidente@unina.it (A.E.)
*
†
Correspondence: famacias@uca.es; Tel.: +34-956012770
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 7 June 2018; Accepted: 20 June 2018; Published: 25 June 2018
Abstract: (+)-epi-Epoformin (
and sphaeropsidins A and C, a nor-ent-pimarane and two pimaranes, from the culture filtrates of
Diplodia quercivora, a fungal pathogen for cork oak in Sardinia, Italy. Compound possesses a plethora
of biological activities, including antifungal, zootoxic and phytotoxic activity. The last activity and the
peculiar structural feature of suggested to carry out a structure activity relationship study, preparing
1), is a fungal cyclohexene epoxide isolated together with diplopimarane
1
1
eight key hemisynthetic derivatives and the phytotoxicity was assayed. The complete spectroscopic
characterization and the activity in the etiolated wheat coleoptile bioassay of all the compounds is
reported. Most of the compounds inhibited growth and some of them had comparable or higher
activity than the natural product and the reference herbicide Logran. As regards the structure-activity
relationship, the carbonyl proved to be essential for their activity of
1, as well as the conjugated
double bond, while the epoxide could be altered with no significant loss.
Keywords: Diplodia quercivora; oak; epi-epoformin; cyclohexeneoxide; etiolated wheat coleoptile
bioassay; phytotoxicity; allelopathy; SAR
1. Introduction
(+)-epi-Epoformin (1, Figure 1) is a cyclohexene epoxide, isolated together with some
nor-ent-pimaranes and pimarane diterpenes, namely diplopimarane and sphaeropsidins A and C,
from culture filtrates of the fungus Diplodia quercivora, isolated from declining Quercus canariensis trees
in Tunisia as a canker-causing agent [1]. The fungi belonging to Diplodia genus are well known as
pathogens of forest plants and as producers of several bioactive metabolites belonging to different
classes of natural compounds [2].
Compound
leaf of crepe myrtle (Lagerstroemia indica L. Pers.) [
Its total enantioselective and asymmetric synthesis [4–7
1
was also previously reported from an unidentified fungus isolated from a diseased
] and showed inhibition of lettuce seed germination.
] was also developed and the assignment of its
3
absolute configuration was determined by time-dependent density together with that of other close
naturally occurring cyclohexene oxides [8].
When isolated from D. quercivora,
bioassays. In leaf-puncture tests on holm oak (Quercus ilex L.), cork oak (Quercus suber L.), and tomato
(Lycopersicum esculentum Mill.) leaves, caused necrotic lesions on holm and cork oak (area lesion sizes
1 was tested for its phytotoxic activity using different
1
Molecules 2018, 23, 1529; doi:10.3390/molecules23071529
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1529
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of 6.7 and 9.5 mm2, respectively), while inducing irregular necrotic lesions (13.5 mm2 area) on tomato.
In a tomato cutting bioassay, caused, to different extents, stewing on stem at 0.2 and 0.1 mg/mL.
1
It also showed activities against phytopathogenic fungi and oomycetes Phytophthora cinnamomi and
Phytophthora plurivora were the most sensitive target organisms followed by Athelia rolfsii, whereas
Diplodia corticola was the most resistant as its mycelial growth was only slightly inhibited (39.7%
inhibition) [
larval mortality at 200 µg/ mL after 36 h to metabolites exposure [1].
More recently, was also used in a preliminary screening to evaluate the potential of plant and
1]. Finally, when assayed on brine shrimps (Artemia salina L.) 1 caused more than 50%
1
fungal metabolites, belonging to different classes of natural compounds, for the biological control
of several rusts species belonging to the genera Puccinia and Uromyces, which are pathogens of very
important crops including legumes. Compound 1 showed significant inhibition of all the species tested
also exhibiting an effective penetration [9]. The potential fungicide activity of 1 also in plants against
U. pisi and P. triticina was successively confirmed [10].
In this manuscript, 1 was isolated and tested with eight hemisynthetic derivatives (2–9) in order to
obtain clues about the structure-activity (SAR) requirements of these compounds, where we acetylated
and oxidized the hydroxy group, open the epoxide, reduce the carbonyl group or saturated the
double bond.
Figure 1. Structure of (+)-epi-epoformin (1) and derivatives (2–9).
2. Results and Discussion
The structure-activity relationship study of (+)-epi-epoformin (
of its hemisynthetic derivatives. Although was previously described in the literature as a fungal
phytotoxic compound [11], there is no information available regarding its activity in etiolated wheat
1) was carried out preparing eight
1

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coleoptiles. Thus, the phytotoxic activity of the parent compound (
1
) and its eight hemisynthetic
derivatives was assayed using this test.
The culture filtrates of D. quercivora were extracted and purified as reported in detail in the
Section 3.2. Compound
was obtained as a white solid and identified comparing its physical ([α]D20
and spectroscopic (1H and 13C-NMR and ESI-MS) properties with those previously reported [1,3].
The acetylation of to obtain , in a moderate yield (67%), was successfully carried out using
acetic anhydride [12]. Its NMR spectra (Section 3.4) showed that an acetyl group was incorporated to
the compound by the new singlet at
2.13 (H-9) in the 1H-NMR and the corresponding carbons of the
acetyl group in the 13C-NMR [13], with a carbonyl at
193.5 (C-8) and the carbon of the methyl at
5.72) was also observed for H-4 when the 1H-NMR
14]. In addition, the OH broad band of disappeared in the
. HSQC spectrum
was employed to assign each carbon to their corresponding hydrogens and the HMBC showed the
correlations to elucidate the position of the acetyl group: the signal at 2.13 (H-9) correlated with that
1
)
1
2
δ
δ
δ
20.7. A significant downfield shift (from
spectrum of was compared to that of
δ
4.67 to δ
1
2
[
1
FTIR spectrum. 2D NMR experiments [15] allowed confirming the structure of
2
δ
at δ 64.5 (C-4), while the signal at δ 5.72 (H-4) correlated with that at δ 169.8 (C-8).
On the other hand, the oxidation of the hydroxyl group with Dess-Martin periodinane [16] was
repeated several times changing the reaction times. The highest yield was 44% for
obtained after 16 hours, recovering a 51% of the starting material ( ). A longer reaction time did
not significantly increase the yield and less starting material would be recovered. The success on
3, which was
1
synthesizing 3 was confirmed firstly by the absence of any hydroxyl group and the presence of two
narrow bands at 1737 and 1715 cm−1 corresponding to the two conjugated carbonyls in the FTIR
spectrum [17]. The NMR experiments showed two signals of conjugated carbonyl carbons in the
13C-NMR at
In the HMBC the correlation of the signal at
observed allowing to assign that signal to the C-1. Due to the proximity of the signals at
and 53.7 (C-6) the HSQC experiment was indispensable to assign them to their corresponding signals
in the H-NMR at 3.79 (H-5) and δ 3.84 (H-6). These signals were assigned to their positions in
δ
192.3 (C-1) and 191.2 (C-4) and the signal of the H-4 of
1
disappeared in the 1H-NMR.
2.02 (H-7) was
54.1 (C-5)
δ
192.3 (C-1) with the methyl signal at
δ
δ
δ
1
δ
the compounds thanks to the correlations in the COSY experiment, where the signal at
δ 3.79 (H-5)
correlated with the signal at δ 6.43 (H-3).
Following the procedure described by Nicolaou et al. [18] we attempted the opening of the
epoxide to obtain a dihydroxylated compound in C-4 and C-5. Unexpectedly, the integrals of the
1H-NMR of the product showed that there was only one hydrogen at C-6 at
δ
4.77 (H-6), too high for
position to the carbonyl at C-1. After studying all the NMR spectra we determined
that the structure was that of , obtained with a high yield (83%), but the compound did not ionize
hydrogens in
α
4
properly to observe the mass corresponding to the product. The presence of iodine in the compound
was confirmed by the presence of an I− ion in the MS (ES−), with m/z 126.9045, acting as a leaving
group in an intramolecular nucleophilic substitution, which leads to the corresponding neutral epoxide.
The stereochemistry of the hydroxyl at C-5 was confirmed by the coupling constants value of H-5 at
δ
2.80 with
(axial-axial).
In order to study the influence of the double bond at C-2 in the bioactivity of
δ 4.77 (H-6) and δ 4.21 (H-4) being J5,6 = 4.1 Hz (equatorial-equatorial) and J4,5 = 7.7 Hz
1, this compound was
treated with carbon supported palladium. After several tries, only traces of the expected compound
with the epoxide at C-5 and the reduced double bond at C-2 were found. Instead, the major compounds
were, unexpectedly,
opening of the epoxide that occurred at the same time that the reduction of the double bond. By using
carbon supported platinum and were again the major products. Interestingly, by using Pd/C the
yields were slightly higher for obtaining compounds and (29 and 33%, respectively) than by using
5 and 6, with different stereochemistry at C-5 and derived from the reductive
5
6
5
6
Pt/C (19% and 21%, respectively). Only by direct infusion and solving the compounds in MeOH (0.1%
formic acid) was possible to ionize these compounds, obtaining the [M + Na]+ ions (m/z 167.3 for
C7H12O3Na). The lack of conjugation with a double bond in
5 and 6 moved the carbonyl bands to

Molecules 2018, 23, 1529
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higher wavenumbers in the FTIR spectrum, from the starting 1674 cm−1 in
1
to 1704 and 1711 cm−1
1
in
5
and
6
, respectively. The H-NMR spectra of both compounds showed that the typical signal
of H2-3 moved to higher field, being located at
stereochemistry of the hydroxyl at H-5 could be assigned by using the values of coupling constants of
H-5 with H-4 in both compounds (J4,5 = 4.3 Hz in and J4,5 = 8.8 Hz in indicating equatorial-axial and
axial-axial couplings, respectively), NOE- and ROE-difference experiments were needed to assign the
relative position of the methyl at C-2. By irradiating H-2, H-4 and H-5 in with a ROE-1D experiment,
it was confirmed that H-2 (δ 2.78) was close in space to H-4 (δ 4.02) and H-5 (δ 4.18). In addition, H-4
was close in the space to H-6a ( 2.43). On the other hand, H-4, H-5 and H-3b were irradiated in with
a NOE-1D experiment. In this case, it was observed that the signal for H-3b ( 1.35) was close in space
with H-5 ( 3.65). The latter was close to H-6b ( 2.75) and H-4 ( 3.88) was close in space to H-2 ( 2.46)
and H-3a ( 2.22), thus confirming the opposite stereochemistry of H-5 in . There was one signal for
each hydrogen at C-6 in the 1H-NMR of both compounds (
2.91 and 2.43 for , and 2.75 and 2.46
for compound ). In the case of , this was also observed for the hydrogens at C-3 ( 2.22 and 1.35).
The COSY and the HSQC experiments were used to assign each pair of signals to their corresponding
δ 1.99 in 5 and δ 2.22 and 1.35 in 6. Although the
5
6
5
δ
6
δ
δ
δ
δ
δ
δ
6
δ
δ
5
δ
δ
6
6
δ
δ
carbons, while the HMBC allowed differentiating the close carbons at
δ 74.0 (C-5, correlated with both
H-6) and δ 73.8 (C-4 correlated with both H-3) in 6.
The opening of the epoxide was performed using basic and acidic media (0.1 M NaOH, 0.1 M HCl
and 0.05 M H2SO4) and none of them worked properly. Surprisingly, while using acyl chlorides with
DMAP to acetylate the hydroxyl at C-4 using the Yamaguchi esterification [19], the epoxide opened
spontaneously, yielding both compounds 7 and 8 with high yields (39% and 45%, respectively) and
no amount of the desired esters. At first, we checked the literature to compare the spectra of our
trihydroxylated compounds with the already reported gabosines A and N [20], but the signals were
different. Therefore, the spectra were analyzed to determine their structures. Firstly, it was observed
the opening of the epoxide in the shift of the signals to lower fields, from
to 4.05 and 4.57 ( ) or 3.84 and 4.38 ( ). H-5 and H-6 were assigned by observing the COSY, where
H-5 and H-6 were correlated among themselves, and the HMBC where H-5 correlated with 69.3
(C-4), and H-6 correlated with 190.6 (C-1) and 74.6 (C-5) in the spectrum of . The same correlations
were found in , H-5 and H-6 were correlated in the COSY, and in the HMBC H-5 correlated with
71.4 (C-4), and H-6 correlated with 189.7 (C-1) and 78.3 (C-5). Regarding the stereochemistry,
the analysis of the NOE-difference experiments and the coupling constants [21] indicated that H-5 and
H-6 of were close in the space, H-5 had a high coupling constant with H-4 (J = 7.1 Hz) and a lower
constant with H-6 (J = 3.5 Hz), indicating the 4S,5R,6R stereochemistry. Regarding , the coupling
δ 3.78 (H-5) and δ 3.53 (H-6)
δ
7
δ
8
δ
δ
δ
7
8
δ
δ
δ
7
8
constants of H-5 were with H-4 (J = 8.2 Hz) and H-6 (J = 11.3 Hz), both high, indicating axial-axial
coupling in both cases and the 4S,5R,6S stereochemistry.
Finally,
1 was treated with NaBH4 and CeCl3 in MeOH to reduce the carbonyl without affecting
the double bond or the epoxide, obtaining
9
in a moderate 39% yield. The FTIR showed clearly the
presence of an OH broad band at 3234 cm−1 and the missing narrow band of the carbonyl. Regarding
the 1H-NMR, all the signals shifted to a higher field. The lack of the carbonyl was clearly observed
in the 13C-NMR with only one quaternary signal at
δ
135.2 (C-2). The close signals of the epoxide
hydrogens and carbons at positions 5 and 6 were assigned with the aid of the COSY, HSQC and
HMBC experiments. The signal at 3.30 (H-5) correlated with 3.36 (H-6) in the COSY, with 51.9
in the HSQC, and was a dd with J = 3.6 (H-5 with H-4) and 1.5 Hz (H-5 with H-6). On the other
hand, the signal at 3.36 (H-6) correlated with the signals at 3.30 (H-5) and 4.25 (H-1) in the COSY,
with signals at 53.0 (C-6) in the HSQC, and with 135.2 (C-2) in the HMBC and was a dd with J
= 3.5 (H-6 with H-1) and 1.5 Hz (H-6 with H-5). The signal for H-1 was assigned at 4.25 after the
assignment of the signal at 4.46 to H-4 by observing the correlation of 4.46 (H-4) with
in the COSY. The stereochemistry was assigned using the NOESY experiment, where
and
structure and stereochemistry shown in Figure 1 for 9.
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
δ
5.51 (H-3)
4.46 (H-4)
δ
δ
4.25 (H-1) where correlated, meaning that they were close in the space. These data confirm the

Molecules 2018, 23, 1529
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SAR Study
The results from the etiolated wheat coleoptile bioassay of compounds
In the case of and , a stimulatory activity was observed at 30 and 10
allelopathic compounds can stimulate growth at lower doses and inhibit growth at higher doses [22
According to their IC50 values (Table 1), the most active compounds were 3 and then 4. Compound 3
was one order of magnitude more active than
and comparable to the synthetic herbicide Logran®,
however, the activity profile was inconsistent with the increasing concentration. The IC50 for was
found at the lowest concentration tested (10 M) and this value did not change significantly at higher
concentrations. The test solutions of were clear and no precipitate was observed; however, this profile
1
–9
are shown in Figure 2.
1
2
µM. It has been reported that
,
23].
4
3
µ
3
is typically linked with a problem of solubility at the highest concentration. It is unknown if this or the
nature of the compound caused the strange profile.
Figure 2. Activity of compounds 1–9 in the etiolated wheat coleoptile bioassay. Results are given in
% from the control coleoptile length. Positive values indicate a higher growth than the control and
negative values a lower growth than the control.
Table 1. IC50 and AlogP values for compounds 1–9. IC50 was calculated only for those that reached the
50% of inhibition in the bioassay. a Tested concentration at which the inhibition was
compound which not reached 50% inhibition.
−
50%. b Tested
Compound
AlogP
IC50 (µM)
R2
1
2
3
4
5
6
7
8
−0.12
457
326
0.9737
0.9630
-
0.9913
1.000
-
0.9887
0.9916
-
0.26
0.13
1.00
10.0 a
117
−0.43
−0.43
−0.88
−0.88
−0.36
-
762
b
-
307
580
b
9
-
Logran
33.1
0.9898

Molecules 2018, 23, 1529
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Compound
4
, containing iodine in the structure was the most active and three times more active
than the next compounds
results, since the other dihydroxylated compound with the same stereochemistry (
with different stereochemistry ( ) were much less active or not active at all. However, the lack of
a double bond removing the -unsaturated system could also play a role. Since the reaction for
removing the iodine did not work, we could not explore further this hypothesis.
AlogP values varied between 0.88 and 1. Only compounds and followed the Lipinski’s rule
of five [24 25], having the appropriate molecular weight and AlogP. As predicted by Lipinski, these
two compounds were part of the most active compounds of the collection. However, , violating both
2
and
7. The halogen in this structure seems to have a key role in these
6) and another one
5
α-β
−
2
4
,
7
requirements, also exhibited a remarkable activity. The stereochemistry seems to play an important
role in this compound, since its epimer 8 had almost half the activity.
The least active compounds were
5, 6 and 9, showcasing that the double bond and the carbonyl are
both indispensable for the observed activity. The epoxide, on the other hand, proved to be superfluous,
since the opening of the epoxide did not result in a drop in activity (compounds 4, 7 and 8) and the
compound preserving the epoxide and lacking the carbonyl had nil activity (9).
In summary, the addition of iodine in the molecule (compound
appropriate stereochemistry (compound ) had a positive effect in the bioactivity, while the acetylation
(compound ) of the hydroxyl naturally present had little effect and the reduction of the carbonyl
(compound 9) or the double bond (compounds 5 and 6) had a negative effect.
A possible explanation of the higher activity of derivatives and was that
structure and derivative by elimination of HI via E2 could be converted into the corresponding
hemiquinone derivative and thus in the corresponding quinone. This could also occur in derivative
but the leaving group from C-6 is OH− with respect to the good one I− in
. The hemiquinone
generated from , being also an enolic compound could be stabilized from the hydrogen bond between
OH-6 and O=C-1. Because of the low oxidation level of derivatives and , as well as , they
would be more difficult to convert in the corresponding hemiquinone.
4) and an extra hydroxyl with the
7
2
3
4
3 has a hemiquinone
4
7
4
7
2
,
5
,
6
,
8
9
1
This hypothesis on the mode of action of epi-epoformin is in full agreement with the SAR study
carried out using some derivatives of sphaeropsidones, two epimeric phytotoxic cyclohexene epoxides
isolated from Diplodia cupressi (a cypress pathogen in the Mediterranean basin) [26] and the testing
their phytotoxic and antifungal activities on non-host plants and on five fungal pathogenic species
belonging to the genus Phytophthora [27]. Successively, when sphaeropsidones and the same and
new other derivatives were tested by haustorium-inducing activity in Orobanche cumana, O. crenata
and Striga hermonthica, the results obtained suggested that the activity is due to the possibility to
convert the natural sphaeropsidones and natural and hemisynthetic derivatives into the corresponding
3-methoxyquinone [28].
This hypothesis on the mode of action of sphaeropsidone, that as above reported could work
also for epi-epoformin, is in full agreement with the results obtained using natural and synthetic
quinones as sorghum xenognosin and dimethoxybenzoquinones, in studies carried out on haustoria
and the chemistry in host recognition parasitic angiosperms. Quinone/hydroquinone structures
serve as cofactors in many metabolic pathways, playing critical chemical roles in oxidation/reduction
processes [29,30].
3. Materials and Methods
3.1. General Experimental Procedures
The purities of the compounds were determined by 1H-NMR spectroscopy and every compound
1
was purified in the HPLC prior bioassays. H-NMR and 13C-NMR spectra were recorded on Agilent
™
(Santa Clara, CA, USA) 400 and 500 MHz spectrometers using CDCl3 (MagniSolv™, Merck, Darmstadt,
Germany) as solvent. The residual peaks of the solvent at 7.26 ppm (1H-NMR) and 77.0 ppm
(13C-NMR) were used as the internal reference. Carbon multiplicities were determined by DEPT

Molecules 2018, 23, 1529
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spectra [16]. DEPT, COSY, HSQC, HMBC, and NOESY experiments were performed using Varian
vnmrj microprograms. Exact masses were measured on a UPLC-QTOF ESI (Waters Synapt G2,
Manchester, UK) high-resolution mass spectrometer (HRTOFESIMS). Mass spectra were recorded in
the negative- or positive-ion mode in the range m/z 100–2000, with a mass resolution of 20,000 and
an acceleration voltage of 0.7 kV. FTIR spectra were obtained on Perkin-Elmer (Waltham, MA, USA)
Spectrum TWO IR spectrophotometer. Major absorptions in the infrared are given as wavenumbers υe
in cm−1.Optical rotations were measured in CHCl3 on a JASCO (Easton, PA, USA) P-2000 polarimeter.
HPLC in the isocratic mode was performed on a Merck Hitachi D-7000 equipped with a LiChroCART
250-10 Si 60 (10 µm) column. Column chromatography (CC) was performed using silica gel (Merck,
Geduran® Si 60, 0.063–0.200 mm) and C18-reversed phase silica gel (Sigma-Aldrich, C18 phase 90 A pore
size, St. Louis, MO, USA). Analytical and preparative TLC were performed on silica gel (Kieselgel 60,
F254, 0.25 and 0.5 mm respectively) and on reversed phase (Merck, Kieselgel 60 RP-18, F254, 0.20 mm)
plates. The spots were visualized by exposure to UV radiation (253 nm), or iodine vapour, or by
spraying first with 10% H2SO4 in MeOH and then with 5% phosphomolybdic acid in EtOH, followed
◦
by heating at 110 C for 10 min. Either Sigma-Aldrich Co., Merck or Alfa Aesar (Ward Hill, MA, USA)
supplied the reagents and the solvents. Seeds for the etiolated wheat coleoptile bioassay were kindly
supplied by Fitó (Barcelona, Spain).
3.2. Fungal Strain
The fungal strain of D. quercivora used in this study was originally isolated from a symptomatic
branch of Q. canariensis Willd. collected in a natural area in Tunisia as previously described [1].
3.3. Extraction and Purification of (+)-epi-Epoformin (1) from D. quercivora
The fungus was and grown in vitro as previously described [1]. The culture filtrates (6.7 L) were
acidified to pH 4 with 2 M HCl and extracted exhaustively with EtOAc. The organic extracts were
combined, dried with Na2SO4, and evaporated under reduced pressure to give a brown oil residue
(1.14 g). This latter was fractionated through chromatography column on silica gel, eluted with
CHCl3:i-PrOH (95:5). Eight homogeneous fraction groups were collected. The residue of the fourth
fraction (474.6 mg) was purified by CC on reverse phase eluted with Me2CO:H2O (7:3), yielding
1
(276.1 mg) as a white solid. The spectroscopic data of
1
are as follows: HRMS, m/z (M+) calcd for
C7H9O3 141.0552, found 141.0601 [M + H]+; IR υemax 3357 (O–H), 1674 (C=O) cm–1, [α]2D0 = +139.3◦
1
(
c = 0.08). H-NMR (400 MHz, CDCl3,
δ, ppm): 6.46 (brs, 1H, H-3), 4.67 (brs, 1H, H-4), 3.78 (m, 1H,
H-5), 3.53 (m, 1H, H-6), 1.86 (s, 3H, H-7).
3.4. Synthesis of (+)-epi-Epoformin (1) Derivatives 2–9
(
1R,2S,6R)-4-Methyl-5-oxo-7-oxabicyclo[4.1.0]hept-3-en-2-yl acetate (
was dissolved in dry CH3CN (2 mL) in a 10 mL pear-shaped flask, in N2 atmosphere at 0 ◦C. Then
4-dimethylaminopyridine (10.5 mg, 0.085 mmol) and acetic anhydride (29 L, 0.308 mmol) were
added and stirred slowly reaching room temperature (Scheme 1). After 10 minutes, the reaction was
quenched with 2 mL brine and extracted 3 with EtOAc. The organic layers were combined, dried
2). Compound 1 (10.8 mg, 0.077 mmol)
µ
×
with anhydrous Na2SO4 and concentrated under reduced pressure in a rotatory evaporator obtaining
14.7 mg of crude extract. After purification in column chromatography using n-hexane:acetone, 9:1,
9.4 mg of 2 (67% yield) were obtained as a light-yellow oil. Spectroscopic data are as follows: HRMS,
m/z (M+) calcd for C9H11O4 183.0657, found 183.0504 [M + H]+; IR υemax 2926 (C–H), 1741 (C=O), 1687
◦
1
(C=O) cm–1, [α]D20 = +102.8 (c = 0.13). H-NMR (500 MHz, CDCl3,
δ, ppm): 6.37 (m, 1H, H-3), 5.72 (dd,
J = 5, 1.2 Hz, 1H, H-4), 3.72 (m, 1H, H-5), 3.53 (dd, J = 3.6, 1.1 Hz, 1H, H-6), 2.13 (s, 3H, H-9), 1.86 (dd,
J = 1.4, 1.2 Hz, 3H, H-7). 13C-NMR (125 MHz, CDCl3,
δ, ppm): 193.5 (s, C-1), 169.8 (s, C-8), 136.6 (s,
C-2), 134.3 (d, C-3), 64.5 (d, C-4), 55.0 (d, C-5), 52.9 (d, C-6), 20.7 (q, C-9), 16.0 (q, C-7).

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(1R,6S)-3-Methyl-7-oxabicyclo[4.1.0]hept-3-ene-2,5-dione (
3). Compound
1
(9.8 mg, 0.070 mmol) was
dissolved in CH2Cl2 (1 mL) in a 10 mL pear-shaped flask. Then Dess-Martin periodinane (45.9 mg,
0.105 mmol) was added in small portions while stirring at rt. After 16 hours, the reaction was
quenched with 1 mL of aqueous saturated Na2S2O3 and extracted
×
3 with CH2Cl2. The organic
layers were combined and washed with 1 mL aqueous saturated NaHCO3, dried with anhydrous
Na2SO4 and concentrated under reduced pressure in a rotatory evaporator. The crude was purified in
column chromatography in a gradient of n-hexane:EtOAc from 1:0 to 2:3, and later in the HPLC using
hexane:acetone 9:1 with a retention time of 12 min. Compound
3
was obtained as a colorless oil in
a 44% yield (4.3 mg) and 5 mg of
1
(51%) were recovered. Spectroscopic data are as follows: HRMS,
m/z (M+) calcd for C7H7O3 139.0395 [M + H]+, found 139.0401; IR υemax 2925 (C–H), 1737 (C=O), 1715
(C=O) cm–1, [α]D20 = +5.8◦ (c = 0.02). H-NMR (500 MHz, CDCl3,
δ, ppm): 6.43 (brs, 1H, H-3), 3.84 (dd,
1
J = 3.7, 0.7 Hz, 1H, H-6), 3.79 (dd, J = 2.5, 0.7 Hz, 1H, H-5), 2.02 (d, J= 1.4 Hz, 3H, H-7). 13C-NMR
(125 MHz, CDCl3, δ, ppm): 192.3 (s, C-1), 191.2 (s, C-4), 146.8 (s, C-2), 133.3 (d, C-3), 54.1 (d, C-5), 53.7
(d, C-6), 16.4 (q, C-7).
(4S,5R,6R)-4,5-Dihydroxy-6-iodo-2-methylcyclohex-2-en-1-one (4). Compound 1 (9.8 mg, 0.070 mmol) was
dissolved in acetone (1 mL) in a 10 mL pear-shaped flask. Then NaI (37 mg, 0.252 mmol), NaOAc
(2.3 mg, 0.252 mmol) and AcOH (14 L, 0.252 mmol) were added and stirred at rt. After 7 hours,
the reaction was quenched with 1 mL of aqueous saturated Na2S2O3 and evaporated the acetone
under reduced pressure. The resultant aqueous solution was extracted 3 with EtOAc. The organic
µ
×
layers were combined and washed with 1 mL H2O, 1 mL aqueous saturated NaHCO3 and 1 mL brine,
dried with anhydrous Na2SO4 and concentrated under reduced pressure in a rotatory evaporator.
The crude was purified in column chromatography in a gradient of n-hexane:EtOAc from 1:0 to 2:3,
and furnished 13.8 mg of a fraction with a mixture of two compounds. This fraction was later purified
in the HPLC with n-hexane:acetone 7:3 obtaining two peaks at 15 (4) and 17 min. A total of 8.2 mg of 4
(83% yield) as a colorless oil were isolated. Spectroscopic data are as follows: HRMS, m/z (M−) calcd
for I 126.9050, found 126.9045 [M]−; IR υemax 3264 (O–H), 2925 (C–H), 1683 (C=O) cm–1, [α]D20 = +67.3◦
1
(c = 0.05). H-NMR (400 MHz, CDCl3,
δ
, ppm): 6.59 (m, 1H, H-3), 4.77 (d, J = 4.1 Hz, 1H, H-6), 4.21 (dt,
J = 7.7, 2.1 Hz, 1H, H-4), 2.80 (dd, J = 7.7, 4.1 Hz, 1H, H-5), 1.87 (dd, J = 2.1, 1.5 Hz, 3H, H-7). 13C-NMR
(100 MHz, CDCl3, δ, ppm): 192.3 (s, C-1), 143.9 (d, C-3), 133.8 (s, C-2), 73.6 (d, C-4), 73.5 (d, C-5), 35.2
(d, C-6), 15.9 (q, C-7).
(2R,4S,5R)-4,5-Dihydroxy-2-methylcyclohexan-1-one (5) and (2R,4S,5S)-4,5-dihydroxy-2-methylcyclohexan-1-
one (6). Compound 1 (9.8 mg, 0.070 mmol) was disolved in EtOAc (2 mL) in a 10 mL pear-shaped
flask and N2 atmosphere. Then carbon supported Pt (23 mg, 0.004 mmol) was added followed by H2
atmosphere and a H2 balloon was connected while stirring at rt. After 10 minutes, no more starting
material was observed in TLC and the crude of reaction was filtered through celite. Then, it was
purified by column chromatography with a gradient of n-hexane:acetone from 1:0 to 2:3, furnishing a
major fraction of 4.4 mg containing the mixture of reduced products. This fraction was purified in the
HPLC with n-hexane:acetone 7:3 observing two peaks at 25 (
mg (19% yield) and 2.2 mg (21%), respectively, both being colorless oils. Alternatively, using Pd/C(3
mg, 0.003 mmol) instead of Pt/C, and 9.2 mg of (0.066 mmol), 2.7 mg of compound (29% yield)
and 3.2 mg of (33% yield) were obtained. Spectroscopic data of
are as follows: MS, m/z (M+) calcd
5) and 32 min. (6), with a total mass of 1.9
1
6
6
5
for C7H12O3Na 167.0684, found at low resolution 167.3 [M + Na]+; IR υemax 3385 (O–H), 2926 (C–H),
1
1704 (C=O) cm–1, [α]D20 = +13.1◦ (c = 0.03). H-NMR (400 MHz, CDCl3,
δ
, ppm): 4.18 (ddd, J = 4.9
,
4.3, 3.8 Hz, 1H, H-5), 4.02 (dd, J = 8.4, 4.3 Hz, 1H, H-4), 2.91 (dd, J = 14.4, 3.8 Hz, 1H, H-6b), 2.78 (m,
1H, H-2), 2.43 (dd, J = 14.4, 4.9 Hz, 1H, H-6a), 1.99 (dd, J = 8.4, 4.0 Hz, 2H, H-3), 1.08 (d, J = 6.8 Hz,
3H, H-7). 13C-NMR (100 MHz, CDCl3,
40.1 (d, C-2), 36.9 (t, C-3), 14.8 (q, C-7). Spectroscopic data of
δ
, ppm): 211.5 (s, C-1), 74.0 (d, C-5), 69.0 (d, C-4), 44.6 (t, C-6),
6
are as follows: MS, m/z (M+) calcd
for C7H12O3Na 167.0684, found at low resolution 167.3 [M + Na]+; IR υemax 3392 (O–H), 2930 (C–H),
◦
1
1711 (C=O) cm–1, [α]D20 = +11.5 (c = 0.03). H-NMR (400 MHz, CDCl3,
δ, ppm): 3.88 (ddd, J = 13.3, 8.8,

Molecules 2018, 23, 1529
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4.5 Hz, 1H, H-4), 3.65 (ddd, J = 12.2, 8.8, 5.3 Hz, 1H, H-5), 2.75 (ddd, J = 13.7, 5.3 Hz, 1H, H-6b), 2.46 (m,
1H, H-2), 2.46 (m, 1H, H-6a), 2.22 (ddd, J = 13.2, 5.7, 4.5 Hz, 1H, H-3a), 1.35 (td, J = 13.2, 11.4, 1H, H-3b),
1.06 (d, J = 6.6 Hz, 3H, H-7). 13C-NMR (100 MHz, CDCl3, δ, ppm): 207.9 (s, C-1), 74.0 (d, C-5), 73.8 (d,
C-4), 47.0 (t, C-6), 42.4 (d, C-2), 37.1 (t, C-3), 13.8 (q, C-7).
(4S,5R,6R)-4,5,6-Trihydroxy-2-methylcyclohex-2-en-1-one (
7) and (4S,5R,6S)-4,5,6-trihydroxy-2-methyl-
cyclohex-2-en-1-one ( ). Compound (7.1 mg, 0.051 mmol) was dissolved in dry CH3CN (2 mL)
8
1
in a 10 mL pear-shaped flask. Then 4-dimethylaminopyridine◦(6.9 mg, 0.056 mmol) and hexanoyl
chloride (29 µL, 0.204 mmol) were added while stirring at 0 C and gradually reached rt. After
5 hours and 30 min. no more starting material was observed in TLC and the reaction was quenched
with 2 mL NaHCO3 and extracted 3 with EtOAc. The organic layers were combined, dried over
×
anhydrous Na2SO4 and the solvent evaporated in a rotatory evaporator. The crude was purified by
column chromatography with a gradient of n-hexane:acetone from 1:0 to 2:3, furnishing two fractions.
These fractions were purified in the HPLC with n-hexane:acetone 7:3 observing two peaks at 16 (
17 min. ( ), with a total mass of 3.2 mg (39% yield) and 3.6 mg (45%), respectively, both as colorless
oils. The same compounds and similar yields were obtained when using butanoyl chloride (22 L,
are as follows: HRMS, m/z (M+) calcd
7) and
8
µ
0.204 mmol) and the same procedure. Spectroscopic data of
7
for C7H10O4 157.0506, found 157.0505 [M
[
−
H]−; IR υemax 3372 (O–H), 2926 (C–H), 1682 (C=O) cm–1,
1
α]2D0 = +65.7◦ (c = 0.03). H-NMR (400 MHz, CDCl3,
δ
, ppm): 6.68 (dq, J = 2.3, 1.4 Hz, 1H, H-3), 4.59
(ddd, J = 7.1, 2.3, 2.1 Hz, 1H, H-4), 4.57 (d, J = 3.5 Hz, 1H, H-6), 4.05 (d, J = 7.1, 3.5 Hz, 1H, H-5), 1.88
(dd, J = 2.1, 1.4 Hz, 3H, H-7). 13C-NMR (100 MHz, CDCl3,
δ, ppm): 190.6 (s, C-1), 144.1 (d, C-3), 134.3
(s, C-2), 74.6 (d, C-5), 69.3 (d, C-4), 61.4 (d, C-6), 15.7 (q, C-7). Spectroscopic data of
8
are as follows:
HRMS, m/z (M+) calcd for C7H10O4 157.0506, found 157.0540 [M
−
H]−; IR υemax 3370 (O–H), 2925
◦
1
(C–H), 1691 (C=O) cm–1, [α]D20 = +28.2 (c = 0.03). H-NMR (400 MHz, CDCl3,
δ, ppm): 6.70 (dq, J = 2.1,
1.5 Hz, 1H, H-3), 4.51 (ddd, J = 8.2, 2.1, 2.1 Hz, 1H, H-4), 4.38 (d, J = 11.3 Hz, 1H, H-6), 3.84 (d, J = 11.3,
8.2 Hz, 1H, H-5), 1.89 (dd, J = 2.1, 1.5 Hz, 3H, H-7). 13C-NMR (100 MHz, CDCl3,
δ, ppm): 189.7 (s, C-1),
144.2 (d, C-3), 135.1 (s, C-2), 78.3 (d, C-5), 71.4 (d, C-4), 66.9 (d, C-6), 15.8 (q, C-7).
(1S,2R,5S,6R)-3-Methyl-7-oxabicyclo[4.1.0]hept-3-ene-2,5-diol ( ). Compound (9.8 mg, 0.070 mmol) was
9
1
dissolved in MeOH (2 mL) containing 0.4 M CeCl3 (0.8 mmol) in a 10 mL pear-shaped flask. Then
NaBH4 (3.8 mg, 0.102 mmol) were added in small portions and stirred at rt. The reaction was quenched
after 5 min. with 2 mL saturated aqueous NH4Cl, evaporated the MeOH in a rotatory evaporator and
extracted
×
3 with EtOAc. The organic layers were combined, dried over anhydrous Na2SO4 and the
solvent evaporated. Product was purified in the HPLC with n-hexane:acetone 7:3 observing a peak at
34 min., corresponding to
Spectroscopic data of
9, with a total mass of 3.8 mg (39% yield) as an amorphous colorless solid.
9
are as follows: HRMS, m/z (M+) cal◦cd for C7H11O3 143.0708, found 143.0681
1
[M + H]+; IR υemax 3234 (O–H), 2916 (C–H) cm–1, [α]D20 = +0.8 (c = 0.03). H-NMR (400 MHz, CDCl3,
δ
,
ppm): 5.51 (dd, J = 5.1, 1.6 Hz, 1H, H-3), 4.46 (ddd, J = 5.1, 3.6, 0.9Hz, 1H, H-4), 4.25 (d, J = 3.5, 1.5 Hz,
1H, H-1), 3.36 (dd, J = 3.5, 1.5 Hz, 1H, H-6), 3.30 (dd, J = 3.6, 1.5 Hz, 1H, H-5), 1.83 (dd, J = 1.6, 0.9 Hz,
3H, H-7). 13C-NMR (100 MHz, CDCl3,
δ, ppm): 135.2 (s, C-2), 121.6 (d, C-3), 66.3 (d, C-1), 63.1 (d, C-4),
53.0 (d, C-6), 51.9 (d, C-5), 21.2 (q, C-7).

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Scheme 1. Synthesis of compounds 2–9.
1H- and 13C-NMR Spectra of the compounds are available in the Supplementary Materials.
3.5. Etiolated Wheat Coleoptile Bioassay
The compounds
The conditions for this bioassay were reported previously [31
1
–
9
were tested for their bioactivity in an etiolated wheat coleoptile bioassay.
32] and replicated in this study, using
,
the same herbicide (Logran®) as positive control and the buffer solution as negative control. Wheat
(Triticum aestivum) was the ‘catervo’ variety. All the samples were solved in a 0.5% of dimethyl
sulfoxide and gave clear solutions at all the tested concentrations (10−3–10−5 M). The results are shown
in Figure 2.
3.6. Calculation of IC50 and logP
The bioactivity data were fitted to a sigmoidal dose-response model using the GraphPad Prism
v.5.00 software package (GraphPad Software, San Diego, CA, USA) [33] to obtain the IC50 values
shown in Table 1. The AlogP was calculated using the ALOGPS v.2.1 software (Helmholtz Zentrum
München, Munich, Germany), based on computed atom contributions [34–36].
4. Conclusions
(+)-epi-Epoformin (
procedures in the literature. Then, eight derivatives of
for their bioactivity in the etiolated wheat coleoptile bioassay. The derivatives were compared with the
starting material to look for structure-activity relationships. The -unsaturated system in proved
1) was successfully isolated from extracts of D. quercivora following the
1
were synthesized and tested altogether
α
-β
1
to be essential to obtain active derivatives and the inclusion of iodine and an extra hydroxyl in the
molecules increased the activity, while the acetylation of the hydroxyl did not have a significant effect.
The results described here illustrate that natural products, especially those provided by fungi, are
valuable resources which need to be investigated further as natural herbicides against weeds.
Supplementary Materials: Supplementary data are available online.
Author Contributions: All the authors contributed in the design of the study, the analysis of the data and writing
of the manuscript. A.C. (Antonio Cala) and M.M. performed the experiments. A.C. (Alessio Cimmino) purified
the epi-epoformin. J.M.G.M., F.A.M. and A.E. revised and finalized the manuscript. All authors read and approved
the final manuscript.
Funding: “This research was partly funded by Ministerio de Economía, Industria y Competitividad’ (MINEICO),
Spain, Project AGL2017-88-083-R.” “This research was carried out in the frame of Programme STAR 2017,
financially supported by UniNA and Compagnia di San Paolo.” APC was sponsored by MDPI. A.Ca. would like
to thank the financial support of the University of Cádiz in the ‘Plan Propio de Investigación 2017’.

Molecules 2018, 23, 1529
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Acknowledgments: The authors are very grateful to L. Maddau, Dipartimento di Agraria, Università di Sassari,
Sassari, Italy to providing the culture filtrates of the fungus D. quercivora. We also thank Fitó for providing the
seeds for the etiolated wheat coleoptile bioassay free of charge. A.E. is associated to the Istituto di Chimica
Biomolecolare del CNR, Pozzuoli, Italy.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–9 are available from the authors.
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