Bioactivity studies of oxysporone and several derivatives

Article abstract of DOI:10.1016/j.phytol.2014.07.005

Recently oxysporone, a phytotoxic dihydrofuropyranone, was isolated along with two closely related compounds, afritoxinones A and B, from liquid cultures of Diplodia africana, an invasive fungal pathogen of Phoenicean juniper. In this study, eight derivatives were hemisynthesized and assayed for their phytotoxic and antifungal activities in comparison to the parent compound. Each compound was tested on non-host plants and on four destructive plant pathogens such as Athelia rolfsii, Diplodia corticola, Phytophthora cinnamomi and P. plurivora. The results on the phytotoxic activity showed that the dihydrofuropyranone carbon skeleton and both the double bond the hydroxy group of dihydropyran ring appeared to be structural features important in conferring activity. Although the data concerning the antifungal activity did not allow to extract any structure-activity relationships, it should be underlined that the conversion of oxysporone into the corresponding 4-O-benzoyl derivative led to a compound showing a good antifungal activity towards three out of the four organisms tested.

Full text of DOI:10.1016/j.phytol.2014.07.005

Phytochemistry Letters 10 (2014) 40–45
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Bioactivity studies of oxysporone and several derivatives
Anna Andolfi a, Lucia Maddau b, Benedetto T. Linaldeddu b, Bruno Scanu b,
Alessio Cimmino a, Sara Basso a, Antonio Evidente a,
*
a
`
Dipartimento di Scienze Chimiche, Universita di Napoli Federico II, Napoli, Italy
b
`
Dipartimento di Agraria, Sezione di Patologia vegetale ed Entomologia, Universita degli Studi di Sassari, Sassari, Italy
A R T I C L E I N F O
A B S T R A C T
Article history:
Recently oxysporone, a phytotoxic dihydrofuropyranone, was isolated along with two closely related
compounds, afritoxinones A and B, from liquid cultures of Diplodia africana, an invasive fungal pathogen
of Phoenicean juniper. In this study, eight derivatives were hemisynthesized and assayed for their
phytotoxic and antifungal activities in comparison to the parent compound. Each compound was tested
on non-host plants and on four destructive plant pathogens such as Athelia rolfsii, Diplodia corticola,
Phytophthora cinnamomi and P. plurivora. The results on the phytotoxic activity showed that the
dihydrofuropyranone carbon skeleton and both the double bond the hydroxy group of dihydropyran ring
appeared to be structural features important in conferring activity. Although the data concerning the
antifungal activity did not allow to extract any structure–activity relationships, it should be underlined
that the conversion of oxysporone into the corresponding 4-O-benzoyl derivative led to a compound
showing a good antifungal activity towards three out of the four organisms tested.
Received 24 January 2014
Received in revised form 14 July 2014
Accepted 15 July 2014
Available online 30 July 2014
Keywords:
Diplodia africana
Juniperus phoenicea
Oxysporone
SAR
ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1. Introduction
Pestalotia longiseta (Speg.), the causal agent of tea grey blight
(Nagata and Ando, 1989) and by Pestalotiopsis oenotherae
Diplodia africana Damm & Crous was detected as the main
pathogen involved in the aetiology of cankers and branch dieback
of Phoenicean juniper trees on Caprera Island, within the La
Maddalena Archipelago in Sardinia, Italy (Linaldeddu et al., 2011).
For this pathogen, the production of two new phytotoxic
dihydrofuropyran-2-ones, named afritoxinone A and B (2 and 3,
Fig. 1), along with six others known metabolites such as
oxysporone (1, Fig. 1), sphaeropsidin A, epi-sphaeropsidone,
R-(ꢀ) mellein, (3R,4S)-4-hydroxymellein and (3R,4R)-4-hydroxy-
mellein was recently reported (Evidente et al., 2012). Among these
secondary metabolites, oxysporone proved to be the most
phytotoxic compound.
This latter was firstly isolated from Fusarium oxysporum Schltdl.
and recognized as the compound responsible for the antibiotic
activity of earthworm casts used in some parts of Nigeria to treat
chronic dysentery (Adegosan and Alo, 1979). Successively,
oxysporone was also identified as a phytotoxin produced by
Venkatas., Grand & Van Dyke a pathogen of evening primrose
(Venkatasubbaiah et al., 1991). To date, no information is available,
to our knowledge, on the antifungal activity of this metabolite.
Recently, the absolute configuration, which is strongly related
with the biological activity of naturally occurring compounds
(Evidente et al., 2011, 2013) was also determined for 1 as depicted
in Fig. 1. The absolute configuration was assigned by chiroptical
methods such as optical rotatory dispersion, electronic circular
dichroism and vibrational circular dichroism (Mazzeo et al., 2013).
Both biological and chemical properties make oxysporone an
attractive compound with potential biotechnological applications
in agriculture, especially as new agrochemicals with a lower
environmental impact. In this study eight derivatives of oxyspor-
one (4–11, Fig. 2) were prepared by chemical transformation of 1 in
order to obtain new insights on the structure-activity relationships
and generate derivatives with improved biological properties.
Compounds 4–7 and 9 are new derivatives, 8, 10, and 11 were
previously reported (Edwards et al., 2001; Salvatore et al., 1994).
Oxysporone (1) and its derivatives were tested for their
phytotoxicity on non-host plants and antifungal activity against
four destructive plant pathogens belonging to three different
classes such as Ascomycetes, Basidiomycetes and Oomycetes.
*
`
Corresponding author at: Dipartimento di Scienze Chimiche, Universita di
Napoli Federico II, Napoli, Italy. Tel.: +39 081 2539178/6741330.
E-mail address: evidente@unina.it (A. Evidente).
http://dx.doi.org/10.1016/j.phytol.2014.07.005
1874-3900/ß 2014 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

A. Andolfi et al. / Phytochemistry Letters 10 (2014) 40–45
41
(Curzi) C.C. Tu & Kimbr. strain SR1 (Basidiomycetes) together two
Oomycetes Phytophthora cinnamomi Rands strain PH030 and
Phytophthora plurivora T. Jung & T.I. Burgess strain PH089 were
used as organisms test in the antifungal assay. The strain BL38 of D.
corticola was originally isolated from a cankered branch of cork oak
collected in a natural forest in Tuscany (Italy). The strain SR1 of
A. rolfsii was isolated from the collar rot of an artichoke plant in
northern Sardinia (Italy). The strain PH030 of P. cinnamomi was
isolated from necrotic roots of a holm oak seedling in nursery in the
central Sardinia. The strain PH089 of P. plurivora was isolated from
a rhizosphere soil sample collected from a declining chestnut tree
in an orchard in central Sardinia. All strains were identified on the
basis of morphological characters and analysis of internal
transcribed spacer (ITS) rDNA. DNA extraction, polymerase chain
reaction mixtures, amplification conditions and DNA sequencing
were carried out as previously reported (Linaldeddu et al., 2013).
Representative ITS sequences of all 4 strain were deposited in
GenBank (accession numbers: JX894198; KF724852; KF724851;
KF724853). Pure cultures were maintained on potato-dextrose
agar (Fluka, Sigma–Aldrich Chemic GmbH) and stored at 4 8C in the
collection of the Dipartimento di Agraria, Sezione di Patologia
OH
H
4
3
2
5
6
7
8
O
1
O
O
H
1
H
5
H
H
4
3
2
O
O
O
O
O
O
H3CO
H3CO
H
3
2
Fig. 1. Structures of oxysporone, afritoxinones A and B (1, 2 and 3, respectively).
2. Materials and methods
2.1. General experimental procedures
`
IR spectra were recorded as deposit glass film on a Perkin-Elmer
Spectrum One FT-IR spectrometer, and UV spectra were measured
in MeOH, unless otherwise noted, on a Perkin–Elmer Lambda 25
UV/Vis spectrophotometer. 1H NMR spectra were recorded at 600
and 400 MHz in CDCl3, on Bruker spectrometers. The same solvent
was used as internal standard. HR ESI and APCI MS were recorded
on Agilent Technologies 1100 LC/MSD TOF and on Agilent
Technologies 6120 Quadrupole LC/MS instrument, respectively.
Analytical and preparative TLC were performed on silica gel,
Kieselgel 60, F254 plates, 0.25 and 0.5 mm respectively, and on
reversed phase, DC Kieselgel 60 RP-18, F254, 0.25 mm plates. The
spots were visualized by exposure to UV radiation (253), or by
spraying first with 10% H2SO4 in MeOH and then with 5%
phosphomolybdic acid in EtOH, followed by heating at 110 8C
for 10 min. Column chromatography was performed on silica gel,
Kieselgel 60, 0.063–0.200 mm (Merck).
vegetale ed Entomologia, Universita degli Studi di Sassari, Italy.
2.2. Production, extraction and purification of oxysporone (1)
Oxysporone was isolated from culture filtrate of D. africana
strain DA1 grown on a mineral defined liquid media named M1-D
(Pinkerton and Strobel, 1976) as recently reported in details
(Evidente et al., 2012). The purification of the organic extract
obtained by its culture filtrate (12 l), carried out as previously
described (Evidente et al., 2012), allowed to obtain oxysporone (1)
as homogeneous oils (357.6 mg, 29.7 mg/l).
2.3. 4-O-Acetyloxysporone (4)
Oxysporone (1, 30.5 mg), dissolved in pyridine (60
converted into the corresponding 4-O-acetyl derivative (4) by
acetylation with Ac2O (60 l) at room temperature for 2 h. The
ml), was
m
2.1.1. Fungal strains
reaction was stopped by addition of MeOH, and the azeotrope
formed by addition of benzene was evaporated in a N2 stream.
The oily residue (38.2 mg) was purified by TLC on silica gel, eluent
Two fungal strains namely Diplodia corticola A.J.L. Phillips,
A. Alves & J. Luque strain BL38 (Ascomycetes) and Athelia rolfsii
OH
O
H
4
4
OAc
3
2
OAc
H
5
g
O
O
H
O6
O
HO
10
4
O
f
3
2
H
O
O
5
O
O
O
O
H
H
b
4
9
a
O
OH
H
H
c
4
4
3
2
7
5
6
O
8
O
1
O
O
O
O
H
H
1
6
O
d
e
2'
Br
O
5'
O
O
O
H
4
4
O
5
O
h
O
8 OCH3
O
O6
O
HO
H
7
11
8
Fig. 2. Reagent and condition to convert 1 into 4–11: (a) Ac2O, pyridine, rt, 2 h; (b) H2, Pt 5%, MeOH, rt, 4 h; (c) Corey’s reagent, CH2Cl2, rt, 1 h; (d) p-Br-BzCl, DMAP, MeCN, rt,
4 h; (e) Jones’s reagent, Me2CO, 0 8C, 10 min; (f) H2, Pt 5%, MeOH, rt, 5 h; (g) Jones reagent, Me2CO, 0 8C, 10 min; (h) CH2N2, MeOH, rt.

42
A. Andolfi et al. / Phytochemistry Letters 10 (2014) 40–45
CHCl3-i-PrOH (95:5), yielding derivative 4 (25.8 mg, 69%, Rf 0.7) as
a homogeneous oil. 4: UV max nm (log ): 206 (3.00); IR max 1789,
l
e
n
1731, 1652, 1230, 1011 cmꢀ1; 1H NMR: see Table 1; HRESIMS (+),
m/z: 221.0437 [C9H10NaO5, calcd. 221.0426, M+Na]+.
IUPAC NAME of 4: Acetic acid 2-oxo-2,3,3a,7a-tetrahydro-4H-
furo[2,3-b]pyran-4-yl ester.
2.4. 2,3-Dihydrooxysporone (5)
Oxysporone (1, 15.0 mg), dissolved in MeOH (750
added to a presaturated suspension of Pt (5%) in MeOH (750
m
l), was
l).
m
Hydrogenation was carried out at room temperature and
atmospheric pressure under stirred conditions. After 4 h the
reaction was stopped by filtration and clear solution then
evaporated under reduced pressure. The residue (17.4 mg) purified
by preparative TLC on silica gel, eluent CHCl3-i-PrOH (93:7), giving
5 (11.0 mg, 73%, Rf 0.3) as a homogeneous solid. 5: UV lmax nm
(log e ;
): 205 (2.91); IR nmax 3426, 1767, 1165 cmꢀ1 1H NMR: see
Table 1; HRESIMS (+), m/z: 339 [2M+Na]+, 181.0487 [C7H10NaO4,
calcd. 181.0477, M+Na]+.
IUPAC NAME of 5: 4-Hydroxy-tetrahydro-furo[2,3-b]pyran-2-
one.
2.5. 4,O-Dehydrooxysporone (6)
To oxysporone (1, 15.1 mg), dissolved in CH2Cl2 (3.0 ml),
Corey’s reagent (Corey and Suggs, 1975) (75.1 mg) was added
and the reaction mixture was stirred at room temperature for 1 h.
The reaction was then stopped by filtration on a glass fennel and
clear solution obtained evaporated under reduced pressure. The
residue (36.5 mg) purified by preparative TLC on silica gel, eluent
CHCl3-i-PrOH (93:7), giving
6
(4.3 mg 30%, Rf 0.36) as
a
homogeneous solid. 6: UV lmax nm (log
e
): 205 (3.77); IR nmax
1794, 1738, 1681, 1005 cmꢀ1; 1H NMR: see Table 1; HRESIMS (ꢀ):
m/z 153.0142 [C7H5O4, calcd. 153.0153, M-H]ꢀ; APCIMS (+), m/z
155 [M+H]+; APCIMS (ꢀ), m/z 153 [M ꢀ H]ꢀ.
IUPAC NAME of 6: 3a,7a-Dihydro-3H-furo[2,3-b]pyran-2,4-
dione.
2.6. 4-O-p-Bromobenzoyloxysporone (7)
To oxysporone (1, 10.0 mg), dissolved in CH3CN (1.0 ml) DMAP
(23.0 mg,) and p-bromobenzoyl chloride (25.0 mg) were added.
The reaction mixture was stirred at room temperature for 4 h, and
then evaporated under reduced pressure. The residue (43.2 mg)
was purified by TLC on silica gel, eluent CHCl3-i-PrOH (97:3), giving
7 (10.6 mg, 42%, Rf 0.8) as a white solid. 7: UV
(4.42), 245 (4.28); IR nmax 1798, 1715, 1655, 1619, 1266,
1013 cmꢀ1 1H NMR: see Table 1; HRESIMS (+), m/z 378
lmax nm (log e): 203
;
[M+2+K]+, 376 [M+K]+, 362 [M+2+Na]+, 360.9676 [C14H11BrNaO5,
calcd. 360.9688, M+Na]+; ESMS (ꢀ), m/z 338 [M+2ꢀH]ꢀ, 336
[MꢀH]ꢀ, 201 [p-BrC6H4 – CO2 + 2]ꢀ, 199 [p-BrC6H4 – CO2]ꢀ.
IUPAC NAME of 7: 4-Bromo-benzoic acid 2-oxo-2,3,3a,7a-
tetrahydro-4H-furo[2,3-b]pyran-4-yl ester.
2.7. 2-(4-Pyronyl)acetic acid (8)
To oxysporone (1, 10.0 mg), dissolved in Me2CO (2.0 ml), at 0 8C,
Jones’s reagent (Bowers et al., 1953) was added under stirring until
to have a orange sovranatant. After 10 min the reaction was
stopped by addition of i-PrOH and ice. The suspension was diluted
with iced H2O to obtain a brilliant green solution, which was
extracted with EtOAc (3 ꢁ 2 ml). The organic extracts were
combined, dried (Na2SO4), filtered and evaporated under reduced
pressure. The residue (33.1 mg) was purified by TLC on reverse
phase, eluent MeOH-H2O (1:1), giving 8 (4.1 mg, 40%, Rf 0.78) as

A. Andolfi et al. / Phytochemistry Letters 10 (2014) 40–45
43
amorphous solid. 8: UV
n
l
max nm (log
e
): 204 (3.56), 251 (3.51); IR
for symptoms after 7 days. The effect of the toxins on the leaves
were observed up to 10 days. Lesions were estimated using APS
Assess 2.0 software (Lamari, 2002) following the tutorials in the
user’s manual. The lesion size was expressed in mm2.
max 3400, 1740, 1653, 1082 cmꢀ1; [lit. 13:
n
max (KBr)/cmꢀ1 3600–
3300, 1725 and 1638]. 1H NMR was similar to that previously
reported in D2O (Salvatore et al., 1994). 1H NMR: see Table 1;
ESIMS (+), m/z 177 [M+Na]+; APCIMS (+), m/z 155 [M+H]+; APCIMS
(ꢀ), m/z 153 [MꢀH]ꢀ.
2.11.2. Tomato cutting assay
IUPAC NAME of 8: (4-Oxo-4H-pyran-3-yl)-acetic acid.
Tomato cuttings were taken from 21-day-old seedlings and
each compound was assayed at 0.05, 0.1 and 0.2 mg/ml. Cuttings
were placed in the test solutions (2 ml) for 72 h and then
transferred to distilled water. Symptoms were visually evaluated
up to 7 days.
2.8. 4-O-Acetyl-2,3-dihydrooxysporone (9)
Compound 4 (15.0 mg) dissolved in MeOH (750
to a presaturated suspension of Pt (5%) in MeOH (750
m
l), was added
l).
m
Hydrogenation was carried out at room temperature and
atmospheric pressure under stirred conditions. After 5 h the
reaction was stopped by filtration and clear solution was then
evaporated under reduced pressure. The residue (18.5 mg) was
purified by preparative TLC on silica gel, eluent CHCl3-i-PrOH
(97:3), giving 9 (8.1 mg 53%, Rf 0.4) as a homogeneous solid. 9: UV
2.11.3. Antifungal assays
Oxysporone (1), its derivatives (4–9) were tested on four
different plant pathogens including two fungal species (Athelia
rolfsii and Diplodia corticola) and two Oomycetes (Phytophthora
cinnamomi and P. plurivora) which have a great impact in both
agriculture and natural ecosystems. Preliminary, the sensitivity of
all species to oxysporone and its derivatives was evaluated by
transferring mycelial plugs (6-mm diameter) of each isolate, taken
from the margin of actively growing colonies of 3-day-old, to CA
l
max nm (log e): 203 (4.01); IR n
max 1733, 1243 cmꢀ1; 1H NMR see
Table 1; HRESIMS (+), m/z 223.0583 [C9H12NaO5, calcd. 223.0582,
M+Na]+.
IUPAC NAME of 9: Acetic acid 2-oxo-hexahydro-furo[2,3-
medium supplemented with 100 mg/ml of each compound. MeOH
b]pyran-4-yl ester.
(0.5% v/v) and DMSO (0.5% v/v) were used as a negative control.
Metalaxyl-M (mefenoxam; p.a. 43.88%; Syngenta), PCNB and
Toclofos-methyl (Rizolex; p.a. 50%; Siapa), synthetic fungicides to
which Oomycetes, Ascomycetes and Basidiomycetes are sensitive,
were used as positive controls. Each disk was placed in the centre
of a Petri dish with mycelia in contact with the medium. The plates
were incubated at 20 or 25 8C depending on fungal species in the
dark until the target fungi used as negative control covered the
plate’s surface. Then the two perpendicular diameters of all fungi
were measured and the results were reported as inhibition of
growth. Three replicates were tested for each isolate and each
assay was performed twice. Based on the in vitro antifungal
preliminary screening, EC50 (effective dose required for 50%
inhibition growth) values of the more active compounds were
evaluated in comparison with metalaxyl-M, PCNB and toclofos-
methyl. The experiment was performed using the same medium
used in the preliminary test amended with each compound at
2.9. 2-(2,3-Dihydro-4-pyronyl)acetic acid (10)
To compound 5 (5.0 mg) in Me2CO (1.0 ml), at 0 8C, Jones’s
reagent (Bowers et al., 1953) was added under stirring as
previously reported to convert
(13.2 mg) of the work-up reaction was purified by TLC on reverse
1 into for 8. The residue
phase, eluent MeOH–H2O (1:1), giving 10 (1.4 mg, 30%, Rf 0.70) as
amorphous solid. 8: UV
lmax nm (log e): 268 (3.72); IR nmax 3416,
1725, 1659, 1612, 1601 cmꢀ1 [lit: (Scott et al., 1972) lmax (EtOH)
267 nm (e
7960); IR nmax (CHCl3) 3510, 1718, 1672, 1621]; 1H
NMR: see Table 1; ESIMS (+), m/z 179 [M+Na]+; ESIMS (ꢀ), m/z 155
[MꢀH]ꢀ; APCI MS (+), m/z 157 [M+H]+; APCIMS (ꢀ), m/z 155
[MꢀH]ꢀ.
IUPAC NAME of 10: (4-Oxo-5,6-dihydro-4H-pyran-3-yl)-acetic
acid.
concentration of 0.1, 1, 10, 50 and 100
mg/ml. MeOH (0.5% v/v) and
2.10. Methyl ester of 2-(4-pyronyl)acetic acid (11)
DMSO (0.5% v/v) were used as a negative control. Three replicate
Petri dishes were used for each treatment. The dishes were
inoculated, incubated and colony growth was determined as
described above.
To compound 8 (2.5 mg) dissolved in MeOH (1.0 ml), was
slowly added an ethereal solution of CH2N2 until a yellow colour
was persistent. The solvent was evaporated under a N2 stream and
the residue (3.1 mg) purified by preparative TLC on silica gel,
eluent CHCl3-i-PrOH (93:7), giving 11 (1.2 mg, 48.0%, Rf 0.5) as a
2.11.4. Analysis of data
Each experiment in this study was repeated twice with three
replicates. All data were processed with XLSTAT software
white solid. 11: UV lmax nm (log e): 205 (2.91), 248 (2.95); IR nmax
1731, 1650, 1610, 1438 cmꢀ1; 1H NMR see Table 1; ESIMS (+), m/z
191 [M+Na]+; APCIMS (+), m/z 169 [M+H]+, 137 [M+CH3O]+.
IUPAC NAME of 11: (4-Oxo-4H-pyran-3-yl)-acetic acid methyl
ester.
(Addinsoft, Damremont, Paris, France). Probit analysis was used
´
to calculate the effective concentration values that inhibited
mycelial growth by 50% (EC50).
3. Results and discussion
2.11. Biological assays
Eight derivatives of oxysporone were hemisynthezised in order
to carry out a structure-activity relationships study aimed to
obtain compounds with improved activities and specificity.
Firstly 1 was converted into the corresponding 4-O-acetyl
derivative (4, Fig. 2) by usual acetylation carried out with acetic
anhydride and pyridine; 4 showed the reversible modification of
the hydroxy group at C-4 of dihydropyran ring. A different
modification of the same hydroxy group was obtained by
conversion of 1 into the corresponding 4-O-p-bromobenzoyl
derivative (7, Fig. 2) by reaction with p-bromobenzoyl chloride
carried out in CH3CN. 7 differed from 4 for the aromatic and bulky
nature of the acyl group.
2.11.1. Leaf puncture assay
Young cork oak (Quercussuber L.), holm oak (Q. ilex L.) and
grapevine (Vitisvinifera L.) leaves were utilized for this assay. Each
compound was assayed at 1.0 mg/ml. Compounds were first
dissolved in MeOH, and then a stock solution with sterile distilled
water was made. A droplet (20
the adaxial sides of leaves that had previously been needle
punctured. Droplets (20 l) of MeOH in distilled water (4%)
ml) of test solution was applied on
m
were applied on leaves as control. Each treatment was repeated
three times. The leaves were kept in a moist chamber to prevent
the droplets from drying. Leaves were observed daily and scored

44
A. Andolfi et al. / Phytochemistry Letters 10 (2014) 40–45
The dihydropyran ring was modified by catalytic hydrogenation
compounds 4 and 6 retained the phytotoxicity, whereas the
derivatives 5, 7, 8, 9, 10 and 11 were inactive. Specifically,
the monoacetyl derivative (4) of oxysporone exhibited the same
moderate activity as that shown by 6 on all plant species tested
(lesion sizes ranging from 3.50 to 7.83, and from 4.28 to 7.86 mm2,
respectively).
In tomato cuttings bioassay, the compounds 1, 4, and 6 were
active. Oxysporone (1) caused wilting within 48 h from the
application at 0.2 mg/ml. Stewing or wilting symptoms were also
observed with this compound at 0.1 and 0.05 mg/ml. Both 4 and 6
caused stewing on stem at 0.2 and 0.1 mg/ml. None of the other
compounds caused any visible symptoms in this bioassay at the
highest concentration used.
of its double bond. The reaction was carried out both on
oxysporone (1) and on its acetylated derivative (4), yielding the
corresponding 2,3-dihydroderivative 5 and 9 (Fig. 2).
Oxysporone showed a different reactivity in respect to some
oxidant reagents. In fact, when the reaction was conducted with
Corey’s reagent (Corey and Suggs, 1975), the expected oxidation of
secondary hydroxy group at C-4 was observed, yielding the
corresponding
a,b-unsaturated ketone (6, Fig. 2). Instead, when
the reaction was performed with Jones’s reagent (Bowers et al.,
1953), the derivative 6 was not obtained and the only product of
the oxidation was 2-(4-pyronyl) derivative of acetic acid (8, Fig. 2).
A probable reaction mechanism, which justify the conversion of 1
into 8, is in agreement with the literature data (Edwards et al.,
2001). The strong acid conditions probably induced the enolization
of the ketone group at C-4 of 6 and the protonation of the carbonyl
of the furanone ring. This latter rearranged into the 2-(4-
pyronyl)acetic acid (8) being the driving force the stability of
the 4-pyrone ring. This compound, named xylaric acid, had been
firstly isolated from Xylaria spp. (Salvatore et al., 1994). Then it was
also isolated from Xylaria grammica (Mont.) Fr. along with
grammicin (Edwards et al., 2001) and from Pestalotiopsis sp.
HC02 isolated from Chondracris rosee gut together pestalotines A
and B (Zhang et al., 2008). The acid 8 was converted into the
corresponding methyl ester 11 (Fig. 2) by reaction with an ethereal
solution of diazomethane.
As already observed for oxysporone, the oxidation conducted
with Jones’s reagent on the 2,3-dihydrooxysporone (5, Fig. 2) gave
the expected 2-(2,3-dihydro-4-pironyl)acetic acid (10), probably
through a reaction mechanism similar to that hypothesized for
formation of 8. 10, also known as desoxypatulinic acid, was
previously obtained by synthesis (Woodward and Singh, 1950) and
by hydrogenation of patulin (Engel et al., 1949). It was also
successively isolated from cultures of Penicillium patulum (Scott
et al., 1972) and from an unidentified marine fungus reported as
Penicillium sp. (Wu et al., 2010).
From the analysis of the structures and the activities displayed
by all compounds, structure–activity relationships and some
structural features related to the phytotoxic activity can be
extracted.
The dihydrofuropyran carbon skeleton is a structural feature
important for the activity considering that the oxidative opening of
the dihydrofuran ring into the corresponding 2-substituted acetic
acid joined with the conversion of the pyran ring into the
corresponding semiquinone as in 8 and 11 and the corresponding
2,3-dihydro derivative 10 determine the complete loss of activity.
The double bond of the dihydropyran ring present in 1 appears to
be also an important structural feature for phytotoxic activity. In
fact, derivatives as 5 and 9 lacking this double bond were inactive.
This was also confirmed by the inactivity of derivative 10. The
hydroxy group at C-4 seems also to be somehow associated with
the phytotoxic effects. In fact, the acetylation of this functional
group as in derivative 4 led to a decrease of activity, and the
introduction of a bulky substituent in the same position as in
derivative 7 resulted in the total loss of activity. The oxidation by
Corey’s reagent of hydroxy group at C-4 seems only to decrease the
bioactivity of 1 as observed testing derivatives 6.
All compounds, with the exception of 10 and 11, obtained in
small quantities, were preliminary screened for antifungal
The structures of all derivatives 4–11 were determined by
comparing their spectroscopic data, essentially 1H NMR (see
Table 1) and ESI and/or APCIMS, with those of oxysporone.
Two bioassays were used to investigate the phytotoxic activity
of oxysporone (1), its derivatives (4–11). In the leaf-puncture
bioassay, the phytotoxicity was evaluated on holm oak, cork
oakand grapevineleaves. As shown in Table 2, oxysporone (1)
confirmed its strong toxicity on all species tested causing necrotic
lesions ranging from 15.81 mm2 on cork oak leaves to 23.04 mm2
on grapevine leaves. Among oxysporone (1) derivatives,
activities in vitro against four plant pathogens at 100 mg/ml (see
experimental). The results summarized in Table 2 showed that
among all the tested compounds only compound 7 displayed
significant activities (35.6–100% inhibition rates) against all
pathogens tested at 100
mg/ml. Oxysporone, the other derivatives
4–6 and 8–9 were inactive at the same concentration. Fungal
sensitivity to 7 varied according to the species. Athelia rolfsii and
Phytophthora plurivora appeared to be more sensitive (100%
inhibition) to this compound. Diplodia corticola showed to be
less sensitive (inhibition <40%), whereas on P. cinnamomi the
Table 2
Biological data for oxysporone (1) and its derivatives (4–11)a
Bioassay
Target
Leaf puncture bioassayb
Cuttingsc
Antifungal activityd
Holm oak
Cork oak
Grapevine
Tomato
Diplodia
corticola
Phytophthora
Phytophthora
plurivora
Athelia
rolfsii
cinnamomi
Compound
1
4
19.58 ꢂ 1.62
15.81 ꢂ 1.75
23.04 ꢂ 0.97
Wilting
Stewing
na
na
na
na
na
35.6
na
na
nt
na
na
na
na
77.2
na
na
nt
na
na
na
na
100
na
na
nt
na
na
na
na
100
na
na
nt
4.05 ꢂ 0.53
3.50 ꢂ 0.47
7.83 ꢂ 1.85
5
na
na
na
6
4.28 ꢂ 0.37
4.42 ꢂ 0.92
7.86 ꢂ 0.66
Stewing
na
7
na
na
na
na
na
na
na
na
na
na
na
na
na
na
na
8
na
9
na
10
11
na
na
nt
nt
nt
nt
na, inactive; nt, not tested.
a
PCNB, Toclofos-Methyl and Metalaxyl-M were used as positive controls
b
c
The compounds were tested at concentration of 1 mg/ml. Data are expressed as median area lesions ꢂ error standard (mm2)
Symptoms developed within 2 days on tomato at 0.2 mg/ml;
d
Data expressed as percentage of linear growth inhibition at 100 mg/ml

A. Andolfi et al. / Phytochemistry Letters 10 (2014) 40–45
45
Table 3
Acknowledgment
EC50 Values of derivative 7, toclofos-methyla, PCNB and metalaxyl-M against three
pathogens belonging to Oomycetes and Basidiomycetes.
NMR spectra were recorded by Mrs Dominique Melck, who is
acknowledged, in the laboratory of Istituto di Chimica Biomole-
colare del CNR, Pozzuoli, Italy.
Target
EC50 (mg/ml)
Phytophthora
cinnamomi
Phytophthora
plurivora
Athelia
rolfsii
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Conflicts of interest
Zhang, Y.L., Ge, H.M., Li, F., Song, Y.C., Tan, R.X., 2008. New phytotoxins metabolites
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The authors declare no conflicts of interest.