Structure-activity relationship in the interaction of substituted perinaphthenones with Mycosphaerella fijiensis

Article abstract of DOI:10.1021/jf901052e

The levels of native fungitoxic perinaphthenone phytoalexins in susceptible Musa varieties (banana), which are commercially grown in large plantations, are too low to provide plants with long-lasting protection against highly pathogenic fungi. Novel strategies for plant protection are necessary to reduce crop losses and to prevent the development of resistant fungal strains. The synthesis of novel fungicides based on the structures of perinaphthenone natural products is considered to be a promising strategy. Thirteen substituted perinaphthenones, among them two known natural products (1, 2) and 11 synthetics (3-13), were evaluated for their activity against Mycosphaerella fijiensis, and their half-maximal inhibitory concentrations (IC₅₀) were calculated to establish structure-activity relationships (SAR). A SAR trend was hypothesized, leading to the design of a new compound, 4-methoxy-2-nitro-1H- phenalen-1-one (14); the new compound displayed significantly enhanced in vitro activity against M. fijiensis compared to other perinaphthenone derivatives. The activity of 14 was comparable to that of two commercial fungicides.

Full text of DOI:10.1021/jf901052e

J. Agric. Food Chem. 2009, 57, 7417–7421 7417
DOI:10.1021/jf901052e
Structure-Activity Relationship in the Interaction of
Substituted Perinaphthenones with Mycosphaerella fijiensis
†
IDALGO,† LUISA
´
D
UQUE,‡ JAIRO
S
AEZ,‡ RAFAEL
§
A
RANGO,† JESUS
GIL,
ꢀ
W
ILLIAM
H
,‡
B
ENJAMIN
R
OJANO,† BERND
SCHNEIDER
,
AND
F
ELIPE
O
TALVARO
*
ꢀ
†Universidad Nacional de Colombia sede Medellı
Colombia, ‡Instituto de Quı
´
n, Autopista Norte, Calle 64 Cra 65, AA 1027 Medellı
´
n,
´
mica, Universidad de Antioquia, Calle 67 # 53-108, AA 1226 Medellın,
´
::
::
::
Colombia, and §Max Planck Institut fur Chemische Okologie, Beutenberg Campus, Hans-Knoll-Strasse 8,
07745 Jena, Germany
The levels of native fungitoxic perinaphthenone phytoalexins in susceptible Musa varieties (banana),
which are commercially grown in large plantations, are too low to provide plants with long-lasting
protection against highly pathogenic fungi. Novel strategies for plant protection are necessary to
reduce crop losses and to prevent the development of resistant fungal strains. The synthesis of
novel fungicides based on the structures of perinaphthenone natural products is considered to be
a promising strategy. Thirteen substituted perinaphthenones, among them two known natural
products (1, 2) and 11 synthetics (3-13), were evaluated for their activity against Mycosphaerella
fijiensis, and their half-maximal inhibitory concentrations (IC50) were calculated to establish
structure-activity relationships (SAR). A SAR trend was hypothesized, leading to the design
of a new compound, 4-methoxy-2-nitro-1H-phenalen-1-one (14); the new compound displayed
significantly enhanced in vitro activity against M. fijiensis compared to other perinaphthenone
derivatives. The activity of 14 was comparable to that of two commercial fungicides.
KEYWORDS: Mycosphaerella fijiensis; banana; perinaphthenone; phenalenone; phototoxicity; reactive
oxygen species
INTRODUCTION
plants tofungal infection would be greatly reduced. Here, another
promising approach, namely, designing synthetic fungitoxic
agents based on the perinaphthenone structure, is reported.
Recently, some natural perinaphthenones, among them
compounds 1 and 2 in Musa acuminata cv. ’Yangambi’, were
identified as the most potent class of compounds isolated from
this plant; they are active against the ascomycetous fungal
pathogen Mycosphaerella fijiensis. As a result, the perinaphthe-
none nucleus has come to be seen as an interesting structural
motif for the development of a new class of fungicides (7). One
reason for the interest in the perinaphthenone moiety is its
plausible phototoxic mode of action (8-10): perinaphthenone,
a well-known photosensitizer (1), is presumed to generate singlet
molecular oxygen (1O2) and act as a catalyst for the production of
other reactive oxygen species, which may in turn cause cell
damage to the pathogen (8).
Bananas are among the most important crops worldwide; they
are a staple food in many developing countries and a crucial
economic factor for the communities that produce them (1).
Nearly all currently grown cultivars of Musa (banana) are derived
from two species, Musa acuminata (A genome) and Musa
balbisiana (B genome), and belong to the “Cavendish” subgroup.
The commercial cultivation of genetically closely related clones of
the Cavendish banana in monocultures has boosted outbreaks of
invading insects and nematodes and resulted in devastating
fungal infections. The appearance of phytopathogenic fungi such
as Fusarium oxysporum and Mycosphaerella fijiensis and their
global proliferation has caused the Panama disease and Black
Sigatoka disease, among others. Detrimental crop losses have led
to a debate about the future of banana production (1-3).
Phenalenones (perinaphthenones) and their phenyl derivatives
and oxidative transformation products have been identified as
native defense compounds and are reported as phytoalexins of
Musa (4-6). Resistant Musa varieties accumulate perinaphthe-
nones to much higher levels in their tissue than susceptible
varieties do (7). Hence, the enhancement of the biosynthetic
potential for native perinaphthenones by either conventional
breeding or genetic modification is considered to be a possible
way to improve Musa plants; the susceptibility of such improved
To produce singlet oxygen (3O2f1O2), the photon-excited
perinaphthenone must undergo intersystem crossing to a triplet
state from which deactivation can occur by different competing
energy transfer processes such as phosphorescence, thermal
decay, or radical reactions with other compounds (12). Therefore,
suitable experimental conditions have to be designed for each
particular plant pathosystem (13) to prove the involvement
of 1O2. Previous bioassays with natural perinaphthenones active
against M. fijiensis were conducted in the dark (7). Because
phototoxicity cannot be assessed under conditions of light
exclusion, it was not clear if the tested compounds displayed
*Corresponding author (telephone 05742195653; fax 05742330120;
e-mail pipelion@quimica.udea.edu.co).
© 2009 American Chemical Society
Published on Web 07/24/2009
pubs.acs.org/JAFC

7418 J. Agric. Food Chem., Vol. 57, No. 16, 2009
Hidalgo et al.
photodynamic activity. However, when there is more than one
mode of action, light exclusion is suitable for distinguishing
phototoxicity from other activities. This paper reports the activity
of two known natural (1, 2) and 12 synthetic (3-14) perinaphthe-
nones against M. fijiensis in the dark and under light-controlled
conditions. Compounds 1-13 were chosen to gain information
on structure-activity relationship (SAR) trends, which led to the
design of a new compound, 4-methoxy-2-nitro-1H-phenalen-1-
one (14).
preparative TLC using CH2Cl2/MeOH (40:1, Rf 0.20) as eluent to afford
an orange powder (102 mg, 55%): 1H NMR (C3D6O, 500.13 MHz) δ 8.51
(dd, J = 7.9, 1.2 Hz, H-9), 8.38 (d, J = 9.0 Hz, H-3), 7.91 (dd, J=7.4, 1.2
Hz, H-7), 7.80 (d, J = 9.4 Hz, H-6), 7.48 (dd, J = 7.9, 7.4 Hz, H-8), 6.84 (d,
J = 9.4 Hz, H-5), 6.82 (d, J = 9.0 Hz, H-2); 13C NMR (C3D6O, 125.75
MHz) δ 176.6 (C-4), 175.2 (C-1), 138.2 (C-6), 135.6 (C-3), 132.9 (C-7),
131.0 (C-9b), 128.8 (C-9), 128.1 (C-6a), 127.7 (C-5), 127.3 (C-9a), 123.7
(C-8), 118.0 (C-2), 116.6 (C-3a); HREIMS, m/z 196.053394 (calcd for
C13H8O2, 196.052430).
2-Amino-1H-phenalen-1-one (9). A balloon filled with hydrogen
was fitted to a 10 mL round-bottom flask charged with the catalyst (10%
Pd/C, 2.5 mg) and a solution of 13 (5 mg) in MeOH (5 mL). The mixture
was stirred at 25 °C for 10 min, after which the catalyst was filtered and the
solvent evaporated. Preparative TLC (n-hexane/ethyl acetate 2:1, Rf 0.62)
afforded the desired compound (4 mg, 98%) as a deep red solid: 1H NMR
(C3D6O, 500.13 MHz) δ 8.61 (dd, J = 7.3, 1.2 Hz, H-9), 8.33 (dd, J=8.0,
1.2 Hz, H-7), 7.88 (dd, J = 7.9, 1.5 Hz, H-6), 7.83 (dd, J = 7.3, 8.0 Hz,
H-8), 7.59 (dd, J=7.3, 1.5 Hz, H-4), 7.56 (dd, J = 7.3, 7.9 Hz, H-5), 6.89
(s, H-3); 13C NMR (C3D6O, 125.75 MHz) δ 180.9 (C-1), 143.0 (C-2), 136.3
(C-7), 133.1 (C-6a), 131.1 (C-3a), 130.5 (C-9), 129.2 (C-9a), 128.2 (C-5),
127.9 (C-4), 127.8 (C-6), 127.5 (C-8), 124.7 (C-9b), 110.0 (C-3); LC-
HRESIMS (Micromass Quattro II tandem quadrupole mass spectro-
meter, Micromass Ltd., Manchester, U.K.), m/z 194.06097 (calcd for
C13H8NO, 194.06059).
MATERIALS AND METHODS
General Experimental Procedures. 1H NMR, 13C NMR, DEPT
135, 1H-1H COSY, HMBC, and HMQC spectra of synthetic compounds
were recorded ona BrukerDRX 500 NMR spectrometerequipped with an
ATM inverse probe (5 mm) or a Bruker AV 500 NMR spectrometer
equipped with a TCI cryoprobe (5 mm) (Bruker-Biospin, Karlsruhe,
Germany). The spectra were referenced to internal TMS in all cases.
For routine measurements, a Bruker AMX III 300 spectrometer was
employed. If not otherwise indicated, HREIMS was run on a Micromass
MasSpec mass spectrometer (Micromass Ltd., Manchester, U.K.) at 70 eV
with a direct insertion probe.
Biological Material. M. fijiensis was isolated from naturally infected
banana leaves supplied by the Asociacio
´
n de Bananeros de Colombia
(AUGURA) from Apartado, Colombia, according to the method re-
2-Bromo-1H-phenalen-1-one (11). The method of Imanzadeh
et al. (23) for bromination was adapted in this case. Flame-dried neutral
alumina (1.4 g), N-bromosuccinimide (303 mg, 1.7 mmol), and peri-
naphthenone (186 mg, 1 mmol) were ground in a mortar at room
temperature until a uniform color was perceived. The mixture was then
transferred to a test tube and heated at 45 °C for 3 h. Extraction with
CH2Cl2 (20 mL) and preparative TLC (n-hexane/dichloromethane 1:1,
Rf 0.28) afforded a bright yellow solid (150 mg, 58%, 98% based on
recovered perinaphthenone): 1H NMR (C3D6O, 500.13 MHz) δ 8.63 (d,
J = 7.4 Hz, H-9), 8.48 (s, H-3), 8.46 (d, J = 8.2 Hz, H-7), 8.27 (d, J =
8.2 Hz, H-6), 8.02 (d, J = 7.1 Hz, H-4), 7.92 (dd, J = 8.2, 7.4 Hz, H-8),
7.74 (dd, J = 8.2, 7.1 Hz, H-5); 13C NMR (C3D6O, 125.75 MHz) δ 178.6
(C-1), 144.2 (C-3), 136.7 (C-7), 133.5 (C-6), 133.3 (C-6a), 133.0 (C-4), 132.3
(C-9), 129.4 (C-9a), 128.7 (C-3a), 128.5 (C-8), 128.2 (C-5), 127.4 (C-9b),
126.0 (C-2); HREIMS, m/z 257.967261 (calcd for C13H779BrO,
257.968026).
´
ported by Quinones et al. (8) Briefly, isolates of M. fijiensis were obtained
from leaves infected with Black Sigatoka by discharging ascospores over a
2% aqueous potato dextrose agar (PDA) solution and were maintained on
PDA in test tubes held at 25 ( 2 °C. Isolates of M. fijiensis were
characterized by PCR amplification of the internally transcribed spacer
region of rDNA to differentiate it from M. musicola (14). The following
oligonucleotide sequences were employed: MF137 50GGCGCCCC-
CGGAGGCCGTCTA30 (specific for M. fijiensis) and MM137 50 GGC-
GCCCCCGGAGGTCTCCTT30 (specificfor M. musicola) in conjunction
with the nonspecific primer R635 50GGTCCGTGTTTCAAGA-
CGG30 (14). Strains of M. fijiensis were classified according to the system
of Canas et al. (15) and are maintained in the Unidad de Biotecnologı
Vegetal UNALMED-CIB (Medellın, Colombia) under vouchers 060124
and 080105.
´
a
´
Synthetic Methods. 3-Hydroxyperinaphthenone (3) and perinaphthe-
none (10) were purchased from Aldrich (Milwaukee, WI) and, prior to
being bioassayed, purified by preparative TLC using Et2O/n-hexane (2:1)
as an eluent (Rf 0.21 and 0.68, respectively). 2-Hydroxyperinaphthenone
(1) (7), 2-methoxyperinaphthenone (2) (7), 4-methoxyperinaphthenone
(6) (16), 6-hydroxyperinaphthenone (7) (17), and 6-methoxyperinaphthe-
none (8) (17) were prepared and purified according to reported methods.
Compounds 4 (18), 5 (19), 9 (20), 11 (21), 12 (22), and 13 (20) were
previously reported, but their synthesis followed different procedures and/
or their spectroscopic characterization is described here for the first time.
3-Methoxy-1H-phenalen-1-one (4). An ethanol-containing so-
lution of diazomethane was prepared from Diazald using a standard
procedure. This solution was added dropwise to 3-hydroxy-1H-phenalen-
1-one (3) (5 mg, 0.026 mmol) until gas evolution had ceased. The reaction
mixture was dried under a stream of nitrogen gas and the compound
purified by preparative TLC (silica gel 60 F254, 1 mm layer thickness,
Merck, Darmstadt, Germany) using CH2Cl2-/MeOH (40:1, Rf 0.70) as
eluent to afford the desired compound (5 mg) as a yellow solid: 1H NMR
(C3D6O, 500.13 MHz) δ 8.45 (dd, J = 7.2, 1.3 Hz, H-9), 8.30 (dd, J=8.1,
1.3 Hz, H-7), 8.23 (dd, J=7.3, 1.2 Hz, H-4), 8.21 (dd, J = 8.3, 1.2 Hz, H-
6), 7.80 (dd, J = 8.1, 7.2 Hz, H-8), 7.71 (dd, J = 8.3, 7.3 Hz, H-5), 6.08 (s,
H-2), 4.07 (s, -OCH3); 13C NMR (C3D6O, 125.75 MHz) δ 184.9 (C-1),
166.8 (C-3), 134.9 (C-7), 133.1 (C-6), 133.1 (C-6a), 129.5 (C-9a), 129.4 (C-
9), 128.0 (C-9b), 127.6 (C-8), 127.2 (C-5), 126.8 (C-4), 125.7 (C-3a), 104.3
(C-2), 56.7 (-OCH3); HREIMS, m/z 210.068131 (calcd for C14H10O2,
210.068080).
2-Chloro-1H-phenalen-1-one (12). Concentrated (65%) HNO3
(186 μL) was added to perinaphthenone (10) (100 mg, 0.56 mmol)
dissolved in concentrated (36%) HCl (5 mL), and the mixture was stirred
for 3 h at room temperature. Liquid-liquid partition between H2O and
CH2Cl2 (3 ꢀ 15 mL) followed by preparative TLC (n-hexane/dichloro-
methane 2:3, Rf 0.65) afforded the desired compound in 55% yield as a
yellow solid: 1H NMR (C3D6O, 500.13 MHz) δ 8.66 (dd, J = 7.4, 1.2 Hz,
H-9), 8.49 (dd, J = 8.1, 1.2 Hz, H-7), 8.28 (dd, J = 8.3, 1.1 Hz, H-6), 8.27
(d, J = 0.5 Hz, H-3), 8.05 (ddd, J = 7.1, 1.1, 0.5 Hz, H-4), 7.95 (dd, J=8.1,
7.4 Hz, H-8), 7.77 (dd, J = 8.3, 7.1 Hz, H-5); 13C NMR (C3D6O, 125.75
MHz) δ 178.7 (C-1), 140.1 (C-3), 136.8 (C-7), 133.9 (C-2), 133.4 (C-6),
133.3 (C-6a), 133.1 (C-4), 132.1 (C-9), 129.9 (C-9a), 128.5 (C-8), 128.2 (C-
5), 128.1 (C-3a), 127.1 (C-9b); HREIMS, m/z 214.018414 (calcd for
C13H735ClO, 214.018543).
2-Nitro-1H-phenalen-1-one (13). The method reported by
Dokunikhin et al. (20) was employed in which a mixture of concentrated
(65%) nitric acid (42 μL) and concentrated (98%) sulfuric acid (121 μL)
was added dropwise to a cooled solution (0 °C) of perinaphthenone (10)
(100 mg, 0.6 mmol) dissolved in concentrated sulfuric acid (2 mL). The
reaction mixture was stirred for 1 h at room temperature, H2O (10 mL) was
added, and the mixture was extracted with CH2Cl2 (3 ꢀ 15 mL). The
organic layer was dried and concentrated in vacuo. Preparative TLC (n-
hexane/ethyl acetate 1:1, Rf 0.65) afforded the desired compound (yellow
solid) in 30% yield (55% based on recovered perinaphthenone): 1H NMR
(C3D6O, 500.13 MHz) δ 8.77 (s, H-2), 8.71 (dd, J = 7.3, 1.2 Hz, H-9), 8.59
(dd, J=8.1, 1.2 Hz, H-7), 8.03 (dd, J = 7.3, 8.1 Hz, H-8), 7.91 (dd, J=7.2,
8.3 Hz, H-5), 7.50 (dd, J=7.3, 1.1 Hz, H-6), 7.40 (dd, J=7.2, 1.1 Hz, H-4);
13C NMR (C3D6O, 125.75 MHz) δ 175.2 (C-1), 147.9 (C-2), 138.8 (C-3),
137.7 (C-7), 137.6 (C-4), 136.6 (C-6), 133.3 (C-6a), 132.4 (C-9), 130.5 (C-
9a), 129.0 (C-8), 128.6 (C-5), 127.8 (C-9b), 125.2 (C-3a); HREIMS, m/z
225.043327 (calcd for C13H7NO3, 225.042593).
4-Hydroxy-1H-phenalen-1-one (5). 4-Methoxyperinaphthenone
(6) (200 mg, 0.95 mmol) was treated with 47% HBr (2.8 mL) in acetic acid
(20 mL) and refluxed under nitrogen for 9 h. Acetic acid was removed
under vacuum, and the crude mixture was extracted with CH2Cl2/H2O.
The organic layer was dried (Na2SO4), concentrated, and purified by

Article
J. Agric. Food Chem., Vol. 57, No. 16, 2009 7419
enhanced activity of the methoxylated compounds. That com-
pound 1 and its O-methyl analogue 2 (Figure 2a) show nearly
equal activities in the bioassay can be rationalized in terms of a
strong intramolecular hydrogen bond present in 1, which gives it
an even lower polarity than its O-methyl counterpart 2 as revealed
by the Rf values (0.77 for 2 and 0.88 for 1) in normal phase silica
gel (CH2Cl2/MeOH 40:1). It is worth noting that compounds 3, 5,
and 7 offer the possibility of strong intermolecular hydrogen
bonding (24), which seems to diminish the activity. That light had
no effect on the activities of compounds 1-8 against M. fijiensis
prompted us to explore other SAR trends in the perinaphthenone
series.
Polycyclic aromatic R,β-unsaturated ketones are well recog-
nized for their Michael acceptor ability (25); therefore, peri-
naphthenones seemed to be a good candidate for this mode of
action. Accordingly, electron-withdrawing groups attached at the
C-2 position of perinaphthenone should increase the activity,
and, conversely, the opposite effect should be observed for
electron-donating groups. To explore this possibility, peri-
naphthenone derivatives 9-13 (Figure 1) were chosen with
substituents that affect the electron density of the double bond
by either inductive or conjugation effects, and these were assayed
in the dark. Electron-withdrawing groups attached to the C-2
position ofthe perinaphthenone nucleus dramaticallyincrease the
activity of perinaphthenones (Figure 2b). 2-Nitroperinaphthe-
none (13) was the most active compound in this series (Figure 2b),
as expected for a conjugated nitroalkene under a Michael
acceptor mode of action (26). Compounds 10-13 were the only
ones in this series for which activity was enhanced under light
(Tukey variance analysis, 95% confidence). Specifically, com-
pounds 11-13 with electron-withdrawing groups at C-2 dis-
played activity enhancement with both strains of M. fijiensis.
This confirms previous evidence indicating that electron-with-
drawing groups in position C-2 of perinaphthenone result in
molecules with high quantum yields of 1O2 production (27). In the
case of perinaphthenone (10), significant differences between
activities in the light and the dark were observed only for strain
060124 (Figure 2b).
Figure 1. Synthetic perinaphthenones (1-14) assayed in this work.
Compounds 1 and 2 occur in M. acuminata var. Yangambi (7).
4-Methoxy-2-nitro-1H-phenalen-1-one (14). Concentrated
HNO3 (30 μL) was added to 4-methoxyperinaphthenone (6) (70 mg,
0.3 mmol) dissolved in acetic acid (7 mL), and the mixture was stirred for
3 h at room temperature. Evaporation of the solvent was followed by
liquid-liquid partition between H2O and CH2Cl2. Preparative TLC
purification (n-hexane/ethyl acetate 2:1, Rf 0.14) afforded 14 (yellow solid,
28 mg, 33%, 65% based on recovered 6): 1H NMR (C3D6O, 500.13 MHz)
δ 8.94 (s, H-2), 8.70 (dd, J=7.6, 1.2 Hz, H-9), 8.54 (d, J = 9.3 Hz, H-6),
8.47 (dd, J=7.9, 1.2 Hz, H-7), 7.85 (dd, J = 7.6, 7.9 Hz, H-8), 7.74 (d, J=
9.3 Hz, H-5), 4.30 (s, -OCH3); 13C NMR (C3D6O, 125.75 MHz) δ 174.4
(C-1), 164.5 (C-4), 146.1 (C-2), 140.7 (C-6), 137.5 (C-7), 133.2 (C-9), 132.8
(C-3), 130.7 (C-9a), 129.1 (C-9b), 128.7 (C-6a), 126.7 (C-8), 115.3 (C-5),
110.2 (C-3a), 57.9 (-OCH3); HREIMS, m/z 255.053327 (calcd for
C14H9NO4, 255.053158).
Bioassays. A microtiter well method (7) was applied according to
which measures of the mycelial growth were taken after 8 days of
incubation (logarithmic growth phase of the fungi) in the dark or under
light-controlled conditions. For the light-controlled experiments, the
plates were placed at a distance of 1 m from 4 ꢀ 2 white light tubes
(Sylvania F32T8, 20 W, Daylight 154) in an incubation chamber with a
12 h photoperiod. In all cases, the working inoculum concentration was
adjusted to 2 ꢀ 105 mycelial fragments/mL. Statistical analysis was
performed according to the method of Lazzaro et al. (9). The IC50 value
was obtained by interpolation in the corresponding myceliar growth
versus phenalenone concentration plots. All statistical analyses were done
using the SAS software version 9.1.
Comparison of the O-methyl derivatives 4, 6, and 8 with the
unsubstituted perinaphthenone 10 revealed that the O-methyl
group always increased activity, compound 6 being the most
active. This finding demonstrated the relevance of a methoxyl
substituent at C-3, C-4, or C-6 in this nucleus and offered us an
opportunity to explore whether an additional electron-withdraw-
ing group at C-2 of O-methyl derivatives 4, 6, or 8 could have a
synergistic effect. Therefore, 2-nitro-4-methoxyperinaphthenone
(14) (Figure 1) was synthesized, and its activity was measured
under light-controlled experiments (Figure 2b).
RESULTS AND DISCUSSION
To check for photodynamic activity, 2-hydroxyperinaphthe-
none (1) and 2-methoxyperinaphthenone (2) (Figure 1), both of
which occur in M. acuminata var. ’Yangambi’ (7), were assayed
for their activity against M. fijiensis (mycelial growth inhibition)
under light-controlled conditions using a slight modification of a
reported protocol (7) (Figure 2a). The activity of natural com-
pounds 1 and 2 did not significantly change under the influence of
light. This made us ask whether hydroxyl and methoxyl groups
generally prevent the photodynamic activities of perinaphthe-
nones or if substituents attached to the ortho position relative to
the carbonyl group were responsible. To address this question,
compounds 3-8 (Figure 1) were synthesized and assayed under
the same conditions (Figure 2a). With the exception of com-
pounds 1 and 2, replacing a hydroxyl group with a methoxyl
group increases the activity by >3-fold. Because no significant
effect of light was observed in this series (Tukey variance analysis,
95% confidence), an increase in lipophilicity may explain the
The IC50 value of compound 14 was the lowest among all tested
perinaphthenone compounds, and its in vitro activity was com-
parable to that of the commercial fungicides benomyl and
propiconazole (Figure 2b). Moreover, the activity of 2-nitro-4-
methoxyperinaphthenone (14) was significantly enhanced in
the presence of light. Comparison of the IC50 value of the
2-nitro-4-methoxy-substituted perinaphthenone 14 with the
values of the 4-methoxy derivative 6 and the 2-nitro derivative
13 (Figure 2) suggests that the two functional groups affect
the Michael acceptor capacity of the perinaphthenone struc-
ture without disturbing the photodynamic properties of the
core nucleus. In summary, these findings demonstrate that
the activity of perinaphthenones against M. fijiensis can be
enhanced by introducing electron-withdrawing groups at C-2
and a methoxyl substituent at C-4. The effects of other types
of substituents at C-4 on the activity against M. fijiensis deserve
more attention.

7420 J. Agric. Food Chem., Vol. 57, No. 16, 2009
Hidalgo et al.
Figure 2. Antifungalactivity ofcompounds1-14 onMycosphaerellafijiensis. Theeffectonmycelialgrowthwasmeasuredinthe darkandunderphotoperiods
of 12 h during 8 days of incubation. Commercial fungicides (benomyl and propiconazole) were tested for reference purposes.
ACKNOWLEDGMENT
yields of singlet oxygen O2(1Δg) sensitization. J. Photochem. Photo-
biol. A: Chem. 1994, 79, 11–17.
(12) Flors, C.; Ogilby, P. R.; Luis, J. G.; Grillo, T. A.; Izquierdo, L. R.;
Gentili, P.; Bussotti, L.; Nonell, S. Phototoxic phytoalexins. Pro-
cesses that compete with the photosensitized production of singlet
oxygen by 9-phenylphenalenones. Photochem. Photobiol. 2006, 82,
95–103.
We thank Sybille Lorenz for mass spectrometric analyses and
Emily Wheeler for editorial assistance.
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Received March 30, 2009. Revised manuscript received June 10, 2009.
Accepted July 14, 2009. This research was financially supported
by Universidad de Antioquia, Universidad Nacional de Colombia
::
sede Medellın, and the Max Planck Institut fur Chemische
::
Okologie.