Phenalenone-type compounds from Musa acuminata var. "Yangambi km 5" (AAA) and their activity against Mycosphaerella fijiensis

Article abstract of DOI:10.1021/np070091e

Two perinaphthenone-type compounds (1 and 2) were isolated together with four known phenylphenalenones (3-6) from the rhizomes of Musa acuminata var. "Yangambi km 5". The structures of the new phenalenones were assigned as 2-hydroxy-1H-phenalen-1-one (1) and 2-methoxy-1H-phenalen-1-one (2) on the basis of their spectroscopic data and were confirmed by synthesis. Compounds 1 and 2 displayed significantly enhanced activity against Mycosphaerella fijiensis in comparison with other phenylphenalenones.

Full text of DOI:10.1021/np070091e

J. Nat. Prod. 2007, 70, 887-890
887
Phenalenone-Type Compounds from Musa acuminata var. “Yangambi km 5” (AAA) and Their
Activity against Mycosphaerella fijiensis
Felipe Ota´lvaro,*,† Juliana Nanclares,‡ Luz Estella Va´squez,§ Winston Quin˜ones, Fernando Echeverri, Rafael Arango,| and
Bernd Schneider*,3
Departamento de Ingenier´ıa, UniVersidad Nacional de Colombia sede Palmira, Carrera 32 Chapinero V´ıa Candelaria, A.A 237, Palmira-Valle
del Cauca, Colombia, Instituto de Qu´ımica, UniVersidad de Antioquia, A.A 1226, Medell´ın, Colombia, Unidad de Biotecnolog´ıa Vegetal,
Corporacio´n para InVestigaciones Biolo´gicas, Carrera 72 A # 78 B-141, Medell´ın, Colombia, Laboratorio de Qu´ımica de Productos Naturales,
Edificio SIU 234-235, Apartado Ae´reo 1226, Medell´ın-Antioquia, Colombia, Escuela de Biociencias, Facultad de Ciencias, UniVersidad
Nacional de Colombia sede Medell´ın, Autopista Norte, Carrera 64 Calle 65, A.A 3840, Medell´ın, Colombia, and Max-Planck Institut fu¨r
Chemische O¨ kologie, Beutenberg Campus, Hans-Kno¨ll-Strasse 8, 07745, Jena, Germany
ReceiVed March 2, 2007
Two perinaphthenone-type compounds (1 and 2) were isolated together with four known phenylphenalenones (3-6)
from the rhizomes of Musa acuminata var. “Yangambi km 5”. The structures of the new phenalenones were assigned
as 2-hydroxy-1H-phenalen-1-one (1) and 2-methoxy-1H-phenalen-1-one (2) on the basis of their spectroscopic data and
were confirmed by synthesis. Compounds 1 and 2 displayed significantly enhanced activity against Mycosphaerella
fijiensis in comparison with other phenylphenalenones.
Black leaf streak (also known as Black Sigatoka), caused by the
ascomycetous fungal pathogen Mycosphaerella fijiensis, is currently
one of the predominant diseases in Musa spp., attacking almost all
cultivars of banana and plantains.1 One of the reasons for the
susceptibility of Musa species is the almost exclusive cultivation
of seedless, sterile, and genetically uniform clones, especially the
Cavendish banana. Efforts to breed resistant cultivars that can
compete with Cavendish in terms of crop yield and market
acceptance have had limited success. Hence, Cavendish continues
to be the most consumed banana in industrial countries.1 The
frequent application of fungicides is the main control measure where
a high incidence of pathogens exists. Since these cultivars develop
resistance and because of environmental issues, new, environmen-
tally friendly chemotherapeutic agents that can successfully protect
the plant are being sought. Conventional breeding and genetic
engineering may also offer ways to improve the resistance to fungal
infection by Musa species.1
“Yangambi km 5”, a Musa dessert-type variety that produces
small fruits, has shown resistance to several diseases and plagues
including black leaf streak, Yellow Sigatoka disease (caused by
Mycosphaerella musicola), and the borer nematode (Radopholus
similis).2 In the case of black leaf streak pathosystem, the incompat-
ibility of Musa “Yangambi” and Mycosphaerella fijiensis activates
fungal and plant metabolism. Incubating M. fijiensis with tricycla-
zole increased the concentration of 2,4,8-trihydroxytetralone, a
fungal pentaketide metabolite. Musa plants, upon inoculation with
M. fijiensis, respond with an increase of phenylalanine ammonia-
lyase activity.3 Activation of the phenylpropanoid pathway is in
accordance with the well-documented occurrence of phenylphe-
nalenone-type compounds in other Musaceae.4-7
nalenones with different resistance traits of M. fijiensis8 suggested
that these phytoalexins could also be involved in the resistance of
“Yangambi”.
This paper reports the occurrence of two new and four known
phenalenone-type compounds in “Yangambi” and their activity in
response to in vitro cultures of M. fijiensis.
Ethyl acetate extracts from the rhizomes of Musa acuminata cv.
“Yangambi” (group AAA) were analyzed for the occurrence of
phenylphenalenone-type compounds. Apart from compounds 1 and
2, the known phenylphenalenones 2-hydroxy-9-phenyl-1H-phe-
nalen-1-one (3, anigorufone),9 2-methoxy-9-phenyl-1H-phenalen-
1-one (4, methoxyanigorufone),10 2-hydroxy-4-phenyl-1H-phenalen-
1-one (5, 4′-dehydroxyirenolone, isoanigorufone),11 and 2-hydroxy-
4-(4-hydroxyphenyl)-1H-phenalen-1-one (6, 4′-methoxyirenolone)12
were isolated and identified by comparison with authentic standards.
Compound 4 was obtained as the major compound (5 mg kg-1
fresh material) followed by compound 1 (1 mg kg-1). The
concentration levels of compounds 2, 3, 5, and 6 varied in the range
0.2-0.5 mg kg-1
.
Hence, it was anticipated that the “Yangambi” variety, as a
member of the Musa genus, would also produce phytoalexins
belonging to the phenylphenalenone structural class. Furthermore,
the correlation of different accumulation profiles of phenylphe-
* Corresponding authors. (F.O.) Tel: 05722717000, ext 35710. Fax:
05722717008. E-mail: lfotalvarot@palmira.unal.edu.co. (B.S.) Tel: 049
3641 571600. Fax: 049 3641 571600. E-mail: schneider@ice.mpg.de.
† Universidad Nacional de Colombia sede Palmira.
‡ Universidad de Antioquia.
§ Corporacio´n para Investigaciones Biolo´gicas.
Sede de Investigacio´n Universitaria-Universidad de Antioquia.
| Universidad Nacional de Colombia sede Medell´ın.
3 Max-Planck Institut fu¨r Chemische O¨ kologie.
The 1H NMR and 1H-1H COSY spectra of compound 1
displayed eight signals corresponding to two ABX spin systems
attributable to H-4-H-5-H-6 and H-7-H-8-H-9 and two singlets
10.1021/np070091e CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 04/12/2007

888 Journal of Natural Products, 2007, Vol. 70, No. 5
Notes
Table 1. 1H (500 MHz) and 13C NMR (125 MHz)
Spectroscopic Data of Compounds 1 and 2 Measured in
Acetone-d6
Those compounds were 1H-phenalen-1-one (7, perinaphthenone),
2-hydroxy-9-(4-hydroxyphenyl)-1H-phenalen-1-one (9, hydroxy-
anigorufone), 9-phenyl-1H-phenalen-1-one (11), and 9-(4-meth-
oxyphenyl)-1H-phenalen-1-one (13). Due to insufficient amounts
being obtained, compounds 5 and 6 were not assayed.
1
2
δ
H (mult.;
HH in Hz)
δ
H (mult.;
HH in Hz)
Compounds 1 and 2, together with perinaphthenone (7), were
the most active antifungal compounds assayed. The complete
inhibition of mycelial growth was observed at 50-100 ppm for
the three compounds, and this remained in effect for 15 days
(bioassay method 1; Figure 1a). 3-Hydroxyperinaphthenone (8)
displayed very low activity. Phenylphenalenones with an unsub-
stituted phenyl ring, such as anigorufone (3), methoxyanigorufone
(4), and 9-phenylphenalenone (11), showed a moderate activity
especially on the mycelia. The activity was significantly reduced
for compounds 9 and 13 and was even lower for compounds 10
and 12, all of which are substituted by OH or OCH3 in the p-position
of the phenyl ring. With regard to its effects on spore germination
(bioassay method 3; Figure 1b), compound 2 was as active as
perinaphthenone (7) at 100-50 ppm and even slightly more active
than 7 at 10 ppm. Compound 1 was also active at 100 ppm. The
remaining compounds assayed had very little effect on spores,
except for hydroxyanigorufone (9) and 9-phenylphenalenone (11)
at 100 ppm. Compounds 3, 7, and 11 displayed levels of bioactivity
that are similar to those reported earlier,13 but compound 9 was
more active in the present bioassays (Figure 1a,b), and compound
13 was more active on mycelia growth (Figure 1a). In order to
calculate the half-maximal inhibitory concentration (IC50) values
of compounds 1 and 2 for M. fijiensis, a 96-well microtiter plate
photometric bioassay was conducted14 (see bioassay method 2 in
the Experimental Section), revealing values of 23.4 ppm for
2-hydroxy-1H-phenalen-1-one (1) and 19.3 ppm for 2-methoxy-
1H-phenalen-1-one (2). All compounds were tested by bioassay
methods 1 and 2, and similar results were obtained; however,
bioassay method 2 required less of the test compounds.
position
J
δC
J
δC
1
2
3
3a
4
5
6
6a
7
8
9
9a
9b
OCH3
180.9
150.0
114.2
128.9
130.8
127.6
130.2
132.4
136.9
127.2
131.5
127.9
124.9
180.1
154.8
113.8
129.9
130.5
128.5
130.2
133.5
136.5
128.2
130.9
129.9
125.8
56.2
7.20 s
7.20 s
7.84 d (7.0)
7.66 dd (8.2, 7.0)
8.06 d (8.2)
7.82 d (7.1)
7.64 dd (8.2, 7.1)
8.03 d (8.2)
8.43 d (8.0)
7.89 dd (7.5, 8.0)
8.64 d (7.5)
8.37 d (8.0)
7.85 dd (7.5, 8.0)
8.56 d (7.5)
3.89 s
Scheme 1. Synthesis of Compounds 1 and 2 from
Perinaphthenone (7)
of H-3 and -OH; the latter disappeared after addition of D2O. The
signal at low field (δ 8.64; Table 1) suggested a proton in the peri
position to an electron-withdrawing group. This proton signal was
attributed to H-9, and HMBC and HMQC correlations were used
to assign the other H and 13C NMR signals as shown in Table 1.
1
For example, the doublet of H-9 and the singlet at δ 7.20 (H-3)
exhibited HMBC correlations through three bonds with the C-1
carbonyl (δ 180.9). HMBC cross signals of H-3, H-4, H-6, H-7.
and H-9 with a carbon atom at δ 124.9 were used to assign the
latter to C-9b. This pattern suggested a phenalenone molecule
without a phenyl ring. The structure of compound 1 was confirmed
as 2-hydroxy-1H-phenalen-1-one by EIMS and HREIMS. The
synthesis of compound 1 was achieved by epoxidation of peri-
naphthenone, followed by acid treatment of the intermediary
epoxide (Scheme 1).
Compounds 1 and 2 constitute the first phenalenones lacking a
phenyl group that have been isolated from the genus Musa. As far
as we know, only one related compound of that type has been
reported for plants, namely, 4-hydroxy-2-methoxyphenalen-1-one,
which was previously isolated from Strelitzia reginae.15 The activity
of the two compounds 1 and 2 against M. fijiensis is the most potent
reported for a secondary metabolite isolated from Musa and
emphasizes their key role in the Yangambi cultivar’s defense against
M. fijiensis. However, further experiments are required to clarify
if these compounds are constitutive metabolites or if they are
produced de noVo after plants have been infected with the pathogen.
The 1H NMR spectrum of 2 was very similar to that of 1, but a
singlet integrating for three protons appeared at δ 3.89 instead of
the exchangeable singlet of the hydroxyl group, suggesting that 2
Experimental Section
is a methoxy derivative of 1. All H and 13C NMR signals were
1
General Experimental Procedures. The melting point of compound
1 was measured without correction on a Bu¨chi apparatus. UV spectra
were obtained using a Shimadzu UV-160A spectrophotometer. 1H
NMR, 1H-1H COSY, HMBC, and HMQC spectra were recorded on a
Bruker Avance DRX 500 NMR spectrometer equipped with an inverse
detection microprobe head (2.5 mm) or a 5 mm TXI CryoProbe. A
broadband-decoupled microprobe head (2.5 mm) was used for measur-
ing 13C NMR spectra at 125.75 MHz. The spectra were referenced to
internal TMS in all cases. For routine measurements, Bruker Avance
400 or Bruker AMXIII 300 spectrometers were employed. EIMS and
HREIMS were run on a Micromass MasSpec mass spectrometer at 70
eV with a direct insertion probe.
Plant Material and Fungi. Healthy rhizomes of Musa acuminata
cv. “Yangambi” (group AAA) were supplied by Instituto Colombiano
Agropecuario (ICA), Colombia, in March 2003. M. acuminata cv.
“Yangambi” is continuously maintained at the same institute. My-
cosphaerella 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 reported by Quin˜ones
et al.13 Briefly, isolates of M. fijiensis were obtained from ascospores
after discharge from leaves infected with Black Sigatoka and were
maintained on potato dextrose agar (PDA) in test tubes held at 28 ( 2
°C.
assigned unambiguously from the HMBC and HMQC spectra of 2
(Table 1). HREIMS was used to confirm the structure of compound
2 as 2-methoxy-1H-phenalen-1-one. Methylation of compound 1
by diazomethane resulted in a quantitative yield of compound 2
(Scheme 1).
Quin˜ones et al. have reported the biological activity against M.
fijiensis of four natural phenylphenalenones and four 9-phenylphe-
nalenone-related compounds lacking an OH group at the C-2
position.13 Commercial perinaphthenone (7) was also assayed and
displayed significant mycelial growth and spore germination
inhibition; its effects on spore germination were even more effective
than those of some commercial fungicides.13 The activity of
compounds 1 and 2 (synthetic samples) against M. fijiensis was
examined, under the conditions reported by Quin˜ones et al. (see
bioassay methods 1 and 3 in the Experimental Section), along with
synthetic compound 3 and a sample of isolated compound 4. In
addition, 3-hydroxy-1H-phenalen-1-one (8, 3-hydroxyperinaph-
thenone), 2-hydroxy-9-(4-methoxyphenyl)-1H-phenalen-1-one (10),
and 9-(4-hydroxyphenyl)-1H-phenalen-1-one (12) were assayed. In
order to evaluate reproducibility of the bioassay, some phenalenones
that had been tested previously were also subjected to the assay.13

Notes
Journal of Natural Products, 2007, Vol. 70, No. 5 889
Figure 1. Antifungal activity of compounds 1-4 and 7-13 on Mycosphaerella fijiensis. (a) Effect on the mycelia weight measured after
7 and 15 days of incubation as determined by bioassay method 1. (b) Effect on spores. Germination and length of the germ tubes were
established microscopically for 50-150 ascospores after an incubation period of 24 h (determined by bioassay method 3).
Extraction and Isolation. Freshly collected rhizomes of M. acumi-
nata cv. “Yangambi” (20 kg) were washed, chopped, and pressed to
eliminate water, which was discarded. The remaining plant material
(approximately 10 kg) was immediately extracted with ethanol (5 L)
in a percolator system. This crude extract was evaporated to 25% of
its initial volume to remove the organic solvent and was extracted with
EtOAc. The EtOAc extract was evaporated (∼3 g of dried extract
obtained) and subjected to column chromatography (Sephadex LH-
20) using n-hexane-CH2Cl2-MeOH (5:3:1) as eluent. Fourteen
fractions were separated and subjected to TLC to detect phenylphen-
alenones using diethyl ether-n-hexane (7:1). TLC analysis afforded
fractions four to nine as being the most interesting, due to the
appearance of colored spots after spraying with a mixture of sulfuric
acid in acetic acid (1:9) and heating (90-100 °C). Pure compounds
were obtained by preparative reversed-phase HPLC of these fractions.
A LiChrospher 100 RP-18 column (10 µm; 250 × 10 mm; flow rate
3.5 mL min-1; UV 254 nm) was used employing a MeCN (0.1%
TFA)-H2O (0.1% TFA) gradient of 30:70% f 90:10% in 50 min f
30:70% in 5 min (total run time 55 min); 20, 4, 10, 100, 3, and 3 mg
of compounds 1-6 were obtained respectively from this extract.
2-Hydroxy-1H-phenalen-1-one (1,2-hydroxyperinaphthenone):
orange powder, mp 176-178 °C; tR 18.4 min; UV-vis (MeOH) λmax
(log ꢀ) 230 (4.42), 260 (4.29), 330 (3.96), 360 (3.89), 430 (3.87) nm;
fluorescence emission not detected in the range 400-700 nm; 1H NMR
and 13C NMR (see Table 1); EIMS m/z 196 [M]+ (100), 168 (59), 139
(46); HREIMS m/z 196.0528 (calcd for C13H8O2, 196.0524).
(40 wt % in MeOH). After 5 min cooling, the solution was allowed to
warm at ambient temperature and then stirred for 45 min before a
second equal portion of tert-butylhydroperoxide and TritonB was added.
Formation of the epoxide was observed by TLC (CH2Cl2) during a
period of 2 h. p-Toluenesulfonic acid monohydrate (200 mg) was then
added, and the resulting mixture was stirred overnight. The product
was purified by column chromatography on silica gel 60 (particle size
0.040-0.063 mm) (Merck, Darmstadt, Germany), using CH2Cl2 as an
eluent to obtain 145 mg of 2-hydroxy-1H-phenalen-1-one (1) as an
orange powder (70% yield). All reagents were purchased from Aldrich
and used as supplied.
2-Methoxy-1H-phenalen-1-one (2). An ethanol-containing solution
of diazomethane was prepared from Diazald using a standard procedure.
This solution was added dropwise to 2-hydroxy-1H-phenalen-1-one (1)
(50 mg, 0.25 mmol) until gas evolution had ceased. The reaction mixture
was dried under a stream of nitrogen gas to afford 51 mg of the desired
compound as a yellow oil. Analytical data of compounds 1 and 2
matched those of compounds isolated from the plant material.
Anigorufone (3), hydroxyanigorufone (9), 2-hydroxy-9-(4-methoxy-
phenyl)-1H-phenalen-1-one (10), 9-phenyl-1H-phenalen-1-one (11),
9-(4-hydroxyphenyl)-1H-phenalen-1-one (12), and 9-(4-methoxyphen-
yl)-1H-phenalen-1-one (13) were synthesized according to a reported
method16 and purified by column chromatography (silica gel 60, particle
size 0.040-0.063 mm) (Merck) using CH2Cl2 as eluent and preparative
TLC (silica gel 60 F254, 1 mm layer thickness, Merck, Darmstadt,
Germany) using Et2O-n-hexane (7:1) as eluent before being used in
bioassays. Purity was checked by analytical HPLC using conditions
described by Kamo et al.17
2-Methoxy-1H-phenalen-1-one (2,2-methoxyperinaphthenone):
yellow oil; tR 19.7 min; UV-vis (MeOH) λmax (log ꢀ) 230 (4.20), 260
(4.16), 330 (3.74), 360 (3.82), 420 (3.80) nm; fluorescence emission
Perinaphthenone (7) and 3-hydroxyperinaphthenone (8) were pur-
chased from Aldrich (Milwaukee, WI) and, prior to bioassays, purified
by preparative TLC using Et2O-n-hexane (2:1) as an eluent (Rf 0.68
and 0.21, respectively).
Bioassays. Inhibition of Mycelial Growth (Method 1). M. fijiensis
was cultivated on potato dextrose agar (PDA; Becton Dickinson) at 27
°C. Aqueous suspensions of conidia were obtained by washing the
1
λmax 520 nm; H NMR and 13C NMR (see Table 1); HREIMS m/z
210.0686 (calcd for C14H10O2, 210.0681).
Synthetic Methods. 2-Hydroxy-1H-phenalen-1-one (1,2-hydroxy-
perinaphthenone). To a cooled solution (ice-water bath) containing
200 mg of perinaphthenone (97 wt %) in benzene were added 100 µL
of aqueous tert-butylhydroperoxide (70 wt %) and 100 µL of TritonB

890 Journal of Natural Products, 2007, Vol. 70, No. 5
Notes
surface of isolated colonies with sterile distilled water. These conidia
were dispersed in PDA medium in Petri dishes and incubated for 4
days. Isolated small colonies weighing 2 ( 1 mg were placed in Petri
dishes (20 colonies each) with PDA medium containing different
concentrations (10, 50, 100 ppm) of the compounds dissolved in EtOH
(3.7% max. in the medium). Colony weight was determined after 7
and 15 days of incubation (10 colonies per day) and compared to the
weight of colonies grown on a blank medium containing the same
concentration of EtOH but not the compound to be assayed. Experi-
ments were performed in quadruplicate and analyzed by one-way
analysis of variance (ANOVA), with excellent reproducibility.
Microtiter Well Method (Method 2). The method reported by
Pela´ez et al.14 was used for this bioassay in which fresh monosporic
subcultures of different M. fijiensis strains grown for 15-20 days at
27 °C in PDA were used as the source to obtain the inoculum. Mycelia
from these subcultures were resuspended in sterile distilled water. The
suspension was then fragmented by vortexing with glass beads of 6
mm diameter (Schott, Wertheim, Germany) during 1-2 min. Filtration
with sterile “etamine” cloth (100 µm pores) afforded uniform myceliar
fragments. The concentration of myceliar fragments was measured in
a Neubauer counting chamber (1/10 mm deep, bright line; Boeco,
Boeckel & Co, Hamburg, Germany) and adjusted to 2 × 104, 2 × 105,
and 2 × 106 myceliar fragments/mL with water, to test the best working
inoculum dilution. Growth inhibition was determined in sterile, flat-
bottomed 96-well microtiter plates with low-evaporation lids (Falcon-
Becton-Dickinson). Each well was filled with 50 µL of Sabouraud broth
(BBL Becton-Dickinson), 50 µL of the fungal inoculum, and 50 µL of
the different compound solutions in order to obtain final concentrations
of 0, 5, 10, 20, 30, 40, and 50 ppm. Wells filled with 50 µL of
Sabouraud medium, 50 µL of H2O, and 50 µL of inoculum were used
as the positive control. Blanks consisted of 50 µL of Sabouraud medium
and 100 µL of H2O. The microtiter plates were incubated at 27 °C for
several days. Myceliar growth was measured using a spectrophotometer
(Biorad model 550). Readings of the OD595 nm for the suspensions in
each well were taken daily and up to 17 days. IC50 is defined as the
concentration that is able to inhibit 50% of growth.
Acknowledgment. We thank Dr. A. Svatos for mass spectroscopic
analyses, A. Gallego for collaboration with bioassays, and E. Wheeler
for editorial assistance. This research was financially supported by
Universidad de Antioquia (Programa de Sostenibilidad), Instituto
Colombiano para el Desarrollo de la Ciencia y Tecnolog´ıa “Francisco
Jose´ de Caldas” (COLCIENCIAS, grant 2213-12-17825), Corporacio´n
para Investigaciones Biologicas (CIB), Universidad Nacional de
Colombia (grants 2010100516 and 20101006122), and the Max-Planck-
Institut fu¨r Chemische O¨ kologie. F.O. thanks COLCIENCIAS and Max-
Planck-Institut fu¨r Chemische O¨ kologie for fellowships.
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Inhibition of Spore Germination (Method 3). Ascospores dis-
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excellent reproducibility.
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NP070091E