Synthesis and antifungal activity of Musa phytoalexins and structural analogs

Article abstract of DOI:10.3390/50700974

Several perinaphthenone/phenylphenalenone compounds were synthesized to establish a relationship between structure and antifungal activity against Mycosphaerella fijiensis. Substitutions on the unsaturated carbonyl system or addition of a phenyl group reduced antibiotic activity.

Full text of DOI:10.3390/50700974

Molecules 2000, 5, 974-980
molecules
ISSN 1420-3049
© 2000 by MDPI
http://www.mdpi.org
Synthesis and Antifungal Activity of Musa Phytoalexins and
Structural Analogs
Winston Quiñones, Gustavo Escobar, Fernando Echeverri*, Fernando Torres, Yoni Rosero,
Victor Arango, Gloria Cardona and Adriana Gallego
Department of Chemistry, Universidad de Antioquia, P. O. Box 1226, Medellin, Colombia
Tel.: (57+4)2105658, Fax: (57+4)2330120, E-mail: echeveri@catios.udea.edu.co
Received: 18 January 2000; revised form 14 July 2000 / Accepted: 14 July 2000 / Published: 26 July 2000
Abstract: Several perinaphthenone/phenylphenalenone compounds were synthesized to es-
tablish a relationship between structure and antifungal activity against Mycosphaerella fijien-
sis. Substitutions on the unsaturated carbonyl system or addition of a phenyl group reduced
antibiotic activity.
Keywords: phenylphenalenones, synthesis, antifungal activity, Mycosphaerella fijiensis.
Introduction
Phytoalexins are natural antibiotic compounds proposed as fungicides or templates for production of
new pesticides [1]. Recently, the search for novel antifungal compounds has received special attention
as a result of an enhanced microbial resistance to current pesticides.
Banana plants are affected by the pathogenic fungi Fusarium oxysporum var. cubensis type 4 and
Mycosphaerella fijiensis, causal agents of the diseases named Black Sigatoka and Panama Disease, re-
spectively. These diseases can drastically reduce banana production by as much as ca. 20% [2]. Under
colonization by these microorganisms or treatment of the leaves with kanamycin, banana plants produce
two types of phytoalexins, 9-phenylphenalenones (also known as musanolones [3]) and 4-
phenylphenalenones [4], The main aim of the present work was to synthesize several structurally-related
compounds and to determine the relationship between phytoalexin structure and their antifungal effects
against M. fijiensis.
Results and Discussion
The planned synthetic approach involved a 1,4-addition of a Grignard reagent to a perinaphthenone,
t
followed by reduction with DDQ, epoxidation with BuOOH and finally enolization induced by acid
treatment (Figure 1).

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Molecules 2000, 5
O
O
R
R-MgX
+
1
1) tBuOOH/Triton B
2) p-TSA
OH
O
R
Figure 1. Synthesis of phenylphenalenones.
Previously, we have used this reaction to unambiguously confirm the structure of 4´-methoxy-
irenolone, a natural phytoalexin [5]; in addition, aryl Grignard reagents have also been used for the
synthesis of extended phenalenones [6]. Other compounds such as irenolone [4-(4’-
hydroxy)phenylphenalenone)], and 4-phenylphenalenone were obtained from natural sources, as de-
scribed elsewhere [1,5]. The results of the synthesis are summarized in Table 1; products derived from
addition to the 2-en-1-one system were obtained in less than 5% yield.
Table 1. Structure and yield of phytoalexins and structural analogues.

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Molecules 2000, 5
Activity was measured by the effect of the compounds on the weight of an established Mycosphae-
rella fijiensis colony (Figure 2). In this figure the results for each compound are represented by six bars
(doses at 10.0, 50.0 and 100.0 ppm and measurements at 7, 12 and 15 days). When the antibiotic activ-
ity detected was very low (e.g. compound 3), the corresponding bars are not displayed. Compound 7
was only tested at 50 and 100 ppm due to limited availability.
Perinaphthenone (1H-phenalen-1-one, 1) was the most active antifungal compound assayed. Com-
plete mycelial growth inhibition was observed at 50-100 ppm and its action remained over 15 days. This
action was similar to benomyl, while 9-phenylperinaphthenone (2) showed slightly reduced activity. Ad-
ditional substitution on the side phenyl group (compounds 3, 4 and 5) drastically reduces antibiotic
properties. Natural phytoalexins (compounds 6-9) were less effective when compared to the results ob-
tained above, although 9-phenylphenalenone 6 exhibited significant activity during the first days of ex-
perimentation.
10 ppm
100 ppm
Day 7
%
Day 15
50 ppm
120
100
O
OH
Fungicide
O
F
O
O
1
2
6
OH
80
I
N
H
I
B
I
MeO
O
O
MeO
O
OMe
4
7
60
40
OH
OH
O
O
O
HO
O
3
O
OH
5
8
9
T
I
O
N
20
0
COMPOUNDS
Figure 2. Antifungal activity on M. fijiensis growth (mycelial weight).
Concerning the effects on spore germination, perinaphthenone (1) at 50-100 ppm, was a more active
antibiotic than the commercial fungicides tested (benomyl, propioconazole, tridermorph), as shown in
Figure 3. The other compounds, including the phytoalexins, displayed only moderate effects.
%
120
O
F
Germination
100
80
Germ Tube Lenght
1
I
N
H
I
B
I
Propioconazole, Tridemorph,
Benomyl, 100 ppm after 15 days
60
40
2
5
6
7
4
8
T
I
O
N
20
0
3
9
COMPOUNDS
Figure 3. Antifungal activity on M. fijiensis spores.

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Molecules 2000, 5
These results indicate the important antifungal properties of synthetic substances 1 and 2. Although
perinaphthenone (1) is an efficient singlet oxygen sensitizer [7], very limited information is available
about its mode of action. Formation of singlet oxygen and damage to biomolecules (DNA, proteins,
fatty acids etc) could be an explanation (Figure 4).
HO
H
*
O
HO
HO
O
RH
R
3O2
1O2
DNA, Proteins , Enzymes
Cellular Damage
Figure 4. Singlet oxygen and biological effects by perinaphthenone.
However, more complex biochemical interactions could be involved, since the growth inhibition pro-
file observed is very diferent from the spore germination one: e.g. 9-phenyl-perinaphthenone (2) is pow-
erful as an inhibitor whereas in germination it is practically inactive. Although it has been reported that a
hydroxyphenyl group in the phenylphenalenone moiety is essential for antibiotic activity against Colle-
totrichum musa [9], this action seems to be dependent on other factors, especially molecular shape and
the inductive effect of substituents.
Perinaphthenone, with its planar tricyclic system, may act as a DNA intercalating agent. On the other
hand, steric influence effects are displayed in the reduced activity of phenylperinaphthenone 2 (Figures 2
and 3). Some derivatives (compounds 3-5) do not exhibit significant antifungal activity, probably due to
modifications in their insertion ability or a lack of stabilization of potential free radicals on the side ring
due to inductive effects of the substituents. Other natural products such as hypericin and perylenequi-
none displayed an activity related to presence of free radicals [8] and oxygen activation. Finally, the
natural phytoalexins assayed (compounds 6-9) displayed low antifungal activity, probably due to poor
solubilization and incorporation to the agar media. Further investigations concerning the in vivo effects
of this kind of phenalenones are ongoing, since they represent a new type of antifungal compounds
which display long lasting antibiotic activity.
Experimental
General
400 MHz 1H and 100 MHz 13C NMR spectra were recorded for CDCl3. solutions on in a Bruker 400
Avance instrument. MS were registered on a VG Micromass ZAB-2F at 70 eV; IR on a Perkin Elmer
1600 (FTIR) instrument. Perinaphthenone (1) was obtained from a commercial source (Aldrich). All
anhydrous reactions were carried out under an argon atmosphere using freshly dried solvents.

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Molecules 2000, 5
General synthetic procedures
A solution of 1 (0.47g, 0.003 mol) in THF (5 mL) was added to the appropriate Grignard reagent
(0.004 mol; prepared from the respective bromide and Mg in THF (10 ml)). After stirring for 20 min at
-70°C the reaction mixture was allowed to reach room temperature and then was quenched with an
aqueous saturated solution of NH4Cl. The aqueous phase was extracted with dichloromethane and the
combined organic extracts were washed with brine, dried over Na2SO4 and evaporated to dryness. The
crude material dissolved in CH2Cl2 was treated with DDQ (1.0 eq), refluxed for 3h and then allowed to
reach room temperature. After water addition the CH2Cl2 phase was separated and purified by HPLC
(hexane-EtOAc 99:1)
The preparation of the 2-hydroxyphenylphenalenone type compounds was carried out as follows: a
solution of the compound (3 mmol, obtained as above) in benzene (15 mL) was cooled at 0°C and
treated with Triton B (3 mmol, benzene) and t-butylhydroperoxide (3 mmol) for 3h. The reaction was
quenched with NH4Cl, the organic phase was separated, dried and evaporated. The residue (2.5 mmol)
was dissolved in diethyl ether and several crystals of p-toluenesulfonic acid were added. After workup
the final product was purified by HPLC, affording deep-yellow solids.
Satisfactory spectroscopic data were obtained for all compounds.
Spectral data
Perinaphthenone (1)
1H NMR: d 8.57 (dd, J = 1.1 and 7.4 Hz, 1H, H-9), 8.13 (dd, J= 0.8 and 8.0 Hz, 1H, H-7), 7.95 (d,
J= 8.5 Hz, 1H, H-6), 7.72 (d, J= 7.6 Hz ,1H, H-8), 7.72 (d, J= 7.6 Hz,1H, H-4), 7.68 (d, J= 9.8 Hz,1H,
H-3), 7.53 (dd, J= 7.6 and 8.2 Hz, 1H, H-5), 6.68 (d, J= 9.8 Hz ,1H, H-3); 13C NMR: d 185.16 (C-1),
141.38 (C-3), 134.51 (C-3a), 131.67 (C-9a), 131.55 (C-9), 131.08 (C-4), 129.80 (C-6a), 128.96 (C-7),
128.73 (C-6), 127.29 (C-5), 126.99 (C-8), 126.60 (C-9a), 126.23 (C-2)
9-Phenylperinaphthenone (2)
1H NMR: d 8.18 (d, J= 8.3 Hz, 1H, H-7), 8.05 (d, J= 8.2 Hz, 1H, H-6), 7.79 (d, J= 7.0 Hz, 1H, H-
4), 7.70 (d, J= 9.7 Hz, 1H, H-3), 7.62 (dd, J= 7.1 and 8.2 Hz, 1H, H-5), 7.61 (d, J= 8.3 Hz, 1H, H-8),
7.45-7.34 (m, 5H, H-2',3',4',5',6'), 6.62 (d, J= 9.7 Hz, 1H, H-2); 13C NMR: d 186.13 (C-1), 148.07 (C-
9), 143.32 (C-1'), 140.75 (C-3), 134.09 (C-7), 132.18 (C-9a), 132.11 (C-6), 132.01 (C-8), 131.76 (C-
4), 130.94 (C-2), 128.94 (C-3a), 128.81 (C-9b), 128.67 (C-3',5'), 128.27 (C-6'), 127.55 (C-2',4'),
126.76 (C-6a), 126.48 (C-5).
9-(p-Methoxyphenyl)perinaphthenone (3)
1H NMR: d 8.15 (d, J= 8.3 Hz, 1H, H-7), 8.03 (d, J= 8.2 Hz, 1H, H-6), 7.76 (d, J= 6.4 Hz, 1H, H-
4), 7.68 (d, J= 9.7 Hz, 1H, H-3), 7.62 (dd, J= 7.0 Hz and 7.9 Hz, 1H, H-5), 7.61 (d, J= 8.3 Hz , 1H, H-
8), 7.34 (dd, J = 2.1 and 6.6 Hz, 2H, H-3',5'), 7.03 (dd, J= 2.1 and 6.6 Hz, 2H, H-2',6'), 6.62 (d, J= 9.7
Hz, 1H, H-2), 3.90 (s, 3H, -OCH3); 13C NMR: d 186.26 (C-1), 159.51 (C-4'), 147.96 (C-9), 140.64 (C-
3), 135.36 (C-1'), 134.08 (C-7), 132.38 (C-6), 132.11 (C-8), 132.01 (C-9a), 131.72 (C-4), 131.07 (C-

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Molecules 2000, 5
2), 129.86 (C-2',6'), 128.95 (C-3a), 128.86 (C-9b), 126.59 (C-5), 126.41 (C-6a), 114.23 (C-3',5'),
55.66 (-OCH3)
9-(2’,4’-Dimethoxyphenyl)-perinaphthenone (4)
1H NMR: d 8.19 (d, J= 8.4 Hz, H-7), 8.05 (d, J= 8.0 Hz, H-6), 7.78 (d, J= 6.8 Hz, H-4), 7.70 (d, J=
9.6 Hz, H-3), 7.62 (dd, J= 7.2 and 8.4 Hz, H-5), 7.60 (d, J= 8.4 Hz, H-8), 7.16 (d, J= 8.0 Hz, H-6’),
6.66 (dd, J= 2.4 and 9.7 Hz, H-5’), 6.62 (d, J= 9.6 Hz ,H-2), 6.58 (d, J= 2.4 Hz, H-3’), 3.92 (s, -
OCH3), 3.73 (s, -OCH3); 13C NMR: d 185.66 C-1, 160.49 C-2’, 157.52 C-4’, 143.46 C-9, 140.07 C-3,
133.67 C-7, 132.12 C-6’, 131.68 C-6, 131.57 C-8, 130.98 C-9a, 130.28 C-4, 129.28 C-2, 128.39 C-3a,
128.22 C-9b, 126.96 C-5, 126.02 C-1’, 104.57 C-5’, 98.77 C-3’, 55.57-OCH3, 55.35-OCH3.
9-(3’,4’-Methylenedioxy)perinaphtenone (5)
1H NMR: d 8.15 (d, J= 8.3 Hz, 1H, H-7), 8.03 (d, J= 8.1 Hz, 1H, H-6), 7.78 (d, J= 6.8 Hz, 1H, H-
4), 7.69 (d, J= 9.7 Hz, 1H, H-3), 7.63 (dd, J= 7.2 Hz and 8.1 Hz, 1H, H-5), 7.60 (d, J= 8.3 Hz, 1H, H-
8), 6.93 (d, J= 7.8 Hz, 1H, H-6'), 6.88 (s, 1H, H-2'), 6.86 (d, J= 7.8 Hz, 1H, H-5'), 6.62 (d, J= 9.7 Hz,
13
1H, H-2), 6.04 (s, 2H, -O-CH2-O-); C NMR : d 186.12 (C-1), 148.02 (C-4'), 147.67 (C-3'), 147.45
(C-9), 140.68 (C-3), 137.03 (C-1'), 134.09 (C-7), 132.25 (C-6'), 132.15 (C-6), 132.11 (C-8), 131.97
(C-9a), 130.97 (C-4), 130.22 (C-2), 128.87 (C-3a), 126.87 (C-9b), 126.58 (C-5), 126.11 (C-6a),
109.46 (C-5'), 108.82 (C-2'), 101.46 (O-CH2-O-)
Antifungal activity
Inhibition of mycelial growth [10]
Mycosphaerella fijiensis was initially isolated from naturally infected banana plantations located in
Uraba (Colombia); cultures were routinely grown in PDA medium. Sterile distilled water suspensions of
conidia were obtained by washing the surface of the sporulating colony growing in PDA at 26°C; these
conidia were dispersed in PDA medium in petri dishes and incubated at 26°C for 4 days. Some of these
colonies were placed in petri dishes with PDA medium containing different concentrations of the com-
pounds to be assayed or with EtOH only. After incubation in the dark at 26°C, radial mycelial growth
was measured after 7, 9, 12 and 15 days of incubation. The inhibition of fungal growth was estimated as
a percentage of the control values. In addition, colonies were recovered, agar traces were eliminated
and the mycelia were weighed.
Inhibition of spore germination
Ascospores discharged from infected banana leaves were used [11]; germination and length of the
germ tubes were established microscopically in 50-150 ascospores. Appropriate controls of fungal cul-
tures without compounds and controls with or without 1% EtOH were run. The following reference
compounds were used to determine the threshold of detection: propiconazole, tridemorph and benomyl.
Experiments were done in quadruplicate, and data were analyzed by one-way analysis of variance
(ANOVA)

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Molecules 2000, 5
Acknowledgements: The authors thank Colciencias (Colombia) and the Universidad de Antioquia for
financial support; G.E. is indebted to Colciencias for a fellowship.
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g
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