Natural Products as Sources of New Fungicides: Synthesis and Antifungal Activity of Zopfiellin Analogues

Article abstract of DOI:10.1111/j.1747-0285.2012.01343.x

The synthesis of a series of cyclooctadiene anhydrides, analogues of the natural compound zopfiellin, was performed to assay their in vitro and in vivo antifungal activity on a set of plant pathogenic fungi. Most of the synthesized compounds possessed a b

Full text of DOI:10.1111/j.1747-0285.2012.01343.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01343.x
Chem Biol Drug Des 2012; 79: 780–789
Research Article
Natural Products as Sources of New Fungicides:
Synthesis and Antifungal Activity of Zopfiellin
Analogues
Loana Musso1,*, Sabrina Dallavalle1,
Gandolfina Farina2 and Elio Burrone2
pounds may be used as agricultural chemicals (4). Alternatively,
they may constitute useful starting points as lead molecules that
could be used for the rational design of structural analogues with
improved biological and chemico-physical properties (e.g. solubility,
1Dipartimento di Scienze Molecolari Agroalimentari, Universitꢀ degli
Studi di Milano, via Celoria 2, 20133 Milano, Italy
2Dipartimento di Protezione dei Sistemi Agroalimentare e Urbano e
Valorizzazione delle Biodiversitꢀ, Universitꢀ degli Studi di Milano,
Via Celoria 2, 20133 Milano, Italy
environmental profile, toxicity) (5).
Through the analysis of recent literature concerning the antifungal
activity of substances isolated from natural sources, we selected a
molecule, named zopfiellin (Figure 1), that could be used as a lead
compound for the synthesis of new fungicides.
*Corresponding author: Loana Musso, loana.musso@unimi.it
The synthesis of a series of cyclooctadiene anhy-
drides, analogues of the natural compound zopfiel-
lin, was performed to assay their in vitro and in
vivo antifungal activity on a set of plant patho-
genic fungi. Most of the synthesized compounds
possessed a broad spectrum of activity. In particu-
lar, the anhydrides 2 and 5a were very effective
against the Oomycete diseases such as Phytoph-
thora infestans and Pythium ultimum, reaching a
level of activity well comparable with that of com-
mercial fungicides in use. Preliminary in vivo eval-
uation of their protectant activity is also reported.
Zopfiellin is a secondary metabolite of the ascomycete Zopfiella
curvata (Fuckel) Winter, identified in a discovery programme for the
individuation of new types of fungicidal agents undertaken by Nis-
san Chemical Industries Ltd (Tokyo, Japan) (6).
Zopfiellin contains a disubstituted cyclooctadiene ring fused with
two maleic anhydride moieties. This compound is endowed with
interesting antifungal activity in particular against Botrytis cinerea
(IC80 10 lM), Colletotrichum gloeosporioides (IC80 10 lM) and Collet-
otrichum fragariae (IC80 10 lM) (7,8). At present, the mode of action
of zopfiellin is still unknown, although preliminary studies performed
by Wedge and co-workers (8) indicated that the antifungal activity of
this molecule may be associated with physiological processes involv-
ing oxaloacetate metabolism and strongly influenced by the pH of
the medium, being maximal at pH 5.0. The authors assumed that at
low pH, the closed-ring bis-anhydride form is able to cross the
plasma membrane and penetrate into the fungi, whereas the carbox-
ylate form, present at higher pH values, is too polar to move across
the lipophilic plasma membrane. Therefore, intramolecular ring clo-
sure of zopfiellin from tetracarboxylate to an anhydride form occur-
ring at pH below 6 is apparently required for uptake by the fungus.
Key words: antifungal activity, cyclooctadiene anhydrides, natural
compounds, synthesis, zopfiellin
Received 23 September 2011, revised 9 January 2012 and accepted for
publication 10 January 2012
Plant pathogens are estimated to cause yield reductions of almost
20% in the principal food and cash crops worldwide (1). Although
these losses may be attenuated by the use of disease-tolerant culti-
vars, crop rotation or sanitation practices, fungicides are still essen-
tial for effective control of most plant diseases and therefore to
maximize crop yields. Despite the choice of effective fungicides avail-
able, new antifungal chemicals are still needed to obtain improved
yields and quality benefits. An important impetus for maintaining
research programmes in this field is the growing demand by the pub-
lic for crop protection agents with low use rates, a benign environ-
mental profile and low toxicity to humans and wildlife.
Naturally occurring substances in fungi, bacteria and higher plants
are important sources of molecules with antifungal properties (2,3).
If sufficient quantities can be obtained from natural sources or by
synthesis and their overall properties are acceptable, such com-
Figure 1: Structure of zopfiellin.
780

Cyclooctadiene Anhydride Analogues of Zopfiellin
Although two approaches to the synthesis of zopfiellin have been
published (9,10), currently, the only method to obtain this compound
is the isolation from a fermentation broth of a suitable fungal strain
(11). Besides, some cyclooctadiene derivatives obtained by various
chemical modifications of the natural compound showed high fungi-
cidal activity (11). These facts justify further studies on this class of
molecules to identify the essential structural requirements for their
antifungal activity.
plete, the resultant pale yellow suspension was stirred for 30 min at
room temperature and then cooled to 0 ꢀC. With continued stirring,
dimethyl acetylenedicarboxylate (DMAD) (193 lL, 1.57 mmol) was
slowly added with the temperature being kept below 5 ꢀC. The reac-
tion mixture was then stirred at room temperature for 2 h. Glacial
acetic acid (1 mL) and 2 N aqueous HCl (2 mL) were added at 0 ꢀC;
the aqueous layer was separated and washed three times with Et2O.
The organic layer was washed once with water, dried (Na2SO4) and
evaporated under reduced pressure to leave a reddish gum, which
was purified by flash chromatography (hexane ⁄ ethyl acetate, 3:1) to
In this article, we report the preparation of structurally and synthet-
ically simplified zopfiellin analogues and the evaluation of their
antifungal activity. Recently, when this work was already in an
advanced stage, Krohn and co-workers (12) reported the isolation of
a new compound containing a disubstituted cyclooctadiene ring
fused with two maleic anhydride moieties. The interesting biological
activity showed by this molecule further supports the interest in
this kind of structures as potential antifungal scaffolds.
1
give 294 mg (57%) of the title product as a pale yellow oil. H NMR
d: 13.25 (s, 1H, OH), 7.33 (dd, 1H, J = 1.5, 7.4 Hz, Ar), 7.27 (ddd, 1H,
J = 7.4, 7.8, 1.5 Hz, Ar), 7.23 (ddd, 1H, J = 7.8, 7.2 1.5 Hz, Ar), 7.04
(dd, 1H, J = 7.8, 1.5 Hz, Ar), 3.77 (s, 3H, -OCH3), 3.69 (s, 3H, -OCH3),
3.60 (s, 3H, -OCH3), 3.14 (m, 1H, >CHCH3), 2.77 (dd, 1H, J = 13.0,
7.8 Hz, -CH2CHCH3), 2.74 (dd, 1H, J = 13.0, 11.9 Hz,-CH2CHCH3), 1.24
(d, 3H, J = 7.0 Hz, >CHCH3,). Anal. Calcd for C19H20O7: C, 63.33; H,
5.59. Found: C, 63.16; H, 5.44.
Materials and Methods
5-Hydroxy-10-methyl-1,3-dioxo-1,3,10,11-
Chemistry
tetrahydro-2-oxa-benzo[a]cyclopenta[e]
1H and 13C NMR spectra were recorded on CDCl3 solutions (where
not otherwise stated) at room temperature on a Bruker AMX-300
spectrometer operating at 300 MHz for 1H and 75 MHz for 13C.
Chemical shifts are reported as d values in parts per million
(p.p.m.) and are indirectly referenced to tetramethylsilane (TMS) via
cyclooctene-4-carboxylic acid methyl ester (5b)
To a solution of 4b (150 mg, 0.42 mmol) in ethanol (3 mL) at 0 ꢀC,
a solution of NaOH (133 mg, 3.33 mmol) in water (3 mL) was
added. The resulting mixture was stirred for 24 h at room tempera-
ture, then it was cooled to 0 ꢀC, and an excess of 2 N aqueous
HCl (5 mL) was added. The precipitated solid was filtered, washed
with water and dried under vacuum to obtain 94 mg (68%) of the
1
the solvent signal (7.26 for H, 77.0 for 13C) in CDCl3. Coupling con-
stants (J) are given in Hz.
1
title compound as a white solid. mp 175–176 ꢀC; H NMR d: 13.61
Solvents were routinely distilled prior to use; anhydrous tetrahydrofu-
ran (THF) and ether (Et2O) were obtained by distillation from sodium-
benzophenone ketyl; dry methylene chloride (DCM) and toluene were
obtained by distillation from CaCl2. All reactions requiring anhydrous
conditions were performed under a positive nitrogen flow, and glass-
ware was oven-dried. Isolation and purification of the compounds
were performed by flash column chromatography on silica gel 60
(230–400 mesh); when necessary, deactivated silica gel was used.
(Deactivated silica gel: a suspension of 110 g of SiO2 in 400 mL of a
4% aqueous solution of KH2PO4 was evaporated under reduced pres-
sure and dried at 110 ꢀC overnight.) Analytical thin-layer chromatog-
(s, 1H, OH); 7.49 (m, 2H, Ar); 7.35 (m, 2H, Ar); 3.84 (s, 3H, -OCH3);
3.74 (m, 1H, -CHCH3); 2.89 (dd, 1H, J = 4.1, 19.3 Hz); 2.62 (dd, 1H,
J = 13.0, 19.3 Hz); 1.43 (d, 3H, J = 7.0). Anal. Calcd for C17H14O6:
C, 64.97; H, 4.49. Found: C, 64.89; H, 4.52.
3-Benzyl-2-cyano-hept-2-enoic acid methyl ester
(7)
A solution of 1-phenyl-2-hexanone 6 (2 g, 12.6 mmol), methyl cya-
noacetate (2.3 mL, 25.2 mmol), acetic acid (3 mL) and ammonium
acetate (980 mg, 12.6 mmol) in dry toluene (10 mL) was refluxed
for 40 min. The mixture was cooled, washed with water, dried
(Na2SO4) and concentrated under vacuum. The crude material was
purified by flash chromatography (hexane ⁄ ethylether, 5:1) to give
1.89 g (55%) of product as a yellow oil. 1H NMR d: 7.27 (m,
5H+4H, Ar), 7.16 (m, 1H, Ar), 4.19 (s, 2H, -CH2Ph), 3.89 (s, 2H, -
CH2Ph), 3.85 (s, 3H, -OCH3), 3.83 (s, 3H, -OCH3), 2.66 (t, 2H,
J = 7.8 Hz, CH2C=C<), 2.45 (t, 2H, J = 7.8 Hz, -CH2C=C<), 1.49 (m,
2H); 1.36 (m, 6H), 0.88 (t, 3H, J = 7.4 Hz, CH3), 0.87 (t, 3H,
J = 7.4 Hz,-CH3). Anal. Calcd for C17H21NO2: C, 75.25; H, 7.80; N,
5.16. Found: C, 75.09; H, 7.69; N, 5.22.
raphy (TLC) was conducted on Fluka TLC plates (silica gel 60 F254
,
aluminium foil), and spots were visualized by UV light and ⁄ or by
means of dyeing reagents. Melting points were determined on a Stu-
art Scientific SMP3 instrument and are uncorrected.
Compounds 2, 4a, 5a and 16 were prepared according to the liter-
ature procedures (13). Compounds 13a and 17a,c,d, were pre-
pared according to the literature procedures (14).
5-Hydroxy-10-methyl-9,10-dihydro-
3-Benzyl-2-cyano-heptanoic acid methyl ester
(8)
benzocyclooctene-6,7,8-tricarboxylic acid
trimethyl ester (4b)
Compound 7 (400 mg, 1.55 mmol) was hydrogenated in ethanol solu-
tion (6 mL) using 10% palladium on charcoal (15 mg) as a catalyst.
The mixture was stirred for 4 h under normal pressure of hydrogen.
The catalyst was filtered off, and the solution was concentrated
To a suspension of sodium hydride (77 mg, 1.93 mmol, 60% disper-
sion in mineral oil) in dry toluene (2 mL) under nitrogen atmosphere,
2-methoxycarbonyl-4-methyl-1-tetralone 3b (312 mg, 1.43 mmol) dis-
solved in dry toluene (4 mL) was added; when the addition was com-
Chem Biol Drug Des 2012; 79: 780–789
781

Musso et al.
under vacuum to give 401 mg (80%) of product that was directly uti-
lized without further purification. Yellow oil. H NMR (mixture of dia-
sphere to a suspension of sodium hydride (60 mg, 1.35 mmol, 60%
dispersion in mineral oil) in dry toluene (4 mL). When the addition
had been completed, the resultant pale yellow suspension was stir-
red for 30 min at room temperature and then cooled to 0 ꢀC. With
continued stirring, DMAD (150 lL, 1.1 mmol) was slowly added
with the temperature being kept below 5 ꢀC. The reaction mixture
was then stirred at room temperature for 6 h. Glacial acetic acid
(1 mL) and 2 N aqueous HCl (1 mL) were added at 0 ꢀC; the aque-
ous layer was separated and washed three times with Et2O. The
organic layer was washed once with water and once with brine,
dried (Na2SO4) and evaporated under reduced pressure. The crude
material (570 mg) was purified by flash chromatography (hex-
ane ⁄ Et2O, 6:4) to give 168 mg (42%) of product as a white solid.
mp 95–96 ꢀC; 1H NMR d: 13.15 (s, 1H, OH), 7.38 (dd, 1H,
J = 7.4 Hz, 1.5, Ar), 7.28 (ddd, 1H, J = 7.4, 7.8, 1.8 Hz, Ar), 7.21
(ddd, 1H, J = 7.4, 7.8 Hz, Ar), 7.07 (dd, 1H, J = 1.5, 7.8 Hz, Ar),
3.78 (s, 3H, -OCH3), 3.76 (s, 3H, -OCH3), 3.57 (s, 3H, -OCH3), 3.35
(m, 2H, >CH(CH2)3CH3+ -CHHCH<), 2.71 (dd, 1H, J = 18.2, 14.1 Hz,
CHHCH<), 1.66–1.18 (m, 6H, -CH2-); 0.88 (t, 3H, J = 7.0 Hz, -CH3).
Anal. Calcd for C22H26O7: C, 65.66; H, 6.51. Found: C, 65.79; H,
6.65.
1
stereoisomers): d 7.38–7.14 (m, 10H, Ar), 3.75 (s, 3H, -OCH3), 3.67 (s,
3H, -OCH3), 3.37 (d, 1H, J = 3.3 Hz, -CH<), 3.04 (dd, 1H, J = 13.0,
3.3 Hz, >CH2Ph), 2,75 (d, 2H, J = 7.4 Hz, >CH2Ph), 2.50 (dd, 1H,
J = 13.0, 11.1 Hz, >CH2Ph), 2.48 (m, 1H+1H, >CH(CH2)3CH3), 1.60–
1.19 (m, 6H+6H, (CH2)3CH3), 0.89 (t, 3H, J = 7.0 Hz, -CH3), 0.87 (t, 3H,
J = 7.0 Hz, -CH3). Anal. Calcd for C17H23NO2: C, 74.69; H, 8.48; N,
5.12. Found: C, 74.75; H, 8.51; N, 4.98.
3-Butyl-3,4-dihydro-2H-naphthalen-1-one (9)
A solution of cyanoester 8 (300 mg, 1.16 mmol) in a mixture of acetic
acid (2 mL), water (1.5 mL) and sulphuric acid (1.5 mL) was heated at
reflux for 40 h. The mixture was cooled, diluted with water (3 mL)
and washed with Et2O. The organic layer was washed with brine and
concentrated under reduced pressure to give 168 mg (72%) of prod-
1
uct as a yellow oil. H NMR d: 8.00 (dd, 1H, J = 1.5, 7.8, Ar), 7.46
(ddd, 1H, J = 1.5, 7.4, 7.8 Hz, Ar), 7.26 (m, 2H, Ar), 3.01 (m, 1H), 2.75
(m, 2H), 2.28 (dd, 1H, J = 11.9, 16.0 Hz), 2.18 (m, 1H, >CHCH2-), 1.34
(m, 6H, -CH2-), 0.91 (t, 3H, J = 7.0 Hz, -CH3). Anal. Calcd for C14H18O:
C, 83.12; H, 8.97; Found: C, 82.97; H, 8.89.
11-Butyl-5-hydroxy-1,3-dioxo-1,3,10,11-tetrahydro-
2-oxa-benzo[a]cyclopenta[e]cyclooctene-4-
carboxylic acid methyl ester (12)
3-Butyl-1-oxo-1,2,3,4-tetrahydro-naphthalene-2-
carboxylic acid methyl ester (10)
Under nitrogen atmosphere, sodium hydride (136 mg, 60% disper-
sion in mineral oil, 3.40 mmol) was washed with dry hexane
(2 mL); the solvent was removed, and sodium hydride was placed
under dry toluene; dimethyl carbonate (1 mL, 12 mmol) was added
to the stirred suspension. The mixture was heated at 80 ꢀC, and
with continued stirring, compound 9 (300 mg, 1.48 mmol) in tolu-
ene (4 mL) was added dropwise in 10 min, and then toluene
(10 mL) was added to dilute the mixture. After 4 h at 90 ꢀC, the
mixture was cooled to 0 ꢀC, and 0.5 N aqueous HCl (10 mL) was
added. Ice water was added, and the aqueous layer was separated
and washed three times with Et2O. The organic phase was then
washed with saturated aqueous sodium hydrogen carbonate and
water, dried (Na2SO4) and evaporated under reduced pressure to
give the crude product, which was purified by flash chromatography
(hexane ⁄ ethyl acetate, 19:1) to obtain 266 mg (69%) of the title
A solution of NaOH (133 mg, 3.33 mmol) in water (3 mL) was
added at 0 ꢀC to a solution of 11 (168 mg, 0.42 mmol) in ethanol
(3 mL). The mixture was stirred for 24 h at room temperature, then
it was cooled to 0 ꢀC, and an excess of 2 N aqueous HCl (5 mL)
was added. The precipitated solid was filtered, washed with water
and dried under vacuum to obtain 94 mg (63%) of the title com-
pound as a white powder. mp 147–148 ꢀC; 1H NMR: d 13.48 (s,
1H, OH), 7.48 (m, 1H, Ar), 7.42 (d, 1H, J = 7.8 Hz, Ar) 7.32 (m, 1H,
Ar), 7.20 (m, 1H, Ar), 3.85 (s, 3H, -OCH3), 3.10 (dd, 1H, J = 12.2,
14.5 Hz, -CHHCH<), 3.04 (m, 1H, >CH(CH2)3CH3), 2.88 (dd, 1H,
J = 12.2, 4.1 Hz, -CHHCH<), 1.77 (m, 2H, -CH2-), 1.61 (m, 2H, -CH2-),
1.39 (m, 2H, -CH2-), 0.92 (t, 3H, J = 7.0 Hz, -CH3). Anal. Calcd for
C20H20O6: C, 67.41; H, 5.66. Found: C, 67.35; H, 5.53.
1
product as a yellow oil. H NMR d: 12.45 (s, 1H, enolic OH); 8.00
10-Butoxy-4,5-dihydro-2-oxa-benzo[a]cyclopenta[e]
cyclooctene-1,3-dione (13b)
(d, 1H, J = 7.8, Ar), 7.77 (dd, 1H, J = 1.1, 7.0 Hz, Ar), 7.50 (m, 2H,
Ar), 7.20–7.35 (m, 3H, Ar), 7.15 (d, 1H, J = 7.0 Hz, Ar), 3.82 (s, 3H,
-OCH3), 3.78 (s, 3H, -OCH3), 3.38 (d, 1H, J = 10.4 Hz), 2.99 (dd, 1H,
J = 4.1, 16.4 Hz, -CH2CH<,), 2.97 (m, 2H, -CH2CH<), 2.80 (m, 1H,
CH2CH<), 2.75 (m, 1H, -CH2CH<), 2.57 (m, 1H, -CH2CH<), 1.37 (m,
6H+6H, -(CH2)3CH3 ketone+enol), 0.89 (m, 3H+3H, -CH3 ke-
tone+enol). Anal. Calcd for C16H20O3: C, 73.82; H, 7.74. Found:
73.91; H, 7.69.
n-Butanol (60 lL, 0.66 mmol) was added dropwise to a suspen-
sion of 2 (40 mg, 0.16 mmol) in dry toluene (0.5 mL); thionyl
chloride (50 lL, 0.66 mmol) was then added at 0 ꢀC. The result-
ing mixture was stirred at room temperature for 24 h. After this
period, the mixture was washed with water and brine. The sol-
vent was evaporated under reduced pressure, and the crude
material was purified by flash chromatography (hexane ⁄ Et2O, 3:1)
to give 47 mg (96%) of the title product as a pale yellow solid.
mp. 113–112 ꢀC; 1H NMR d: 7.39 (m, 2H, Ar), 7.25 (m, 2H, Ar),
5.60 (s, 1H, -CH=C<), 4.07 (m, 2H, -OCH2CH2CH2CH3), 3.50–2.50
(m, 4H, -CH2CH2-), 1.80 (m, 2H, -CH2-), 1.50 (m, 2H, -CH2-), 1.00
(t, 3H, J = 7.4 Hz, -CH3). Anal. Calcd for C18H18O4: C, 72.47; H,
6.08. Found: C, 72.33, H, 5.98.
9-Butyl-5-hydroxy-9,10-dihydro-benzocyclooctene-
6,7,8-tricarboxylic acid trimethyl ester (11)
A solution of 2-methoxycarbonyl-4-butyl-1-tetralone 10 (260 mg,
1 mmol) in dry toluene (2 mL) was added under nitrogen atmo-
782
Chem Biol Drug Des 2012; 79: 780–789

Cyclooctadiene Anhydride Analogues of Zopfiellin
2-Butyl-10-methoxy-4,5-dihydro-2-aza-
benzo[a]cyclopenta[e]cyclooctene-1,3-dione
(17b)
(m, 2H, -CH2-), 1.50 (m, 2H, -CH2-), 1.00 (t, 3H, J = 7.0 Hz,-CH3).
Anal. Calcd for C18H19NO3: C, 72.71; H, 6.44; N, 4.71. Found: C,
72.65; H, 6.31; N, 4.86.
Freshly distilled n-butylamine (780 lL, 7.8 mmol) was added to a
solution of 13a (180 mg, 0.70 mmol) in ethanol (4 mL); the mixture
was stirred for 24 h at room temperature. The solvent was removed
under reduced pressure, and the residue was dissolved in DCM; the
resulting solution was washed twice with 1 N aqueous HCl and
once with brine. In vacuo concentration of the organic phase fol-
lowed by flash column chromatography with deactivated silica gel
of the residue (hexane ⁄ ethyl acetate, 95:5) gave 174 mg (80%) of
product as an orange powder. mp. 97-98 ꢀC; 1H-NMR d: 7.37 (m,
2H, Ar), 7.25 (m, 2H, Ar), 5.59 (s, 1H, -CH=C<), 3.98 (s, 3H, -CH3),
3.37 (m, 2H, -CH2N<), 2.75 (m, 4H, -CH2CH2-), 1. 50 (m, 2H, -CH2-),
1.25 (m, 2H, -CH2-), 0.90 (t, 2H, J = 7.0 Hz, CH3). Anal. Calcd for
C19H21NO3: C, 73.29; H, 6.80; N, 4.50. Found: C, 73.35; H 6.88; N,
4.43.
10-Butoxy-2-butyl-4,5-dihydro-2-aza-
benzo[a]cyclopenta[e]cyclooctene-1,3-dione
(18b)
To a solution of 13b (82 mg, 0.83 mmol) in ethanol (4 mL), freshly
distilled n-butylamine (900 lL, mmol) was added; the mixture was
then stirred for 24 h at room temperature. The solvent was
removed under reduced pressure; the residue was dissolved in
DCM and washed twice with 1 N aqueous HCl and once with
brine. Concentration of the organic phase in vacuo followed by
flash column chromatography on deactivated silica gel of the resi-
due (hexane ⁄ ethyl acetate, 95:5) gave 293 mg (99%) of product as
a yellow powder. mp 106–107 ꢀC; 1H NMR d: 7.37 (m, 2H, Ar),
7.25 (m, 2H, Ar), 5.59 (s, 1H, -CH=C<), 4.05 (m, 2H, -CH2O-), 3.37
(m, 2H, -CH2N<), 2.37 (m, 4H, -CH2CH2-), 1.75 (m, 2H, -CH2-), 1.50
(m, 4H, -CH2-), 1.25 (m, 2H, -CH2-), 0.90 (t, 3H, J = 7.0 Hz,-CH3,),
0.60 (t, 3H, J = 7.0 Hz,-CH3). Anal. Calcd for C22H27NO3: C, 74.76;
H, 7.70; N, 3.96. Found: C, 74.68; H, 7.65; N, 4.01.
2-(2-Hydroxyethylamino)-10-methoxy-4,5-
dihydro-2-aza-benzo[a]cyclopenta[e]cyclooctene-
1,3-dione (17e)
To a solution of 13a (80 mg, 0.31 mmol) in methanol (2 mL), 2-hy-
droxyethylhydrazine (24 lL, 0.35 mol) was added; the mixture was
stirred for 24 h at room temperature. Concentration of the mixture
in vacuo followed by flash chromatography of the residue on deacti-
vated silica gel (hexane ⁄ ethyl acetate, 1:2) gave 79 mg (86%) of
2-Benzyl-10-butoxy-4,5-dihydro-2-aza-
benzo[a]cyclopenta[e]cyclooctene-1,3-dione
(18c)
1
product as a yellow powder. mp. 113–114 ꢀC; H-NMR d: 7.40 (m,
To a solution of 13b (53 mg, 0.17 mmol) in toluene (1.5 mL), benzyl-
amine (50 lL, 0.46 mmol) was added. The mixture was then refluxed
for 1 h. Ethanol was removed under reduced pressure; the residue
was dissolved in DCM and washed twice with 1 N aqueous HCl and
once with brine. Concentration of the organic phase in vacuo fol-
lowed by flash column chromatography on deactivated silica gel of
the residue (hexane ⁄ ethyl acetate, 95:5) gave 36 mg (52%) of the
2H, Ar); 7.20 (m, 2H, Ar); 5.60 (s, 1H, -CH=C<); 3.99 (s, 3H, -OCH3);
3.50 (m, 2H, -CH2CH2OH); 3.00 (m, 2H, -CH2CH2OH); 2.85 (m, 4H, -
CH2CH2). Anal. Calcd for C17H18N2O4: C, 64.96; H, 5.77; N, 8.91.
Found: C, 64.89; H, 5.65; N, 9.03.
1
2-Dimethylamino-10-methoxy-4,5-dihydro-2-
azabenzo[a]cyclopenta[e]cyclooctene-1,3-dione
(17f)
title compound yellow powder. mp 130–131 ꢀC; H NMR d: 7.25 (m,
9H, Ar); 5.57 (s, 1H, -CH=C<); 4.52 (s, 2H, -CH2Ph); 4.00 (m, 2H, -
CH2O-); 3.75–2.75 (m, 4H, -CH2CH2-); 1.75 (m, 2H, -CH2-); 1.50 (m,
4H, -CH2-); 1.00 (t, 3H, J = 7.0 Hz, -CH3). Anal. Calcd for C25H25NO3:
C, 77.49; H, 6.50; N, 3.61. Found: C, 77.55; H, 6.68; N, 3.55.
To a solution of 13a (98 mg, 0.38 mmol) in methanol (2 mL), 1,1-
dimethylhydrazine (30 lL, 0.39 mmol) was added; the resulting mix-
ture was stirred for 6 h at room temperature. Concentration of the
mixture in vacuo followed by flash chromatography of the residue
on deactivated silica gel (hexane ⁄ ethyl acetate, 1:1) gave 104 mg
(94%) of product as a yellow sticky oil. 1H NMR d: 7.40 (m, 2H,
Ar), 7.20 (m, 2H, Ar), 5.60 (s, 1H, -CH=C<), 3.97 (s, 3H, -OCH3),
3.40–2.85 (m, 4H, -CH2CH2), 2.85 (s, 6H, -N(CH3)2). Anal. Calcd for
C17H18N2O3: C, 68.44; H, 6.08; N, 9.39. Found: C, 68.56; H, 6.15; N,
9.27.
Evaluation of in vitro antifungal activity
The activity of the compounds was assayed as growth inhibition by
the commonly used 'poisoned food' technique. Different solutions of
each compound were prepared by dissolving the appropriate
amount of compound in DMSO and Tween-20 (2%). Equal volumes
of DMSO-containing diluted compounds were added to sterile cool
agar media [potato dextrose agar for Phytophthora infestans (Mont.)
de Bary, Pythium ultimum Trow, B. cinerea Pers., Cercospora betico-
la Sacc., Rhizoctonia solani J.G. Kꢀhn] to give suitable concentra-
tions for each substance. A zero-concentration treatment, containing
the same percentage of DMSO and Tween-20 to ensure equivalent
concentrations of these components in all the treatments, was pre-
pared for each fungus. The final DMSO concentration did not
exceed 0.3% of the final volume in both control and treated cul-
tures. Compound-amended agar media were dispersed aseptically
onto 9-cm-diameter plastic Petri dishes (10 mL ⁄ dish). Each dish was
inoculated with two mycelial discs cut from the periphery of
10-Butoxy-4,5-dihydro-2-aza-benzo[a]cyclopenta[e]
cyclooctene-1,3-dione (18a)
To a suspension of 13b (100 mg, 0.33 mmol) in ethanol (5 mL),
33% aqueous ammonia (1 mL) was added; the mixture was then
refluxed for 20 min. The solvent was removed under reduced pres-
sure, and the residue was crystallized by Et2O to obtain 88 mg
1
(89%) of product as a yellow powder. mp 165–166 ꢀC; H NMR d:
7.40 (m, 2H, Ar), 7.25 (m, 2H, Ar), 7.05 (bs, 1H, >NH), 5.57 (s, 1H,
-CH=C<), 4.00 (m, 2H, -OCH2-), 3.75–2.75 (m, 4H, -CH2CH2-), 1.80
Chem Biol Drug Des 2012; 79: 780–789
783

Musso et al.
actively growing colonies. Two replicates were used for each con-
centration, together with dishes containing toxicant-free media. The
growth inhibition was calculated from mean differences between
treated and control cultures as a percentage of the latter. The
growth was determined after 24 h of incubation at 24 ꢀC for
P. infestans and P. ultimum, 3 days for B. cinerea, 6 days for
R. solani and 9 days for C. beticola.
hypothesis of their use as a scaffold. A set of phytopathogenic
fungi causing widespread diseases on crops were used (P. infe-
stans, P. ultimum, B. cinerea, C. beticola and R. solani). As reported
in Table 2, these molecules showed a broad spectrum of activity.
Interestingly, the ester 4a was found to be the less active com-
pound, highlighting the relevance of anhydride moiety for the anti-
fungal activity. Moreover, anhydride 5a showed higher activity than
2 against the fungal species tested, especially at the lowest con-
centrations.
The results were compared with those obtained with three standard
fungicides: Euparenꢁ (tolilfluanide, 50%, Bayer S.p.A., Italy),
Sideralꢁ (procymidone, 50%, Sipcam S.p.A., Italy) and tetraconaz-
oleꢁ (Isagro S. p. A., Italy).
To assess the influence of substituents on the cyclooctadiene
ring, we synthesized some compounds bearing alkyl chains
at position 10, 11 of compound 2. We exploited again the
reaction of ring expansion of carbocyclic b-ketoesters in the
presence of acetylenic esters, extending for the first time this
methodology to 2-carboxymethyltetralones substituted at position
3 or 4.
Evaluation of in vivo antifungal activity
The tests of direct protectant activity of the compounds were per-
formed on the following pathogen–host combinations: Puccinia gra-
minis Pers. ⁄ wheat cv Irnerio (wheat rust), Erysiphe graminis
D.C. ⁄ wheat cv Irnerio (powdery mildew), Plasmopara viticola Berl et
de Toni ⁄ vine cv dolcetto (downy mildew) and P. infestans (Mont) de
Bary ⁄ tomato cv Marmande (downy mildew). Inoculation with a suit-
able spore suspension was performed 7 days after the treatment
on grapevine and tomato and 24 h after the treatment on wheat.
The area of inoculated leaves covered by disease symptoms was
assessed on a 100-point scale from 0 to 100, in which 100 corre-
sponded to completely infected leaves and 0 to not visible symp-
toms. The results were compared with those obtained with two
standard fungicides in commercially available formulations, benal-
axyl and tetraconazole, and the activity was expressed as per cent
inhibition of infection in comparison with untreated control.
The ester 4b was prepared by reacting the 2-methoxycarbonyl-4-
methyltetralone 3b (15) with DMAD. After mild alkaline hydrolysis
and acidification, compound 5b was obtained in 68% yield,
Scheme 1.
To prepare 11-substituted derivatives, it was necessary to synthe-
size the suitable tetralone functionalized at position 3, as
reported in Scheme 2. Among the existing alternatives for the
synthesis of these substrates, a Cope condensation appeared to
be the most efficient and practical one (15,16). The first step
involved the condensation of ketone 6 with ethyl cyanoacetate
to obtain 7 in 55% yield; catalytic reduction of the double bond
gave compound 8 in 80% yield and then converted into tetr-
alone 9 by means of acidic treatment. Finally, the desired
b-keto-ester was obtained in 69% yield by treatment with
dimethyl carbonate. Compound 12 was obtained in 63% yield.
All the intermediates and the final product were obtained as
stereoisomeric mixtures.
Results and Discussion
Assuming that less polar molecules than zopfiellin could show a
better uptake, we considered a cyclooctadiene skeleton fused to an
aromatic ring and only one maleic anhydride moiety as a possible
scaffold (Figure 2).
We then capitalized on the presence of the carbonyl group in com-
pound 2 to prepare derivatives substituted at position 5. The intro-
duction of alkyl chains linked through an oxygen atom was carried
out by treatment of ketone 2 with thionyl chloride and methanol
(14) or n-butanol, respectively (Scheme 3). All the attempts to
extend this procedure to functionalized alcohols, such as EtO(CH2-
CH2)O(CH2CH2)OH, 4-chlorobutanol and 2-dimethylaminoethanol,
failed.
This compound was easily prepared following a reported procedure
(13), exploiting the Michael addition of 2-methoxycarbonyltetralone
to DMAD resulting in the (n + 2) ring-expanded compound 4a
(Scheme 1). By mild alkaline hydrolysis and acidification, compound
5a was obtained in 76% yield, whilst acid hydrolysis and decarbox-
ylation led to keto-anhydride 2 in 76% yield (Figure 2).
The antifungal activity of compound 2 and of the intermediates 4a
and 5a was assessed by in vitro experiments to validate the
To assess the relevance of the aromatic ring on our scaffold 2,
we tested the cyclooctenone 16 (13), obtained by reacting
methyl 2-oxo-cyclohexanecarboxylate 14 with DMAD. The inter-
mediate 15 was directly hydrolysed and decarboxylated under
acidic conditions to give the desired compound. All the attempts
to obtain the anhydride analogue of 5a and 5b, avoiding decar-
boxylation by basic hydrolysis of compound 15, failed
(Scheme 4).
Finally, we tested the imido derivative 17a and the hydrazino deriv-
ative 17d (14) to explore the relevance of the anhydride group for
the antifungal activity. As the activity was maintained in both
Figure 2: Cyclooctadiene scaffold.
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Chem Biol Drug Des 2012; 79: 780–789

Cyclooctadiene Anhydride Analogues of Zopfiellin
Scheme 1: Synthesis of compounds 2, 4a and 5a,b.
Scheme 2: Synthesis of compound 12.
compounds, we prepared new derivatives bearing different substitu-
ents at the imido nitrogen.
possible product from the reaction of maleic anhydride and
hydrazine.
All these compounds were obtained by reacting the anhydride 13a
with a suitable amine or hydrazine in ethanol (Scheme 5). The
groups introduced and the corresponding yields are reported in
Table 1.
Evaluation of antifungal activity
The antifungal activity of the synthesized compounds was assessed
by in vitro experiments on a set of phytopathogenic fungi causing
widespread diseases on crops (P. infestans, P. ultimum, B. cinerea,
C. beticola and R. solani).
To confirm the structure of 17d, it was reacted with benzalde-
hyde (17). The formation of the corresponding imino derivative
excluded the presence of a maleic hydrazide derivative, the other
The activity data for the compounds 2, 4a and 5a against different
fungi are reported in Table 2.
Chem Biol Drug Des 2012; 79: 780–789
785

Musso et al.
Scheme 3: Synthesis of compounds 13a,b.
Scheme 4: Synthesis of compound 16.
Scheme 5: Synthesis of compounds 17a–f and 18a–c.
As mentioned in the previous paragraph, these compounds showed
a broad spectrum of activity; in particular, oomycetes were the most
sensitive target organisms followed by B. cinerea. Both anhydrides
5a and 2 showed high activity against P. infestans and P. ultimum.
In particular, compound 5a completely inhibited the growth of both
these pathogens showing the same effectiveness of the standard
compound, tolilfluanide, at 100 lg ⁄ mL. Moreover, compound 5a
showed higher activity than 2 against all the other fungal species
tested, especially at the lowest concentrations. The ester 4a
resulted to be the less active one; moreover, its scarce solubility
made difficult to test the compound at concentrations higher than
250 lg ⁄ mL m. The anhydride 5b, bearing a methyl group at posi-
tion 10 of the cyclooctadiene ring, was less active than 5a against
the oomycete organisms, but showed almost the same activity
against the other species. Surprisingly, anhydride 12 with a butyl
chain as in zopfiellin generally showed weaker fungicidal activity.
The anhydride 16 was less active than 2 (at 250 and 125 lg ⁄ mL);
it is possible to hypothesize that the presence of an aromatic ring
plays an important role in terms of lipophilicity or in rigidifying the
structure.
The introduction of an alkyl chain as an enolether group in position
5 led to a substantial decrease in antifungal activity against
786
Chem Biol Drug Des 2012; 79: 780–789

Cyclooctadiene Anhydride Analogues of Zopfiellin
Table 1: Structure and yields of compounds 17a–f and 18a–c
esting activity against the tested pathogens, and the N-benzyl deriv-
ative 18c was not soluble enough to be tested.
n
R
R'
Yield (%)
On the contrary, the introduction of a hydrazino group (17d–f) had
a strong increasing effect and enlarged the spectrum of activity.
The activity increased from the less polar (17f) to the more polar
17d, bearing the simple aminoimido moiety; this compound com-
pletely inhibited the mycelial growth of P. infestans at 62.5 lg ⁄ mL;
it also induced 100% inhibition of P. ultimum and 70.9% of B. cine-
rea at 125 lg ⁄ mL.
17a
17b
17c
17d
17e
17f
CH3
CH3
CH3
CH3
CH3
CH3
n-butyl
n-butyl
n-butyl
H
88
80
74
90
86
94
89
99
52
n-butyl
benzyl
NH2
NHCH2CH2OH
N(CH3)2
H
18a
18b
18c
n-butyl
benzyl
Preliminary in vivo evaluation of protectant
activity
The most active compounds 5a, 2 and 17d were selected for a
preliminary in vivo evaluation of their protectant activity.
P. infestans and P. ultimum (13a and 13b versus 2). On the con-
trary, the ethers 13a and 13b resulted to be more active than 2
at 125 lg ⁄ mL against B. cinerea, C. beticola and R. solani.
The introduction of a simple imido group (17a) did not improve the
activity with respect to the corresponding anhydride 13a (Table 3).
Moreover, with the only exception of B. cinerea, the imido deriva-
tive of butyl enolether 18a resulted in much less active than 17a,
the imide bearing a methyl enolether group.
The tests were performed on pathogen–host combinations chosen
among some of the most economically relevant and widespread
ones: P. graminis ⁄ wheat cv Irnerio (wheat rust), E. graminis ⁄ wheat
cv Irnerio (powdery mildew), P. viticola ⁄ vine cv dolcetto (downy mil-
dew) and P. infestans ⁄ tomato cv Marmande (downy mildew).
The N-butylimido derivative 17b showed the highest activity
against C. beticola and R. solani, whereas the N-butylimido deriva-
tive 18b, because of its very poor solubility, could only be tested
at a low concentration (25 lg ⁄ mL), at which it was not active.
Compound 17c, featuring a N-benzyl group, did not show any inter-
No phytotoxic effect was shown by all compounds at the concentra-
tions used. Compound 5a resulted to be the most active: at
1000 lg ⁄ mL, it inhibited 60% of wheat rust, 40% of downy mildew
on grapevine and 10% of wheat powdery mildew (Table 4). Com-
pound 2 was active against wheat rust (50% inhibition) and scarcely
Table 2: Per cent inhibition of mycelial growth induced by compounds 2, 4a, 5a,b, 12, 16 and 13a–b
Oomycetes
Ascomycota
Basidiomycota
Concentration
(lg ⁄ mL)
Phytophthora
infestans
Pythium
ultimum
Botrytis
cinerea
Cercospora
beticola
Rhizoctonia
solani
Compound
4a
250
125
500
250
125
125
62.5
125
62.5
500
250
125
250
125
62.5
100
50
38.5
20.1
100
100
100
66.1
50.3
28.5
18.8
100
100
100
87.2
35.7
10.0
41.2
40.0
16.7
13.5
100
⁄
34.3
20.6
⁄
42.0
32.0
76.0
74.0
67.0
72.8
55.6
9.88
⁄
68.6
35.0
0
3
0
12.2
25.4
45.6
30.1
10.6
29.7
18.7
23.4
15.6
41.7
4.8
0
7.4
0
0
29.5
29.5
36.4
34.1
⁄
16.2
15.2
44.4
32.2
20.2
26.0
15.6
25.0
23.2
30.3
15.2
10.1
4.5
5a
⁄
100
88.9
66.9
32.4
25.5
⁄
90.4
67.1
70.9
48.1
21.5
46.8
45.4
19.5
16.7
100
⁄
5b
12
2
16
0
0
0
13a
13b
25.8
20.7
44.6
30.8
⁄
100
⁄
9.34
6.4
23.1
10.6
⁄
125
62.5
100
25
aa
ba
ca
⁄
⁄
10
⁄
⁄
100
90.6
aa, Euparenꢁ (tolilfluanide, 50%); b, Sideralꢁ (procymidone, 50%); c, tetraconazole. The concentrations reported for the standards are referred to the pure
compound.
Chem Biol Drug Des 2012; 79: 780–789
787

Musso et al.
Table 3: Per cent inhibition of mycelial growth induced by compounds 17a–f and 18a–b
Oomycetes
Ascomycota
Basidiomycota
Concentration
(lg ⁄ mL)
Phytophthora
infestans
Pythium
ultimum
Botrytis
cinerea
Cercospora
beticola
Rhizoctonia solani
Compound
17a
250
125
62.5
125
62.5
25
125
62.5
125
62.5
125
62.5
125
62.5
25
65.7
55.7
44.3
19.9
11.1
0
100
100
52.7
38.2
26.1
22.4
10.3
8.1
64.6
51.8
41.8
23.1
12.8
2.5
100
89.7
61.4
37.9
66.9
50.4
23.8
16.7
0
22.2
19.0
26.9
14.5
8.0
0
70.9
32.3
55.6
34.6
35.8
⁄
27.3
25.6
25.6
56.2
46.2
8.5
50.4
46.7
48.4
42.2
39.1
⁄
11.4
9.1
4.7
⁄
⁄
16.6
8.9
7.5
52.6
23.1
0
32.6
16.8
13.5
8.33
11.5
⁄
17b
17c
17d
17e
17f
18a
58.5
52.2
0
14.3
13.3
2.1
⁄
18b
aa
ba
ca
0
100
⁄
100
25
10
100
⁄
⁄
⁄
100
⁄
⁄
⁄
100
90.6
aa: Euparenꢁ (tolilfluanide, 50%); b: Sideralꢁ (procymidone, 50%); c: tetraconazole. The reported standard concentrations are referred to the pure compound.
In particular, we focused on cyclooctadiene structures carrying only
one anhydride moiety. The synthesis of such compounds was car-
ried out exploiting the reaction of ring expansion of carbocyclic b-
ketoesters in the presence of acetylenic esters. For the first time,
we extended this methodology to 2-carboxymethyltetralones substi-
tuted at position 3 or 4 to obtain, in good yields, cyclooctadienes
bearing alkyl chains at allylic or homoallylic position with respect to
the anhydride moiety.
Table 4: In vivo protectant activity of compounds 5a, 2 and
17d expressed as per cent inhibition of infection
Wheat
Grapevine Tomato
Concentration Puccinia Erysiphe Plasmopara Phytophthora
Compound
(lg ⁄ mL)
graminis graminis viticola
infestans
2
1000
500
1000
500
1000
500
50
10
60
20
0
10
0
10
0
10
0
0
0
40
10
0
0
0
0
0
0
5a
The analysis of the in vitro growth inhibition data on a set of
phytopathogen fungi showed that most of the synthesized com-
pounds possessed a broad spectrum of activity. Even if the syn-
thesized compounds resulted to be less active than zopfiellin
against B. cinerea (7), they were very effective against the oomy-
cetes P. infestans and P. ultimum, the anhydrides 5a and 2
reaching a level of activity well comparable with that of com-
mercial fungicides in use.
17da
0
0
0
Benalaxyl
100
100
100
Tetraconazole 100
100
100
aCompound 17d was dispersed in water ⁄ Tween-20.
(10%) against wheat powdery mildew. Surprisingly, compound 17d
resulted completely inactive versus all the tested diseases.
The replacement of the anhydride moiety with the simple aminoimi-
do group afforded the most active derivative (17d).
None of the compounds controlled the development of downy mil-
dew caused by P. infestans infection on tomato plants, notwith-
standing the good in vitro inhibition of this pathogen by 5a, 2 and
17d.
However, the encouraging results of the activity in vitro were not
confirmed by in vivo protectant tests on the most active compounds
5a, 2 and 17d. Further studies are necessary to improve the pene-
tration or transport properties of these compounds.
Conclusions
Acknowledgements
In summary, we performed the synthesis of structurally and synthet-
ically simplified derivatives of the natural compound zopfiellin, with
the aim to establish whether these compounds could maintain anti-
fungal activity and could be useful as new lead compounds.
The authors thank Dr Luigi Mirenna, Department of Biology and
Agronomy of ISAGRO Research, for the in vivo tests and the Univer-
sity of Milano (FIRST funds) for financial support.
788
Chem Biol Drug Des 2012; 79: 780–789

Cyclooctadiene Anhydride Analogues of Zopfiellin
1,2,5,6-tetracarboxylates through photochemical [2 + 2]cycloaddi-
tion of 3-substituted cyclobutene-1,2-dicarboxylates and thermal
isomerization, Chem Commun;1999(17):1753–1754.
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