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Keywords:

  • antifungal activity;
  • cyclooctadiene anhydrides;
  • natural compounds;
  • synthesis;
  • zopfiellin

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgements
  7. References

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 broad spectrum of activity. In particular, the anhydrides 2 and 5a were very effective against the Oomycete diseases such as Phytophthora infestans and Pythium ultimum, reaching a level of activity well comparable with that of commercial fungicides in use. Preliminary in vivo evaluation of their protectant activity is also reported.

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 cultivars, crop rotation or sanitation practices, fungicides are still essential for effective control of most plant diseases and therefore to maximize crop yields. Despite the choice of effective fungicides available, 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 public for crop protection agents with low use rates, a benign environmental 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 compounds 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, 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.

image

Figure 1.  Structure of zopfiellin.

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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 Nissan 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 μm), Colletotrichum gloeosporioides (IC80 10 μm) and Colletotrichum fragariae (IC80 10 μm) (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 involving 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 carboxylate form, present at higher pH values, is too polar to move across the lipophilic plasma membrane. Therefore, intramolecular ring closure of zopfiellin from tetracarboxylate to an anhydride form occurring at pH below 6 is apparently required for uptake by the fungus.

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 fungicidal activity (11). These facts justify further studies on this class of molecules to identify the essential structural requirements for their antifungal activity.

In this article, we report the preparation of structurally and synthetically 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.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgements
  7. References

Chemistry

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 δ values in parts per million (p.p.m.) and are indirectly referenced to tetramethylsilane (TMS) via the solvent signal (7.26 for 1H, 77.0 for 13C) in CDCl3. Coupling constants (J) are given in Hz.

Solvents were routinely distilled prior to use; anhydrous tetrahydrofuran (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 glassware 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 pressure and dried at 110 °C overnight.) Analytical thin-layer chromatography (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 Stuart Scientific SMP3 instrument and are uncorrected.

Compounds 2, 4a, 5a and 16 were prepared according to the literature procedures (13). Compounds 13a and 17a,c,d, were prepared according to the literature procedures (14).

5-Hydroxy-10-methyl-9,10-dihydro-benzocyclooctene-6,7,8-tricarboxylic acid trimethyl ester (4b)

To a suspension of sodium hydride (77 mg, 1.93 mmol, 60% dispersion in mineral oil) in dry toluene (2 mL) under nitrogen atmosphere, 2-methoxycarbonyl-4-methyl-1-tetralone 3b (312 mg, 1.43 mmol) dissolved in dry toluene (4 mL) was added; when the addition was complete, 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 μL, 1.57 mmol) was slowly added with the temperature being kept below 5 °C. The reaction 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 give 294 mg (57%) of the title product as a pale yellow oil. 1H NMR δ: 13.25 (s, 1H, OH), 7.33 (dd, 1H, = 1.5, 7.4 Hz, Ar), 7.27 (ddd, 1H, = 7.4, 7.8, 1.5 Hz, Ar), 7.23 (ddd, 1H, = 7.8, 7.2 1.5 Hz, Ar), 7.04 (dd, 1H, = 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, = 13.0, 7.8 Hz, -CH2CHCH3), 2.74 (dd, 1H, = 13.0, 11.9 Hz,-CH2CHCH3), 1.24 (d, 3H, = 7.0 Hz, >CHCH3,). Anal. Calcd for C19H20O7: C, 63.33; H, 5.59. Found: C, 63.16; H, 5.44.

5-Hydroxy-10-methyl-1,3-dioxo-1,3,10,11-tetrahydro-2-oxa-benzo[a]cyclopenta[e]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 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 (68%) of the title compound as a white solid. mp 175–176 °C; 1H NMR δ: 13.61 (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, = 4.1, 19.3 Hz); 2.62 (dd, 1H, = 13.0, 19.3 Hz); 1.43 (d, 3H, = 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 cyanoacetate (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 δ: 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, = 7.8 Hz, CH2C=C<), 2.45 (t, 2H, = 7.8 Hz, -CH2C=C<), 1.49 (m, 2H); 1.36 (m, 6H), 0.88 (t, 3H, = 7.4 Hz, CH3), 0.87 (t, 3H, = 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.

3-Benzyl-2-cyano-heptanoic acid methyl ester (8)

Compound 7 (400 mg, 1.55 mmol) was hydrogenated in ethanol solution (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 under vacuum to give 401 mg (80%) of product that was directly utilized without further purification. Yellow oil. 1H NMR (mixture of diastereoisomers): δ 7.38–7.14 (m, 10H, Ar), 3.75 (s, 3H, -OCH3), 3.67 (s, 3H, -OCH3), 3.37 (d, 1H, = 3.3 Hz, -CH<), 3.04 (dd, 1H, = 13.0, 3.3 Hz, >CH2Ph), 2,75 (d, 2H, = 7.4 Hz, >CH2Ph), 2.50 (dd, 1H, = 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, = 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 product as a yellow oil. 1H NMR δ: 8.00 (dd, 1H, = 1.5, 7.8, Ar), 7.46 (ddd, 1H, = 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, = 11.9, 16.0 Hz), 2.18 (m, 1H, >CHCH2-), 1.34 (m, 6H, -CH2-), 0.91 (t, 3H, = 7.0 Hz, -CH3). Anal. Calcd for C14H18O: C, 83.12; H, 8.97; Found: C, 82.97; H, 8.89.

3-Butyl-1-oxo-1,2,3,4-tetrahydro-naphthalene-2-carboxylic acid methyl ester (10)

Under nitrogen atmosphere, sodium hydride (136 mg, 60% dispersion 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 toluene (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 product as a yellow oil. 1H NMR δ: 12.45 (s, 1H, enolic OH); 8.00 (d, 1H, = 7.8, Ar), 7.77 (dd, 1H, = 1.1, 7.0 Hz, Ar), 7.50 (m, 2H, Ar), 7.20–7.35 (m, 3H, Ar), 7.15 (d, 1H, = 7.0 Hz, Ar), 3.82 (s, 3H, -OCH3), 3.78 (s, 3H, -OCH3), 3.38 (d, 1H, = 10.4 Hz), 2.99 (dd, 1H, = 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 ketone+enol). Anal. Calcd for C16H20O3: C, 73.82; H, 7.74. Found: 73.91; H, 7.69.

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 atmosphere 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 stirred for 30 min at room temperature and then cooled to 0 °C. With continued stirring, DMAD (150 μL, 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 aqueous 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 (hexane/Et2O, 6:4) to give 168 mg (42%) of product as a white solid. mp 95–96 °C; 1H NMR δ: 13.15 (s, 1H, OH), 7.38 (dd, 1H, = 7.4 Hz, 1.5, Ar), 7.28 (ddd, 1H, = 7.4, 7.8, 1.8 Hz, Ar), 7.21 (ddd, 1H, = 7.4, 7.8 Hz, Ar), 7.07 (dd, 1H, = 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, = 18.2, 14.1 Hz, CHHCH<), 1.66–1.18 (m, 6H, -CH2-); 0.88 (t, 3H, = 7.0 Hz, -CH3). Anal. Calcd for C22H26O7: C, 65.66; H, 6.51. Found: C, 65.79; H, 6.65.

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)

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 compound as a white powder. mp 147–148 °C; 1H NMR: δ 13.48 (s, 1H, OH), 7.48 (m, 1H, Ar), 7.42 (d, 1H, = 7.8 Hz, Ar) 7.32 (m, 1H, Ar), 7.20 (m, 1H, Ar), 3.85 (s, 3H, -OCH3), 3.10 (dd, 1H, = 12.2, 14.5 Hz, -CHHCH<), 3.04 (m, 1H, >CH(CH2)3CH3), 2.88 (dd, 1H, = 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, = 7.0 Hz, -CH3). Anal. Calcd for C20H20O6: C, 67.41; H, 5.66. Found: C, 67.35; H, 5.53.

10-Butoxy-4,5-dihydro-2-oxa-benzo[a]cyclopenta[e]cyclooctene-1,3-dione (13b)

n-Butanol (60 μL, 0.66 mmol) was added dropwise to a suspension of 2 (40 mg, 0.16 mmol) in dry toluene (0.5 mL); thionyl chloride (50 μL, 0.66 mmol) was then added at 0 °C. The resulting mixture was stirred at room temperature for 24 h. After this period, the mixture was washed with water and brine. The solvent 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 δ: 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, = 7.4 Hz, -CH3). Anal. Calcd for C18H18O4: C, 72.47; H, 6.08. Found: C, 72.33, H, 5.98.

2-Butyl-10-methoxy-4,5-dihydro-2-aza-benzo[a]cyclopenta[e]cyclooctene-1,3-dione (17b)

Freshly distilled n-butylamine (780 μL, 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 followed 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 δ: 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, = 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.

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-hydroxyethylhydrazine (24 μL, 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 deactivated silica gel (hexane/ethyl acetate, 1:2) gave 79 mg (86%) of product as a yellow powder. mp. 113–114 °C; 1H-NMR δ: 7.40 (m, 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.

2-Dimethylamino-10-methoxy-4,5-dihydro-2-azabenzo[a]cyclopenta[e]cyclooctene-1,3-dione (17f)

To a solution of 13a (98 mg, 0.38 mmol) in methanol (2 mL), 1,1-dimethylhydrazine (30 μL, 0.39 mmol) was added; the resulting mixture 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 δ: 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.

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 pressure, and the residue was crystallized by Et2O to obtain 88 mg (89%) of product as a yellow powder. mp 165–166 °C; 1H NMR δ: 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 (m, 2H, -CH2-), 1.50 (m, 2H, -CH2-), 1.00 (t, 3H, = 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.

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 μL, 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 residue (hexane/ethyl acetate, 95:5) gave 293 mg (99%) of product as a yellow powder. mp 106–107 °C; 1H NMR δ: 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, = 7.0 Hz,-CH3,), 0.60 (t, 3H, = 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-Benzyl-10-butoxy-4,5-dihydro-2-aza-benzo[a]cyclopenta[e]cyclooctene-1,3-dione (18c)

To a solution of 13b (53 mg, 0.17 mmol) in toluene (1.5 mL), benzylamine (50 μL, 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 followed by flash column chromatography on deactivated silica gel of the residue (hexane/ethyl acetate, 95:5) gave 36 mg (52%) of the title compound yellow powder. mp 130–131 °C; 1H NMR δ: 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, = 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.

Evaluation of in vitroantifungal 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 beticola Sacc., Rhizoctonia solani J.G. Kühn] to give suitable concentrations 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 prepared for each fungus. The final DMSO concentration did not exceed 0.3% of the final volume in both control and treated cultures. 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 actively growing colonies. Two replicates were used for each concentration, 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.

The results were compared with those obtained with three standard fungicides: Euparen® (tolilfluanide, 50%, Bayer S.p.A., Italy), Sideral® (procymidone, 50%, Sipcam S.p.A., Italy) and tetraconazole® (Isagro S. p. A., Italy).

Evaluation of in vivo antifungal activity

The tests of direct protectant activity of the compounds were performed on the following pathogen–host combinations: Puccinia graminis 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 suitable 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 corresponded to completely infected leaves and 0 to not visible symptoms. The results were compared with those obtained with two standard fungicides in commercially available formulations, benalaxyl and tetraconazole, and the activity was expressed as per cent inhibition of infection in comparison with untreated control.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgements
  7. References

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).

image

Figure 2.  Cyclooctadiene scaffold.

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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 decarboxylation led to keto-anhydride 2 in 76% yield (Figure 2).

image

Figure Scheme 1:.  Synthesis of compounds 2, 4a and 5a,b.

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The antifungal activity of compound 2 and of the intermediates 4a and 5a was assessed by in vitro experiments to validate the hypothesis of their use as a scaffold. A set of phytopathogenic fungi causing widespread diseases on crops were used (P. infestans, 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 compound, highlighting the relevance of anhydride moiety for the antifungal activity. Moreover, anhydride 5a showed higher activity than 2 against the fungal species tested, especially at the lowest concentrations.

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 β-ketoesters in the presence of acetylenic esters, extending for the first time this methodology to 2-carboxymethyltetralones substituted at position 3 or 4.

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 synthesize 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 tetralone 9 by means of acidic treatment. Finally, the desired β-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.

image

Figure Scheme 2:.  Synthesis of compound 12.

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We then capitalized on the presence of the carbonyl group in compound 2 to prepare derivatives substituted at position 5. The introduction 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(CH2CH2)O(CH2CH2)OH, 4-chlorobutanol and 2-dimethylaminoethanol, failed.

image

Figure Scheme 3:.  Synthesis of compounds 13a,b.

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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 intermediate 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 decarboxylation by basic hydrolysis of compound 15, failed (Scheme 4).

image

Figure Scheme 4:.  Synthesis of compound 16.

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Finally, we tested the imido derivative 17a and the hydrazino derivative 17d (14) to explore the relevance of the anhydride group for the antifungal activity. As the activity was maintained in both compounds, we prepared new derivatives bearing different substituents at the imido nitrogen.

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.

image

Figure Scheme 5:.  Synthesis of compounds 17a–f and 18a–c.

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Table 1.   Structure and yields of compounds 17a–f and 18a–c
nRR’Yield (%)
17aCH3H88
17bCH3n-butyl80
17cCH3benzyl74
17dCH3NH290
17eCH3NHCH2CH2OH86
17fCH3N(CH3)294
18an-butylH89
18bn-butyln-butyl99
18cn-butylbenzyl52

To confirm the structure of 17d, it was reacted with benzaldehyde (17). The formation of the corresponding imino derivative excluded the presence of a maleic hydrazide derivative, the other possible product from the reaction of maleic anhydride and hydrazine.

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).

The activity data for the compounds 2, 4a and 5a against different fungi are reported in Table 2.

Table 2.   Per cent inhibition of mycelial growth induced by compounds 2, 4a, 5a,b, 12, 16 and 13a–b
CompoundConcentration (μg/mL)OomycetesAscomycotaBasidiomycota
Phytophthora infestansPythium ultimumBotrytis cinereaCercospora beticolaRhizoctonia solani
  1. aa, Euparen® (tolilfluanide, 50%); b, Sideral® (procymidone, 50%); c, tetraconazole. The concentrations reported for the standards are referred to the pure compound.

4a25038.534.342.012.216.2
12520.120.632.025.415.2
5a500100/76.045.644.4
250100/74.030.132.2
12510010067.010.620.2
5b12566.188.972.829.726.0
62.550.366.955.618.715.6
1212528.532.49.8823.425.0
62.518.825.5/15.623.2
2500100/68.641.730.3
25010090.435.04.815.2
12510067.10010.1
1625087.270.937.44.5
12535.748.1000
62.510.021.5000
13a10041.246.825.829.59.34
5040.045.420.729.56.4
13b12516.719.544.636.423.1
62.513.516.730.834.110.6
aa100100100///
ba25//100//
ca10///10090.6

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 Bcinerea. 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 μg/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 μg/mL m. The anhydride 5b, bearing a methyl group at position 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 μg/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 Pinfestans and Pultimum (13a and 13b versus 2). On the contrary, the ethers 13a and 13b resulted to be more active than 2 at 125 μg/mL against Bcinerea, Cbeticola and Rsolani.

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 Bcinerea, the imido derivative of butyl enolether 18a resulted in much less active than 17a, the imide bearing a methyl enolether group.

Table 3.   Per cent inhibition of mycelial growth induced by compounds 17a–f and 18a–b
CompoundConcentration (μg/mL)OomycetesAscomycotaBasidiomycota
Phytophthora infestansPythium ultimumBotrytis cinereaCercospora beticolaRhizoctonia solani
  1. aa: Euparen® (tolilfluanide, 50%); b: Sideral® (procymidone, 50%); c: tetraconazole. The reported standard concentrations are referred to the pure compound.

17a25065.764.622.227.316.6
12555.751.819.025.68.9
62.544.341.826.925.67.5
17b12519.923.114.556.252.6
62.511.112.88.046.223.1
17c2502.508.50
17d12510010070.950.432.6
62.510089.732.346.716.8
17e12552.761.455.648.413.5
62.538.237.934.642.28.33
17f12526.166.935.839.111.5
62.522.450.4///
18a12510.323.858.511.414.3
62.58.116.752.29.113.3
18b250004.72.1
aa100100100///
ba25//100//
ca10///10090.6

The N-butylimido derivative 17b showed the highest activity against Cbeticola and Rsolani, whereas the N-butylimido derivative 18b, because of its very poor solubility, could only be tested at a low concentration (25 μg/mL), at which it was not active. Compound 17c, featuring a N-benzyl group, did not show any interesting activity against the tested pathogens, and the N-benzyl derivative 18c was not soluble enough to be tested.

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 completely inhibited the mycelial growth of Pinfestans at 62.5 μg/mL; it also induced 100% inhibition of Pultimum and 70.9% of Bcinerea at 125 μg/mL.

Preliminary in vivoevaluation of protectant activity

The most active compounds 5a, 2 and 17d were selected for a preliminary in vivo evaluation of their protectant activity.

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 mildew) and P. infestans/tomato cv Marmande (downy mildew).

No phytotoxic effect was shown by all compounds at the concentrations used. Compound 5a resulted to be the most active: at 1000 μg/mL, it inhibited 60% of wheat rust, 40% of downy mildew on grapevine and 10% of wheat powdery mildew (Table 4). Compound 2 was active against wheat rust (50% inhibition) and scarcely (10%) against wheat powdery mildew. Surprisingly, compound 17d resulted completely inactive versus all the tested diseases.

Table 4. In vivo protectant activity of compounds 5a, 2 and 17d expressed as per cent inhibition of infection
CompoundConcentration (μg/mL)WheatGrapevineTomato
Puccinia graminisErysiphe graminisPlasmopara viticolaPhytophthora infestans
  1. aCompound 17d was dispersed in water/Tween-20.

21000501000
50010000
5a10006010400
500200100
17da100001000
5000000
Benalaxyl100  100100
Tetraconazole100100100  

None of the compounds controlled the development of downy mildew caused by Pinfestans infection on tomato plants, notwithstanding the good in vitro inhibition of this pathogen by 5a, 2 and 17d.

Conclusions

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgements
  7. References

In summary, we performed the synthesis of structurally and synthetically simplified derivatives of the natural compound zopfiellin, with the aim to establish whether these compounds could maintain antifungal activity and could be useful as new lead compounds.

In particular, we focused on cyclooctadiene structures carrying only one anhydride moiety. The synthesis of such compounds was carried out exploiting the reaction of ring expansion of carbocyclic β-ketoesters in the presence of acetylenic esters. For the first time, we extended this methodology to 2-carboxymethyltetralones substituted 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.

The analysis of the in vitro growth inhibition data on a set of phytopathogen fungi showed that most of the synthesized compounds possessed a broad spectrum of activity. Even if the synthesized compounds resulted to be less active than zopfiellin against Bcinerea (7), they were very effective against the oomycetes P. infestans and P. ultimum, the anhydrides 5a and 2 reaching a level of activity well comparable with that of commercial fungicides in use.

The replacement of the anhydride moiety with the simple aminoimido group afforded the most active derivative (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 penetration or transport properties of these compounds.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgements
  7. References

The authors thank Dr Luigi Mirenna, Department of Biology and Agronomy of ISAGRO Research, for the in vivo tests and the University of Milano (FIRST funds) for financial support.

References

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  2. Abstract
  3. Materials and Methods
  4. Results and Discussion
  5. Conclusions
  6. Acknowledgements
  7. References
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