Fungistatic activity of bicyclo[4.3.0]-γ-lactones

Article abstract of DOI:10.1021/jf105019u

Five optically active and sixteen racemic lactones (nine of them new) of bicyclo[4.3.0]nonane structure were synthesized. IC₅₀ values for the following phytopathogens were determined: Aspergillus ochraceus AM 456, Fusarium culmorum AM 282, Fusarium oxysporum AM 13, Fusarium tricinctum AM 16. Effect of compound structures, especially stereogenic centers, on fungistatic activity has been discussed. The highest fungistatic activity was observed for trans-7,8-dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one (3c), IC₅₀ = 30.1 μg/mL (0.10 μM/mL), and cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2- one (3b), IC₅₀ = 72.2 μg/mL (0.47 μM/mL), toward F. oxysporum AM 13.

Full text of DOI:10.1021/jf105019u

ARTICLE
pubs.acs.org/JAFC
Fungistatic Activity of Bicyclo[4.3.0]-γ-lactones
Teresa Olejniczak,*,† Filip Boratyꢀnski,† and Agata Biazoꢀnska‡
†Department of Chemistry, University of Environmental and Life Sciences, Norwida 25, 50-375 Wroczaw, Poland
‡Faculty of Chemistry, Wroczaw University, F. Joliot-Curie 14, 50-383 Wroczaw, Poland
ABSTRACT: Five optically active and sixteen racemic lactones (nine of them new) of bicyclo[4.3.0]nonane structure were
synthesized. IC50 values for the following phytopathogens were determined: Aspergillus ochraceus AM 456, Fusarium culmorum AM
282, Fusarium oxysporum AM 13, Fusarium tricinctum AM 16. Effect of compound structures, especially stereogenic centers, on
fungistatic activity has been discussed. The highest fungistatic activity was observed for trans-7,8-dibromo-cis-3-oxabicyclo-
[4.3.0]nonan-2-one (3c), IC50 = 30.1 μg/mL (0.10 μM/mL), and cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b), IC50
72.2 μg/mL (0.47 μM/mL), toward F. oxysporum AM 13.
=
KEYWORDS: lactones, IC50, HLADH, Fusarium oxysporum, Fusarium culmorum
’ INTRODUCTION
’ MATERIALS AND METHODS
Species of Aspergillus, Fusarium, and Penicilium are commonly
found in soil and air. Not only are they saprophytes and plant
pathogens but they also may attack people with low immunore-
sistance.1ꢀ4 Under unfavorable conditions numerous strains may
produce mycotoxins.5,6 Especially troublesome and difficult to
combat are phytopathogens of the genus Fusarium, for example
F. culmorum and F. tricinctum species are responsible for head blight
infection,7ꢀ10 and F. oxysporum is the main cause of wilt.11,12 The
microorganisms of the genus Fusarium cause huge loss in agricul-
ture and product storage, and also they produce a wide range of
toxic compounds, which are dangerous for humans, like trichocetin
toxins (e.g., T-2 toxin, deoxynivalenone), zearalenone and
fumonisins.8,10,13,14 These compounds may attack liver, kidney,
circulatory, endocrine or digestive systems, skin and blood.8
Lactones are a very interesting group of compounds, because
of different properties and biological activities. They have been
identified as natural ingredients of plant oils, vegetables, fruits
and food products.15ꢀ19 Terpenoid lactones are characterized
with attractive, mainly fruity fragrances and usually demonstrate
sweet, coconut or herbal taste.20,21
Compounds with lactone ring show a wide range of biological
activities, among which the most widely known are fungistatic,22ꢀ24
bacteriostatic25,26 and cytostatic27ꢀ30 ones. Moreover numerous
insect pheromones consist of a lactone ring in their structure.31 The
lactones synthesized by our group proved also to have deterrent
activity toward insects.32ꢀ34 Therefore, it was purposeful to check
whether they also indicate fungistatic activity toward fungi strains
present in soil or in stored grain, vegetables and fruits.
Analytical Methods. Composition and purity of products were
established by thin layer chromatography (TLC) and gas chromatogra-
phy (GC). TLC was carried out on silica gel Kieselgel 60 F254 (Merck)
with hexane:acetone:isopropanol:ethyl acetate (60:1:3:1) as a develop-
ing system. Compounds were visualized by spraying the plates with 1%
Ce(SO4)2, 2% H3[P(Mo3O10)4] in 10% H2SO4, followed by heating.
Gas chromatography analyses were performed on an Agilent instrument,
fitted with a flame ionization detector (FID), using a Carbowax column
(30 m, 0.53 mm, 0.88 μm film thickness) or a chiral column: CP-
cyclodextrin (25 m ꢁ 0.25 mm, 0.25 μm film thickness); H2 at a flow
rate of 2 mL/min was used as a carrier gas.
Molecular mass was confirmed on a Varian Chrompack GC MS CP-
3800 Saturn 2000 GC/MS/MS with ionization energy of 70 eV,
using HP-1 column (cross-linked methyl silicone gum, 25 m ꢁ 0.32
mm ꢁ0.25 μm film thickness), and HRMS analysis was conducted on a
micrOTOF-Q Bruker.
Optical rotations were measured on an Autopol IV automatic
polarimeter (Rudolph) with thermostatic system, in chloroform solu-
tions, at concentrations given in g/100 mL.
X-ray data for compound 3c were collected on a Kuma KM4CCD
diffractometer (Mo KR radiation; λ = 0.71073 Å) at 100 K using an
Oxford Cryosystem device. Data reduction and analysis were carried out
with the CrysAlice “RED” program. The analytical correction for
absorption was applied. The space groups were determined using the
XPREP program. The structure was solved by direct methods using the
XS program and refined using all F2 data, as implemented by the XL
program.40 Non-hydrogen atoms were refined with anisotropic displa-
cement parameters. All H atoms were placed at calculated positions.
Before the last cycle of refinement all H atoms were fixed and were
allowed to ride on their parent atoms.
We have synthesized 9-carbon lactones with structures similar
to natural phthalides,35ꢀ39 and their structural analogues. Fungi-
static activity of these compounds was determined for
the following strains: Aspergillus ochraceus AM 456, Fusarium
culmorum AM 282, F. oxysporum AM 13, F. tricinctum AM 16 and
Penicillium citrinum AM 354. Our study concerned structural
aspects of fungistatic activity, so that we synthesized both racemic
and optically active bicyclic lactones and we were searching for
functional groups or other parts of molecules that are important
with respect to their biological activity.
Structures of the substrates and the products were determined based
on 1H NMR, 13C NMR, COSY, HMQC and IR spectra. NMR spectra
were recorded at 500 MHz using CDCl3 solution, on a Bruker Avance
Received: December 31, 2010
Accepted: April 26, 2011
Revised:
April 25, 2011
Published: April 26, 2011
r
2011 American Chemical Society
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DRX-500 spectrometer. IR spectra were measured on a Mattson FTIR-
300 Thermo Nicolet spectrometer.
J = 5.0, 8.8 Hz, one of CH2-4); 13C NMR (151 Mz, CDCl3) δ (ppm)
22.4 (CH2-6), 22.8 (CH2-8), 23.3 (CH2-7), 27.1 (CH2-9), 35.3 (CH-5),
39.4 (CH-1), 71.7 (CH2-4), 178.6 (C-2); IR (film, cmꢀ1) 2942 (s),
2254 (s), 1766 (s), 1210 (m), 989 (m); GCꢀEIMS 141 [M þ 1].
The cis-diols: 4,5-bis(hydroxymethyl)cyclohexene 16 and 1,2-bis-
(hydroxymethyl)cyclohexane 17 were prepared in good yields by the
literature41 procedures.
Chemicals. The chemicals used for the synthesis were purchased
from Aldrich, Sigma, and Fluka. PDA for fungistatic tests was purchased
from BTL in Poland. DDAC, didecyldimethylammonium chloride (50%
solution in propan-2-ol/water 2:3), was purchased from Merck.
Enzymes and Coenzymes. The following alcohol dehydro-
genases were used: HLADH recombinant from Escherichia coli
(EVO), PADH 3000, screening kit (Codexis), LKADH from Lactoba-
cillus kefir (Fluka), YADH from Baker yeast (Sigma-Aldrich).
(()-4,4-Dimethyl-cis-3-oxabicyclo[4.3.0]non-7-en-2-one (4a). To
magnesium (215 mg, 8.96 ꢁ 10ꢀ3 mol) activated with iodine was added
anhydrous diethyl ether (10 mL), followed by slow addition of methyl
iodide (1.272 g, 8.96 ꢁ 10ꢀ3 mol). The reaction was continued until all
the magnesium had reacted, and then cis-1,2,3,6-tetrahydrophthalic
anhydride 1 (0.6809 g, 4.48 ꢁ 10ꢀ3 mol) was added dropwise at room
temperature. After 3 h the reaction mixture was poured into 6% HCl
mixed with ice and extracted with diethyl ether (5 ꢁ 50 mL), and the
ethereal phase was washed with 6% HCl (2 ꢁ 30 mL) and brine until
neutral. The extract was dried over MgSO4, the solvent was evaporated off
and the reaction mixture was purified by column chromatography using
hexane:acetone 4:1 as eluent to give 0.5212 g of lactone 4a (yield 70%):
1H NMR (500 MHz, CDCl3) δ (ppm) 1.38 (s, 6, C-4(CH3)2),
1.75ꢀ1.95 (m, 1, one of CH2-6), 2.00ꢀ2.35 (m, 3, one of CH2-9, one
of CH2-6, H-5), 2.36ꢀ2.56 (m, 1, H-1), 2.95ꢀ3.18 (m, 1, one of CH2-9),
5.63ꢀ5.89 (m, 2, H-7, H-8); 13C NMR (151 Mz, CDCl3) δ (ppm) 21.9
(CH2-6), 22.3 (CH2-9), 23.1 (CH3ꢀC-4), 27.1 (CH3ꢀC-4), 37.7
(CH-1), 40.8, (CH-5), 85.0 (C-4), 125.0 (CH-8), 125.3 (CH-7), 178.5
(C-2); IR (film, cmꢀ1) 3019 (s), 1782 (m), 1736 (w), 1216 (s);
GCꢀEIMS 167 [M þ 1].
Coenzymes. Nicotinamide adenine dinucleotide (NADþ) and flavin
mononucleotide (FMN) were purchased from Sigma Chemical Co.
Microorganisms. The phytopathogens Aspergillus ochraceus AM
456, Fusarium culmorum AM 282, F. oxysporum AM 13, F. tricinctum AM
16, and Penicillium citrinum AM 354 were obtained from the collection of
Institute of Botany of Medical University of Wroczaw.
Synthesis of Lactones. (()-cis-3-Oxabicyclo[4.3.0]non-7-en-2-
one (3a). LiAlH4 (0.20 g, 5.3 ꢁ 10ꢀ3 mol) in anhydrous diethyl ether
(15 mL) was placed in a 2-neck flask equipped with a dropping funnel, a
condenser and a magnetic stirrer. To the stirred mixture cis-1,2,3,6-
tetrahydrophthalic anhydride 1 (820 mg, 5.4 ꢁ 10ꢀ3 mol) in 15 mL of
diethyl ether was added dropwise (the substrate was dissolved in diethyl
ether with heat and then cooled). After 20 min the reaction mixture was
poured into 25 g of ice mixed with 50 mL of 6% hydrochloric acid and
extracted three times with 50 mL of diethyl ether. The combined
ethereal extracts were washed with brine and dried over anhydrous
MgSO4. After evaporation of the solvent the pure product 3a
(0.5434 g) was isolated by column chromatography (eluent hexane:
acetone 4:1 v/v) with yield 73%.
(()-4,4-Dimethyl-cis-3-oxabicyclo[4.3.0]nonan-2-one (4e). Lac-
tone 4e was obtained (0.4990 g) with yield 73%, by the same procedure
as lactone 4a, using magnesium (195 mg, 8.12 ꢁ 10ꢀ3 mol), methyl
iodide (1.1540 g, 8.1 ꢁ 10ꢀ3 mol) and cis-1,2-cyclohexanedicarboxylic
anhydride 2 (0.6257 g, 4.06 ꢁ 10ꢀ3 mol).
(ꢀ)-(1S,5R)-cis-3-Oxabicyclo[4.3.0]non-7-en-2-one ((ꢀ)-3a). cis-
4,5-Bis(hydroxymethyl)cyclohexene 16 (0.223 g, 1.4 mmol), NADþ
(73 mg, 0.11 mmol) and FMN (0.8 g, 2.03 mmol) were dissolved in
40 mL of 0.1 M glycineꢀNaOH buffer in an Erlenmeyer flask (100 mL).
pH of the mixture was readjusted to 9 with 20% NaOH. Then the
HLADH (8 mg) was added and the mixture was kept at room
temperature with periodic adjustment of pH to 9. The progress of
oxidation was monitored by GC. When the reaction was completed (7
days), the pH was raised to 12 and the mixture was three times extracted
with CHCl3 (50 mL) to remove the unreacted diol. The aqueous phase
was acidified to pH 3, then extracted with CHCl3 (5 ꢁ 50 mL), and the
extract was dried (MgSO4). After solvent evaporation, the crude product
was purified by column chromatography (eluent hexane:acetone
3:1 v/v) to give 130 mg (yield 58%) of (ꢀ)-3a: ee = 90% (GC, chiral
(ꢀ)-(1R,5S)-4,4-Dimethyl-3-cis-oxabicyclo[4.3.0]nonan-2-one ((ꢀ)-
4e). To 30 mg (13.58 ꢁ 10ꢀ4 mol) of magnesium activated with iodine
was added anhydrous diethyl ether (10 mL), followed by slow addition of
methyl iodide (0.1725 g, 13.58 ꢁ 10ꢀ4 mol). The reaction was continued
until all the magnesium had reacted, and then (þ)-(1S,5R)-cis-3-
oxabicyclo[4.3.0]nonan-2-one ((þ)-3e) (80 mg, 5.70 ꢁ 10ꢀ4 mol)
was added dropwise at room temperature. After 3 h the reaction mixture
was poured into 2% HCl mixed with ice and extracted with diethyl ether
(5 ꢁ 30 mL), and the ethereal phase was washed with 6% HCl (2 ꢁ
30 mL) and brine until neutral. The extract was dried over MgSO4, the
solvent was evaporated off and the crude product without purification
was oxidized with dichromate pyridinium (0.215 g, 0.57 mmol) in
CH2Cl2 (15 mL). When the substrate was consumed (TLC, GC, 36
h), the reaction mixture was filtered through Florisil, the solvent was
evaporated off and the crude product was purified by column chroma-
column); [R] = ꢀ58.3° (c = 2.0, CHCl3), lit.41 [R] = ꢀ67.1°. 1H
20
589
20
589
NMR (500 MHz, CDCl3) δ 1.77ꢀ2.05 (m, 1, one of CH2-6),
1.90ꢀ2.90 (m, 3, one of CH2-6, H-5, one of CH2-9), 2.74ꢀ2.80 (m,
2, one of CH2-9, H-1), 4.00 (dd, 1, J = 2.0, 8.8 Hz, one of CH2-4), 4.30
(dd, 1, J = 5.1, 8.8 Hz, 1H, one of CH2-4), 5.60ꢀ5.70 (m, 2, H-8, H-7);
IR (film, cmꢀ1) 1771 (s), 1134 (m); GCꢀEIMS 139 [M þ 1].
(()-cis-3-Oxabicyclo[4.3.0]nonan-2-one (3e). Lactone 3e was ob-
tained (0.5313 g) with yield 69%, by the same procedure as lactone 3a,
using LiAlH4 (0.23 g, 6.0 ꢁ 10ꢀ3 mol) and cis-1,2-cyclohexanedicar-
boxylic anhydride 2 (850 mg, 5.5 ꢁ 10ꢀ3 mol).
tography using hexane:acetone 4:1 as eluent to give 48 mg of lactone
20
589
(ꢀ)-4e (yield 50%): ee = 91% (GC, chiral column); [R] = ꢀ35.2°
20
589
(c = 1.2, CHCl3), lit.41 [R] = ꢀ40.1°; 1H NMR (500 MHz, CDCl3) δ
(ppm) 1.00ꢀ1.17 (m, 1, one of CH2-6), 1.33ꢀ1.35 (two s, 6, C-4-
(CH3)2), 1.45ꢀ1.65 (m, 5, one of CH2-6, CH2-7, CH2-8), 1.65ꢀ1.79
(m, 2, CH2-9), 1.98ꢀ2.24 (m, 1, H-1), 2.96 (t, 1, J = 6.3 Hz, H-5); 13
C
(þ)-(1S,5R)-cis-3-Oxabicyclo[4.3.0]nonan-2-one ((þ)-3e). 3e was
obtained by the same procedure as lactone 3a, using cis-1,2-bis-
(hydroxymethyl)cyclohexane 17 (0.335 g, 2.1 mmol), NADþ (110
mg, 0.165 mmol), FMN (1.2 g, 3.05 mmol) and 0.1 M glycineꢀNaOH
NMR (151 Mz, CDCl3) δ (ppm) 22.5 (CH2-8), 22.8 (CH2-6), 22.9
(CH2-7), 23.6 (CH3ꢀC-4), 25.1 (CH3ꢀC-4), 26.1 (CH2-9), 39.9
(CH-1), 43.5 (CH-5), 84.0 (C-4), 177.6 (C-2); IR (film, cmꢀ1) 2937
(m), 2254 (m), 1758 (s), 911 (s), 731 (s); GCꢀEIMS 169 [M þ 1].
(()-cis-7,8-Epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b). Lactone
3a (0.2503 g, 1.8 ꢁ 10ꢀ3 mol) dissolved in dichloromethane (15 mL) was
placed in a round-bottom flask fitted with a magnetic stirrer, and 70% m-
chloroperbenzoic acid (MCPBA) (0.70 g, 2.8 ꢁ 10ꢀ3 mol) was added to
itportionwise. After8 hofthe reaction 20%Na2SO3 solution (15mL) was
added and the mixture was extracted with diethyl ether (3 ꢁ 50 mL). The
organic layer was washed with 10% NaHCO3 solution (5 mL) and brine
buffer (60 mL, pH = 9) at room temperature. A seven-day reaction gave
20
589
212 mg of (þ)-3e (yield 65%): ee = 91% (GC, chiral column;) [R]
=
20
589
þ42.5° (c = 2.2, CHCl3), lit.41 [R] = þ48.8°; 1H NMR (500 MHz,
CDCl3) δ (ppm) 0.80ꢀ0.98 (m, 1, one of CH2-6), 1.05ꢀ1.30 (m, 5,
CH2-8, CH2-7, one of CH2-6), 1.34 (d, 1, J = 10.2 Hz, one of CH2-9),
1.45ꢀ1.95 (m, 1, one of CH2-9), 2.10 (dd, 1, J = 11.0, 23.3 Hz, H-1),
2.35ꢀ2.70 (m, 1, H-5), 3.92 (d, 1, J = 8.8 Hz, one of CH2-4), 4.16 (dd, 1,
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until neutral and dried over MgSO4. After evaporation of solvent the
product mixture was purified by column chromatography using hexane:
aceton 2:1 as eluent to give 0.2482 g of 3b (yield 89%).
(m, 1, H-1), 2.89ꢀ3.04 (m, 1, one of CH2-9), 3.05ꢀ3.25 (m, 1, H-5),
3.85ꢀ4.06 (m, 2, H-7, H-8); 13C NMR (151 Mz, CDCl3) δ (ppm) 22.9
(CH3ꢀC-4), 25.5 (CH3ꢀC-4), 25.9 (CH2-6), 26.7 (CH2-9), 36.4
(CH-1), 38.8 (CH-5), 47.1 (CH-7), 50.8 (CH-8), 83.6 (C-4) and
176.9 (C-2); IR (film, cmꢀ1) 2977 (s), 1766 (s), 1274 (m), 1112 (m);
GCꢀEIMS 326, 327 [M þ 1]þ; HRMS 248.92, 350.92 [M þ Na]þ.
(()-trans-7-Bromo-8-hydroxy-cis-3-oxabicyclo[4.3.0]nonan-2-one
(3d). To lactone 3a (0.5941 g, 4.3 ꢁ 10ꢀ3 mol) dissolved in 15 mL of
(ꢀ)-(1S,5R,7S,8R)-cis-7,8-Epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one
((ꢀ)-3b). Epoxide (ꢀ)-3b was obtained (48.3 mg) with yield 88%, by the
same procedure as lactone 3b, using lactone (ꢀ)-3a (52 mg, 3.77 ꢁ 10ꢀ4
mol) and 70% MCPBA (0.14 g, 0.56 ꢁ 10ꢀ3 mol): ee = 91% (GC, chiral
column); [R] = ꢀ11.1° (c = 2.0, CHCl3) lit.42 [R] = ꢀ12.9°; 1H
20
589
20
589
NMR (500 MHz, CDCl3) δ (ppm) 1.66 (dd, 1, J = 11.4, 15.0 Hz, one of
CH2-6), 2.17ꢀ2.26 (m, 1, one of CH2-6), 2.31ꢀ2.37 (m, 1, one of
CH2-9), 2.51 (dd, 1, J = 7.2, 13.9 Hz, one of CH2-9), 2.62ꢀ2.70 (m, 1,
H-1), 3.08ꢀ3.11 (m, 1, H-5), 3.18 (t, 2, J = 7.0 Hz, H-7, H-8), 3.94 (dd, 1,
J = 1.1, 9.0 Hz, one of CH2-4), 4.17 (dd, 1, J = 5.1, 9.0 Hz, one of CH2-4);
13C NMR (151 Mz, CDCl3) δ (ppm) 20.5 (CH2-6), 24.6 (CH2-9), 30.5
(CH-1), 34.7 (CH-5), 49.6 (CH-7), 51.6 (CH-8), 71.2 (CH2-4), 178.5
(C-2); IR (film, cmꢀ1) 1771 (s), 1158 (m); GCꢀEIMS 155 [M þ 1].
(()-4,4-Dimethyl-cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one
(4b). Lactone 4b was obtained (0.6024 g) with yield 76%, by the same
procedure as lactone 3b, using lactone 4a (0.7234 g, 4.35 ꢁ 10ꢀ3 mol)
THF:H2O solution (7:3) NBS (N-bromosuccinimide) (0.85 g, 4.7 ꢁ
10ꢀ3 mol) was added portionwise and the mixture was stirred until the
substrate was consumed (TLC). Next 30 mL of water and 150 mL
of diethyl ether were added. The organic layer was washed with brine
(3 ꢁ 20 mL) until neutral and dried over anhydrous MgSO4. Then the
solvent was evaporated off and the mixture was purified by column
chromatography, using a mixture of hexane:acetone 2:1 v/v as eluent.
The bromohydrine 3d (0.7783 g) was isolated with yield 77%: 1H NMR
(500 MHz, CDCl3) δ (ppm) 1.94ꢀ2.10 (m, 1, one of CH2-6),
2.11ꢀ2.25 (m, 1, one of CH2-6), 2.26ꢀ2.56 (m, 3, CH2-9, H-1),
2.57ꢀ2.73 (m, 1, H-5), 2.76ꢀ2.94 (m, 1, H-8), 3.95ꢀ4.17 (m, 1,
H-7), 4.22 (dd, 1, J = 3.8, 7.6 Hz, one of CH2-4), 4.28 (dd, 1, J = 5.3, 9.1
Hz, one of CH2-4); 13C NMR (151 Mz, CDCl3) δ (ppm) 25.0 (CH2-6),
28.3 (CH2-9), 31.6 (CH-5), 36.3 (CH-1), 50.2 (CH-7), 68.0 (CH-8),
71.1 (CH2-4), 179.0 (C-2); IR (film, cmꢀ1) 3446 (w), 1767 (m);
GCꢀEIMS 236, 237 [M þ 1]þ; HRMS 234.99, 236.99 [M þ 1]þ.
(()-trans-7-Bromo-8-hydroxy-cis-4,4-dimethyl-3-oxabicyclo[4.3.0]
nonan-2-one (4d). Bromohydrine 4d (0.5795 g) was isolated with yield
75%, by the same procedure as lactone 3d, using 0.3950 g (2.8 ꢁ 10ꢀ3
mol) of 4a and 0.53 g (2.99 ꢁ 10ꢀ3 mol) of NBS: 1H NMR (500 MHz,
CDCl3) δ (ppm) 1.38 and 1.42 (two s, 6, C-4(CH3)2), 1.87 (ddd, 1, J =
4.3, 4.4, 14.5 Hz, one of CH2-6), 2.20ꢀ2.32 (m, 3, one of CH2-6, one of
CH2-9, ꢀOH), 2.40ꢀ2.70 (m, 2, H-1, one of CH2-9), 2.96 (dd, 1, J =
7.0, 7.1 Hz, H-5), 4.06ꢀ4.12 (m, 1, H-8), 4.25ꢀ4.32 (m, 1, H-7); 13C
NMR (151 Mz, CDCl3) δ (ppm) 18.7 (CH3ꢀC-4), 20.5 (CH3ꢀC-4),
20.9 (CH2-6), 22.0 (CH2-9), 32.6 (CH-1), 34.8 (CH-5), 45.7 (CH-7),
63.3 (CH-8), 80.2 (C-4), 174.1 (C-2); IR (film, cmꢀ1) 3444 (w), 2253
(m), 1760 (m), 906 (s), and 730 (s); GCꢀEIMS 263, 264 [M þ 1]þ;
HRMS 285.00, 287.00 [M þ Na]þ.
1
and 70% MCPBA (1.18 g, 4.8 ꢁ 10ꢀ3 mol): H NMR (500 MHz,
CDCl3) δ (ppm) 1.22 (s, 1, one of CH2-6), 1.31 and 1.40 (two s, 6,
C-4(CH3)2), 1.56ꢀ1.74 (m, 1, one of CH2-6), 2.10ꢀ2.25 (m, 1, one of
CH2-9), 2.31ꢀ2.47 (m, 1, one of CH2-9), 2.75ꢀ2.97 (m, 1, H-1),
3.00ꢀ3.21 (m, 1, H-5), 3.25 (t, 2, J = 4.0 Hz, H-7, H-8); 13C NMR (151
Mz, CDCl3) δ (ppm) 20.9 (CH2-6), 22.5 (CH3ꢀC-4), 23.5 (CH3ꢀC-
4), 26.7 (CH2-9), 35.4 (CH-1), 38.8 (CH-5), 49.7 (CH-7), 51.6 (CH-8),
84.0 (C-4), 178.1 (C-2); IR (film, cmꢀ1) 3019 (w), 2399 (w), 1773 (s),
1215 (m), 756 (s); GCꢀEIMS 183 [M þ 1]þ; HRMS 183.1 [M þ 1]þ.
(()-trans-7,8-Dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one (3c).
The lactone 3a (0.3020 g, 2.12 ꢁ 10ꢀ3 mol) was dissolved in tetrachlor-
omethane (15 mL) and was placed in a sealed round-bottom flask fitted
with septum and a magnetic stirrer. Then 0.7 mL (13.6 ꢁ 10ꢀ3 mol)
of bromine was added dropwise using a syringe. The reaction mixture was
stirred at room temperature for 2 h, and then 20 mL of 10% sodium
thiosulfate solution was added. The product was extracted with diethyl
ether (3 ꢁ 50 mL) and washed with brine and water until neutral. The
organic layer was dried over MgSO4, the solvent was evaporated off and
the crude product was purified by column chromatography using
dichloromethane:methanol (399:1) as eluent. 0.5079 g of lactone 3c
was obtained (yield 78%).
(()-8-Hydroxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (6). A round-
bottom flask equipped with a magnetic stirrer was charged with 1.050
g (6.3 ꢁ 10ꢀ3 mol) of cis-1,2,3,6-tetrahydrophthalic anhydride 1
dissolved in 25 mL of dichloromethane, and 1.875 g (7.5 ꢁ 10ꢀ3 mol)
of 70% MCPBA was added to it portionwise. The mixture was stirred for
12 h, then 25 mL of 20% Na2SO3 solution was added and the mixture
was extracted with methylene chloride (4 ꢁ 50 mL). The organic layer
was washed with 10% Na2SO3 (10 mL) solution and brine until neutral
and dried over MgSO4. The solvent was evaporated off, and the mixture
was purified by column chromatography, using hexane:acetone 5:3 as
eluent. 0.7184 g of the cis-4,5-epoxy-cis-1,2-cyclohexanedicarboxylic
anhydride (5) was isolated (yield 65%). Next LiAlH4 (0.60 g, 15.9 ꢁ
10ꢀ3 mol) was placed in a 2-neck flask equipped with a condenser, a
dropping funnel and a magnetic stirrer, and then 20 mL of diethyl ether
was added, followed by slow addition of cis-4,5-epoxy-cis-1,2-cyclohex-
anedicarboxylic anhydride 5 (530 mg, 3.15 ꢁ 10ꢀ4 mol), previously
dissolved in 15 mL of diethyl ether. The mixture was refluxed until all the
substrate was consumed (3 h, TLC, GC) and poured into 50 mg of ice
mixed with 50 mL of 6% HCl solution. The product was extracted with
diethyl ether (3 ꢁ 50 mL), and the organic layer was washed with brine
and dried over MgSO4. After evaporation of solvent the pure product
was isolated by column chromatography (hexane:acetone 2:1) to give
0.199 g of lactone 6 (yield 45%): 1H NMR (500 MHz, CDCl3) δ (ppm)
1.09ꢀ1.12 (dt, 1, J = 3.8, 8.6 Hz, one of CH2-6), 1.24ꢀ1.27 (m, 1, one of
CH2-7), 1.54ꢀ1.60 (m, 3, CH2-9, one of H-6), 1.82ꢀ1.98 (m, 1, one of
CH2-7), 2.32ꢀ2.46 (m, 1, CH2-1), 2.60ꢀ2.67 (m, 1, H-5), 3.65ꢀ3.84
(m, 1, H-8), 4.22 (dd, 1, J = 3.8, 7.6 Hz, one of CH2-4), 4.32 (dd, 1,
(þ)-(1S,5R,7R,8R)-trans-7,8-Dibromo-cis-3-oxabicyclo[4.3.0]nonan
-2-one ((þ)-3c). Product (þ)-3c was obtained (121 mg) with yield
70%, by the same procedure as lactone 3c, using lactone (ꢀ)-3a (80 mg,
5.80 ꢁ 10ꢀ4 mol) and bromine (0.1 mL, 19.5 ꢁ 10ꢀ4 mol):
20
589
1
[R] =þ27.8° (c = 1.1, CHCl3); H NMR (500 MHz, CDCl3) δ
(ppm) 1.23 (s, 1, one of CH2-6), 1.54 (s, 1, one of CH2-6), 1.73ꢀ2.36
(m, 2, one of CH2-9, H-1), 3.95ꢀ4.16 (m, 1, H-5), 4.17ꢀ4.40 (m, 2,
H-7, H-8), 4.53 (d, 1, J = 1.6 Hz, one of CH2-4), 4.62 (d, 1, J = 3.0 Hz,
one of CH2-4); 13C NMR (151 Mz, CDCl3) δ (ppm) 25.4 (CH2-6),
29.6 (CH2-9), 32.8 (CH-5), 36.9 (CH-1), 47.1 (CH-7), 52.2 (CH-8),
70.5 (CH2-4), 176.9 (C-2); IR (film, cmꢀ1) 2360 (w), 1767 (m), 1167
(w); GCꢀEIMS 299, 300 [M þ 1]þ; HRMS 298.91, 300.90 [M þ 1]þ.
Crystal data for 3c: C8H10Br2O2, M = 297.98, colorless block, crystal
dimensions 0.32 ꢁ 0.21 ꢁ 0.13 mm, monoclinic, space group P21/c,
a = 6.646(2), b = 13.691(4), c = 10.087(3) Å, β = 99.12(3)°, V =
906.2(5) Å3, Z = 4, Dc = 2.184 Mg mꢀ3, μ(Mo KR) = 8.90 mmꢀ1, T =
100(2) K, R = 0.025, wR = 0.062 (1699 reflections with I > 2σ(I)) for
109 variables. CCDC reference number: 794604.
(()-trans-7,8-Dibromo-cis-4,4-dimethyl-3-oxabicyclo[4.3.0]nonan-
2-one (4c). Lactone 4c was obtained (1.1320 g) with yield 80%, by the
same procedure as lactone 3c, using 0.7216 g (4.30 ꢁ 10ꢀ3 mol) of 4a
and 1 mL (19.5 ꢁ 10ꢀ3 mol) of bromine: 1H NMR (500 MHz, CDCl3)
δ (ppm) 1.32ꢀ1.50 (two s, 6, C-4(CH3)2), 1.72ꢀ1.90 (m, 1, one
of CH2-6), 2.10ꢀ2.41 (m, 2, one of CH2-6, one of CH2-9), 2.44ꢀ2.60
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Journal of Agricultural and Food Chemistry
ARTICLE
J = 7.6, 9.2 Hz, one of CH2-4); 13C NMR (151 Mz, CDCl3) δ (ppm)
22.4 (CH2-6), 25.3 (CH2-7), 28.7 (CH2-9), 32.1 (CH-5), 36.7 (CH-1),
69.5 (CH-8), 72.3 (CH2-4), 176.2 (C-2); IR (film, cmꢀ1); 3443 (s),
2938 (s), 2248 (m), 1768 (s), 1172 (m), 1010 (m); GCꢀEIMS 157
[M þ 1]þ; HRMS 157.98 [M þ 1]þ.
(()-trans-9-Bromo-cis-2-oxabicyclo[4.3.0]nonan-3-one (12). To
ester 9 (0.723 g, 4.3 ꢁ 10ꢀ3 mol) dissolved in 15 mL of a mixture of
THF:H2O (7:3) was added 0.8 g (5.3 ꢁ 10ꢀ3 mol) of NBS portionwise.
The mixture was stirred until all the substrate was fully consumed (TLC)
and then acidified with 10% HCl and stirred for a further 3 h. Thirty
milliliters of water and 150 mL of diethyl ether were added to the reaction
mixture, and the organic layer was separated, washed with brine until
neutral (3 ꢁ 20 mL) and dried over MgSO4. The solvent was evaporated
off, and the crude product was purified by column chromatography with
hexane:acetone 9:1 as eluent to give 0.6602 g of bromolactone 12 (yield
70%): 1H NMR (500 MHz, CDCl3) δ (ppm) 1.28ꢀ1.39 (m, 1, one of
CH2-6), 1.48ꢀ1.60 (m, 1, one of CH2-7), 1.71ꢀ1.88 (m, 2, one of CH2-
7, one of CH2-6), 1.90ꢀ2.11 (m, 2, CH2-8), 2.28 (dd, 1, J = 3.3, 16.9 Hz,
one of CH2-4), 2.61 (dd, 1, J = 6.8, 16.9 Hz, one of CH2-4), 2.70ꢀ2.83
(m, 1, H-5), 4.42ꢀ4.50 (m, 1, H-9), 4.60 (dd, 1, J = 4.3, 4.4 Hz, H-1); 13C
NMR (151 Mz, CDCl3) δ (ppm) 18.5 (CH2-7), 26.1 (CH2-6), 29.3
(CH2-8), 32.5 (CH2-4), 36.5 (CH-5), 48.2 (CH-9), 81.3 (CH-1), 175.8
(C-3); IR (film, cmꢀ1) 1785 (s), 1173 (s), 1019 (s); GCꢀEIMS 220, 221
[M þ 1]þ; HRMS 240.98, 242.98 [M þ Na]þ.
(()-Cyclohex-2-en-1-ol (8). A solution of cyclohex-2-en-1-one (7)
(7.0 g, 72.8 mmol) in 15 mL of dry diethyl ether was added dropwise to a
stirred LiALH4 (1.4 g, 36.4 mmol) in 35 mL of diethyl ether, and the
stirring was continued at room temperature. When reaction was com-
pleted (3 h, TLC, GC), the mixture was diluted with diethyl ether
(150 mL) and washed successively with an aqueous solution of 10% HCl
(15 mL) and water (until neutral). After solvent evaporation, the product
was purified by column chromatography (hexane:acetone 3:1 v/v) to
1
give 6.6 g of 8 (yield 93%): H NMR (500 MHz, CDCl3) δ (ppm)
1.55ꢀ1.62 (two m, 3, CH2-5, one of CH2-4), 1.84ꢀ1.91 (m, 1, one of
CH2-6), 2.27ꢀ2.32 (m, 2, one of CH2-4, one of CH2-6), 4.13ꢀ4.17 (m,
1, H-1), 5.69ꢀ5.71 (m, 1, H-3), 5.80 (dt, 1, J = 3.2, 10.1 Hz, H-2); IR
(film, cmꢀ1) 3336 (s), 2934 (s), 1055 (s); GCꢀEIMS 99 [M þ 1].
(()-Ethyl(cyclohex-2-ene-1-yl)acetate (9). A mixture of cyclohex-2-
en-1-ol (8) (5.3 g, 54.1 mmol), ethyl orthoacetate (66.4 mL, 0.41 mol)
and propionic acid (0.03 mL) was heated at 138 °C in a flask equipped
with a distillation head. The reaction was continued until all the substrate
was consumed. The excess of ethyl orthoacetate was distilled off, and the
residue was purified by column chromatography (hexane:acetone 19:1)
(()-trans-9-Acetyl-cis-2-oxabicyclo[4.3.0]nonan-3-one (13). Hy-
droxy lactone 11 (0.4250 g, 2.72 ꢁ 10ꢀ3 mol) was dissolved in
2.5 mL of anhydrous pyridine. To the stirred mixture was slowly added
0.7 mL of acetyl chloride (0.77 g, 9.81 ꢁ 10ꢀ3 mol). Progress of the
reaction was checked by TLC. After 4 h the reaction mixture was diluted
with 150 mL of diethyl ether and the pyridine was washed off with 6%
HCl solution (3 ꢁ 50 mL) and brine (3 ꢁ 20 mL). The organic layer of
intensively yellow color was dried over MgSO4, the solvent was
evaporated off and the residue was purified three times by column
chromatography using the following eluents: (1) hexane:acetone 4:1;
(2) dichloromethane:methanol 399:1; (3) hexane:acetone 2:1. 0.3945 g
1
to give 7.2 g of ester 9 (yield 80%): H NMR (500 MHz, CDCl3) δ
(ppm) 1.14ꢀ1.29 (m, 1, one of CH2-6), 1.23 (t, 3, J = 7.0 Hz, ꢀCH3),
1.52ꢀ1.81 (two m, 3, CH2-5, one of CH2-6), 1.92ꢀ1.95 (m, 2, CH2-4),
2.27 (dd, 1, J = 8.1, 15.2 Hz, one of ꢀCH2ꢀC(O)ꢀ), 2.36 (dd, 1, J = 6.8,
15.2 Hz, one of ꢀCH2ꢀC(O)ꢀ), 2.52ꢀ2.56 (m, 1, H-1), 4.09 (q, 2, J =
7.1 Hz, >C(O)ꢀOꢀCH2ꢀ), 5.50 (dddd, 1, J = 0.9, 2.0, 4.0, 10.1 Hz,
CH-2); 5.50 (ddd, 1, J = 3.4, 5.8, 10.1 Hz, CH-3); IR (film, cmꢀ1) 2998
(s), 1748 (s), 1164 (s) 1036 (s); GCꢀEIMS 169 [M þ 1].
1
of pure product 13 was obtained (yield 73%): H NMR (500 MHz,
CDCl3) δ (ppm) 1.34ꢀ1.59 (m, 1, one CH2-6), 1.60ꢀ1.76 (m, 4, one of
CH2-8, CH2-7, one of CH2-6), 1.79ꢀ1.93 (m, 1, one of CH2-8), 2.07 (s,
3, C(O)CH3), 2.33 (dd, 1, J = 7.2, 17.1 Hz, one of CH2-4), 2.53 (dd, 1, J
= 7.5, 17.0 Hz, one of CH2-4), 2.65ꢀ2.82 (m, 1, H-5), 4.36 (t, 1, J = 5.9
Hz, H-1) 4.96 (m, 1, H-9); 13C NMR (151 Mz, CDCl3) δ (ppm) 17.7
(CH3C(O)ꢀ), 20.9 (CH2-7), 25.5 (CH2-8), 26.5 (CH2-6), 34.0
(CH2-4), 34.5 (CH2-5), 70.4 (CH-9), 79.4 (CH-1), 169.8 (>CdO),
175.9 (C-3); IR (film, cmꢀ1) 1783 (s), 1730 (s), 1240 (m), 1021 (m);
GCꢀEIMS 199 [M þ 1].
(()-cis-Ethyl(7-oxabicyclo[4.1.0]hept-2-yl)acetate (10). To ester 9
(1.5235 g, 9.06 ꢁ 10ꢀ3 mol) dissolved in CH2Cl2 (20 mL), m-
chloroperbenzoic acid (2.290 g, 9.29 ꢁ 10ꢀ3 mol) was added portion-
wise. The reaction mixture was stirred for 8 h, then the solvent was
evaporated off and the residue was dissolved in 1.5 mL of DMF and
purified by column chromatography (eluent hexane:acetone 25:1) to
give 1.3848 g of pure 10 (yield 83%): 1H NMR (500 MHz, CDCl3) δ
(ppm) 1.10ꢀ1.15 (m, 1, one of CH2-4), 1.24 (t, 3, J = 7.2 Hz, ꢀCH3),
1.52ꢀ1.81 (two m, 3, CH2-5, one of CH2-4), 1.72ꢀ1.91 (m, 2, CH2-6),
2.38ꢀ2.40 (m, 2, ꢀCH2ꢀC(O)ꢀ), 2.55ꢀ2.58 (m, 1, H-3), 3.12ꢀ3.15
(m, 1, H-7); 3.20 (t, 1, J = 4.1 Hz, H-8), 4.18 (q, 2, J = 7.2 Hz,
>C(O)ꢀOꢀCH2ꢀ); 13C NMR (151 Mz, CDCl3) δ (ppm) 14.3
(ꢀCH3), 19.6 (CH2-5), 23.6 (CH2-6), 25.0 (CH2-4), 31.9
(ꢀCH2ꢀC(O)ꢀ), 37.9 (CH-3), 53.2 (CH-7), 55.1 (CH-8), 60.4
(ꢀOꢀCH2ꢀ), 172.7 (ꢀC(O)ꢀOꢀ); IR (film, cmꢀ1) 1748 (s),
1182 (s), 1152 9 (s), 1032 (s); GCꢀEIMS 185 [M þ 1].
(()-trans-9-Butyryl-cis-2-oxabicyclo[4.3.0]nonan-3-one (14). 14
was obtained by the same procedure as lactone 13, using hydroxy lactone
11 (0.3133 g, 2.0 ꢁ 10ꢀ3 mol) and butyryl chloride (0.6 mL, 0.504 g, 4.75
ꢁ 10ꢀ3 mol) as reagents. The product was purified by column
chromatography using hexane:acetone in 4:1 ratio to give 0.2450 g of
pure 14 (65% yield): 1H NMR (500 MHz, CDCl3) δ (ppm) 0.94 (t, 3,
J = 7.4 Hz, CH3-40), 1.40ꢀ1.70 (m, 5, CH2-6, CH2-7, one of CH2-8),
1.65 (sext, 2, J = 7.4 Hz, CH2-30), 2.30 (t, 2, J = 7.4 Hz, CH2-20), 2.31
(dd, 1, J = 6.9, 17.0 Hz, one of CH2-4), 2.53 (dd, 1, J = 7.4, 17.0 Hz, 1H,
one of CH2-4), 2.64ꢀ2.76 (m, 1, H-5), 4.35 (t, 1, J = 5.8 Hz, H-1),
4.95ꢀ5.06 (m, 1, H-9); 13C NMR (151 Mz, CDCl3) δ (ppm) 13.4
(CH3-40), 17.7 (CH2-30), 18.3 (CH2-7), 25.6 (CH2-8), 26.4 (CH2-6),
34.0 (CH2-20), 34.7 (CH2-4), 36.1 (CH-5), 69.9 (CH-9), 79.4 (CH-1),
172.5 (C-3), 176.0 (>CdO); IR (film, cmꢀ1) 1785 (m), 1735 (m) and
1153 (m); GCꢀEIMS 227 [M þ 1]þ; HRMS 226.98 [M þ 1]þ.
(()-trans-9-(p-Methoxyphenyl)acetoxy-cis-2-oxabicyclo[4.3.0]
nonan-3-one (15). 15 was obtained by the same procedure as lactone
13, using hydroxy lactone 11 (0.4923 g, 3.15 ꢁ 10ꢀ3 mol) and
4-methoxyphenylacetyl chloride (0.9 mL, 0.672 g, 3.65 ꢁ 10ꢀ3 mol).
After purification by column chromatography using hexane:acetone in
4:1 ratio 0.6780 g of pure 15 was obtained (yield 63%): 1H NMR (500
MHz, CDCl3) δ (ppm) 1.30ꢀ1.85 (three m, 6, CH2-6, CH2-7, CH2-8),
2.27 (dd, 1, J = 6.3, 16.8 Hz, one of CH2-4), 2.27 (dd, 1, J = 7.3, 16.8 Hz,
1H, one of CH2-4), 2.55ꢀ2.68 (m, 1, H-5), 3.56 (s, 2, ꢀCH2ꢀPh-),
(()-trans-9-Hydroxy-cis-2-oxabicyclo[4.3.0]nonan-3-one(11). Epoxy
ester 10 (1.2430 g, 6.75 ꢁ 10ꢀ3 mol) was dissolved in 20 mL of a mixture
of THF:H2O:HClO4 (10:5:0.5 v/v) and stirred at room temperature. The
reaction was stopped after 4 h, when the intermediate diol ester was no
longer observed by TLC and GC. The reaction mixture was neutralized
with 20% solution of NaHCO3, extracted with diethyl ether (4 ꢁ 50 mL)
and purified by column chromatography to give 0.8231 g of 11 (yield
78%): 1H NMR (500 MHz, CDCl3) δ (ppm) 1.34ꢀ1.52 (m, 2, one of
CH2-6, ꢀOH), 1.54ꢀ1.74 (m, 4, one of CH2-8, CH2-7, one of CH2-6),
1.86ꢀ1.92 (m, 1, one of CH2-8), 2.31 (dd, 1, J = 9.3, 17.1 Hz, one of CH2-
4), 2.48 (dd, 1, J = 7.8, 17.1 Hz, one of CH2-4), 2.70ꢀ2.85 (m, 1, H-5),
3.82 (ddd, 1, J = 4.3, 6.5, 8.9 Hz, H-9), 4.28 (t, 1, J = 6.5 Hz, H-1); 13C
NMR (151 Mz, CDCl3) δ (ppm) 17.9 (CH2-7), 25.4 (CH2-6), 29.5
(CH2-8), 33.8 (CH2-4), 34.4 (CH-5), 69.3 (CH-9), 84.1 (CH-1), 177.1
(C-3); IR (film, cmꢀ1) 3441 (s), 2935 (s), 2252 (m), 1772 (s), 1179 (m),
1010 (m); GCꢀEIMS 157 [M þ 1].
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Journal of Agricultural and Food Chemistry
ARTICLE
Scheme 1a
a (i) LiAlH4, Et2O; (ii) 2 mol of MeMgI, Et2O; (iii) MCPBA, CH2Cl2; (iv) Br2, CCl4; (v) NBS, THF:H2O.
3.77 (s, 3, ꢀOꢀCH3), 4.31 (t, 1, J = 5.8 Hz, H-1), 4.98ꢀ5.05 (m, 1, CH-
9), 6.80ꢀ7.20 (four m, 4, -C6H4-); 13C NMR (151 Mz, CDCl3) δ
(ppm) 17.7 (CH2-7), 25.6 (CH2-8), 26.2 (CH2-6), 33.9 (CH2-4), 34.8
(CH-5), 40.4 (ꢀCH2ꢀPh), 55.1 (ꢀOꢀCH3), 70.4 (CH-9), 79.1 (CH-
1), 113.8 (CHd30, CHd50), 125.7 (CHd20, CHd60), 130.1 (CHd10),
158.6 (CHd40), 170.7 (>CdO), 176.0 (C-3); IR (film, cmꢀ1) 3019 (s),
1782 (m), 1736 (w), 1216 (s); GCꢀEIMS 305 [M þ 1]þ; HRMS
305.98 [M þ 1]þ.
Synthesis of cis-3-oxabicyclo[4.3.0]nonan-2-one deriva-
tives (3aꢀe) and their gem-dimethyl derivatives (4aꢀe).
Starting from cis-1,2,3,4-tetrahydrophthalic anhydride (1) in the
reduction with lithium aluminum hydride racemic cis-3-oxabicyclo-
[4.3.0]non-7-en-2-one (3a) was obtained (Scheme 1). The struc-
ture of the product was established by means of IR, 1H NMR, and
13C NMR. Formation of a lactone ring in compound 3a was
confirmed by the presence of a single band at 1771 cmꢀ1 in the IR
spectrum, the signal of a carbonyl carbon atom of δ = 175 ppm in
the 13C NMR, and two signals at 4.0 and 4.3 ppm (doublet of
Antifungal Assays. The proper amounts of tested lactones were
dissolved in DMSO and added to sterilized potato dextrose agar (PDA)
(0.01 mL of DMSO for 1 mL of agar) in 9 cm Petri dishes to give lactone
concentrations of 50, 100, 150, 200, 250, or 300 μg/mL. After transfer-
ring a mycelium of a fungus to the nutrient prepared in such a way, the
testing dishes were incubated at 27 °C and 60ꢀ80% of relative humidity.
When the mycelium of the fungi in the control dishes (nutrient with
DMSO, without lactones added) reached 5.5ꢀ6.0 cm of diameter, a
diameter of growth zone in control samples and in experimental
dishes were measured. All the lactones were tested at concentration of
150 μL/mL and at three more concentrations, which were chosen
individually. Each test was repeated three times. Didecyldimethylamm-
monium chloride (DDAC) was used as reference compound. IC50 (the
half maximal inhibitory concentration) values for each tested compound
were determined based on four measured values, as a percent of
inhibition of growth and calculated according to the formula (1 ꢀ Da/
Db) ꢁ 100%, where Da is the diameter of growth zone in experimental
dish (cm) and Db is diameter of growth zone in the control dish (cm).
Statistical analysis was done using ANOVA (p = 0.05), and the means
were compared by calculating the least significant difference (LSD,
Tukey).
1
doublets) in the H NMR, which indicate that there are two
protons at C-4. The presence of a double bond was proved by the
1
multiplet at 5.60ꢀ5.70 ppm in the H NMR spectrum and two
signals at 125.0 and 125.3 ppm in the 13CNMR spectrum.
4,4-Dimethyl-cis-3-oxabicyclo[4.3.0]non-7-en-2-one (4a) the
structural analogue of lactone 3a with gem-dimethyl group
at C-4 was obtained in a Grignard reaction of two equivalents
of methylmagnesium iodide with one equivalent of anhydride
1
1. The signals of the methyl groups were found in the H
NMR spectrum as a singlet of six protons at 1.38 ppm, whereas
the double bond protons were visible as a multiplet at
5.63ꢀ5.89 ppm.
In the next stage of the synthesis, double bonds of lactones 3a
and 4a were oxidized with m-chloroperbenzoic acid to give cis-
1
epoxides 3b42,43 and 4b (Scheme 1). In the H NMR spectra
the signals of protons at C-7 and C-8 are represented by
separate triplets of a coupling constant J = 7.0 Hz for cis-7,8-
epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b) and J = 4.0 Hz
for 4,4-dimethyl-cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-
one (4b). Bromination of unsaturated lactones 3a and 4a
proceeded highly stereoselectively and gave trans-dibromo deri-
vatives: trans-7,8-dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one
(3c) and trans-7,8-dibromo-4,4-dimethyl-cis-3-oxabicyclo[4.3.0]
nonan-2-one (4c) (Scheme 1). In these compounds, similarly
to epoxy lactones, the signals of protons at C-7 and C-8 in the
’ RESULTS AND DISCUSSION
In the first stage of our work we synthesized racemic lactone
derivatives of cis-3-oxabicyclo[4.3.0]nonan-2-one and cis-2-
oxabicyclo[4.3.0]nonan-3-one structures.
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The presence of the hydroxyl group of hydroxy lactone 6 was
confirmed by IR (3443 cmꢀ1). The location of the hydroxyl
group at C-8 was established by means of spectral data: 1H NMR,
1
13C NMR and HMCQ. The signal from H-8 in the H NMR
spectrum was observed as not a diagnostic signal, so its position
was appointed by HMCQ, where we observed coupling between
multiplet from H-8 (3.65ꢀ3.84 ppm) and C-8 (69.5 ppm).
The second hydroxy lactone, trans-9-hydroxy-cis-2-oxabicyclo-
[4.3.0]nonan-3-one (11), was obtained in a 4-step synthesis
(Scheme 3), in which allyl alcohol 8, obtained in the reduction
of ketone 7, was subjected to Claisen rearrangement leading to
γ,δ-unsaturated ethyl ester 9. This ester was oxidized with
m-chloroperbenzoic acid to epoxide 10, obtained as a mixture
of diastereoisomers cis:trans in a 9:1 ratio. The structure of cis
epoxy ester 10 is confirmed by the coupling constant (J = 4.1 Hz)
1
of protons at C-7 and C-8 in the H NMR. Under acidic
conditions both epoxy esters underwent cyclization (via dihy-
droxy esters) to a single, diastereoisomerically pure hydroxy
lactone 11. Relative configuration of lactone 11 was established
by 1H NMR spectrum. Contrary to the small coupling constant
(0ꢀ5 Hz) of diequatorial or axialꢀequatorial orientation of
protons in a six-membered cyclohexane ring, the coupling con-
stant of neighboring axial protons is in the range 6ꢀ14 Hz. The
coupling constant (J = 6.5 Hz) between H-1 (δ = 4.28) and H-9
(δ = 3.82) in hydroxy lactone 11 convinced us that the OH group
is located in the equatorial position.
Additionally, the presence of a doublet of doublets of doublets
(δ = 3.82) with the large coupling constants at H-9 with
neighboring axial proton H-8 (J = 8.9 Hz) and equatorial proton
H-8 (J = 4.3 Hz) proved the equatorial orientation of the hydroxy
group. It indicates that the OH group is situated trans to the
γ-lactone ring.
Figure 1. Crystal structure of trans-7,8-dibromo-cis-3-oxabicyclo-
[4.3.0]nonan-2-one (3c). Crystal data: C8H10Br2O2; M = 297.98;
colorless block; crystal dimensions 0.32 ꢁ 0.21 ꢁ 0.13 mm; monoclinic;
space group P21/c; a = 6.646(2), b = 13.691(4), c = 10.087(3) Å; β =
99.12(3)°; V = 906.2(5) Å3; Z = 4; Dc = 2.184 Mg mꢀ3; μ = 8.900
mmꢀ1; T = 100(2) K; R = 0.025, wR = 0.062 (1699 reflections with I >
2σ(I)) for 109 variables; CCDC reference number 794604.
1H NMR overlap and give nondiagnostic multiplets. For dibromo
lactone 3c X-ray analysis has been done (Figure 1).
The crystal structure confirms the mechanism of bromine
addition to cis-3-oxabicyclo[4.3.0]non-7-en-2-one (3a).
We also obtained bromohydrine derivatives of lactones 3a and
4a in the addition reactions. The mechanism of bromohydrine
addition to unsaturated bond leads to trans product.
Synthesis of cis-2-Oxabicyclo[4.3.0]nonan-3-one Deriva-
tives: 12ꢀ15. In order to check how the protection of the
hydroxyl group in lactone 11 would influence the fungistatic
activity, this lactone was transformed into three esters: 13, 14 and
15, in which the hydroxyl group was esterified with acetic acid
chloride, butyric acid chloride and p-methoxyphenylacetic acid
chloride, respectively (Scheme 3). Additionally, trans-9-bromo-
cis-2-oxabicyclo[4.3.0]nonan-3-one (12) was synthesized, in
which the hydroxyl group was replaced with the more bulky
bromine atom. This lactone was synthesized in the reaction of
γ,δ-unsaturated ester 9 with N-bromosuccinimide (Scheme 3).
Fungistatic Activity of Racemic Lactones 3aꢀe, 4aꢀd, 6
and 11ꢀ15. The racemic lactones were subjected to the tests of
activity toward three phytopathogenic species: Aspergillus ochra-
ceus AM 456, Fusarium culmorum AM 282 and Penicillium
citrinum AM 354. The tested compounds were the most active
toward F. culmorum, so the next two microorganisms of the genus
Fusarium were subjected to the tests: F. oxysporum AM 13 and
F. tricinctum AM 16. The half maximal inhibitory concentration
(IC50) was calculated based on degree of growth inhibition on
the cultivation medium with tested compound added in compar-
ison with a control sample (Table 1a,b).
In both syntheses we isolated single diastereoisomer, which
was confirmed by IR and 13C NMR spectra. Because the
significant diagnostic signals in 1H NMR spectra were multiplets,
the relative configuration of 3d and 4d was established on the
basis of additional spectroscopic methods (COSY and HMQC
for 4d). The COSY spectra of 3d and 4d indicated the correla-
tions between multiplets from H-7 (ꢀCHꢀBr) and two signals
from CH2-6. The other correlation was observed for signals of
H-8 (ꢀCHꢀOH) and two multiplets from CH2-9. We observed
correlation between H-7 and H-8 too. In the HMQC spectrum of
4d the correlation between signals from proton H-7 (4.28 ppm)
and carbon atom C-7 (45.7 ppm), as well as signals from proton
H-8 (4.09 ppm) and carbon atom C-8 (63.3 ppm), confirmed
these assignations.
The saturated lactones 3e and 4e were obtained in an
analogous way to their unsaturated analogues 3a and 4a.
Synthesis of Hydroxy Lactones 6 and 11. Two isomeric
hydroxy lactones of different position of oxygen atom in the five-
membered ring were synthesized. The first one, 8-hydroxy-cis-3-
oxabicyclo[4.3.0]nonan-2-one (6), was obtained in a two-step
synthesis (Scheme 2). In the first step cis-1,2,3,4-tetrahy-
drophthalic anhydride 1 was oxidized with m-chloroperbenzoic
acid to cis-epoxy anhydride 5.
Lactones of cis-3-oxabicyclo[4.3.0]nonan-2-one structure
(3aꢀe) and their gem-dimethyl derivatives (4aꢀe) inhibit my-
celium growth of A. ochraceus AM 456, F. culmorum AM 282 and
F. oxysporum AM 13 stronger than of F. tricinctum AM 16 and
P. citrinum AM 354.
Lactones cis-3-oxabicyclo[4.3.0]non-7-en-2-one (3a) (entry 3),
cis-3-oxabicyclo[4.3.0]nonan-2-one (3e) (entry 7), 4,4-dime-
thyl-cis-3-oxabicyclo[4.3.0]non-7-en-2-one (4a) (entry 8) and
Compound 5 was subjected to the reduction with lithium
aluminum hydride which gave a mixture of two hydroxy-cis-3-
oxabicyclo[4.3.0]nonan-2-ones with a hydroxyl group at either
C-7 or C-8 in a 15:85 ratio. Isomerism of these hydroxy lactones
was confirmed by GCꢀMS [Mþ 156]. Only the major product 6
was isolated.
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Scheme 2a
a (i) MCPBA, CH2Cl2; (ii) LiAlH4, Et2O.
Scheme 3a
a (i) LiAlH4, Et2O; (ii) CH3C(OC2H5)3, C2H5COOH; (iii) MCPBA, CH2Cl2; (iv) THF:HClO4:H2O; (v) NBS, THF:H2O; (vii) CH3C(O)Cl, Py;
(vii) C3H7C(O)Cl, Py; (viii) CH3OPhCH2C(O)Cl, Py.
4,4-dimethyl-cis-3-oxabicyclo[4.3.0]nonan-2-one (4e) (entry 12)
inhibit growth of mycelium of A. ochraceus AM 456, F. culmorum
AM 282 and F. oxysporum AM 13 at IC50 = 134.5ꢀ314.3 μg/mL
(0.80ꢀ2.25 μM/mL). An increase of fungistatic activity is attrib-
uted to the presence of the gem-dimethyl group (4a, 4e). We
analyzed the influence of double bond on two pairs of lactones:
3a, 3e and 4a, 4e. Fungistatic activity of those lactones is similar,
with the exception of activity of saturated lactones 3e, 4e toward
F. culmorum AM 282.
The highest fungistatic activity was observed for trans-7,8-
dibromo-cis-3-oxabicyclo[4.3.0]nonan-2-one (3c) (entry 5). This
lactone inhibits growth of F. oxysporum AM 13 by 50% at a
concentration of 30.1 μg/mL (0.10 μM/mL), whereas for the
other Fusarium species this degree of inhibition is achieved for
concentrations of 81.5ꢀ110.5 μg/mL (0.28ꢀ0.37 μM/mL). The
structural analogue of 3c with a gem-dimethyl group at C-4 (4c)
(entry 11) exhibits two or three times lower activity. Activity of
IC50 below 100 μg/mL toward F. oxysporum AM 13, F. culmorum
AM 282 was also observed for cis-7,8-epoxy-cis-3-oxabicyclo-
[4.3.0]nonan-2-one (3b) (entry 4), whereas its gem-dimethyl
derivative (4b) (entry 10) was less effective. Replacement of
one of the bromine atoms at C-8 with a hydroxyl group in trans-
7-bromo-8-hydroxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3d) and
in trans-7-bromo-8-hydroxy-4,4-dimethyl-cis-3-oxabicyclo[4.3.0]
nonan-2-one (4d) negatively affects the fungistatic activity.
We expected lower activity of 8-hydroxy-cis-3-oxabicyclo-
[4.3.0]nonan-2-one (6) (entry 13) compared to lactone 3d.
Such a correlation was observed for A. ochraceus AM 456 and
P. citrinum AM 354; however, the IC50 values for the
tested Fusarium strains were lower. An attempt to synthesize
7-bromo-cis-3-oxabicyclo[4.3.0]nonan-2-one was unsuccessful,
therefore we included derivatives of cis-2-oxabicyclo[4.3.0]nonan-
3-one in the tests. Additionally, this allowed us to check whether the
location of oxygen atom in the lactone ring has an influence on the
IC50 value. A negative effect of hydroxyl group on fungistatic activity
was confirmed in tests with hydroxy lactone 11 (entry 14), which to
a small degree inhibited growth of the tested phytopathogens.
When the hydroxyl group is protected with an ester moiety
(13ꢀ15) or replaced with bromine (12), the fungistatic activity
toward F. culmorum AM 282 and F. tricinctum AM 16 is increased,
however, this considerably decreases the fungistatic activity
toward F. oxysporum AM 13.
Influence of Stereogenic Centers on Fungistatic Activity.
The derivatives of the cis-3-oxabicyclo[4.3.0]nonan-2-one struc-
ture, especially lactones 3b and 3c, showed higher activity toward
F. oxysporum AM 13 and F. culmorum AM 282 than those of cis-2-
oxabicyclo[4.3.0]nonan-3-one.
The methods of obtaining of two enantiomers of cis-3-
oxabicyclo[4.3.0]non-7-en-2-one (3a) and cis-3-oxabicyclo-
[4.3.0]nonan-2-one (3e) described in the literature include,
among others, hydrolysis of anhydrides by means of lipases
and oxidation of meso diols using HLADH.41ꢀ43 HLADH
isolated from horse liver is not commercially available, therefore
we used the enzyme recombinated in Escherichia coli.
In the screening tests we have checked whether cis-1,2-
bis(hydroxymethyl)cyclohexane 17 can be oxidized to both
enantiomers of cis-lactone 3e using commercial alcohol dehydro-
genases (Scheme 4). The results are presented in Table 2.
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Table 1. The Half Maximal Inhibitory Concentration (IC50) for Racemic Lactones (a) in μg/mL and (b) in μM/mL against
A. ochraceus AM 456, F. culmorum AM 282, F. oxysporum AM 13, F. tricinctum AM 16, and P. citrinum AM 354a
(a) IC50 [μg/mL]
entry
compd
A. ochraceus AM 456
F. culmorum AM 282
F. oxysporum AM 13
F. tricinctum AM 16
P. citrinum AM 354
1
1
286.7
342.3
170.9
141.0
219.0
140.9
186.8
159.3
142.1
120.9
220.4
159.7
212.5
>350
244.3
226.7
282.5
172.8
51.3
310.2
291.5
184.0
90.4
220.3
240.5
282.0
72.2
289.4
302.9
220.7
156.5
110.5
211.2
221.9
204.7
180.5
191.0
162.4
212.6
161.1
219.5
173.4
146.0
111.0
181.6
27.9
>350
>350
>350
312.5
305.9
292.0
>350
274.5
>350
254.8
212.1
291.8
>350
212.5
281.7
282.4
>350
263.6
41.6
2
2
3
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
6
4
5
81.5
30.1
6
172.4
147.3
182.6
141.5
154.7
166.3
134.5
120.7
>350
133.1
144.9
172.8
159.2
24.5
141.0
314.3
188.2
95.3
7
8
9
10
11
12
13
14
15
16
17
18
80.2
170.4
179.1
126.6
195.3
184.1
299.7
218.6
262.5
24.2
11
12
13
14
15
DDAC
(b) IC50 [μM/mL]
F. oxysporum AM 13
entry
compd
A. ochraceus AM 456
F. culmorum AM 282
F. tricinctum AM 16
P. citrinum AM 354
1
1
1.89
2.23
1.25
0.91
0.73
0.60
1.33
0.96
0.78
0.38
0.85
0.95
1.36
2.24
1.12
1.25
1.25
0.57
0.14
2.04
18.88
1.33
0.59
0.28
0.73
1.05
1.10
0.77
0.47
0.63
0.80
0.77
2.24
0.61
0.80
0.76
0.52
0.07
1.45
1.56
2.04
0.47
0.10
0.60
2.25
1.13
0.52
0.25
0.65
1.06
0.81
1.25
0.84
1.64
0.96
0.86
0.07
1.90
1.96
1.60
1.01
0.37
0.90
1.58
1.23
0.99
0.58
0.62
1.26
1.03
1.41
0.79
0.80
0.49
0.60
0.08
2.30
2.27
2.54
2.03
1.03
1.24
2.50
1.65
1.92
0.78
0.81
1.73
2.24
1.36
1.28
1.55
1.54
0.87
0.11
2
2
3
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
6
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
11
12
13
14
15
DDAC
a Statistical analysis was done using ANOVA (p = 0.05), and the means were compared by calculating the least significant difference (LSD, Tukey). IC50
was calculated on the basis of standard curve with standard deviation (1.825ꢀ2.134.
The most effective enzyme, which was used in the next part of
our work, was HLADH, which oxidized the meso diol 17 to (þ)-
isomer of (1S,5R)-cis-3-oxabicyclo[4.3.0]nonan-2-one (3e). Al-
cohol dehydrogenase from Lactobacillus kefir (LKADH) oxidized
the diol to the (ꢀ)-enantiomer, however, with an ee of 82% only.
Five enantiomerically enriched lactones were obtained via
enzymatic oxidation of the respective diols (Scheme 5). For
enantiomerically enriched lactones (ꢀ)-(1S,5R)-cis-3-oxabicyclo-
[4.3.0]non-7-en-2-one ((ꢀ)-3a) and (þ)-(1S,5R)-cis-3-
oxabicyclo[4.3.0]nonan-2-one ((þ)-3e) absolute configuration
(1S,5R) were assigned by literature date.41(ꢀ)-(1S,5R,7S,8R)-
cis-7,8-Epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one ((ꢀ)-3b) was
obtained by oxidation of enantiomerically enriched lactone
(ꢀ)-(1S,5R)-cis-3-oxabicyclo[4.3.0]non-7-en-2-one ((ꢀ)-3a)
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with m-chloroperbenzoic acid, and its configuration was estab-
lished on sign of optical rotation and literature date.42
The gem-dimethyl group was introduced in a two-step synthe-
oxidized to (ꢀ)-lactone 4e. Absolute configuration (1S,5R) was
assigned by known literature mechanism44 and a sign of optical
rotation.41
sis, including a Grignard reaction leading to diol, which was next
Configuration of stereogenic centers of new lactone (þ)-
(1S,5R,7R,8R)-trans-7,8-dibromo-cis-3-oxabicyclo[4.3.0]nonan-
2-one (3c) was established on the basis of absolute configuration
of substrate, spectroscopic methods and X-ray diffraction study.
The fungistatic activity of the obtained 5 isomers of ee =
89ꢀ91% was determined toward F. culmorum 282 and F. oxyspor-
um 13. The IC50 values and differences in activities of optical
isomers compared to the racemic substrates are presented in
Table 3.
Scheme 4
Enantiomerically enriched 3a, 3b, 4e showed essential increase
in fungistatic activity compared to racemic compounds. Optically
active (þ)-(1S,5R,7R,8R)-trans-7,8-dibromo-cis-3-oxabicyclo-
[4.3.0]nonan-2-one (3c) was less active than the racemic mixture
against the strains of F. oxysporum AM 13 and F. culmorum
AM 282.
Table 2. Oxidation of 1,2-Bis(hydroxymethyl)cyclohexane
(17) Using Commercially Available Alcohol Dehydrogenases
time
conversion
lactol
[%]
3e
ee of
entry biocatalyst [days]
of 17 [%]
[%]
3e [%]
In summary, we have synthesized and determined anti-
fungical activity of three groups of lactones: two of them with
cis-3-oxabicyclo[4.3.0]nonan-2-one skeleton and the third group
1
2
3
4
5
6
HLADH
PADH I
PADH II
PADH III
YADH
1
7
95
100
100
2
67
35
5
28
65
95
0
(þ) 92
(þ) 92
(þ) 92
0
14
1
2
7
11
11
36
42
43
6
11
11
0
0
0
Table 3. The Half Maximal Inhibitory Concentration (IC50)
Measured in μg/mL for Chiral Lactones against F. culmorum
AM 282 and F. oxysporum AM 13a
14
1
0
0
36
42
43
2
(ꢀ) 26
(ꢀ) 26
(ꢀ) 25
(þ) 95
(þ) 54
(þ) 16
0
7
0
F. culmorum AM 282
F. oxysporum AM 13
14
1
0
b
b
4
entry compd
IC50[μg/mL]
ΔIC50
IC50[μg/mL]
ΔIC50
7
26
100
0
0
26
90
0
1
2
3
4
5
(ꢀ)-3a
(ꢀ)-3b
(þ)-3c
(þ)-3e
(ꢀ)-4e
171.0
59.7
þ13
þ31
ꢀ9
222.0
51.9
þ60
þ20
ꢀ17
þ53
þ17
14
1
10
0
90.5
47.5
7
14
46
0
5
9
(þ) 58
(þ) 34
0
169.4
ꢀ22
þ14
261.0
14
1
2
44
0
120.6
162.3
LKADH
0
a Statistical analysis was done using ANOVA (p = 0.05), and the means
were compared by calculating the least significant difference (LSD,
Tukey). IC50 was calculated on the basis of standard curve with standard
7
0
83a
0
0
0
14
19
24
(ꢀ) 82
a : additional products.
deviation (1.825ꢀ2.134. b ΔIC50 = IC50racemate ꢀ IC50enantiomer
.
Scheme 5a
a (i) LiAlH4, Et2O; (ii) HLADH; (iii) MCPBA, CH2Cl2; (iv) Br2, CCl4; (v) MeMgI, Et2O; (vi) dichromate pyridine, CH2Cl2.
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with cis-2-oxabicyclo[4.3.0]nonan-3-one. These compounds inhibited
growth of the fungi by 50% at concentrations from 30 μg/mL
(0.10 μM/mL) to above 350 μg/mL (1.92 μM/mL) and
were active toward A. ochraceus AM 456, F. culmorum AM 282,
F. oxysporum AM 13, and F. tricinctum AM 16. The strain
of P. citrinum AM 354 was found to be very resistant to these
lactones. The tested lactones, due to their structural differences,
allowed us to assess how the structure affects the biological
activity. An impact of the following factors was determined:
configuration of stereogenic centers; location of lactone
function in the five-member γ-lactone ring of [4.3.0] structure;
the presence and location of gem-dimethyl moiety; the
presence of epoxide ring, bromine atoms, hydroxyl group and
ester group.
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cals with fungicides for the suppression of Fusarium wilt of cyclamen.
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ynivalenol, nivalenol, and acetylated derivated by Australian isolates of
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As a result of the biological tests it was found that the compounds
cis-7,8-epoxy-cis-3-oxabicyclo[4.3.0]nonan-2-one (3b), its gem-
dimethyl derivative 4b and trans-7,8-dibromo-cis-3-oxabicyclo-
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282, F. oxysporum AM 13 and F. tricinctum AM 16.
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Corresponding Author
*Tel: 048 71 320 52 67. Fax: 048 71 328 35 76. E-mail: teresa.
olejniczak@up.wroc.pl.
Funding Sources
This work was supported by the Polish State Committee for
Scientific Research, Grant No. 2200/B/P01/2007/33.
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