Preparation of 2H-furo[2,3-c]pyran-2-one derivatives and evaluation of their germination-promoting activity

Article abstract of DOI:10.1021/jf0633241

The butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), has recently been identified as the germination stimulant present in smoke that promotes the germination of seeds from a wide range of plant species. In this paper, we describe the preparation of a number of analogues of 1 and compare their efficacy in promoting seed germination of three highly smoke-responsive plant species, Lactuca sativa L. cv. Grand Rapids (Asteraceae), Emmenanthe penduliflora Benth. (Hydrophyllaceae), and Solanum orbiculatum Poir. (Solanaceae). The results show that the methyl substituent at C-3 in 1 is important for germination-promoting activity while substitution at C-7 reduces activity. In contrast, bioactivity is mostly retained with analogues substituted at C-4 or C-5.

Full text of DOI:10.1021/jf0633241

J. Agric. Food Chem. 2007, 55, 2189−2194 2189
Preparation of 2H-Furo[2,3-c]pyran-2-one Derivatives and
Evaluation of Their Germination-Promoting Activity
GAVIN R. FLEMATTI,*,† ETHAN D. GODDARD-BORGER,† DAVID J. MERRITT,‡,§
|
EMILIO L. GHISALBERTI,† KINGSLEY W. DIXON,‡,§ AND ROBERT D. TRENGOVE
School of Biomedical, Biomolecular and Chemical Sciences and School of Plant Biology,
The University of Western Australia, Crawley, WA 6009, Kings Park and Botanic Garden,
West Perth, WA 6005, and School of Biological Sciences and Biotechnology, Murdoch University,
Murdoch, WA 6150, Australia
The butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), has recently been identified as the germination
stimulant present in smoke that promotes the germination of seeds from a wide range of plant species.
In this paper, we describe the preparation of a number of analogues of 1 and compare their efficacy
in promoting seed germination of three highly smoke-responsive plant species, Lactuca sativa L. cv.
Grand Rapids (Asteraceae), Emmenanthe penduliflora Benth. (Hydrophyllaceae), and Solanum
orbiculatum Poir. (Solanaceae). The results show that the methyl substituent at C-3 in 1 is important
for germination-promoting activity while substitution at C-7 reduces activity. In contrast, bioactivity is
mostly retained with analogues substituted at C-4 or C-5.
KEYWORDS: Butenolide; seed germination; seed dormancy; smoke; germination stimulant
INTRODUCTION
significantly impairing the germination activity would be useful
in the design of labeled analogues suitable for determining the
site and mode of action.
Plant-derived smoke has been reported to promote the
germination of a variety of wild species from regions as diverse
as South Africa (1), Australia (2), North America (3), and
Europe (4). A number of important crop species including lettuce
(5), maize (6), and celery (7) have also shown significant
increases in germination when treated with smoke, as have
selected weed species (8, 9). There have been many attempts
to identify the active agent in smoke, and recently, the
butenolide, 3-methyl-2H-furo[2,3-c]pyran-2-one (1), was identi-
fied as the key stimulant (10). Confirmation of the germination
activity of 1 has been demonstrated with a variety of smoke-
responsive plant species with activities observed at below ppb
concentrations (<10-9 M), thus providing a new class of potent
germination stimulants.
With the identity of the germination stimulant in smoke now
known, a better understanding of the way in which 1 acts in
promoting seed germination can be investigated. To date, little
is known about the mechanism of action of 1 in stimulating
seed germination. Furthermore, nothing is known about the
activity of analogues of 1. The preparation of analogues of 1
and assessment of their ability to promote germination will give
some insight into the molecular aspects of 1 that are important
for its germination-promoting activity. In addition, knowledge
of which part of the molecule that can be elaborated without
In this paper, we report on the synthesis of a number of
analogues of 1 and the germination activity of these analogues
with seeds of three highly smoke-responsive plant species,
Lactuca satiVa L. cv. Grand Rapids (Asteraceae), Emmenanthe
penduliflora Benth. (Hydrophyllaceae), and Solanum orbicu-
latum Poir. (Solanaceae). The germination-promoting abilities
of the analogues have been compared with the parent butenolide
(1), and some structure-activity relationships are discussed.
MATERIALS AND METHODS
General. Melting points (mp) were determined using a Kofler hot-
stage apparatus and are uncorrected. High-resolution mass spectra
(HRMS) were recorded using a VG Autospec mass spectrometer using
electron impact (70 eV) ionization. 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded using a Bruker ARX-300,
Bruker AV-500, or Bruker AV-600 spectrometer. Chemical shifts are
measured on the δ scale (in ppm) in d6-acetone with residual acetone
used as the internal standard (1H, δ 2.04; and 13C, δ 29.8). The signals
are described as singlet (s), doublet (d), triplet (t), and quartet (q).
* To whom correspondence should be addressed. E-mail: gflematt@
chem.uwa.edu.au.
† School of Biomedical, Biomolecular and Chemical Sciences, The
University of Western Australia.
‡ Kings Park and Botanic Garden.
§ School of Plant Biology, The University of Western Australia.
| Murdoch University.
10.1021/jf0633241 CCC: $37.00
© 2007 American Chemical Society
Published on Web 02/23/2007

2190 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Flematti et al.
Solvents used were of technical grade and were distilled before use.
Millipore (MP) water was obtained by passage through a Milli-Q
ultrapure water system (Millipore, Australia).
Maltol (7), 2-chloropropionyl chloride, and 2-chloroacetyl chloride
were obtained from Sigma-Aldrich. Pyromeconic acid (3), allomaltol
(8), 2,6-dimethyl-3-hydroxy-4H-pyran-4-one (9), and 3-hydroxy-6-
(methoxymethyl)-4H-pyran-4-one (13) were prepared from kojic acid
(2) (Merck) as described by Ellis et al. (11). Bromomaltol (15) was
prepared from 7 as described by Looker et al. (12).
Statistical Analyses. Data generated were statistically analyzed by
analysis of variance (ANOVA). Percentage germination data were
transformed (arcsinex) to conform to ANOVA assumptions (untrans-
formed data appears in Figures). Mean comparisons were made using
Fisher’s protected least significant difference, at the 95% confidence
level (p < 0.05).
Germination Testing. For testing, 1-2 mg of each analogue was
dissolved in MP water to give a stock concentration of 10 mg/L. A
series of 10-fold dilutions were performed to give four test solutions
of concentrations 1 mg/L and 100, 10, and 1 µg/L.
into water (100 mL) and stirred until one phase was formed. The
aqueous solution was filtered and extracted with dichloromethane (3
× 20 mL). The organic extract was washed with 1 M NaHCO3 (2 ×
20 mL), dried (Na2SO4), filtered, and evaporated to dryness. The residue
was extracted with 0.2 M potassium carbonate solution (2 × 50 mL)
by heating gently, and the resulting yellow solution was filtered and
extracted with dichloromethane (3 × 15 mL). The organic extract was
washed with brine, dried (Na2SO4), filtered, and evaporated to dryness
to give a yellow residue. The residue was purified by silica gel
chromatography (20-30% ethyl acetate/light petroleum) to afford the
appropriate butenolide compound (typical yields, 1-25%).
3,7-Dimethyl-2H-furo[2,3-c]pyran-2-one (10). Compound 7 was
converted to the thione and esterified with 2-chloropropionyl chloride
as described above. The thiono-maltol ester (350 mg, 1.5 mmol) was
refluxed in acetic anhydride to afford 10 as a white solid (45 mg, 0.27
1
mmol, 18%), which crystallized from hexane (mp 136-137 °C). H
NMR (500.1 MHz, d6-acetone): δ 7.59 (1H, d, J ) 5.5 Hz, H-5);
6.73 (1H, d, J ) 5.5 Hz, H-4); 2.32 (3H, s, CH3); 1.84 (3H, s,
CH3). 13C NMR (125.8 MHz, d6-acetone): δ 171.2 (CdO); 149.6
(C-5); 140.8 (C-3a); 138.9 (C-7a); 137.3 (C-7); 103.7 (C-4); 99.4
(C-3); 13.8 (CH3); 7.7 (CH3). HRMS calculated for C9H8O3, 164.0473;
found, 164.0478.
Grand Rapids Lettuce Seed. Grand Rapids lettuce seeds (Waltham
strain) were obtained from R.B. Dessert Seed Co. and were air-dried
and stored at -18 °C in sealed laminated foil sachets until required.
For testing, 2.5 mL of each test solution was applied to three replicate
Petri dishes (90 mm) lined with two layers of Whatman #1 filter paper
(7 cm). MP water served as a control for each experiment. In a dark
room, 40-50 seeds were added to the Petri dishes, which were sealed
and stored in a light-proof container and incubated at 19 ( 2 °C. All
manipulations involving the seed were carried out in a dark room, and
germinants, based on the appearance of a radicle, were scored after 48
h of incubation.
E. penduliflora and S. orbiculatum. The S. orbiculatum seeds were
collected in the Shark Bay region (Western Australia) in November
2004 and were stored under ambient laboratory conditions (c. 23 °C
and 50% relative humidity) until required. E. penduliflora was sourced
from garden-collected material in California from Rancho Santa Ana
Botanic Garden. Solutions were tested by adding 2.5 mL to two pieces
of Whatman #1 filter paper (7 cm) in Petri dishes (9 cm). MP water
served as the control. Approximately 20-25 seeds of each species were
added to each Petri dish, and each solution was tested in triplicate.
The Petri dishes were sealed with a layer of plastic wrap and incubated
at 19 ( 2 °C in a light-proof container for 6 days before counting.
General Method for Converting Pyran-4-one Derivatives to
Pyran-4-thiones. Phosphorus pentasulphide (1.5 equiv) in tetrahydro-
furan was added to a stirred solution of the pyran-4-one compound
dissolved in tetrahydrofuran (10 mL) following the general method of
Scheeren et al. (13). Solid sodium hydrogen carbonate (6 equiv) was
added, and the reaction mixture was stirred at room temperature and
monitored by thin-layer chromatography for completion (ca. 3 h). The
reaction mixture was added cautiously to water (100 mL), and the
resulting aqueous mixture was extracted with ethyl acetate (4 × 20
mL). The combined organic extract was washed with 0.2 M NaHCO3
(2 × 20 mL) and saturated brine, then dried (Na2SO4), filtered, and
evaporated to dryness under reduced pressure. The residue was purified
by rapid silica filtration (60% ethyl acetate/light petrol) to yield the
pure thione (typical yields, 60-80%).
General Method for Esterification. Triethylamine (1.2 equiv) was
added to a stirred solution of the thione (1 equiv) in dichlorometh-
ane (10 mL) at 0 °C. A solution of the acyl chloride (1.5 equiv) diluted
in dichloromethane (1 mL) was added dropwise to the solution, and
the reaction mixture was stirred for a further 10 min at 0 °C. The
solution was evaporated to dryness under reduced pressure, and the
resulting residue was purified by rapid silica filtration (dichloromethane)
to afford the ester (typical yields 80-90%). The ester was prone to
hydrolysis when stored and so was used immediately in the cyclization
reaction.
General Method for Butenolide Cyclization. A mixture of
anhydrous sodium acetate (3 equiv) and triphenyl phosphine (1.1 equiv)
in acetic anhydride (20 mL) was heated at reflux for 5 min. A solution
of the thione ester (1 equiv) diluted with acetic anhydride (2 mL) was
added dropwise to the refluxing mixture. The mixture was refluxed
for a further 30 min and allowed to cool. The dark mixture was poured
3,5-Dimethyl-2H-furo[2,3-c]pyran-2-one (11). Compound 8 was
converted to the thione and esterified with 2-chloropropionyl chloride
as described above. The thioester (700 mg, 3 mmol) was refluxed in
acetic anhydride to afford 11 as a light yellow solid (57.4 mg, 0.35
1
mmol, 12%), which crystallized from hexane (mp 101-102 °C). H
NMR (500.1 MHz, d6-acetone): δ 7.71 (1H, s, H-7); 6.58 (1H, s, H-4);
2.28 (3H, s, CH3), 1.83 (3H, s, CH3). 13C NMR (125.8 MHz,
d6-acetone): δ 171.5 (CdO); 159.9 (C-5); 142.4 (C-3a); 142.4 (C-7a);
127.7 (C-7); 101.1 (C-4); 98.4 (C-3); 19.8 (CH3); 7.5 (CH3). HRMS
calculated for C9H8O3, 164.0473; found, 164.0476.
3,5,7-Trimethyl-2H-furo[2,3-c]pyran-2-one (12). Compound 9 was
converted to the thione and esterified with 2-chloropropionyl chlor-
ide as described above. The thioester (207 mg, 0.84 mmol) was re-
fluxed in acetic anhydride to afford 12 as a light yellow solid (3.0
mg, 0.02 mmol, 2%), which crystallized from hexane (mp 105-106
°C). 1H NMR (600.1 MHz, d6-acetone): δ 6.49 (1H, s, H-4); 2.28
(3H, s, CH3); 2.27 (3H, s, CH3); 1.79 (3H, s, CH3). 13C NMR (150.9
MHz, d6-acetone): δ 171.5 (CdO); 159.5 (C-5); 142.4 (C-7a); 138.2
(C-7); 136.7 (C-3a); 100.7 (C-4); 97.8 (C-3); 19.7 (CH3); 13.8
(CH3); 7.6 (CH3). HRMS calculated for C10H10O3, 178.0630; found,
178.0629.
5-Methoxymethyl-3-methyl-2H-furo[2,3-c]pyran-2-one (14). Com-
pound 13 was converted to the thione and esterified with 2-chloropro-
pionyl chloride as described above. The thioester (140 mg, 0.5 mmol)
was refluxed in acetic anhydride to afford 14 as a light yellow solid (2
mg, 0.01 mmol, 2%). 1H NMR (600.1 MHz, d6-acetone): δ 7.75 (1H,
s, H-7); 6.78 (1H, t, J ) 0.8 Hz, H-4); 4.27 (2H, d, J ) 0.8 Hz, CH2);
3.40 (3H, s, OCH3); 1.87 (3H, s, CH3). 13C NMR (150.9 MHz, d6-
acetone): δ 171.3 (CdO); 158.8 (C-5); 142.6 (C-7a); 141.4 (C-3a);
127.6 (C-7); 101.3 (C-4); 100.3 (C-3); 71.0 (CH2); 58.8 (OCH3); 7.6
(CH3). HRMS calcd for C10H10O4, 194.0579; found, 194.0586.
4-Bromo-3,7-dimethyl-2H-furo[2,3-c]pyran-2-one (16). Compound
15 was converted to the thione and esterified with 2-chloropropionyl
chloride as described above. The thioester (90 mg, 0.3 mmol) was
refluxed in acetic anhydride to afford 16 as a light yellow solid (1.2
mg, 0.01 mmol, 2%). 1H NMR (500.1 MHz, d6-acetone): δ 7.85 (1H,
s, H-5); 2.32 (3H, s, CH3); 2.07 (3H, s, CH3). 13C NMR (125.8 MHz,
d6-acetone): δ 170.7 (CdO); 148.5 (C-5); 138.2 (C-3a); 137.7 (C-7);
137.2 (C-7a); 102.4 (C-3); 100.3 (C-4); 13.7 (CH3); 8.3 (CH3). HRMS
calcd for C9H7BrO3, 241.9579; found, 241.9570.
2H-Furo[2,3-c]pyran-2-one (17). Pyromeconic acid thione (4) (200
mg, 1.6 mmol) was esterified with 2-chloroacetyl chloride as described
above. Attempts to purify the ester by rapid silica filtration were
unsuccessful; therefore, the ester reaction mixture was evaporated to
dryness under reduced pressure, and the residue was used directly in
the cyclization step. Acetic anhydride was added, and the mixture was
filtered through a plug of cotton wool. The filtered solution was added
dropwise to a mixture of refluxing acetic anhydride, triphenylphosphine,
and sodium acetate as described above. Workup and purification

Preparation of 2H-Furo[2,3-c]pyran-2-one
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2191
Scheme 1. Synthesis of 1 from 2
afforded the desmethyl butenolide (17) as a light yellow solid (14.2
mg, 0.1 mmol, 7%), which crystallized from hexane (mp 105-106
1
°C). H NMR (300.1 MHz, d6-acetone): δ 7.92 (1H, d, J ) 1.5 Hz,
H-7); 7.70 (1H, d, J ) 5.4 Hz, H-5); 6.90 (1H, dd, J ) 5.4, 0.5 Hz,
H-4); 5.40 (1H, dd, J ) 1.5, 0.5 Hz, H-3). 13C NMR (75.5 MHz, d6-
acetone): δ 170.5 (CdO); 151.1 (C-5); 146.1 (C-3a); 144.1 (C-7a);
129.4 (C-7); 105.5 (C-4); 90.7 (C-3). HRMS calculated for C7H4O3,
136.0160; found, 136.0164.
7-Methyl-2H-furo[2,3-c]pyran-2-one (18). Compound 7 was con-
verted to the thione (100 mg, 0.7 mmol) and esterified with 2-chloro-
acetyl chloride as described above. The reaction mixture was evaporated
to dryness under reduced pressure, and the residue was used directly
in the cyclization step. Acetic anhydride was added, and the mixture
was filtered through a plug of cotton wool. The filtered solution was
added dropwise to a mixture of refluxing acetic anhydride, triphen-
ylphosphine, and sodium acetate as described above. Workup and
purification afforded 18 as a light yellow solid (1.3 mg, 0.01 mmol,
1
1%). H NMR (600.1 MHz, d6-acetone): δ 7.67 (1H, d, J ) 5.4 Hz,
H-5); 6.82 (1H, d, J ) 5.4 Hz, H-4); 5.34 (1H, s, H-3); 2.36 (3H, s,
CH3). 13C NMR (150.9 MHz, d6-acetone): δ 170.6 (CdO); 150.7
(C-5); 146.0 (C-3a); 140.2 (C-7a); 138.9 (C-7); 105.1 (C-4); 90.1
(C-3); 14.0 (CH3). HRMS calculated for C8H6O3, 150.0317; found,
150.0314.
Scheme 2. Synthesis of Analogues of 1 from Variants of 3
5-Methyl-2H-furo[2,3-c]pyran-2-one (19). Compound 8 was con-
verted to the thione (200 mg, 1.4 mmol) and esterified with 2-chloro-
acetyl chloride as described above. The ester reaction mixture was
evaporated to dryness under reduced pressure, and the residue was used
directly in the cyclization step. Acetic anhydride was added, and the
mixture was filtered through a plug of cotton wool. The filtered solution
was added dropwise to a mixture of refluxing acetic anhydride,
triphenylphosphine, and sodium acetate as described above. Workup
and purification afforded 19 as a light yellow solid (1.7 mg, 0.01 mmol,
1
1%). H NMR (600.1 MHz, d6-acetone): δ 7.84 (1H, d, J ) 1.4 Hz,
Scheme 3. Synthesis of 3-Desmethyl Analogues from Variants of 3
H-7); 6.67 (1H, q, J ) 0.7 Hz, H-4); 5.26 (1H, d, J ) 1.4 Hz, H-3);
2.32 (3H, d, J ) 0.7 Hz, CH3). 13C NMR (150.9 MHz, d6-acetone): δ
170.9 (CdO); 161.3 (C-5); 147.8 (C-3a); 143.4 (C-7a); 128.9 (C-7);
102.6 (C-4); 89.2 (C-3); 14.0 (CH3). HRMS calculated for C8H6O3,
150.0317; found, 150.0312.
3-Methylfuro[2,3-c]pyridin-2(3H)one (21). Compound 1 (15 mg,
0.1 mmol) was heated with a concentrated NH4OH solution (28% NH3,
3 mL) on a steam bath for 5 h following the method of Hwang et al.
(14). The resulting solution was evaporated to dryness under reduced
pressure and purified by silica gel chromatography (20% MeOH/ethyl
acetate/1% triethylamine) to give 21 as a light brown solid (1.5 mg,
1
0.01 mmol, 10%). H NMR (600.1 MHz, d6-acetone): δ 8.14 (1H, s,
Scheme 4. Synthesis of Some Nitrogen Containing Analogues of 1
H-7); 7.97 (1H, d, J ) 4.9 Hz, H-5); 7.05 (1H, d, J ) 4.9, H-4); 3.90
(1H, q, J ) 7.2 Hz, H-3); 1.48 (3H, d, J ) 7.2 Hz, CH3). 13C NMR
(150.9 MHz, d6-acetone): δ 178.7 (CdO); 153.3 (C-7a); 142.0 (C-5);
140.9 (C-7); 135.2 (C-3a); 124.3 (C-4); 44.6 (C-3); 17.4 (CH3). HRMS
calculated for C8H7NO2, 149.0477; found, 149.0479.
3,6-Dimethylfuro[2,3-c]pyridin-2(6H)one (22). Compound 1 (10.6
mg, 0.07 mmol) was heated in a sealed tube with aqueous methylamine
solution (24%, 3 mL) overnight at 70 °C following the method of Liu
et al. (15). The resulting solution was evaporated to dryness under
reduced pressure and purified by silica gel chromatography (10%
MeOH/ethyl acetate/1% triethylamine) to give 22 as a light brown solid
(1.6 mg, 0.01 mmol, 14%). 1H NMR (600.1 MHz, d6-acetone): δ 7.38
(1H, dd, J ) 6.9, 1.4 Hz, H-5); 7.32 (1H, d, J ) 1.4 Hz, H-5); 6.54
(1H, d, J ) 6.9, H-4); 3.81 (3H, s, N-CH3); 1.79 (3H, s, CH3). 13C
NMR (150.9 MHz, d6-acetone): δ 172.0 (CdO); 144.2 (C-3a); 143.7
(C-7a); 135.8 (C-5); 114.5 (C-7); 103.3 (C-4); 86.0 (C-3); 44.1
(N-CH3); 7.4 (CH3). HRMS calcd for C9H9NO2, 163.0633; found,
163.0628.
metabolite (16) that is commercially available. In the syn-
thesis, 2 was converted over four steps to 3 (11), which
was subsequently treated with phosphorus pentasulphide to give
4 (Scheme 1). Esterification of 4 with 2-chloropropionyl
chloride in the presence of triethylamine afforded the ester 5,
which was refluxed in acetic anhydride with triphenyl phos-
phine and sodium acetate to give 1. The yield of 1 was low
(22%) mainly due to the formation of the transesterification
product 6, which can be recovered and hydrolyzed back to 4 if
required.
RESULTS AND DISCUSSION
Synthesis of Analogues. The synthesis of the butenolide, 1,
has only recently been reported (10) and, to date, represents
the only method for preparing this new class of compound. The
overall synthesis was achieved in seven steps from 2, a fungal
For preparing analogues of 1, the use of alternative starting
materials would provide a simple route. In particular, substituting
3 for simple variants such as 7, 8, and 9 should provide

2192 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Flematti et al.
Figure 1. Germination activity of the butenolide (1) and some 3-methyl analogues. Error bars represent the standard error of the mean (SEM), and MP
water served as the control (0 g/L).
µ
analogues with methyl substituents at the corresponding posi-
tions. Conveniently, 7 is commercially available, while 8 and
9 are easily prepared from 2 (11).
Following the butenolide annulation methods in Scheme 1,
7 provided the 3,7-dimethyl analogue (10) in moderate yield
(Scheme 2). Similar treatment of 8 and 9 returned the corre-
sponding 3,5-dimethyl (11) and the 3,5,7-trimethyl (12) ana-
logues, respectively. Attempts to treat 2 in a similar fashion
proved unsuccessful, as did treatment of the acetyl-protected
2. Alternatively, conversion of 2 into the methyl ether (13) and
subsequent treatment provided the 5-methoxymethyl analogue
(14).
While use of this methodology on different starting materials
allowed for easy elaboration of the C-5 and C-7 positions of
the 2H-furo[2,3-c]pyran-2-one skeleton, it was difficult to
substitute at the C-4 position. A simple pyrone that would lead

Preparation of 2H-Furo[2,3-c]pyran-2-one
J. Agric. Food Chem., Vol. 55, No. 6, 2007 2193
Figure 2. Germination activity of 3-desmethyl analogues and nitrogen analogues of 1. Error bars represent SEM, and MP water served as the control
(0 g/L).
µ
to an analogue substituted at C-4 was 15 (12). Hence, use of
15 returned the 16 derivative containing a substituent at C-4.
The butenolide analogues prepared so far all contain a methyl
group at C-3. To determine the importance of this methyl group
for biological activity, 2-chloroacetyl chloride was used as the
esterifying agent instead of 2-chloropropionyl chloride. Reflux-
ing the chloroacetyl-thione ester in acetic anhydride afforded
the desmethyl butenolide (17) in very low yields, lower than
obtained using the chloropropionyl-thione ester; however,
enough material was obtained to enable characterization and
germination testing. Similar treatment of 7 and 8 gave the
7-methyl (18) and the 5-methyl (19) analogues, respectively,
in similar low yields.
butenolide in concentrated ammonium hydroxide yielded the
pyridine 21. In an analogous reaction using aqueous methyl-
amine solution, the expected N-methyl-pyridone 22 was ob-
tained.
Germination-Promoting Activity of Analogues. The ana-
logues were tested for germination-promoting activity with the
three highly smoke-responsive species L. satiVa Grand Rapids
lettuce, E. penduliflora, and S. orbiculatum. Grand Rapids
lettuce was used as a key bioassay species for the isolation and
identification of 1 (10) and represents an excellent test species
for assessing the germination-promoting activity of butenolide
analogues. Likewise, E. penduliflora has been used extensively
for assessing the activity of smoke extracts (17) and for probing
the mode of action of smoke-induced seed germination (18).
The third species, S. orbiculatum, is a West Australian native
species that has recently been found to be highly responsive to
smoke and, now, to 1. Typical control germination for this
Compound 3 and its analogues can be easily converted to
the corresponding pyridone derivatives upon treatment with
amines (11). It was thought that similar treatment of 1 may
provide the pyridone analogue 20. However, heating the

2194 J. Agric. Food Chem., Vol. 55, No. 6, 2007
Flematti et al.
Floristic Region, South Africa. S. Afr. J. Bot. 2004, 70, 559-
581.
species is less than 10%, while butenolide-treated seeds are
promoted to germinate in excess of 90% even at 1 ppb, thus
providing a reliable and sensitive test species for evaluating the
bioactivity of analogues.
(2) Roche, S.; Dixon, K. W.; Pate, J. S. Seed ageing and smoke:
Partner cues in the amelioration of seed dormancy in selected
Australian native species. Aust. J. Bot. 1997, 45, 783-815.
(3) Keeley, J. E.; Fotheringham, C. J. Smoke-induced seed germina-
tion in California chaparral. Ecology 1998, 79, 2320-2336.
(4) Pe´rez-Ferna´ndez, M. A.; Rodr´ıguez-Echeverr´ıa, S. Effect of
smoke, charred wood, and nitrogenous compounds on seed
germination of ten species from woodland in central-western
Spain. J. Chem. Ecol. 2003, 29, 237-251.
(5) Drewes, F. E.; Smith, M. T.; van Staden, J. The effect of a plant
derived smoke extract on the germination of light-sensitive
lettuce seed. Plant Growth Regul. 1995, 16, 205-209.
(6) Modi, A. T. Short-term preservation of maize landrace seed and
taro propagules using indigenous storage methods. S. Afr. J. Bot.
2004, 70, 16-23.
(7) Thomas, T. H.; Van Staden, J. Dormancy break of celery (Apium
graVeolens L.) seeds by plant derived smoke extract. Plant
Growth Regul. 1995, 17, 195-198.
(8) Adkins, S. W.; Peters, N. C. B. Smoke derived from burnt
vegetation stimulates germination of arable weeds. Seed Sci. Res.
2001, 11, 213-222.
(9) Doherty, L. C.; Cohn, M. A. Seed dormancy in red rice (Oryza
satiVa). XI. Commercial liquid smoke elicits germination. Seed
Sci. Res. 2000, 10, 415-421.
(10) Flematti, G. R.; Ghisalberti, E. L.; Dixon, K. W.; Trengove, R.
D. A compound from smoke that promotes seed germination.
Science 2004, 305, 977.
(11) Ellis, B. L.; Duhme, A. K.; Hider, R. C.; Hossain, M. B.; Rizvi,
S.; Van der Helm, D. Synthesis, physicochemical properties, and
biological evaluation of hydroxypyranones and hydroxypyridi-
nones: Novel bidentate ligands for cell-labeling. J. Med. Chem.
1996, 39, 3659-3670.
(12) Looker, J. H.; Prokop, R. J.; Serbousek, W. E.; Clifton, M. D.
Bromomaltol: Structure and conversion to novel pyridone and
pyridine derivatives. J. Org. Chem. 1979, 44, 3408-3410.
(13) Scheeren, J. W.; Ooms, P. H. J.; Nivard, R. J. F. A general
procedure for the conversion of a carbonyl group into a thione
group with tetraphosphorus decasulfide. Synthesis 1973, 149-
151.
The germination responses of the three test species used for
evaluating the activity of the analogues of 1 were broadly
similar, although the sensitivity of each species to the analogues
varied somewhat. The L. satiVa bioassay was more sensitive to
the germination-promoting effects of the analogues, while E.
penduliflora was the least sensitive of the three species, requiring
higher concentrations of analogues for promotion of germination
to be observed. The S. orbiculatum gave the largest range
between control (<10%) and butenolide-like enhancement
(>80%), indicating that this species could provide a useful assay
for evaluating the activity of analogues of 1 in future work.
For each of the test species, the natural stimulant (1) showed
significantly higher germination activity than the control
(Figures 1 and 2) for each of the concentrations tested (1 mg/L
to 1 µg/L). A number of the analogues were also found to
promote germination. In particular, the 3,5-dimethyl analogue
(11) showed similar levels of activity to those observed for 1
(Figure 1). In contrast, introduction of a methyl at C-7, as in
10, resulted in a 100-fold reduction in activity. A similar effect
was observed for the 3-desmethyl variant (17) and analogues
with a methyl at C-7 (18) and C-5 (19). Introduction of a
bromine at C-4 had little effect as shown by comparison of the
activities of the 3,7-dimethyl (10) and 3,7-dimethyl-4-bromo
analogues (16). Replacement of the pyran oxygen by nitrogen,
as in 21 and 22, does not eliminate the activity. It is also worth
noting that none of the starting materials or intermediates used
in the preparation of the butenolide analogues showed germina-
tion stimulation on these test species.
It appears that some variation of substitution at C-5 can be
tolerated, since introduction of a methyl at this position does
not reduce activity. Although the analogue containing a meth-
oxymethyl (14) at C-5 had reduced activity, it is still active at
100 µg/L. Importantly, substitution at C-5 is rather simple
and may lend itself to the introduction of various labeling
groups, such as fluorescent dyes or affinity labels (19). In
addition, the fact that the 3,5-dimethyl analogue (11) shows
similar levels of activity to the parent butenolide (1) may be of
some interest since the synthesis of 11 is simpler to that
developed so far for 1.
In summary, the results (Figures 1 and 2) show that the
methyl group at C-3 is important for germination-promoting
activity of 1 and substitution at C-7 reduces germination activity.
Substitution at C-5 and C-4 can be tolerated more and does not
affect bioactivity to the same extent as substitution at C-7.
Furthermore, replacement of the pyran oxygen with a nitrogen
atom gives pyridine/pyridone analogues that also have germina-
tion activity. In terms of labeling the butenolide, substitution
through C-5 appears to be more promising and should provide
analogues that retain some germination-promoting ability.
(14) Hwang, D. R.; Proctor, G. R.; Driscoll, J. S. Pyridones as
potential antitumour agents II: 4-Pyridones and bioisosteres of
3-acetoxy-2-pyridone. J. Pharm. Sci. 1980, 69, 1074-1076.
(15) Liu, Z. D.; Piyamongkol, S.; Liu, D. Y.; Khodr, H. H.; Lu, S.
L.; Hider, R. C. Synthesis of 2-amido-3-hydroxypyridin-4(1H)-
ones: Novel iron chelators with enhanced pFe3+ Values. Biomed.
Chem. 2001, 9, 563-573.
(16) Beelik, A. Kojic acid. AdV. Carbohydr. Chem. 1956, 11, 145-
183.
(17) Preston, C. A.; Becker, R.; Baldwin, I. T. Is ‘NO’ news good
news? Nitrogen oxides are not components of smoke that elicits
germination in two smoke-stimulated species, Nicotiana attenu-
ata and Emmenanthe penduliflora. Seed Sci. Res. 2004, 14, 73-
79.
(18) Egerton-Warburton, L. M. A smoke-induced alteration of the
sub-testa cuticle in seeds of the post-fire recruiter, Emmenanthe
penduliflora Benth. (Hydrophyllaceae). J. Exp. Bot. 1998, 49,
1317-1327.
(19) Reizelman, A.; Wigchert, S. C. M.; del-Bianco, C.; Zwanenburg,
B. Synthesis and bioactivity of labelled germination stimulants
for the isolation and identification of the strigolactone receptor.
Org. Biomol. Chem. 2003, 1, 950-959.
ACKNOWLEDGMENT
Thanks to Samantha Clarke for assistance with germination
testing and Lucy Commander for collection of S. orbiculatum
seeds. Thanks also to Simon Brayshaw for assistance with
preparing analogues.
Received for review November 16, 2006. Revised manuscript received
January 22, 2007. Accepted January 24, 2007. Funding for G.R.F. and
D.J.M. for this research was provided by the Australian Research
Council.
LITERATURE CITED
(1) Brown, N. A. C. Smoke seed germination studies and a guide
to seed propagation of plants from the major families of the Cape
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