New class of algicidal compounds and fungicidal activities derived from a chromene amide of amyris texana

Article abstract of DOI:10.1021/jf101626g

A chromene amide, N-[2-(2,2-dimethyl-2H-1-benzopyran-6-yl)ethyl]-N,3- dimethylbutanamide, was isolated from the EtOAc extract of the leaves of Amyris texana and found to have moderate antifungal activity against Colletotrichum spp. and selective algicidal activity against Planktothrix perornata, a cyanobacterium (blue-green alga) that causes musty off-flavor in farm-raised channel catfish (Ictalurus punctatus). To improve the selective algicidal activity and provide water solubility, a series of chromene analogues were synthesized and evaluated for algicidal activity using a 96-well microplate rapid bioassay. In addition, the chromene analogues were evaluated for antifungal and phytotoxic activities. Hydrochloride salts of a chromene amine analogue showed improved water solubility and selectivity toward P. perornata with activity comparable to that of the naturally occurring chromene amide.

Full text of DOI:10.1021/jf101626g

9476 J. Agric. Food Chem. 2010, 58, 9476–9482
DOI:10.1021/jf101626g
New Class of Algicidal Compounds and Fungicidal Activities
Derived from a Chromene Amide of Amyris texana
‡
K
UMUDINI M. MEEPAGALA,*,† KEVIN K. SCHRADER,† CHARLES L. BURANDT
,
†
†
D
AVID E. WEDGE
,
AND
STEPHEN O. DUKE
†Natural Products Utilization Research Unit, Agricultural Research Service, U.S. Department of
Agriculture, P.O. Box 8048, University, Mississippi 38677-8048, and ‡Research Institute of Pharmaceutical
Sciences, National Center for Natural Products Research, University, Mississippi 38677-8048
A chromene amide, N-[2-(2,2-dimethyl-2H-1-benzopyran-6-yl)ethyl]-N,3-dimethylbutanamide, was
isolated from the EtOAc extract of the leaves of Amyris texana and found to have moderate
antifungal activity against Colletotrichum spp. and selective algicidal activity against Planktothrix
perornata, a cyanobacterium (blue-green alga) that causes musty off-flavor in farm-raised channel
catfish (Ictalurus punctatus). To improve the selective algicidal activity and provide water solubility, a
series of chromene analogues were synthesized and evaluated for algicidal activity using a 96-well
microplate rapid bioassay. In addition, the chromene analogues were evaluated for antifungal and
phytotoxic activities. Hydrochloride salts of a chromene amine analogue showed improved water
solubility and selectivity toward P. perornata with activity comparable to that of the naturally
occurring chromene amide.
KEYWORDS: Chromene amide; chromenes; algicide; fungicide
Column chromatography was carried out with kieselgel 60 (particle size=
0.063-0.2 mm, Merck) with mixtures of hexane and acetone in various
amounts. All solvents were of reagent grade and used without further
purification. 1H and 13C NMR spectra were recorded either on a Bruker
AMX NMR spectrometer (Palo Alto, CA) operating at 400 MHz for 1H
NMR and at 100 MHz for 13C NMR or on a Varian Mercury AS400
INTRODUCTION
Off-flavor problems cause significant economic losses to catfish
farmers in the United States. Planktothrix perornata f. attenuata
[Skuja], a cyanobacterium (blue-green alga) common in catfish
production ponds in the southeastern United States, produces the
monoterpene 2-methylisoborneol (MIB), which is absorbed into
catfish flesh and imparts a musty off-flavor, rendering them
unpalatable and unmarketable. Algicides such as copper sulfate
and diuron [N0-(3,4-dichlorophenyl)-N,N-dimethylurea] that are
currently used to control P. perornata have broad-spectrum
toxicity toward other beneficial phytoplankton, such as the green
alga Selenastrum capricornutum, as well as low biodegradability.
Therefore, there is an urgent need to find algicides with high
selectivity, minimum toxicities toward nontarget organisms, and
biodegradability to control P. perornata in catfish production
ponds.
spectrometer operating at 400 MHz for 1H NMR and at 100 MHz for 13
C
NMR. The HR-ESIMS was measured by using either a Bruker QTOF
micromass spectrometer or a JEOL AccuTOF (JMS-T100LC) (Peabody,
MA) mass spectrometer. GC-MS analysis was carried out on an HP5790
MSD spectrometer (Hewlett-Packard) equipped with a GC 5890 using a
DB-1 column (20 m ꢀ 0.2 mm, 0.18 μm film thickness). The oven was
temperature programmed from 60 °C (5 min) to 280 °C (20 min) at 5 °C/
min with helium as the carrier gas.
Plant Material. Leaves of A. texana were collected in Cameron
County in southern Texas, in June 2002, by Dr. Charles Burandt at the
University of Mississippi. A voucher specimen (BUR 190204 a) is
deposited at the University of Mississippi herbarium. The leaves were
air-dried, ground, and stored at room temperature until they were
extracted.
Extraction and Isolation of the Bioactive Constituents. Air-dried
and ground A. texana leaves (500 g) were extracted repeatedly at room
temperature (25-28 °C) with ethyl acetate (2 L ꢀ 3) by stirring with a
magnetic stirrer. The resulting extracts were filtered through filter paper
(Whatman no. 1) and combined, and the solvent was evaporated at 40 °C
under reduced pressure to afford a dark green residue (21.6 g). This residue
(20.0 g) was subjected to column chromatography on a silica gel column
(3.8 cm ꢀ 81 cm) eluting with hexane and then with increasing amounts of
acetone (up to 100%). Fractions of 400 mL were collected, and the elution
profile of the column was monitored by TLC plates sprayed with
anisaldehyde and Dragendroff spray reagents and by I2 vapor. The
fractions with similar TLC profiles were combined to yield 34 fractions.
Each fraction was tested for the presence of antifungal constituents by
TLC bioautography by spraying with Colletotrichum fragariae spores (1)
Amyris texana P. Wilson (Rutaceae), also known as “Texas
torchwood”, is native to Texas and Mexico, distributed mainly in
the arid regions in the southwestern United States. As part of our
continuing efforts to search for natural product-based agrochem-
icals, the leaves of A. texana were extracted and investigated for
algicidal, antifungal, and phytotoxic constituents.
MATERIALS AND METHODS
General Experimental Procedures. Extracts were analyzed on silica
gel TLC plates GF with fluorescent indicator (250 μm, Analtech, Newark,
DE). Iodine vapor, UV light (at 254 and 365 nm), and Dragendorff and
anisaldehyde spray reagents were used for the detection of compounds.
*Corresponding author [phone (662) 915-1036; fax (662) 915-1035;
e-mail kmeepaga@olemiss.edu.].
pubs.acs.org/JAFC
Published on Web 08/09/2010
© 2010 American Chemical Society

Article
J. Agric. Food Chem., Vol. 58, No. 17, 2010 9477
[2-(2,2-Dimethyl-2H-chromen-6-yl)ethyl]isopropylamine Hydrochlor-
ide (6). Isopropylamine (5 g, 80 mmol) and 5 (1 g, 30 mmol) were reacted
in a closed tube at 80 °C for 1 h. The reaction mixture was cooled, and excess
amine was removed by flushing with N2 gas. The solid obtained was
partitioned between ethyl acetate (50 mL) and 5% aqueous NaOH (50 mL).
The organic layer was washed with water and saturated NaCl and dried over
anhydrous Na2SO4 to afford a gum. This gum was dissolved in ethanol
(10 mL), treated with concentrated HCl (1 mL), diluted with diethyl ether
(20 mL), and then left to stand for 12 h at 4 °C. The white crystals obtained
were filtered to obtain 6 (yield, 700 mg, 20 mmol, 66%): 1H NMR (CDCl3)
δ 1.34 (6H, s), 1.44 (3H, d, J = 6.4 Hz), 3.04 (2 H, m), 3.17 (2H, m), 3.31
(1H, septet, J=6.4 Hz) 5.52 (1H, d, J=10.0 Hz), 6.16 (1H, d, J=10.0 Hz),
6.61 (1H, d, J=8.0 Hz), 6.8 (1H, d, J=2.0 Hz), 6.91 (1H, dd, J=6.4,
2.0 Hz); 13C NMR (CDCl3) δ 19.1, 27.9, 31.6, 46.4, 50,5, 76.1, 116.4, 121.4,
121.9, 126.5, 128.8, 129.1, 131.0, 151.9; HRMS(ESI-TOF) m/z 246.1847
[M þ H]þ (calcd for C16H24ON, 246.1857).
Figure 1. Structure of the natural chromene amide N-[2-(2,2-dimethyl-
2H-chromen-6-yl)ethyl]-3,N-dimethylbutyramide.
and algicidal constituents using a rapid bioassay according to the
published methods (2).
[2-(2,2-Dimethyl-2H-chromen-6-yl)ethyl]methylamine Hydrochloride
(7). In a closed tube, 5 (600 mg, 2.2 mmol) was reacted with a 2 M
solution of methylamine in THF (5 mL) in a procedure similar to that for 6
to afford the amine as an oil after the workup of the reaction. The oil was
dissolved in methanol (7 mL) and treated with concentrated HCl (0.7 mL)
and then allowed to stand overnight at 4 °C to afford white crystals of the
hydrochloride salt 7 (yield, 420 mg, 1.9 mmol, 87%): 1H NMR (CDCl3) δ
1.24 (6H, s), 2.58 (3H, s), 2.99 (4H, br m), 5.43 (1H, d, J = 9.6 Hz), 6.09 (1
H, d, J = 10.0 Hz), 6.53 (1H, d, J = 8.0 Hz), 6.72 (1H, br s), 6.82 (1H, br d,
J = 6.8 Hz), 9.35 (1H, br s); 13C NMR (CDCl3) δ 27.8, 31.4, 33.0, 50.7,
76.1, 116.5, 121.4, 121.9, 126.4, 128.2, 129.0, 131.0, 151.8; HRMS(ESI-
TOF) m/z 218.154372 [M þ H]þ (calcd for C14H20ON, 218.154489).
Compounds 8, 9, and 10 were synthesized according to the published
methods and characterized by spectroscopic data (5).
Chromene Amide N-[2-(2,2-Dimethyl-2H-chromen-6-yl)ethyl]-3,
N-dimethylbutyramide (1). The pale yellow oily mass in fraction 23 was
further purified by silica column chromatography with 10% EtOAc in
hexane to yield an oil. The structure was confirmed as 1 (Figure 1), a
chromene amide by comparison of 1H and 13C spectroscopic data with
those reported in the literature (3). The chromene amide was also tested for
algicidal and phytotoxic activity in addition to fungicidal activity.
Syntheses of Analogues. [4-(1,1-Dimethyl-prop-2-ynyloxy)phenyl]-
acetic Acid Methyl Ester (2). Methyl-4-hydroxyphenylacetate (16.6 g,
100 mmol) was heated under reflux with KI (24.4 g, 140 mmol), anhydrous
K2CO3 (24.4 g, 170 mmol), and 3-chloro-3-methyl-1-butyne (25 g, 240 mmol)
in dry acetone (122 mL) under N2 for 48 h. The mixture was allowed to cool
to room temperature and filtered, and the residue was washed with acetone.
The combined acetone solution was evaporated to afford a gum, which was
dissolved in diethyl ether (200 mL) and partitioned between 1 M aqueous
NaOH (300 mL ꢀ 2). The ether layer was dried over anhydrous MgSO4 and
evaporated to afford a pale yellow oil (yield, 11.5 g, 49 mmol, 49.5%): 1H
NMR (CDCl3) δ 1.65 (6H, s), 2.58 (1H, s), 3.58 (2H, s), 3.69 (3 H, s), 7.17
(2H, d, J = 8.4 Hz), 7.20 (2H, d, J = 8.4 Hz); 13C NMR (CDCl3) δ 29.6,
40.4, 51.9, 72.3, 72.9, 86.1, 121.4, 128.3, 129.7, 154.7, 172.1; HRMS(ESI-
TOF) m/z 233.11889 [M þ H]þ (calcd for C14H17O3, 233.11777).
(2,2-Dimethyl-2H-chromen-6-yl)acetic Acid Methyl Ester (3). 2 (11 g,
47 mmol) and N,N-diethylaniline (40 mL) were refluxed under N2 with
stirring (210-220 °C) for 75 min. The reaction mixture was allowed to cool
to room temperature and diluted with diethyl ether (300 mL). The ether
solution was washed with 6 M aqueous HCl (300 mL ꢀ 2), followed by
saturated aqueous NaCl, and dried over anhydrous Na2SO4. Ether was
removed under reduced pressure to afford a pale yellow oil, which was
purified by silica gel column chromatography to afford 3 (yield, 10.5 g, 45
mmol, 95.7%): 1H NMR (CDCl3) δ 1.41 (6H, s), 3.50 (2 H, s), 3.68 (3H, s),
5.59 (1H, d, J = 10.0 Hz), 6.28 (1H, d, J = 9.6 Hz), 6.73 (1H, d, J = 8.4
Hz), 6.88 (1H), 6.99 (1H, dd, J = 8.0, 1.6 Hz); HRMS(ESI-TOF) m/z
233.1175 [M þ H]þ (calcd for C14H17O3, 233.1178).
6-Bromomethyl-2,2-dimethyl-2H-chromene (11). 10 (1.89 g, 9.9 mmol)
was reacted in a similar procedure as for the synthesis of 5 with CBr4 (12.1
g, 36 mmol) and Ph3P (3.8 g, 14 mmol) in CH2Cl2 (15 mL). Ether (30 mL)
was added to the mixture and filtered. The filtrate was evaporated, and
the product was purified by silica gel column chromatography eluting
with 8% EtOAc in hexane to afford 11 (1.1 g, 4.3 mmol, 44%) as a pale
yellow oil, which decomposed readily upon exposure to air. 1H NMR
(CDCl3) δ 1.39 (6H, s), 3.74 (2H, s), 5.55 (1H, d, J=10.0 Hz), 6.23 (1H, d,
J = 10.0 Hz), 6.66 (1H, d, J = 8.4 Hz), 6.74 (1H, br s), 6.88 (1H, d,
J = 8.4 Hz); HRMS(ESI-TOF) m/z 252.01489 [M þ H]þ (calcd for
C12H14OBr, 252.014976).
(2,2-Dimethyl-2H-chromen-6-ylmethyl)isopropylamine (12). 11 (600 mg,
2.3 mmol) was treated with isopropylamine (5 mL) in a closed con-
tainer and heated at 80 °C for 1 h. Excess amine was evaporated with
N2, and the solid was partitioned between aqueous 5% NaOH and EtOAc.
The EtOAc layer was washed with H2O and saturated NaCl, dried over
anhydrous Na2SO4, and evaporated to obtain a gum as the free amine: 1H
NMR (CDCl3) δ 1.08 (6H, d, J=6.0 Hz), 1.40 (6H, s), 2.84 (1H, septet,
J=6.4 Hz), 3.65 (2H, s), 5.58 (1H, d, J=10.4 Hz), 6.29 (1 H, d, J=
10.0 Hz), 6.70 (1H, d, J=8.0 Hz), 6.92 (1H, d, J=1.2 Hz), 7.02 (1H, br d,
J=8.0 Hz).
The free amine was treated with concentrated HCl (1 mL), diluted with
ether (15 mL), and left for 12 h at 4 °C. The solid was filtered and dried to
yield 12 (420 mg, 1.5 mmol, 68%) as white needle-like crystals: HRMS-
(ESI-TOF) m/z 232.1702 [M þ H]þ (calcd for C15H22ON, 232.1701).
Compounds 13 and 14 were synthesized according to the published
methods and characterized by spectroscopic data (5).
2-(2,2-Dimethyl-2H-chromen-6-yl)ethanol (4). 3 (10 g, 43 mmol) and
LiAlH4 (3.8 g, 100 mmol) indry diethyl ether (100 mL) wererefluxed under
N2 for 30 min. Excess LiAlH4 was destroyed by adding ethyl acetate (20 mL).
The reaction mixture was cooled in ice, and 20% NaOH (5 mL) was added
with stirring. The ether layer was decanted, the residue was washed with
diethyl ether (2 ꢀ 100 mL), and the combined ether layer was dried over
anhydrous MgSO4 and then evaporated under reduced pressure to afford a
colorless oil (yield, 8.5 g, 0.04 mol, 93%). The identity of the compound was
established by comparison of 1H and 13C NMR with those reported in the
literature (4).
3-(1,1-Dimethylprop-2-ynyloxy)benzoic Acid Methyl Ester (15). Meth-
yl 3-hydroxybenzoate (3.5 g, 25 mmol) was heated under reflux with KI
(6.0 g), anhydrous K2CO3 (6.0 g), and 3-chloro-3-methyl-1-butyne (6.8
mL, 60.5 mmol) in dry acetone (300 mL) under N2 for 48 h. The mixture
was allowed to cool to room temperature and filtered, the residue was
washed with acetone, and the solvent was evaporated to yield a semisolid.
This was partitioned between 5% NaOH (100 mL) and ether (100 mL ꢀ
2). The ether layer was dried with anhydrous Na2SO4 and evaporated to
dryness to yield 15 as pale yellow oil, which crystallized to yield needle-like
crystals (3.8 g, 14 mmol, 40%): 1H NMR (CDCl3) δ 1.64 (6H, s), 2.59
(1H, s), 3.88 (3H, s), 7.30-7.41 (2H, m), 7.71 (1H, d, J=7.6 Hz), 7.86
(1H, s); 13C NMR (CDCl3) δ 29.7, 52.3, 73.0, 74.7, 122.6, 124.2, 129.1,
131.3, 155.8, 167.0; HRMS(ESI-TOF) m/z 219.10223 [MþH]þ (calcd for
C13H15O3, 219.10212).
6-(2-Bromoethyl)-2,2-dimethyl-2H-chromene (5). To a magnetically
stirred solution of 4 (2.04 g, 10 mmol) and CBr4 (4.15 g, 10 mmol) in
CH2Cl2 (15 mL) at 0 °C was added Ph3P (3.93 g, 15 mmol), and the mixture
was stirred for 30 min. Diethyl ether (25 mL) was added to the mixture, and
the resulting precipitate was removed by filtration. The filtrate was evapo-
rated, and the product, 5, was purified by silica gel chromatography eluting
with 8% ethyl acetate in hexane (yield, 2.1 g, 7 mmol, 78%): 1H NMR
(CDCl3) δ 1.42 (6H, s), 3.05 (2 H, t, J=7.6 Hz), 3.55 (2H, t, J=7.6 Hz),
5.61 (1H, d, J=9.6 Hz), 6.28 (1H, d, J=10 Hz), 6.72 (1H, d, J=8.0 Hz),
6.81 (1H, s), 6.96(1H, d, J=8.0 Hz); HRMS(ESI-TOF) m/z 267.03848
[M þ H]þ (calcd for C13H16 BrO, 267.038451).

9478 J. Agric. Food Chem., Vol. 58, No. 17, 2010
Meepagala et al.
2,2-Dimethyl-2H-chromene-5-carboxylic Acid Methyl Ester (16) and
2,2-Dimethyl-2H-chromene-7-carboxylic Acid Methyl Ester (17). 15 (3.2 g,
14.6 mmol) was refluxed with N,N-diethylaniline (15 mL). After 1 h,
the reaction was allowed to cool to room temperature, diluted with
ether (75 mL), washed with 6 M HCl (4 ꢀ 30 mL) and saturated NaCl,
and dried over anhydrous Na2SO4. The solvent was removed, and the
products were separated by silica gel column chromatography using
90% hexane in toluene to yield 16 as the major product (1.2 g, 5.5
mmol, 37%) and elution with 80% hexane in toluene to yield 17 as the
minor product (800 mg, 3.6 mmol, 24%). 16: 1H NMR (CDCl3) δ 1.41
(6H, s), 3.86 (3 H, s), 5.73 (1H, d, J = 10.0 Hz), 6.94 (1H, d, J = 8.0
Hz), 6.10 (1H, t, J = 8.0 Hz), 7.24 (1H, d, J = 10.0 Hz), 7.44 (1H, d,
J = 7.6 Hz); HRMS(ESI-TOF) m/z 219.1037 [M þ H]þ (calcd for
C13H15O3, 219.10213).
151.6, 173.4; HRMS(ESI-TOF) m/z 269.11113 [M þ Na]þ (calcd for
C15H18NaO3, 269.11536).
3-(2,2-Dimethyl-2H-chromen-6-yl)propan-1-ol (23). 22 (2.8 g, 11 mmol)
was refluxed with LiAlH4 (600 mg, 16 mmol) in dry diethyl ether (100 mL)
for 3 h. Excess LiAlH4 was destroyed with EtOAc, and the reaction
mixture was treated with concentrated NaOH (5 mL) and stirred for
30 min. The ether layer was decanted, and the residue was washed with
ether. The combined ether layer was dried over MgSO4 and evaporated to
1
yield 23 (2.3 g, 10 mmol, 90%) as a pale yellow oil: H NMR (CDCl3)
δ 1.41 (6H, s), 1.84 (2H, multiplet) 2.59 (2H, t, J=8 Hz), 3.64 (2H, t, J=
6 Hz), 5.58 (1H, d, J=9.6 Hz), 6.27 (1H, d, J=9.6 Hz), 6.70 (1H, d, J=
8.0 Hz), 6.80 (1H, d, J=1.6 Hz), 6.93 (1H dd, J=6.9, 1.6 Hz).
[4-(1,1-Dimethylprop-2-ynyloxy)-3-methoxyphenyl]acetic Acid Ethyl
Ester (24). Ethyl 4-hydroxy-3-methoxyphenylacetate (2 g, 9.52 mmol)
was heated under reflux with KI (2.4 g), anhydrous K2CO3 (2.4 g), and
3-chloro-3-methyl-1-butyne (2.6 mL, 24 mmol) in dry acetone (12 mL) under
N2 for 48 h. The mixture was allowed to cool to room temperature and
filtered, and the residue was washed with acetone. The combined acetone
solution was evaporated to afford a gum, which was dissolved in diethyl ether
(100 mL) and partitioned between 1 M aqueous NaOH (100 mL ꢀ 2). The
ether layer was dried over anhydrous MgSO4 and evaporated to afford 24 as
a pale yellow solid (1.15 g, 4.1 mmol, 43%): 1H NMR (CDCl3) δ 1.21 (3H, t,
J = 7.2 Hz) 1.60 (6H, s), 2.49 (1H, s), 3.51 (2H, s), 3.75 (3H, s), 4.10 (2H, q,
J = 7.2 Hz), 6.74 (1H, d, J = 8.8 Hz), 6.80 (1H, s), 7.30 (1H, d, J = 8.8 Hz);
13C NMR (CDCl3) δ 14.4, 29.5, 41.3, 55.9, 61.0, 73.6, 74.1, 86.6, 113.4, 121.3,
130.1, 143.9, 153.0, 171.8; HRMS(ESI-TOF) m/z 277.14184, [M þ H]þ
(calcd for C16H21O4, 277.14398).
17: 1H NMR (CDCl3) δ 1.41 (6H, s), 3.85 (3 H, s), 5.70 (1H, d, J = 10.0
Hz), 6.32 (1H, d, J = 10.0 Hz), 6.99 (1H, t, J = 7.6 Hz), 7.42 (1H, s), 7.50
(1H, d, J = 7.6 Hz); HRMS(ESI-TOF) m/z 219.1032 [M þ H]þ (calcd for
C13H15O3, 219.10213).
(2,2-Dimethyl-2H-chromen-5-yl)methanol (18). 16 (300 mg, 1.3 mmol)
was dissolved in dry ether (50 mL) and refluxed with LiAlH4 (80 mg, 2.1
mmol) for 1 h. Excess LiAH4 was destroyed with EtOAc, treated with NaOH
(20%, 1 mL), and stirred, and the organic layer was decanted. The residue
was washed with ether (50 mL), and the combined ether layer was dried over
anhydrous Na2SO4 and evaporated. The product was purified by silica gel
column chromatography eluting with 10% EtOAc in hexane to yield 18 (220
mg, 1.1 mmol) as a colorless, viscous oil: 1H NMR (CDCl3) δ 1.43 (6H, s),
3.20 (1H, br s), 4.58 (2H, s), 5.66 (1H, d, J = 10.0 Hz), 6.56 (1H, d, J = 10
Hz), 6.75 (1H, d, J = 8.0 Hz), 6.83 (1H, d, J = 7.2 Hz), 7.06 (1H, t, J = 8.0
Hz); 13C NMR (CDCl3) δ 27.8, 62.3, 75.5, 116.4, 118.75, 119.3, 120.7, 128.7,
131.2, 136.1, 153.0.
(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)acetic Acid Ethyl Ester
(25). 24 (1 g, 3.6 mmol) was refluxed with N,N-diethylaniline (5 mL)
under N2 for 90 min. The reaction was cooled to room temperature,
acidified with aqueous HCl, and extracted with EtOAc (100 mL ꢀ 2). The
EtOAc layer was washed with water, dried over anhydrous MgSO4, and
evaporated. The product was purified by silica gel column chromatogra-
phy using 5% EtOAc in hexane to yield 25 (730 mg, 2.6 mmol, 73%): 1H
NMR (CDCl3) δ 1.25 (3H, t, J = 7.2 Hz), 1.45 (6H, s), 3.48 (2H, s), 3.84
(3H, s), 4.14 (2H, q, J = 7.2 Hz), 5.58 (1H, d, J = 9.6 Hz), 6.26 (1H, d, J =
9.6 Hz), 6.33 (1H, s), 6.69 (1H, s); HRMS(ESI-TOF) m/z 277.14384, [M þ
H]þ (calcd for C16H21O4, 277.14398).
(2,2-Dimethyl-2H-chromen-7-yl)methanol (19). 17 (250 mg, 1.1 mmol)
was reacted with LiAlH4 (80 mg, 2 mmol) in a similar manner as above to
yield 19 (180 mg, 0.94 mmol) as a colorless, viscous oil: 1H NMR (CDCl3)
δ 1.41 (6H, s), 3.0 (1H, br s), 4.50 (2H, s), 5.67 (1H, d, J = 10.0 Hz), 6.29
(1H, d, J = 10 Hz), 6.75 (1H, s), 6.78 (1H, d, J = 8.4 Hz), 6.91 (1H, t, J =
7.6 Hz); 13C NMR (CDCl3) δ 27.9, 64.7, 76.2, 114.8, 119.2, 120.4, 120.0,
126.3, 130.5, 142.3, 152.9.
3-(4-Hydroxyphenyl)propionic Acid Methyl Ester (20). A solution of
4-hydroxybenzenepropanoic acid (5 g, 30 mmol) in dry MeOH (25 mL) was
treated with concentrated H2SO4 (0.25 mL) and refluxed for 24 h. The sol-
vent was removed under vacuum, and the oil was partitioned with EtOAc
and aqueous NaHCO3. The organic layer was washed with water and satu-
rated NaCl, dried over MgSO4, and evaporated to yield 20 (4.2 g, 23 mmol,
92%) as a white solid: 1H NMR (CDCl3) δ 2.61 (2H, t, J=7.6 Hz), 2.87 (2H,
t, J = 8 Hz), 3.67 (3H, s), 6.71 (1H, s), 6.76 (2H, d, J=7.6 Hz), 7.02 (2H, d,
J = 8 Hz).
2-(8-Methoxy-2,2-dimethyl-2H-chromen-6-yl)ethanol (26). 25 (700 mg,
2.7 mmol) was refluxed with LiAlH4 (200 mg, 5 mmol) in dry ether (10 mL)
for 1 h. Excess LiAlH4 was destroyed with EtOAc, and the reaction
mixture was treated with concentrated NaOH (2 mL) and stirred for 30
min. The ether layer was decanted, and the residue was washed with ether.
The combined ether layer was dried over MgSO4 and evaporated to yield
26 (590 mg, 2.5 mmol, 93%) as a pale yellow oil: 1H NMR (CDCl3) δ 1.32
(6H, s), 2.62 (2H, t, J = 6.4 Hz), 3.64 (2H, t, J = 6.4 Hz), 3.70 (3H, s), 5.48
(1H, d, J = 8.4 Hz), 6.14 (1H, d, J = 8.4 Hz), 6.37 (1H, s), 6.52 (1H, s); 13C
NMR (CDCl3) δ 27.7, 38.7, 56.3, 63.4, 76.5, 113.4, 119.3, 122.0, 122.4,
130.9, 131.2, 140.4, 148.0.
3-[4-(1,1-Dimethylprop-2-ynyloxy)phenyl]propionic Acid Methyl Ester
(21). 20 (4.1 g, 22.8 mmol) was heated under reflux with KI (6.0 g),
anhydrous K2CO3 (6.0 g), and 3-chloro-3-methyl-1-butyne (6.8 mL, 60.5
mmol) in dry acetone (30 mL) under N2 for 48 h. The reaction mixture was
filtered, and the solvent was evaporated to yield a gum. The gum was
dissolved in ether (100 mL) and washed with 1 M NaOH (30 mL). The ether
layer was dried over anhydrous MgSO4 and evaporated to yield 21 (4.2 g, 17
mmol) as a pale yellow oil, which soon crystallized upon cooling to needle-
like crystals: 1H NMR (CDCl3) δ 1.59 (6H, s), 2.54 (1H, s), 2.58 (2H, t, J =
7.6 Hz), 2.87 (2H, t, J = 7.6 Hz), 3.61 (3H, s), 7.05-7.11 (4H, m); 13C NMR
(CDCl3) δ 29.8, 30.4, 36.0, 51.7, 72.5, 74.12, 86.4, 121.8, 128.9, 135.2, 154.1,
173.5; HRMS(ESI-TOF) m/z 247.133531, [M þ H]þ (calcd for C15H19O3,
247.13342).
6-(2-Bromoethyl)-8-methoxy-2,2-dimethyl-2H-chromene (27). 26 (480
mg, 2.1 mM) was treated with CBr4 (850 mg, 2.56 mM) and Ph3P (870 mg,
3.1 mM) in dry CH2Cl2 (8 mL) at 0 °C under N2. After 30 min, solvent was
evaporated, and the residue was applied to a silica gel column and eluted
with 2.5% EtOAc in hexane to yield 27 (480 mg, 1.6 mmol, 77%) as a
yellow oil: 1H NMR (CDCl3) δ 1.48 (6H, s), 3.07 (2H, t, J = 7.6 Hz), 3.54
(2 H, t, J = 7.6 Hz), 3.80 (3H, s), 5.63 (1H, d, J = 10.0 Hz), 6.29 (1H, d,
J=9.6 Hz), 6.49 (1H, d, J=1.6 Hz), 6.63 (1H, d, J=1.6 Hz); HRMS(ESI-
TOF) m/z 297.04516, [M þ H]þ (calcd for C14H18BrO2, 297.04902).
Isopropyl[2-(8-methoxy-2,2-dimethyl-2H-chromen-6-yl)ethyl]amine
Hydrochloride (28). 27 (308 mg, 1.04 mmol) was refluxed with isopropy-
lamine (2 mL) in CH3CN (8 mL) for 3 h. The solvent was evaporated, and
the crystalline solid was partitioned between dilute HCl and ether. The acidic
layer was basified with NaOH and extracted with ether (50 mL ꢀ 2). The or-
ganic layer was washed with water, dried over NaSO4, and evaporated to
yield the free amine (250 mg) as a gum. This gum was dissolved in ethanol
(10 mL) and treated with concentrated HCl (0.5 mL). The solvent was eva-
porated under vacuum, and the crystalline product was recrystallized from
ethanol and ether to give 28 as a white crystalline solid (220 mg, 70 mmol,
69%): 1H NMR (CDCl3) δ 1.44 (6H, s), 1.50 (6H, d, J = 6.4 Hz), 3.10 (2H,
br m), 3.26 (2H, br m), 3.49 (1H, septet, J=5.2 Hz), 5.60 (1H, d, J=10.0 Hz),
3-(2,2-Dimethyl-2H-chromen-6-yl)propionic Acid Methyl Ester (22).
21 (4.0 g, 16.3 mmol) was refluxed with N,N-diethylaniline (20 mL) under
N2 for 90 min. The reaction mixture was cooled, acidified with 5% HCl,
and extracted with EtOAc. The organic layer was washed with water, dried
over anhydrous MgSO4, and evaporated to yield a pale yellow oil. The
product was purified by silica gel column chromatography using 5%
EtOAc in hexane to afford (22) (3.3 g, 13.4 mmol): 1H NMR (CDCl3) δ
1.40 (6H, s), 2.57 (2H, t, J = 7.6 Hz), 2.83 (2H, t, J = 7.6 Hz), 3.65 (3H, s),
5.58 (1H, d, J = 9.6 Hz), 6.27 (1H, d, J = 9.6 Hz), 6.68 (1H, d, J = 8.0 Hz),
6.79 (1H, d, J = 1.6 Hz), 6.91 (1H dd, J = 6.9, 1.6 Hz); 13C NMR (CDCl3)
δ 28.1, 30.3, 36.1, 51.7, 76.2, 116.4, 121.4, 122.5, 126.3, 129.0, 131.0, 132.8,

Article
J. Agric. Food Chem., Vol. 58, No. 17, 2010 9479
6.21 (1H, d, J=9.6 Hz), 6.51 (1H, s), 6.64 (1H, s), 9.67 (1H br s); HRMS-
(ESI-TOF) m/z 276.20056, [M þ H]þ (calcd for C17H26O2N, 276.19635).
6-(3-Bromopropyl)-2,2-dimethyl-2H-chromene (29). Compound 23
(1.2 g, 5.5 mmol) was treated with CBr4 (2.28 g, 6.88 mM) and PPh3
(2.17 g, 8.26 mM) in dry CH2Cl2 (10 mL) at 0 °C under N2. After 30 min,
solvent was evaporated, and the residue was applied to a silica gel column
and eluted with 2.5% EtOAc in hexane to yield 29 as a yellow oil (1.1 g, 3.9
mmol, 70%): 1H NMR (CDCl3) δ 1.43 (6H, s), 2.14 (2H, quintet, J = 6.8
Hz), 2.69 (2H, t, J = 7.2 Hz), 3.40 (2H, t, J = 6.4 Hz), 5.63 (1H, d, J = 9.6
Hz), 6.31 (1H, d, J = 10 Hz), 6.72 (1H, d, J = 8 Hz) 6.82 (1H, d, J = 1.6
Hz), 6.95 (1H, dd, J = 6.4, 1.6 Hz); 13C NMR (CDCl3) δ 28.0, 33.1, 33.2,
34.3, 76.1, 116.2, 121.2, 122.3, 126.3, 129.0, 131.0, 132.6, 151.3; HRMS-
(ESI-TOF) m/z 281.05452 [M þ H]þ (calcd for C14H18OBr, 281.05410).
[3-(2,2-Dimethyl-2H-chromen-6-yl)propyl]isopropylamine Hydrochlo-
ride (30). 29 (500 mg, 1.7 mmol) was refluxed with isopropylamine
(2 mL, 23 mmol) and CH3CN (8 mL) for 3 h. Excess amine was evaporated
under reduced pressure, and the residue was partitioned between aqueous
5% NaOH and EtOAc. The EtOAc layer was washed with H2O and
saturated NaCl, dried over anhydrous Na2SO4, and evaporated to obtain
a gum as the free amine. The free amine was isolated by silica gel column
chromatography using 30% EtOAc in hexane as a viscous oil (270 mg,
61%): 1H NMR (CDCl3) δ 1.04 (6H, d, J = 6 Hz), 1.42 (6H, s), 1.76 (2H,
quintet, J = 7.2 Hz), 2.55 (2H, t, J = 8.0 Hz), 2.61 (2H, t, J = 7.6 Hz),
2.74-2.80 (1H, m) 5.58 (1H, d, J = 10.0 Hz), 6.28 (1H, d, J = 10.0 Hz),
6.69 (1H, d, J = 8.4 Hz), 6.79 (1H, d, J = 1.6 Hz), 6.91 (1H, dd, J = 8.0,
1.6 Hz); 13C NMR (CDCl3) δ 23.1, 27.9, 32.1, 32.9, 74.1, 48.7, 75.9, 116.0,
121.0, 122.4, 126.1, 128.8, 130.7, 134.3, 150.9; HRMS(ESI-TOF) m/z
260.201426 [M þ H]þ (calcd for C17H25NO, 260.20143). The free amine
(250 mg) was dissolved in ethanol (2 mL), treated with concentrated HCl
(1 mL), diluted with diethyl ether (5 mL), and kept for 12 h at 4 °C. The
hydrochloride salt of the amine 30 was crystallized as white needle-like
crystals (220 mg).
published methods (8). Phytotoxic activities were ranked on a scale from 1
to 5 visually, with 1 representing no inhibition and 5 representing complete
inhibition of germination. The compounds were further evaluated for
activity on plant cell integrity by determination of electrolyte leakage from
cucumber (Cucumis sativus) cotyledon disks according to a published
method (9).
RESULTS AND DISCUSSION
Preliminary bioautography of the ethyl acetate extract of the
leave of A. texana indicated the presence of antifungal com-
pounds against C. fragariae. Bioassay-guided fractionation of the
extract led to the isolation of the chromene amide 1 (Figure 1) as
the major antifungal constituent. Algicidal assay of this com-
pound showed selective activity toward P. perornata, with an
LCIC of 10.0 μM and an IC50 of 2.3 μM compared to those for S.
capricornutum with an LCIC of 100.0 μM and an IC50 of 12.6 μM.
However, the low water solubility and low activity of 1 preclude
further research including efficacy studies as a useful agent to
control P. perornata in catfish production ponds. To identify the
structural featuresresponsible for the algicidal activity ofthis type
of compound and also to develop water-soluble analogues, a
series of chromene derivatives based on the natural chromene
amide were synthesized and evaluated (Figure 2).
Acid hydrolysis of the parent amide is expected to yield an
amine, which could then be converted to an acid salt to yield a
water-soluble analogue. Due to limited availability of the parent
compound, a synthetic procedure was developed to synthesize
these analogues. The general synthetic procedure is shown in
Figure 3. Thus, the corresponding methyl or ethyl esters of
phenolic acids were reacted with 3-chloro-3-methyl-1-butyne to
obtain the propynyloxy ether, which was subsequently cyclized to
obtain the corresponding chromene derivative (Figure 3). The
reduction of ester yielded the corresponding alcohol, which was
converted to its bromide. Treatment of the bromide with amines
yielded chromene amines, which were crystallized as hydrochlor-
ide salts.
The treatment of bromide 5 with methylamine yielded the amine
analogue 7. The hydrochloride salt of this amine gave a crystalline
water-soluble compound. This compound showed improved activ-
ity toward P. perornata (LOEC=1.0 μM) but showed reduced selec-
tivity based upon LCIC values when compared to S. capricornutum
(Table 1). The chromene analogues 3 and 4 did not show toxicity
toward P. perornata. These results indicated that the amide in
the side chain is not a prerequisite for algicidal activity of the
chromenes, but the amine group is important for activity. A
previous study with anthraquinone analogues showed that the
amine groups on the side chain can have a great influence on
the selective algicidal activity of the analogues (10). In this study,
the isopropylamine analogue yielded the optimum activity and
selectivity toward P. perornata. Treatment of bromide 5 with
isopropylamine followed by treatment with HCl yielded the
corresponding amine analogue 6. The hydrochloride salt 6
showed enhanced selectivity toward P. perornata compared to
S. capricornutum, but no improvement on the activity compared
to the parent amide 1 (Table 1). The primary amine analogue 31
of this compound was prepared by treatment of bromide 5 with
sodium azide and subsequent reduction with H2 and Pd/C. This
amine showed improved toxicity toward both P. perornata and S.
capricornutum, but with reduced selectivity.
2-(2,2-Dimethyl-2H-chromen-6-yl)ethylamine (31). 5 (3 g, 11.24 mM)
was reacted with NaN3 (0.952 g, 14 mmol) in anhydrous DMF (40 mL)
under N2 at room temperature for 3 h. The reaction mixture was added to
water (100 mL), extracted with diethyl ether (250 mL), and evaporated to
dryness to obtain the azide. This azide was dissolved in absolute ethanol
(20 mL) and hydrogenated with Pd/C (10%) at 10 psi for 4 h. The mixture
was filtered through Celite, and the solvent was evaporated to give a
viscous oil. 31 was obtained as a viscous oil after silica gel column
chromatography using 30% EtOAc in hexane as the solvent (1.9 g,
83%): 1H NMR (CDCl3) δ 1.42 (6H, s), 1.78 (2H, quintet, J = 7.6 Hz),
2.64 (2H, t, J = 7.6 Hz), 2.71 (2H, t, J = 7.6 Hz), 5.63 (1H, d, J = 10.0
Hz), 6.31 (1H, d, J = 10.0 Hz), 6.71 (1H, d, J = 8.4 Hz), 6.80 (1H, d, J =
1.6 Hz), 6.93 (1H, dd, J = 8.0, 1.6 Hz).
The free amine was dissolved in ethanol (10 mL) and treated with
concentrated HCl (1 mL), diluted with ether (25mL), and left at 4 °C for 48
h to yield the hydrochloride salt as a white powder.
Bioassay for Evaluating Algicidal Activity. Algicidal activities of
the compounds were tested in 96-well microplates in a dose-response
format according to the previously published methods (2). The lowest
observed effect concentration (LOEC), the lowest complete inhibition
concentration (LCIC), and the 50% inhibition concentration (IC50) were
determined by graphing the absorbance data. Whereas previous studies
have used the traditional approach designation of Oscillatoria perornata f.
attenuata [Skuja], we used the modern approach designation of P.
perornata f. attenuata [Skuja] in the current study.
Bioassay for Fungicidal Activity against Plant Pathogenic Fungi.
Bioautography on silica gel TLC plates was used to detect the presence of
antifungal constituents in the extracts, column fractions, and purified
compounds and synthesized analogues according to published methods (6).
To evaluate the quantitative fungicidal activity, the active compounds
identified by bioautography were evaluated in a dose-response format in
the 96-well microbioassay using modifications of published methods (6, 7).
Chromene derivatives were evaluated at three concentrations (1.0, 10.0, and
100.0 μM) and compared to the commercial fungicide captan for activity
against Colletotrichum fragariae, Colletotrichum gloeosporioides, Colletotri-
chum acutatum, Botrytis cinerea, and Fusarium oxysporium.
To study the influence of the length of the side chain on the
activity, isopropylamine analogues of chromenes with one-car-
bon (12) and three-carbon (30) side chains were prepared.
Hydrochloride salts of compounds 30 and 12 showed equal or
less activity, respectively, compared to the two-carbon chain
analogue 6, and both compounds lacked selectivity.
Bioassay for Phytotoxic Activity. The compounds were evaluated
for phytotoxicity on a monocot and a dicot using lettuce (Lactuca sativa
cv. Iceberg) and bent grass (Agrostis stolonifera cv. Pencross) according to

9480 J. Agric. Food Chem., Vol. 58, No. 17, 2010
Meepagala et al.
Figure 2. Structures of chromene derivatives.
Figure 3. General synthetic procedure for water-soluble chromene derivatives.
To investigate the influence of functional groups on the chro-
mene ring on algal toxicity, the methoxy analogue 28 was prepared
and tested for algicidal activity. The hydrochloride salt of this
amine had lower activity compared to that of the demethoxy
analogue 6 and lacked selectivity. The halogenated chromene ring
analogues were not prepared as the intention of this work was to
find compounds very close to natural products in nature. To
investigate the influence of the position of the side chain on the
chromene ring on algicidal activity, analogues 32 and 33 were
prepared and assayed. Cyclization of propynyloxy ether 15 yielded
a mixture of chromene analogues, which were separated by
column chromatography and converted to the corresponding
isopropylamine analogues 32 and 33. Algicide bioassay results of
these compounds indicate that toxicity is enhanced when the side
chain is at the 7-position compared to when the side chain is at the
5-position; however, selective toxicity is reduced (Table 1).

Article
J. Agric. Food Chem., Vol. 58, No. 17, 2010 9481
Figure 4. Fungal growth inhibition of Botrytis cinerea at 72 h against chromene derivatives. Captan was used as the positive control.
Table 1. Algicide Activity of Chromene Derivatives
test organism
fungicidal activity on TLC bioautography. Therefore, the structural
requirements for algicidal and fungicidal activities should be differ-
ent. The analogues that showed significant antifungal activity were
further evaluated for fungicidal activity by microbioassay in a dose-
response manner according to the published methods (6, 7, 11).
Captan was used as the positive standard, and the fungal strains
used were C. fragariae, C. acutatum, C. gloeosporioides, B. cinerea,
and F. oxysporum. The data for B. cinerea are shown in Figure 4.
According to the microbioassay, B. cinerea was the most sensitive to
the chromenes among the fungal strains that we tested. The natural
chromene amide was the most active against B. cinerea, with 80%
inhibition at 100.0 μM. Among the synthetic chromene analogues,
17, 23, 4, 19, 9, and 16 at 100.0 μM showed >50% growth inhibition
of B. cinerea at 72 h after treatment. Both 3, an ester, and 10, an
alcohol with one CH2 group in the side chain at the 6-position of the
aromatic ring, showed about 30% growth inhibition at 72 h after
treatment on the same fungal strain. Compound 4, having a chain
with two CH2 groups at C-7, showed higher activity than 23 with
three CH2 groups at C-7. Between the isomers 18 and 19, where the
only difference is the position of the CH2OH moiety, the chromene
analogue 19 was more active. Therefore, the side chain at the
7-position of the chromene ring shows better antifungal activity.
None of our chromene analogues showed better or comparable
activity to that of captan, a widely used commercial fungicide. The
synthetic chromenes did not show significantly higher activity than
the natural product 1. Thus, a different strategy is needed to develop
more potent fungicides that can compete with the commercial
fungicides that are currently available.
Planktothrix perornata
Selenastrum capricornutum
test compound
LOECa (μM) LCICb (μM) LOEC (μM)
LCIC (μM)
chromene amide 1
3
10
>100
>1000
10
10
>100
>1000
10
10
>100
>1000
10
100
>100
>1000
1000
10
4
6
7
1.0
10
10
12
28
30
31
32
33
10
10
10
10
10
1
100
100
10
10
100
100
10
10
10
10
10
10
100
10
10
10
1
1
a LOEC, lowest observed effect concentration. b LCIC, lowest complete inhibition
concentration.
These results suggest that a two-carbon side chain is necessary
for highest algicidal activity among these analogues. Extending
the carbon chain of 6 by one carbon as in 30 did not improve the
algicidal activity. For the amine group, the isopropylamine
moiety as in 6, 30, and the methylamine moiety as in 7 had the
same level of activity as the natural product 1. The analogue 28
with a methoxy group in the aromatic ring was less active.
Therefore, we can conclude that a two-carbon side chain with
an isopropylamine moiety had the highest selective algicidal
activity among the chromene derivatives that we tested against
P. perornata when compared to results for S. capricornutum.
It has been reported that 4, a compound isolated from Eutypa
lata, which is the fungus responsible for dying-arm disease in
grapevines, and the corresponding aldehyde of 4 are phytotoxic (5).
However, in the bioassays carried out in our laboratory following
published procedures (8), none of our chromene analogues showed
any phytotoxicity at the concentrations that we tested (0.1-1000
μM). We have also performed assays on effects on cellular integrity
with cucumber cotyledon disks for 4, 6, and 10 and found no
phytotoxicity (data not shown).
Chromenes occur in many plants belonging to diverse fami-
lies (12-17). These compounds exhibit a diverse array of biological
activities such as antifeedent, fungicidal, phytotoxic, cytotoxic, larvi-
cidal, and phytotoxic properties. Plants in general produce second-
ary metabolites as part of their defense mechanism. Hence, we can
postulate that the naturally occurring chromene amide 1 as a natural
fungicide produced by A. texana acts as a defense compound. Des-
pite the numerous biological activities reported for chromenes, algi-
cidal activity has not previously been reported in the literature. This
study is the first describing the algicidal activity of chromenes.
ACKNOWLEDGMENT
TLC bioautography of the chromene analogues when tested
against C. fragariae indicated that only the analogues that had
CH2OH and ester moieties in the side chain were significantly
antifungal. Interestingly, none of the amine derivatives showed
We express our gratitude to Robert Johnson, for performing
the phytotoxicity assays; to Hope Harris and Alicia Thurber, for
assistance in isolation and syntheses; and to Linda Robertson,

9482 J. Agric. Food Chem., Vol. 58, No. 17, 2010
Meepagala et al.
Ramona Pace, and Dewayne Harries, for performing the fungi-
cidal and algicidal bioassays.
(10) Schrader, K. K.; Nanayakkara, N. P. D.; Tucker, C. S.; Rimando,
A. M.; Ganzera, M.; Schaneberg, B. T. Novel derivatives of 9,10-
anthraquinone are selective algicides against the musty-odor cyano-
bacterium Oscillatoria perornata. Appl. Environ. Microbiol. 2003, 69,
5319–5327.
LITERATURE CITED
(1) Wedge, D.; Nagle, D. G. A new 2D-TLC bioautography method for
the discovery of novel antifungal agents to control plant pathogens.
J. Nat. Prod. 2000, 63, 1050–1054.
(2) Schrader, K. K.; de Regt, M. Q.; Tucker, C. S.; Duke, S. O. A rapid
bioassay for selective algicides. Weed Technol. 1997, 11, 767–774.
(3) De la Fuente, G.; Reina, M.; Timon, I. Chromene amides from
Amyris texana. Phytochemistry 1991, 30, 2677–284.
(4) Wattanapiromsakul, C.; Waterman, P. G. Flavanone, triterpene and
chromene derivatives from the stems of Paramignya griffithii.
Phytochemistry 2000, 55, 269–273.
(11) Wedge, D. E.; Galindo, J. C. G.; Macias, F. A. Fungicidal activity of
natural and synthetic sesquiterpene lactone analogs. Phytochemistry
2000, 53, 747–757.
(12) Morandim, A. A.; Bergamo, D. C. B.; Kato, M. J.; Cavalheiro, A. J.;
Bolzani, V. da S.; Furlan, M. Circadian rhythm of anti-fungal
prenylated chromene in leaves of Piper aduncum. Phytochem. Anal.
2005, 16, 282–286.
(13) Chen, J. J.; Duh, C. Y.; Chen, I.-S. Cytotoxic chromenes from
Myriactis humilis. Planta Med. 2005, 71, 370–372.
(14) Iqbal, M. C. M.; Jayasinghe, U. L. B.; Herath, H. M. T. B.;
Wijesekara, K. B.; Fujimoto, Y. A fungistatic chromene from
Ageratum conyzoides. Phytoparasitica 2004, 32, 119–126.
(15) Amelot, M. E. A.; Avendano, M.; Aubert, L.; Avila, J. L. Repellency
and feeding deterrence activity of Ageratum conyzoides against the
stored grain pests Tribolium castaneum and Sitophilus oryzae. Active
plant parts and composition. Ciencia 2003, 11, 61–76.
(16) Menut, C.; Bessiere, J. M.; Ntalani, H.; Verin, P.; Henriques, A. T.;
Limberger, R. Aromatic plants from Brazil. VII. Two chromene
derivatives from Calyptranthes tricona. Phytochemistry 2000, 53,
975–979.
(5) Smith, L. R.; Mahoney, N.; Molyneux, R. J. Synthesis and struc-
ture-phytotoxicity relationships of acetylenic phenols and chro-
mene metabolites, and their analogues, from the grapevine pathogen
Eutypa lata. J. Nat. Prod. 2003, 66, 169–176.
(6) Meepagala, K. M.; Sturtz, G.; Wedge, D. E. Antifungal constituents
of the essential oil fraction of Artemisia dracunculus L. var. dracun-
culus. J. Agric. Food Chem. 2002, 50, 6989–6992.
(7) Oliva, A.; Meepagala, K. M.; Wedge, D. E.; Harries, D.; Hale, A. L.;
Aliotta, G.; Duke, S. O. Natural fungicides from Ruta graveolens L.
leaves, including a new quinolone alkaloid. J. Agric. Food Chem. 2003,
51, 890–896.
(8) Dayan, F. E.; Romagni, J. G.; Duke, S. O. Investigating the mode
of action of natural phytotoxins. J. Chem. Ecol. 2000, 26, 2079–
2094.
(17) Saxena, R. C.; Sharma, M.; Kumar, M. L.; Bansal, S. K.; Shrivastava,
D. Developmental effects of 6,7-dimethoxy-2,2-dimethyl chromene on
the preimaginal stages of Anopheles stephensi. Proc. Acad. Environ.
Biol. 1994, 3, 181–184.
(9) Kenyon, W. H.; Duke, S. O.; Vaughn, K. C. Sequence of effects
of acifluorfen on physiological and ultrastructural parameters
in cucumber cotyledon discs. Pestic. Biochem. Physiol. 1985, 24,
240–250.
Received for review April 28, 2010. Revised manuscript received July 26,
2010. Accepted July 28, 2010.