Synthesis and antifungal activity of β mrifluoroalkyl aminovinyl ketone derivatives

Article abstract of DOI:10.1021/jf902190w

Ten β-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effects on several phytopathogenic fungi, an oomycete and plants were assessed. The various compounds were fungitoxic at the 10-100 μM range, with (Z)-3-amino-4,4,4-trifluoro-1-(4-chlorophenyl)but-2-en1-one exhibiting the highest inhibitory effect on most of the test pathogens. Alternarla alternata and Neurospora crassa were the most tolerant and sensitive fungi to the compounds, respectively. We propose that (Z)-3-amino-4,4,4- trifluoro-1-phenylbut-2-en-1-one is the minimal structural requirement for a β-trifluoroalkyl aminovinyl ketone fungitoxic derivative. 2009 American Chemical Society.

Full text of DOI:10.1021/jf902190w

J. Agric. Food Chem. 2009, 57, 8303–8307 8303
DOI:10.1021/jf902190w
Synthesis and Antifungal Activity of β-Trifluoroalkyl
Aminovinyl Ketone Derivatives
‡
GARY
G
ELLERMAN,*,† NATALI
P
O
ARIENTE,† ZAHI
P
AZ,‡ ANAT
S
HNAIDERMAN
,
AND
,‡
DED
Y
ARDEN
*
†Department of Biological Chemistry, Ariel University Center of Samaria, Ariel 40700, Israel, and
‡Department of Plant Pathology and Microbiology, The Robert H. Smith Faculty of Agriculture, Food
and Environment, The Hebrew University of Jerusalem, Rehovot 76100, Israel
Ten β-trifluoroalkyl aminovinyl ketone derivatives were synthesized, and their inhibitory effects on
several phytopathogenic fungi, an oomycete and plants were assessed. The various compounds
were fungitoxic at the 10-100 μM range, with (Z)-3-amino-4,4,4-trifluoro-1-(4-chlorophenyl)but-2-en-
1-one exhibiting the highest inhibitory effect on most of the test pathogens. Alternaria alternata and
Neurospora crassa were the most tolerant and sensitive fungi to the compounds, respectively. We
propose that (Z)-3-amino-4,4,4-trifluoro-1-phenylbut-2-en-1-one is the minimal structural require-
ment for a β-trifluoroalkyl aminovinyl ketone fungitoxic derivative.
KEYWORDS: Antifungal; β-trifluoroalkyl aminovinyl ketone derivatives; structure-activity relationship;
Alternaria alternata; Penicillium digitatum; Rhizoctonia soliani; Botrytis cinerea; Sclerotinia sclerotior-
um; Neurospora crassa; Pythium aphanidermatum
INTRODUCTION
compounds consists of two simple stages using inexpensive and
commercially available compounds.
In many agricultural practices, fungicides are extensively used
to prevent and curb the progression of plant diseases that have
severe adverse effects on crop yields and quality. In some cases,
the prolonged and repeated use of many fungicides has resulted in
the proliferation of plant pathogen isolates that are resistant to
the fungicides employed. Therefore, identifying new classes of
antifungal compounds based on structures not previously used in
combating plant diseases may assist in the control of plant
pathogens that are tolerant or resistant to known compounds.
Sclerotinia sclerotiorum and Botrytis cinerea are necrotrophic
phytopathogenic filamentous ascomycetes known to attack more
than 400 and 200 plant species, respectively (1-7). Diseases
caused in economically important plants by these fungi occur
worldwide, cause considerable damage, and have proven difficult
to control (culturally or chemically), and host resistance to these
fungi is inadequate. Annual losses of crops from diseases caused
by S. sclerotiorum and B. cinerea are in the multimillion dollar
range (e.g., http://www.whitemoldresearch.com). In addition,
their ubiquitous prevalence along with the intensive use of
fungicides for their control has resulted in repeated appearance
of fungicide-resistant strains (8-11). In order to expand the range
of chemicals that could potentially be harnessed to control these
fungi, we have synthesized a group of (Z)-β-trifluoroalkyl amino-
vinyl ketones and assessed their fungitoxic activity. We have
determined that some of these compounds can curb fungal
growth at the 10-100 μM range. The potential utilization of
these molecules is supported by the fact that synthesis of these
MATERIALS AND METHODS
Analytical HPLC was performed on a 250 ꢀ 4.2 mm Lichro-
prep RP-18 column from Merck, with a 1 mL/min flow and
detection at 214 nm. The eluents were triply distilled water and
HPLC-grade CH3CN (containing 0.1% TFA) or MeOH. Mass
spectra were measured in the positive and negative modes using a
quadrupole mass spectrometer equipped with an electrospray
ionization source and cross-flow inlet. 1H and 13C NMR spectra
were recorded at 300 and 75 MHz, respectively in CDCl3, unless
otherwise indicated. Assignments in the final products were
supported by 2D COSY, HMBC and HMQC spectroscopy. All
chemical shifts are reported with respect to TMS. Chromatogra-
phy was carried out by standard flash chromatography and TLC
on silica gel (Merck 7735).
Compounds and Syntheses. Preparation of Trifluoroacetoni-
trile. Sulfuric acid (2 mL) was slowly added to 100 mL of methanol while
stirring in an ice bath. Trifluoroacetic acid (118 gr, 78 mL) was added to
the cooling methanol solution. The mixture was stirred while heating
(40-45 °C) for 6 h. After cooling to rt, water was added and the resulting
trifluoromethyl ester organic phase was separated and dried over anhy-
drous sodium sulfate. After filtration, 1 L of a 2.0 M solution of ammonia
in methanol was added to the filtrate, and the mixture was stirred
overnight. Solvent and unreacted ammonia were evaporated, and the
white solid was dried under vacuum (98 g) and used for the next step
without any further purification. The obtained trifluoroacetamide was
rapidly mixed with phosphorus pentaoxide (P2O5) (in 3-fold excess) in a
500 mL flask (the condensation reaction starts immediately). The reaction
mixture was heated to 150 °C, and the obtained gaseous trifluoroacetoni-
trile was directly bubbled into the following reaction.
*Authors to whom correspondence should be addressed. G.G.: tel,
(972) 03 9371442; fax, 972-3-9066634; e-mail, Garyg@ariel.ac.il. O.Y.:
tel, (972) 08 9489298; fax: (972) 08 9468785; e-mail, Oded.Yarden
@huji.ac.il.
General Procedure for Preparation of β-Trifluoroalkyl
Aminovinyl Ketones 5a-j. 4 mmol of acetophenone was added in a
dropwise manner to a mixture of 10 mmol of sodium tert-butoxide in
© 2009 American Chemical Society
Published on Web 08/27/2009
pubs.acs.org/JAFC

8304 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Gellerman et al.
100 mL of dry THF. Then, trifluoroacetonitrile (produced from 15 g of
amide and 45 g of P2O5) was bubbled in for 2 h to the reaction vessel
equipped with a dry ice-acetone condenser. After bubbling, the mixture
was stirred for 5 h at rt. A solution (10 mmol) of commercial HCl-dioxane
4 N was added dropwise, and after 10 min the mixture was filtrated and
dioxane was evaporated under vacuum. The crude product was recrys-
tallized from hexane.
chromatography on silica gel using n-hexane-EtOAc (8:2) as eluent
yielded 1.02 of 5g (84% yield) as a colorless oil: νmax (KBr) 1665, 1570,
1360, 1240 cm-1; HRMS (DI, m/z) calcd for C11H7F6NO (MHþ) 284.048,
found 284.051; 1H NMR (CDCl3) δ 6.21 (s, 1, CHdC), 7.6 (t, 1, J=8.82
Hz, H-3), 7.8 (d, 2, J=8.82 Hz, H-2), 8.09 (d, 2, J=8.82 Hz, H-4), 8.19 (s, 1,
H-6); 13C NMR (δ, ppm, CDCl3) 189.7, 160.7, 138.2, 133.2, 131.5, 130.9,
129.6, 126.2, 124.2, 94.0.
Preparation of (Z)-4-(3-Amino-4,4,4-trifluorobut-2-en-
oyl)benzonitrile (5a). Compound 5a was prepared from 3 and
4-cyanoacetophenone (1 g, 6.9 mmol) following the general procedure
described above. Purification by column chromatography on silica gel
using n-hexane-EtOAc (8:2) as eluent, yielded 1.44 g of 5a (87% yield) as
a colorless oil: νmax (KBr) 2880, 1670, 1635, 1270 cm-1; HRMS (DI, m/z)
calcd for C11H7F3N2O (MHþ) 241.05, found 241.04; 1H NMR (CDCl3)
δ 6.19 (s, 1, CHdC), 7.68 (d, 2, J=10.7 Hz, H-2), 8.14 (d, 2, J=10.7 Hz,
H-3); 13C NMR (δ, ppm, CDCl3) 189.7, 160.7, 142.2, 132.7, 131.5, 130.6,
118.4, 115.7, 95.5.
Preparation of (Z)-3-Amino-1-(3-bromophenyl)-4,4,4-tri-
fluorobut-2-en-1-one (5h). Compound 5h was prepared from 3 and
3-bromoacetophenone (1 g, 5.0 mmol) following the general procedure
described above. Purification by column chromatography on silica gel
using n-hexane-EtOAc (8:2) as eluent yielded 1.32 g of 5h (89% yield) as a
colorless oil: νmax (KBr) 1675, 1640, 1410. 1305, 1060 cm-1; HRMS (DI,
m/z) calcd for C10H7BrF3NO (MHþ) 294.295, found 293.972, 295.974
(1:1); 1H NMR (CDCl3) δ 6.19 (s, 1, CHdC), 7.38 (t, 1, J=6.25, 1H, H-4),
7.69 (d, 1 J=12.5 Hz, H-3), 7.81 (d, 1, J=12.5 Hz, H-2), 8.09 (s, 1, H-6). 13C
NMR (δ, ppm, CDCl3) 189.7, 160.7, 140.1, 137.4, 133.4, 131.5, 128.9,
123.6, 95.5.
Preparation of (Z)-3-Amino-4,4,4-trifluoro-1-(4-fluorophe-
nyl)but-2-en-1-one (5b). Compound 5b was prepared from 3 and
4-fluoroacetophenone (1 g, 7.3 mmol) following the general procedure
described above. Purification by column chromatography on silica gel
using n-hexane-EtOAc (8:2) as eluent yielded 1.28 g of 5b (85% yield) as a
colorless oil: νmax (KBr) 1670, 1650, 1190 cm-1; HRMS (DI, m/z) calcd for
C10H7F4NO (MHþ) 234.046, found 234.052; 1H NMR (CDCl3) δ 6.19 (s,
1, CHdC), 7.06 (t, 2, J=5.35 Hz, H-3), 7.66 (m, 2, H-2); 13C NMR (δ,
ppm, CDCl3) 189.7, 168.7, 160.7, 133.5, 131.5, 116, 95.5.
Preparation of (Z)-3-Amino-1-(4-chlorophenyl)-2-ethyl-
4,4,4-trifluorobut-2-en-1-one (5i). Compound 5i was prepared from
3 and 4-chlorobutyrophenone (1 g, 5.5 mmol) following the general
procedure described above. Purification by column chromatography on
silica gel using n-hexane-EtOAc (8:2) as eluent yielded 1.17 g of 5i (68%
yield) as a colorless oil: νmax (KBr) 1665, 1630, 1390 cm-1; HRMS (DI, m/
z) calcd for C12H11ClF3NO (MHþ) 277.048, found 277.043:277.044 (3:1);
1H NMR (CDCl3) δ 0.98 (t, 3, J=9.31 Hz, CH2CH3), 2.00 (q, 2, J=9.31
Hz, CdCCH2CH3), 7.51 (d, 2, J=10.34, H-3), 7.86 (d, 2, J=10.34, H-4);
13C NMR (δ, ppm, CDCl3) 190.5, 152.5, 140.1, 136, 131.3, 129.3, 125.3,
112.9, 12.6, 11.6.
Preparation of (Z)-3-Amino-4,4,4-trifluoro-1-(4-chloro-
phenyl)but-2-en-1-one (5c). Compound 5c was prepared from 3
and 4-chloroacetophenone (1 g, 6.5 mmol) following the general procedure
described above. Purification by column chromatography on silica gel
using n-hexane-EtOAc (8:2) as eluent yielded 1.38 of 5c (81% yield) as a
colorless oil: νmax (KBr) 1670, 1590, 1380, 1060 cm-1; HRMS (DI, m/z)
calcd for C10H7ClF3NO (MHþ) 250.017, found 250.020: 252.018 (3:1); 1H
NMR (CDCl3) δ 6.19 (s, 1, CHdC), 7.41 (d, 2, J=10.7 Hz, H-3), 7.82 (d, 2,
J=10.7 Hz, H2). 13C NMR (δ, ppm, CDCl3) 189.7, 160.7, 140.1, 136,
131.5, 131.3, 129.3, 95.0.
Preparation of (Z)-3-Amino-1-(4-chlorophenyl)-4,4,5,5,5-
pentafluoropent-2-en-1-one (5j). Compound 5j was prepared from
gaseous 2,2,3,3,3-pentafluoropropanenitrile (prepared from commercial
CF3CF2CONH2 in the same manner as for 3 and 4-chlorobutyrophenone
(1 g, 6.9 mmol) following the general procedure. Purification by column
chromatography on silica gel using n-hexane-EtOAc (8:2) as eluent
yielded 1.18 g of 5j (57% yield) as a colorless oil: νmax (KBr) 1660, 1600,
1470, 1290 cm-1; HRMS (DI, m/z) calcd for C11H17ClF5NO (MHþ)
300.014, found 300.016:302.017 (3:1); 1H NMR (CDCl3) δ 6.23 (s, 1,
CHdC), 7.40 (d, 2, J=10.7 Hz, H-3), 7.84 (d, 2, J=10.7 Hz, H-4; 13C
NMR (δ, ppm, CDCl3) 191.2, 161.6, 141.7, 1364, 136.9, 133.8, 130.6,
127.1, 97.3.
Preparation of (Z)-3-Amino-4,4,4-trifluoro-1-(4-hydroxy-
phenyl)but-2-en-1-one (5d). Compound 5d was prepared from 3 and
4-hydroxyacetophenone (1 g, 7.4 mmol) following the general procedure
described above. Purification by column chromatography on silica gel
using n-hexane-EtOAc (6:4) as eluent yielded 1.19 g of 5d (76% yield) as a
white powder; νmax (KBr) 3500-3180 (bs), 1660, 1520, 1450, 1230 cm-1
;
Fungal Strains and Culturing Conditions. The fungi Sclerotinia
sclerotiorum (isolate 1980 (12)), Botrytis cinerea (isolate BcI16 (13)),
Penicillium digitatum (14), Alternaria alternata and Rhizoctonia solani
(both from the fungal collection of the Dept. of Plant Pathology and
Microbiology) and Neurospora crassa (strain 74-OR23-1A, The Fungal
Genetics Stock Center (15)) were used during this study. In addition, the
oomycete Pythium aphanidermatum (from the Dept. of Plant Pathology
and Microbiology) was also used as a test organism. Strains were cultured
on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI), unless
otherwise stated.
HRMS (DI, m/z) calcd for C10H8F3NO2 (MHþ) 232.05, found 232.04; 1H
NMR (CDCl3) δ 6.19 (s, 1, CHdC), 6.89 (d, 2, J=10.7 Hz, H-3), 7.82 (d, 2,
J=10.7 Hz, H-2); 13C NMR (δ, ppm, CDCl3) 189.7, 164.3, 160.7, 131.3,
130.5, 125.5, 116.4, 94.5.
Preparation of (Z)-tert-Butyl-4-(3-amino-4,4,4-trifluoro-
but-2-enoyl)phenyl carbamate (5e). Compound 5e was prepared
from 3 and 4-BOC-amino acetophenone (1 g, 4.3 mmol) following the
general procedure described above. Purification by column chromatogra-
phy on silica gel using n-hexane-EtOAc (6:4) as eluent yielded 1.01 g of 5e
(72% yield) as a colorless oil: νmax (KBr) 1660, 1500, 1350, 1120 cm-1
;
HRMS (DI, m/z) calcd for C15H17F3N2O3 (MHþ) 331.119, found 331.126;
1H NMR (CDCl3) δ 1.5 (s, 9, tBu), 6.19 (s, 1, CHdC), 7.45 (d, 2, J=10.7
Hz, H-3), 7.85 (d, 2, J=10.7 Hz, H-2); 13C NMR (δ, ppm, CDCl3) 189.7,
164.3, 160.7, 131.5, 131.3, 130.5, 116.4, 90.0.
Scheme 1. General Synthesis of β-Trifluoroalkyl Aminovinyl Ketone
Derivatives 5a-j
Preparation of (Z)-3-Amino-4,4,4-trifluoro-1-(naphthalen-
2-yl)but-2-en-1-one (5f). Compound 5f was prepared from 3 and
acetonaphthone (1 g, 5.9 mmol) following the general procedure described
above. Purification by column chromatography on silica gel using
n-hexane-EtOAc (7:3) as eluent yielded 1.11 g of 5f (74% yield) as a
colorless oil: νmax (KBr) 1655, 1570, 1360, 1240 cm-1; HRMS (DI, m/z)
calcd for C14H10F3NO (MHþ) 266.071, found 266.077; 1H NMR (CDCl3)
δ 6.19 (s, 1, CHdC), 7.58 (m, 3, H-3,7,8), 7.99 (m, 3, H-4,5,6), 8.40 (s, 1,
H-2); 13C NMR (δ, ppm, CDCl3) 189.7, 160.7, 135.6, 134.7, 132.5, 131.5,
129.5, 128.6, 128.5, 128.3, 127.7, 126.9, 124.2, 93.0.
Preparation of (Z)-3-Amino-4,4,4-trifluoro-1-(3-(trifluoro-
methyl)phenyl)but-2-en-1-one (5g). Compound 5g was prepared
from 3 and 3-trifluoromethyl acetonaphthone (1 g, 4.29 mmol) follow-
ing the general procedure described above. Purification by column

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8305
Assessment of Fungitoxicity. Initial toxicity against the fungi
of new leads and for providing access to diverse chemical entities
with novel structures and properties (17). We decided to explore
additional compounds that share some similarity with the cin-
namic acid backbone in a scaffold hopping strategy toward the
generation of novel antifungal compounds. In order to identify
potential antifungal compounds, we designed and synthesized
β-trifluoroalkyl aminovinyl ketone molecules that differed in the
R1, R2, and R3 substituents as well as in the length of the
fluorocarbon chain in the scaffold (structure 5 in Scheme 1).
Several synthetic routes to the synthesis of fluoroalkyl-contain-
ing β-aminovinyl ketones starting from unsymmetrical fluoro-
alkyl-containing β-diketones and amines have been previously
described (18-21). Unfortunately, these reactions result in poor
to moderate yields. We adopted a different approach for tri-
fluoroalkyl β-aminovinyl ketone synthesis: namely, condensation
of trifluoroacetonitrile (22) with acetophenones in the presence of
a strong base in dry aprotic solvents. After examining several
alternatives, sodium tert-butoxide (NaOtBu) and dry THF were
chosen as a base and solvent of choice respectively. This general
method was applied very successfully in the preparation of 5a-j
(Scheme 1). Thus, we initially prepared gaseous CNCF3 (3) from
the corresponding trifluoroacetamide by dehydration with P2O5
at high temperature. Compound 3 was then directly bubbled into
the reaction mixture of the appropriate acetophenone in the
presence of NaOtBu under nitrogen atmosphere in dry THF.
The reaction vessel was equipped with an acetone/dry ice cold-
finger to avoid the undesired escape of gaseous 3. After comple-
tion (monitored by TLC and HPLC), the reactions were
quenched with dilute HCl, resulting in crude products that after
(excluding S. sclerotiorum) and the oomycete was assessed by culturing
the test organsims on Petri dishes containing 10 mL of PDA amended with
either 10 or 100 μM of the ten original test compounds. Stock solutions of
the different compounds were prepared in dimethyl sulfoxide (DMSO),
and aliquots (not exceeding 0.001% v/v, a concentration that had no
observable effect on the test organisms) were added to warm (50 °C) sterile
medium prior to dispensing to the Petri dishes. S. sclerotiorum and B.
cinerea were used for the advanced phase of tests, where the effects of
compound concentrations (0-50 μM) were tested. The two fungi were
cultured at 17 and 22 °C, respectively, for 48 h, after which radial growth
was measured and compared to that of the fungal strains cultured in
medium not amended with the antifungal compounds.
RESULTS AND DISCUSSION
Phenyl R,β-unsaturated carbonyl derivatives of cinnamic and
coumaric are known backbones found in potent compounds in
antifungal discovery (16). In recent years research based on these
core structures has become an important tool for the generation
Figure 1. Structure of the compounds prepared in this study.
Figure 2. Effect of nine β-trifluoroalkyl aminovinyl ketone derivatives on radial growth of six test organisms. Test organisms were cultured on potato dextrose
agar in the presence of either 10 or 100 μM concentrations (empty and full bars, respectively) of the various compounds. Y-error bars indicate standard
deviation.

8306 J. Agric. Food Chem., Vol. 57, No. 18, 2009
Gellerman et al.
exhibited very similar sensitivity to 5d and 5c. Commercially
available 4d, that lacks the trifluoroprop-1-en-2-amine part in
the backbone, was the least active compound and, interest-
ingly, at the lower concentrations may have even stimulated the
radial growth of S. sclerotiorum. This was also evident at the
lower concentrations of 5j. Overall, the most effective com-
pound (in terms of curbing radial growth) was 5h, even though
the sensitivity of B. cinerea to this compound was significantly
higher, at all concentrations, than that of S. sclerotiorum.
Based on the results obtained it appears that (Z)-3-amino-
4,4,4-trifluoro-1-phenylbut-2-en-1-one is the minimal scaffold
for presenting β-trifluoroalkyl aminovinyl ketone fungitoxic
activity, preferably substituted with a weak electron with-
drawal halogen atom at para or meta positions. These findings
emphasize the potential that some of these molecules have for
fungicide development. Some of these compounds are poten-
tial leads to be considered for practical use, therefore, future
full analysis of their biological activities need to be determined.
One of these is their potential phytotoxicity. To that end, we
performed preliminary tests on effect of the presence of the
compounds on seed germination of alfalfa (Medicago sativa L.)
and on induced phytotoxic response on leaves of tomato
(Solanum lycopersicum L.), which are both hosts of B. cinerea
and S. sclerotiorum. In all cases, no significant phytotoxic
effects were observed (Gellerman et al., unpublished results).
Work is in progress to assess the phytotoxic effects of this
family of compounds.
Taken together, we have determined that a novel group of
compounds, sharing the β-trifluoroalkyl aminovinyl ketone scaf-
fold, exhibit antifungal activities in vitro. On the one hand, their
activity is evident at relatively high concentrations (similar to
those determined for most cinnamic acid derivatives described by
Bisogno et al. (16)), even though at the highest tested concentra-
tions fungal inhibition was not complete. On the other hand, the
ease of synthesis of these stable compounds along with their
apparent lack of phytotoxicity makes these compounds attractive
for future consideration as potential components of antifungal
intervention.
Figure 3. Effect of five β-trifluoroalkyl aminovinyl ketone derivatives on
radial growth of Sclerotina sclerotiorum and Botrytis cinerea. Y-error bars
indicate standard deviation.
subsequent purification by flash chromatography on silica gel
(EtOAc/ n-hexane), pure 5a-i were obtained in good yields
(68-89%). Even a compound with the longer flourocarbone
chain (5j) was prepared in the same manner as 5a-i, using
gaseous 2,2,3,3,3-pentafluoropropanenitrile (initially prepared
from commercial CF3CF2CONH2 and P2O5) and 4-chlorobutyr-
ophenone in 57% yield .
Coumaric and cinnamic acids (Figure 1), but not caffeic0acid,
have been shown to be inhibitory to fungal (Aspergillus spp0)
growth (23). Apparently, the location of the substituent on the
cinnamic acid backbone plays crucial role in determining the
antifungal activity of the compound. This data prompted us to
examine other phenyl R,β-unsaturated carbonyl scaffolds sub-
stituted with different functional groups at various locations and
study their potential antifungal activity.
Almost all of the synthesized compounds exhibited some
degree of toxicity toward the filamentous fungi tested
(Figure 2). Nonetheless, significant differences in toxicity were
observed among these compounds. For example, the highest
toxicity levels were evident when with (Z)-3-amino-4,4,4-tri-
fluoro-1-(4-chlorophenyl)but-2-en-1-one (5c) was present in
the growth medium, while 5j, 5i and 5e, respectively, were
significantly less toxic. The fungi least susceptible to most of
the compounds tested were A. alternata and P. digitatum. None
of the compounds tested inhibited radial growth of A. alternata
more than 50% (even at a 100 μM concentration) and only 5c
inhibited P. digitatum by more than 50%. In contrast, N. crassa
appeared to be the most sensitive to the compounds at the
higher concentrations tested, 5c, 5h and 5b inhibited radial
growth almost entirely. The oomycete P. aphanidermatum was
sensitive to higher concentrations of 5d, 4d, 5c and 5h. The
other compounds tested inhibited its radial growth only to a
limited extent. Based on the outcome of the initial assessment
of toxicity of the compounds to the test organisms, we decided
to focus our analysis on five of the original compounds tested
5d, 4b, 5c, 5h and 5j and their toxicity to the omnivorus
phytopathogen B. cinerea and an additional broad-host patho-
gen related to B. cinerea: S. sclerotiorum. This analysis allowed
us to determine if these phylogenetically related pathogens
differ in their sensitivity to the structurally related tested
compounds. In most instances, the fungal response to the
tested compounds was dose dependent (Figure 3). Both fungi
ACKNOWLEDGMENT
O.Y. is the incumbent of the Buck Family Chair in Plant
Pathology.
Supporting Information Available: NMR spectra of com-
pounds 5a-c,h and HRMS of compounds 5c,f-h. This material
is available free of charge via the Internet at http://pubs.acs.org.
LITERATURE CITED
(1) Boland, G. J.; Hall, R. Index of plant hosts of Sclerotinia sclerotior-
um. Can. J. Plant Pathol. 1994, 16, 93–108.
(2) Bolton, M. V.; Bart, P. H.; Thomma, J.; Nelson, B. D. Sclerotinia
sclerotiorum (Lib.) de Bary: biology and molecular traits of a
cosmopolitan pathogen. Mol. Plant Pathol. 2006, 7, 1–16.
(3) Choquer, M.; Fournier, E.; Kunz, C.; Caroline Levis, C.; Pradier,
J.-M.; Simon, A.; Viaud, M. Botrytis cinerea virulence factors:new
insights into a necrotrophicand polyphageous pathogen. FEMS
Microbiol. Lett. 2007, 277, 1–10.
(4) Erental, A.; Dickman, M. B.; Yarden, O. Sclerotial development in
Sclerotinia sclerotiorum: awakening molecular analysis of a “dor-
mant” structure. Fung. Biol. Rev. 2008, 22, 6–16.
(5) Van Kan, J. A. L. Licensed to kill: the lifestyle of a necrotrophic
plant pathogen. Trends Plant Sci. 2006, 11, 247–253.
(6) Purdy, L. H. Sclerotinia sclerotiorum: history, diseases and sympto-
matology, host range, geographic distribution and impact. Phyto-
pathology 1979, 69, 875–880.

Article
J. Agric. Food Chem., Vol. 57, No. 18, 2009 8307
(7) Tu, J. C. An integrated control of white mold (Sclerotinia
sclerotiorum) of beans, with emphasis on recent advances in biolo-
gical control. Bot. Bull. Acad. Sinica 1997, 38, 73–76.
(8) Gossen, B. D.; Rimmer, S. R.; Holley, J. D. First report of resistance
to benomyl fungicide in Sclerotinia sclerotiorum. Plant Dis. 2001, 85,
1206.
(9) Ma, Z. H.; Michailides, T. J. Advances in understanding molecular
mechanisms of fungicide resistance and molecular detection of
resistant genotypes in phytopathogenic fungi. Crop Prot. 2005, 24,
853–863.
(16) Bisogno, F.; Mascoti, L.; Sanchez, C.; Enriz, R. Structure-antifungal
activity relationship of cinnamic acid derivatives. J. Agric. Food
Chem. 2007, 55, 10635–10640.
(17) Narasimhan, B.; Belsare, D.; Pharande, D.; Dhake, D. Esters, amides
and substituted derivatives of cinnamic acid: synthesis, antimicrobial
activity and QSAR investigations. Eur. J. Med. Chem. 2004, 39, 827–834.
(18) Pashkevich, K. I.; Filyakova, V. I.; Postovskii, I. Ya. Competitive
amination of carbonyl groups in unsymmetrical polyfluoroalkylaryl-
containing β-diketones. Izv. Akad. Nauk SSSR, Ser. Khim. 1981, 10,
2346–2349.
(19) Filyakova, V. I.; Ratner, V. G.; Karpenko, N. S.; Pashkevich, K. I.
Reaction of fluoroalkyl-containing β-diketones with amines. Izv.
Akad. Nauk, Ser. Khim. 1996, 9, 2278–2284.
(10) Mendoza, L.; Araya-Maturana, R.; Cardona, W.; Delgado-Castro,
T.; Garcia, C.; Lagos, C.; Cotoras, M. In vitro sensitivity of Botrytis
cinerea to anthraquinone and anthrahydroquinone derivatives.
J. Agric. Food Chem. 2005, 53, 10080–10084.
(11) Yarden, O.; Katan, T. Mutations leading to substitutions at amino
acids 198 and 200 of beta tubulin that correlate with benomyl-
resistance phenotypes of field strains of Botrytis cinerea. Phyto-
pathology 1993, 83, 1478–1483.
(12) Godoy, G.; Steadman, J. R.; Dickman, M. B.; Dam, R. Use of
mutants to demonstrate the role of oxalic acid in pathogenicity of
Sclerotinia sclerotiorum on Phaseolus vulgaris. Physiol. Mol. Plant
Pathol. 1990, 37, 179–191.
(13) Swartzberg, D., Kirshner, B., Elad, Y., Granot, D. 2008. Botrytis
cinerea induces senescence and is inhibited by autoregulated expres-
sion of the IPT gene. Eur. J. Plant Pathol. 2008, 120, 289-297.
(14) Yarden, O.; Katan, J.; Aharonson, N. A rapid bioassay for the
determination of carbendazim residues in soil. Plant Pathol. 1985, 34,
69–74.
(20) Boltacheva, N. S.; Filyakova, V. I.; Charushin, V. N. Fluoroalkyl-
containing lithium 1,3-diketonates in reactions with amines and
ammonium salts. Russ. J. Org. Chem. 2005, 41, 1452–1457.
(21) Sosnovskikh, V. Ya.; Gor0kii, A. M. Condensation of nitriles of
polyhalogenated carboxylic acids and benzonitrile with 2-hydroxy-
4,6-dimethylacetophenone. Russ. Chem. Bull. 1998, 47, 354–356.
(22) Sosnovskikh, V. Ya.; Ovsyannikov, I. S. Condensation of trichloro-
and trifluoroacetonitrile with 4-hydroxy-4-methyl-2-pentanone and
20-hydroxyacetophenone. Zh. Org. Khim. 1993, 29, 89–93.
(23) Kim, J. H.; Campbell, B. C.; Mahomey, N. E.; Chan, K. L.;
Molyneux, R. J. Identification of phenolics for control of Aspergillus
flavus using Saccharomyces cerevisiae as a target-gene bioassay.
J. Agric. Food Chem. 2004, 52, 7814–7821.
Received March 19, 2009. Revised manuscript received August 17, 2009.
Accepted August 17, 2009. This research was supported by BARD, the
United States-Israel Binational Research and Development Fund (to O.Y.).
(15) McCluskey, K., The fungal genetics stock center: From molds to
molecules. Adv. Appl. Microbiol. 2003, 52, 245–262.