Fungicidal Properties of Some Novel Trifluoromethylphenyl Amides

Article abstract of DOI:10.1002/cbdv.201800618

Trifluoromethylphenyl amides (TFMPAs) were designed and synthesized as potential pesticides. Thirty-three structures were evaluated for fungicidal activity against three Colletotrichum species using direct bioautography assays. Active compounds were subsequently tested against C. fragariae, C. gloeosporioides, C. acutatum, Phomopsis obscurans, P. viticola, Botrytis cinerea and Fusarium oxysporum. The study identified 2-chloro-N-[2,6-dichloro-4-(trifluoromethyl)phenyl]acetamide (7a) as showing the strongest antifungal activity, and the broadest activity spectrum in this set against Colletotrichum acutatum (at 48 and 72 h) and Phomopsis viticola (at 144 h). The presence of triethylamine in its complex with N-[2,6-dichloro-4-(trifluoromethyl)phenyl]-2,2,3,3,3-pentafluoropropanamide (7b′) played an important role in the bioactivity, and depending on the concentration or fungal species it showed higher or lower activity than the parent amide. X-Ray crystallography has shown that the complex (7b′) is an ion pair, (C₁₀H₂Cl₂F₈NO)^? (C₆H₁₆N)⁺, where a proton is transferred from the amide nitrogen to the triethylamine nitrogen and then connected by hydrogen bonding to the acyl oxygen (N?H 0.893 ?; H???O 1.850 ?; N???O 2.711 ?; N?H???O 161.2(13)°). Although none of these compounds were better than standards, this work revealed some potential lead structures for further development of active novel compounds.

Full text of DOI:10.1002/cbdv.201800618

Title: Fungicidal properties of some novel trifluoromethylphenyl amides
Authors: Maia Tsikolia, Ulrich R. Bernier, David E. Wedge, Nurhayat
Tabanca, Khalil A. Abboud, and Kenneth J. Linthicum
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201800618
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800618

10.1002/cbdv.201800618
Chemistry & Biodiversity
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Fungicidal properties of some novel trifluoromethylphenyl amides
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Maia Tsikoliaa*, Ulrich R. Bernierb, David E. Wedgec, Nurhayat Tabancad, Khalil A.
Abboude, and Kenneth J. Linthicumb
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a
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Emerging Pathogens Institute, Department of Entomology and Nematology, University
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of Florida, Gainesville, FL 32610-0009 USA
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U.S. Department of Agriculture-Agricultural Research Service, Center for Medical,
Agricultural, and Veterinary Entomology, Gainesville, FL, 32608 USA
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United States Department of Agriculture, Agricultural Research Service, Natural
Products Utilization Research Unit, The University of Mississippi, University, MS 38677 USA
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U.S. Department of Agriculture-Agricultural Research Service, Subtropical Horticulture
Research Station, 13601 Old Cutler Rd., Miami, FL 33158 USA
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Center for X-ray Crystallography, Department of Chemistry, University of Florida,
Gainesville, FL 32611 USA
Abbreviations: TFMPA, trifluoromethylphenyl amides; THF, tetrahydrofuran; CH2Cl2,
dichloromethane; Et3N, triethylamine; TLC, thin layer chromatography; SI, Supporting
Information.
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* Correspondence to: Maia Tsikolia, Email: m.tsikolia@gmail.com; Address: Emerging
Pathogens Institute, 2055 Mowry Road, Department of Entomology and Nematology, University
of Florida, Gainesville, FL 32610-0009.
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Abstract
Trifluoromethylphenyl amides (TFMPAs) were designed and synthesized as potential
pesticides. Thirty three structures were evaluated for fungicidal activity against three
Colletotrichum species using direct bioautography assays. Active compounds were subsequently
tested against C. fragariae, C. gloeosporioides, C. acutatum, Phomopsis obscurans, P. viticola,
Botrytis cinerea and Fusarium oxysporium. The study identified 2-chloro-N-(2,6-dichloro-4-
(trifluoromethyl)phenyl)acetamide (7a) as showing the strongest antifungal activity, and the
broadest activity spectrum in this set against Colletotrichum acutatum (at 48 and 72 h) and
Phomopsis viticola (at 144 h). The presence of triethylamine in its complex with N-(2,6-
dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (7b′) played an important
role in the bioactivity, and depending on the concentration or fungal species it showed higher or
lower activity than the parent amide. X-Ray crystallography have shown that the complex (7b′)
is an ion pair, (C10H2Cl2F8NO)- (C6H16N)+, where a proton is transferred from the amide nitrogen
to the triethylamine nitrogen and then connected by hydrogen bonding to the acyl oxygen [N-H
0.893 Å; H…..O 1.850 Å; N…..O 2.711 Å; N-H…..O 161.2(13)]. Although none of these
compounds were better than standards, this work revealed some potential lead structures for
further development of active novel compounds.
Keywords: Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Botrytis cinerea,
Phomopsis viticola, P. obscurans, micro-dilution broth assay, bioautography assay, X-ray
crystallography.
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Introduction
Fungi are the main cause of plant diseases and result in significant loss of agricultural
production worldwide.[1,2] Fungicides thus are an important component of the agrochemical
armamentarium.[3,4] The resistance to fungicides remains one of the major problems in crop
protection programs,[4] and development of new fungicides is an important research objective.[5]
Amide derivatives, such as mandipropamid, dimethomorph, flumorph, mepronil, tiadinil etc.
have been developed over the last several decades as chemicals that kill or inhibit the growth of
fungi.[6,7] The presence of fluorine can increase the bioactivity of potential insecticides and
fungicides[8-10] and can make them highly effective. Flutolanil, fluopyram, fluoxastrobin and
fluxapyroxad are examples of fluorine-containing amide fungicides mainly used in plant
protection[11-13] with a medium to high risk of resistance, except fluopicolide, for which
resistance formation has not yet been observed.
In our previous work[14] we studied the fungicidal activity of twenty trifluoromethylphenyl
amides (TFMPAs 1-4, Figure 1) that were designed following an extensive literature search for
compounds with insect repellent or pesticidal activity using the SciFinder database.[15] These
twenty TFMPAs were evaluated against three Colletotrichum species using direct bioautography
assays and the active compounds were subsequently tested against C. fragariae, C.
gloeosporioides, C. acutatum, Phomopsis obscurans, P. viticola, Botrytis cinerea and Fusarium
oxysporium using a 96-well micro-dilution broth assay. 2,2,2-Trifluoro-N-(2-
(trifluoromethyl)phenyl)acetamide (4c), was the most potent fungicide against P. obscurans
within this original set[14] (Figure 1). The initial twenty TFMPAs[14] were subsequently used as
the basis for designing additional fluorine-containing amides (5-12) as potential leads for broad-
spectrum pesticides and repellents[16] (Figure 2, Table 1).
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All 33 structures were evaluated for pesticidal properties, which included evaluation of
insecticidal activity against mosquito species, as well as fungicidal effects against plant
pathogens. The present work describes the fungicidal activity studies for these 33 TFMPAs. An
X-ray crystallography study was performed for complex N-(2,6-dichloro-4-
(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide with triethylamine (7b′, Figure 3),
which was formed in an effort to synthesize amide 7b.
Results and Discussion
Design and Synthesis
Fluorine-containing N-(substituted)phenyl amide derivatives were designed with the aim to
develop novel compounds as the broad-spectrum activity pesticides. Substituents[14-23] were
selected by structural similarity to the active compounds from our previous work[14] (Figure 2,
Table 1), and also using a literature search of the SciFinder database.[15] Previous reports on the
biological activity gave important information on the impact of the substituted groups on the
properties of the tested compounds. There are many reports on the pesticidal activity of
compounds with trifluoromethyl groups attached to the phenyl ring or fluoroalkyl substituents
attached to the amide carbonyl carbon. The amide groups can improve stability and provide
intermolecular hydrogen bonds with biological targets.[14]
Out of 33 TFMPAs sixteen compounds were synthesized (7b′, 8-12, Figure 2, Table 1)
twelve of which were novel structures. Fourteen compounds (5-7) were prepared previously[16]
and three compounds (8a, 8f and 11a) were purchased from commercial sources. Acid chlorides
were prepared in situ by overnight reaction of the corresponding carboxylic acids with a 20–25%
excess of thionyl chloride at 25 C.[14,16] Anhydrides were purchased from commercial sources.
Reaction of 1.05 equivalents of acid chloride or acid anhydride with one equivalent of
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corresponding trifluoromethylphenyl amines in tetrahydrofuran (THF) (10 mL) at 0-25 C (8b-e,
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8g-h, 9e-f, 10a, 11b), or at 66 C (9a-d), or in toluene at 110 C (12a) led to the production of
TFMPAs 8-12 in yields of 68-99% (Figure 2). Triethylamine (Et3N) was used as a base for the
preparation of amide 12a (see Supporting Information (SI) for compounds yields, melting points,
NMR, mass spectral data).
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X-Ray crystallography
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Complex 7b′ was formed in an attempt to synthesize 7b using triethylamine (Et3N) as the base.
We found it interesting to study the crystal structure of 7b′ along with the pesticidal activity of
this complex in comparison with the activity of free 7b and other compounds. A complex ratio of
1:1 was confirmed by X-ray crystallography and NMR analysis (see SI text and Figures S1 and
S2 for 1H and 13C NMR of 7b′ in comparison with 7b). According to the X-ray crystallography
results, 7b′ is an ion pair, (C10H2Cl2F8NO)-(C6H16N)+, where proton (H2) is transferred from the
amide nitrogen (N1) to the nitrogen (N2) of triethylamine and then connected by hydrogen
bonding to the acyl oxygen (O1); H2 is involved in strong H-bonding with O1 of the molecule
[N2-H2 0.893(15) Å; H2…..O1 1.850(15) Å; N2…..O1 2.711(1) Å; N2-H2…..O1 161.2(13)]
(Figures 3 and S3, Tables S1-S6).
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Direct bioautography assay for activity against plant pathogenic fungi
All 33 TFMPAs were pre-screened against three Colletotrichum species using a matrix
bioautography technique. Bioautography of compounds 5-7 indicated that 6a, 7a, 7b and 7b′
had antifungal activity against Colletotrichum test species, while compounds 5a-f and 6b-f were
inactive against these species. Compounds possessing antifungal activity produced a clear zone
of inhibition and different compounds showed different sizes of the inhibitory zone (Table 2).
Compound 6a exhibited good, clear antifungal zones against C. acutatum and C. fragariae,
while it had diffuse zones against C. gloeosporioides, which indicated potentially selective
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activity. Bioautography of compounds 8a-12a revealed four compounds 8f, 8h, 10a and 11a
with only mild antifungal activity (Table 2).
Microdilution broth assay
Active antifungal compounds 6a, 7a, 7b, 7b′, 8f, 8h, 10a and 11a were subjected to a
secondary screening using a liquid broth culture bioassay (micro-dilution broth assay) in a
concentration-response format (Figures 4 and 5). Against C. acutatum at 48 h, compounds 7a and 7b
were more active at all concentrations than the azoxystrobin standard, but less active than the
captan standard (Figure 4A). At 0.3 µM concentration, compound 7a showed higher activity than
both azoxystrobin and captan standards. Against B. cinerea at 72 h, at all concentrations,
compounds 6a, 7a, 7b and 7b′ had little or no activity (Figure 4B). Compounds 7a and 7b′
produced 95% and 89% fungal growth inhibition of P. viticola at 72 h, respectively, at 30 μM
and were comparable to the captan and azoxystrobin standards (88% and 100% inhibition,
respectively, Figure 4C, Table 3). It should be noted that in our previous work[14] the most
potent fungicide 2,2,2-trifluoro-N-(2-(trifluoromethyl)phenyl)acetamide (4c, Figure 1), with
~44% growth inhibition was less active than both standards at 120 h, at 30.0 μM concentration
against P. viticola. Compounds 8f, 8h, 10a and 11a were less active than the standards, against
P. obscurans at 144 h (Figure 5), and poorly active against the other test fungi (data not shown).
In our previous research[14] compound 4c (Figure 1) was comparable to captan standard against
P. obscurans, producing ~72% inhibition at 3.0 μM, at 120 h exposure. Against C. fragariae
(Table 3) at 48 h, at 30.0 μM, 7a, 7b and 7b′ showed higher activity (~35-55% inhibition) than
the azoxystrobin standard (~29 % inhibition), but they were much less active than the captan
standard (~98% inhibition). Against C. gloeosporioides and F. oxysporum (data not shown),
tested compounds (6a, 7a, 7b, 7b′, 8f, 8h, 10a and 11a) were less active than both standards at
30 µM or showed no activity (Table 3).
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It appears from Figure 4A that as concentration of 6a increases from 3.0 to 30.0 µM, growth
inhibition decreases (fungal growth increases). Such a response is typical for compounds from
the solution as the concentration increases. This is a misinterpretation of spectrophotometer
measured turbidity caused by the molecule as it precipitates out of solution. The growth of the
organism may or may not be increased due to loss of solubility from the aqueous broth and,
accordingly, only data at concentrations below 30.0 µM is valid. Also, there was at least one
observation when 7b demonstrated the solubility problems associated with testing lipophilic
compounds.
Fungal growth inhibition against three Colletotrichum species and F. oxysporium (data not
shown) generally decreased with time for 6a, 7a, 7b and 7b′. The fungi may metabolize these
compounds, or these compounds are not stable in the bioassay system, or there could be other
reasons (an effect of adaption, or fungi can induce defense mechanisms such as increased efflux
etc.). Further study is needed to identify the inhibition mechanism for these species. B. Cinerea
and the two Phomopsis species do not demonstrate this effect.
Structure activity relationship
Active compounds were not only those with the halogenated acyl (6a, 7a, 7b, 7b′, 8f and
8h), but also those with aliphatic acyl substituents (10a and 11a). On the other hand, 2,6-
dichloro-4-(trifluoromethyl)phenyl) amide moiety produced most potent compounds (7a, 7b,
7b′); Acording to our previous work,[14] 2,6-dichloro-4-(trifluoromethyl)phenyl) amide 1c (figure
1), which is structurally similar to 7b, but with a shorter trifluoroacetyl substituent, was not
active against Colletotrichum species. N-ortho-(Trifluoromethyl)phenyl amide 4c with the same
trifluoroacetyl group (figure 1) was the best fungicide according to our previous work;[14] while,
in this study, N-ortho-(trifluoromethyl)phenyl amide 5b, with a longer fluorinated chain,
pentafluoropropionyl, attached to the nitrogen atom, was not active in bioautography assay
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against Colletotrichum species. Also, none of the N-ortho-(trifluoromethyl)phenyl amides 5a-f
and 9a-f were active, as well.
The presence of triethylamine in 7b′ resulted in significant alteration of the biological
properties of the active ingredient 7b. As shown on Figure 4A and B, at 0.3 µM 7b′ promoted
fungal growth in C. acutatum and B. cinerea while 7b inhibited growth. Against C. acutatum at
30 µM both 7b and 7b′ showed almost the same activity; at 3 µM 7b remained active while
activity of 7b′ dramatically decreased; at 3 and 30 µM concentrations against P. viticola 7b′ was
more active than 7b (Figure 4C); at 0.3 µM 7b′ showed less activity than 7b although 7b′ was
more potent than captan standard at all concentrations. Thus, the presence of triethylamine in the
7b′ complex significantly influenced the bioactivity and depending on the concentration or
fungal species it performed better or was less active than the original amide (7b).
Conclusions
2,6-Dichloro-4-trifluoromethyl phenyl amides 7a, 7b and 7b′ with the
pentafluoropropionyl or chloroacetyl substituents showed the best activity out of 33 compounds,
and against Colletotrichum and Phomopsis they were comparable to standards. For amides: 7b
and 1c,[14] 5b and 4c,[14] this study, once more, showed that a small change in the structure can
produce a significant change in the biological (in this case fungicidal) properties of the molecule.
The fungicidal activity of the complex 7b′ differ from the parent molecule 7b. Overall, this study
revealed several 2,6-dichloro-4-trifluoromethyl phenyl amides with the antifungal activity and
broad antifungal spectrum with potential for further study and development of the lead
compounds for the control of Phomopsis infections. The synthesis of a larger number of
structural analogues is needed to establish proper structure-activity relationships for these leads.
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Supporting Information
Supporting Information is available for compounds yields, melting points, NMR, mass
spectral and X-ray crystallography data.
Experimental Section
Synthesis
Sixteen compounds (7b′, 8-12, twelve novel) were synthesized. Three Compounds were
purchased from commercial sources: 8a, 8f (SynQuest Labs, Inc. Alachua, FL, USA) and 11a
(Matrix Scientific, Columbia, SC, USA). Fourteen compounds (groups 5-7, Table 1) we
synthesized previously.[16]
General methods and materials
Melting points were determined on a hot-stage apparatus and are uncorrected. Nuclear
Magnetic Resonance (NMR) analyses were performed at NMR Facility of the University of
Florida in Gainesville, FL, USA. NMR spectra were recorded in CDCl3 or acetone-d6 with TMS
(tetramethylsilane) as the internal standard for 1H (500 MHz) and CDCl3 or acetone-d6 as the
internal standard for 13C (125 MHz). Accurate masses were measured at the Mass Spectrometry
Facility of the Department of Chemistry, University of Florida, using a 6220 TOF-MS (Agilent
Technologies) equipped with an electrospray and atmospheric pressure chemical ionization
source. Direct analysis in real time (DART) was performed with the aid of an Ionsense DART
source (Ionsense, inc). Samples were dissolved in dichloromethane and solutions introduced via
direct injection. All reactions were carried out under argon atmosphere in anhydrous THF,
CH2Cl2 or toluene, from Acros Organics, NJ, USA. The progress of a reaction was monitored by
thin layer chromatography (TLC).
Preparation of 7b′
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Route 1: To a solution of 2,6-dichloro-4-(trifluoromethyl)phenyl amine (10 mmol) in THF
(12 mL), acid anhydride (10.5 mmol) was added at 0 C in the presence of Et3N (10.1 mmol) and
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the mixture was stirred continuously for 2 h at 0-25 C. The reaction mixture was diluted and
extracted with ethyl acetate (40 mL), washed with sat. aq. NaHCO3 (3 x 60 mL) and the organic
layer dried over anhydrous Na2SO4. Evaporation of the solvent and recrystallization from
hexane/ethyl acetate gave the product N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-
pentafluoropropanamide with Et3N (7b′ ) in 96% yield. Route 2: To a solution of N-(2,6-
dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide (0.5 mmol) in THF or
CH2Cl2 (1 mL) was added Et3N (0.55 mmol) and stirred continuously at 25 C. The mixture was
concentrated and recrystallized from hexane/ethyl acetate to give desired product N-(2,6-
dichloro-4-(trifluoromethyl)phenyl)-2,2,3,3,3-pentafluoropropanamide with triethylamine (7b′).
According to the NMR analysis and X-ray crystallography data, ratio for this 7b′ complex
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[7b:Et3N] is 1:1 (Figures 3, S1-S3, SI text).
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Preparation of 8b-e, 8g-h, 9e-f, 10a, 11b
To a solution of trifluoromethylphenyl amine (10 mmol) in THF (12 mL), acid chloride (10.5
mmol, for 8c-e, 9e, 9f, 10a ,11b) or acid anhydride (10.5 mmol, for 8b, 8g-h) was added at 0 C
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and the mixture was stirred continuously for 0.2-4 h at 25 C (Figure 2, route A). The reaction
mixture was diluted and extracted with ethyl acetate (40 mL), washed with sat. aq. NaHCO3 (3 x
60 mL) and the organic layer was dried over anhydrous Na2SO4. Evaporation of the solvent and
recrystallization from hexane (for 9f, 11b) or hexane/ethyl acetate (for 8b, 8d-e, 8g-h), or
purification by silica gel column chromatography using hexane/ethyl acetate as a eluent (for 8c,
10a) gave pure compounds 8b-e, 8g-h, 9e-f, 10a, 11b in yields of 70-86 %.
Preparation of 9a-d
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To a solution of 2-trifluoromethylphenyl amine (10 mmol) in THF (12 mL), acid anhydride
(10.5 mmol) was added at 0C, the mixture was stirred continuously and then refluxed (66 C)
for 16 h (Figure 2, route B). The reaction mixture was diluted and extracted with ethyl acetate
(40 mL), washed with sat. aq. NaHCO3 (3 x 60 mL) and the organic layer dried over anhydrous
Na2SO4. Evaporation of the solvent and recrystallization from hexane/ethyl acetate (9a, 9c)
hexane (9b), or purification by silica gel column chromatography using hexane/ethyl acetate as a
eluent (9d) gave the pure compounds 9a-d in yields of 73-97 %.
Preparation of 12a
To a solution of 2,6-Dichloro-4-(trifluoromethyl)aniline (10 mmol) in toluene (12 mL), 3-
cyclohexylpropanoyl chloride (10.5 mmol) was added in the presence of Et3N (10.5 mmol) and
stirring continued for 6 h under reflux (110C) (Figure 2, route C). The reaction mixture was
diluted and extracted with ethyl acetate (40 mL), washed with sat. aq. NaHCO3 (3 x 60 mL) and
the organic layer dried over anhydrous Na2SO4. Evaporation of the solvent and recrystallization
from hexane resulted in compound 12a in 76% yield. See the Supporting Information for yields,
melting points, NMR and mass spectral data.
X-ray crystallography
Crystals were grown using the following procedure: complex 7b′ (20 mg) was dissolved in
hot hexane (0.5 mL) in a 3 mL vial and left at 23C with a loose lid for slow evaporation of
solvent from the solution of the compound until saturation was reached and crystals formed.
The structure (7b′) was solved and refined in SHELXTL2014,[24] using full-matrix least-
squares refinement (Bruker-AXS, Madison, Wisconsin, USA). The non-H atoms were refined
with anisotropic thermal parameters and all of the H atoms were calculated in idealized positions
and refined riding on their parent atoms. The amine proton H2 was obtained from a Difference
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Fourier map and refined freely. In the final cycle of refinement, 4598 reflections (of which 4144
are observed with I > 2(I)) were used to refine 269 parameters and the resulting R1, wR2 and S
(goodness of fit) were 2.45%, 6.33% and 1.051, respectively.
Fungicidal bioassay
Isolates of Colletotrichum acutatum Simmonds, C. fragariae Brooks, and C. gloeosporioides
(Penz.) Penz. and Sacc. were obtained from B. J. Smith, (USDA, ARS, Poplarville, MS).
Cultures of Phomopsis viticola (Sacc.) Sacc. and P. obscurans (Ellis & Everh.) were obtained
from Mike Ellis (The Ohio State University, Columbus, OH), and B. cinerea Pers. and F.
oxysporum Schlechtend were isolated at USDA-ARS, NPURU (Oxford, MS). The three
Colletotrichum species and P. obscurans were isolated from strawberry (Fragaria x ananassa
Duchesne), while P. viticola and B. cinerea were isolated from commercial grape (Vitis vinifera
L.) and F. oxysporum from orchid (Cynoches sp.).
Direct bioautography assay for activity against plant pathogenic fungi
Matrix bioautography was used to screen of TFMPAs onto a silica plate. Colletotrichum
species were used as test organisms to identify the antifungal activity. Conidia of C. fragariae,
C. acutatum and C. gloeosporioides suspensions were adjusted to 3.0x105 conidia/mL with
liquid potato-dextrose broth (PDB, Difco, Detroit, MI) and 0.1% Tween-80. Using a 50 mL
chromatographic sprayer, each glass silica gel thin layer chromatography (TLC) plate with
fluorescent indicator (250 mm, Silica Gel GF Uniplate, Analtech, Inc., Newark, DE) was sprayed
lightly (until damp) three times with the conidial suspension. Inoculated plates were placed in a
30 x 13 x 7.5 cm moisture chamber (398-C, Pioneer Plastics, Inc. Dixon, KY) and incubated in a
growth chamber at 24  1oC with 12 h photoperiod under 60  5 µmols•m-2•sec-1 light with a Li-
Cor Quantum/Radiometer/Photometer (Model LI-250 Light Meter, Lincoln, NE USA).
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The diameter of the clear zones of fungal growth inhibition were measured 4 d after
treatment.[14] Clear zones appearing against a dark background on the TLC plate represent
regions where fungal mycelia or reproductive stroma are not present. Direct bioautography is an
effective technique in evaluating extracts or pure compounds that are lipophilic.[25] Sensitivity of
each fungal species in response to each test compound was determined by comparing the mean
diameter of the inhibitory zones.
Microdilution broth assay
A standardized 96-well micro-dilution broth assay developed by Wedge and Kuhajek
(1998)[26] and validated by Abril et al.[27] was used to evaluate antifungal activity towards B.
cinerea, C. acutatum, C. fragariae, C. gloeosporioides, P. viticola, P. obscurans and F.
oxysporum. Azoxystrobin and captan are registered for control of anthracnose (caused by
Colletotrichum spp.) and Botrytis fruit rot (caused by B. cinerea).[28] For this reason, captan
(Phthalimide, multi-site inhibitor) and azoxystrobin (strobilurin class, Quinone outside inhibitor
(QoI) with different modes of action were used as internal fungicide standards in all assays.
Each test fungus was challenged in a dose-response format using test compounds where the
final treatment concentrations were 0.3, 3.0 and 30.0 µM. Sixteen wells containing broth and
inoculum served as positive controls. Eight wells containing solvent at the appropriate
concentration and broth without inoculum were used as negative controls. The experiments were
repeated three times. Fungal growth was then evaluated by measuring absorbance of each well
at 620 nm using a microplate photometer (Packard Spectra Count, Packard Instrument Co.,
Downers Grove, IL). Mean absorbance values and standard errors were used to evaluate fungal
growth at 48 h and 72 h except for P. obscurans and P. viticola the data were recorded at 144 h.
Means for percent inhibition of each fungus at each dose of test compound relative to untreated
positive growth controls were used to evaluate fungal growth. The SAS system analysis of
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variance procedure (Statistical Analysis System, Cary, North Carolina) was used to identify
significant factors, and Fisher’s protected LSD was used to separate means.[29]
Acknowledgements
We thank J. Linda Robertson and Ramona Pace for assistance with the bioautography and
microtiter assays as well as the Nucleic Magnetic Resonance, Mass Spectrometry facilities and
the Center for X-Ray Crystallography of the University of Florida. K. A. A. wishes to
acknowledge the National Science Foundation and the University of Florida for funding of the
purchase of the X-ray equipment.
This work was supported by the Deployed War-Fighter Protection Research Program and
funded by the United States Department of Defense through the Armed Forces Pest Management
Board, Agreement 60-0208-4-001 and under USDA Specific Cooperative Agreements 58-0208-
0-068 and 58-0208-5-001.
Author Contribution Statement
M. T., N. T., K. A. A. and D. E. W. performed the experiments, analyzed the data and wrote the
paper. U. R. B and K. J. L. contributed samples/reagents/materials/analysis tools and analyzed
the data. M. T., N. T. and U. R. B conceived and designed the experiments.
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Graphical abstract:
Development of new fungicides is an important research objective. This work revealed some lead
structures for further development of active novel compounds.
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Table 1. The 33 trifluoromethylphenyl amides used in this study.
ID
Compound name
2-chloro-N-(2-
Structure
R
1.
2.
5a
(trifluoromethyl)phenyl)acetamide
2,2,3,3,3-pentafluoro-N-(2-
(trifluoromethyl)phenyl)propanamide
5ba
3.
4.
5.
6.
5c
5d
5e
5fa
N-(2-(trifluoromethyl)phenyl)heptanamide
N-(2-(trifluoromethyl)phenyl)octanamide
N-(2-(trifluoromethyl)phenyl)decanamide
N-(2-(trifluoromethyl)phenyl)undecanamide
2-chloro-N-(3-
(trifluoromethyl)phenyl)acetamide
7.
6a
2,2,3,3,3-pentafluoro-N-(3-
(trifluoromethyl)phenyl)propanamide
8.
6ba
9.
6c
6da
N-(3-(trifluoromethyl)phenyl)heptanamide
N-(3-(trifluoromethyl)phenyl)octanamide
N-(3-(trifluoromethyl)phenyl)decanamide
10.
11.
6e
12.
6fa
7a
N-(3-(rifluoromethyl)phenyl)undecanamide
2-chloro-N-(2,6-dichloro-4-
(trifluoromethyl)phenyl)acetamide
13.
N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-
2,2,3,3,3-pentafluoropropanamide
14.
15.
7b
N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-
2,2,3,3,3-pentafluoropropanamide with
triethylamine
7b′a
438
439
440
441
442
443
444
445
446
447
448
449
450
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452
Table 1. The 33 trifluoromethylphenyl amides used in this study (contd).
ID
8a
Compound name
Structure
R
16.
17.
18.
19.
20.
21.
22.
23.
N-(3,5-bis(trifluoromethyl)phenyl)acetamide
N-(3,5-
8ba
8ca
8da
8e
bis(trifluoromethyl)phenyl)hexanamide
N-(3,5-bis(trifluoromethyl)phenyl)hex-5-
enamide
N-(3,5-bis(trifluoromethyl)phenyl)-3-
cyclohexylpropanamide
N-(3,5-bis(trifluoromethyl)phenyl)-2-
methylbenzamide
N-(3,5-bis(trifluoromethyl)phenyl)-2-
chloroacetamide
8f
N-(3,5-bis(trifluoromethyl)phenyl)-2,2,2-
trifluoroacetamide
8g
N-(3,5-bis(trifluoromethyl)phenyl)-
2,2,3,3,3-pentafluoropropanamide
8h
453
454
455
456
457
458
459
460
461
462
463
464
465
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467
Table 1. The 33 trifluoromethylphenyl amides used in this study (contd).
ID
9a
Compound name
Structure
R
24.
25.
N-(2-(trifluoromethyl)phenyl)acetamide
9b
9c
N-(2-(trifluoromethyl)phenyl)propionamide
N-(2-(trifluoromethyl)phenyl)butyramide
N-(2-(trifluoromethyl)phenyl)pentanamide
N-(2-(trifluoromethyl)phenyl)hex-5-enamide
26.
27.
28.
9d
9ea
3-cyclohexyl-N-(2-
(trifluoromethyl)phenyl)propanamide
29.
30.
9fa
3-cyclohexyl-N-(3-
(trifluoromethyl)phenyl)propanamide
10aa
31.
32.
11a
N-[4-(trifluoromethyl)phenyl]acetamide
3-cyclohexyl-N-(4-
(trifluoromethyl)phenyl)propanamide
11ba
3-cyclohexyl-N-(2,6-dichloro-4-
(trifluoromethyl)phenyl)propanamide
33.
12aa
468
469
470
aNovel compounds. See references for other compounds: 5-7;[14] 8a;[17] 8e;[18] 8f,g;[19] 8h;[20]
9a;[21] 9b,c,d;[22] 11a.[23]
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Chemistry & Biodiversity
Table 2. Antifungal activity results of amides 6a, 7a, 7b, 8f, 8h, 10a and 11a using direct
bioautography against three Colletotrichum Species.
a
Mean zone diameter of fungal growth inhibition in mm (SD)
Compounds
C. acutatum
12 mM
C. fragariae
12 mM
C. gloeosporioides
Concentration
12 mM
Applied
6a
4 µL
8 µL
4 µL
8 µL
4 µL
8 µL
5.5 (0.7)
9.5 (0.7)
9.0 (0)
10.5 (0.7)
2.0 (0)
2.0 (0)
2.0 (0)
2.0 (0)
13.0 (0.7)
11.0 (1.4)
10.5 (0.7)
12 (0)
6.0 (0)
9.5 (2.1)
10.0 (1.4)
8.5 (0.7)
2.7 (0)
3.0 (0)
2.0 (0)
2.0 (0)
8.0 (1.4)
12.5 (0.7)
12.5 (0.7)
11.5 (0.7)
4.3 (0.6)
3.0 (0)
Diffuse
11.5 (0.7)
9.00 (0)
8.5 (0.7)
2.0 (0)
Diffuse
Diffuse
12.5 (0.7)
8.5 (0.7)
3.0 (0)
7a
7b
7b′
8f
3.0 (0)
8h
2.3 (0.5)
3.0 (0)
2.0 (0)
3.0 (0)
10a
11a
3.3 (0.5)
4.3 (1.5)
2.0 (0)
3.7 (1.1)
Diffuse
3.0 (0)
Diffuse
Agrochemical standards
C. acutatum
C. fragariae
2 mM
C. gloeosporioides
2 mM
Concentration
Applied
2 mM
2 µL
2 µL
2 µL
Benomyl
Diffuse
12.5 (0.7)b
Diffuse
21.5 (2.1)
13.5 (0.7)
28.5 (0.7)
Diffuse
Captan
15.0 (4.2)
Diffuse
Azoxystrobin
a Mean inhibitory zone diameter and standard deviation (SD) were used to determine the level of
antifungal activity against each fungal species,
Diffuse: Diffuse zone is indicated by the growth inhibitory zone that appears thinly populated
with mycelia and reproductive hyphae and the zone margin is not sharply contrasted.
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Table 3. Mean fungal growth inhibition (%) of compounds 6a, 7a, 7b, 7b′ 8f, 8h, 10a and 11a
against fungal plant pathogens at 30 µM.
Mean fungal growth inhibition, % (SEM)a
Compound
B. cinerea
at 72 h
P. viticola
at 144 h
C. acutatum
P. obscurans
C. fragariae
C. gloeosporioides
at 48 h
at 144 h
at 48 h
at 48 h
6a
7a
7b
7b′
29.2 (8.2)
66.8 (5.4)
44.5 (5.9)
47.3(4.1)
bNA
40.0 (13.1)
40.4 (11.0)
45.5 (34.2)
59.7 (22.6)
58.6 (25.7)
57.6 (20.5)
18.4 (5.1)
35.4 (4.8)
42.1 (2.1)
54.7 (1.2)
0.0 (3.9)
4.2 (2.2)
7.5 (2.8)
17.3 (11.3)
49.0 (9.0)
23.2 (33.7)
12.8 (16.2)
95.3 (1.9)
51.4 (4.1)
89.5 (2.6)
8f
NA
20.9 (2.3)
18.0 (2.9)
17.4 (0.9)
17.4 (0.9)
99.9 (0.5)
88.4 (8.8)
42.9 (16.9)
59.4 (3.4)
63.6 (4.0)
62.4 (3.9)
98.0 (9.4)
95.9 (1.7)
NA
NA
8h
NA
NA
NA
NA
10a
NA
NA
NA
NA
11a
NA
NA
NA
NA
Azoxystrobin
Captan
41.8 (10.6)
86.9 (12.6)
96.2 (0.8)
98.4 (1.2)
29.0 (3.1)
97.8 (6.4)
31.3 (6.3)
81.5 (8.9)
aSEM, Standard error of the mean.
bNA, not applicable
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Figure 1. TFMPAs 1-4 we synthesized and tested in our previous work.[14]
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Figure 2. Synthesis of TFMPAs 8-12.
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10.1002/cbdv.201800618
Chemistry & Biodiversity
Figure 3. X-ray crystal structure for the complex 7b′.
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Chemistry & Biodiversity
A. Colletotrichum acutatum growth response at 48 h
80.0%
0.3 µM
3.0 µM
30 µM
50.0%
20.0%
(10.0%)
(40.0%)
(70.0%)
(100.0%)
6a
7a
7b
7b' AzoxystrobinCaptan
B. Botrytis cinerea growth response at 72 h
80.0%
50.0%
0.3 µM
3.0 µM
30 µM
20.0%
(10.0%)
(40.0%)
(70.0%)
(100.0%)
6a
7a
7b
7b' AzoxystrobinCaptan
C. Phomopsis viticola growth response at 144 h
80.0%
50.0%
0.3 µM
3.0 µM
30 µM
20.0%
(10.0%)
(40.0%)
(70.0%)
(100.0%)
6a
7a
7b
7b' AzoxystrobinCaptan
Figure 4. Growth inhibition of various fungi using a 96 well micro-dilution broth assay in a
3-point dose response to 6a, 7a, 7b and 7b′ compared to the commercial fungicide standards
Azoxystrobin and captan. Colletotrichum acutatum (A), Botrytis cinerea (B), and Phomopsis
viticola (C) exposures.
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Chemistry & Biodiversity
Phomopsis obscurans growth response at 144 h
80.0%
0.3 µM
3.0 µM
30 µM
50.0%
20.0%
(10.0%)
(40.0%)
(70.0%)
(100.0%)
8f
8h
10a
11a Azoxystrobin Captan
Figure 5. Growth inhibition of Phomopsis obscurans at 144 h using a 96 well micro-dilution
broth assay in a 3-point dose response to 8f, 8h, 10a and 11a compared to the commercial
fungicide standards azoxystrobin and captan.
29
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