Insecticidal, repellent and fungicidal properties of novel trifluoromethylphenyl amides

Twenty trifluoromethylphenyl amides were synthesized and evaluated as fungicides and as mosquito toxicants and repellents. Against Aedes aegypti larvae, N-(2,6-dichloro-4-(trifluoromethyl)phenyl)-3,5dinitrobenzamide (1e) was the most toxic compound (24 h LC5


Introduction
The goal of this research is to discover new mosquito insecticides, repellents, and fungicides by synthesizing inexpensive novel compounds which may be active or lead to the discovery of additional active compounds based on structure-activity analysis. Compounds with a broad spectrum of activities would be ideal and could result in new products for eventual commercial use. Our approach is to evaluate a set of compounds with similar chemical base structures and varied substitutions. In this study, fluorinecontaining chemicals were the focus because over the past decade they have become increasingly important in controlling agricultural pests. Compounds within this class are effective insecticides and fungicides [1]. Examples of pesticides that contain fluorine as a trifluoromethyl group include fipronil, flonicamid, and flubendiamide (Fig. 1). The inclusion of fluorine atoms or a trifluoromethyl group into small molecules can significantly increase their biological activity by promoting electrostatic interactions with biological targets, increasing metabolic stability, and improving cellular membrane permeability and bioavailability [2][3][4][5][6][7][8].
The design of target molecules was based on an extensive literature search [9]. Previous reports of compounds with insecticidal, mosquito repellent or fungicidal activity provided valuable information on potential base structures. We then synthesized compounds comprised of trifluoromethylphenyl moieties attached to the amide nitrogen of the base structures. The trifluoromethyl groups were located in the ortho-, meta-, or para-positions on the N-phenyl ring, since there are reports describing promising insecticidal and repellent properties in all three different ring substitution positions. The amide groups within the molecule were retained since they are known to improve stability and provide the ability to establish intermolecular hydrogen bonds with biological targets. The addition of a fluorine or trifluoromethyl on the aryl ring increases lipophilicity and can strongly polarize the parent structure [5,10], and thus should significantly influence the biological activity of the molecule. A total of 20 trifluoromethylphenyl amides (14 of which were novel) were designed and synthesized based on the aforementioned criteria.
All compounds were evaluated for toxicity against Aedes aegypti larvae and adults, for repellency against adult female Ae. aegypti and Anopheles albimanus, and for fungicidal activity against Colletotrichum fragariae, C. gloeosporioides, C. acutatum, Phomopsis obscurans, P. viticola, Botrytis cinerea and Fusarium oxysporum. Selected compounds were evaluated for toxicity against Drosophila melanogaster.

General methods and materials
Melting points were determined on a hot-stage apparatus and are uncorrected. Nuclear Magnetic Resonance (NMR) analyses were performed at the NMR Facility of the University of Florida in Gainesville, FL, USA. NMR spectra were recorded in CDCl 3 or DMSO-d 6 with TMS (tetramethylsilane) as the internal standard for 1 H (500 MHz) and CDCl 3 or DMSO-d 6 as the internal standard for 13 C (125 MHz). Accurate masses were measured at the Mass Spectrometry Facility of the University of Florida, using a 6220 TOF-MS (Agilent Technologies) equipped with an electrospray and atmospheric pressure chemical ionization source. Samples were dissolved in dichloromethane and solutions introduced via direct injection. All reactions were carried out under argon atmosphere in anhydrous THF obtained from Acros Organics, NJ, USA. The progress of a reaction was monitored by thin layer chromatography (TLC).
2.1.2.2. Preparation of 1b, 1d, and 1e. To a solution of 2,6-dichloro-4-(trifluoromethyl)phenyl amine (10 mmol) in THF (12 mL), NaH (10.4 mmol) was added and stirred continuously for 40 min at 0°C (Fig. 2, route B). Acid chloride 5 (10.5 mmol) was then added and stirred continuously for 48-72 h at 25°C. The reaction was quenched with water (10 mL), extracted with ethyl acetate (40 mL), washed with sat. aq. NaHCO 3 (3 Â 60 mL) and dried over anhydrous Na 2 SO 4 . Evaporation of the solvent and recrystallization from ethanol resulted in compounds 1b, 1d and 1e with yields of 72-80%.   To a solution of trifluoromethylphenyl amine (10 mmol) in THF (12 mL), acid chloride 5 or acid anhydride 6 (for 2a,c, 3a,c and 4a,c) (10.5 mmol) was added at 0°C and stirred continuously for 1-2 h at 25°C to produce compounds 2b-2e, 3b-3e and 4b-4e, and for 16-24 h at 65°C to produce compounds 2a, 3a and 4a (Fig. 2, route C). The reaction mixture was diluted and extracted with ethyl acetate (40 mL), washed with sat. aq. NaHCO 3 (3 Â 60 mL) and the organic layer dried over anhydrous Na 2 SO 4 . Evaporation of the solvent and recrystallization from ethanol or ethanol/water gave pure compounds 2a-2e, 3b-3e and 4a-4e in 84-97% yields and chromatography on silica gel using hexanes/ethyl acetate as eluent gave pure compound 3a in 89% yield.       7.39 (t, J = 7.7, 0.9 Hz, 1H). 13  Adult topical assays were performed against female Ae. aegypti. The mosquito rearing and application methods were conducted as previously described by Pridgeon et al. [16]. Prior to the application of the test compounds, 5-to 8-day-old adult mosquitoes were collected from a screened cage using a vacuum aspirator (BioQuip Products, Rancho Dominguez, CA, USA) and cold anesthetized at 4°C for an hour. Ten female mosquitoes were sorted into 3.5 oz clear plastic cups (Solo Cup Company, Lake Forest, IL, USA) using pointed featherweight forceps (BioQuip Products, Rancho Dominguez, CA, USA). The opening to each cup was sealed with a double layer of mesh tulle fabric and secured with a rubber elastic band.
The majority of the chemicals were solubilized in DMSO to a 4 M stock solution, except for 1a, 1e, 2e, and 4e, which were solubilized at 2 M, and 1d and 3e which were solubilized at 1 M. The stock solutions were diluted with acetone to produce a 5% DMSO/acetone treatment solution and 0.5 lL was applied to the thorax of each mosquito using a 700 series syringe and a PB 600 repeating dispenser (Hamilton, Reno, NV, USA). Ten mosquitoes each were treated with one of the four concentrations that were tested for each chemical. After the topical application, mosquitoes were transferred back into a plastic assay cup, held as described above, and provided with a 10% sucrose solution daily. Mortality data was recorded at 24, 48 and 72 h post topical application for determination of LD 50 . Fipronil (50 nmol) was used as a positive control, while acetone and untreated mosquitoes were used as negative controls. Three replicates were completed and the data were analyzed using PoloPlus probit analysis software v2.0 (LeOra Software, Petaluma, CA, USA) to calculate the LC 50 and LD 50 .

Contact toxicity bioassays with D. melanogaster
Susceptible (Oregon-R) and cyclodiene-resistant (rdl; 1675) strains of D. melanogaster were used to determine the contact toxicity of the trifluoromethylphenyl amides. The Oregon-R strain was originally donated by Doug Knipple, Cornell University, Ithaca NY, USA, and has been maintained in culture at the University of Florida since 2009. The rdl strain, 1675, was purchased from the Bloomington Drosophila Stock Center at Indiana University, Bloomington, IN. Both strains were reared in plastic vials on artificial media purchased from Carolina Biological Supply, Burlington, NC. Toxicity bioassays used a surface-contact method in which compounds were dissolved in acetone and a 100 lL aliquot was applied to glass vials (40 cm 2 ) that were evenly coated by manual rotation of the vial for a duration of 2 min. Twenty female flies were then added to the vials, which were stoppered with cotton balls containing 10% sucrose solution. Ethanol was used as the negative control and fipronil was used as the positive control. Fipronil produced 100% mortality at concentrations equal or less than that of the experimental inhibitors. Mortality was determined at 24 h post treatment and analyzed by PoloPlus. Six compound concentrations were used in triplicate to construct dose-response curves to determine one LC 50 value. LC 50 values were averaged (n = 3) using GraphPad InStat™ (GraphPad Software, San Diego, CA, USA) to determine mean LC 50 values for each compound to D. melanogaster. The mean LC 50 values were statistically analyzed using an unpaired t-test (two tail) with significance being represented by P < 0.05. Statistical analyses were performed using GraphPad InStat™.

Repellency bioassays with Ae. aegypti mosquitoes
The mosquito species used for testing were Ae. aegypti (Orlando strain, 1952) and An. albimanus (El Salvador, 1974) from colonies maintained at USDA-ARS CMAVE in Gainesville, FL. Newly emerged mosquitoes were maintained on 10% sugar water and kept in laboratory cages at an ambient temperature of 28 ± 1°C and RH of 35-60%. Nulliparous 6-to 8-day-old female mosquitoes were pre-selected from stock cages using a hand-draw box and trapped in a collection trap [17]. After 500 (±10%) females were collected in the trap, they were transferred to a test cage (approximately 59,000 cm 3 with dimensions 45 Â 37.5 Â 35 cm) and allowed to acclimate for 17.5 (±2.5) min before initiating testing [18].
To evaluate the minimum effective dosage (MED) [19], a 1 mL solution of an appropriate concentration of each amide was transferred into a 2-dram vial containing a muslin cloth patch (5 x 10 cm). The MED is a measurement used to estimate the concentration level of repellent, which fails to prevent mosquito bites, equivalent to an ED 99 . There were two series of dosages used. The series of high dosage was 25.000, 12.500, 6.250, and 3.125 lmol/ cm 2 . The lower dosage series consisted of cloths treated with 2.500, 1.250, 0.625, 0.313, 0.156, 0.078, 0.039, and 0.020 lmol/ cm 2 . Prior to the start of testing, the cloth was removed from the vial and affixed with staples onto two sections of card stock (5 Â 2.5 cm). Approximately 5 cm of masking tape was affixed to the edges of the card stock. After the cloth and card stock were treated, they were placed on a drying rack and allowed to dry for at least 3 min prior to testing. The MED calculation was initiated using the middle range (0.313 lmol/cm 2 ) treated cloth and followed by use of higher or lower dosage treatments as necessary until all subjects had evaluated the cloths and pinpointed the dosage at the 1% (5 bites) failure point. If the 2.500 lmol/cm 2 cloth was not efficacious (>5 bites per min), then a higher dosage series was used to determine the MED. There were 3 volunteers (all male) that tested each cloth. During each test, all volunteers wore a patch treated with a specific compound and tested it for a 1 min interval. Patches were then rotated among the volunteers. DEET was the positive control for these tests and cloth treated with acetone, which was the solvent used in this bioassay, served as the negative control. No patch was evaluated more than 10 min after the 3 min drying period in order to avoid any bias that may result from evaporative loss of treatment from the cloth. All procedures were approved by the University of Florida Human Use Institutional Review Board and informed consent was provided by all participants (Project #636-2005).
Each volunteer participating in the bioassay test wore a specially designed sleeve that exposed only a small area of the forearm to the mosquitoes. The hand of each human volunteer was protected by a powder-free latex glove (Diamond Grip, Microflex Corporation, Reno, NV). The gloved hand and arm were then placed inside a knee-high stocking (Leggs Everyday Knee Highs, Winston-Salem, NC). A plastic sleeve constructed of polyvinyl was then placed over the arm and stocking. The sleeve had a lengthwise Velcro seam to allow sealing over the arm. There was a window cut into the sleeve (4 Â 8 cm opening) approximately half way between the wrist and elbow. This window allowed odors from the volunteer's skin surface to escape from the sleeve through the opening, over which the treated cloth was placed.
The arm, sleeve and cloth were inserted into the mosquito cage for 1 min to determine if the compound and dosage on cloth were repellent to the mosquitoes. The number of fed mosquitoes was determined by shaking the arm briskly after 1 min and counting the number of mosquitoes that remained biting through the cloth. During the testing process, no more than 10 compounds were assayed in succession with a caged population of test mosquitoes before allowing a 15 min recovery period. This was necessary because following prolonged and repeated repellent exposure, mosquitoes fatigue and exhibit decreased response to attractant (skin) odors.

Fungicidal bioassay
2.2.4.1. Direct bioautography assay for activity against plant pathogenic fungi. Pure compounds were initially evaluated for their antifungal activity against three important plant pathogenic fungi (Colletotrichum species) using direct-bioautography. Pathogen production and bioautography procedures described by Wedge et al. [20], were used to evaluate antifungal activity against fungal plant pathogens. Technical grade commercial fungicides azoxystrobin, and captan (Chem Service, Inc., West Chester, PA, USA) were used as fungicide standards at 2 mM in 2 lL of 95% ethanol. The test compounds and commercial fungicides were applied onto TLC plates at 12 mM in 4 lL of 95% ethanol and tested against all three Colletotrichum species. Conidia of C. fragariae, C. acutatum and C. gloeosporioides suspensions were adjusted to 3.0 Â 10 5 conidia/ mL with liquid potato-dextrose broth (PDB, Difco, Detroit, MI, USA) and 0.1% Tween-80. Using a 50 mL chromatographic sprayer, each glass silica gel TLC plate treated with fluorescent indicator (250 mm, Silica Gel GF Uniplate) (Analtech, Inc., Newark, DE, USA) was sprayed lightly (until damp) three times with the conidial suspension. Inoculated plates were placed in a 30 Â 13 Â 7.5 cm moisture chamber (398-C; Pioneer Plastics, Inc., Dixon, KY, USA) and incubated in a growth chamber at 24 ± 1°C with 12 h photoperiod under 60 ± 5 lmols m À2 sec À1 light. Inhibition of fungal growth was measured 4 d after treatment. Sensitivity of each fungal species to each test compound was determined by comparing the size of inhibitory zones. Fungal growth inhibition means for extracts and pure compounds were analyzed separately by ANOVA using SAS software, Ver. 8 (Statistical Analysis System, Cary, NC, USA). Mean separations were performed based on Fisher's Protected Least Significant Difference (LSD) (P = 0.05). Statistical comparisons were made for fungal growth across compounds, and for each compound across fungal growth. Means and standard deviations of inhibitory zone size were used to evaluate antifungal activity of pure compounds.

Microdilution broth assay. A standardized 96-well microdilution broth assay developed by Wedge and
Kuhajek [20] was used to evaluate antifungal activity towards B. cinerea, C. acutatum, C. fragariae, C. gloeosporioides, P. viticola, P. obscurans and F. oxysporum. The commercial fungicides captan and azoxystrobin were used as the positive controls and 95% ethanol as the negative control in all assays. Solutions of tested compounds and positive controls were prepared in 95% ethanol. Each fungus was challenged in a dose-response format using test compounds where the final treatment concentrations were 0.3, 3.0 and 30.0 lM in microtiter plates (Nunc MicroWell, untreated; Fisher Scientific, Roskilde, Denmark) covered with a plastic lid and incubated in a growth chamber, as described previously [21]. Sixteen wells containing broth and inoculum served as growth controls; eight wells containing solvent at the appropriate concentration and broth without inoculum were used as negative controls. Experiments were conducted in triplicate. Mean absorbance values and standard errors were used to evaluate fungal growth at 48 and 72 h except for P. obscurans and P. viticola, for which the data were recorded at 120 h. Means for percent inhibition of each fungus at each dose of test compound relative to the untreated positive growth controls were used to evaluate fungal growth inhibition. The SAS system analysis of variance procedure was used to identify significant factors, and Fisher's protected LSD was used to separate means [22]. 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, USA).
In toxicity bioassays against Ae. aegypti adults, compounds 2a-2e and 4a-e, demonstrated 610% mortality at any time point and in series 1a-e and 3a-e only 1c and 3b showed some activity. The controls: water and DMSO were zero and fipronil was always 100% at any time point. Compound 1c at 100 and 50 nmol doses resulted in approximately 80%, 90%, and 100% mortality after 24, 48, and 72 h, respectively (Table 3), and the 100 nmol dose of compound 3b produced 30%, 70%, and 80% mortality after 24, 48, and 72 h post-application, respectively. The LD 50 values for 1c were 19.182 nM for 24 h, 13.389 nM for 48 h, and 12.077 nM for 72 h, compared to fipronil which was determined to have a 24 h LD 50 of 0.787 10 À4 nM (Table 4). Because only compound 1c produced sufficient mortality in screening assays to merit evaluation of the LD 50 , additional adult topical testing was not conducted on the remaining compounds.
The experimental trifluoromethylphenyl amides were found to be minimally toxic to D. melanogaster through contact exposure. Compounds 1c and 4c were the only compounds found to be toxic to either strain, Oregon-R or the GABA receptor mutant, 1675 (Table 5). Compound 1c was found to have near identical LC 50 values for the Oregon-R and 1675 strains of 5.6 and 4.9 lg/cm 2 , respectively. Compound 4c was found to have LC 50 values of 15.3 lg/ cm 2 and 20.6 lg/cm 2 for the Oregon-R and 1675 strains, respectively. The mean LC 50 values for compounds 1c and 4c were not significantly different (P > 0.05) between the two fly strains. LC 50 values of other experimental compounds were found to be greater than 25 lg/cm 2 and were deemed to be non-toxic. Fipronil was highly toxic with LC 50 values of 0.004 and 0.017 lg/cm 2 for the Oregon-R and 1675 strain, respectively, a statistically significant 4-fold difference between fly strains. These data demonstrate that the toxicity of experimental compounds 1c and 4c are able to circumvent resistance from the GABA receptor mutation present in 1675 (rdl).
The toxicity bioassays with Ae. aegypti adults (Table 3) and D. melanogaster (Table 5) resulted in poor to no activity for most of the compounds. In both sets of assays, the most active compound was 1c. This compound has a 2,6-dichloro-substitution, a trifluromethyl group located in the para-position relative to the amide   aegypti with an MED of 0.039 lmol/cm 2 which in this study was lower than for DEET (0.091 lmol/cm 2 ). Compound 4a had an MED comparable to that of DEET ( Table 6) The presence of a 2,6-dichloro-substitution on the phenyl ring increased the repellent activity of para-trifluoromethylphenyl amide with the trifluoromethyl group attached to the carbonyl carbon (1c) compared to 2c, and, in contrast, decreased the repellent activity of the amide with alkyl group attached to the carbonyl carbon (1a) compared to 2a (Table 6). Bioautography indicated that six trifluoromethylphenyl amides had antifungal activity against C. acutatum, C. fragariae and C. gloeosporioides (Table 7). Antifungal activity was evident by the presence of clear zones with a dark background where fungal mycelia or reproductive stroma were not present on the TLC plate. Compounds 1e, 3a and 4c appeared to be the most effective against all three Colletotrichum species and generated clear zones of fungal growth inhibition. Some of the compounds showed diffuse zones in those regions on the bioautography plate where the fungal growth is visually interspersed with few mycelia. Compounds 2a, 2b and 3b showed selective activity against C. gloeosporioides. The six most active antifungal compounds identified by bioautography were subsequently evaluated using a 96-well microbioassay for activity against C. acutatum, C. fragariae, C. gloeosporioides, B. cinerea, P. obscurans, P. viticola and F. oxysporum. Compound 4c had the most fungicidal activity with 70% growth inhibition at 3.0 lM against P. obscurans (Fig. 3). Compounds 1e, 3b and 4c at 30 lM reduced P. obscurans growth by more than 60%. Secondary screening of active compounds using this microbioassay system showed Mosquitoes were treated with a 0.5 lL solution of each concentration. a LD 50 is an estimate, and therefore a range is given. b Number of insects tested in total. c Slope standard error of the mean. d Degrees of freedom.   dz, diffuse zone. 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. n/a, not applicable. a Mean inhibitory zones and standard error of the mean (SEM) were used to determine the level of antifungal activity against each fungal species. Fig. 3. Growth inhibition of P. obscurans and P. viticola after 120 h using 96 well microdilution broth assay in a dose-response format using the commercial fungicide azoxystrobin and captan as standards.
that five out of six amides, except 3a, inhibited P. viticola growth by 50% at 30 lM, after 120 h exposure (Fig. 3). P. obscurans appeared to be approximately 10 times more sensitive to 4c, because 4c produced 72.8% inhibition at 3.0 lM in P. obscurans and in P. viticola 4c at 30.0 lM produced only 44.3% growth inhibition. While 4c was more active than captan at 3.0 lM, it did not produce 100% growth inhibition even at the higher concentration of 30.0 lM. Captan is an excellent fungicide with a multisite mode of action that is applied to crops such as strawberry at relative high rates, in the range of 1.64 kg (ai/ha), and azoxystrobin, a QoI (quinone outside inhibitor) is applied at 0.131 oz (ai/ha) [23]. Therefore, the most active compound in this series, 4c, is currently not suitable for commercial application with its therapeutic threshold of 3.0 lM; however, it is the analog of choice for further structural activity studies against P. obscurans. Compound 4c contains two trifluoromethyl groups: one in ortho-position of the phenyl ring and another attached to the carbonyl carbon. The same compound was the most active repellent against female Ae. aegypti and An. albimanus.

Conclusions
Twenty trifluoromethylphenyl amides (14 of which were novel) were designed, synthesized and evaluated for insecticidal, repellent and fungicidal activity.
Seven compounds 1b, 1e, 2b, 2e, 3a, 3b, and 3e produced 100% mortality in first instar Ae. aegypti larvae at a concentration of 100 lM after 24-72 h, although the LC 50 for the most active 1e was $143 times higher than for fipronil. Compound 1c was the most active compound in this series against female Ae. aegypti, but the LD 50 for this compound at 24 h was $23,055 times higher than that for fipronil. The same compound, 1c, had highest activity against D. melanogaster; the LC 50 for the 1675 strain was $288 times and the LC 50 for the OR strain $1,400 times higher than that for fipronil. However, unlike fipronil, there was no cross resistance to 1c and 4c in the rdl strain of D. melanogaster.
Compound 4c was the most potent repellent against Ae. aegypti with an MED 2.3 times lower than that of DEET; although 3.5 times higher than DEET for An. albimanus. Compound 4a had an MED comparable to DEET against Ae. aegypti.
None of the active trifluoromethylphenyl amides produced inhibition in the range of azoxystrobin, and none of them showed significant inhibition against C. acutatum, C. fragariae, C. gloeosporioides, B. cinerea and F. oxysporum. We found, that compounds 3b and 4c have the potential to control Phomopsis species.
The structure-activity relationships based on the bioassay results against Ae. aegypti larvae showed that the presence of a trifluoromethyl group in the para-or meta-positions of the phenyl ring of amides increased their larvicidal activity, compared to ortho-trifluoromethylphenyl amides. The presence of a 2,6-dichloro-substitution in para-trifluoromethylphenyl amides increased the larvicidal activity of amides with an aromatic group attached to the carbonyl carbon, and decreased the larvicidal activity of compounds with an alkyl group attached to the carbonyl carbon.
According to repellency bioassay results against female Ae. aegypti and An. albimanus, ortho-trifluoromethylphenyl amides with a trifluoromethyl or an alkyl group attached to the carbonyl carbon produced higher repellent activity than meta-or para-trifluoromethylphenyl amides. Addition of a 2,6-dichloro-substitution decreased the repellent activity of para-trifluoromethylphenyl amide with alkyl group attached to the carbonyl carbon and increased the repellent activity of para-trifluoromethylphenyl amide with trifluoromethyl group attached to the carbonyl carbon.
Although none of the novel compounds synthesized and evaluated were potent insecticides, this study revealed the potential structures that could serve as the basis for further design to find new derivatives with a broad spectrum of activity for controlling pest insects and pathogenic fungi.