Regioselective Claisen–Schmidt Adduct of 2-Undecanone from Houttuynia cordata Thunb as Insecticide/Repellent against Solenopsis invicta and Repositioning Plant Fungicides against Colletotrichum fragariae

The U.S. Department of Agriculture (USDA) has established research programs to fight the phytopathogen Colletotrichum fragariae and the invasive red imported fire ant, Solenopsis invicta. C. fragariae is known to cause anthracnose disease in fruits and vegetables, while S. invicta is known for its aggressive behavior and painful stings and for being the cause of significant damage to crops, as well as harm to humans and animals. Many plants have been studied for potential activity against C. fragariae and S. invicta. Among the studied plants, Houttuynia cordata Thunb has been shown to contain 2-undecanone, which h is known for its antifungal activity against Colletotrichum gloesporioides. Based on the mean amount of sand removed, 2-undecanone showed significant repellency at 62.5 µg/g, similar to DEET (N,N-diethyl-meta-toluamide), against S. invicta. The 2-Undecanone with an LC50 of 44.59 µg/g showed toxicity against S. invicta workers. However, neither H. cordata extract nor 2-undecanone had shown activity against C. fragariae despite their known activity against C. gloesporioides, which in turn motivates us in repositioning 2-undecanone as a selected candidate for a Claisen–Schmidt condensation that enables access to several analogs (2a–f). Among the prepared analogs, (E)-1-(3-methylbenzo[b]thiophen-2-yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f) showed promising activity against C. fragariae, revealing a distinctive structural activity relationship (SAR). The generated analogs revealed a clear regioselectivity pattern through forming the C=C alkene bond at position C-1. These data open the window for further lead optimization and product development in the context of managing C. fragariae and S. invicta.


Introduction
The U.S. Department of Agriculture (USDA) is actively engaged in ongoing research programs to control the spread of the aggressive red imported fire ant (RIFA) and to find

Digging Bioassay
The mean weight (g) of treated sand removed by red imported fire ant workers in a digging bioassay with different concentrations of 2-undecanone and DEET is presented in Figure 1. Repellency was determined based on the minimum repellent effective dose (µg/g) where the quantity of sand removed was similar to that of the ethanol control. Based on the amount of sand removed, 2-undecanone (F 3,8 = 13.36; p = 0.002; Figure 1a) and DEET (F 3,8 = 16.24; p < 0.001; Figure 1b), both showed a significantly higher repellency than ethanol at dosages of 125 and 62.5 µg/g, whereas the activity at 31.25 µg/g was similar to ethanol.
In the present study, we evaluated H. cordata extract and 2-undecanone against S. invicta and C. fragariae and used the rationale repositioning of 2-undecanone as a selected candidate for the Claisen-Schmidt condensation to enable access to several analogs (2a-f) for further evaluation against C. fragariae.

Digging Bioassay
The mean weight (g) of treated sand removed by red imported fire ant workers in a digging bioassay with different concentrations of 2-undecanone and DEET is presented in Figure 1. Repellency was determined based on the minimum repellent effective dose (µg/g) where the quantity of sand removed was similar to that of the ethanol control. Based on the amount of sand removed, 2-undecanone (F3, 8 = 13.36; p = 0.002; Figure 1a) and DEET (F3, 8 = 16.24; p < 0.001; Figure 1b), both showed a significantly higher repellency than ethanol at dosages of 125 and 62.5 µg/g, whereas the activity at 31.25 µg/g was similar to ethanol.

Toxicity Bioassay
Toxicity data of 2-undecanone and bifenthrin at 24 h post treatment against RIFA workers are given in Table 1. 2-Undecanone with LC50 values of 44.59 µg/g showed toxicity against red imported fire ant workers. Bifenthrin with an LC50 value of 0.03 µg/g was more toxic against RIFA than 2-undecanone.

Toxicity Bioassay
Toxicity data of 2-undecanone and bifenthrin at 24 h post treatment against RIFA workers are given in Table 1. 2-Undecanone with LC 50 values of 44.59 µg/g showed toxicity against red imported fire ant workers. Bifenthrin with an LC 50 value of 0.03 µg/g was more toxic against RIFA than 2-undecanone.

Chemistry
2-Undecanone is known for its fungal activity against C. gloesporioides, however, neither H. cordata extract nor 2-undecanone had shown activity against C. fragariae, which motivated us in repositioning 2-undecanone as a selected candidate for the Claisen-Schmidt condensation ( Figure 2).

Direct Antifungal Bioautography Assay
A direct bioautography assay helps to determine the potential of 2-undecanone and its analogs as antifungal agents against anthracnose disease in fruits and vegetables, and it provides valuable information for further studies on their mechanism of action and potential for agricultural applications. Compound 2b showed zones of inhibition of 0.5, 0.91, and 1.2 cm at the tested concentrations of 5, 10, and 15 µL of 10.0 mg/mL prepared stock solution, respectively, while Compound 2f showed zones of inhibition of 0.7, 1.0, and 1.5 cm at the tested concentrations of 5, 10, and 15 µL of 10.0 mg/mL prepared stock solution, respectively ( Table 2).

Direct Antifungal Bioautography Assay
A direct bioautography assay helps to determine the potential of 2-undecanone and its analogs as antifungal agents against anthracnose disease in fruits and vegetables, and it provides valuable information for further studies on their mechanism of action and potential for agricultural applications. Compound 2b showed zones of inhibition of 0.5, 0.91, and 1.2 cm at the tested concentrations of 5, 10, and 15 µL of 10.0 mg/mL prepared stock solution, respectively, while Compound 2f showed zones of inhibition of 0.7, 1.0, and 1.5 cm at the tested concentrations of 5, 10, and 15 µL of 10.0 mg/mL prepared stock solution, respectively ( Table 2). Table 2. Inhibition zones (cm) of the parent compound 1 and the generated analogs 2a-2f against C. fragariae using 10 mg/mL stock solutions.

Discussion
Insecticidal, repellent, and behavioral effects of 2-undecanone have been well established [29,30]. Whang and Tonelli [31] reported that 2-undecanone from its crystalline inclusion compound with α-cyclodextrin repelled 100% of cockroaches for the first 2 days after application. Ali et al. [32] reported that the biting deterrent activity against Aedes aegypti L. of 2-undecanone at 8.5 µg/cm 2 was similar to DEET at 4.8 µg/cm 2 . Ntalli et al. [33] reported that 2-undecanone caused 87.8% and 98.9% mortality of the larvae and adults of Tenebrio molitor L., respectively, at 7 days post exposure to wheat treated at a dose of 1000 µL/kg. Data from the present study demonstrated that 2-undecanone significantly suppressed ant digging activity. It repelled the ants, and the minimum effective dose of repellency was 62.5 µg/g below which the repellency of this natural product failed. The level of ant activity in this treatment and the DEET treatment were similar, suggesting that 2-undecanone can offer promise against the red imported fire ant. The toxicity data from the present study showed that 2-undecanone was toxic to the red imported fire ants. Taken together, our data further support the idea that 2-undecanone can be a promising pest management tool. The Claisen-Schmidt condensation is a valuable chemistry tool invented by Rainer Ludwig Claisen and J. G. Schmidt [34]. The reaction is between an aldehyde or ketone having an α-hydrogen with an aromatic carbonyl compound lacking an α-hydrogen [34]. An example is the synthesis of dibenzylideneacetone (1E,4E)-1,5diphenylpenta-1,4-dien-3-one) [35], where dibenzylideneacetone is used as a component in sunscreens and as a ligand in organometallic chemistry [36]. Using our lead 2-undecanone as a ketone model enables us to generate a library of six analogs. The reaction in general was of a reasonable yield for most of the presented aromatic aldehydes, therefore, the feasibility and the success rate are quite high as a practical approach ( Figure 2). As predicted, with respect to regioselectivity, our products are mainly formed at the 1-position. The model molecules have been examined through ChemDraw 21.0 for their Log P and pKa, where they showed a Log P of~2.5-3.5 and pKa of~8.5-9.5 confirming their favorable drug-like properties. Also, they have shown considerable solubility in the assay aqueous condition at a maximum concentration of 20 µM. Among the prepared analogs, 2b and 2f have shown increased activity against C. fragariae in comparison with the parent molecule 1, which did not show any antifungal activity against C. fragariae. The two active analogs of 2-undecanone, (E)-1-(3-methylbenzo[b]thiophen-2-yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f), demonstrated faint clear zones on the TLC plate, which in turn highlighted the importance of such reactions in the development of fungicidal products. Moreover, compounds 2a-b showed a distinctive SAR, where the introduction of a methyl group at C-3 of the benzothiophene ring (2b) is a promoter for increasing activity against C. fragariae in comparison to the unsubstituted derivative (2a).

General Experimental Procedures
The 1 H and 13 C-NMR spectra were recorded in DMSO-d 6 and CDCl 3 on a Bruker 400 and 500 MHz spectrometer operating at 400, 500 MHz for 1 H and 100, 150 MHz for 13 C NMR. Chemical shift (δ) values are presented in ppm and in reference to the residual solvent signals of DMSO-d 6 and CDCl 3 at δ H /δ C 2.50/39.5 and 7.25/70.2, respectively. The coupling constant (J) is reported in Hz.
The LC analysis of the generated analogs (2a-2f) was conducted using an Agilent 1100 HPLC system, RP-C18 column (150 × 4.6 mm; particle size of 5 µm; Luna) with column oven temperature set at 25 • C, and a gradient system of eluent water (A) and acetonitrile (B). The gradient condition was as follows: 0-2 min (5% B), 2-5 min (5% B → 50% B), 5-10 min (50% B → 100% B), and 10-15 min (100% B). The flow rate of the solvent was 1.0 mL/min, and the injection volume was 25 µL. All analyses were carried out at a wavelength of 254 nm with a run time of 15 min. HPLC-grade acetonitrile and water solvents were used. Acetic acid was added as a modifier to achieve a final concentration of 0.1% in each solvent.
Preparative HPLC purification of the generated analogs (2a-2f) was carried out using an Agilent 1100 HPLC system, RP-C18 column (250 × 10 mm; particle size of 10 µm; Luna) with column oven temperature set at 25 • C and a gradient system of eluent water (A) and acetonitrile (B). The gradient condition was as follows: 0-2 min (5% B), 2-5 min (5% B → 50% B), 5-10 min (50% B → 100% B), and 10-15 min (100% B). The flow rate of the solvent was 3.0 mL/min, and the injection volume was 25 µL. All analyses were carried out at a wavelength of 254 nm with a run time of 15 min. HPLC-grade acetonitrile and water solvents were used. Acetic acid was added as a modifier to achieve a final concentration of 0.1% in each solvent.
Other common chromatographic techniques, such as thin layer chromatography (TLC) on precoated silica gel G 254 aluminum plates and silica gel flash column chromatography, were also engaged in the purification of the synthesized compounds.

Imported Fire Ants
RIFA used in these bioassays were collected from Washington County, MS 38748 (33 • 09 31.2 N 90 • 54 56.4 W). A plastic tray was used to contain the colony, and the tray walls were coated with Insect-a-Slip (BioQuip Products, 2321 Gladwick Street, Rancho Dominguez, CA 90220, USA) to prevent the fire ants from crawling out. A 25% solution of water and sugar was provided to the ant colonies, and crickets were added weekly as food sources. A water-filled test tube plugged with cotton was added to serve as a moisture source. The laboratory conditions were set at 27 ± 2 • C and 60 ± 10% RH with a photoperiod regimen of 12:12 h (L:D). Ants were maintained under this setting for one month before starting the bioassays. The venom alkaloid and hydrocarbon indices of the workers were used for the identification of the imported fire ant species [37,38].

Digging Bioassay
The digging bioassay used in this study for testing repellency was described by Ali et al. [37]. Briefly, the bioassay consisted of a 150 mm × 15 mm Petri dish (Fisher Scientific Co. LLC, 2775 Horizon Ridge CT, Suwanee, GA 30024, USA) and four 2 mL Nylgene Cryoware Cryogenic vials (Thermo Fisher Scientific, Rochester, NY 14825, USA), which were glued to the lower side of the Petri dish. The inner walls of the Petri dish were coated with Insect-a-Slip (BioQuip Products, 2321 Gladwick Street, Rancho Dominguez, CA 90220, USA). Four grams of sand (Premium Play Sand, Plassein International, Longview, TX, USA) of uniform size (500 microns) were weighed in fluted aluminum (45 mL size) dishes (Fisher Scientific, 300 Industry Drive, Pittsburgh, PA 15275, USA), and each treatment was added at 100 µL/g of sand. The control sand received treatment with ethanol only. After the solvent had evaporated, the sand was moistened by adding 0.6 µL/g of de-ionized water and was filled into the treatment vials, which were then screwed to the Petri dish. Each vial contained a mean weight of 3.6 g of sand on a dry weight basis. Fifty imported fire ant workers were released per Petri dish. The experiments were conducted at 25 ± 2 • C temperature and 50 ± 10% relative humidity for a 12 h (L:D) photoperiod. After 24 h, sand from the treatment vials was collected back into aluminum dishes, oven dried at 190 • C for 1 h, and weighed. Both 2-undecanone and DEET (N,N-diethyl-meta-toluamide) were tested. DEET was tested as a positive control for comparison whereas ethanol was included as a negative control. Serial doses were started from a dose of 125 µg/g until the failure of the treatment. The stock solution and concentrations were prepared in ethanol. Overall, three sets of experimental replicates were run on 3 different days. Sand removal data were analyzed using Analysis of Variance (ANOVA) and means were separated using Ryan-Einot-Gabriel-Welsch multiple range test (p ≤ 0.05; SPSS, ver. 25), (Figure 1).

Toxicity Bioassay
The toxicity bioassay used for determining mortality in imported fire ants was described by Ali et al. [37]. Briefly, 3 g of sand was weighed into fluted 42 mL aluminum dishes and treated with 300 µL of treatment solution. Control sand was treated with ethanol only. The solvent was evaporated, and 0.6 µL/g of de-ionized water was added to moisten the sand. Treatment sands were transferred into 60 × 15 mm stackable Petri dishes (KORD-VALMARK, Mfg by Bioplast Manufacturing, L.L.C. 128 Wharton Road, Bristol, PA 19007, USA). Ten fire ant workers were released per treatment/replicate. A wet cotton swab tip was added to the Petri dish 1 h post release of the workers to ensure continuous availability of the moisture. The number of dead workers was recorded at 24 h post treatment. LC 50 values were calculated using probit analysis (SAS 2012), (Table 1).

Direct Bioautography Assay
The direct bioautography assay was conducted to assess the antifungal activity of 2-undecanone and its six analogs against the phytopathogen C. fragariae. First, the inoculum of C. fragariae was prepared by harvesting fresh conidia from a 7-10-day old fungal culture grown on 1 2 strength potato dextrose agar (PDA). The conidia were collected by flooding the plate with 10 mL of sterile water and scrapping it with a sterile L-shaped spatula. The resulting spore suspension was filtered through a sterile double Mira cloth (Calbiochem-Novabiochem Corp., La Jolla, CA, USA) to remove mycelia and agar pieces, and the spores were counted using a Countess 3 cell counter. The spore suspension was centrifuged at 1968 RCF for 10 min, and the supernatant was discarded. The spore concentration was then adjusted to 3 × 10 5 spores mL −1 by adding the required volume of PDB-TLC media (12.5 g PDB, 0.5 g agar, 0.5 mL Tween 80 in 500 mL of water) to obtain the inoculum for the bioautography assay.
Subsequently, 10 µL of each compound (2-undecanone and its six analogs 2a-f) was spotted twice onto a silica gel plate (Analtech, Inc., Newark, DE, USA, Silica Gel GHLF, 250 microns). The inoculum of C. fragariae was then uniformly sprayed onto the silica gel plate using a hand sprayer, ensuring that the spores were evenly distributed across the plate. The TLC plate was placed in a moisture chamber box with 99.9% humidity and incubated at approximately 26 • C for 4 days, which provides optimal conditions for the growth of C. fragariae. After incubation, the antifungal activity of the compounds was evaluated by the presence of clear zones on the TLC plate, which indicates the inhibition of fungal growth. Captan (>98%, Chem Service, Inc., West Chester, PA, USA) and fludioxonil (>99.5%, Chem Service, Inc., West Chester, PA, USA), which are technical-grade fungicides, were used as positive controls at a concentration of 2 mg/mL in ethanol, with 1 µL of each control applied on the plate to compare the antifungal activity of the test compounds.

Conclusions
H. cordata Thunb, has been shown to contain 2-undecanone, which is known for its fungal activity against C. gloesporioides; however, it does not possess activity against C. fragariae. Repositioning 2-undecanone via a Claisen-Schmidt condensation enables access to six analogs (2a-f). Among these analogs, (E)-1-(3-methylbenzo[b]thiophen-2yl)dodec-1-en-3-one (2b) and (E)-1-(5-bromothiophen-2-yl)dodec-1-en-3-one (2f) showed promising activity against C. fragariae, revealing an interesting SAR and clear regioselectivity pattern through the formation of the C=C alkene bond at position C-1. Alongside that, 2-Undecanone significantly suppressed the digging activity at 62.5 µg/g, similar to DEET, against red imported fire ants, and it showed toxicity with an LC 50 of 44.59 µg/g against fire ant workers. These data suggested that 2-undecanone may play a useful role in the integrated management of C. fragariae and S. invicta. Data Availability Statement: Data relevant to the paper will be available from the authors upon request.