Larvicidal and Nematicidal Activities of 3-Acylbarbituric Acid Analogues against Asian Tiger Mosquito, Aedes albopictus, and Pine Wood Nematode, Bursaphelenchus xylophilus

Widespread concern for the occurrence of resistant strains, along with the avoidance of the use of highly toxic insecticides and their wide environmental dispersal, highlights the need for the development of new and safer pest control agents. Natural products provide inspiration for new chemical entities with biological activities, and their analogues are good lead compounds for the development of new pest control agents. For this purpose, we evaluated the larvicidal and nematicidal activities of 48 3-acylbarbituric acid analogues against the Asian tiger mosquito, Aedes albopictus and the pine wood nematode, Bursaphelenchus xylophilus, organisms of increasing global concern. Among the 48 3-acylbarbituric acid analogues, four compounds—10, 14d, 14g and 19b—showed >90% larvicidal activity against Ae. albopictus at 10 μg/mL concentration, and one (compound 10) showed the strongest larvicidal activity against Ae. albopictus, with a LC50 value of 0.22 μg/mL. Only compound 18 showed strong nematicidal activity against pine wood nematode. Most active compounds possessed similar physicochemical properties; thus, actives typically had ClogP values of around 1.40–1.50 and rel-PSA values of 16–17% and these similar cheminformatic characteristics reflect their similar structure. This study indicates that active 3-acylbarbituric acids analogues have potential as lead compounds for developing novel mosquito control agents.


Physicochemical Property-Larvicidal Activity Relationships
The barbiturate library, whose synthesis and characterization have been previously documented [16], comprised a series of substituted systems, varying in ring and nitrogen groups R 1 , R 2 , and R 3 (which include a range of aliphatic and aromatic substituents, with different degrees of side chain functionality) as shown in Figure 1. Of interest is that only a small subset of the examined compounds exhibited larvicidal activity against Ae. albopictus (>70% mortality at 10 µg/mL), and the most active systems, shown in Figure 2, included the structurally related group-14c, 14d, 14e, 14f, 14g-with a disubstituted pendant aryloxypropanoyl side chain with methyl/and or chloro groups. All aliphatic side chains were inactive, with the exception of the cyclohexylpropyl system 2b, although of interest was the high activity of the complex system 10. By contrast, there was a much weaker spectrum of nematicidal activity against Bursaphelenchus xylophilus, for which only compound 18 was active, with no closely related aromatic substituted systems showing any activity. This activity pattern is of interest, because it offers the possibility of selective activity against either Ae. Albopictus or Bursaphelenchus xylophilus. With the exception of compound 10, all active compounds possessed similar physicochemical properties (Table 3); thus, actives typically had ClogP values of around 1.40-1.50 and rel-PSA values of 16-17% and this reflects their similar structure. Of interest is that of the remaining inactive compounds, only compounds 7, 13, 14a, 15c, 16, 20 and 24 possessed this combination of values of ClogP and rel-PSA, suggesting that compounds which are similar in structure are broadly similar in activity, and the converse. The physicochemical properties of barbituric acid analogues and antibacterial activity relationships has been discussed in a previous study [16], where barbiturates with antibacterial activity were found to possess physicochemical properties as follows: −3.0 < ClogD 7.4 < 2.0; 0 < ClogP < 3.0; 60 < PSA < 120 Å 2 ; 10 < rel-PSA < 30% and 270 < MSA < 650 Å 3 as well as acceptable molecular weight (< 400 Da), rotatable bonds (usually less than 6), and appropriate numbers of proton-donor (1-2) and -acceptor groups (4-6).
Interestingly, 3-acylbarbituric acids with positive larvicidal or nematicidal activities possess a much narrower range of physicochemical properties. This result suggests that the physicochemical properties of active barbituric acids might provide higher permeability of compounds to the target sites not only of bacteria, but also nematodes and insects. The modes of action of structurally different larvicides or nematicides have been reported; for example, a recent study showed that the mode of action of four flavonoids and two fatty acids isolated from Millettia pinnata seed against three mosquito species-Aedes aegypti, Ae. albopictus, and Culex pipens pallens-was closely related to the acetylcholinesterase or octopaminergic systems [21]. Avermectin and emamectin benzoate, widely used to control pine wood nematode in Korea, are known to act as agonists of GABA receptors on neuromuscular cells [22]. Wang et al. [23] reported that the mode of action of Huanong AVM, an analogue of avermectin, against pine wood nematode might be related to PIP Kinase family members. However, in the work reported here, the detailed mode of action of active barbituric acids was not determined, and future work will be required to investigate this in detail.  Compound 10 is particularly interesting, because its chemical structure and physicochemical properties are different from the other active barbituric acids. It will be necessary in a future study to examine the structure-activity relationship of compound 10 and its analogues to know the essential pharmacophore for larvicidal activity against mosquito. Table 3. Physiocochemical properties of all barbiturates (see Figure 2).  Compound 10 is particularly interesting, because its chemical structure and physicochemical properties are different from the other active barbituric acids. It will be necessary in a future study to examine the structure-activity relationship of compound 10 and its analogues to know the essential pharmacophore for larvicidal activity against mosquito. Table 3. Physiocochemical properties of all barbiturates (see Figure 2).

Chemicals
Chemical structures of 48 barbituric acids analogues are shown in Figure 1. Their synthesis and characterization have been previously documented [16]. Temephos (purity 95.6%) was used as a positive control and was purchased from Sigma-Aldrich (Milwaukee, WI, USA).

Insects
Ae. albopictus were reared in the laboratory, without exposure to any insecticides. Ae. albopictus adults were supplied with a 10% sugar solution. A live mouse was used for blood under a Korea National Institute of Health Institutional Animal Care and Use Committee (KCDC-020-11-2A) protocol approved for this study. Larvae were reared in plastic pans (24 cm × 35 cm × 5 cm) at 26 ± 1 • C, with a relative humidity of 60 ± 5%, under a 16:8 h light:dark cycle. Ae. albopictus larvae were supplied with sterilized diet (Super TetraMin ® , Sewha Pet Food CO. Seoul, Korea) for food.

3.3.Collection of the Pine Wood Nematode
Pine wood nematode, B. xylophilus, was isolated from infected Pinus densiflora wood collected in the Haman-region, Gyeongsangnam-do province, Korea. Baermann funnel method was used to extract the pine wood nematode. The colony was maintained on a lawn of Botrytis cinerea cultured on potato dextrose agar medium (PDA) in the dark at 28 • C.

Larvicidal Activity Test
A larvicidal activity test followed the method of a previous study with slight modification [24]. Briefly, barbituric acids were serially diluted from an initial 0.01% (w/v) stock solution prepared in acetone. One mL of each compound was suspended in 200 mL of water in 270 mL paper cups. Ten early third instar larvae of Ae. albopictus were moved individually into a paper cup by using a glass pipette. A set of paper cups treated with 1 mL of acetone only was used as control. Treated and control larvae were kept at the same conditions used for colony maintenance, and dead and live larvae were counted 48 h after treatment. Food was not supplied to larvae during the bioassay. All treatments were replicated four times.

Nematicidal Activity Test
To test the nematicidal activity of barbituric acids, test compounds were dissolved in ethanol (1 mg/mL). Barbituric acid solutions (1 µL) were added to the wells of a 96-well plate (Falcon, USA) containing 50~150 nematodes (mixture of juvenile and adult nematodes, male:female:juvenile ≈ 1:1:2) in 99 µL of water. The total volume of the solution in each well was 100 µL, and the concentration of the test barbituric acids was 10 µg/mL. Four wells treated with ethanol (1 µL) only, in the same volume as the test samples, served as controls. In four adjacent wells (i.e., in a column) on the plate, nematodes were treated with barbituric acids and a set of other barbituric acids was placed in the wells of every next column. All experiments were repeated four times. The order of barbituric acids was randomly determined. The tested plates were kept in the dark at 25 ± 1 • C and 60% relative humidity. Pine wood nematode mortality was investigated after 48 h of treatment as follows: 10 µL of the test suspension was transferred to 100 µL of fresh water with a micropipette. Ten minutes after transfer, nematode mortality was observed under a microscope. Nematodes were classified as dead if their bodies were motionless and straightened.

Statistical Analysis
The percentages of mortality of Ae. albopictus mosquito larvae and pine wood nematode were transformed to arcsine square-root values prior to analysis of variance (ANOVA). Treatment mean values were compared and separated using Scheffe's test. Mean ± SE values of untransformed data have been reported. The LC 50 was estimated by probit analysis [25].

Conclusions
In this study, larvicidal and nematicidal activities of 48 3-acylbarbituric acids analogues against Asian tiger mosquito and pine wood nematode were evaluated. Among test compounds, compounds 10, 14d, 14g and 19b showed very strong larvicidal activities, and only compound 18 exhibited strong nematicidal activity. Structure-activity relationships study indicated that all active compounds except compound 10 possessed similar physicochemical properties. Further studies including safety of active 3-acylbarbituric acids analogues to humans and non-target organisms, formulations, and their modes of action are necessary to develop the practical use of 3-acylbarbituric acids analogues as novel mosquito-control agents.