Semisynthesis and insecticidal activity of some novel fraxinellone-based thioethers containing 1,3,4-oxadiazole moiety

Two series of novel fraxinellone-based thioethers containing 1,3,4-oxadiazole moiety were prepared as insecticidal agents against the oriental armyworm, Mythimna separata Walker. The structural assignment was based on the spectroscopic and X-ray analysis data. Among all the target compounds, compounds 4b, 4k, 5b, 5j and 5k exhibited more potent insecticidal activity with final mortality rates (FMRs) of more than 65%, especially 4k with the FMR of 75.9%, when compared with toosendanin. Some interesting results of structure–activity relationships are also discussed.


Introduction
The oriental armyworm (Mythimna separata Walker, Lepidoptera: Noctuidae), a typical long-distance migratory insect, is one of the most serious pests of cereal crops in countries including China, India, Australia and New Zealand [1,2]. Seasonal outbreaks of this pest can cause significant economic damage to cereal crops in China and other countries [3]. A recent outbreak of M. separata has been reported in northeast and central China during 2012, which caused losses of approximately 10 million acres of crops [4]. Synthetic chemical pesticides play a crucial role in agriculture with the characteristics of high-efficiency,   quick-fix and broad-spectrum insecticides. Although they have been extensively used to control insect pest outbreaks, the overuse and improper application of synthetic chemical pesticides over the years has resulted in enhancement of pest resistance, environmental problems and negative impacts on human health [5][6][7]. Hence the discovery and development of effective, selective and eco-friendly pesticides is necessary in the future.
Fraxinellone (1, figure 1), a naturally occurring degraded limonoid, isolated from Fagaropsis glabra, [8] Dictamnus albus, [9] Melia azadarach [10] and Dictamnus dasycarpus, [11] exhibits a variety of interesting activities both in the fields of medicinal chemistry and agrochemistry, such as anti-inflammatory [12], vascular relaxing activity [13] and insecticidal activity [14][15][16]. The total synthesis of fraxinellone can be easily achieved, which has been reported early in 1972 [17]. Previously, we have studied the insecticidal activity of some fraxinellone-based hydrazones and esters [18,19] (I-IV, figure 1) modified at the C-4 or C-10 position in the A ring of fraxinellone, and N-phenylpyrazole fraxinellone hybrid compounds [20] (V, figure 1), and found some compounds against M. separata displayed higher insecticidal activity than positive control toosendanin. To the best of our knowledge, little attention has been paid to the introduction of active N-heterocyclic moieties on the furyl-ring of fraxinellone as insecticidal agents. 1,3,4-Oxadiazoles are an important class of N-heterocyclic compounds with a wide range of biological activities [21] including antimicrobial, analgesic, anticancer activities, especially insecticidal and herbicidal activities [22,23]. In a continuation of our programme aimed at the development of fraxinellone-based insecticidal agents, herein we prepared two series of novel fraxiellone-based thioethers containing 1,3,4-oxadiazole moiety (VI and VII, figure 1) as insecticidal agents against M. separata.

Instrument and materials
The intermediate 2-mercapto-5-aryl-1,3,4-oxadiazoles a-k (scheme 1) were synthesized as previously reported [24]. Other reagents were of analytically grade and purchased from commercial resources. Fraxinellone (1) was isolated from Dictamnus dasycarpus and its purity was more than 99% as measured with reverse phase high-performance liquid chromatography (RP-HPLC). Analytical thinlayer chromatography (TLC) and preparative thin-layer chromatography (PTLC) were prepared by silica gel plates using silica gel GF 254

General procedure for synthesis of compounds 2 and 3
To a stirred suspension solution of AlCl 3 (1.0 mmol) in dry CH 2 Cl 2 (5 ml) at RT, chloroacetyl chloride (1.1 mmol) was added. The mixture was then stirred for 10 min, and a solution of compound 1 (1.0 mmol) in dry CH 2 Cl 2 (5 ml) was added dropwise to the above mixture. When the reaction was complete according to TLC analysis, the reaction mixture was poured into ice water (15 ml) and extracted with CH 2 Cl 2 (40 ml × 3). The combined organic phase was washed with saturated brine (40 ml), dried over anhydrous Na 2 SO 4 , concentrated in vacuo, and then purified by PTLC to give the pure products 2 (35% yield) and 3 (45% yield).  2.3. General procedure for synthesis of compounds 4a-k and 5a-k A mixture of the corresponding 2-mercapto-5-aryl-1,3,4-oxadiazole (0.3 mmol), 2 or 3 (0.2 mmol, 61.6 mg), K 2 CO 3 (0.3 mmol, 41.5 mg) and KI (0.05 mmol, 8.3 mg) in acetone (10 ml) was stirred at room temperature. After the reaction was complete according to TLC analysis, the solvent was removed and the residue was dissolved in CH 2 Cl 2 and filtered. The filtrate was concentrated in vacuo and purified by PTLC to give pure products 4a-k and 5a-k. The example data of 4a-d and 5a-d are described as follows, whereas the data of other compounds 4e-k and 5e-k are shown in the electronic supplementary material.

X-ray crystallography
The structures of compounds 4e and 5d were unambiguously confirmed by X-ray crystallography. Crystallographic data (excluding structure factors) of compounds 4e and 5d were deposited at the Cambridge Crystallographic Data Centre (CCDC) with deposition numbers of CCDC 1552786 and 1552787, respectively.

Biological assay
Growth inhibitory activity of compounds 1-3, 4a-k and 5a-k against M. separata was evaluated by leafdipping method as described previously [19,25]. For each compound, 30 pre-third-instar larvae of same size and level of health (10 larvae per group) were chosen as the tested pests. Solutions of compounds 1-3, 4a-k and 5a-k and toosendanin (used as a positive control) were prepared in acetone at the concentration of 1 mg ml −1 . The larvae of tested groups were fed with compound-coated leaves (fresh corn leaf discs (1 × 1 cm) were dipped into the corresponding solution for 3 s, then taken out and dried at RT), whereas the blank control group (CK) was fed with acetone alone. Several treated leaf discs were kept in each dish.                    Once the treated leaves were consumed, the corresponding ones were added to the dish. The experiment was carried out at 25 ± 2°C; relative humidity (RH) 65-80%, and on 12 h/12 h (light/dark) photoperiod. After 48 h, untreated fresh leaves were added to all dishes until the adult emergence. The corrected mortality rate values of the tested compounds were calculated by the following formula: corrected mortality rate (%) = (T -C) × 100/(1 -C); Where T is the mortality rate in the treated group, and C is the mortality rate of CK.

Synthesis
As shown in scheme 1, compounds a-k were synthesized based on previously reported literature [24]. Using different hydrazides as starting materials, compounds a-k were prepared by cyclization reaction of different hydrazides with carbon disulfide in the presence of KOH in EtOH at reflux temperature,

Insecticidal activity
The growth inhibitory activity of compounds 1-3, 4a-k and 5a-k against M. separata was tested at 1 mg ml −1 . Toosendanin, a commercial insecticide derived from Melia azedarach, was used as the positive control at 1 mg ml −1 , and corn leaves treated with acetone alone were used as a blank control. As shown in table 1, compounds 4b, 4c, 4j, 4k, 5b-d, 5j, 5k exhibited higher insecticidal activity than toosendanin and their precursor fraxinellone. For example, the final mortality rates (FMRs) of compounds 4b, 4c, 4j,  4k, 5b-d, 5j, 5k were 72.4%, 55.2%, 62.1%, 75.9%, 69.0%, 62.1%, 55.2%, 65.5% and 69.0%, respectively. In particular, compound 4k showed the most potent insecticidal activity, which was about 24% higher than toosendanin. The symptoms for the tested M. separata during the different periods of larval, pupation and adult were recorded by the same methods as our previous reports [19,25]. For example, in treated   groups, due to overfeeding of treated leaves in the beginning, some larvae died slowly with thin and wrinkled bodies ( figure 5). This phenomenon maybe results from these fraxinellone derivatives effects to nutritional or digestive interference [26]. During the pupation stage, some of the larvae did not successfully moult to normal pupae, and died ( figure 6). In the last stage of emergence, many malformed moths appeared with shrunken or immature wings ( figure 7). These results suggest that the fraxinellone derivatives containing the 1,3,4-oxadiazole probably affected the insect moulting hormone, which was crucial for the growth of M. separata. On the other hand, as displayed in figure 8, the percentages of FMRs  of compounds 4b, 4j, 4k, 5b, 5c, 5j, 5k and toosendanin at three different growth stages of M. separata were investigated. We found that at least 50% of FMRs for compounds 4b, 4k, 5c, 5j, 5k and toosendanin were at the larval period except for compounds 4j and 5b.  Finally, we also discovered some interesting results of structure-activity relationships of the tested compounds.

Conclusion
In summary, we have prepared two series of novel fraxinellone-based thioethers containing 1,3,4oxadiazole and evaluated for their insecticidal activity against a cereal crop-threatening agricultural insect pest, M. separata. The structures of key compounds 4e and 5d were assigned by X-ray crystallography. Among all target compounds, compounds 4b, 4k, 5b, 5j and 5k exhibited more potent insecticidal activity with FMRs of more than 65%. The results suggested that the introduction of 3/4pyridinyl or o-fluoro/chlorophenyl units on the 1,3,4-oxadiazole ring to the compound 2 or 3 could afford more promising compounds. This will lay the foundations for further structural modification and application of fraxinellone as novel pesticidal agents in agriculture.
Ethics. The study was approved by the Research Ethics Committee of Zhengzhou University, Henan Province, PR China.