Cantharidin and Its Anhydride-Modified Derivatives: Relation of Structure to Insecticidal Activity

Cantharidin is a natural compound of novel structure with ideal insecticidal activity. However, the relationship of structure to insecticidal activity of cantharidin and its derivatives has not been ever clarified. To explore what determines the insecticidal activity structurally of cantharidin-related compounds, two series target compounds 6 and 7 were synthesized by replacing the anhydride ring of norcantharidin with an aromatic amine or fatty amine with different electron density, respectively. The structures of these compounds were characterized by 1H NMR, 13C NMR and HRMS-ESI. A bioassay showed that compounds 6 (a–m) lacked any larvicidal activity against Plutella xylostella; whereas their ring-opened partners 7 (a–m) provided a variety of larvicidal activities against P. xylostella, and compound 7f indicated the highest larvicidal activity with LC50 value of 0.43 mM. The present work demonstrated that the form of the compound (cyclic or ring-opened) or their ability to hydrolyze facilely was the key to determine whether it exhibits larvicidal activity. Moreover, it revealed that the improvement of insecticidal activity required a reasonable combination of both aliphatic amide and aromatic amide moieties, and the type of substituent Y on the aniline ring was critical.


Introduction
The blister beetle Mylabris phalerata (Coleoptera: Meloidae) has been used for centuries as a traditional Chinese medicine. Cantharidin 1 (Figure 1), the active ingredient in the blister beetle toxin, has served as a valuable drug against several kinds of cancer. Similar to what occurs when a variety of human cancer cell lines are co-cultured with cantharidin, insect cell lines Spex-VII and Sf9 can be induced to undergo apoptosis by this toxin [1][2][3][4]. Among the novel applications of cantharidin and its analogs were preliminary studies of the anti-insect activity of this natural product. Cantharidin has been confirmed to be toxic to arthropods in a diversity of orders: Coleoptera, Diptera, Hymenoptera, Homoptera, Lepidoptera and Acarina [5][6][7]. Apoptosis-like symptoms of insect tissues (midgut, Malpighian tubules, testes, fat body and epidermis) due to cantharidin poisoning of insects were observed under the transmission electron microscope [8][9][10]. Additionally, the activities of acid phosphatase, alkaline phosphatase and polyphenol oxidase in cantharidin-treated Mythimna separata significantly declined [11].
Whereas the procedure for synthesizing cantharidin under assay conditions is complex and difficult [12][13][14], its analogs are much easier to be synthesized and are promising substitutes for cantharidin. However, cantharidin derivatives possess different structures and are likely to have either higher or lower insecticidal effects.
Our study is designed to explore the relationships between the structures and insecticidal activities of cantharidin analogs. Two series of anhydride-modified cantharidin analogs were designed and synthesized with norcantharidin 3 (Figure 1) as the parent compound. Among them, 6b, 6g, 6m, 7a, 7b, 7e and 7f, seven derivatives, were new compounds. Their insecticidal activities were evaluated against the pre-third-instar larvae of Plutella xylostella, in vivo.

Chemistry
As shown in Scheme 1, the target analogs 6 were synthesized by the reaction of norcantharidin 3 and various primary amines in the presence of triethylamine as the binding acid agent [15], and the analogues 7 were synthesized by replacing the anhydride ring of norcantharidin 3 with an aromatic amine or fatty amine of different electron density [16].
Compound 5 was synthesized to imitate the synthetic procedure of phthalimide [17], as shown in Scheme 2. As can be seen from Schemes 3 and 4, compounds 1 and 3 underwent a rapid conversion to dicarboxylic acid (compounds 2 and 4) under assay conditions, or through the slow but facile way that the hydrolyzing occurred at room temperature.
Thus the hydrolytic stabilities of analogs 6 presented in Table 1 were examined. We noted that analogs 6 were stable under assay conditions, showing no decomposition even under infrared radiation (typically no decomposition after 12 h treatment).

Insecticidal Activity
After a period of starvation, released larvae wriggled around and attempted to eat the nearby treated leaves. They would not present a series of symptoms until a certain amount of the drug was taken. All active compounds caused the same symptoms.
Unlike most commercial pesticides currently used, the poisoned larvae gradually lost their locomotive ability and were powerless to resist mechanical irritations. Meanwhile, compared with the healthy larvae (Figure 2A,B), a darker patch often appeared anteriorly on each dying larva ( Figure 2C), which would spread gradually to the whole bodies until they died ( Figure 2D). Wet, green frass might stick to the anal areas of dying larvae ( Figure 2D), and mucus was frequently observed at the end of the abdomen of dead larvae, which either was kept between the fourth pair of prolegs and caudal prolegs ( Figure 2E), or it glued dying larvae to leaves or filter papers ( Figure 2F) indicating the experimental drugs might disrupt the digestive system in larvae. In addition, cantharidin is well known as a strong inhibitor of serine/threonine protein phosphatases (PPs) [18], a broad class of PPs associated with signaling and control of numerous cellular processes in many organisms [19]. The toxicity associated with cantharidin is derived from its ability to inhibit the family of PPs. However, the catalytic domain of all PP subfamilies is highly conserved in animals, plants, protozoans and all eukaryotes [20]. Inhibitors, such as cantharidin and norcantharidin, bind to a hydrophobic pocket of the PP active site [21]. The structural similarity between cantharidin and norcantharidin has been apparent to animal scientists and similar mechanisms of action on animal serine/threonine PPs have been confirmed [22]. Like all other eukaryotes, the family of serine/threonine protein phosphatases belonging to insects and related arthropods should be considered since it is highly similar to that of mammals. Hence, it is deduced that PPs are the potential target sites of insecticidal activity of cantharidin and its related compounds. Meanwhile, because of the high conservation of the catalytic domain of all PP subfamilies, cantharidin and its derivatives are indeed toxic to all eukaryotes, not just insects.

SAR
Among the sixteen compounds listed in Table 1, only cantharidin 1 and norcantharidin 3 showed any significant larvicidal activities with 100% mortality at concentrations of 500 μg mL −1 , respectively. Interestingly, the replacement of the anhydride oxygen atom of norcantharidin 3 with nitrogen (compound 5) resulted in complete loss of activity. Meanwhile, Table 1 showed that the target compounds 6 also lacked any larvicidal activities. Indeed, to result in any larvicidal activity, no modifications of the cyclic anhydride are tolerated, consequently resulting in larvicidal activity. We thought the replacement of the O-atom with N (as N-H and N-R, where R = alkyl of aryl) would furnish us with a better understanding of the relationship between the electronic effect and larvicidal activities; however no larvicidal activity was demonstrated for the modified cyclic anhydride.
To examine the electronic effect of substituent Y on the aniline ring, the electron-donating substituent -OCH 3 and electron-withdrawing substituents -CF 3 , -OCF 3 , F and -CO 2 H were introduced. Compounds with electron-withdrawing substituents displayed higher larvicidal activities against P. xylostella than compounds with electron-donating substituents, as seen in the comparison of the compounds 7d (Y = 2'-OCH 3  Compounds with electron-withdrawing substituents showed excellent larvicidal activities against P. xylostella, while compounds with electron-donating substituents display lower larvicidal activity. In addition, as shown in Table 2, compound 2, 4 and 7f were the most active compounds. All of their larvicidal activities against P. xylostella at 500 μg mL −1 were 100% after 48 h, while the larvicidal activity of the parent compounds 1 and 2 were 100% at the same concentration after 48 h as shown in Table 1. These results indicated that compounds 2, 4 and 7f displayed comparable larvicidal activity with their corresponding lead compound against P. xylostella at 500 μg mL −1 . Therefore, we carried out further insecticidal activity assay for compounds 2, 4 and 7f, and cantharidin 1 and norcantharidin 2 were used as a control to make a judgment on the larvicidal potency of these compounds. As shown in Table 3, it was found that the LC 50 values of compounds 4 and 7f against P. xylostella were 0.70 mM and 0.43 mM respectively, while that of the parent compound norcantharidin was 0.84 mM. Moreover, the LC 50 of compounds 2 was the same as that of its parent compound cantharidin 1 with value of 0.06 mM. The larvicidal activity values of anhydride analogs in Table 1 were compared with those of their ring-opened dicarboxylic acid partners in Table 2. The anhydride analogs indicated no any larvicidal activity, except 1 and 3, as mentioned above. However, their ring-opened dicarboxylic acid partners all suggested various larvicidal activities. On the one hand, the larvicidal activities of cantharidin 1 and norcantharidin 3 were equal to that of their ring-opened partners, compounds 2 and 4 with LC 50 values of 0.06 mM, 0.84 mM, 0.06 mM, and 0.70 mM in Table 3, respectively. And the larvicidal activities of cantharidin 1 and norcantharidin 3 appeared to be the results of the spontaneous hydrolyzing process to ring-opened compounds 2 and 4. On the other hand, compared with 1 and 3, compounds 6 were so stable that they could not be hydrolyzed to their ring-opened partners naturally, which leads to the absence of larvicidal activity. Therefore, we conjectured that the active form of cantharidin and its derivatives is the dicarboxylic acid analog, a ring-opened compound.

General Experimental Procedures
Melting points of all compounds were determined on a WRS-2 apparatus (Shanghai Precision & Scientific Instrument Co. Ltd., Shanghai, China) and are uncorrected. 1 H NMR and 13 C NMR spectra were obtained using a Bruker AVANCE-500 MHz spectrometer in CDCl 3 or DMSO solution with tetramethylsilane as the internal standard. Chemical shift values (δ) were given in parts per million (ppm). High-resolution mass spectrometry (HRMS) data were obtained on a Bruker micrOTOF-Q II instrument.
Cantharidin 1 was isolated from Mylabris phalerata (Chinese blister beetle), which was bought from the Chinese herbal medicine market in Xian, China. Norcantharidin 3 was purchased from Alfa Aesar Chemical Co. Ltd. (Haverhill, MA, USA). All primary amine reagents were of analytical reagent grade. Solvents were dried and purified using standard techniques immediately before use. Silica gel for TLC and CC was obtained from Qingdao Haiyang Chemical Co. Ltd. (Qingdao, China).

General Synthetic Procedure for Compound 2
To a solution of cantharidin 1 (390 mg, 2 mmol) in water (10 mL) was dropwise added a solution of sodium hydroxide (180 mg, 4.5 mmol) in water (5 mL). This was stirred at 100 °C for 2 h. After the mixture had cooled to room temperature, a solution of hydrogen chloride (4.5 mmol) in water (9 mL) was added dropwise to the mixture and stirred overnight at room temperature. The white slurry was filtered and the cake washed with dichloromethane. The crude solid was recrystallized from methanol to give 2 as white solid (400 mg, 93%). mp: 232-233 °C. 1

General Synthetic Procedure for Compound 4
A mixture of norcantharidin 3 (1.0 g, 5.95 mmol) and water (15 mL) was refluxed for 8 h. After the mixture had cooled to room temperature, the reaction was concentrated in vacuo and filtered. The resulting precipitate was recrystallized from methanol to obtain compound 4 as white solid (960 mg, 87%). mp: 137-138 °C. 1

General Synthetic Procedure for the Target Compounds 6 (a-m)
Primary amine (1 equiv, 5.59 mmol) was added to a stirred mixture of norcantharidin 3 (1.0 g, 5.95 mmol), triethylamine (1.5 mL) and toluene (15 mL). The reaction was carried out at reflux temperature, and the progress was monitored by TLC. After completion, the mixture was moved to vacuo. The residue was either recrystallized from EtOAc/Hexane (1:1, v/v) or purified by column chromatography on a silica gel using EtOAc and hexane (EtOAc/Hexane, 2:5, v/v) as the eluent to afford the target compounds 6. The yields, physical properties, 1 H NMR, 13

General Synthetic Procedure for the Target Compounds 7 (a-m)
A mixture of norcantharidin 3 (1.0 g, 5.95 mmol), primary amine regent (1 equiv, 5.95 mmol) and THF (10 mL) was stirred for several hours at room temperature, and the progress of reaction was monitored by TLC. After norcantharidin disappeared, the reaction was concentrated under reduced pressure and diluted with acetone (100 mL). The resulting precipitate was either recrystallized from methanol or purified by column chromatography (MeOH/CH 2 Cl 2 , 1:4, v/v) to afford the desired products 7. The yields, physical properties, 1 H NMR, 13