Total Synthesis and Antidepressant Activities of Laetispicine and Its Derivatives

The first total synthesis of laetispicine (1a), an amide alkaloid isolated from the stems of Piper laetispicum C.DC (Piperaceae), and the synthesis of some of its derivatives were described. Based on the evaluation of antidepressant activities in the forced swimming test, compounds 1h and 1i were identified as potent and safe antidepressant lead compounds.


Introduction
Natural products have traditionally played an important role in drug discovery and are the basis of many important therapeutics that have found broad use in the clinic [1]. Extensive studies to search among natural products for new antidepressants that possess both high efficacy and safety have been carried out [2,3]. Laetispicine (Figure 1) was first isolated in 2002 by Pan and co-workers from Piper laetispicum C.DC., a herb growing in China and parts of southeast Asia [4,5]. Piper laetispicum C.DC. is one of the species in pepper family and had been used for invigorating circulation and reducing stasis, detumescence and as an analgesic agent in China for a long time [5]. Pan et al. reported that laetispicine was effective in producing antidepressant and antinociceptive effects and hypothesized that OPEN ACCESS laetispicine was possibly acting on the monoaminergic neurotransmission system to mediate the antidepressant and antinociceptive disorders [6]. The structure of laetispicine is unique. Unlike other antidepressants, laetispicine does not contain an acyclic or aromatic amine. Moreover, it contains an unconjugated allylbenzene motif that has little precedent in natural products. However, molecules containing 1,3-benzodioxole have been shown to induce oxidative damage in vivo [7]. To better understand the SAR of laetispicine and explore the potential of laetispicine derivatives as new potent and safe antidepressants, we developed a synthetic approach that would facilitate the synthesis of laetispicine analogues. By using this strategy, laetispicine and eight of its derivatives were synthesized efficiently. Here, we first report the synthesis of laetispicine and several derivatives and their preliminary antidepressant activities evaluated in a forced swimming test.

Results and Discussion
As shown in Scheme 1, our approach to the synthesis of laetispicine and its derivatives involves the modified Julia-Kocienski olefination as the key step. By varying the combination of different fragments 8a-i, laetispicine and eight of its analogues were obtained. Our SAR study was mainly focused on replacing the 1,3-benzodioxole ring with halogen and alkoxy groups.

Scheme 1.
Retro-synthetic analysis of laetispicine and its derivatives.

Scheme 4. Synthesis of compounds 1a-i.
Reagents and conditions: (g) 6, 8a-i (1.1 equiv.), KHMDS (1.2 equiv.), DME, −60 °C, 2 h. Laetispicine (1a) and its analogues 1b-i were evaluated for their antidepressant activities in forced swimming test in mice [6,14]. The immobility time of forced swimming mice exposed to 1b-i (10 mg/Kg each) are shown in Table 1. Among the eight derivatives tested, the compounds containing alkoxybenzene moieties showed less antidepressant activities than laetispicine (1e, 1g vs. 1a, Table 1), or even lost their antidepressant activities entirely (1c, 1d, 1f vs. 1a, Table 1). Compound 1b with no substituent showed similar antidepressant activities as laetispicine. These results suggested that the 1,3-benzodioxole moiety was replaceable, and it was interesting to note that by introducing the halogen atom in the phenyl ring better antidepressant activities than that of laetispicine were achieved (1h, 1i vs. 1a, Table 1). Furthermore, these two compounds with potent antidepressant activities were tested in patch clamp assay to measure their potential to block hERG potassium channel ( Table 2). The results showed that 1h and 1i had weak inhibition on hERG current (IC 50 > 100 μM) when compared to the positive reference compound cisapride. Table 1. Effects of compound 1b-i on the forced swimming test in mice (means ± SEM of eight animals).

5-(4-Fluorophenethylsulfonyl)-
The forced swimming test adopted here is a modification of the method described by Porsoltetal [14]. Briefly, mice were individually forced to swim for 15 min in glass cylinders (height: 20 cm, diameter: 14 cm), containing 10 cm of water at 25 °C, which is a pre-test, and then mice were removed and dried before being returned to cages. Twenty-four hours later, mice were placed in the cylinders again for a 6-min test in the same system depicted above. The duration of immobility was recorded during the last 4 min of the 6-min testing period.

Statistical Analyses
Data obtained were expressed as mean ± SEM and analyzed by analysis of variance (ANOVA) followed by Bonferroni's test. p-values less than 0.05 (p < 0.05) were used as the significant level. The percent of inhibition was determined using the following formula: Inhibition (%) = 100 × [(control − experiment) / control].

hERG Inhibition
A CHO cell line stably expressing hERG potassium channels were voltage clamped using automated QPatch electrophysiology system. Test items were dissolved in DMSO and diluted with external recording buffer. Cells were exposed to test concentration for approximately 5 min or till a steady state block was reached at 20-35 °C. Each cell acted as its own control. Cisapride (1 M) was used as an internal positive control to confirm the sensitivity of the test system to hERG inhibition. The extent of inhibition of channel was expressed as a percentage of the control response (minus the test compound).

Conclusions
In conclusion, we have synthesized laetispicine (1a) and eight of its derivatives. The synthetic route achieved a good yield and high E/Z stereoselectivity (7 steps, 22.1% overall yield and E:Z ratio > 14:1) for laetispicine (1a). This methodology may potentially be applicable to the synthesis of other analogues of this family and facilitate further SAR research on laetispicine derivatives. Based on the outcomes of a forced swimming test and hERG channel binding studies, compounds 1h and 1i were identified as potential new lead antidepressant compounds. Further studies based on this kind of scaffold are in progress and will be reported in due course.