New Cassane Diterpenoids from Caesalpinia sappan and Their Antiplasmodial Activity

One new cassane diterpene possessing an unusual N bridge between C-19 and C-20 named caesalsappanin R (1), as well as another new diterpene caesalsappanin S (2), were isolated from the seeds of Caesalpinia sappan with methanol extract. Their structures were determined by spectroscopic analysis and examined alongside existing data from prior studies. Their biological activities were profiled by their antiplasmodial activity.


Introduction
Caesalpinia sappan has been a part of traditional Chinese herbal medicine and is widely used in the treatment of dysmenorrheal, blood stagnation, and tetanus. Previous phytochemical investigations indicated that this genus contains an abundant source of cassane diterpenes with different structure types, and most of them showed in vitro or in vivo pharmacological impacts such as antiproliferative [1][2][3], antiplasmodial [4,5], antibacterial [6], antihelmintic, and antineoplastic activity [7]. As a continuation of our project towards new bioactive diterpenes discovery from the genus Caesalpinia [2,3,8], we examined the chemical constituents of C. sappan and obtained two new cassane diterpenes, designated caesalsappanin R (1) and caesalsappanin S (2) (Figure 1). Compound 1 is a rather unusual cassane diterpenoid lactone-type skeleton, consisting of an N bridge between C-19 and C-20. In this paper, we detail the separation and structural determination of the novel agents and the examination of their antiplasmodial activity.

Purification of Compounds 1-2
The seeds of C. sappan were extracted with MeOH three times. The two cassane-type diterpenoids were isolated and purified via silica gel chromatography, Sephadex LH-20 gel chromatography and semi-HPLC.

Purification of Compounds 1-2
The seeds of C. sappan were extracted with MeOH three times. The two cassane-type diterpenoids were isolated and purified via silica gel chromatography, Sephadex LH-20 gel chromatography and semi-HPLC.

In Vitro Antiplasmodial and Larvicidal Activities of Compounds 1-2
The two compounds were tested against the chloroquine-resistant strain K1 of P. falciparum (Table 2). Compound 1 exhibited relatively good antiplasmodial activity in vitro with IC50 values of 3.6 μM, compared with chloroquine. On the other hand, compound 2 showed only weak activity against the chloroquine-resistant K1 strain of P. falciparum. It appears that the presence of the N bridge in cassane-type diterpenoids may play an important role in enhancing activity against the chloroquine-resistant K1 strain of P. falciparum in vitro. Furthermore the toxic activity of compounds 1 and 2 against mosquito larvae was evaluated. Both compounds displayed only low activity.

In Vitro Antiplasmodial and Larvicidal Activities of Compounds 1-2
The two compounds were tested against the chloroquine-resistant strain K1 of P. falciparum (Table 2). Compound 1 exhibited relatively good antiplasmodial activity in vitro with IC 50 values of 3.6 µM, compared with chloroquine. On the other hand, compound 2 showed only weak activity against the chloroquine-resistant K1 strain of P. falciparum. It appears that the presence of the N bridge in cassane-type diterpenoids may play an important role in enhancing activity against the chloroquine-resistant K1 strain of P. falciparum in vitro. Furthermore the toxic activity of compounds 1 and 2 against mosquito larvae was evaluated. Both compounds displayed only low activity.

General Experimental Procedures
Optical rotation data were measured with a Perkin-Elmer 341 digital polarimeter (PerkinElmer, Norwalk, CT, USA). UV and IR data spectra were recorded on Shimadzu UV2550 and FTIR-8400S spectrometers (Shimadzu, Kyoto, Japan). NMR spectra were obtained using a Bruker AV III 600 NMR spectrometer with chemical shift values presented as δ values having TMS (Tetramethylsilane) as the internal standard. HRESIMS was performed using an LTQ-Orbitrap XL spectrometer (Thermo Fisher The LC 50 were calculated from toxicity data by using probit analysis [18]. Chloroquine was included as a standard for comparison. Data are presented as means ± SEM. Statistical analyses were done by means of the Student's t-test. A P value of less than 0.05 was considered a significant difference.

Conclusions
In conclusion, two new cassane-type diterpenoids (1 and 2) were isolated and characterized by spectrometric analysis (1 and 2D NMR, HRESIMS). Compound 1 exhibited active antiplasmodial activity in vitro with IC 50 at 3.60 µM. In addition, the compounds that we had reported also showed antiplasmodial activities; caesalsappanins A, G, H, and I displayed antiplasmodial activities with IC 50 values of 7.4, 0.78, 0.52, and 2.5 µM, respectively [3]. Therefore, we believe that this plant is an important source for the diverse structure of cassane-type diterpenoids and should be further investigated for the antiplasmodial activity.