Carapanosins D—F from the Seeds of Andiroba (Carapa guianensis, Meliaceae) and Their Effects on LPS-Activated NO Production

A novel nor-phragmalin-type limonoid, named carapanosin D (1), and two novel mexicanolide-type limonoids, carapanosins E (2) and F (3), were isolated from the seed oil of andiroba (Carapa guianensis Aublet), a traditional medicine in Brazil and Latin American countries. Their structures were unambiguously determined on the basis of spectroscopic analyses using one-dimensional (1D) and two-dimensional (2D) NMR techniques and High resolution Fast Atom Bombardment Mass Spectrometry (HRFABMS). Compounds 1–3 were evaluated for their effects on the production of nitric oxide (NO) in Lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that compounds 2 and 3 have high potency as inhibitors of macrophage activation.


Introduction
Meliaceae plants are a well-known source of structurally diverse limonoids with a wide range of bioactivities, such as antimalarial and antifeedant. Limonoids in the plant kingdom occur mainly in the Meliaceae, Rutaceae, and Simaroubaceae families [1]. Andiroba is one of the Meliaceae plants in the rain forests of South America, and its woody four-cornered nut has four cells, each of which contains two to three seeds with oil-rich kernels. Limonoids, most of which are highly oxidized tetranortriterpenoids, can be classified in terms of seco form and cyclization patterns of rings A-D in the triterpene skeleton. Extracts from its flowers, bark, and seeds have been used for centuries by the Amazonian people and exhibit analgesic [2], anti-malarial [3], anti-inflammatory [4], anti-allergic [5], and anti-plasmoidal [6] activities, and also acute and subacute toxicities [7].
Our series of studies on the components of the seed oil of C. guianensis revealed the structures of carapanolides A and B [8], guianolide A and B [9], carapanolides C-I [10], carapanolides J-L [11], carapanolides M-S [12], carapanolides T-X [13], and carapanosins A-C [14] in the seed oil of andiroba. Last year, we reported the absolute structure of guianolactones A and B from the seed oil of C. guianasis (Meliaceae) [15]. Our recent study of the seed oil of C. guianasis revealed the structures of an unusual 19-nor-phragmalin-type limonoid, named carapanosin D and two novel mexicanolide-type limonoids, named carapanosins E and F. We herein describe the isolation and structural determination of three new limonoids and the effects of 1-3 on the production of nitric oxide (NO) in Lipopolysaccharide LPS-activated mouse peritoneal macrophages. The structures of 1-3 were determined on the basis of NMR spectroscopy, including one-dimensional (1D) and two-dimensional (2D) ( 1 H, 1 H-COSY, NOESY, HSQC, HMBC) NMR, and Fast Atom Bombardment (FABMS).

Results and Discussion
The seeds of Carapa guianensis were dissolved in MeOH , and the extract was separated by silica gel column chromatography (CC), medium-pressure liquid chromatography (MPLC), and reverse-phased HPLC to obtain three novel limonoids, 1, 2, and 3 ( Figure 1).

General Procedures
Melting points were determined on a Yanagimoto micro-melting point apparatus (YANAKO Measuring Instrument Trading Corporation, Kyoto, Japan) and were uncorrected. Optical rotations were measured using a JASCO DIP-1000 digital polarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were recorded using a PerkinElmer 1720X FTIR spectrophotometer (PerkinElmer Japan Co. Ltd., Yokohama, Japan). All NMR experiments were measured with a Varian INOVA 600

General Procedures
Melting points were determined on a Yanagimoto micro-melting point apparatus (YANAKO Measuring Instrument Trading Corporation, Kyoto, Japan) and were uncorrected. Optical rotations were measured using a JASCO DIP-1000 digital polarimeter (JASCO Corporation, Tokyo, Japan). IR spectra were recorded using a PerkinElmer 1720X FTIR spectrophotometer (PerkinElmer Japan Co. Ltd., Yokohama, Japan). All NMR experiments were measured with a Varian INOVA 600 spectrometer (Varian Medical Systems, Tokyo, Japan) with standard pulse sequences, operating at 600 and 150 MHz. CDCl 3 was used as the solvent and Tetramethylsilane (TMS) as the internal standard. FABMS were recorded on a JEOL-7000 mass spectrometer (70 eV) (JEOL Ltd., Tokyo, Japan). Column chromatography (CC) was carried out on silica gel 60 (70-230 mesh) (Merck Chemicals B.V., Tokyo, Japan) and MPLC was carried out with silica gel (230-400 mesh) (Merck Chemicals B.V., Tokyo, Japan). HPLC was completed using a JASCO PU-1586 instrument (JASCO Corporation, Tokyo, Japan) equipped with a differential refractometer (RI 1531). Fractions obtained from column chromatography were monitored by thin-layer chromatography (TLC) (silica gel 60 F 254 ) (Merck Chemicals B.V., Tokyo, Japan).

Plant Material
The seed oil (2.03 kg) of Andiroba (Carapa guianensis A ublet , Meliaceae) was collected in the Amazon, Brazil, in March, 2013. It was kindly provided by Mr. Akira Yoshino (who is a representative person of the "NGO Green Heart love Amazon project"). A voucher specimen (CGS-01-2) was deposited in the Herbarium of the Laboratory of Medicinal Chemistry, Osaka University of Pharmaceutical Sciences.