Four New Limonoids from the Barks of Toona ciliata

Four new limonoids, toonayunnanaes F − I (1 − 4), and six known compounds (5 − 10) were isolated from the barks of Toona ciliata. Their structures were elucidated by thoroughly analyzing of NMR and HRMS data, and single-crystal X-ray diffraction of 1. The oxetane ring moiety in 1 was rare in limonoids and other natural products. Compound 1 showed nitric oxide (NO) inhibitory effect with an IC50 38.45 ± 0.41 µM in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages.


Introduction
The genus Toona, belonging to the family Meliaceae, contains 15 species mainly distributed in the regions of tropical Asia and Africa, of which four species and six varieties grow in China [1]. Toona ciliata Roem. var. ciliata is a timber tree mainly found in the south of China, such as Yunnan, Sichuan, Guangdong, and Hainan provinces [2]. Its bark has been used in Chinese folk medicine to treat dysentery, fever, and menstrual disorders [3]. Our previous phytochemical investigations on T. ciliata var. henryi and T. ciliata var. yunnanensis have led to the isolation of a series of limonoids with potential biological activities such as cytotoxicity, anti-inflammatory, and anti-multidrug resistance (MDR) activities [4][5][6][7][8]. As a part of our continuous research for bioactive limonoids from Toona plants, four new limonoids, toonayunnanaes F − I (1 − 4), and six known compounds (5 − 10) were isolated from the barks of T. ciliata. Their structures were elucidated by thoroughly analyzing of NMR and In memory of Professor Jun Zhou.
Pan-Pan Zhang and Yun-Ge Bu have contributed equally to this work.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s1365 9-020-00274 -w) contains supplementary material, which is available to authorized users.  HRMS data, and single-crystal X-ray diffraction of 1.

Results and Discussion
The molecular formula of toonayunnanae G (2) was established as C 28 H 38 O 6 based on the HRESIMS ion at m/z 493.2551 [M + Na] + (calcd. 493.2561) and its 13 C NMR data. Apart from the signals for an acetoxy substituent, the remaining 26 carbon resonances observed in the 1D NMR spectra indicated that 2 might also be a ring-intact limonoid [8][9][10]. Comparison of its NMR data ( Table 1) with those of the known toonaciliatone F (5) [13] revealed that the main differences were the absence of a double bond and an acetoxy group. The chemical shift of C-3 (δ C 217.8) implied the presence of a keto carbonyl rather than an α,βunsaturated ketone moiety in ring A, which was confirmed by the HMBC correlations of H 2 -1 (δ H 1.90, 1.77), H 2 -2 (δ H 2.76, 2.31), H 3 -28, and H 3 -29 to C-3. The HMBC correlations of H-6 (δ H 5.31) to C-5, C-7 (δ C 72.2), and an ester carbonyl carbon (δ C 169.8) and of H-7 (δ H 3.90) to C-6 (δ C 72.1), C-8, C-9, and C-30 (Fig. 3) allowed the assignment of an acetoxyl at C-6 and a hydroxy group at C-7. The relative configuration of 2 was deduced to be identical to that of 5 by a ROESY experiment. The ROESY cross-peaks of H 3 -19/H-6, H 3 -29/H-6, H 3 -30/H-6, H-6/H-7, and H-7/H 3 -30 revealed the α-orientations of 6-OAc and 7-OH. Therefore, the structure of 2 was assigned as depicted.
Additionally, the inhibitory effects on NO generation in LPS-activated RAW 264.7 macrophages of compounds 1 − 4 were evaluated at the concentrations of 50 μM and below. Compound 1 showed NO inhibitory effect with an IC 50 38.45 ± 0.41 µM.

General Experimental Procedures
Optical rotations were measured on a JASCO P-1020 automatic digital polarimeter at room temperature. IR spectra were recorded on a Bruker Tensor 27 spectrometer using KBr pellets. UV spectra were recorded on a Shimadzu UV-2450 spectrophotometer (Shimadzu, Tokyo, Japan). High-resolution electrospray ionization mass spectrometry (HRESIMS) was obtained on an Agilent 6529B Q-TOF mass instrument using electrospray ionization. The 1D and 2D nuclear magnetic resonance (NMR) spectra were obtained on Bruker AVANCE III 500 MHz or Bruker AVIIIHD 600 MHz spectrometers in CDCl 3 with TMS as an internal standard. Analytical HPLC was conducted on an Agilent 1260 infinity system equipped with a DAD-UV detector. Preparative HPLC was carried out using a Shimadzu LC-6A system (Shimadzu, Tokyo, Japan) equipped with a Shimpack RP-C 18 column (20 × 200 mm, i.d. 10 μm, Shimadzu, Tokyo, Japan) with a flow rate of 10.0 mL/min, detected by a binary channel UV detector. Silica gel (200-300 mesh, Qingdao Haiyang Chemical Co. Ltd., China), MCI (Mitsubishi, Japan), and RP-C 18 silica (40-63 μm, FuJi, Japan) were used for column chromatography.

Extraction and Isolation
The air-dried and powder bark of T. ciliata (29 kg) was extracted with 95% EtOH three times (3 × 6.