(22 E ,24 S )-24-Propylcholest-5en-3 α -acetate: A New Steroid from the Stembark Aglaia angustifolia (Miq.) (Meliaceae)

: A new propylcholesterol-type steroid, namely (22 E ,24 S )-24-propylcholest-5en-3 α -acetate ( 1 ), has been isolated from the stembark of Aglaia angustifolia (Miq.). The structure of 1 was determined on the basis of spectroscopic data including 1D- and 2D-NMR as well as high resolution mass spectroscopy analysis. Compound 1 showed weak activity against the MCF-7 breast cancer cell line.


Introduction
The genus Aglaia is the largest genus in subtropical and tropical angiosperm plants belonging to the Meliaceae family, which consists of more than 130 species and is widely distributed in the Southern mainland of China, the Indo-Malaysian region, and the Pacific Islands [1,2]. Some parts of the stem of the Aglaia plant have been used in traditional medicine for the treatment of skin diseases, fever, and wounds [3,4]. During the course of our continuous investigation for biologically active compounds from Indonesian Aglaia plants, we already isolated several bioactive compounds from A. smithii [5], A. eximia [6][7][8], A. argentea [9,10], and A. elliptica [11,12]. In further investigation on Aglaia plants that grow in Indonesia, we found that A. angustifolia has not been studied phytochemically previously. In this paper, we report the isolation and structural elucidation of a new propylcholesterol-type steroid, (22E,24S)-24-propylcholest-5en-3α-acetate (1) (Figure 1), along with its cytotoxic activity against MCF-7 breast cancer lines.

Extraction and Isolation
The dried stembark of A. angustifolia (1.97 kg) was extracted successively with n-hexane (3 × 4 L), ethyl acetate (3 × 4 L), and methanol (3 × 4 L) at rt to give a crude n-hexane extract (19.5 g), ethyl acetate (20.4 g), and methanol (35 g) after removal of the solvent. The n-hexane extract (19.0 g) was separated by vacuum liquid chromatography on Merck GF 254 silica gel using a 10% gradient of n-hexane-ethyl acetate-methanol solvent to give eight fractions (A-G). Fraction B (1.3 g) was subjected to column chromatography on silica gel using n-hexane:ethyl acetate (98:2) as a solvent to give ten subfractions (B1-B10). Subfraction B5 (44.2 mg) was further separated by column chromatography on silica gel using n-hexane:dichloromethane:ethyl acetate (90:9:1) to give 1 (5.6 mg).  The 13 C NMR spectrum showed 32 carbon resonances, which were classified by their chemical shifts, DEPT, and HMQC spectra as 7 methyl groups (2 tertiary, 3 secondary, 1 primary, and 1 acetyl), 12 methylene carbons, 9 methane carbons (1 oxygenated and 1 olefinic carbons), and 4 quaternary carbons (1 olefinic and 1 oxygenated carbons). These functionalities accounted for 2 out of the total 6 hydrogen deficiency indexes. The remaining four hydrogen deficiency indexes were consistent with the tetracyclic steroidal structure [13] with acetyl and olefinic as additional groups. The significant difference side chain structure of 1 with other sterol type steroids was the n-propyl side chain of 1 at C-24 instead of the ethyl group of other sterols like stigmasterol [10,11,13]. It was indicated by 13 C NMR spectrum of 1, an additional chemical shift (29.9 ppm) at C-28 with δ H (1.26 (1H, m, H-28a) and 1.57 (1H, m, H-28b)) and showed as CH 2 with DEPT spectra of 1. A comparison of the NMR data of 1 with 24ξ-n-propylcholesterol compounds, isolated from a cultured marine chrysophyte [14], and with (24R)-24-propylidenecholesterol isolated from Aureoumbra lagunensis, the Texas brown tide alga [15], and with a stereospecific synthesis 24-propylcholesterol isolated from the Texas brown tide [16], indicated that the structure of 1 is very similar to the synthetic compound (22E,24S)-24-propylcholest-5-en-3β-ol. The main difference was the absence of a hydroxyl group and the presence of an acetyl group, which suggested that 1 was an acetyl derivative of (22E,24S)-24-propylcholest-5-en-3β-ol. The 1 H-1 H COSY spectrum of 1 (Figure 2 The HMBC correlations from the tertiary, secondary, and primary methyl protons to their neighboring carbons enabled the assignment of the two tertiary methyls at C-10 and C-13, secondary methyls at C-20 and C-25 (2×), as well as a primary methyl at C-29, respectively. Furthermore, the olefinic proton at δ H 5.37 (1H, d, J = 6.0 Hz, H-6) was correlated to olefinic carbon at δ C 139.8 at methylene carbon at δ C 32.1, indicating an olefinic moiety was located at C-5 and C-6 (∆ 5,6 ), similar to stigmast-5en-3α-acetate [10,11]. Correlations from oxygenated methine proton at δ H 4.60 (H-3) and methyl acetyl at δ H 2.03 (Me-32) to carbonyl ester at δ C 170.9 (C-31) were used to assign an acetyl group that was located on C-3.

General Experimental Procedures
The optical rotation was obtained with an Autopol IV automatic polarimeter. Mass spectra were measured with a Waters Xevo QTOFMS (Waters, Milford, MA, USA). NMR data were recorded on a Bruker Avance-600 spectrometer at 600 MHz for 1 H and 150 MHz for 13 C using TMS as an internal standard (Billerica, MA, USA). Chromatographic separations were carried out on silica gel (60, 70-230, and 200-400 mesh) (Merck, Darmstadt, Germany). TLC plates were precoated with silica gel GF 254 (Merck, Darmstadt, Germany, 0.25 mm), and detection was achieved by spraying with 10% H 2 SO 4 in ethanol, followed by heating.

Plant Material
The stem bark of A. angustifolia (Miq.) was collected from Bogor Botanical Garden, Bogor, Indonesia in February 2017. The plant was identified by Mr. Ismail, the staff of the Bogoriense Herbarium, Indonesian Science Institute, Bogor, Indonesia. A voucher specimen (II.K.57a) was deposited at the herbarium.

Cytotoxic Bioassay
The cytotoxicity of the compounds against MCF-7 human breast cancer cells was measured using the PrestoBlue cell viability assay. The cells were maintained in a Roswell Park Memorial Institute (RPMI) medium supplemented with 10% (v/v) Fetal Bovine Serum (FBS) and 1 µL/mL antibiotics. Cultures were incubated at 37 • C in a humidified atmosphere of 5% CO 2 . Tumor cells were seeded in 96-well microliter plates at 1.7 × 10 4 cells per well. After 24 h, compounds were added to the wells. After 96 h, cell viability was determined by measuring the metabolic conversion of resazurin substrate into pink fluorescent resorufin product resulting from reduction in viable cells. The PrestoBlue assay results were read using a multimode reader at 570 nm. All compounds were tested at eight concentration (7.81; 15.63; 31.25; 62.50; 125.00; 250.00; 500.00; 1000.00 µg/mL) in 100% DMSO with a final concentration of DMSO of 2.7% in each well. Each concentration of the compounds was tested in two parallels experiments. IC 50 values were calculated by the linear regression method using Microsoft Excel software.