Tecomastane, a new megastigmane from the flowers of Tecoma stans

Abstract Tecoma stans is a tropical plant that is widely used in folk medicine. Little is known about the chemical constituents of flowers of this plant. From flowers of the native plant in Vietnam, 12 compounds were isolated and elucidated, including one new compound tecomastane (1) and eleven known compounds, (3S,5R,6S,7E)-5,6-epoxy-3-hydroxy-7-megastigmane-9-one (2), bosciallin (3), chakyunglupulin B (4), (2S,6R)-2,6-dimethyloctane-1,8-diol (5), cleroindicin F (6), rengyoxide (7), 3,4-dihydroxybenzoic acid (8), methyl 3,4-dihydrobenzoate (9), 3,5-dihydroxybenzoic acid (10), luteolin (11), and indole-3-carboxylic acid (12). Compound 5 was a new natural product. The chemical structures of isolated compounds were identified by interpretation of their spectroscopic data (1D, 2D NMR, and HRESIMS) and by comparison with the literature. Compounds 1–7 and 10–12 were evaluated for alpha-glucosidase inhibition and antimicrobial activity against antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Graphical Abstract

NMR data of compound 4 was consistent with those of pubinernoid A in the same deuterated solvent (Huang et al. 2006).In 2015, Kim and co-workers isolated chakyunglupulin B having the identical NMR data with those of pubinernoid A (Kim et al. 2015).The difference is their chemical structures.Particularly, the ether linkage between two hydroxy groups at C-2 and C-7 occurred in the chemical structure of pubinernoid A (Figure S2).Kim and co-workers used the Mosher method to define the presence of the hydroxy group at C-1 and its absolute configuration.Based on complete NMR data of 4, we concluded that NMR data of 4 were identical with those of pubinernoid A and chakyunglupulin B. This finding indicated that pubinernoid A and chakyunglupulin B are the same compounds.With no ether linkage, Chakyunglupulin B had two tautomerised isomers existed (4 A and 4B, Figure S2).The downfield chemical shift of H 3 -11 and the strong HMBC correlation of this group to carbon at d C 183.6 proposed that 4 A should be the major tautomer that is similar to that of pubinernoid A.
(2S,6R)-2,6-Dimethyloctane-1,8-diol (5) was prepared from (R)-citronellol by Gramatica et al. (1986).In 1997, Berkowitz and Wu synthesized this compound as an intermediate for archaebacterial lipids.From both reports, the absolute stereochemistry of 5 was defined based on its negative specific rotation.The specific rotation of 5 is À7.2 (c 2.0, CHCl 3 ), which was similar to that in the literature, indicating its absolute configuration as (2S,6R).To the best of our knowledge, compound 5 is a newly reported natural compound.Complete NMR assignments of 5 were defined using 2D NMR (Table S2).Compounds 6 and 7 had the cyclohexylethanoid-type skeleton, which was found previously in the Tecoma plants, i.e., Tecoma capensis (Guiso et al. 1997).Isolates 1-7 and 12 were isolated for the first time from the genus Tecoma.

In vitro alpha-glucosidase inhibition and antimicrobial activity assays
Compounds 1-7 and 10-12 were evaluated the alpha-glucosidase inhibition.Compounds 4, 10, and 11 showed stronger activity than the positive control (acarbose, IC 50 360 ± 3.1 mM) with IC 50 values of 67.1, 157.9, and 88.3 mM, respectively.These compounds were also evaluated the antimicrobial against antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii.Only compounds 11 and 12 inhibited S. aureus with the inhibition zones of 11 and 12 mm at the concentration of 50 mg/mL.The positive control, apramycin showed the inhibition zone of 21 mm, 20 mm, and 21 mm against Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii, respectively.Thin-layer chromatography (TLC) was carried out on pre-coated silica gel 60 F 254 or silica gel 60 RP-18 F 254S (Merck), and spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating.Gravity column chromatography was performed on silica gel 60 (0.040-0.063 mm, HiMedia)

Source of plant material
Flowers of Tecoma stans (L.) were collected in Ho Chi Minh City, Vietnam between April and June 2020.The scientific name of the plant was authenticated by Dr. Dang Van Son, Institute of Tropical Biology, Vietnam.A voucher specimen (No.UP020) was deposited with the Department of Chemistry, Ho Chi Minh University of Education.

Extraction and isolation
Dried T. stans (4.5 kg) flowers were crushed and extracted for 24 h with acetone (3 Â 10 L) at ambient temperature.The filtrated solution was evaporated to dryness under reduced pressure to obtain a crude extract (207 g).This was successively partitioned by n-hexane, n-hexane: EtOAc (1:1, v/v), and EtOAc to afford extracts H (18 g), followed that in a previous report (Tran et al. 2021).All samples were analyzed in triplicate at five different concentrations around the IC 50 values, and the mean values were retained.The following equation was used to calculate the inhibition percentage (%): Inhibition (%) ¼ [1 -(Asample/Acontrol)] Â 100.

Antibacterial activity assay
The agar well diffusion method was used to evaluate the antibacterial activity of the isolated compounds on antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, Acinetobacter baumannii.These two bacterial pathogens were cultured in Nutrient Broth at 37 C for 18 hours.After that, the bacterial cultures were diluted with sterilized 0.9% NaCl to obtain the bacterial solutions of 1.5 Â 10 8 CFU/ml and 100 mL of each bacterial solution was spread on Mueller-Hinton agar (MHA) plate.Then, holes with a diameter of 8 mm were punched aseptically with tips to make wells on the surface of the MHA plates.The compounds were prepared in DMSO at the concentration of 1 mg/ml and 50 mL of each compound solution was introduced into the wells.The plates were incubated at 37 C for 16-18 hours and the antibacterial activity of each compound was recorded by measuring the diameters of inhibition zones surrounding the wells.DMSO was used as a control in this experiment.

3. 1 .
General experimental procedures NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR) using residual solvent signals as internal references: acetone-d 6 at d H 2.05, d C 29.84 and chloroform-d at d H 7.26, d C 77.18.