Phenolic and bisamide derivatives from Aglaia odorata and their biological activities

Abstract Three new compounds (1–3), including two bisamide derivatives (1 and 2) and a lignin (3), along with 15 known compounds were isolated from Aglaia odorata. Compound 2 was a pair of enantiomers and successfully resolved into the anticipated enantiomers. Their structures were elucidated by extensive spectroscopic analysis, electronic circular dichroism (ECD) calculations, and X-ray crystallography. Three compounds showed excellent inhibitory activities on α-glucosidase with IC50 values ranging from 54.48 to 240.88 μM, better than that of the positive control (acarbose, IC50 = 590.94 μM). Moreover, compounds 3, 13, and 15 presented moderate inhibitory activities against butyrylcholinesterase. Compound 17 exhibited potent PTP1B inhibitory activity with an IC50 value of 179.45 μM. Representative active compounds were performed for the molecular docking study. Herein, we described the isolation, structure elucidation, the inhibitory effects on three enzymes, and molecular docking of the isolates from the title plant. Graphical Abstract


Introduction
The genus Aglaia is composed of about 120 species, and mainly distributed in the South of Asian, such as Thailand, Malaysia, China, and Philippines (Satasook et al. 1994).Several species have long been used for the treatment of coughs, diarrhea, inflammation, and injuries in those countries (Nugroho et al. 1997).Cyclopenta[b]benzofuran (Liu et al. 2013), triterpenoids (Xie et al. 2007), bisamides (Greger et al. 2008), lignans (Sianturi et al. 2016), and flavones (Puripattanavong et al. 2000) have been isolated from this genus.Among them, some compounds exhibited excellent biological activities, including anticancer (Saifah et al. 1993), anti-inflammatory (Proksch et al. 2005), and insecticidal activities (Chaidir et al. 1999;Wang et al. 2004).Therefore, this genus has attracted considerable interests in the areas of natural products and pharmacology.
Aglaia odorata, named Mi Zailan, is utilized as a traditional Chinese medicine with the properties of improving blood circulation and curing dizziness, traumatic injuries, and bruises (Editorial board of the State Administration of traditional Chinese Medicine 1999; Li et al. 2007;Li et al., 2020).Moreover, this plant is widely planted in the south of China due to its special aroma of the flowers, and thus the flowers were also used as the spice of tea (Li et al. 2007).However, there is no systematic chemical constituent investigation of Aglaia odorata on the a-glucosidase inhibitory activity.Thus, to investigate the potential a-glucosidase inhibitory compounds in A. odorata, bioactivity-guided identification was also conducted.Accordingly, three new compounds (1-3), including a pair of enantiomers (2), and 15 known compounds (Figure 1) were isolated from the active ethyl acetate part of A. odorata.Herein, we described the isolation, structure elucidation, the inhibitory effects on three enzymes, and molecular docking of the isolates from the title plant.

Results and discussion
The leaves and twigs of A. odorata were extracted with 95% EtOH at room temperature to give a crude extract, which was suspended in H 2 O and partitioned with ethyl acetate (EA).Both fractions of water and EA were applied to a-glucosidase inhibitory evaluation.The EA part showed good inhibitory effect on a-glucosidase with the inhibitory rate of 88.8% at the concentration of 500 lg/mL.Thus, the active EA part was purified by a series of column chromatographic methods to afford compounds 1-18, including three new compounds (1-3) as shown in Figure 1.
Fortunately, the crystal of compound 1 was acquired from ethyl acetate solution, the X-ray crystallographic data of 1 with Cu Ka radiation [Flack parameter ¼ 0.02 (5)] not only confirmed the planar structure but also defined its absolute configuration as 2'S, 2S (Figure S1).Therefore, agladorin A (1) was assigned as an epimer of (-)-odorinal (4).
According to a line of the CD spectra and the small optical activities for compound 2, compound 2 was speculated to be a pair of enantiomers.Luckily, the enantiomers, (þ)-2 and (À)-2, with the opposite Cotton effects in the CD spectra and optical activities, were resolved with the help of the Phenomenex LUX column (Figure S19).The CD spectrum of (À)-2 showed a positive Cotton effect at k max 228 nm (De þ 57.6) and a negative Cotton effect at k max 279 nm (De À 61.6), while the CD spectrum of (þ)-2 exhibited the opposite CD spectrum of (À)-2.The absolute configurations of (þ)-2 and (À)-2 were determined by comparing the calculated ECD results (Figure S19) with relevant experimental data (Liu et al. 2020;Zeng et al. 2021).
Compound (3) was isolated as a white powder.).Further HMQC spectrum along with 1 D NMR data implied the presence of 20 carbon signals, including two methoxy group, four methylenes, four olefinic methines, and 10 olefinic quaternary carbons.Thus, compound 3 is a lignan with a similar skeleton as that of cynwilforone C (Jiang et al. 2019).
The planar structure of 3 consisted of units A and B by analysing the 2 D NMR data (Figure S20).In unit A, a phenyl group with an ABX spin system could be observed from the above-mentioned data, further confirmed by the 1 HÀ 1 H COSY cross-peaks between H-3 0 /H-4 0 and HMBC correlation from H-6 0 to C-7 0 .The 3-hydroxypropanoyl group was assigned at C-5 0 from the HMBC correlations of H-4 0 , H-6 0 , and H 2 -9 0 to C-7 0 (d C 198.2), along with the 1 HÀ 1 H COSY cross-peaks between H 2 -8 0 /H 2 -9 0 .The connection of 3-hydroxypropanoyl group to C-5 0 was further verified by the nuclear Overhauser effect (NOE) correlations between H 2 -8 0 with H-4 0 and H-6 0 (Figure S20).Thus, the 3-hydroxypropanoyl group was assigned at C-5 0 in unit A.
Molecular docking was used to predict binding sites between the active compound and the enzyme.Three most active compounds against these three enzymes (a-glucosidase, BuChE, and PTP1B) were applied to the docking study, and the results implied that those compounds (13 and 17) were docked well with those enzymes (Figure S60 and Table S2).Compound 13 with the most potent inhibitory activity on a-glucosidase, was selected for the docking study on a-glucosidase in Figure S60 (Zhang and Zhao 2016).As shown in Figure S60, compound 13 was perfectly packed in a hydrophobic pocket formed by Phe673, Trp423, Trp525, and Trp562 of a-glucosidase.Given that all those amino acids have aromatic group, they might have a p-p interaction between 13 and those amino acids.In addition, two hydrogen bonds were observed between compound 13 and the a-glucosidase (PDB:7jty).One was formed between the hydroxyl group of 13 and the carboxyl group of Asp451, while another was observed between the hydroxyl group of 13 and His 698.These interactions could be responsible for the good inhibitory effect of compound 13 on a-glucosidase.
Compound 13 was also applied for the docking study on BuChE (Meden et al. 2019).In Figure S60, 13 was packed well in the hydrophobic pocket formed by twelve amino acid residues of the protein, Trp430, Met437, Gly78, Gly439, His438, Trp82, Phe329, Gly115, Ala328, Gly117, Gly116, and Ser198.In addition, two hydrogen bonds were observed between 13 and the protein of BuChE (Figure S60 and Table S2).Moreover, compound 17 was selected for the docking study on PTP1B (Liu et al. 2008).Compound 17 was packed in a hydrophobic pocket formed by eleven amino acid residues of the protein (PDB: 3eb1), Trp179, Phe182, Val184, Asp181, Pro180, Glu115, Lys120, Gln266, Gly220, Lys116, and Arg221 (Figure S60).Furthermore, three hydrogen bonds were observed between 17 and the aldehyde group of Gly183, the hydroxy group of Ser216, and the sulfide group of Cys215.The hydrophobic and hydrogen bond interactions contributed to its excellent inhibitory effect against PTP1B.

General experimental procedure
Optical rotations were tested on a JASCOP-1020 polarimeter.UV spectra were applied on a Shimadzu UV-2401PC spectrometer.IR spectra were tested on a Bruker FT-IR Tensor-27 infrared spectrophotometer with KBr disks. 1 H, 13 C NMR and 2 D NMR spectra were recorded on INOVA-400 MHz, and Bruker Avance III 600 MHz spectrometers using TMS as an internal standard.ESIMS and HR-ESIMS analysis were carried out on Waters Xevo TQS and Agilent 1290 UPLC/6540 Q-TOF mass spectrometers, respectively.Column chromatography was performed on silica gel (300-400 mesh; Qingdao Marine Chemical Co. Ltd., China), Sephadex LH-20 (40-70 lm, Amersham Pharmacia Biotech AB, Uppsala, Sweden), MCI gel (75-150 lm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Lichroprep RP-C18 gel (40-63 lm, Merck, Darmstadt, Germany).HPLC separation was performed on an instrument consisting of a Waters 600 controller, a Waters 600 pump, and a Waters 2487 dual k absorbance detector with preparative columns, an X-bridge (250 Â 10 mm) column and Phenomenex LUX column (250 Â 4.6 mm).All solvents used for general chromatography were analytical grade (Sinopharm Chemical Reagents Co. Ltd., China), and the solvents used for HPLC were HPLC grade (Merck Chemicals, Darmstadt, Germany).Fractions were monitored by TLC (GF 254, Qingdao Marine Chemical Co., Ltd.), and spots were visualized by heating silica gel plates immersed in 5% H 2 SO 4 in ethanol.

Plant material
The leaves and twigs of A. odorata (Meliaceae) were collected in Liguo Town, Ledong County, Hainan Province, China, in October 2019 and were identified by Mr. Jun Zhang.A voucher specimen (Y20191001) was deposited at the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences.

a-Glucosidase inhibitory assay
The inhibitory activity of a-glucosidase (Sigma) was conducted by modification of the procedure previously reported (Chen et al. 2016).Shortly, 10 lL (500 lg/mL as the initial concentration) of the samples and 20 lL of the enzyme (0.5 U/mL) were successively added to 96-well plates with 10 mM buffer (pH ¼ 7.0) 70 lL, and the inhibitors were incubated with the enzyme at 37 C for 15 min.Then, the substrate p-nitrophenyl a-D-glucopyranoside (pNPG) (0.25 mM) 20 lL was added and the enzymatic reaction was performed at 37 C for 30 min, after that sodium carbonate (80 mM) 80 lL as a stop buffer was added.Finally, the absorbance of the solvent was measured at 405 nm.Acarbose was used as a positive control.It was considered that the compound had inhibitory activity on a-glycosidase when the inhibition rate was close to or exceeded the activity of acarbose.Then, the potential molecule was diluted by five times, and the IC 50 value was measured and calculated by the same method.

BuChE inhibitory assay
The BuChE inhibitory activities of all compounds were performed using the methodology described previously (Li et al. 2020).The measurement was conducted using a 96-well plate.Buffer (pH ¼ 7.0) 150 lL, test compounds with a series of concentrations (10 lL), BuChE (10 lL), and DTNB (5, 5 0 -dithiobis) 20 lL, were added into the wells in sequence.The mixture was then incubated at 25 C for 15 min.Then, 20 lL of butyrylthiocholine iodide (BTC) was added, and after that the reaction was initiated.After incubation at 37 C for 10 min, the absorption was tested at 405 nm.Tacrine was used as a positive control.Inhibition ratio was tested according to [(A blank -A sample )/A blank ] Â 100%, in which, A sample and A blank were the absorbance of the test compound and the blank control, respectively.The anti-cholinesterase activity was evaluated by the value of IC 50 .

Protein tyrosine phosphatase 1B assay
The inhibitory activity of PTP1B was determined by little modification of the procedure previously reported (Tahtah et al. 2016).Shortly, before the assay, working buffer which contains MOPS (34.5 mM), DTT (2 mM), EDTA (0.69 mM), BSA (2 mg/mL) and NaCl (2 M), was prepared.10 lL of the samples (dissolved in DMSO), and 10 lL of PTP1B enzyme (12.5 mg/L in working buffer) was added to 96-well plates containing 70 lL working buffer, and the inhibitors were incubated with the enzyme at 37 C for 15 min.Then, the substrate p-NPP (100 mM in working buffer) 10 lL was added and the enzymatic reaction was performed at 37 C for 30 min, after that 100 lL of Na 2 CO 3 (0.1 M) as a stop buffer was added.Finally, the absorbance of the mixture was tested at 405 nm.Suramin was used as a positive control.

Molecular docking of a-glucosidase
Molecular docking analysis of a-glucosidase with compounds 12, 13, and 18 was performed using LeDock (version 1) (Zhang and Zhao 2016).The coordinates for a-glucosidase (PDB entry code: 7jty) enzyme was obtained from the Protein Data Bank (Karade et al. 2021).Water was removed and polar hydrogens were added for the accurate calculation of partial charges.The binding pocket was set at x between À19.578 and À3.599, y between 16.81 and 28.254, z between À18.487 and À4.958 for the protein.All the parameters were set as the default.The best docked complex for a-glucosidase with compound was selected on the basis of binding free energy value.

Molecular docking of BuChE
Molecular docking analysis of BuChE with compound 13 was performed using LeDock (version 1).The coordinates for BuChE (PDB entry code: 6qac) enzyme was obtained from the Protein Data Bank (Meden et al. 2019).Water was removed and polar hydrogens were added for the accurate calculation of partial charges.The binding pocket was set at x between 8. 175 and 27.786, y between 35.032 and 53.424, z between 34.78 and 47.48 for the protein.All the parameters were set as the default.The best