A new neolignan and a new phenolic acid from the heartwood of Mansonia gagei J.R. Drumm

Abstract One new neolignan (1) and one new phenolic compound (2), together with four known compounds (3–6) were isolated from the heartwood of Mansonia gagei. Their structures were elucidated by extensive spectroscopic analyses, including 1D and 2D NMR and HRESIMS. The absolute configuration of 2 was established based on the DP4+ protocol and by comparison of experimental and calculated ECD spectra. All isolated compounds were evaluated by DPPH assay for antioxidant activity, while compounds 3–6 were assayed using the MTT-based colorimetric assay for cytotoxicity against lung cancer cell line A549. In terms of antioxidant activity, 1 and 3 exhibited stronger activity (IC50 14.91 ± 1.10 and 17.46 ± 0.16 μM, respectively) than the positive control, ascorbic acid (IC50 30.20 ± 0.47 μM). Among the compounds tested for cytotoxicity, compound 3 showed the highest activity, with an IC50 value of 26.04 ± 2.95 µM. Graphical Abstract

The antioxidant activity of isolated compounds 1-6 was determined using the DPPH radical scavenging assay (Figure 1).Interestingly, neolignan 1 and 3 exhibited a potent free-radical scavenging effect (IC 50 14.91 and 17.46 lM, respectively) and were twice as strong as ascorbic acid (IC 50 30.20 lM) (Supplementary material, Table S3).Moreover, the cytotoxic activity of 3-6 against the human lung carcinoma cell line A549 was evaluated using the MTT method.Among the compounds tested, 3 showed the highest cytotoxicity with an IC 50 value of 26.04 ± 2.95 mM, followed by 6 with an IC 50 value of 59.66 ± 0.99 mM, while 4 and 5 showed no cytotoxicity against A549 cells (Supplementary material, Table S4).

General experimental procedures
Optical rotations were measured on a JASCO P-1010 polarimeter (JASCO, Easton, MD, USA).The experimental ECD data were recorded on a JASCO J-815 spectropolarimeter.
HRESIMS spectra were recorded on a Bruker micrOTOF mass spectrometer.The NMR spectra were measured using a JEOL 500 MHz spectrometer in methanol-d 4 and CDCl 3 with TMS as an internal standard.Silica gel 60 G (Merck), silica gel 70 À 230 mesh (Merck) and Sephadex LH-20 (GE Chemical Corporation) were used for the column chromatography.TLC was performed using silica gel GF254 plates (Merck).

Plant material
The dried heartwood of Mansonia gagei was collected from Saraburi province, Thailand in 2019.The identity of this plant was compared with voucher specimen No. 43281 at the herbarium of the Royal Forestry Department of Thailand.

ECD and NMR calculations
Initial structures of 2a and 2b were constructed in Chem3D and subsequently subjected to a conformational search implemented in the MOE software using the MMFF94S force field (Halgren 1996).All conformers in the lowest 5 kcal/mol energy window (21 and 16 conformers for 2a and 2b, respectively) were used for Density Functional Theory (DFT) optimizations.The DFT, time dependent (TD) DFT calculations and NMR calculations using the gauge including atomic orbitals (GIAO) method were performed with the Gaussian 09 package (Frisch et al. 2016).The first optimization was performed by the B3LYP level of theory using the 6-31 G basis set in the gas phase.The optimized geometries in the lowest 2.5 kcal/mol energy window (4 and 7 conformers for 2a and 2b, respectively) were further optimized at the CAM-B3LYP/6-311 þ G ÃÃ level in MeOH for the ECD calculation or in CHCl 3 for the NMR calculation, using CPCM model.TMS was also optimized at the same level in CHCl 3 to calculate the chemical shifts.The frequency analysis and TD-DFT ECD calculation were performed at the same level of theory in MeOH, while the magnetic shielding constants were computed at the mPW1PW91/6-31 þ G ÃÃ level in CHCl 3 (PCM model).The chemical shifts were calculated according to the following equation: where r ref and r x are the NMR isotropic magnetic shielding tensors for TMS and the corresponding nucleus, respectively.The resultant theoretical ECD spectra and NMR shielding tensors for the individual conformers were averaged according to their Boltzmann populations at 293.15 K.The experimental and computed NMR data were analysed by the DP4þ method to assign the relative stereochemistry (Grimblat et al. 2015).The calculated ECD spectra were visualized with SpecDis software ver.1.71 (Bruhn et al. 2017).The half-band width and UV correction were 0.26 eV and þ10 nm, respectively.

Biological assays
Assay for cytotoxicity was performed as previously described (Sakunrangsit et al. 2016), and the DPPH assay followed the method described in the literature (Rankovi c et al. 2012).

Conclusion
Chemical investigation of M. gagei heartwood resulted in the isolation of a new neolignan (1) and a new phenolic acid (2), along with four known compounds (3-6).Their structures were unambiguously established by spectroscopic analyses (HRESIMS, 1D and 2D NMR), as well as by comparison to literature data.The stereochemistry of 2 was elucidated through DP4þ parameter and ECD calculations.Both neolignans 1 and 3 showed excellent antioxidant activities, with IC 50 values of 14.91 ± 1.10 and 17.46 ± 0.16 mM, respectively.Compound 3 also showed a potent cytotoxicity to the human lung cancer cell line, with an IC 50 value of 26.04 ± 2.95 mM.

Disclosure statement
No potential conflict of interest was reported by the author(s).
103.1  and 98.3), one carbonyl carbon (d C 171.7), one methoxy carbon (d C 56.4), one oxygenated methine carbon (d C 71.0) and one oxygenated quaternary carbon (d C 92.1).These NMR data were similar to those of phenol A (R€ odel and Gerlach 1995), indicating that 2 is a derivative of a phenolic compound.The HMBC cross-peak from the methoxy group to C-4 (d C 165.5) suggested that the methoxy group is attached at C-4.Moreover, the HMBC long-range correlation (Supplementary material, FigureS16) from H-5 (d H 6.43) to C-7 (d C 171.7) implied the occurrence of a carboxy group located at C-1 (d C 103.1).In addition, the COSY correlation between H 3 -4 0 (d H 0.92) and H-3 0 (d H 4.21), along with the HMBC correlation from H 3 -1 0 (d H 1.80) to C-2 0 (d C 92.1) and C-3 0 (d C 71.0), indicated a sec-butyl moiety, identified as C-1 0 /C-2 0 /C-3 0 /C-4 0 with the C2'-C-2 linkage, which was further supported by the HMBC correlation of H 3 -1 0 and H 3 -8 to C-2 (d C 150.0).These results suggest the connections of five substituents on the phenyl ring of 2, including hydroxy, carboxyl, sec-butyl, methyl and methoxy groups.