New benzofuran neolignans with neuroprotective activity from Phyllanthodendron breynioides

Abstract A pair of undescribed dihydrobenzofuran neolignan enantiomers, (+/−)-phybrenan A (1a/1b), two new benzofuran neolignans, phybrenan B and C (2 and 3), along with four known neolignans (4 − 7) were obtained from the plants of Phyllanthodendron breynioides P. T. Li. The planar structures of all isolates were demonstrated by the analysis of detailed spectroscopic evidence (NMR, HRMS, and IR), and the absolute configurations of novel neolignans were elucidated by combined calculated and experimental ECD data analysis. The neuroprotective activities of all benzofuran neolignans against sodium nitroprusside (SNP)-induced cell death were examined in rat pheochromocytoma PC12 cells. The results exhibited that three compounds (4 − 6) possessed remarkable neuroprotective activities at 10 µM, better than the positive drug edaravone. Graphical Abstract


Introduction
Lignans and neolignans, widely distributed in the plant kingdom, are important components of naturally occurring phenols composed by the oxidative coupling of two phenylpropanoid units (Teponno et al. 2016).Among them, benzofuran neolignans, an important subgroup of this kind of natural phenols, are characterized by the existence of a furan ring generated by the C-8 À C-5 0 carbon bond and the oxygen bridge between C-7 and C-4 0 (Xu et al. 2022).Over the years, numerous benzofuran neolignans have been isolated, and their structural diversity mainly stems from different substitutions pattern in the aromatic moieties (C6 units).Their diverse structures confer them various bioactivities, such as anti-inflammatory, anticancer, and neuroprotective properties (Teponno et al. 2016), which attracted the considerable attention of natural product chemists and pharmacologists.For example, (7 R,8S)-dehydrodiconiferyl alcohol (DDA), a benzofuran neolignan glycoside obtained from Clematis armandii, could inhibit LPS-stimulated inflammatory response via modulating JNK signaling and inhibiting NF-jB activation in BV2 cell (Liu et al. 2016).Boehmenan, another dihydrobenzofuran neolignan from the same plant C. armandii, was found to inhibit human epidermoid carcinoma cells growth by stemming the p70S6/S6 kinase pathway (Pan et al. 2017).Moreover, one of the dihydrobenzofuran neolignan enantiomers from R. idaeus, (À)-rasidasin II, was demonstrated to reduce H 2 O 2 -induced apoptosis in SH-SY5Y cells by attenuating mitochondrial dysfunction and reactive oxygen species (ROS) generation (Zhou et al. 2018).Notably, our group also has reported that a series of benzofuran neolignans from Aristolochia fordiana exhibited significantly protective effect against glutamate-induced injury via maintaining the Bcl-2 protein as well as the Nrf2/HO-1 signaling pathway (Tang et al. 2015).To obtain natural neuroprotective lead compounds, phytochemical investigations on other plants were carried out in depth.
Euphorbiaceae, containing approximately 7500 species, is one of the largest families of flowering plant (Xu et al. 2021), which is well-known for producing metabolites with various structures and wide biologic activities.Phyllanthodendron breynioides P. T. Li, one of species of Euphorbiaceae, is an erect shrub native to the Guangxi Zhuang Autonomous Region of China.The chemical compositions of P. breynioides have never been studied so far.In our continuing efforts toward the discovery of active natural products from Euphorbiaceae family (Wang et al. 2020;Hu et al. 2021;Huang et al. 2021;Weng et al. 2021;Wu et al. 2021;Yan et al. 2021;2022;Yuan et al. 2022), a fraction of the ethanolic extract of the leaves and twigs of P. breynioides showed neuroprotective activities against SNP-induced oxidative injury in rat pheochromocytoma PC12 cells.Subsequent chemical studies led to the separation of four new benzofuran neolignans (1a, 1 b, 2, and 3) and four known ananlgues (4 À 7) (Figure 1).And their neuroprotective activities of were also evaluated.Herein, we report the isolation, structural elucidation, and neuroprotective activities of all benzofuran neolignans.
Considering the specific rotation value of compound 1 was close to zero, an effort of chiral resolution of 1 was carried out by HPLC.Subsequently, a pair of enantiomers 1a and 1 b showing the opposite rotation values ([a] 20 D À37.0 for 1a, [a] 20 D þ31.0 for 1 b) as well as the mirror image-like ECD curves (Figure S1.3A in Supplemental Material) were obtained through the chiral resolution.Analysis of the ECD spectra of dihydrobenzofuran neolignans could well establish their absolute configurations.According to the well-known reversed helicity rule, the positive/negative 1 L b band CD (260 À 310 nm) in the 7-hydroxy-2,3-dihydrobenzo[b]furan chromophore system was caused by P/M helicity of heterocyclic ring (Goel et al. 2013;Kruakaew et al. 2022).Therefore, the negative Cotton effect at 266 nm (De À3.14) in the experimental ECD curve of 1a (Figure S1.3A in Supplemental Material) proved the presence of an M- helicity, and then the absolute configurations of 1a was identified as (7 R,8 R) (Tshitenge et al. 2017).In contrary to 1a, 1 b was identified as the 7S,8S absolute configurations based on the positive Cotton effect at 266 nm.Thus, 1a/1b were determined as shown and were named (À)-phybrenan A and (þ)-phybrenan A, respectively.
Compound 2 ([a] 20 D ¼ À68.0) was isolated as yellowish gum.Its molecular formula C 19 H 18 O 5 was classified by the HRESIMS ion at m/z 349.1041 The NMR data of 2 presented resemblances to those of minor oxidative by-product of perseal G (Tsai et al. 2001), except for the absence of the methoxy group and the presence of one 1-oxopropyl group in 2 rather than the 1-oxo-2-hydroxypropyl unit in this known neolignan.The HMBC correlations from H-2 0 , H-6 0 , and H-8 0 to C-7 0 and 1 HÀ 1 H COSY correlation of H-8 0 /H-9 0 further proved the presence and location of the 1-oxopropyl group (Figure S1.1 in Supplemental Material).The aforementioned data established the plane structure of 2 as a new dihydrobenzofuran neolignan.In the same way as 1a/1b, the trans relative configuration of H-7 and H-8 in compound 2 was established based on the large 1 HÀ 1 H coupling constants (J 7,8 ¼ 8.9 Hz).In addition to applying the reversed helicity rule to determine the absolute configuration, a calculating ECD method was adopted for 2 (Figure S1.3B in Supplemental Material).The tendency of the experimental ECD spectra of 2 exhibited a well resemblance to the calculated ECD spectra of (7 R,8R)-2a in the region of 190 À 400 nm, indicating that compound 2 displayed the same configuration as (7 R,8R).Therefore, compound 2 was demonstrated as shown and was named (À)-phybrenan B.
Compound 3, yellowish gum, had a molecular formula C 20 H 20 O 5 , as established by the HRESIMS ion at m/z 341.1389 341.1384).The spectroscopic data of 3 was very similar to eupomatenoid-7 (5) (Cheng et al. 2001), except for the presence of a 2-oxopropyl in 3 instead of a trans-propenyl at C-1 0 in 5.This was further supported by the key HMBC correlation from H 3 -9 0 to C-8 0 as well as H 2 -7 0 to C-1 0 , C-2 0 , C-6 0 , and C-8 0 (Figure S1.1 in Supplemental Material).Thus, the structure of compound 3 was determined as a new benzofuran neolignan and was given a trivial name phybrenan C.
The neuroprotective activities of 1a, 1b, and 2 À 7 against SNP (700 mM) induced cell death were screened in rat pheochromocytoma PC12 cells by the MTT assay.The preliminary screening (Figure 2A) showed that compounds 4 À 6 exhibited better neuroprotective effects than the positive control edaravone at a concentration of 10 mM.All compounds were non-toxic at the tested concentration.Particularly, these three active neolignans also exhibited remarkable effects in a concentration-dependent manner from 2.5 À 10 mM (Figure 2B).According to the activity results, the preliminary structure-activity relationships were summarized as follows: a) The presence of a trans double bond at the C 3 side chain was a prerequisite for the activity (1a, 1 b, and 4 À 6 vs 2, 3, and 7); b) Compared with 5-OH, the existence of 4-OH was favorable for the activity, as exampled by 4 À 6 vs 1a and 1 b; c) The difference of configuration had slight influence on the activity (1a vs 1 b).

Plant material
The leaves and twigs of P. breynioides were collected from Baise city, Guangxi Zhuang Autonomous Region, P. R.China and was authenticated by one of the authors (G.-H.Tang).A voucher specimen (accession number: YPBS-202006) has been deposited at the School of Pharmaceutical Sciences, Sun Yat-sen University.

ECD calculation of 2
The details of ECD calculations for compound 2 are presented in Supporting Material (S2 in Supplemental Material).

Neuroprotective bioassays
The neuroprotective activities of benzofuran neolignans against SNP (700 mM) induced cell death were examined in rat pheochromocytoma PC12 cells by the MTT assay (Weng et al. 2022), and the positive control was the drug edaravone.

Conclusion
In the current study, four new benzofuran neolignans (1a, 1 b, 2, and 3) with four known ananlgues (4 2 7) were first obtained from the P. breynioides.The structures were demonstrated by the analysis of detailed spectroscopic evidence (HRMS, IR, NMR and ECD).Among these, benzofuran neolignans 4 À 6 exhibited better potent neuroprotective effects than the positive control edaravone.Consequently, the first investigation of the chemical constituents of P. breynioides not only provided scientific evidence for the potential activities of this plant, but also enriched the structural diversity of benzofuran neolignans.Additionally, the remarkable neuroprotective effects of benzofuran neolignans 4 À 6 proved that neolignans could be as privileged structural motif in neuroprotective drug discovery and further investigation still required for the neuroprotective mechanism of these benzofuran neolignans.

Disclosure statement
No potential conflict of interest was reported by the authors.