New Benzofuran Oligomers from the Roots of Eupatorium heterophyllum Collected in China

The chemical constituents of two root samples of Eupatorium heterophyllum DC. collected in Yunnan Province, China, were investigated. Five new oligomeric benzofurans (1–5), nine new benzofuran/dihydrobenzofuran derivatives, and a new thymol analog were isolated, and their structures were determined using extensive spectroscopic techniques, such as 1D and 2D NMR spectroscopy and DFT calculations of the CD spectra. Most of the new compounds, including oligomeric benzofurans (1–5), were obtained from only one of the root samples. Furthermore, this is the first example that produces oligomeric benzofurans in this plant. These results imply that diversification of secondary metabolites in E. heterophyllum is ongoing. Plausible biosynthetic pathways for 1–5 are also proposed.


Introduction
Plants in the Hengduan mountains area are useful for studying plant diversity and evolution. We have been studying inter-and intra-specific diversity in chemical constituents of some Asteraceae plants growing in this area, including Ligularia [1,2], Cremanthodium [3], Saussurea [4,5], and Eupatorium [6][7][8][9]. To date, the presence of intra-specific diversity has been discovered at various levels in most species, affording numerous new compounds produced by limited populations within a species.
Eupatorium heterophyllum DC. (Asteraceae) is a perennial herb widely distributed in the Hengduan Mountains, including in the Yunnan, Sichuan, and Gansu provinces and the Xizang Autonomous Region of China [10]. Only our group has previously reported detailed phytochemical studies on this plant [6][7][8][9]. Sesquiterpene lactones were isolated as major constituents from several samples of the aerial parts of E. heterophyllum collected in northwestern Yunnan and southwestern Sichuan [7], and benzofurans were isolated from the roots of this species in northwestern Yunnan [6] and northern Sichuan [8]. The characteristics of the chemical compositions are similar to those of E. chinense [11][12][13], suggesting that E. heterophyllum and E. chinense are related chemotaxonomically. Intra-specific diversity in the root chemicals of E. heterophyllum was observed in minor constituents, and a variety of benzofuran/dihydrobenzofuran derivatives, propynyl thiophenes, acetylenic compounds, and oxygenated thymol were obtained. Benzofurans are a significant group of heterocyclic compounds with wide ranges of biological activities [14], such as antibacterial [15], antifungal [16,17], and antifeedant activities [18,19]. These findings prompted us to conduct additional phytochemical studies on this plant growing in other locations. In this study, two additional root samples of E. heterophyllum were collected at different locations in Yunnan Province of China as part of our ongoing research into the chemical diversity of the genus Eupatorium. Fifteen new compounds, including five benzofuran different locations in Yunnan Province of China as part of our ongoing research into the chemical diversity of the genus Eupatorium. Fifteen new compounds, including five benzofuran oligomers (1-5), were isolated from the MeOH extract of the roots of the collected samples. Herein, we report the isolation and structural elucidation of these compounds. Plausible biosynthetic pathways for 1-5 are also proposed.

Samples
Two E. heterophyllum samples were collected in Lanping County (sample 1) and Lijiang City (sample 2) of Yunnan Province. These sampling locations were approximately 50-100 km southeast from those of our previous Yunnan samples [6]. The dried roots of each sample were cut into pieces and extracted with MeOH, and the compounds were separated using silica-gel column chromatography and normal-phase HPLC to yield 49 compounds, 15 of which were new. The structures of the new compounds (1-5, 14, 17, 21, 25, 26, 31, 32, 34, 36, and 39) were elucidated as follows.

Structure Elucidation
Compound 1 was obtained as yellow amorphous powder. The [M+Na] + peak at m/z 489.1527 in HRFABMS revealed that its molecular formula is C26H26O8. The presence of hydroxy (3416 cm −1 ) and conjugated carbonyl (1651 cm −1 ) groups suggested using the IR spectrum. The 1 H NMR spectrum (Table 1) revealed resonances attributable to two hydroxy groups [δH 11.72 (1H, s) and 2.84 (1H, br s)], an aromatic proton [δH 7.14 (1H, s)], two oxymethines [δH 5.34 and 5.00 (each 1H, s)], an exo-methylene [δH 5.10 and 4.94 (each 1H, s)], and two methyls [δH 2.88 and 1.79 (each 3H, s)]. These spectroscopic features were very similar to those of 29 [8], except for the appearance of a singlet aromatic proton signal and the disappearance of a pair of ortho-coupled aromatic proton signals in the 1 H NMR spectrum of 1. Only 13 carbon resonances, including a carbonyl, three methines, a methylene, two methyls, and six quaternary carbons (Table 1), were detected in the 13 C NMR and HSQC spectra that 1 exhibited. Moreover, 1 exhibited adequate negative optical rotation ([α] 11 D −23.9). These observations and the molecular formula of 1 suggest that 1 is a homodimer of 29 with a symmetrical structure, in which the benzene rings of each monomeric unit are directly connected. The structure of the monomeric unit and its connection to another unit via C-6 and C-6' was established from the 1 H 1 H COSY and HMBC correlations shown in Figure 1, which were further supported by the downfield shift of C-6 to δC 130.3 and the NOESY correlation between 5-OH and H-7'. The relative stereochemistry of 1 was proposed to be 2,3-trans, based on the small JH-2-H-3 (br s) observed [20,21], as well as the NOE correlation between H-2 and 3-OH; H-3 and H-11/H3-12. Therefore, the relative structure of compound 1 was established, as shown in Figure 1.    13 C NMR spectrum of compound 2 revealed twenty-six carbon signals, including two carbonyls, seven methines, two methylene, four methyls, and eleven quaternary carbons (Table 1). These observations, along with the 1 H 1 H COSY and HMBC correlations (Figure 2), indicated that 2 is composed of two 2,3-dihydrobenzofuran moieties, similar to 29 [8]. The connection of the two benzofuran moieties via an ether bond between C-5 and C-6' in 2 was confirmed by the disappearance of the 5-OH signal in the 1 H NMR spectrum and the NOESY correlations between H-7' and H-6/H 3 -14. Based on a similar consideration as 1, the relative stereochemistry of C-2/C-3 and C-2'/C-3' was discovered to be trans. Therefore, the relative structure of compound 2 was determined as depicted in Figure 2. (1H, d, 8.8 Hz), and 6.66 (1H, s)], four oxymethines [δH 5.29 (1H, dd, 7.1, 2.6 Hz), 5.21 (1H, br s), 5.04 (1H, s), and 4.95 (1H, br s)], two exo-methylenes [δH 5.10, 5.04, 4.92 and 4.92 (each 1H, s)], and four methyls [δH 2.86, 2.69, 1.78 and 1.75 (each 3H, s)]. The 13 C NMR spectrum of compound 2 revealed twenty-six carbon signals, including two carbonyls, seven methines, two methylene, four methyls, and eleven quaternary carbons (Table 1). These observations, along with the 1 H 1 H COSY and HMBC correlations (Figure 2), indicated that 2 is composed of two 2,3-dihydrobenzofuran moieties, similar to 29 [8]. The connection of the two benzofuran moieties via an ether bond between C-5 and C-6' in 2 was confirmed by the disappearance of the 5-OH signal in the 1 H NMR spectrum and the NOESY correlations between H-7' and H-6/H3-14. Based on a similar consideration as 1, the relative stereochemistry of C-2/C-3 and C-2'/C-3' was discovered to be trans. Therefore, the relative structure of compound 2 was determined as depicted in Figure 2. Compound 3 was obtained as yellow amorphous powder, and its molecular formula, C39H38O12, was calculated from the [M + Na] + ion peak observed at m/z 721.2260 in HR-FABMS. The 1 H and 13 C NMR spectra of 3 (Table 1) revealed signals attributable to the three 2,3-dihydrobenzofuran moieties, indicating that 3 is a trimeric benzofuran derivative. The comparison of the NMR data of 3 with those of 2 implied that 3 shares a common structure with 2 and is connected to another 2,3-dihydrobenzofuran unit ( Figure 3). The linkages of the benzofuran units via oxygen atoms between C-5 and C-6' and between C-5' and C-6" were determined by NOESY correlations between H3-14 and H-7'/H-7"; H3-14' and H-7". Therefore, the relative structure of compound 3 was determined, as shown in Figure 3. Compound 3 was obtained as yellow amorphous powder, and its molecular formula, C 39 H 38 O 12 , was calculated from the [M + Na] + ion peak observed at m/z 721.2260 in HR-FABMS. The 1 H and 13 C NMR spectra of 3 (Table 1) revealed signals attributable to the three 2,3-dihydrobenzofuran moieties, indicating that 3 is a trimeric benzofuran derivative. The comparison of the NMR data of 3 with those of 2 implied that 3 shares a common structure with 2 and is connected to another 2,3-dihydrobenzofuran unit ( Figure 3). The linkages of the benzofuran units via oxygen atoms between C-5 and C-6' and between C-5' and C-6" were determined by NOESY correlations between H 3 -14 and H-7'/H-7"; H 3 -14' and H-7". Therefore, the relative structure of compound 3 was determined, as shown in Figure 3.  Because compounds 1-3 were considered oligomers of 29, their absolute configurations were determined by comparing their ECD spectra to that of 29 [8]. As shown in Figure 4, the experimental ECD spectra of 1-3 and 29 are in good agreement with the theoretical ECD spectrum of (2S,3R)-29. Therefore, the absolute configurations of 1-3 were established to be (2S,3R,2'S,3'R)-1, (2S,3R,2'S,3'R)-2, and (2S,3R,2'S,3'R,2"S,3"R)-3, respectively. The calculated ECD spectra of (2R,3R)-29 and each optimized conformer of (2S,3R)-29 are shown in Supplementary Materials: Figures S19 and S20.  Because compounds 1-3 were considered oligomers of 29, their absolute configurations were determined by comparing their ECD spectra to that of 29 [8]. As shown in Figure 4, the experimental ECD spectra of 1-3 and 29 are in good agreement with the theoretical ECD spectrum of (2S,3R)-29. Therefore, the absolute configurations of 1-3 were established to be (2S,3R,2'S,3'R)-1, (2S,3R,2'S,3'R)-2, and (2S,3R,2'S,3'R,2"S,3"R)-3, respectively. The calculated ECD spectra of (2R,3R)-29 and each optimized conformer of (2S,3R)-29 are shown in Supplementary Materials: Figures S19 and S20.
Compounds 4 and 5 exhibited significantly weaker Cotton effects in their experimental ECD spectra than the calculated spectra of (10S,2'S,3'R)-4 and (9'R)-5 ( Figure S33a), respectively, indicating that they are racemates. To confirm this, chiral HPLC analyses of 4 and 5 were performed, resulting in the detection of enantiomers in a ratio of approximately 1:1 for 4 and 5 ( Figure S33b). parts. The conformation of the dihydropyran ring was established by the NOE correlations shown in Figure 6.
Compounds 4 and 5 exhibited significantly weaker Cotton effects in their experimental ECD spectra than the calculated spectra of (10S,2'S,3'R)-4 and (9'R)-5 ( Figure S33a), respectively, indicating that they are racemates. To confirm this, chiral HPLC analyses of 4 and 5 were performed, resulting in the detection of enantiomers in a ratio of approximately 1:1 for 4 and 5 ( Figure S33b). Compound 14 was obtained as colorless amorphous powder. Based on the [M+H] + peak at m/z 291.1231 in its HRFABMS, its molecular formula was determined to be C16H18O5. The 1 H and 13 C NMR data (Table 3) were similar to those of known compound 13 [25]; however, the appearance of signals attributed to a propionyl group [δH 2.31 (2H, q, 7.6 Hz), 1.12 (1H, t, 7.6 Hz); δC 174.1, 27.7, 9.0] in the NMR spectrum of 14 instead of those attributed to an isobutanoyl group in 13 suggested that 14 was a 3-propionyloxy analog of 13 ( Figure 7). The 2,3-cis nature was indicated by J2,3 (6.4 Hz) [20,21] and the NOE correlation between H-2 and H-3 ( Figure 8). Therefore, the structure of compound 14 was determined as shown in Figure 8.
Compound 17 was obtained as yellowish amorphous powder. Its molecular formula was determined to be C18H20O6 by the [M + Na] + peak at m/z 355.1160 in its HRFABMS. Compound 14 was obtained as colorless amorphous powder. Based on the [M+H] + peak at m/z 291.1231 in its HRFABMS, its molecular formula was determined to be C 16 H 18 O 5 . The 1 H and 13 C NMR data (Table 3) were similar to those of known compound 13 [25]; however, the appearance of signals attributed to a propionyl group [δ H 2.31 (2H, q, 7.6 Hz), 1.12 (1H, t, 7.6 Hz); δ C 174.1, 27.7, 9.0] in the NMR spectrum of 14 instead of those attributed to an isobutanoyl group in 13 suggested that 14 was a 3-propionyloxy analog of 13 ( Figure 7). The 2,3-cis nature was indicated by J 2,3 (6.4 Hz) [20,21] and the NOE correlation between H-2 and H-3 ( Figure 8). Therefore, the structure of compound 14 was determined as shown in Figure 8. was also observed in the 1 H NMR spectrum of 17. Therefore, 17 was determined to be an 11-O-angeloyl derivative of compound 19, as supported by the HMBC correlations shown in Figure 7, particularly from H 2 -11 to C-1'.    Compound 21 was obtained as yellow amorphous powder. Its molecular formula was determined to be C 13 H 12 O 4 based on the [M + H] + peak at m/z 233.0814 in its HRFABMS. Careful comparison of the 1 H NMR data of 21 (Table 3) with those of platypodantherone [27] revealed that 21 was a 6-O-demethyl derivative of platypodantherone because of the absence of a methoxy signal and the appearance of a hydrogen-bonded phenolic hydroxy signal (δ H 12.99). This structure was confirmed by the HMBC from H 2 -12 to C-3 and other 2D NMR correlations (Figure 7).
The molecular formulae of compounds 34 and 36 were determined to be C 18 in 36), which was the same value as J 2β,3α in 33 [6] and 7-hydroxytoxol [29,30], respectively. This stereochemistry was supported using the NOESY correlations between H-3 and H 3 -12 ( Figure 8). Compound 39 was obtained as white amorphous powder. Its molecular formula was determined to be C 15 H 20 O 4 by the quasi-molecular ion at m/z 247.1334 [M-H 2 O+H] + in its HRFABMS. The 1 H and 13 C NMR spectra of 39 were closely related to those of 3,9β-epoxy-9α-isobutanoyloxymentha-1,3,5-trien-8a-ol, a recently reported thymol derivative [8], except for the disappearance of the signals attributable to an isobutanoyloxy group substituted at C-9 and the appearance of those of a 2-methylbutanoyloxy group ( Table 5). The relative configuration of the furan moiety was determined by the NOESY correlation between H-9 and H 3 -10 ( Figure 8). A detailed analysis of the 1 H and 13 C NMR spectra of 39 revealed that it was a mixture of C-2' epimers (ca. 3:1 based on the integration of 1 H NMR signals).

Discussion
Plausible biosynthetic pathways for the new benzofuran oligomers (1-5) are depicted in Scheme 1. Compound 1 can be a homocoupling product of two ortho-radicals generated by one-electron oxidation of known compound 29. Similarly, compound 2 is likely to be formed by the radical coupling of 29-derived phenoxy-and ortho-radicals, and subsequent radical coupling of 29 with 2 will afford trimer 3. Compound 4 can be produced by the nucleophilic attack of euparin (15) on an epoxide, derived from another molecule of 15, via ring-opening of the epoxide, followed by the construction of another furan ring. Compound 5 will be yielded via a [4 + 2] cycloaddition reaction between 27-derived aldehyde and 37.

Discussion
Plausible biosynthetic pathways for the new benzofuran oligomers (1-5) are depicted in Scheme 1. Compound 1 can be a homocoupling product of two ortho-radicals generated by one-electron oxidation of known compound 29. Similarly, compound 2 is likely to be formed by the radical coupling of 29-derived phenoxy-and ortho-radicals, and subsequent radical coupling of 29 with 2 will afford trimer 3. Compound 4 can be produced by the nucleophilic attack of euparin (15) on an epoxide, derived from another molecule of 15, via ring-opening of the epoxide, followed by the construction of another furan ring. Compound 5 will be yielded via a [4 + 2] cycloaddition reaction between 27-derived aldehyde and 37. Scheme 1. Plausible biogenetic pathway for 1-5.
In this study, 49 compounds, including 15 new compounds, were isolated from 2 root samples of E. heterophyllum collected in Yunnan Province (Figure 9). The major constituents of both samples were benzofuran/dihydrobenzofuran derivatives, such as 6 and 15. Sample 2 also contained a significant amount of thiophenes (e.g., 43). These characteristics of chemical composition were very similar to those of our previous E. heterophyllum In this study, 49 compounds, including 15 new compounds, were isolated from 2 root samples of E. heterophyllum collected in Yunnan Province (Figure 9). The major constituents of both samples were benzofuran/dihydrobenzofuran derivatives, such as 6 and 15. Sample 2 also contained a significant amount of thiophenes (e.g., 43). These characteristics of chemical composition were very similar to those of our previous E. heterophyllum samples collected in Yunnan and Sichuan provinces [6,8]. However, it is worth noting that sample 2 is the only sample to date that produces oligomeric benzofurans: two dimeric benzofuran diastereomers [9]. Compounds 1-5 were obtained from this sample, but not from sample 1 or other previous samples [6,8], implying that the diversification of secondary metabolites in E. heterophyllum is ongoing.
Eupatorium heterophyllum is generally regarded as a synonym of E. mairei [10]. In contrast, Kawahara et al. have proposed that E. heterophyllum is distinguished from E. mairei and may be a hybrid originating from E. mairei and E. chinense [48]. As described above, the chemical compositions of E. heterophyllum are similar to those of E. chinense [11][12][13]. Moreover, some research groups have recently reported the isolation of various oligomeric and related benzofuran compounds from E. chinense of various origins [49][50][51][52][53]. These findings indicate a close relationship between E. heterophyllum and E. chinense, which would lend support to Kawahara's theory. Further chemical studies on E. heterophyllum collected from other regions are in progress.
Author Contributions: Y.S., X.G. and T.T. conceived and designed the study; Y.H. isolated the chemicals; Y.H., Y.S. and Y.M. analyzed the spectroscopic data; Y.H., Y.S. and T.T. discussed the conclusion and wrote the paper. All authors have read and agreed to the published version of the manuscript.