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

Abstract

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.

1. 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 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.

2. Results and Discussion

2.1. 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.

2.2. 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 ¹H NMR spectrum (

Table 1. ¹ H (500 MHz) and ¹³ C (126 MHz) NMR data of 1–3 in CDCl₃.

	1				2				3
Position	δH	(mult., J in Hz)	δC	Position	δH	(mult., J in Hz)	δC	Position	δH	(mult., J in Hz)	δC
2, 2′	5.00	(1H, br s)	92.9	2	5.04	(1H, br s)	92.2	2	4.97	(1H, br s)	91.86
3, 3′	5.34	(1H, br s)	77.7	3	5.21	(1H, br s)	76.3	3	5.06	(1H, br s)	76.22
4, 4′			119.2	4			128.1	4			126.31
5, 5′			153.4	5			148.8	5			149.28
6, 6′			130.3	6	6.91	(1H, d, 8.8)	122.1	6	6.76	(1H, d, 8.8)	118.89
7, 7′	7.14	(1H, s)	119.8	7	7.03	(1H, d, 8.8)	115.5	7	6.88	(1H, d, 8.8)	114.94
8, 8′			153.4	8			157.2	8			156.35
9, 9′			126.6	9			130.3	9			129.56
10, 10′			141.1	10			141.4	10			141.24 (a)
11, 11′	5.10	(1H, s)	112.6	11	5.10	(1H, s)	112.0	11	5.04	(1H, s)	112.14
	4.94	(1H, s)			4.92	(1H, s)			4.88	(1H, s)
12, 12′	1.79	(3H, s)	17.8	12	1.78	(3H, s)	17.6	12	1.72	(3H, s)	17.51
13, 13′			204.6	13			202.0	13			202.48
14, 14′	2.88	(3H, s)	31.1	14	2.69	(3H, s)	32.1	14	2.49	(3H, s)	32.23
3-OH, 3′-OH	2.84	(1H, br s)		3-OH	4.37	(1H, br s)		3-OH	4.46	(1H, d, 1.2)
5-OH, 5′-OH	11.72	(1H, s)		2′	4.95	(1H, br s)	93.0	2′	5.06	(1H, br s)	92.72
				3′	5.29	(1H, dd, 7.1, 2.6)	77.5	3′	5.24	(1H, dd, 3.4, 1.7)	75.98
				4′			119.2	4′			130.41
				5′			148.0	5′			147.93
				6′			147.2	6′			139.88
				7′	6.66	(1H, s)	106.4	7′	6.81	(1H, s)	107.59
				8′			153.0	8′			157.70
				9′			120.6	9′			126.03
				10′			141.0	10′			141.09
				11′	5.04	(1H, s)	112.5	11′	5.13	(1H, s)	112.23
					4.92	(1H, s)			4.96	(1H, s)
				12′	1.75	(3H, s)	17.7	12′	1.82	(3H, s)	17.74
				13′			203.6	13′			201.64
				14′	2.86	(3H, s)	31.3	14′	2.67	(3H, s)	30.98
				3′-OH	2.93	(1H, d, 7.1)		3′-OH	4.32	(1H, d, 1.7)
				5′-OH	10.49	(1H, s)		2″	4.91	(1H, br s)	93.22
								3″	5.14	(1H, dd, 9.3, 2.4)	77.28
								4″			118.39
								5″			146.83
								6″			146.83
								7″	6.58	(1H, s)	104.86
								8″			152.60
								9″			120.32
								10″			141.09 (a)
								11″	4.98	(1H, s)	112.06
									4.87	(1H, s)
								12″	1.71	(3H, s)	17.67
								13″			204.18
								14″	2.78	(3H, s)	32.22
								3″-OH	4.04	(1H, d, 9.3)
								5″-OH	11.25	(1H, s)

^(a) interchangeable.

) revealed resonances attributable to two hydroxy groups [δ_H 11.72 (1H, s) and 2.84 (1H, br s)], an aromatic proton [δ_H 7.14 (1H, s)], two oxymethines [δ_H 5.34 and 5.00 (each 1H, s)], an exo-methylene [δ_H 5.10 and 4.94 (each 1H, s)], and two methyls [δ_H 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 ¹H 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 ¹³C NMR and HSQC spectra that 1 exhibited. Moreover, 1 exhibited adequate negative optical rotation ([α${]}_D^{11}$ −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 ¹H ¹H COSY and HMBC correlations shown in Figure 1, which were further supported by the downfield shift of C-6 to δ_C 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 J_H-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.

Compound 2 was obtained as yellow amorphous powder. The same molecular formula of C₂₆H₂₆O₈ as compound 1 was determined using HRFABMS and ¹³C NMR data. The IR spectrum indicated the presence of hydroxy (3411 cm⁻¹) and conjugated carbonyl (1656 cm⁻¹) groups. The UV spectrum showed maximum absorptions at 222 and 337 nm.

The ¹ H NMR spectrum (Table 1) revealed three hydroxy groups [δ_H 10.49 (1H, s), 4.37 (1H, br s), and 2.93 (1H, d, 7.1 Hz)], three aromatic protons [δ_H 7.03 (1H, d, 8.8 Hz), 6.91 (1H, d, 8.8 Hz), and 6.66 (1H, s)], four oxymethines [δ_H 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 [δ_H 5.10, 5.04, 4.92 and 4.92 (each 1H, s)], and four methyls [δ_H 2.86, 2.69, 1.78 and 1.75 (each 3H, s)]. The ¹³C 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 ¹H ¹H 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 ¹H 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 ¹H and ¹³C 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.

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.

Compound 4 was obtained as yellow amorphous powder. Its molecular formula was determined to be C₂₆H₂₄O₇ by the [M + H]⁺ peak at m/z 449.1600 in its HRFABMS, indicating 15 degrees of unsaturation. Its ¹H and ¹³C NMR data (

Table 2. ¹H (500 MHz) and ¹³C (126 MHz) NMR data of 4 and 5 in CDCl₃.

	4				5
Position	δH	(mult., J in Hz)	δC	Position	δH	(mult., J in Hz)	δC
2			160.2	2			161.7
3	6.31	(1H, d, 0.8)	102.3	3	6.35	(1H, s)	99.0
4	7.70	(1H, s)	123.5	4	6.94	(1H, s)	106.5
5			116.8	5			158.7
6			160.8	6			115.4
7	6.79	(1H, br s)	99.1	7	7.70	(1H, s)	111.0
8			159.2	8			147.3
9			120.8	9			137.5
10			84.6	10			104.7
11	2.84	(1H, dd, 13.2, 1.2)	43.1	11	7.37	(1H, s)	144.4
	2.45	(1H, dd, 13.2, 8.8)		12	2.82	(1H, dddd, 16.2, 11.2, 6.0, 1.8)	18.9
12	1.74	(3H, s)	27.9		2.44	(1H, dddd, 16.2, 5.4, 2.6, 1.0)
13			204.0	13			203.4
14	2.64	(3H, s)	26.9	14	2.66	(3H, s)	26.7
6-OH	12.42	(1H, s)		5-OH	12.26	(1H, s)
2′			122.8	1′			129.6
3′	3.87	(1H, d, 8.8)	49.8	2′	7.47	(1H, d, 1.9)	110.0
4′	7.30	(1H, d, 1.0)	126.0	3′			146.7
5′			113.9	4′			151.6
6′			165.9	5′			108.1
7′	6.17	(1H, s)	97.9	6′	7.58	(1H, d, 1.9)	127.1
8′			165.5	7′			78.4
9′			120.9	8′			94.8
10′			141.7	9′			71.9
11′	5.46	(1H, s)	113.8	10′	2.29	(1H, ddd, 13.5, 6.0, 2.6)	33.2
	5.10	(1H, t, 1.5)			2.02	(1H, ddd, 13.5, 11.2, 5.4)
12′	1.86	(3H, s)	18.2	11′	1.79	(3H, s)	28.1
13′			201.8	12′			196.1
14′	2.44	(3H, s)	26.1	13′	2.53	(3H, s)	26.2
5′-OH	12.76	(1H, s)		3′-OCH3	3.92	(3H, s)	56.3
				4′-OH	6.28	(1H, s)

), as well as the HMBC correlations (Figure 5) from H-4 to C-3/C-6/C-8/C-13; 6-OH to C-5/C-6/C-7; H-4’ to C-6’/C-8’/C-13’, and 6’-OH to C-5’/C-6’/C-7’, suggested that 4 consists of two 5-acetyl-6-hydroxybenzofuran units, such as euparin (15) [22,23], one of the major constituents of this plant [9]. Furthermore, the ¹H ¹H COSY cross-peak between H₂-11/H-3’, HMBC correlations from H₂-11 to C-2/C-10/C-2’/C-3’/C-9’, and the molecular formula of 4 indicated that these benzofuran units are linked by a tetrahydrofuran ring composed of C-10 (δ_C 84.6), C-11 (δ_C 43.1), C-3’ (δ_C 49.8), C-2’ (δ_C 122.8), and oxygen between C-10 and C-2’. The NOESY correlations of H-3’/H₃-12’, H₃-12/H-11’a, H₃-12’/H-11’b, H-11a/H-3, and H-11a/H-4’ and the coupling constants of H-3’ [δ_H 3.87 (1H, d, 8.8 Hz)], H-11a [δ_H 2.84 (1H, dd, 13.2, 1.2 Hz)], and H-11b [δ_H 2.45 (1H, dd, 13.2, 8.8 Hz)] established the relative configurations as (10S *,2’S *,3’R *)-4 (Figure 5).

Compound 5 was obtained as yellow amorphous powder. The [M + H]⁺ peak at m/z 461.1601 in HRFABMS determined its molecular formula to be C₂₇H₂₄O₇, indicating 16 degrees of unsaturation. The IR spectrum revealed the presence of hydroxy (3245 cm⁻¹) and conjugated carbonyl groups (1672 cm⁻¹), as well as an acetylenic moiety (2225 cm⁻¹).

The ¹H NMR spectrum of 5 exhibited signals derived from two hydroxy groups [δ_H 12.26 and 6.28 (each 1H, s)], six aromatic/olefinic protons [δ_H 7.70 (1H, s), 7.58 (1H, d, 1.9 Hz), 7.47 (1H, d, 1.9 Hz), 7.37 (1H, s), 6.94 (1H, s), and 6.35 (1H, s)], two methylenes [δ_H 2.82 (1H, dddd, 16.2, 11.2, 6.0, 1.8 Hz), 2.44 (1H, dddd, 16.2, 5.4, 2.6, 1.0 Hz), 2.29 (1H, ddd, 13.5, 6.0, 2.6 Hz), and 2.02 (1H, ddd, 13.5, 11.2, 5.4 Hz)], a methoxy group [δ_H 3.92 (3H, s)], and three methyls [δ_H 2.66, 2.53, and 1.79 (each 3H, s)] (Table 2). The ¹³C NMR spectrum revealed 27 carbon signals, including two carbonyls, six methines, two methylenes, four methyls, and thirteen quaternary carbons (Table 2). These observations indicated that the structure of 5 is composed of benzofuran and acetylenic moieties, such as 27 [24] and 37 [6], respectively. The substitution pattern of the benzene rings in 5 were determined from the HMBC correlations shown in Figure 6. Moreover, the ¹H ¹H COSY correlation between H₂-12 and H₂-10’, as well as the HMBC correlations from H₂-10’ to C-10 and from H-11 to C-9’, revealed the presence of a dihydropyran ring linking the benzofuran and acetylenic 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 ¹H and ¹³C NMR data (

Table 3. ¹H (500 MHz) and ¹³C (126 MHz) NMR data of 14, 17, and 21 in CDCl₃.

	14			17			21
Position	δH	(mult., J in Hz)	δC	δH	(mult., J in Hz)	δC	δH	(mult., J in Hz)	δC
2	5.14	(1H, d, 6.4)	89.2			160.6	5.47	(1H, d, 6.1)	88.4
3	6.26	(1H, d, 6.4)	72.2	6.64	(1H, d, 1.0)	102.5	5.67	(1H, d, 6.1)	81.9
4	7.81	(1H, s)	130.0	7.93	(1H, s)	123.7	7.81	(1H, s)	129.2
5			114.7			117.0			114.8
6			166.7			161.1			167.0
7	6.46	(1H, s)	98.9	6.99	(1H, br s)	99.8	6.38	(1H, s)	98.5
8			166.7			159.5			166.8
9			117.8			120.8			118.3
10			138.0			71.3			145.3
11	5.20	(1H, d, 1.0)	114.7	4.53	(1H, d, 11.5)	69.7	4.36	(1H, d, 12.5)	69.2
	5.12	(1H, d, 1.0)		4.38	(1H, d, 11.5)		4.22	(1H, d, 12.5)
12	1.81	(3H, s)	19.2	1.67	(3H, s)	23.9	5.56	(1H, s)	112.9
							5.38	(1H, s)
13			202.7			204.0			202.5
14	2.57	(3H, s)	26.4	2.69	(3H, s)	26.9	2.58	(3H, s)	26.3
1′			174.1			167.9
2′	2.31	(2H, q, 7.6)	27.7			127.1
3′	1.12	(3H, t, 7.6)	9.0	6.09	(1H, qq, 7.3, 1.4)	139.5
4′				1.89	(3H, dq, 7.3, 1.4)	15.8
5′				1.83	(3H, quint, 1.4)	20.5
6-OH	13.04	(1H, s)		12.46	(1H, s)		12.99	(1H, s)

) 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.

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. The ¹H and ¹³C NMR data of 17 (Table 3) were similar to those of known compound 19 [26], except for the presence of additional signals assignable to an angeloyloxy group [δ_H 6.09 (1H, qq, 7.3, 1.4 Hz), 1.89 (3H, dq, 7.3, 1.4 Hz), 1.83 (3H, quint, 1.4); δ_C 167.9, 127.1, 139.5, 15.8, 20.5]. The downfield shift of H₂-11 [δ_H 4.53 (1H, d, 11.5 Hz), 4.38 (1H, d, 11.5 Hz)] was also observed in the ¹H 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 H₂-11 to C-1’.

Compound 21 was obtained as yellow amorphous powder. Its molecular formula was determined to be C₁₃H₁₂O₄ based on the [M + H]⁺ peak at m/z 233.0814 in its HRFABMS. Careful comparison of the ¹H 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 H₂-12 to C-3 and other 2D NMR correlations (Figure 7).

The molecular formulae of compounds 25 and 26 were determined to be C₁₃H₁₄O₄ and C₁₅H₁₆O₅, respectively, using HRFABMS. The structural similarity of compound 25 to that of 11-hydroxy-10,11-dihydro-euparin [28] was deduced from the ¹H NMR data (

Table 4. ¹H (500 MHz) and ¹³C (126 MHz) NMR data of 25, 26, 31, and 32 in CDCl₃.

	25			26			31			32
Position	δH	(mult., J in Hz)	δC	δH	(mult., J in Hz)	δC	δH	(mult., J in Hz)	δC	δH	(mult., J in Hz)	δC
2			166.8			165.7			162.0			154.2
3	6.46	(1H, s)	102.8	6.42	(1H, s)	102.6	6.70	(1H, s)	102.7	7.56	(1H, s)	112.9
4	7.01	(1H, s)	107.4	7.01	(1H, s)	107.5	7.80	(1H, d, 1.5)	115.9	7.93	(1H, d, 1.5)	117.4
5			158.4			158.4			133.8			134.7
6			115.8			115.9	7.48	(1H, d, 1.5)	105.5	7.62	(1H, d, 1.5)	108.0
7	7.78	(1H, s)	111.8	7.78	(1H, s)	111.8			145.3			146.2
8			147.8			147.7			146.8			147.8
9			136.7			136.7			129.4			128.3
10	3.18	(1H, sext, 6.5)	36.9	3.32	(1H, sext, 6.8)	33.7	5.06	(1H, m)	64.0			188.6
11	3.88	(2H, m)	66.0	4.36	(1H, dd, 11.0, 6.8)	66.3
				4.27	(1H, dd, 11.0, 6.3)
12	1.39	(3H, d, 7.1)	14.9	1.39	(3H, d, 7.1)	15.4	1.66	(3H, d, 6.6)	21.4	2.66	(3H, s)	26.7
13			203.7			203.8			197.6			197.1
14	2.68	(3H, s)	26.7	2.68	(3H, s)	26.7	2.66	(3H, s)	26.6	2.68	(3H, s)	26.6
1′						170.9
2′				2.05	(3H, s)	20.9
5-OH	12.16	(1H, s)		12.16	(1H, s)
7-OCH3							4.06	(3H, s)	56.1	4.08	(3H, s)	56.2
10-OH							2.18	(1H, d, 4.9)

); however, the resonance of an aromatic proton (δ_H 7.78) was slightly downfield-shifted in 25 as compared to that of 11-hydroxy-10,11-dihydro-euparin. The HMBC and NOESY correlations (Figure 7 and Figure 8) revealed compound 25 as a 6-acetyl-5-hydroxy-isomer of 11-hydroxy-10,11-dihydro-euparin. The ¹H and ¹³C NMR spectra of 26 were distinguished from those of 25 by the presence of acetate-derived signals [δ_H 2.05 (3H, s); δ_C 170.9, 20.9] and the downfield-shift of H₂-11 [δ_H 4.36 (1H, dd, 11.0, 6.8 Hz), 4.27 (1H, dd, 11.0, 6.3 Hz)] (Table 4). Therefore, 26 was identified as an 11-O-acetyl derivative of 25, which was further supported by the HMBC correlation from H₂-11 to C-1’ (Figure 7).

Compounds 31 and 32 were obtained as yellow amorphous powders. From the [M + H]⁺ peaks at m/z 235.0970 and 233.0814 in their HRFABMS, their molecular formulae were determined to be C₁₃H₁₄O₄ and C₁₃H₁₂O₄, respectively. The presence of the 5-acetyl-7-methoxybenzofuran core in the structures of 31 and 32 was deduced from their ¹H and ¹³C NMR spectra (Table 4) and HMBC correlations (Figure 7). Furthermore, the ¹H ¹H COSY correlations between H₃-12/H-10/10-OH in 31 and the HMBC correlations from H₃-12 to C-2/C-10 in 31 and 32 revealed that a 1-hydroxyethyl group was substituted at C-2 in 31, whereas an acetyl group was substituted in 32. Therefore, the structures of 31 and 32 were established, as shown in Figure 8.

The molecular formulae of compounds 34 and 36 were determined to be C₁₈H₂₂O₅ and C₁₄H₁₆O₄, respectively, using HRFABMS. Their ¹H and ¹³C NMR data (

Table 5. ¹H (500 MHz) and ¹³C (126 MHz) NMR data of 34, 36, and 39 in CDCl₃.

	34			36 (a)		39
Position	δH	(mult., J in Hz)	δC	δH	(mult., J in Hz)	δH (Major/Minor)		(mult., J in Hz)	δC (Major/Minor)
1									141.60
2	5.13	(1H, d, 2.8)	92.1	5.04	(1H, d, 4.0)	6.761		(1H, d, 0.5)	111.67
3	6.17	(1H, d, 2.8)	77.3	5.22	(1H, dd, 7.8, 4.0)				158.47
4	7.62	(1H, d, 1.5)	120.7	7.65	(1H, d, 1.5)				127.58
5			N.D. (b)			7.222		(1H, d, 7.6)	122.68
6	7.59	(1H, d, 1.5)	113.0	7.57	(1H, d, 1.5)	6.848		(1H, dd, 7.6, 0.5)	123.07
7			144.8			2.353		(3H, s)	21.72
8			154.0						79.73
9			N.D. (b)			6.506		(1H, s)	104.85/104.80
10			140.5			1.626		(3H, s)	20.12
11	5.10	(1H, s)	114.1	5.13	(1H, d, 0.8)
	4.98	(1H, s)		4.97	(1H, d, 0.8)
12	1.76	(3H, s)	17.5	1.76	(3H, s)
13			196.4
14	2.56	(3H, s)	26.4	2.58	(3H, s)
1′			176.8						175.28
2′	2.57	(1H, sept, 7.1)	33.9			2.407/2.398		(1H, sext, 7.0)	41.02/40.97
3′	1.20	(3H, d, 7.1)	18.9			1.688		(1H, m)	26.49/26.35
						1.498		(1H, m)
4′	1.17	(3H, d, 7.1)	18.8			0.913/0.907		(3H, t, 7.0)	11.42
5′						1.157/1.164		(3H, d, 7.0)	16.07/16.38
3-OH				2.16	(1H, d, 7.8)
7-OCH3	3.96	(3H, s)	56.2	3.96	(3H, s)
8-OH						2.033/2.036		(1H, s)

^{(a) 13}C NMR was not measured due to a small amount. ^(b) N.D. = not detected.

) suggested structural similarities to the known compound 33 [6]; however, the signals derived from an angeloyloxy group in 33 were replaced by those of an isobutanoyloxy group [δ_H 2.57 (sept, J = 7.1 Hz), 1.20 (d, J = 7.1 Hz), 1.17 (d, J = 7.1 Hz); δ_C 176.8, 33.9, 18.9, 18.8] in 34 and a hydroxy group [δ_H 2.16 (1H, d, 7.8)] in 36. The 2,3-trans nature was indicated by J_2,3 (2.8 Hz in 34 and 4.0 Hz 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 H₃-12 (Figure 8).

Compound 39 was obtained as white amorphous powder. Its molecular formula was determined to be C₁₅H₂₀O₄ by the quasi-molecular ion at m/z 247.1334 [M-H₂O+H]⁺ in its HRFABMS. The ¹H and ¹³C 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 H₃-10 (Figure 8). A detailed analysis of the ¹H and ¹³C NMR spectra of 39 revealed that it was a mixture of C-2’ epimers (ca. 3:1 based on the integration of ¹H NMR signals).

The structures of 34 known compounds, including 24 benzofurans (6 [23], 7 [31], 8 [8], 9 [25], 10 [32], 11 [29], 12 [33], 13 [25], 15 [22,23], 16 [34], 18 [35], 19 [26], 20 [36], 22 [15,37], 23 [8], 24 [8], 27 [24], 28 [38], 29 [8], 30 [39], 33 [6], 35 [40], 37 [6], and 38 [6]), 4 thiophenes (40 [41], 41 [41], 42 [8], 43 [6]), 2 triterpenoids (44 [42] and 45 [43]), and 4 other aromatic compounds (46 [44], 47 [45], 48 [46], 49 [47]) were identified by comparing their NMR data with those reported in the literature (Figure 9).

2.3. 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.

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.

3. Materials and Methods

3.1. General Experimental Procedures

A JASCO P1020 NK digital polarimeter was used to measure the optical rotations. The diffuse reflectance method was used to record IR spectra on a JASCO FT/IR-410 spectrophotometer. UV spectra were obtained on a JASCO V-560 UV/Vis spectrophotometer. ECD spectra were measured on a JASCO J-725N spectrophotometer. ¹H and ¹³C NMR spectra were recorded on a JEOL JNM-AL 400 (¹H: 400 MHz) or Varian Unity plus 500 (¹H: 500 MHz, ¹³C: 126 MHz, respectively) spectrometer using CDCl₃. Chemical shift values are given in δ (ppm), using the solvent peak signals (CDCl₃: TMS) as references, and coupling constants (J) are reported in Hz. A JEOL JMS-700 MStation was used to record mass spectra, including high-resolution spectra. Column chromatography was performed on Silica gel 60 (100–210 mesh, Kanto Chemical Co., Inc., Tokyo, Japan). Preparative HPLC was performed on a JASCO chromatograph (n-hexane–EtOAc, CHCl₃–EtOAc) equipped with a JASCO PU-2086 pump, a JASCO UV-970 detector, a JASCO RI-2031 detector, and various columns: COSMOSIL 5SL-II (10 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan), COSMOSIL 5SL-II (4.6 × 250 mm, Nacalai Tesque Inc., Kyoto, Japan), YMC-Pack Diol-120-NP (4.6 × 250 mm, YMC Co., Ltd., Kyoto, Japan), and Inertsil CN-3 (4.6 × 250 mm, GL Sciences, Tokyo, Japan).

3.2. Plant Materials

Samples were collected in Yunnan Province of P.R. China in August 2014 and authenticated by Dr. Takayuki Kawahara, Forestry and Forest Products Research Institute (Japan Forest Technology Association, General Incorporated Association in present affiliation). Sample 1 was collected in Lanping Bai and Pumi Autonomous County, and sample 2 was collected in Gucheng District, Lijiang City, approximately 40 km distant from one another. The voucher specimen numbers for samples 1 and 2 were 2014-10 and 2014-48, respectively. These were deposited in Kunming Institute of Botany, Kunming, China.

3.3. Extraction and Isolation

The dried roots of sample 2 (48.1 g) were cut into small pieces and extracted twice with MeOH at room temperature. After removal of the solvent under reduced pressure not exceeding 30 °C, a concentrated and combined MeOH extract (7.5 g) was obtained, which was separated on a silica gel column (n-hexane–EtOAc, 1:0, 99:1, 98:2, 95:5, 93:7, 9:1, 85:15, 8:2, 7:3, 1:1; EtOAc–MeOH, 1:0, 9:1, 7:3, 0:1) to afford nine subfractions (Fr. 1–9). 15 (198.2 mg), 6/9 (1100.9 mg, mixture 7:3), and 43 (1230.1 mg, purity 50%) were obtained as Fr. 2, 3, and 8, respectively. Fr. 1 (132.0 mg) was separated using HPLC (COSMOSIL 5SL-II, 10 × 250 mm, n-hexane–EtOAc, 98:2) into six fractions: 1-0 to 1-5. 44 (32.6 mg), 40 (37.8 mg), and 15 (1.2 mg) were obtained as Fr. 1-2, 1-3, 1-5, respectively. Fr. 1-4 (10.0 mg) was separated using HPLC (COSMOSIL 5SL-II, 4.6 × 250 mm, n-hexane–EtOAc, 99:1) into four fractions: 1-4-0 to 1-4-3. Fr. 1-4-0 was 30 (1.9 mg). Fr. 1-4-1 (5.8 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 95:5) to yield 45 (3.6 mg) and 22 (1.3 mg). Fr. 4 (230.9 mg) was separated using HPLC (COSMOSIL 5SL-II, 10 × 250 mm, n-hexane–EtOAc, 85:15) into four fractions: 4-0 to 4-3. Fr. 4-3 was 7 (128.2 mg). Fr. 4-1 (62.7 mg) was purified using HPLC (COSMOSIL 5SL-II, 10 × 250 mm, n-hexane–EtOAc, 95:5) to yield 15 (0.7 mg), 6 (14.2 mg), 9 (10.1 mg), and 13 (11.0 mg). Fr. 4–2 (18.8 mg) was purified using HPLC (COSMOSIL 5SL-II, 10 × 250 mm, n-hexane–EtOAc, 85:15) into five fractions: 4-2-0 to 4-2-4. 41 (3.7 mg) and 33/38 (6.2 mg, mixture 2:1) were obtained as Fr. 4-2-2 and 4-2-3, respectively. Fr. 4-2-1 (1.5 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 95:5) to yield 14 (0.4 mg) and 20 (0.5 mg). Fr. 4-2-4 (2.2 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 9:1) to yield 34 (0.2 mg) and 26 (0.8 mg). Fr. 5 (66.4 mg) was separated using HPLC (COSMOSIL 5SL-II, 10 × 250 mm, n-hexane–EtOAc, 85:15) into seven fractions: 5-0 to 5-6. 6/15 (3.1 mg, mixture 10:3), 9 (1.4 mg), 24 (5.0 mg), 46 (3.3 mg), and 21 (1.6 mg) were obtained as Fr. 5-1, 5-2, 5-4–5-6, respectively. Fr. 5-3 (27.2 mg) was purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 85:15) into five fractions: 5-3-0 to 5-3-4. 7/8 (3.9 mg, mixture 3:2) and 29 (6.5 mg) were obtained as Fr. 5-3-1 and 5-3-4, respectively. Fr. 5-3-2 (5.6 mg) was further purified using Inertsil CN-3 (4.6 × 250 mm, n-hexane–EtOAc, 97:3) to yield 48 (0.8 mg), 35 (1.4 mg), and 23a/23b (0.7 mg/1.3 mg, respectively). Fr. 5-3-3 (3.9 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 9:1) to yield 28 (0.6 mg). Fr. 6 (280.9 mg) was separated using HPLC (COSMOSIL 5SL-II, 10 × 250 mm, n-hexane–EtOAc, 7:3) into eight fractions: 6-0 to 6-7. Fr. 6-1 (47.4 mg) was purified using HPLC (COSMOSIL 5SL-II, 4.6 × 250 mm, n-hexane–EtOAc, 85:15) to yield 6/15/9 (5.6 mg, mixture 10:3:3), 7/29 (4.2 mg, mixture 10:3), and 24 (4.1 mg). Fr. 6-2 (31.9 mg) was purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 8:2) into four fractions: 6-2-0 to 6-2-3. 37/47 (8.7 mg, mixture 10:3) and 49 (1.6 mg) were obtained as Fr. 6-2-2 and 6-2-3, respectively. Fr. 6-2-1 (2.9 mg) was further purified using Inertsil CN-3 (4.6 × 250 mm, n-hexane–EtOAc, 9:1) to yield 4 (1.3 mg). Fr. 6-3 (21.6 mg) was purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 7:3) into four fractions: 6-3-0 to 6-3-3. Fr. 6-3-3 was 1 (2.2 mg). Fr. 6-3-1 (4.7 mg) was further purified using Inertsil CN-3 (4.6 × 250 mm, n-hexane–EtOAc, 9:1) to yield 42 (0.4 mg) and 17 (2.0 mg). Fr. 6-3-2 (2.0 mg) was further purified using Inertsil CN-3 (4.6 × 250 mm, n-hexane–EtOAc, 7:3) to yield 2 (1.1 mg). Fr. 6-4 (43.3 mg) was purified using HPLC (COSMOSIL 5SL-II, 4.6 × 250 mm, CHCl₃–EtOAc, 98:2) into three fractions: 6-4-0 to 6-4-2. Fr. 6-4-2 was 18 (6.0 mg). Fr. 6-4-1 (20.4 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 85:15) to yield 16 (10.2 mg) and 32 (2.2 mg). Fr. 6-5 (48.0 mg) was purified on COSMOSIL 5SL-II (4.6 × 250 mm, CHCl₃–EtOAc, 98:2) to give five fractions: 6-5-0 to 6-5-4. 10 (12.4 mg) and 11 (11.0 mg) were obtained as Fr. 6-5-2 and 6-5-4, respectively. Fr. 6-5-3 (3.0 mg) was further purified using Inertsil CN-3 (4.6 × 250 mm, n-hexane–EtOAc, 7:3) to yield 25 (0.8 mg) and 27 (0.4 mg). Fr. 6-6 (18.7 mg) was purified on COSMOSIL 5SL-II (4.6 × 250 mm, CHCl₃–EtOAc, 98:2) to give five fractions: 6-6-0 to 6-6-4. Fr. 6-6-3 was 31 (1.4 mg). Fr. 6-6-1 (3.1 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 7:3) to yield 5 (1.4 mg). Fr. 6-6-4 (2.1 mg) was further purified using YMC-Pack Diol-120-NP (4.6 × 250 mm, n-hexane–EtOAc, 7:3) to yield 36 (0.4 mg) and 3 (0.8 mg).

Similarly, the MeOH extract (2.8 g) of sample 1 (29.6 g) yielded 6 (66.2 mg), 7 (28.8 mg), 8 (0.3 mg), 9 (8.1 mg), 10 (2.1 mg), 11 (5.4 mg), 12 (0.9 mg), 15 (55.7 mg), 16 (7.5 mg), 19 (7.8 mg), 22 (0.9 mg), 29 (1.1 mg), 33 (0.7 mg), 38 (0.4 mg), 39 (0.3 mg), 40 (1.6 mg), 41 (0.7 mg), 44 (10.2 mg), 46 (0.7 mg), and 47 (0.7 mg).

3.4. Chiral HPLC Analyses of Compounds 4 and 5

Each compound was analyzed using HPLC with a Chiralcel OJ-RH column (4.6 × 150 mm, Daicel, Japan), which was eluted with 50% CH₃CN (for 4) or 20% CH₃CN (for 5) in 50 mM H₃PO₄ at 40 °C (flow rate, 0.8 mL/min and detection, JASCO photodiode array detector MD-2010), resulting in the detection of both enantiomers (4: t_R 13.4 min and t_R 14.9 min; 5: t_R 4.0 min and t_R 4.8 min) with an integral ratio of ca. 1:1.

3.5. Calculation of ECD Spectra

A conformational search was performed using the Monte Carlo method and the MMFF94 force field with Spartan ′14 (Wavefunction, Irvine, CA, USA). The obtained low-energy conformers within 6 kcal/mol were optimized at the B3LYP/6-31G (d,p) level in MeOH (PCM). The vibrational frequencies were also calculated at the same level to confirm their stability, and no imaginary frequencies were found. The energies, oscillator strengths, and rotational strengths of the low-energy conformers were calculated using TDDFT at the CAM-B3LYP/6-31G (d,p) in MeOH (PCM) level, and weight-averaged. The ECD spectra were simulated using GaussView [54] by the overlapping Gaussian function with 0.35 eV exponential half-width, and UV correction was performed (redshifted by 15 nm). All DFT calculations were performed using Gaussian 09 [55].

3.6. Compound Data

• Compound 1

Yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{11}$ -23.9 (c 0.24, CHCl₃); FT-IR 3416, 1651 cm⁻¹; MS (FAB) m/z: 489 [M+Na]⁺; HRMS (FAB) Obs. m/z 489.1527 (Calcd for C₂₆H₂₆O₈Na 489.1525); UV (CH₃OH) λ_max (log ε) 231 (4.24), 260 (4.02), 391 (3.73) nm; ECD (CH₃OH, c = 5.2 × 10⁻⁵ mol/L) λ_max (Δε): 406 (−0.2), 360 (+0.2), 318 (+0.6), 266 (+2.0), 218 (−4.2) nm; ¹H and ¹³C NMR: see Table 1.

• Compound 2

Yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ +5.4 (c 0.10, CHCl₃); FT-IR 3411, 1656, 1600, 1464, 1439, 1254, 1093 cm⁻¹; MS (FAB) m/z: 466 [M]⁺; HRMS (FAB) Obs. m/z 466.1604 (Calcd for C₂₆H₂₆O₈ 466.1628); UV (CH₃OH) λ_max (log ε) 222 (4.39), 337 (3.71) nm; CD (CH₃OH, c = 4.3 × 10⁻⁵ mol/L) λ_max (Δε): 352 (+1.9), 304 (−0.2), 260 (+2.3), 218 (−11.0) nm; ¹H and ¹³C NMR: see Table 1.

• Compound 3

Yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ −59.7 (c 0.08, CHCl₃); FT-IR 3474, 1668, 1596, 1457, 1438, 1251, 1081 cm⁻¹; MS (FAB) m/z: 721 [M + Na]⁺; HRMS (FAB) Obs. m/z 721.2260 (Calcd for C₃₉H₃₈O₁₂Na 721.2261); UV (CH₃OH) λ_max (log ε) 221 (4.56), 335 (3.83) nm; CD (CH₃OH, c = 3.0 × 10⁻⁵ mol/L) λ_max (Δε): 353 (+2.9), 311 (−0.2), 261 (+2.3), 221 (−13.2) nm; ¹H and ¹³C NMR: see Table 1.

• Compound 4

Yellow amorphous powder; FT-IR 3361, 1659, 1651 cm⁻¹; MS (FAB) m/z: 449 [M + H]⁺; HRMS (FAB) Obs. m/z 449.1600 (Calcd for C₂₆H₂₅O₇ 449.1600); UV (CH₃OH) λ_max (log ε) 223 (4.39), 234 (4.48), 266 (4.07), 329 (3.75) nm; ¹H and ¹³C NMR: see Table 2.

• Compound 5

Yellow amorphous powder; FT-IR 3245, 2225, 1672, 1622 cm⁻¹; MS (FAB) m/z: 461 [M + H]⁺; HRMS (FAB) Obs. m/z 461.1601 (Calcd for C₂₇H₂₅O₇ 461.1600); UV (CH₃OH) λ_max (log ε) 243 (4.44), 354 (4.24) nm; ¹H and ¹³C NMR: see Table 2.

• Compound 14

Colorless amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ +45.9 (c 0.05, CHCl₃); FT-IR 3453–2650, 1731, 1651 cm⁻¹; MS (FAB) m/z: 291 [M+H]⁺; HRMS (FAB) Obs. m/z 291.1231 (Calcd for C₁₆H₁₉O₅ 291.1232); ¹H and ¹³C NMR: see Table 3.

• Compound 17

Yellowish amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ +15.9 (c 0.24, CHCl₃); FT-IR 3472, 1713, 1651 cm⁻¹; MS (FAB) m/z: 355 [M + Na]⁺; HRMS (FAB) Obs. m/z 355.1160 (Calcd for C₁₈H₂₀O₆Na 355.1158); ¹H and ¹³C NMR: see Table 3.

• Compound 21

Yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ −189.2 (c 0.17, CHCl₃); FT-IR 3443–2646, 1651 cm⁻¹; MS (FAB) m/z: 233 [M + H]⁺; HRMS (FAB) Obs. m/z 233.0814 (Calcd for C₁₃H₁₃O₄ 233.0814); ¹H and ¹³C NMR: see Table 3.

• Compound 25

Yellowish amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{29}$ +7.0 (c 0.12, CHCl₃); FT-IR 3410, 1650 cm⁻¹; MS (FAB) m/z: 235 [M + H]⁺; HRMS (FAB) Obs. m/z 235.0970 (Calcd for C₁₃H₁₅O₄ 235.0970); ¹H and ¹³C NMR: see Table 4.

• Compound 26

Yellowish amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{29}$ −11.5 (c 0.08, CHCl₃); FT-IR 3541–2635, 1731, 1633 cm⁻¹; MS (FAB) m/z: 277 [M+H]⁺; HRMS (FAB) Obs. m/z 277.1076 (Calcd for C₁₅H₁₇O₅ 277.1076); ¹H and ¹³C NMR: see Table 4.

• Compound 31

Yellow amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ +2.3 (c 0.20, CHCl₃); FT-IR 3485, 1731 cm⁻¹; MS (FAB) m/z: 235 [M + H]⁺; HRMS (FAB) Obs. m/z 235.0970 (Calcd for C₁₃H₁₅O₄ 235.0970); ¹H and ¹³C NMR: see Table 4.

• Compound 32

Yellow amorphous powder; FT-IR 1683, 1595, 1566, 1474, 1360, 1331, 1174 cm⁻¹; MS (FAB) m/z: 233 [M + H]⁺; HRMS (FAB) Obs. m/z 233.0814 (Calcd for C₁₃H₁₃O₄ 233.0814); ¹H and ¹³C NMR: see Table 4.

• Compound 34

Yellowish amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{29}$ −65.2 (c 0.03, CHCl₃); FT-IR 1745, 1681 cm⁻¹; MS (FAB) m/z: 319 [M + H]⁺; HRMS (FAB) Obs. m/z 319.1543 (Calcd for C₁₈H₂₃O₅ 319.1545); ¹H and ¹³C NMR: see Table 5.

• Compound 36

Yellowish amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{29}$ +37.00 (c 0.05, CHCl₃); FT-IR 3410, 1680, 1601, 1497 cm⁻¹; MS (FAB) m/z: 249 [M + H]⁺; HRMS (FAB) Obs. m/z 249.1126 (Calcd for C₁₄H₁₇O₄ 249.1127); ¹H NMR: see Table 5.

• Compound 39

White amorphous powder; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{29}$ +25.34 (c 0.08, CHCl₃); FT-IR 3305, 1746 cm⁻¹; MS (FAB) m/z: 247 [M-H₂O+H]⁺; HRMS (FAB) Obs. m/z 247.1334 (Calcd for C₁₅H₁₉O₃ 247.1334); ¹H and ¹³C NMR: see Table 5.