Sesquineolignan and neolignan enantiomers from Triadica sebifera

Article abstract of DOI:10.1016/j.bioorg.2020.104147

Two pairs of new sesquineolignan enantiomers (1a/1b and 1c/1d), two pair of new 4′,7-epoxy-8,3′-neolignan enantiomers (2a/2b and 3a/3b), and a pair of new 3′,7-epoxy-8,4′-oxyneolignan enantiomers (4a/4b), along with two pairs of known 4′,7-epoxy-8,3′-neol

Full text of DOI:10.1016/j.bioorg.2020.104147

BioorganicChemistry103(2020)104147
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Bioorganic Chemistry
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Sesquineolignan and neolignan enantiomers from Triadica sebifera
Min Yu1, Yong-Li Zhang1, Jin-Long Liu, Su-Juan Wang, Gui-Jie Zhang⁎
College of Pharmacy, Guilin Medical University, Guilin 541199, People’s Republic of China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two pairs of new sesquineolignan enantiomers (1a/1b and 1c/1d), two pair of new 4′,7-epoxy-8,3′-neolignan
enantiomers (2a/2b and 3a/3b), and a pair of new 3′,7-epoxy-8,4′-oxyneolignan enantiomers (4a/4b), along
with two pairs of known 4′,7-epoxy-8,3′-neolignan enantiomers (5a/5b and 6a/6b), were obtained from the
stems and leaves of Triadica sebifera. The structures of the enantiomers were elucidated by spectroscopic ana-
lyses, and their absolute configurations were assigned by the experimental ECD spectra. Among them, com-
pounds 5b, 6a and 6b showed inhibitory activities against NO production in activated microglial BV-2 cells, with
IC50 values of 14.3, 23.2 and 33.3 μM, respectively.
Triadica sebifera
Sesquineolignan ennatiomers
Neolignan enantiomers
NO inhibitors
1. Introduction
2016 in Guilin, Guangxi Province, People’s Republic of China. The plant
material was identified by one of the authors (G.-J. Zhang), a voucher
Triadica sebifera, a tree belonging to the Euphorbiaceae family, is dis-
tributed widely in southern provinces of China [1]. The roots and bark of
this plant have been commonly used in folk medicine to treat dermatitis,
eczema, snakebite, and beriberi [2]. Pharmacological researches have il-
lustrated that the leaves of T. sebifera have anti-inflammatory, analgesic,
and antihypertensive activities [3,4]. Previous phytochemical studies on
this plant have led to the isolation of a few diterpenes [5], triterpenes [6],
flavonoids [7], phenols [7], and coumarins [8]. Over the course of our
continuing search for antineuroinflammatory agents from the genus
Triadica [9–11], the stems and leaves of T. sebifera were investigated. This
work led to the isolation of two pairs of new sesquineolignan enantiomers
(1a/1b and 1c/1d), two pair of new 4′,7-epoxy-8,3′-neolignan en-
antiomers (2a/2b and 3a/3b), and a pair of new 3′,7-epoxy-8,4′-oxy-
neolignan enantiomers (4a/4b), along with two pair of known 4′,7-epoxy-
8,3′-neolignan enantiomers (5a/5b and 6a/6b). More and more en-
antiomers have been reported in natural products [12,13], and the ac-
tivities of some enantiomers are drastically different [12,14]. Hence, it is
mandatory to resolve them and provide pure stereoisomers. Details of the
isolation, structural elucidation, and antineuroinflammatory evaluation of
these enantiomers are reported herein (see Fig. 1).
specimen (No. TS-201608) was deposited at the College of Pharmacy,
Guilin Medical University.
2.2. General experimental procedures
UV absorption spectra were recorded on a PerkinElmer 650 spec-
trophotometer and the IR spectra were acquired on a PerkinElmer
Spectrum Two FT-IR spectrometer. NMR spectra were recorded on a
600 or 400 MHz Bruker AVANCE. HRESIMS were carried out on an
Agilent 6545 Q-TOF LC-MS spectrometer. Experimental ECD spectra
were determined on a JASCO J-1500 spectrometer. Column chroma-
tography (CC) was performed on silica gel (200–300 mesh, Qingdao
Marine Chemical Ltd., People’s Republic of China), RP-C18 silica gel
(50 μm, YMC, Japan), MCI gel (CHP20, 75–150 μm, Mitsubishi
Chemical Ltd., Japan), and Sephadex LH-20 gel (Pharmacia Biotech,
Sweden). TLC analyses were carried out on the precoated silica gel
GF254 plates (Qingdao Marine Chemical Ltd.). Semipreparative re-
versed-phase HPLC (RP-HPLC) was performed using a YMC-Pack ODS-A
column (250 × 20 mm, 5 μm) on an LC3000 instrument (Chuang Xin
Tong Heng Science and Technology Ltd., Beijing, People’s Republic of
China) equipped with a UV3000 detector (Chuang Xin Tong Heng
Science and Technology). Chiral separation was performed using a
2. Materials and methods
Chiralpak AD-H (5 μm, 10
4.6 × 250 mm).
× 250 mm) or ID column (5 μm,
2.1. Plant material
The stems and leaves of Triadica sebifera were collected in August
⁎ Corresponding author.
E-mail address: zhangguijie001@126.com (G.-J. Zhang).
1 These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2020.104147
Received 13 March 2020; Received in revised form 19 May 2020; Accepted 25 July 2020
Availableonline28July2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

M. Yu, et al.
BioorganicChemistry103(2020)104147
Fig. 1. The structures of enantiomers.
2.3. Extraction and isolation
UV (MeOH) λmax (log ε) 204 (4.69), 236 (4.04), 280 (3.44) nm; IR (KBr)
νmax 3399, 2924, 2850, 1600, 1502, 1462, 1423, 1329, 1275, 1214,
The air-dried stems and leaves of T. sebifera (18 kg) were extracted
with 95% aqueous EtOH (3 × 100 L, each 3 h) under reflux. The filtrate
was evaporated under reduced pressure, and the residue (0.78 kg) was
dispersed in H2O, and then sequentially partitioned with petroleum
ether, EtOAc, and n-BuOH.
1123, 1029, 826 cm−1
;
1H and 13C NMR data, see Tables 1 and 2; (+)
HRESIMS m/z 609.2291 [M + Na]+, calcd for C31H38O11Na, 609.2306.
(7S,8R,7′'R,8′'R)-1a: [ ]2D0 − 40.5 (c 0.05, MeOH); ECD (MeOH)
λmax (Δε) 240 (−1.91), 295 (+0.41) nm.
(7R,8S,7′'S,8′'S)-1b: [ ]2D0 + 41.2 (c 0.05, MeOH); ECD (MeOH)
λmax (Δε) 240 (+2.38), 295 (−0.55) nm.
The EtOAc fraction (257 g) was subjected to silica gel (200–300
mesh) CC, eluting with CH2Cl2/MeOH (100:3 to 1:1) to yield seven
fractions (A − K). Fraction H (41 g) was separated into ten subfractions
(H1 − H10) via MCI CC, eluting with a gradient of MeOH/H2O (20:80
to 100:0). Subfraction H3 (5.8 g) was separated by RP-C18 CC (MeOH/
H2O, 20:80 to 80:20) to yield 18 subfractions (H3a − H3r). H3k
(762 mg) was subjected to Sephadex LH-20 CC (MeOH) and then pur-
ified by semi-preparative RP-HPLC (CH3CN/H2O, 20:80, 8 mL/min) to
yield 5 (17.5 mg, tR 65.4 min) and 6 (20.0 mg, tR 59.2 min). Compound
5 was separated by Daicel Chiralpak AD-H (n-hexane/EtOH, 80:20,
3 mL/min) to yield 5a (3 mg, tR 20.9 min) and 5b (1.5 mg, tR 24.8 min).
Compounds 6a (1.5 mg, tR 24.8 min) and 6b (3.0 mg, tR 20.9 min) were
obtained by Daicel Chiralpak ID CC (n-hexane/isopropanol, 65:35,
0.7 mL/min). Subfraction H4 (3.9 g) was separated by RP-C18 CC
(MeOH/H2O, 40:60 to 80:20) to yield 14 subfractions (H4a − H4n).
Subfraction H4g (427 mg) was subjected to Sephadex LH-20 CC
(MeOH) and then purified by semi-preparative RP-HPLC with CH3CN/
H2O (28:72, 8 mL/min) to yield 4 (3 mg, tR 23.0 min). Compounds 4a
(1.5 mg, tR 23.5 min) and 4b (1.1 mg, tR 14.5 min) were obtained by
chiral HPLC using a Daicel Chiralpak AD-H eluting with n-hexane/
isopropanol (75:25, 3 mL/min). Subfraction H4h (443 mg) was purified
by semi-preparative RP-HPLC with CH3CN/H2O (28:72, 8 mL/min) to
yield 1 (10 mg, tR 27.0 min). Compound 1 was separated by Daicel
Chiralpak AD-H (n-hexane/EtOH, 80:20, 3 mL/min) to yield 1b (2 mg,
tR 43.0 min) and 1d (2 mg, tR 48.0 min), and then eluted with n-
hexane/isopropanol (70:30, 3 mL/min) to yield 1a (2 mg, tR 20.5 min)
and 1c (2 mg, tR 17.5 min). Subfraction H4j (175 mg) was purified by
semi-preparative RP-HPLC with CH3CN/H2O (35:65, 8 mL/min) to
yield 2 (3.0 mg, tR 19.0 min) and 3 (4.0 mg, tR 17.0 min). Compounds
2a (1.5 mg, tR 17.0 min) and 2b (1.1 mg, tR 30.5 min) were obtained
using a Daicel Chiralpak AD-H eluting with n-hexane/isopropanol
(65:35, 3 mL/min). Compound 3 was separated by a Daicel Chiralpak
AD-H eluting with n-hexane/isopropanol (65:35, 3 mL/min) to yield 3a
(1.8 mg, tR 24.3 min) and 3b (1.3 mg, tR 40.0 min).
(7R,8S,7′'R,8′'R)-1c: [ ]2D0 − 11.5 (c 0.05, MeOH); ECD (MeOH)
λmax (Δε) 214 (−7.40), 240 (−3.78), 295 (−0.82) nm.
(7S,8R,7′'S,8′'S)-1d: [ ]2D0 + 13.2 (c 0.05, MeOH); ECD (MeOH)
λmax (Δε) 214 (+5.22), 240 (+4.15), 295 (+0.93) nm.
4-hydroxy-3,5′-dimethoxy-9-(3,4,5-trihydroxy)benzoyloxy-4′,7-epoxy-
8,3′-neoligna-9′-ol (2): white powder; [ ]2D0 + 1.5 (c 0.3, MeOH), UV
(MeOH) λmax (log ε) 207 (4.56), 281 (3.81) nm; IR (KBr) νmax 3432,
2924, 1610, 1515, 1460, 1212, 1124, 1032, 579 cm−1
;
1H and 13C
NMR data, see Tables 1 and 2; (+) HRESIMS m/z 513.1749 [M + H]+
,
calcd for C27H29O10, 513.1755.
(7S,8R)-2a: [ ]2D0 + 47.3 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
212 (+6.04), 290 (+1.73) nm.
(7R,8S)-2b: [ ]2D0 − 48.5 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
212 (−3.39), 290 (−0.81) nm.
4-hydroxy-3,5,5′-trimethoxy-9-(3,4,5-trihydroxy)benzoyloxy-4′,7-
epoxy-8,3′-neoligna-9′-ol (3): white powder; [ ]2D0 + 3.4 (c 0.3, MeOH),
UV (MeOH) λmax (log ε) 208 (4.67), 281 (3.73) nm; IR (KBr) νmax 3294,
2922, 1612, 1463, 1323, 1214, 1114 cm−1
;
1H and 13C NMR data, see
Tables 1 and 2; (+) HRESIMS m/z 543.1869 [M + H]+, calcd for
C
28H31O11, 543.1861.
(7S,8R)-3a: [ ]2D0 + 28.3 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
212 (+7.79), 290 (+2.36) nm.
(7R,8S)-3b: [ ]2D0 − 30.8 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
212 (−3.39), 290 (−1.73) nm.
4-hydroxy-3,5′-dimethoxy-3′,7-epoxy-8,4′-oxyneoligna-9,9′-diol (4):
white powder; [ ]2D0 − 10.0 (c 0.1, MeOH), UV (MeOH) λmax (log ε) 207
(4.06), 232 (3.53), 279 (2.94) nm; IR (KBr) νmax 3708, 2920, 2851,
1734, 1466, 1362, 1119 cm−1
;
1H and 13C NMR data, see Tables 1 and
2; (+) HRESIMS m/z 377.1593 [M + H]+, calcd for C20H25O7,
377.1595.
(7S,8S)-4a: [ ]2D0 + 18.2 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
233 (+4.18) nm.
(7R,8R)-4b: [ ]2D0 − 20.8 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
233 (−3.47) nm.
4′'-hydroxy-3,3′',5,5′-tetramethoxy-4′,7-epoxy-4,8′'-oxy-8,3′-sesqui-
neoligna-7′',9,9′,9′'-tetrol (1): white powder; [ ]2D0 − 7.7 (c 0.1, MeOH),
4-hydroxy-3,5′-dimethoxy-4′,7-epoxy-8,3′-neoligna-9,9′-ol (5): color-
less oil; 1H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS m/z
2

M. Yu, et al.
BioorganicChemistry103(2020)104147
Table 1
1H NMR Data for Compounds 1–6 (δ in ppm, J in Hz).
no.
1a
2a
3a
4a
5b
6b
2
6.73 s
6.88 d (1.8)
6.75 d (7.8)
6.82 dd (7.8, 1.8)
5.55 d (6.6)
3.78 m
6.60 s
6.99 d (1.8)
6.83 d (8.1)
6.88 dd (8.1, 1.8)
4.86 d (8.4)
3.99 m
6.93 d (1.6)
6.85 d (8.4)
6.90 dd (8.4, 1.6)
5.53 d (7.2)
3.59 m
6.65 s
5
6
6.73 s
6.60 s
6.65 s
7
5.55 d (6.0)
3.44 m
5.57 d (6.0)
3.79 m
5.53 d (7.6)
3.60 m
8
9a
3.87 m
4.57 dd (10.8, 5.4)
4.47 dd (10.8, 7.8)
6.76 br.s
4.62 dd (10.8, 4.8)
4.46 dd (10.8, 8.4)
6.765 br.s
3.69 dd (12.6, 2.4)
3.48 dd (12.6, 4.8)
6.42 d (1.8)
6.47 d (1.8)
2.58 t (7.2)
3.95 dd (11.6, 6.0)
3.90 m
3.97 dd (11.2, 2.0)
3.90 m
9b
3.77 m
2′
6.72 br.s
6.74 br.s
2.62 t (7.8)
1.81 m
6.67 br.s
6.67 br.s
6′
6.76 br.s
6.771 br.s
6.67 br.s
6.67 br.s
7′
2.63 t (7.8)
1.81 m
2.64 t (7.8)
1.81 m
2.66 t (7.6)
1.88 m
2.67 t (7.6)
1.88 m
8′
1.81 m
9′
3.56 t (6.6)
3.56 t (6.6)
3.56 t (6.6)
3.56 t (6.6)
3.68 t (6.4)
3.69 t (6.4)
1′'
2′'
5′'
6′'
7′'
8′'
9′'
6.99 d (2.4)
6.74 d (8.4)
6.86 dd (8.4, 2.4)
4.98 d (7.2)
4.06 m
7.04 s
7.04 s
7.06 s
7.06 s
3.75 m
3.32 m
OMe-3
OMe-5
OMe-5′
OMe-3′'
3.82 s
3.72 s
3.87 s
3.71 s
3.71 s
3.88 s
3.87 s
3.86 s
3.85 s
3.87 s
3.85 s
3.85 s
3.88 s
3.82 s
3.82 s
3.87 s
a
b
Data were recorded at 600 MHz in MeOH‑d4.
Data were recorded at 400 MHz in CDCl3.
4-hydroxy-3,5,5′-trimethoxy-4′,7-epoxy-8,3′-neoligna-9,9′-ol
(6):
Table 2
13C NMR Data for Compounds 1–6.
white powder; 1H and 13C NMR data, see Tables 1 and 2; (+) HRESIMS
m/z 413.1573 [M + Na]+, calcd for C21H26O7Na, 413.1571.
(7S,8R)-6a: [ ]2D0 − 10.3 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
217 (+2.18), 293 (+0.48) nm.
no.
1a
2a
3a
4a
5b
6b
1
139.8
103.8
154.3
136.7
154.3
103.8
88.5
134.2
110.3
149.1
147.6
116.2
119.5
89.3
133.5
103.7
149.4
136.3
149.4
103.7
89.5
129.6
112.0
149.2
148.3
116.2
121.6
77.6
133.1
108.8
146.6
145.6
114.3
119.4
87.9
132.2
103.1
147.1
134.6
147.1
103.1
88.1
(7R,8S)-6b: [ ]2D0 + 8.7 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
217 (−0.78), 293 (−0.45) nm.
2
3
4
5
6
2.4. NO production measurement and cell viability assay
7
8
55.8
52.5
52.7
79.9
53.8
53.8
The accumulation of nitrite (NO2−) in the culture medium super-
natants was measured using the Griess reaction [9,10]. BV2 cells were
plated in 96-well microtiter plates and treated with each compound at
various concentrations (1, 10, 25, and 50 μM) in the presence of LPS
(100 ng/mL) for 24 h. The absorbance was measured on a plate reader
(Bio-Tek, Winooski, VT, USA) at 540 nm. And cell viability was assessed
by the MTT assay as previously reported [15].
9
65.0
66.9
67.1
62.2
63.9
63.7
1′
137.2
117.9
129.5
147.4
145.3
114.1
32.9
137.2
117.8
128.6
147.6
145.3
114.4
32.8
137.3
117.8
128.5
147.6
145.3
114.5
32.9
135.7
110.5
145.6
132.5
149.9
106.1
32.9
135.4
116.0
127.8
146.5
144.2
112.4
32.0
135.5
115.9
127.7
146.5
144.2
112.4
32.0
2′
3′
4′
5′
6′
7′
8′
35.8
35.7
35.8
35.5
34.6
34.6
9′
62.2
62.2
62.2
62.2
62.3
62.2
1′'
133.4
111.6
148.7
147.1
115.8
120.8
74.5
121.1
110.2
146.6
140.1
146.6
110.2
168.2
121.2
110.2
146.6
140.1
146.6
110.2
168.1
3. Results and discussion
2′'
3′'
Compound 1 was obtained as a white powder. Its molecular for-
mula, C31H38O11, was established by the positive-ion HRESIMS data
(m/z 609.2291 [M + Na]+, calcd 609.2306) and the 13C NMR data
(Table 2) of this compound. The presence of hydroxy (3399 cm−1) and
aromatic ring (1600, 1502, 1462, 1423 cm−1) groups was determined
by their IR absorption bands. Analysis of the low-field region of its 1H
NMR spectrum indicated the presence of a symmetric 1,3,4,5-tetra-
substituted phenyl [δH 6.73 (2H, s)], an asymmetric 1,3,4,5-tetra-
substituted phenyl [δH 6.74 (1H, br.s) and 6.72 (1H, br.s)], and a 1,3,4-
trisubstituted phenyl [δH 6.99 (1H, d, J = 2.4 Hz), 6.86 (1H, dd,
J = 8.4, 2.4 Hz), and 6.74 (1H, d, J = 2.4 Hz)]. The 13C NMR and
HSQC spectra showed 31 carbon resonances, including 18 aromatic
carbons, four methoxy carbons, five methylenes (three oxygenated),
and four methines (three oxygenated). The above data suggested that 1
was a sesquineolignane [16,17]. The HMBC correlations (Fig. 2) from
H-2 (H-6) (δH 6.73) to C-4/C-7, from H-7 (δH 5.55) to C-9/C-3′/C-4′,
from H-8 (δH 3.44) to C-1/C-2′/C-4′, from H-7′ (δH 2.62) to C-2′/C-6′/C-
9′, from H-8′ (δH 1.81) to C-1′, from OMe-3,5 (δH 3.82) to C-3 (C-5), and
4′'
5′'
6′'
7′'
8′'
88.9
9′'
61.7
OMe-3
OMe-5
OMe-5′
OMe-3′'
56.6
56.2
56.7
56.6
56.6
56.8
56.4
56.6
56.0
56.0
56.3
56.3
56.0
56.6
56.3
56.8
a
b
Data were recorded at 151 MHz in MeOH‑d4.
Data were recorded at 101 MHz in CDCl3.
361.1647 [M + H]+, calcd for C20H25O6, 361.1646.
(7S,8R)-5a: [ ]2D0 + 4.8 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
210 (+6.07), 290 (+0.74) nm.
(7R,8S)-5b: [ ]2D0 − 4.7 (c 0.05, MeOH); ECD (MeOH) λmax (Δε)
210 (−5.53), 290 (−0.85) nm.
3

M. Yu, et al.
BioorganicChemistry103(2020)104147
Fig. 2. Key 1H–1H COSY, HMBC and NOESY correlations of 1.
Cotton effect around 240 nm in the ECD spectra (Fig. 4A) revealed an 8′'S
configuration for 1b and 1d, meanwhile, the 8′'R configuration for 1a
and 1c was supported by the negative Cotton effect around 240 nm
[12,20]. The 7′',8′'-threo relative configuration allowed to assign the 7′'S
and 7′'R absolute configuration for 1b/1d and 1a/1c, respectively.
Therefore, the two pairs of enantiomers were assigned as follows:
(−)-(7S,8R,7′'R,8′'R)-4′'-hydroxy-3,3′',5,5′-tetramethoxy-4′,7-epoxy-4,8′'-
oxy-8,3′-sesquineoligna-7′',9,9′,9′'-tetrol (1a), (+)-(7R,8S,7′'S,8′'S)-4′'-
hydroxy-3,3′',5,5′-tetramethoxy-4′,7-epoxy-4,8′'-oxy-8,3′-sesquineoligna-
7′',9,9′,9′'-tetrol (1b), (−)-(7R,8S,7′'R,8′'R)-4′'-hydroxy-3,3′',5,5′-tetra-
methoxy-4′,7-epoxy-4,8′'-oxy-8,3′-sesquineoligna-7′',9,9′,9′'-tetrol (1c),
and (+)-(7S,8R,7′'S,8′'S)-4′'-hydroxy-3,3′',5,5′-tetramethoxy-4′,7-epoxy-
4,8′'-oxy-8,3′-sesquineoligna-7′',9,9′,9′'-tetrol (1d).
Compound 2 was obtained as a white powder. Its molecular formula
was determined to be C27H28O10 by the HRESIMS ion at m/z 513.1749
[M + H]+ (calcd 513.1755). The 1H NMR spectrum displayed a 1,3,4-
trisubstituted [δH 6.88 (1H, d, J = 1.8 Hz), 6.82 (1H, dd, J = 7.8,
1.8 Hz), and 6.75 (1H, d, J = 7.8 Hz)] and two 1,3,4,5-tetrasubstituted
[δH 7.04 (2H, s), 6.76 (2H, br.s)] aromatic rings. The 13C NMR and
HSQC spectra displayed 27 carbons, ascribed to one carbonyl, 18 aro-
matic carbons, two methoxy groups, four methylenes (two oxygenated),
and two methines (one oxygenated). According to the above data, to-
gether with the 2D NMR analysis, the structure of 2 was similar to that
Fig. 3. Stereochemistry of H-7′' and H-8′' in 1. Box indicates conformation that
agrees with measured data.
of
4-hydroxy-3,5′-dimethoxy-4′,7-epoxy-8,3′-neoligna-9,9′-ol
(5)
from OMe-5′ (δH 3.82) to C-5′, together with the 1H–1H COSY correla-
tions (Fig. 2) of H-7/H-8/H2-9 and H2-7′/H2-8′/H2-9′ revealed the
presence of a 4-substituted 3,5,5′-trimethoxy-4′,7-epoxy-8,3′-neoligna-
9,9′-diol moiety. In addition, the HMBC correlations (Fig. 2) from H-2′'
(δH 6.99)/H-6′' (δH 6.86) to C-7′', from H-5′' (δH 6.74) to C-1′'/C-3′',
from H-8′' (δH 4.06) to C-1′', from OMe-3′' (δH 3.87) to C-3′', combined
with the 1H–1H COSY correlations (Fig. 2) of H-7′'/H-8′'/H2-9′ indicated
the presence of an arylglycerol unit. Furthermore, the above two units
were connected through C-4 and C-8′', which was confirmed by the
HMBC correlation (Fig. 2) from H-8′' to C-4. The NOESY correlations
(Fig. 2) of H-7/H-9 (δH 3.77) and H-8/H-2 (H-6), combined with the
coupling constant (J7,8 = 6.0 Hz) indicated a trans configuration be-
tween H-7 and H-8 [17]. The large coupling constant between H-7′' and
H-8′' (J = 7.2 Hz) verified the 7′',8′'-threo configuration for 1 [12,18]
(Fig. 3). Thus, compound 1 was established as 7,8-trans-7′',8′'-threo-4′'-
hydroxy-3,3′',5,5′-tetramethoxy-4′,7-epoxy-4,8′'-oxy-8,3′-sesquineo-
ligna-7′',9,9′,9′'-tetrol.
[21,22], except for the presence of a 3,4,5-trihydroxybenzoyl at C-9 in
2. The above deduction was confirmed by the HMBC correlation from
3
H2-9 (δH 4.57, 4.47) to C-7′' (δC 168.2). The coupling constant of J7,8
(6.6 Hz) along with the NOESY correlations of H-7/H-9 (δH 4.47) and
H-8/H-2 (H-6) indicated the 7,8-trans configuration for 2. Thus, com-
pound 2 was determined to be 7,8-trans-4-hydroxy-3,5′-dimethoxy-9-
(3,4,5-trihydroxy)benzoyloxy −4′,7-epoxy-8,3′-neoligna-9′-ol. Com-
pound 2 was also obtained as a racemate, and the chiral HPLC se-
paration with a Daicel Chiralpak AD-H column afforded a pair of en-
antiomers 2a ([ ]2D0+47.3) and 2b ([ ]2D0−48.5). The two enantiomers
showed typical antipodal ECD curves (Fig. 4B). The positive Cotton
effect at 290 nm in the ECD spectrum of 2a suggested that it had a
7S,8R configuration, on the contrary, its enantiomer 2b had a 7R,8S
configuration [18,19]. Therefore, compounds 2a and 2b were estab-
lished as (+)-(7S,8R)-4-hydroxy-3,5′-dimethoxy-9-(3,4,5-trihydroxy)
benzoyloxy-4′,7-epoxy-8,3′-neoligna-9′-ol (2a) and (−)-(7R,8S) −4-
hydroxy-3,5′-dimethoxy-9-(3,4,5-trihydroxy)benzoyloxy-4′,7-epoxy-
8,3′-neoligna-9′-ol (2b), respectively.
As more and more lignans were reported as partial racemates in
natural products [12,13], compound 1 was subsequently analyzed by
chiral HPLC using a Daicel Chiralpak AD-H column, yielding two pairs of
enantiomers (1a/1b and 1c/1d). Compounds 1a and 1b showed mirror
image-like ECD curves (Fig. 4A) and specific rotations (1a: [ ]2D0 − 40.5;
1b: [ ]2D0 + 41.2), and similarly 1c and 1d displayed opposite ECD curves
(Fig. 4A) and specific rotations (1c: [ ]2D0 − 11.5, 1d:[ ]2D0+13.2). On the
basis of the reversed helicity rule [18,19], the positive Cotton effect at
295 nm indicated a 7S,8R configuration for 1a and 1d, while the nega-
tive Cotton effect at 295 nm of 1b and 1c, in turn, indicated a 7R,8S
configuration for these two stereoisomers. In addition, the positive
Compound 3 was isolated as a white powder. The HRESIMS ion at
m/z 543.1869 [M + H]+ suggested that the molecular formula was
C
28H30O11. The 1H and 13C NMR data of 3 (Tables 1 and 2) showed that
it was an analogue of 2, except for the presence of an additional
methoxy group at C-5. The 13C NMR chemical shift of C-5 in 3 (δC
149.4; ΔδC + 33.2) was deshielded compared to the same position in 2,
and the HMBC correlation from OMe (6H, δH 3.71) to C-3/C-5 also
demonstrated the above deduction. Subsequent chiral separation of 3
afforded a pair of enantiomers 3a and 3b, which showed opposite ECD
4

M. Yu, et al.
BioorganicChemistry103(2020)104147
Fig. 4. Experimental ECD spectra of 1a/1b − 4a/4b and 1c/1d.
curves (Fig. 4B) and optical rotations (3a: [ ]2D0
+ 28.3, 3b:
H-9 (δH 3.48) and H-8 (δH 3.99)/H-2 (H-6), combined with the coupling
[ ]2D0 − 30.8). In the same way, the absolute configurations of 3a and 3b
were determined as 7S,8R and 7R,8S according to the reversed helicity
rule [18,19]. Thus, compounds 3a and 3b were defined as (+)-(7S,8R)-
4-hydroxy-3,5,5′-trimethoxy-9-(3,4,5-trihydroxy)benzoyloxy-4′,7-
epoxy-8,3′-neoligna-9′-ol (3a) and (−)-(7R,8S)-4-hydroxy-3,5,5′-tri-
methoxy-9-(3,4,5-trihydroxy)benzoyloxy-4′,7-epoxy-8,3′-neoligna-9′-ol
(3b), respectively.
3
constant J7,8 (8.4 Hz) indicated the 7,8-trans configuration for 4. The
chiral HPLC purification of 4 afforded a pair of enantiomers 4a and 4b.
The enantiomers also displayed typical antipodal ECD curves (Fig. 4C)
and opposite optical rotations (4a: [ ]2D0 + 18.2, 4b: [ ]2D0 − 20.8). The
absolute configurations of 4a and 4b were assigned as 7S,8S and 7R,8R,
respectively, by analyzing the Cotton effect at 233 nm of the benzo-
dioxane system [20,23]. Therefore, compounds 4a and 4b were as-
signed as (+)-(7S,8S)-4-hydroxy-3,5′-dimethoxy-3′,7-epoxy-8,4′-oxy-
neoligna-9,9′-diol (4a) and (−)-(7R,8R)-4-hydroxy-3,5′-dimethoxy-
3′,7-epoxy-8,4′-oxyneoligna-9,9′-diol (4b), respectively.
Compound 4, a white powder, showed the molecular formula
C
20H24O7 as determined by the HRESIMS ion at m/z 377.1593
[M + H]+ (calcd 377.1595), requiring nine degrees of unsaturation.
The 1H NMR data (Table 1) showed signals of a 1,3,4-trisubstituted
aromatic ring [δH 6.99 (1H, d, J = 1.8 Hz), 6.88 (1H, dd, J = 8.1,
1.8 Hz), and 6.83 (1H, d, J = 8.1 Hz)] and a 1,3,4,5-tetrasubstituted
aromatic ring [δH 6.47 (1H, d, J = 1.8 Hz) and 6.42 (1H, d,
J = 1.8 Hz)]. The 13C NMR and HSQC spectra revealed the existence of
12 aromatic carbons, four methylenes (two oxygenated), two oxyge-
nated methines and two methoxy groups. The above information sug-
gested 4 was a lignan [12,18]. The 13C NMR chemical shifts of C-7 (δC
77.6) and C-8 (δC 79.9), along with the degrees of unsaturation implied
the presence of a 1,4-dioxane ring in 4 [9,10]. Furthermore, the HMBC
correlations from H-2′ (δH 6.42) to C-3′ (δC 145.6)/C-4′ (δC 132.5)/C-6′
(δC 106.1), from H-6′ (δH 6.47) to C-2′ (δC 110.5)/C-4′/C-5′ (δC 149.9),
and from H-7 (δH 4.86) to C-3′ indicated that 4 was a 3′,7-epoxy-8,4′-
oxyneolignane [23]. The NMR data of 4 showed distinct similarity to
(7S,8S)-3-methoxy-3′,7-epoxy-8,4′-oxyneolignan-4,9,9′-triol [23], dif-
fering only by the presence of signals for a methoxy group [δC 56.6, δH
3.86 (s)] at C-5′, which was confirmed by the HMBC correlation from
OMe (δH 3.86) to C-5′. In the NOESY spectrum, the correlations of H-7/
The known compounds 5 and 6 were also obtained as enantiomers.
These enantiomers were identified as (7S,8R)-4-hydroxy-3,5′-di-
methoxy-4′,7-epoxy-8,3′-neoligna-9,9′-ol (5a) [21], (7R,8S)-4-hydroxy-
3,5′-dimethoxy-4′,7-epoxy-8,3′-neoligna-9,9′-ol (5b) [22], (7S,8R)-4-
hydroxy-3,5,5′-trimethoxy-4′,7-epoxy-8,3′-neoligna-9,9′-ol (6a) [24]
and (7R,8S)-4-hydroxy-3,5,5′-trimethoxy-4′,7-epoxy-8,3′-neoligna-9,9′-
ol (6b) [25] by comparing NMR data, specific rotations and ECD data
with values in the literature.
All the isolated enantiomers were tested for their antineuroin-
flammatory activities by an NO inhibition assay in LPS-induced BV-2
microglial cells using the Griess reaction [9,10]. The results displayed
that 5b, 6a and 6b showed inhibitory activities with IC50 values of
14.3
0.3, 23.2
1.4, and 33.3
2.5 μM, respectively, while the
positive control minocycline gave the IC50 values of 13.5
1.1 μM.
Interestingly, compound 5b showed inhibitory effects, however, its
enantiomer 5a was inactive.
5

M. Yu, et al.
BioorganicChemistry103(2020)104147
4. Conclusion
References
In conclusion, seven pairs of lignan enantiomers (1a/1b − 6a/6b
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Declaration of Competing Interest
The authors declare no competing financial interest.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
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6