Bioactive bisbenzylisoquinoline alkaloids from the roots of Stephania tetrandra

Article abstract of DOI:10.1016/j.bioorg.2020.103697

Ten new bisbenzylisoquinoline alkaloids (1–10) and eight known analogues (11–18) were obtained from the roots of Stephania tetrandra. The structures of these compounds were determined by spectroscopic methods, single-crystal X-ray diffraction, electronic

Full text of DOI:10.1016/j.bioorg.2020.103697

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Bioactive bisbenzylisoquinoline alkaloids from the roots of Stephania tetran-
dra
Renzhong Wang, Yanfei Liu, Guoru Shi, Jie Zhou, Jiayuan Li, Li Li, Jiqiao
Yuan, Xuyu Li, Dequan Yu
PII:
S0045-2068(19)32035-8
DOI:
https://doi.org/10.1016/j.bioorg.2020.103697
YBIOO 103697
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
27 November 2019
19 February 2020
23 February 2020
Please cite this article as: R. Wang, Y. Liu, G. Shi, J. Zhou, J. Li, L. Li, J. Yuan, X. Li, D. Yu, Bioactive
bisbenzylisoquinoline alkaloids from the roots of Stephania tetrandra, Bioorganic Chemistry (2020), doi: https://
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Bioactive bisbenzylisoquinoline alkaloids from the roots of Stephania tetrandra
Renzhong Wang, Yanfei Liu, Guoru Shi, Jie Zhou, Jiayuan Li, Li Li, Jiqiao Yuan,
Xuyu Li and Dequan Yu *
State Key Laboratory of Bioactive Substance and Function of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, People’s Republic of China
*
Corresponding Author
E-mail: dqyu@imm.ac.cn (D. Yu). Tel: +86-10-63165224. Fax: +86-10-63017757.
1

ABSTRACT: Ten new bisbenzylisoquinoline alkaloids (1-10) and eight known
analogues (11-18) were obtained from the roots of Stephania tetrandra. The structures
of these compounds were determined by spectroscopic methods, single-crystal X-ray
diffraction, electronic circular dichroism analyses, and chemical method. Compounds
1
, 15, and 16 showed the better anti-inflammatory activities with IC values of 15.26
50
±
2.99, 6.12 ± 0.25, and 5.92 ± 1.89 μM, respectively. Compound 18 possessed
cytotoxic activities against MCF-7, HCT-116, and HepG2 cell lines with IC values
5
0
of 2.81 ± 0.06, 3.66 ± 0.26, and 2.85 ± 0.15 μM, respectively.
Keywords: Stephania tetrandra; Menispermaceae; Bisbenzylisoquinoline alkaloids;
Anti-inflammatory activities; Cytotoxicity
2

M/Z 396
O
4'
N
4a
6
O
O
O
4'
4'
O
4a'
A' B'
a'
3
4'
1'
4a
8a
6
O
O
6'
4a'
4a
8a
O
6'
4a'
8a'
4
8
a
a
6
O
O
6'
O
O
6'
4a'
8a'
3
3
N
B
A
C
N
N
1'
8
a
N
N
H
N
1
15
N
H
8
1'
H
H
O
O
8a' 1'
1
HO
H
1
1
H
α
α'
9'
α'
O
OH
O
α'
9'
4'
O
9
α
α
OH
α
α'
9'
14'
9
'
9
9
14
9
4
O
C'
14
O
O
O
14'
14'
14
1
1
O
O
O
O
1
2
(+)-3a
-
( )-3b
O
6'
4a' 4'
OH
8a
4'
OH
O
4'
O
6'
4'
4a'
4
a
6
O
O
6'
4a'
6'
4a'
4a
6
6
O
6
O
O
3
3
3
3
4
a
4a
8a
O
O
N
N
H
α'
N
N
N
H
N
N
H
α'
8
a'
1'
N
8a'
1'
8a
OR1
8a'
1'
8
a
O
O
O
O
8a'
1'
1
1
H
H
1
H
H
1
α
9
O
O
O
O
α'
α
O
α
α'
α
9'
14'
9'
14'
9'
14'
9
9
9'
14'
9
O
O
O
O
14
1
4
14
14
OR2
O
7
R1= H, R2= CH3
4
5
6
1
0 R1= CH3, R2= H
4 R1= R2= CH3
O
1
4
'
O
4a' 4'
4
'
O
6' 4a'
8a'
4a
8a
6
O
6'
4'
4a'
O
4a'
4a
6
O
4
a
O
6'
3
3
4a
O
6'
6
6
3
3
O
O
O
N
N
H
N
H
N
N
H
α'
N
H
N
H
α'
R3
OR
8a'
1'
N
H
8
a'
1'
1 8a
9
OR1
1'
H
α'
1
8a
O
8a'
1'
8
a
OH
H
1
1
O
O
α'
O
α
O
α
O
α
9
9'
α
9
4
9'
14'
9'
14'
9
9'
14'
O
14'
O
14
O
1
4
O
1
OR2
14
O
O
1
1
1
3 R= CH3, 2'-α O
6 R= CH3, 2'-βO
7 R= H, 2'-βO
8
9
2-βO, 2'-βO
2-βO, 2'-α O
11 R1= R2= R3= CH3
12 R1= H, R2= R3= CH3
8 R1= R2= CH3, R3= H
15
1
Fig. 1. The structures for compounds 1-18
3

1
. Introduction
Bisbenzylisoquinoline alkaloids are structurally diverse and fascinating alkaloid
class, which consist of two benzylisoquinoline units linked by diphenyl ether, benzyl
phenyl ether or biphenyl bonds [1]. To date, nearly 500 bisbenzylisoquinoline
alkaloids have been isolated and structurally characterized from the plants which
mostly distribute the families of Berberidaceae, Menispermaceae, Monimiaceae, and
Ranunculaceae [1,2]. Some of these bisbenzylisoquinoline alkaloids possess various
biological activities, such as antiplasmodial, anti-inflammatory, antibacterial,
antifungal, cytotoxic, and antioxidant activities [3-6]. Stephania tetrandra S. Moore is
a commonly used herb, belonging to the Menispermaceae family. Its roots, named
“
Fen Fang Ji” in Chinese, have been used for treatments of rheumatalgia, dropsy,
dysuria, and eczema [7]. Previous chemical work showed that S. tetrandra afforded a
series of bisbenzylisoquinoline alkaloids [8-10]. Among these, tetrandrine (11) is the
main components, which has been reported to exhibit wide pharmacological activities
[11], such as anti-inflammatory and anticancer.
In order to discover structurally diverse bisbenzylisoquinoline alkaloids with
anti-inflammatory and cytotoxic activities, a detailed chemical investigation on the
roots of S. tetrandra was carried out . In the present study, 18 bisbenzylisoquinoline
alkaloids were successfully isolated, including ten new bisbenzylisoquinoline
alkaloids (1-10) and eight known compounds (11-18) (Fig. 1). The known compounds
were identified as tetrandrine (11) [12], fangchinoline (12) [13,14], tetrandrine
2
'-N-α-oxide (13) [15,16], fenfangjine A (14) [8], isotetrandrine (15) [17], tetrandrine
4

2
'-N-β-oxide (16) [18], fenfangjine C (17) [8], and cycleanorine (18) [19] by
comparison their physical and spectroscopic data with literature values. The isolated
compounds 1-18 were examined for anti-inflammatory activity and cytotoxicity.
Herein, the details of isolation, structure elucidation and biological evaluation of these
compounds are presented.
2
. Materials and methods
2
.1. General experimental procedures
The UV spectra, optical rotations, and ECD spectra were measured with JASCO
V-650, JASCO P-2000, and JASCO J-815 spectrometers, respectively. Melting points
were recorded on an XT5B microscope melting point apparatus. IR spectra were
measured by a Nicolet 5700 spectrometer (FT-IR microscope transmission). The
NMR spectra were acquired by Mercury-400 and Bruker AVⅢHD 600 MHz
spectrometers. HRESIMS/MS data were obtained using Dionex UltiMate 3000
UHPLC equipped with Thermo Scientific Q Exactive Focus Mass spectrometer. The
X-ray crystallographic data were obtained from an Agilent Xcalibur Eos Gemini
diffractometer with Cu-Kα radiation (λ = 1.5418Å). Analytical HPLC was performed
on an Agilent 1260 infinity system equipped with a DAD-UV detector. Preparative
HPLC was conducted on a Shimadazu LC-6AD liquid chromatography system
equipped with a SPD-20A UV detector and an ODS column (C18, 250×10mm, 5um,
CAPCELL PAK-MGⅡ, Kyoto, Japan).
2
.2. Plant material
5

The roots of Stephania tetrandra were collected from Ruichang, Jiangxi
Province, China, in December 2017, and were authenticated by Professor Lin Ma,
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College. A voucher specimen (ID-S-2937) was preserved in the herbarium of
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union
Medical College.
2
.3. Extraction and isolation
The dried and powdered roots of Stephania tetrandra (98 kg) were extracted with
5% EtOH under reflux (100 L × 3). The solution was evaporated under reduced
9
pressure and the residue (8.0 kg) was then added into 1% HCl solution with vigorous
stirring. The acidic solution was filtered, and the acidic filtrate was then basified with
concentrated NH OH to produce free alkaloids and exhaustively extracted with CHCl
4
3
to give a crude alkaloid mixture (2.1 kg), which was subjected to a silica gel column
using gradient elution with petroleum ether-acetone and CHCl -MeOH (1:0 to 25:1
3
and 30:1 to 0:1) to afford seven fractions (A-G). Fraction A (1.3 kg) was
recrystallized in acetone to give 11 (1.2 kg), and the acetone solution was evaporated
and the residue (100 g) was chromatographed further on silica gel eluting with
CHCl -MeOH gradient system (1:0 to 0:1) to afford six fractions (A1-A6). Fraction
3
A3 (34.2 g) was applied to column chromatography over silica gel eluting with
petroleum ether-CH Cl and CH Cl -MeOH gradient system (5:1 and 30:1 to 0:1) to
2
2
2
2
afford five fractions (A3a-A3e). Fraction A3d (29.2 g) was separated by repeated
chromatography, including silica gel column chromatography
6

(EtOAc/MeOH/NH OH gradient), Sephadex LH-20 (CH Cl /MeOH, 1:1) or MPLC
4 2 2
(
ODS, MeCN/H O, 30:70 to 100:0, NH OH, 0.01%), and was further purified by
2
4
preparative HPLC to afford 1 (7.0 mg, MeCN/H O, 55:45, NH OH, 0.01%), 2 (12.0
2
4
mg, MeCN/H O, 65:35, NH OH, 0.01%), and 3 (7.3 mg, MeCN/H O, 56:44, NH OH,
2
4
2
4
0
.01%). Compound 3 was subjected to chiral HPLC using a CHIRALPAK AD-H
column (n-hexane/isopropyl alcohol, 82:18) to afford (+)-3a (3.5 mg) and (‒)-3b (1.3
mg). Fraction A3e (1.6 g) was applied to MPLC (ODS, MeCN/H O, 30:70 to 100:0,
2
NH OH, 0.01%) to obtain six fractions (A3e1-A3e6). A3e1 (100 mg), A3e3 (30 mg),
4
and A3e4 (50 mg) were purified by preparative HPLC (MeCN/H O, 37:63, NH OH,
2
4
0
.01%) to give 13 (8.2 mg), 14 (5.1 mg), and 16 (13.0 mg), respectively. Fraction A4
(40.0 g) was fractionated by silica gel column chromatography using gradient elution
with EtOAc-MeOH (20:1 to 0:1) to afford four fractions (A4a-A4d). Fraction A4c
was crystallized in MeOH to give 12 (1.0 g). Fraction A4d (8.0 g) was
chromatographed using Sephadex LH-20 (CH Cl /MeOH, 1:1), MPLC (ODS,
2
2
MeCN/H O, 30:70 to 100:0, NH OH, 0.01%), and preparative HPLC to yield 4 (4.8
2
4
mg, MeCN/H O, 48:52, diethylamine, 0.01%) and 15 (12.3 mg, MeCN/H O, 64:36,
2
2
diethylamine, 0.01%). Fraction B (400g) was recrystallized in EtOH, and the EtOH
solution was evaporated to give the residue (129 g), which was performed on a silica
gel column chromatography eluting with CH Cl -MeOH and EtOAc-MeOH-NH OH
2
2
4
gradient system (20:1 to10:1 and 20:2:1 to 10:2:1) to afford eight fractions (B1-B8).
After purification by Sephadex LH-20 (CH Cl /MeOH, 1:1), fraction B6 (3.2 g) was
2
2
applied to MPLC (ODS, MeCN/H O, 5:95 to 100:0, diethylamine, 0.01%) to give
2
7

seven fractions (B6a-B6f). Fraction B6a (40.0 mg) was purified by preparative HPLC
MeCN/H O, 18:82, diethylamine, 0.01%) to give 8 (9.2 mg) and 9 (5.0 mg).
(
2
Preparative HPLC was used to purify compounds 7 (19.2 mg) and 17 (14.8 mg) from
B6d (MeCN/H O, 28:72, diethylamine, 0.01%) and B6f (MeCN/H O, 37:63,
2
2
diethylamine, 0.01%). Fraction B6b (63.2 mg) was chromatographed using MPLC
ODS, MeCN/H O, 24:76 to 30:70, diethylamine, 0.01%) and preparative HPLC to
(
2
afford 6 (8.1 mg, MeCN/H O, 28:72, diethylamine, 0.01%) and 10 (6.0 mg,
2
MeOH/H O, 60:40, diethylamine, 0.01%). Fraction B5 (13.4 g) was separated on
2
Sephadex LH-20 (CH Cl /MeOH, 1:1), silica gel column (CH Cl /MeOH, 10:1,
2
2
2
2
EtOAc/MeOH/NH OH, 20:2:1), MPLC (ODS, MeCN/H O, 30:70 to 100:0,
4
2
diethylamine, 0.01%), and preparative HPLC to give 5 (30.0 mg, MeCN/H O, 43:57,
2
diethylamine, 0.01%) and 18 (9.5 mg, MeCN/H O, 63:37, diethylamine, 0.01%).
2
2
.3.1. Fenfangjine J (1)
2
0
colorless crystals (MeOH); mp 260-261℃; [α] +186 (c 0.35, MeOH); UV
D
(MeOH) λmax (log ε) 207 (4.56), 267 (3.37), 293 (3.48) nm; IR νmax 3445, 2933, 1679,
-1
1
582, 1580, 1299, 1122, 828 cm ; ECD (MeOH) λ
(∆ε) 233 (+57.26), 264.5
max
1
13
(
‒9.28), 290.5 (‒4.87) nm; H and C NMR (CDCl ) data see Tables 1 and 2;
3
+
HRESIMS m/z 623.2754 [M+H] (calcd for C H O N , 623.2752 ).
3
7
39
8
2
2
.3.2. Fenfangjine K (2)
colorless crystals (MeOH); mp 252-253℃; [α]
λmax (log ε) 207 (4.42), 287.4 (3.41) nm; IR νmax 3547, 2937, 1600, 1507, 1269, 1230,
2
D
0
‒2 (c 0.55, CHCl ); UV (MeOH)
3
-1
1
126, 1005 cm ; ECD (MeOH) λ (∆ε) 216.5 (+26.65), 240 (+14.80), 291.5 (+1.46)
max
8

1
13
nm; H and C NMR (CDCl ) data see Tables 1 and 2; HRESIMS m/z 623.3117
3
+
[
M+H] (calcd for C H O N , 623.3116).
3
8
43
6
2
2
.3.3. (±)-Fenfangjine L (3)
white amorphous powder; [α]
04 (4.24), 238 (4.25), 311 (2.99), 325 (3.03) nm; IR νmax 3339, 2934, 1606, 1504,
2
D
0
+73 (c 0.40, MeOH); UV (MeOH) λmax (log ε)
2
1
-1
1
13
265, 1227, 862 cm ; H and C NMR (CDCl ) data see Tables 1 and 2; HRESIMS
3
2
0
+
m/z 621.2596 [M+H] (calcd for C H O N , 621.2595); (+)-Fenfangjine L (3a): [α]
D
3
7
37
7
2
+
80 (c 0.35, MeOH); ECD (MeOH) λmax (∆ε) 240.5 (+21.20), 293 (‒0.31), 322.5
2
D
0
(+1.43) nm. (‒)-Fenfangjine L(3b): [α]
‒76 (c 0.13, MeOH); ECD (MeOH) λmax (∆ε)
2
2
41 (‒15.33), 291.5 (+1.28), 322 (‒0.59) nm.
.3.4. Fenfangjine M (4)
2
0
white amorphous powder; [α]
D
+222 (c 0.32, CHCl ); UV (MeOH) λ (log ε)
3 max
2
1
3
04 (4.31), 227 (4.09), 288.8 (3.64) nm; IR νmax 3413, 2939, 1678, 1600, 1506, 1267,
-1
120, 1016 cm ; ECD (MeOH) λ (∆ε) 216 (‒2.23), 233 (+24.72), 264.5 (+2.67),
max
1
13
05 (‒1.31), 337 (+2.51) nm; H and C NMR (CDCl ) data see Tables 1 and 2;
3
+
HRESIMS m/z 621.2593 [M+H] (calcd for C H O N , 621.2595).
3
7
37
7
2
2
.3.5. Fenfangjine N (5)
2
0
white amorphous powder; [α]
D
+327 (c 1.9, CHCl ); UV (MeOH) λ (log ε)
3 max
2
1
05.6 (4.86), 237.8 (4.35), 274.4 (3.98) nm; IR νmax 3396, 2930, 1674, 1509, 1286,
-1
226, 1108, 838 cm ; ECD (MeOH) λ
(∆ε) 216 (+70.76), 239 (‒13.49), 256
max
1
13
(
+13.31), 287 (+24.94) nm; H and C NMR (CDCl ) data see Tables 1 and 2;
3
+
HRESIMS m/z 625.2902 [M+H] (calcd for C H O N , 625.2908).
3
7
41
7
2
9

2
.3.6. Fenfangjine O (6)
2
0
white amorphous powder; [α]
D
+244 (c 0.44, CHCl ); UV (MeOH) λ (log ε)
3 max
2
1
05.2 (4.24), 237 (3.76), 274.4 (3.44) nm; IR νmax 3374, 2938, 1669, 1510, 1284,
-1
260, 1113, 842 cm ; ECD (MeOH) λ (∆ε) 219 (+24.80), 240.5 (‒3.02), 257.5
max
1
13
(
+3.42), 287.5 (+8.19) nm; H and C NMR (CDCl ) data see Tables 3 and 4;
3
+
HRESIMS m/z 641.2854 [M+H] (calcd for C H O N , 641.2857).
3
7
41
8
2
2
.3.7. Fenfangjine P (7)
2
0
white amorphous powder; [α]
D
+222 (c 1.0, CHCl ); UV (MeOH) λ (log ε)
3 max
2
06 (4.13), 285.4 (3.14) nm; IR νmax 3395, 2966, 1513, 1461, 1263, 1235, 1129, 881
-1
1
13
cm ; ECD (MeOH) λ (∆ε) 220 (+100.90), 280.5 (+5.66) nm; H and C NMR
max
+
(
CDCl ) data see Tables 3 and 4; HRESIMS m/z 625.2907 [M+H] (calcd for
3
C H O N , 625.2908).
3
7
41
7
2
2
.3.8. Fenfangjine Q (8)
2
0
white amorphous powder; [α]
D
+289 (c 0.57, CHCl ); UV (MeOH) λ (log ε)
3 max
2
07.4 (4.53), 286.6 (3.43) nm; IR νmax 3388, 2960, 1510, 1460, 1264, 1237, 1128
-1
(∆ε) 221 (+75.43), 275 (+4.02) nm; H and 13C NMR
1
cm ; ECD (MeOH) λ
max
+
(
CDCl ) data see Tables 3 and 4; HRESIMS m/z 641.2858 [M+H] (calcd for
3
C H O N , 641.2857).
3
7
40
8
2
2
.3.9.Fenfangjine R (9)
2
0
white amorphous powder; [α]
D
+199 (c 0.35, CHCl ); UV (MeOH) λ (log ε)
3 max
-1
2
06.6 (4.49), 287 (3.29) nm; IR νmax 3385, 2965, 1511, 1462, 1265, 1235, 1128 cm ;
1
13
ECD (MeOH) λmax (∆ε) 221 (+89.32), 285 (+4.93) nm; H and C NMR (CDCl ) data
3
1
0

+
see Tables 3 and 4; HRESIMS m/z 641.2856 [M+H] (calcd for C H O N ,
3
7
41
8
2
6
41.2857).
2
.3.10 Fenfangjine S (10)
white amorphous powder; [α]
2
D
0
+268 (c 0.1, CHCl ); UV (MeOH) λ (log ε)
3 max
-1
2
07 (4.52), 286.2 (3.33) nm; IR νmax 3406, 2934, 1503, 1454, 1261, 1122, 905 cm ;
1
13
ECD (MeOH) λmax (∆ε) 225 (+86.44), 272.5 (+6.09) nm; H and C NMR (CDCl )
3
+
data see Tables 3 and 4; HRESIMS m/z 625.2907 [M+H] (calcd for C H O N ,
3
7
41
7
2
6
25.2908).
2
.4. Reduction of compounds 7, 8, and 9
Compound 7 (15.8 mg) was dissolved in 10% HCl (30 ml), and then zinc
powder (100 mg) was added. After being stirred at room temperature for 2 h, the
mixture was adjusted to pH 9 with NH OH and then was extracted with CH Cl (3×30
4
2
2
ml). The CH Cl layer was purified by preparative HPLC (MeCN/H O, 58:42,
2
2
2
diethylamine, 0.01%) to yield fangchinoline (12). Based on the same method, the
reduction of compounds 8 and 9 also yielded fangchinoline (12).
2
.5. X-ray crystallographic analysis of compounds 1, 2, and 12.
Single crystals of 1, 2, and 12 were obtained from MeOH. The crystal data were
collected on an Agilent Xcalibur Eos Gemini diffractometer using Cu-Kα radiation (λ
1.5418 Å). The crystal structures were solved by direct methods using the
=
SHELXS-97 program and refined by the full-matrix least-squares difference Fourier
method. Crystallographic data of 1, 2, and 12 have been deposited at the Cambridge
Crystallographic Data Center (CCDC No. 1954074, 1954075, and 1954076
1
1

respectively) and can be obtained free of charge from the CCDC Web site
www.ccdc.cam.ac.uk).
(
2
.5.1. Crystal data of 1
C H N O , M =
622.69,
orthorhombic, a =
7.20673(16) Å,
b =
3
7
38
2
7
3
1
1.8067(5) Å, c = 37.8914(8) Å, U = 3224.10(16) Å , T = 110.35(10), space group
P2 2 2 (no. 19), Z = 4, μ(Cu Kα) = 0.723, 11350 reflections measured, 6112 unique
1
1 1
(
R = 0.0258). The final wR(F ) was 0.0867 (all data). The goodness of fit and flack
int 2
parameters were 1.032 and ‒ 0.01(7), respectively.
2
.5.2. Crystal data of 2
C H N O , M =622.73, orthorhombic, a = 13.2047(3) Å, b = 13.8202(3) Å, c =
3
8
42
2
6
3
1
4
7.9059(4) Å, U = 3267.69(11) Å , T = 115.15(10), space group P2 2 2 (no. 19), Z =
1 1 1
, μ(Cu Kα) = 0.687, 11755 reflections measured, 6183 unique (R = 0.0312). The
int
final wR(F ) was 0.1051 (all data). The goodness of fit and flack parameters were
2
1
.055 and 0.00(10), respectively.
.5.3. Crystal data of 12
2
C H N O , M =608.71,
orthorhombic, a =
7.26130(13) Å,
b
=
3
7
40
2
6
3
1
0.5190(2) Å, c = 39.8162(7) Å, U = 3041.22(10) Å , T = 109.95(10), space group
P2 2 2 (no. 19), Z = 4, μ(Cu Kα) = 0.726, 10594 reflections measured, 5732 unique
1
1 1
(
R = 0.0240) .The final wR(F ) was 0.0949 (all data). The goodness of fit and flack
int 2
parameters were 1.057 and 0.10(15), respectively.
.6. ECD calculation
2
1
2

Conformational analysis was performed by the MOE software package using the
MMFF94s molecular mechanics force field. The conformers with Boltzmann
distribution of over 1% were chosen for ECD calculations. The chosen conformers
were optimized at the WB97XD/6-31g(d) level using the Gaussian 09 program. The
theoretical calculation of ECD was conducted using TDDFT methodology at the
APFD/6-31g(d) level in MeOH. The ECD spectra were simulated by SpecDis and the
final ECD spectra were obtained according to the Boltzmann distribution theory.
2
.7. Anti-inflammatory activity assay
Peritoneal macrophages were obtained from C57BL/6 mice 3 days after
intraperitoneal injection with 1.2 ml of 4% sterile thioglycolate. Then, cells were
5
inoculated into 96-well plates (2×10 cells/well) containing RPMI 1640 medium with
5
5
% fetal bovine serum and were cultured for 2 h under a humidified atmosphere with
% CO at 37 °C. The culture medium was replaced by fresh medium containing
2
-5
compounds (10 M). After incubation for 2h, the macrophages were then incubated
with 1 μg/ml of LPS for 24h. The culture supernatants of macrophages were collected
and Griess reagents were added into supernatants to determine the NO level. The
absorbance was measured at 570 nm with a microplate reader. Dexamethasone (DEX)
was used as a positive control.
2
.8. Cytotoxicity assay
The five human cancer cells (A549, CMF-7, HCT-116, HepG2, and Capan 2)
were cultured in the RPMI1640 medium containing 10% fetal bovine serum and 100
U/mL penicillin under a humidified atmosphere with 5% CO at 37 °C . Cells were
2
1
3

plated into 96-well plates in 100 μL of medium and incubated for 24 h. The medium
was replaced with fresh medium containing the tested compounds at concentrations of
0
.1, 1, and 10 μM, respectively, which were carried out in triplicate. After 72 h, the
mediums were replaced with 100 μL of MTT (0.5 mg/ml in medium), and the cells
were incubated for 4 h. 200 μL of DMSO was then added and the absorbance was
measured at 570 nm with a microplate reader. The inhibition rate was calculated by
the following formula: 1 − mean ODtreated well / mean ODblank well. Paclitaxel was used
as the positive control.
3
. Results and discussion
Compound 1 was obtained as colorless crystals. Its molecular formula was
+
determined to be C H O N by the HRESIMS ion at m/z 623.2754 [M+H] (calcd
3
7
38
8
2
for C H O N , 623.2752), indicating 20 degrees of unsaturation. The IR spectrum
3
7
39
8
2
-1
suggested the presence of carbonyl (1679 cm ) and aromatic ring (1582 and 1508
-1
1
cm ) functionalities. The H NMR spectrum (Table 1) of 1 in the lower field showed
the presence of a para-disubstituted benzene ring at δ 6.50 (1H, dd, J = 8.4, 2.4 Hz,
H
H-10'), 6.79 (1H, dd, J = 8.4, 2.4 Hz, H-11'), 7.09 (1H, dd, J = 8.4, 2.4 Hz, H-13'),
7
.29 (1H, dd, J = 8.4, 2.4 Hz, H-14'), a 1,3,4-trisubstituted benzene ring at δ 6.47
H
(1H, d, J = 1.8 Hz, H-10), 6.86 (1H, d, J = 8.4 Hz, H-13), 6.89 (1H, dd, J = 8.4, 1.8
Hz, H-14), and three aromatic singlet protons at δ 6.35 (1H, s, H-5), 7.21 (1H, s,
H
H-5'), 6.40 (1H, s, H-8'). Six heteroatom-bearing singlet methyls [δ 3.78 (s, 6-OMe);
H
3
2
.23 (s, 7-OMe); 3.91 (s, 12-OMe); 3.48 (s, 6'-OMe); 2.32 (s, 2-N-Me); 3.29 (s,
'-N-Me)] were observed in the higher field. Analysis of the 13C NMR spectrum
1
4

(
Table 2) revealed the presence of 37 carbon signals, including the signals
corresponding to the aforementioned units, four methylenes (δc 44.3, C-3; 22.3, C-4;
1.6, C-α; 37.2, C-α'), two methines (δc 61.6, C-1; 61.2, C-1'), a carbonyl (δc 168.6,
4
1
13
C-4') and 14 aromatic quaternary carbons. Further analysis of the H and C NMR
data of 1 showed a similarity to those of tetrandrine (11) [12], except for the signals of
B'-ring in 1.
In the HMBC spectrum (Fig. 2), the HMBC correlations from H-5' to C-4', C-4a',
and C-8a', from H-8' to C-1', C-4a', and C-8a', from 2'-N-CH (δ 3.28) to C-1' and
3
H
C-4', from H-1' to C-4', C-4a', and C-8a' constructed the B'-ring unit. The locations of
all methoxy groups were corroborated by the ROESY correlations (Fig. 3) of OCH -6
3
(
δ 3.77) with H-5, OCH -12 (δ 3.91) with H-13, and OCH -6' (δ 3.48) with H-5'.
H 3 H 3 H
The MS/MS data of compound 1 displaying a fragment ion peak at m/z 396 (Fig.S5,
Supporting Information) corresponded to the upper half of the molecule, due to the
cleavage of double benzylic bonds. Thus, compound 1 was confirmed as a
head-to-head bisbenzyl-isoquinoline alkaloid [20,21] that contains two diphenyl ether
bridges occurring at C-8/C-7' and C-11/C-12'. The 2D structure of 1 was assigned as
shown (Fig. 2). Fortunately, the crystals were obtained from methanol and the
absolute configuration of 1 was determined as 1S, 1'S by single-crystal X-ray
diffraction (Cu-Kα) analysis (Fig. 4) with a Flack parameter of -0.01(7). Therefore,
the structure of compound 1 was designated as fenfangjine J.
1
5

O
O
O
O
O
O
OH
O
O
O
N
N
N
N
N
N
O
O
HO
O
O
O
O
O
O
1
2
O
5
1
1
H- H COSY
HMBC
1
1
Fig. 2. Key H- H COSY and HMBC correlations of compounds 1, 2, and 5
Compound 2, colorless crystal, was assigned a molecular formula of C H O N
2
3
8
42
6
+
based on its HRESIMS peak at m/z 623.3117 [M+H] (calcd for C H O N ,
3
8
43
6
2
6
23.3116), implying 19 degrees of unsaturation. The UV spectrum (λmax 207 and 287
nm) was very close to those of 11, which indicated the presence of benzylisoquinoline
-1
moieties. Its IR spectrum showed the presence of hydroxy group (3547 cm ) and
-1
13
aromatic ring (1600 and 1507 cm ) functionalities. The C NMR data (Table 2) of 2
1
were similar to those of 11 [12], except for a methylene at δ 62.1. However, the H
C
NMR spectrum (Table 1) of 2 in the lower field displayed only seven aromatic
protons at room temperature. The three aromatic protons of C'-ring not be observed.
1
The variable-temperature (50℃) 400 MHz H NMR spectrum was carried out, which
showed ten aromatic protons [δ 6.95 (1H, dd, J = 8.4, 1.6 Hz, H-14); 6.88 (1H, d, J =
H
8
.4 Hz, H-13); 6.28 (1H, d, J = 1.6 Hz, H-10); 6.74 (1H, s, H-5); 7.10 (2H, brs,
H-10'/14'); 6.70 (2H, brd, H-11'/13'); 6.49 (1H, s, H-5'); 6.48 (1H, s, H-8')]. Compared
1
with 11 [12], the H NMR spectrum of 2 revealed the presence of an additional
methylene group [δ 5.10 (1H, d, J = 9.2 Hz, H-15a), 4.73 (1H, d, J = 9.2 Hz, H-15b)]
H
and the absence of a methoxy group. The HMBC correlations from H-15 to C-7, C-8
and C-8a, from H-5 to C-6, C-7 and C-8a confirmed that the methylene (δ 62.1,
C
C-15) was connected to the C-8 position of the A-ring. Comprehensive analysis of
1
6

1
1
H- H COSY, HSQC and HMBC data (Fig. 2) demonstrated the presence of the two
benzylisoquinoline moieties. The ROESY correlation (Fig. 3) of H -15 with H-5'
2
confirmed that the methylene (δ 62.1, C-15) also was attached at C-6' via a ether
C
bond. In addition, the MS/MS data of 2 showed a fragment ion peak at m/z 388 (Fig.
S17, Supporting Information). These data indicated that 2 was the head-head
connection. Therefore, compound
2
was identified to be
a
unusual
bisbenzylisoquinoline alkaloid with C-15/6' benzyl phenyl ether bridge and C-11/12'
diphenyl ether bridge and named fenfangjine K. The structure of 2 was confirmed by
single-crystal X-ray diffraction analysis (Fig. 4), which also allowed assignment of
the absolute configuration of 2 as 1S, 1'S [Flack parameter 0.00 (10)].
1
2
7
Fig. 3. Key ROESY correlations of compounds 1, 2, and 7.
1
2
12
Fig. 4. X-ray ORTEP drawing of compounds 1, 2, and 12.
1
7

Compound 3 was isolated as a white amorphous powder, which was found to
have a molecular formula of C H O N according to its HRESIMS peak at m/z
3
7
36
7
2
+
6
21.2596 [M+H] (calcd for C H O N , 621.2595), with 21 degrees of unsaturation.
37 37 7 2
The NMR features (Tables 1 and 2) of 3 were similar to those of 11 [12], except for
1
the signals of B- or B'-ring. The H NMR spectrum (Table 1) of 3 revealed the
presence of additional three protons [δ 8.37 (1H, d, J = 5.4 Hz), 7.50 (1H, d, J = 5.4
H
Hz), and 6.08 (1H, s)] in the lower field. While, only one N-CH (δ 2.50) group was
3
H
1
3
observed in the higher field. The C NMR data (Table 2) combined with HSQC data
indicated the presence of two aromatic methines (δ 138.4, 120.0), a downfield
C
chemical shifted aromatic quaternary carbon (δ 157.4), and a oxygenated methine
C
(δC 72.8). All of the data indicated that B- or B'-ring in 3 was an aromatic ring. The
HMBC correlations from H-4' to C-3', C-5', and C-8a', from H-3' to C-4', C-4a', and
C-1', from H-8' to C-1', C-4a', and from H-α' to C-1', C-9', C-10', and C-14' confirmed
that the B'-ring in 3 was a pyridine ring and the hydroxyl group was present at C-α'. In
1
1
addition, other H- H COSY, HMBC, ROESY, and HRESIMS/MS data established
the 2D structure of 3. In the MS/MS spectrum of 3, a weak fragment ion peak at m/z
3
79 (Fig. S30, Supporting Information) corresponded to the upper half of the
molecule, which confirmed that compound 3 was a head-head bisbenzylisoquinoline
with C-8/C-7' and C-11/C-12' diphenyl ether bridges. Compound 3 was analysed by
chiral HPLC using an AD-H column, which afforded the enantiomers (+)-3a and
(‒)-3b with an ee value of approximately 34% for (+)-3a (Fig. S37, Supporting
Information). Finally, the absolute configurations of (+)-3a and (‒)-3b were
1
8

established as 1R, α'R and 1S, α'S by comparing the experimental and calculated ECD
spectra (Fig. 5). Compounds (+)-3a and (‒)-3b were assigned the names
(+)-fenfangjine L and (‒)-fenfangjine L, respectively.
Compound 4 was obtained as a white amorphous powder and possessed the same
+
molecular (C H O N ) as 3 based on the HRESIMS data (m/z 621.2593 [M+H] ,
3
7
36
7
2
calcd for C H O N , 621.2595 ). The IR spectrum revealed the presence of carbonyl
3
7
37
7
2
-1
(
1678 cm ) functionality. The UV spectrum (λ 204, 227, and 288.8 nm) was same
max
1
as those of cheratamine [22]. Comparison of the H NMR data of 4 (Table 1) and
cheratamine revealed a high degree of similarity. The difference mainly resided in the
presence of the methoxy group (δ 3.96) in 4. The HMBC correlation from OCH -12
H
3
(δH 3.96) to C-12 revealed that the methoxy group was attached at C-12. In addition,
the ROESY correlation of OCH -12 (δ 3.96) with H-13 comfirmed the location of
3
H
methoxy group (δ 3.96). The MS/MS data of 4 showed a fragment ion peak at m/z
H
3
80 (Fig. S42, Supporting Information), corresponding to the upper half of the
molecule. The positive optical rotation value and ECD cotton effects of 4 were similar
to those of cheratamine [22], which indicated the absolute configuration of 4 was 1'S.
Thus, compound 4 was elucidated as fenfangjine M.
Compound 5, white amorphous powder, had a molecular formula of C H O N
2
3
7
40
7
+
based on the HRESIMS ion at m/z 625.2902 [M+H] (calcd for C H O N ,
3
7
41
7
2
6
25.2908), indicating 19 degrees of unsaturation. The IR spectrum displayed
-1
-1
absorption bands for hydroxy group (3396 cm ) and carbonyl (1674 cm )
functionalities. Analysis of the 1H NMR data (Table 1) of 5 showed a
1
9

1
8
,3,4-trisubstituted benzene ring at δ 6.68 (1H, d, J = 1.8 Hz, H-10), 6.87 (1H, d, J =
H
.4 Hz, H-13), 6.84 (1H, dd, J = 8.4, 1.8 Hz, H-14), a para-disubstituted benzene ring
at δ 6.33 (1H, dd, J = 8.4, 2.4 Hz, H-10'), 6.73 (1H, dd, J = 8.4, 2.4 Hz, H-11'), 7.14
H
(1H, dd, J = 8.4, 2.4 Hz, H-13'), 7.20 (1H, dd, J = 8.4, 2.4 Hz, H-14'), three aromatic
singlet protons at δ 5.65 (1H, s, H-5), 6.45 (1H, s, H-5'), 5.63 (1H, s, H-8'), two
H
methines at δ 3.96 (1H, br d, J = 10.2 Hz, H-1), 3.66 (1H, dd, J = 11.4, 4.2 Hz, H-1'),
H
three singlet methoxy groups (δ 3.48, 3.60, and 3.93, each 3H), and two N-CH
H
3
groups (δ 2.15 and 2.58, s, each 3H), which were closely related to those of
H
fangchinoline (12) [14], except for the significantly upfield shift of H-5 (∆δ 0.64).
1
3
The C NMR data (Table 2) together with HSQC data showed the presence of a
carbonyl carbon (δc 175.3, C-7) and an oxygenated quaternary carbon (δc 67.7, C-4a).
The HMBC correlations from H-5 to C-6, C-7, C-8a, and C-4, from H-1 to C-4a, C-8,
and C-8a, and from H-3 to C-4a confirmed the carbonyl and hydroxy group were
1
1
located at C-7 and C-4a, respectively. Further analysis of H- H COSY, HMBC (Fig.
2
), and ROESY data, the 2D structure of 5 was established as shown. Additionally,
the MS/MS spectrum exhibited a weak ion peak at m/z 398 (Fig. S53, Supporting
Information), which corresponded to the upper half of compound 5. The absolute
configuration of 5 was elucidated as 1S, 4aR, 1'S based on the experimental and
calculated ECD spectra (Fig. 5). Therefore, compound 5 was named fenfangjine N.
2
0

Calculated ECD of (1S, 4aS, 1'S)-5a
Calculated ECD of (1S, 4aR, 1'S)-5b
Calculated ECD of (1R, 4aS, 1'S)-5c
Calculated ECD of (1R, 4aR, 1'S)-5d
Experimental ECD of 5
Calculated ECD of (1S, α'S)-3
Calculated ECD of (1R, α'R)-3
Calculated ECD of (1S, α'R)-3
Calculated ECD of (1R, α'S)-3
Experimental ECD of (-)-3b
Experimental ECD of (+)-3a
Fig. 5. Calculated and Experimental ECD spectra of compounds 3 and 5.
Compound 6 was found to possess a molecular formula of C H O N as
3
7
40
8
2
+
deduced from the HRESIMS ion at m/z 641.2858 [M+H] (calcd for C H O N ,
3
7
41
8
2
6
41.2857). From comparing the 1D NMR data (Tables 3 and 4) of 6 with those of 5,
the most significant differences resided in the 2'-N-CH (δ 3.34, δc 56.5), a methine
3
H
(
δ 4.38, H-1'; δc 77.9, C-1'), and a methylene (δ 3.96, 3.67, H-3'; δc 59.4, C-3'),
H
H
which were more downfield than those of 5 (respectively at 2,58, 42.6; 3.66, 64.3; and
.29, 2.81, 45.5). These data indicated compound 6 was a 2'-N-oxide of 5. The HMBC
correlations from 2'-N-CH (δ 3.34) to C-1' and C-3', from H-3' to C-1', C-4a', and
3
3
H
2
'-N-CH (δc 56.5), and from H-8' to C-1' confirmed the 2'-N-CH , methine and
3 3
methylene were located at N-2', C-1' and C-3' positions, respectively. The 2'-N-CH of
3
fangchinoline 2'-N-α-oxide (fenfangjine B) [8] and fangchinoline 2'-N-β-oxide
(
fenfangjine C) [8] showed at δ 3.34 and 2.94, respectively, which indicated that
H
2
'-N-oxygen of 6 was α-oriented. In the MS/MS data of 6 (Fig. S64, Supporting
+
Information), an ion peak at m/z 624 [M ‒ 16] was observed due to the loss of
oxygen and a weak ion peak at m/z 398 corresponded to the upper half of molecule
that lost oxygen. The absolute configuration of 6 was assigned as 1S, 4aR, 1'S which
2
1

is the same as that of 5, based on the same ECD curve to those of 5 (Fig. S50 and S61
2
0
Supporting Information) and positive optical rotation value ([α]
D
+244 (c 0.44,
CH Cl)). Hence, the structure of 6 was established as fenfangjine O.
3
Compound 7 was obtained as a white amorphous powder and its molecular
formula was established as C H O N from its HRESIMS peak at m/z 625.2907
3
7
40
7
2
+
1
13
[
M+H] (calcd for C H O N , 625.2908). The H and C NMR data (Tables 3 and
37 41 7 2
4
) of 7 resembled those of fangchinoline (12) [13,14], except for a 2-N-CH3,
methylene of C-3, and methine of C-1 signals. The 2-N-CH of fangchinoline (12)
3
showed at δ 2.33, while that of 7 shifted to lower filed and displayed at δ 2.93. The
H
H
chemical shifts of C-1, 2-N-CH , and C-3 were also downfield shifted to δc 74.9,
3
5
9.8, and 57.1, respectively, comparing to the corresponding signals of fangchinoline
(12) (δc 61.8, 42.6 and 45.3 ). These features suggested that 7 was a 2-N-oxide of
fangchinoline. The 2-N-CH of tetrandrine 2-N-β-oxide (fenfangjine A) [8],
3
tetrandrine 2-N-β, 2'-N-β-dioxide [16], and tetrandrine 2-N-β, 2'-N-α-dioxide [16]
displayed at δH 3.08, 3.06, and 3.06, respectively, indicating 7 was a
fangchinoline-2β-N-oxide. In addition, the ROESY correlation of 2-N-CH with H-14
3
confirmed that the 2-N-oxygen was β-oriented (Fig. 3). In the MS/MS data of 7 (Fig.
S75, Supporting Information), an ion peak at m/z 381 was observed because of the
upper half of molecule losing one oxygen and proton. The positive optical rotation
2
D
0
value of 7 ([α]
+222 (c 1.0, CH Cl)) and its similar ECD spectrum to that of
3
fangchinoline (12) (Fig. S72 and S115, Supporting Information) indicated that 7
possessed the same 1S, 1'S configurations as fangchinoline (12). Finally, the absolute
2
2

configuration of 7 was confirmed by the reduction (Fig. 6) of 7 with zinc powder and
HCl yielding fangchinoline (12) whose absolute configuration was confirmed by
single-crystal X-ray diffraction analysis [Flack parameter 0.10 (15)] (Fig. 4).
Therefore, compound 7 was elucidated as fangchinoline-2β-N-oxide and named
fenfangjine P.
O
O
O
O
O
N
H
N
N
H
N
Zn, 10%HCl
2h, rt
OH
OH
H
H
O
O
O
O
O
O
7
12
Fig. 6. Reduction of compound 7.
Compound 8 was isolated as a white amorphous powder and assigned a
molecular formula of C H O N based on the HRESIMS peak at m/z 641.2858
3
7
40
8
2
+
[
M+H] (calcd for C H O N , 641.2857). The 1D NMR features (Tables 3 and 4) of
37 41 8 2
8
8
were similar to those of 7, except that the 2'-N-CH signal was observed at δ 2.87 in
3 H
. This implied compound 8 was a 2-N, 2'-N-dioxide of fangchinoline. The ROESY
correlation of 2'-N-CH with H-1' revealed that the 2'-N-CH was on the same side as
3
3
the H-1'. The MS/MS data of compound 8 displaying a fragment ion peak at m/z 414
Fig. S86, Supporting Information) corresponded to the upper half of the molecule.
The absolute configurations of C-1 and C-1' were assigned as 1S, 1'S based on the
(
2
0
positive optical rotation value of 8 ([α]
D
+298 (c 0.57, CH Cl)) and its similar ECD
3
spectrum (Fig. S83 and S72, Supporting Information) to that of 7. In addition, the
reduction of 8 with zinc powder and HCl afforded fangchinoline (12). Thus, the
2
3

structure of 8 was confirmed as fangchinoline-2β-N, 2'β-N-dioxide and named
fenfangjine Q.
Compound 9, a white amorphous powder, had the same molecular formula,
+
C H O N , as 8 according to the HRESIMS peak at m/z 641.2856 [M+H] (calcd for
3
7
40
8
2
1
13
C H O N , 641.2857 ). The H and C NMR resonances (Tables 3 and 4) of 9 were
3
7
41
8
2
very close to those of 8. The obvious difference involved the chemical shift of
1
2
'-N-CH , implying the oxygen orientation was different at the 2'-N position. In the H
3
NMR spectrum, the 2'-N-CH signal of 8 showed at δ 2.88, while that of 9 was
3
H
downfield shifted by 0.62 ppm and showed at δ 3.49, which suggested compound 9
H
was a fangchinoline-2β-N, 2'α-N-dioxide [8]. The MS/MS data of compound 9
exhibited an ion peak at m/z 398 (Fig. S97, Supporting Information) due to the upper
half of the molecule losing one oxygen.The ECD spectra of 8 and 9 (Fig. S83 and
S94, Supporting Information) were very similar, and the reduction of 9 with zinc
powder and HCl yielded fangchinoline (12), which indicated compound 9 also
possessed the 1S,1'S configurations. Therefore, the structure of fangchinoline-2β-N,
2
'α-N-dioxide (9) was deduced as shown and named fenfangjine R.
Compound 10 was obtained as a white amorphous powder and was determined
to have same molecular formula, C H O N , as 7 from the HRESIMS data (m/z
3
7
40
7
2
+
6
25.2910 [M+H] , calcd for C H O N , 625.2908). The NMR data (Tables 3 and 4)
37 41 7 2
of 10 were similar to those of 7, except for the chemical shift of one methoxy group.
The location of methoxy group (δ 3.12) was confirmed by the HMBC correlation of
H
OCH -7 (δ 3.12) with C-7. In the MS/MS spectrum, a weak fragment ion peak at m/z
3
H
2
4

3
96 (Fig. S108, Supporting Information) was observed, indicating the upper half of
compound 10 lost the oxygen. In the ROESY spectrum of 10, the correlations of
-N-CH (δ 3.01) with H-14 confirmed the 2-N-oxygen was β-oriented. The positive
2
3
H
2
0
optical rotation value of 10 ([α] +268 (c 0.1, CHCl )) and its similar ECD curve to
D
3
that of 7 (Fig. S105 and S72, Supporting Information) indicated 10 possessed the
same 1S,1'S configurations as 7. Consequently, the structure of 10 was established and
named fenfangjine S.
The anti-inflammatory activities of compounds 1-18 were tested by inhibiting
against NO generation induced by LPS in murine peritoneal macrophage cells.
Dexamethasone (DXM) was used as a positive control. Compounds 1-4 and 14-16
inhibited NO production with 51.94 to 73.31% inhibition at the concentration of 10
μM (Table 5). Other compounds inhibited NO production with inhibitory rates of <
5
0%. Compounds 1, 15, and 16 showed the better anti-inflammatory activities with
IC values of 15.26 ± 2.99, 6.12 ± 0.25, and 5.92 ± 1.89 μM, respectively. Based on a
5
0
preliminary structure-activity relationship analysis, the OCH -7 and OCH -12 might
3
3
be the activating groups. Compounds (1-18) were also evaluated for their cytotoxic
activities against for five human cancer cells (A549, CMF-7, HCT-116, HepG2, and
Capan 2). Only compound 18 exhibited cytotoxic activities against MCF-7, HCT-116,
and HepG2 cell lines with IC values of 2.81 ± 0.06, 3.66 ± 0.26, and 2.85 ± 0.15
5
0
μM, respectively. Other compounds were inactive (IC > 10 μM). (positive control
5
0
taxol, IC < 0.01 μM).
5
0
2
5

In summary, a total of 18 bisbenzylisoquinoline alkaloids, including ten new
bisbenzylisoquinoline alkaloids (1-10) and eight known compounds (11-18), were
obtained from the roots of Stephania tetrandra. The absolute configurations of
compounds 1, 2, and 12 were unambiguously determined by X-ray crystallography.
The reductions of compounds 7-9 only yielded compound 12, which revealed the
absolute configurations of C-1 and C-1' of 7-9 were same as the those of 12.
Compound 3, constituted from a pair of bisbenzylisoquinoline enantiomers, was a
partial racemate and was discovered by chiral HPLC. This case indicated that it is
necessary to increase attention to the partial racemates from natural products, which
are inadvertently regarded as optically pure chemical entities. Among these isolated
compounds, compounds 1-4 and 14-16 showed anti-inflammatory activities and
compound 18 possessed cytotoxicity.
Notes
The authors declare no competing financial interests.
Acknowledgments
This work was financially supported by Chinese Academy of Medical Sciences
(CAMS) Initiative for Innovative Medicine [CAMS-12M-1-010], the State Key
Laboratory of Bioactive Substance and Function of Natural Medicines [No.
GTZA201803], and the Drug Innovation Major Project [2018ZX09711001-008-009].
Appendix A. Supplementary material
2
6

The material (1D and 2D NMR, UV, IR, CD, and HRESIMS/MS data for
compounds 1-10) is provided in the Supporting Information.
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1
Table 1 H NMR Data for Compounds 1-5 in CDCl
3
position 1a
2b
3a
4a
5a
1
3.75 brd (9.0)
3.74 dd (11.2, 2.4) 4.11 brs
3.96 brd (10.2)
2.15 s
2
-N-CH3 2.32 s
2.26 s
2.50 s
3
3.47 m
.89 m
2.90 m
.41 m
6.35 s
3.55 m
2.90 brs
3.92 m
3.62 m
2.63 m
3.45 m
2
2.59-2.67 overlap
2.98 m
2.41 brs
2.52-2.58 overlap
1.90 m
4
2.32 brs
2
2.59-2.67 overlap
6.74 s
1.72 brs
5
6.47 s
6.44 s
5.65 s
α
2.68 dd (13.8, 10.2) 2.76-2.87 overlap
.61 brd (13.8)
2.73 dd (13.8, 3.6)
2.06 brs
3.18 dd (14.4, 10.2)
2.52-2.58 overlap
6.68 d (1.8)
2
1
0
3
4
5
6.47 d (1.8)
6.28 d (1.6)
6.69-6.74 overlap
6.69-6.74 overlap
6.75 dd (7.8, 1.8)
6.93 d (1.8)
1
1
1
6.86 d (8.4)
6.88 d (8.4)
6.92 d (8.4)
6.87 d (8.4)
6.89 dd (8.4, 1.8)
6.95 dd (8.4, 1.6)
5.10 d (9.2)
7.46 dd (8.4, 1.2)
6.84 dd (8.4, 1.8)
4
.73 d (9.2)
1
'
4.74 dd (7.2, 5.4)
3.82 overlap
2.62 s
3.67 dd (9.6, 6.0)
2.46 s
3.66 dd (11.4, 4.2)
2.58 s
2
'-N-CH3 3.29 s
3
'
3.06 m
8.37 d (5.4)
7.50 d (5.4)
3.38 m
3.29 m
2
.54 m
2.77-2.81 overlap 2.81 m
2.87 m 2.90 m
4
'
2.76-2.87 overlap
2
9