Cytotoxic lignans from fruits of Cleistanthus tonkinensis

Article abstract of DOI:10.1016/j.fitote.2019.104432

Seven new lignans, cleistonkinins A– E (1–5), cleistonkisides A and B (6–7) were isolated from the fruits of Cleistanthus tonkinensis (Euphorbiaceae), together with five known aryltetralin lignans, cleisindoside B (8), cleistantoxin (9), cleisindoside D (10), neocleistantoxin (11) and polygamain (12). Their structures were established from spectral analysis, including mass spectrometry and 2D-NMR. The absolute configurations of 4–7 were determined by analysis of their experimental CD spectra and comparison with calculated electronic circular dichroism (ECD) spectra. Compounds 2 and 6 had selective inhibition with moderate cytotoxicity against Pan C1 and A549 cell lines, respectively. Cleistantoxin (9) was significantly active against A549, HeLa, Hep3B, Pan C1 and MCF7 cell lines while it was less cytotoxic against HeLa cells. Neocleistantoxin (11) exhibited remarkable inhibition toward A549, HeLa, MCF7 and Pan C1. This is the first report for cytotoxicity of 9 and 11 against A549, Hep3B and Pan C1 cell lines.

Full text of DOI:10.1016/j.fitote.2019.104432

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Cytotoxic lignans from fruits of Cleistanthus tonkinensis
Lam Hong Nguyen, Van Nam Vu, Dao Phi Thi, Viet Hung Tran,
Marc Litaudon, Fanny Roussi, Van Hung Nguyen, Van Minh
Chau, Huong Doan Thi Mai, Van Cuong Pham
PII:
S0367-326X(19)32027-1
DOI:
https://doi.org/10.1016/j.fitote.2019.104432
FITOTE 104432
Reference:
To appear in:
Fitoterapia
Received date:
Revised date:
Accepted date:
9 October 2019
19 November 2019
19 November 2019
Please cite this article as: L.H. Nguyen, V.N. Vu, D.P. Thi, et al., Cytotoxic lignans
from fruits of Cleistanthus tonkinensis, Fitoterapia (2018), https://doi.org/10.1016/
j.fitote.2019.104432
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© 2018 Published by Elsevier.

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Cytotoxic Lignans from Fruits of Cleistanthus
tonkinensis
Lam Hong Nguyena,b, Van Nam Vua, Dao Phi Thia, Viet Hung Tranc, Marc Litaudond, Fanny
Roussid, Van Hung Nguyena, Van Minh Chaua, Huong Doan Thi Maia,e,* huongdm@imbc.vast.vn
and Van Cuong Phama,e,* phamvc@imbc.vast.vn
aAdvanced Center for Bioorganic Chemistry, Institute of Marine Biochemistry of the Vietnam
Academy of Science and Technology (VAST), 18 Hoang Quoc Viet, Caugiay, Hanoi, Vietnam.
bHanoi University of Pharmacy, 13 Le Thanh Tong, Hoankiem, Hanoi, Vietnam
cInstitute of Drug Quality Control – Ho Chi Minh City , 200 Co Bac Street, Co Giang Ward,
District 1, Ho Chi Minh City, Vietnam
dInstitut de Chimie des Substances Naturelles, CNRS-ICSN, UPR 2301, Univ. Paris-Sud, 91198,
Gif-sur-Yvette, France.
eGraduate University of Science and Technology, VAST, 18 Hoang Quoc Viet, Caugiay, Hanoi,
Vietnam.
*Corresponding authors.
ABSTRACT
Seven new lignans, cleistonkinins A– E (1–5), cleistonkisides A and B (6–7) were isolated
from the fruits of Cleistanthus tonkinensis (Euphorbiaceae), together with five known aryltetralin
lignans, cleisindoside B (8), cleistantoxin (9), cleisindoside D (10), neocleistantoxin (11) and
polygamain (12). Their structures were established from spectral analysis, including mass

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spectrometry and 2D-NMR. The absolute configurations of 4–7 were determined by analysis of
their experimental CD spectra and comparison with calculated electronic circular dichroism
(ECD) spectra. Compounds 2 and 6 had selective inhibition with moderate cytotoxicity against
Pan C1 and A549 cell lines, respectively. Cleistantoxin (9) was significantly active against A549,
HeLa, Hep3B, Pan C1 and MCF7 cell lines while it was less cytotoxic against HeLa cells.
Neocleistantoxin (11) exhibited remarkable inhibition toward A549, HeLa, MCF7 and Pan C1.
This is the first report for cytotoxicity of 9 and 11 against A549, Hep3B and Pan C1 cell lines.
1. Introduction
Cleistanthus genus belongs to the Euphobiaceae family and is distributed in tropical region of
Africa, Asia and Australia [1]. An overview of literature revealed that so far, only eight species
(C. collinus, C. patulus, C. sumafranus, C. sumatranus, C. boivinianus, C. indochinensis, C.
gracilis and C. schlechteri) of the 140 species of Cleistanthus were examined for their chemical
contents [1,2]. Lignans and terpenoids have been found to be major bioactive metabolites of
plants of the Cleistanthus genus [3‒8]. Previously, we reported the isolation and structural
characterization of cleistanone, an unprecedented triterpenoid skeleton from the leaves [9], and a
number of lignans from the fruits of C. indochinensis [10,11]. In the course of a screening
program of the flora in Vietnam, the fruit extract of C. tonkinensis showed significant
cytotoxicity against KB cell line (88.4% inhibition at the concentration of 1 μg/mL). This plant is
used in traditional medicine to treat stomach pain, rheumatism and irregular menstruation [12].
Herein, we describe the isolation and structural elucidation of seven new lignans (1‒7) and five
known compounds, 8–12 from the fruits of C. tonkinensis.
2. Experimental section
2.1. General experimental procedures
Optical rotations were recorded on an Atago Polax-2L polarimeter, using a sodium (589, D
line) lamp. IR spectra were measured on a Nicolet Impact-410 FT-IR spectrometer and CD
spectra on a Chirascan CD spectrometer. NMR spectra were recorded on a Bruker AM500 FT-
1
NMR spectrometer operating at 500.13 MHz and 125.76 MHz for H NMR and 13C NMR
1
spectra, respectively. H NMR chemical shifts were referenced to 7.27 (CDCl3) and 3.31 ppm

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(CD3OD), respectively. Whereas, 13C NMR chemical shifts to the central peak of CDCl3 at 77.0
and CD3OD at 49.0 ppm. The HMBC measurements were optimized to 7.0 Hz long-range
couplings, and NOESY experiments were run with 150 ms mixing time. HR-ESIMS were
recorded on an Agilent 6530 Accurate-Mass Q-TOF LC/MS system.
2.2. Plant material
The fruits of C. tonkinensis was collected in Dai Tu, Thai Nguyen, Vietnam in June 2017 and
identified by Dr. Nguyen The Cuong. A Voucher specimen (VN 2345B) was deposited at the
Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology
(VAST) in Hanoi.
2.3. Extraction and isolation
Dried and ground fruits of C. tonkinensis (5 kg) were extracted with EtOH (5 × 12 L, 18 h
each) at room temperature. The extracts were combined and concentrated under diminished
pressure. The residue was suspended in water (350 mL) and extracted with n-hexane (3 × 500
mL). The aqueous solution was freeze-dried to afford a residue of 168 g, which was subjected to
column chromatography (CC) on silica gel eluting with CH2Cl2/MeOH (from 0% to 100%
MeOH in CH2Cl2) to yield 23 fractions. Fraction 1 (469 mg) was purified by CC on silica gel,
eluted with 0 to 100% EtOAc in n-hexane affording 12 (10.1 mg). Fraction 7 (6.53 g) was
chromatographed on silica gel column (0 to 100% acetone in n-hexane) giving 10 subfractions.
Subfraction 8 (4.1 g) was recrystallized in a mixture of n-hexane/CH2Cl2 (3/7, v/v) yielded 9
(93.3 mg). Fraction 8 (4.2 g) was separated by CC on silica gel (0 to 100% acetone in n-hexane)
to yield 5 subfractions. Subfraction 3 (1.9 g) was subjected to CC on Sephadex LH-20
(CH2Cl2/MeOH: 1/9), to provide 9 (2.0 g) and 4 (251.1 mg). Fraction 11 (8.64 g) was
chromatographed on a silica gel CC (0 to 100% MeOH in CH2Cl2), giving 7 subfractions.
Subfraction 3 (1.09 g) was purified by CC on silica gel (0 to 60% of acetone in CH2Cl2),
followed by CC on Sephadex LH-20 (MeOH) to afford 5 (65.0 mg) and 11 (5.5 mg). Fraction 16
(6.5 g) was separated by CC on silica gel (0 to 100% MeOH in CH2Cl2), followed by CC on
Sephadex LH-20 (CH2Cl2/MeOH: 1/9), to provide 10 (3.3 g). Fraction 17 (26.2 g) was subjected
to a CC on silica gel (0% to 100% MeOH in CH2Cl2) giving 10 subfractions. Subfraction 4 (216
mg) was purified by CC on Sephadex LH-20 (MeOH/CH2Cl2: 9/1), followed by preparative TLC
(CH2Cl2/MeOH: 90/10) affording 6 (18.4 mg). Fraction 18 (19.9 g) was separated by CC on

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silica gel (0 to 100% MeOH in CH2Cl2), followed by CC on Sephadex LH-20 (CH2Cl2/MeOH:
1/9), to furnish 3 (8.2 mg). Fraction 20 (13.8 g) was purified by CC on silica gel (0 to 100%
acetone in n-hexane) to yield 7 subfractions. Subfraction 2 (292 mg) was chromatographed on
Sephadex LH-20 CC (MeOH), followed by preparative TLC (CH2Cl2/MeOH: 9.5/0.5) giving 1
(1.0 mg) and 2 (1.2 mg). Subfraction 5 (230 mg) was subjected to a CC on silica gel (0 to 90%
MeOH in CH2Cl2) to provide 7 (10.8 mg). Finally, subfraction 6 (640 mg) was purified by
Sephadex LH-20 CC (MeOH) affording 8 (60.7 mg).
o
Cleistonkinin A (1): white powder; mp 131‒134 C; IR (KBr) max 3341, 2901, 1502, 1495,
-1
1465, 1231, 1102, 1033, 933 cm ; UV (MeOH): max nm (log) 204 (4.78), 285 (3.70), 309
(3.57); NMR data see Table 1; HRESIMS (negative mode) m/z 321.0753 [M - H]- (calcd for
C19H13O5, 321.0763).
o
Cleistonkinin B (2): white powder; mp 154‒156 C; IR (KBr) max 3341, 2901, 1502, 1495,
-1
1465, 1231, 1102, 1033, 932 cm ; UV (MeOH): max nm (log) 204 (4.78), 285 (3.70), 309
(3.57); NMR data see Table 1; HRESIMS (negative mode) m/z 351.0887 [M - H]- (calcd for
C20H15O6, 351.0869).
o
Cleistonkinin C (3): white microcrystalline; mp 328‒329 C; IR (KBr) max 2920, 1759, 1616,
1531, 1489, 1446, 1398, 1336, 1257, 1228, 1197, 1082, 1037, 1010, 927, 873 cm-1; UV (MeOH):
max nm (log) 204 (4.30), 230 (4.22), 257 (4.07), 332 (3.67), 344 (3.62); NMR data see Table 2;
HRESIMS (positive ion mode) m/z 401.0629 [M + Na]+ (calcd for C21H14NaO7, 401.0637).
o
30
Cleistonkinin D (4): white microcrystalline; mp 328‒330 C; [α]D +88.1 (c 0.042, CHCl3); CD
(MeOH) max (Δ) 226 (-16.5), 257 (+0.5), 278 (-6.6), 346 (+15.9) nm; IR (KBr) max 3547,
2912, 1745, 1656, 1602, 1489, 1440, 1396, 1346, 1240, 1195, 1114, 1051, 1004, 929, 854, 821
-1
cm ; UV (MeOH) max nm (log) 204 (4.30), 230 (4.22), 257 (4.07), 332 (3.67), 344 (3.62);
NMR data see Table 2; HRESIMS (positive mode) m/z 419.0749 [M + Na]+ (calcd for
C21H16NaO8, 419.0743).
o
30
Cleistonkinin E (5): white microcrystalline; mp 270‒271 C; [α]D +234.6 (c 0.108, CHCl3); CD
(MeOH) max (Δ) 203 (-20.1), 225 (-5.5), 239 (-6.3), 261 (+1.9), 278 (-3.2), 357 (+4.3) nm; IR
(KBr) max 3466, 2899, 1726, 1629, 1589, 1475, 1446, 1338, 1269, 1238, 1188, 1099, 1029,
-1
1012, 927, 885 cm ; UV (MeOH) max nm (log) 204 (4.30), 230 (4.22), 257 (4.07), 332 (3.67),

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344 (3.62); NMR data see Table 2; HRESIMS (positive mode) m/z 405.0583 [M + Na]+ (calcd
for C20H14NaO8, 405.0586).
o
30
Cleistonkiside A (6): white microcrystalline; mp 174‒176 C; [α]D -218.18 (c 0.44, CHCl3);
CD (MeOH) max (Δ) 201 (-39.4), 216 (-19.7), 236 (-5.0), 251 (+0.6), 279 (-3.5), 295 (+2.9)
nm; IR (KBr), max 3523, 2900, 1747, 1618, 1504, 1475, 1440, 1338, 1232, 1070, 1033, 997, 925
-1
cm ; UV (MeOH) max nm (log): 204 (4.30), 230 (4.22), 257 (4.07), 332 (3.67), 344 (3.62);
NMR data see Table 3; HRESIMS (negative mode) m/z 659.1601 [M - H]- (calcd for C31H31O16,
659.1612).
o
30
Cleistonkiside B (7): white microcrystalline; mp 184‒185 C; [α]D -101.2 (c 0.083, MeOH);
CD (MeOH) max (Δ) 204 (-13.7), 217 (+0.2), 234 (-3.0), 252 (+0.6), 285 (-1.0), 306 (+0.02)
nm; IR (KBr), max 3329, 2895, 1755, 1643, 1490, 1440, 1382, 1309, 1242, 1165, 1139, 1070,
-1
1012, 933, 869, 806 cm ; UV (MeOH) max nm (log) 204 (4.30), 230 (4.22), 257 (4.07), 332
(3.67), 344 (3.62); NMR data see Table 4; HRESIMS (negative mode) m/z 663.1930 [M - H]-
(calcd for C31H35O16, 663.1925).
2.4. Cytotoxic activity assay
Hep3B (Human hepatoma), Hela (human cervical carcinoma), MCF7 (human breast cancer),
Pan C1 (pancreas epithelioid acarcinoma) and A549 (human lung adenocarcinoma) were kindly
provided by Prof. Jeong-Hyung Lee, Department of Biochemistry, College of Natural Sciences,
o
Kangwon National University, Korea. The cells were cultured at 37 C in RMPI1640 medium
supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL
streptomycin in a 5% CO2 incubator. Cells between 5 and 20 passages were used for the assays.
The cytotoxic activity was measured by using a modified MTT assay [13]. Viable cells were
seeded in 96-well plates at a density of 1.0×105 cells/mL. Cells were treated with various
concentrations of essential oils (0.3 1, 3, 10, and 30 μg/mL) and then incubated at 37oC for 24 h
in fresh RMPI1640 medium. Cells were subsequently incubated at 37 °C with MTT (0.5 mg/mL)
for 4 h. After removal of supernatant, formazan crystals were dissolved in isopropanol and the
optical density was measured at 570 nm. Camptothecin (Andrich, purity >95%) was used as a
positive control.
2.5. TD-DFT electronic CD (ECD) calculation of 4–7

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A conformational search was carried out with Spartan’14 software (Wavefunction Inc.,
Irvine, CA) for the proposed absolute configuration as shown at the molecular mechanics level
(MMFF) [14]. All MMFF minima were re-optimized using DFT calculations at the B3LYP/6-
31G(d) level with the Gaussian 09 program. The geometry was optimized starting from various
initial conformations with vibrational frequency calculations confirming the presence of minima.
The DFT calculations (B3LYP/LanL2DZ) were performed on the lowest-energy conformations
using 30 excited states and a polarizable continuum model (PCM) in methanol. The ECD spectra
of the conformers were combined using Boltzmann weighting with program SpecDis 1.71 [15].
After applying a UV-shift correction, the computed CD spectra were compared with the CD
curves experimentally obtained.
2.6. Acid Hydrolysis of 6 and 7
Each solution of 6 or 7 (each 5.0 mg) in 1N HCl (dioxane/H2O: 1/1, 2.5 mL) was heated at
o
95 C for 5 h. The filtrates from hydrolysate were neutralized with DOWEX HCR-S ion-
exchange resin and filtered. The filtrates were concentrated under reduced pressure and
examined for carbohydrates by silica gel TLC [Kieselgel 60 (Merck Art 5554), i-
PrOH/Me2CO/H2O (5/3/1)] comparing with authentic samples. The Rf values of each sugar were
as follows: glucose, 0.43; and apiose, 0.54. The remaining residue was purified by preparative
30
30
TLC. The sugars were identified as D-glucose {[α]D +53 (c 0.18, H2O)}, and D-apiose {[α]D
+16 (c 0.12, H2O)}.
3. Results and discussion
Dried and ground fruits of C. tonkinensis (5 kg) were extracted with EtOH at room
temperature. The solvent was removed under reduced pressure, and the residue was suspended in
water and extracted with n-hexane. The water layer was freeze-dried and the residue (168 g) was
purified by repeated open column chromatography to give compounds 1‒12 (Figure 1).

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Fig. 1. Structure of compounds 1‒12.
Compound 1 was isolated as white powder. Its negative HRESIMS showed the deprotonated
molecular ion [M - H]- at m/z 321.0753 which together with 13C NMR data are consistent with
1
the molecular formula of C19H14O5. The H NMR spectra of 1 showed the resonances of an ABX
aromatic ring system at H 6.92 (H-2′), 6.91 (H-5′) and 6.89 (H-6′), two singlet aromatic protons
at H 7.19 (H-3) and 7.16 (H-6), and two ortho-coupled spin system at H 7.25 (H-8′) and 7.64
(H-7) in the aromatic region (Table 1). Furthermore, three broad singlets, each of which
integrates to two protons was also observed at H 4.82 (CH2-9), 6.04 (CH2-10) and 6.02 (CH2-9′).
13C NMR data analysis with the aid of HSQC experiment revealed the resonances of 19 carbons,
including two methylenedioxy groups, seven aromatic methines, one oxygenated methylene
group and nine sp2 quaternary carbons (Table 2). Analyses of 2D NMR spectra of 1, especially
HMBC spectrum indicated a naphthalene system in the structure of 1 (Figure 2). The location of
the oxygenated methylene at C-8 was established by correlation of protons at H 4.82 (CH2-9)
with carbons at C 124.5 (C-7), C-8 (C 136.4) and C-8′ (C 124.9). Similarly, the ABX ring was

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linked to C-7′ as demonstrated by cross-peaks of C-7′ at C 139.6 with protons at C 6.92 (H-2′)
and 6.89 (H-6′). Finally, the two methylenedioxy groups at C-4/C-5 and C3′/C4′ were
determined by cross-peaks of CH2-10 protons (H 6.04) with C-4 (C 148.0) and C-5 (C 147.7),
and CH2-9′ protons (H 6.02) with C-3′ (C 147.6) and C-4′ (C 147.0). Compound 1 was reported
here for the first time and named cleistonkinin A.
Fig. 2. Selected HMBC correlations for 1.
Table 1 1H NMR data for 1‒7 (mult. J in Hz).
Cno
1*
2*
3*
4**
5**
6*
7**
2
3
6.94 (1H, s)
7.19 (1H, s)
6.93 (1H, s) 6.86 (1H, s)
6.07 (1H, 6.29 (1H, s)
s)
6.34 (1H, s)
5
6
7
8
9
6.79 (1H, d,
8.0)
7.16 (1H, s)
6.76 (1H, d,
8.0)
7.64 (1H, br. 8.06
(1H, 8.14 (1H, s)
5.12 (1H, 4.71 (1H, dd, 5.27 (1H, d, 5.21 (1H, d,
s)
d, 1.0)
d, 13.5)
6.2, 14.5)
10.0)
3.5)
3.38 (1H, 3.28 (1H, m)
m)
3.18 (1H, m)
2.52 (1H, m)
4.82 (2H, s)
4.83 (2H, s) 5.37 (2H, s)
4.12 (1H, 4.19 (1H, dd,
4.06 (1H, dd,
8.0, 9.0)
t, 9.0)
4.64 (1H, 4.62 (1H, dd,
t, 9.0) 9.0, 8.7)
(2H, 6.05 (2H, br. 5.96 (2H, 6.01 (1H, br.
8.7, 8.5)
4.61 (1H, dd,
8.0, 8.0)
10
6.04 (2H, br. 6.03
5.98 (1H, br. 5.92 (2H, br.

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s)
br. s)
s)
br. s)
s)
s)
s)
6.04 (1H, br.
s)
5.99 (1H, br.
s)
2′
5′
6′
7′
6.92 (1H, d, 6.91
1.0) br. s)
6.91 (1H, d, 6.90
(1H, 6.78 (1H, d, 6.64 (1H,
1.5) s)
6.64 (1H, s)
6.44 (1H, d,
1.0)
(1H, 6.94 (1H, d, 6.87 (1H, 6.95 (1H, d, 6.73 (1H, d, 6.66 (1H, d,
8.0)
d, 8.0)
(1H, 6.88
8.0)
d (8.0)
(1H, 6.59 (1H, 6.67 (1H, d, 6.74 (1H, d, 6.41 (1H, dd,
d, 8.0) 8.0) 8.0) 1.0, 8.0)
4.53 (1H, d, 2.24 (1H, dd,
8.0)
8.0)
8.0)
6.89
(1H, 6.76
dd, 1.5, 8.0)
br. d, 8.0)
dd, 1.5, 8.0)
4.5)
7.5, 13.5)
2.48 (1H, m)
8′
9′
7.25 (1H, d, 7.28
(1H,
(2H,
2.68 (1H, dd, 2.88 (1H, m)
1.5)
d, 1.0)
4.5, 14.5)
3.93 dd (5.0,
8.5)
6.02 (2H, br. 5.99
s)
br. s)
4.28 dd (8.5,
8.5)
10′
6.05 (1H, d, 5.95 (2H, 6.06 (1H, br.
5.98 (1H, br.
s)
5.91 (1H, br.
s)
0.5)
br. s)
s)
5.92 (1H, br.
s)
6.07 (1H, d,
0.5)
6.07 (1H, br.
s)
6.00 (1H, br.
s)
1″
4.38 (1H, d, 3.98 (1H, d,
7.5)
7.0)
2″
3″
3.39 (1H, m)
3.04 (1H, m)
3.53 (1H, dd, 3.03 (1H, m)
9.0, 9.0)
4″
3.37 (1H, dd, 3.01 (1H, m)
9.0, 9.0)
5″
6″
3.39 (1H, m)
4.28 (2H, m)
3.07 (1H, m)
3.35 (1H, m)
3.77 (1H, br.
d, 10.5)
1‴
2‴
4.90 (1H, d,
3.0)
2.87 (1H, d, 3.73 (1H, d,
15.5) 3.0)

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3.10 (1H, d,
15.5)
4‴
5‴
3.54 (1H, d,
9.0)
3.81 (1H, d,
9.0)
3.32 (2H, m)
6-
4.19 (3H, s) 4.21 (3H, s)
3.91 (3H,
OMe
7-OH
s)
5.29 (1H, 5.98 (1H d,
br. s)
6.2)
2′-OH
6.79 (1H, br.
s)
2″-
5.26 (1H, d,
4.0)
OH
3″-
4.94 (1H, d,
5.0)
OH
1‴-
4.06 (3H, s)
OMe
3‴-
4.46 (1H, s)
OH
5‴-
4.74 (1H, t,
5.5)
OH
*Recorded in CDCl3; **Recorded in DMSO-d6
Signals of 1D NMR spectra of cleistonkinin B (2) were close to those of 1 except for the
presence of a methoxy group and a sp2 quaternary carbon instead of the aromatic methine group
which suggested 2 had the same carbon skeleton as 1 with an additional methoxy group. This
observation was confirmed by analyses of 2D NMR spectra. The methoxy group at C-6 was
defined by cross-peaks of C-6 at C 139.4 with the methoxy protons at H 4.19 in the HMBC
spectrum.
Compound 3 was obtained as a white, microcrystalline solid. Its positive HRESI mass
spectrum showed a sodium adduct ion [M + Na]+ at m/z 401.0629. 1D NMR data of 3 suggested
its structural similarity to 2. However, resonance of a sp2 quaternary carbon in place of an
aromatic methine, and an additional carbonyl group at C 169.9 (C-9′) were revealed for 3.

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Analysis of 2D NMR spectra assigned the structure cleistonkinin C (3) which is an aryl
naphthalene lignan lactone. The lactone ring was defined by HMBC cross-peaks of the carbonyl
carbon C-9′ at C 169.9 with CH2-9 protons at H 5.37.
30
Compound 4 was isolated as a white microcrystalline solid and optically active [α]D +88.1 (c
0.042, CHCl3). Its positive HRESI mass spectrum exhibited the sodium adduct molecular ion [M
+ Na]+ at m/z 419.0749, suggesting the molecular formula of C21H16O8. In comparison of 1D
NMR data with 3, the resonances of two sp3 methines were observed for 4, instead of a double
bond in the structure of 3. This was confirmed by correlations of a spin-spin coupling system H-
7 (H 5.12)/H-8 (H 3.38)/CH2-9 (H 4.12 and 4.64) in the COSY spectrum of 4. The chemical
shifts of CH-7 suggested its linkage to oxygen (Tables 1 and 2). The relative configuration of 4
was established by proton coupling constant analysis of H-7 which had anti-coupling constant (J
= 13.5 Hz) indicating a trans-pseudodiaxial relationship between H-7 and H-8. In order to
resolve the absolute configuration of 4, its circular dichroism (CD) spectrum was compared with
calculated electronic circular dichroism (ECD) spectra of the two possible enantiomers, (7S,8R)-
4 and (7R,8S)-4 [16,17]. The ECD quantum chemical calculations were performed using the
Gaussian 09 software [18]. To obtain minimum energy conformers, geometry optimization of
each possible enantiomer of 4 was conducted. The calculated ECD spectra of (7S,8R)-4 and
(7R,8S)-4 were generated using the time-dependent density functional method at the
B3LYP/LanL2DZ level. The calculated ECD spectrum of (7S,8R)-4 displayed Cotton effects in
good agreement with those of the experimental CD spectrum of 4 (Figure 3). The S- and R-
configurations were thus assigned for the chiral carbons C-7 and C-8 of cleistonkinin D (4),
respectively.

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Fig. 3. Calculated ECD and experimental CD spectra of 4.
1H and 13C NMR spectra of 5 exhibited characteristic features of an aryl-dihydronaphthalene
1
lactone type as 4. The H NMR spectrum of 5 presented two singlet protons at H 6.29 (H-3) and
7.14 (H-6), and two ortho-coupled spin systems at H 6.95 (d, J = 8.0 Hz, H-5′) and 6.67 (d, J =
8.0 Hz, H-6′) in the aromatic region which were consistent with the presence of a 1,2,4,5-
tetrasubstitued and a 1,2,3,4-tetrasubstitued benzene rings. The spin-spin coupling system H-7
(H 4.71)/H-8 (H 3.28)/CH2-9 (H 4.19 and 4.62) was observed in the COSY spectrum of 5 as in
the structure of 4. The chemical shifts of C-4, C-5, C-2′, C-3′, C-4′, CH-7 and CH2-9 (Tables 1
and 2) suggested their connection to oxygen. Intensive analysis of 2D NMR data confirmed the
structure of 5. The relative configuration of 5 was similar to that of 4 as indicated by anti-
coupling constant (J = 14.5 Hz) between H-7 and H-8. The absolute configurations of the 5 was
established by comparison of its experimental and calculated electronic circular dichroism
(ECD) spectra (Figure 4). The ECD spectrum of (7S,8R)-5 was in agreement with the
experimental CD spectrum of 5 indicating 7S- and 8R-configurations for cleistonkinin E (5).
Table 2 13C NMR data for 1‒7.
Cno
1*
2*
3*
4**
5**
6*
7**
1
131.1
128.4
102.4
125.4
128.7
97.1
129.0
130.7
98.4
126.3
130.4
104.7
137.3
128.9
108.3
121.5
135.9
105.4
132.1
107.1
147.2
2
3

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4
148.0
147.7
104.2
124.5
136.4
65.5
149.0
133.5
139.4
119.0
134.9
65.8
149.6
136.1
135.7
113.9
139.1
68.3
148.5
139.9
142.5
73.4
149.1
145.4
105.1
71.7
150.4
137.0
141.9
76.4
146.6
107.9
120.2
75.6
5
6
7
8
43.1
42.5
37.6
51.7
9
70.8
70.0
71.9
176.4
101.0
132.2
108.7
147.1
145.5
107.9
121.6
38.5
10
1′
101.1
134.8
110.4
147.6
147.0
108.3
123.2
139.6
124.9
101.1
101.1
134.0
110.4
146.9
147.5
108.2
123.2
139.4
125.7
101.0
101.7
128.5
110.6
147.6
147.5
108.2
123.5
139.8
119.5
169.9
101.2
101.6
128.4
110.5
147.2
147.7
108.3
123.8
145.6
119.6
168.4
102.5
101.8
127.9
146.8
147.5
146.4
107.9
123.4
144.7
119.7
167.9
101.3
101.8
132.9
110.7
147.5
146.9
107.9
124.0
44.3
2′
3′
4′
5′
6′
7′
8′
45.5
36.2
9′
173.9
101.3
98.1
71.9
10′
1″
2″
3″
4″
5″
6″
1‴
2‴
3‴
4‴
5‴
6-OMe
1‴-OMe
101.6
99.1
73.3
73.6
76.8
75.8
69.8
70.2
73.8
76.5
64.2
67.4
166.6
40.4
109.4
76.0
166.8
78.8
73.3
63.4
60.0
60.1
60.9
52.6
60.2

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*Recorded in CDCl3; **Recorded in DMSO-d6
Fig. 4. Calculated ECD and experimental CD spectra of 5.
30
Compound 6 was isolated as a white solid and optically active [α]D -218.1 (c 0.44, CHCl3).
Its HRESIMS in negative mode presented the pseudo molecular ion [M - H]- at m/z 659.1601
which together with the 13C NMR data are consistent with a molecular formula of C31H32O16.
1
The H NMR spectrum of 6 indicated a singlet proton at H 6.34 (H-3), an ABX system
characterized from three protons at H 6.64 (H-2′), 6.73 (H-5′) and 6.74 (H-6′) in the aromatic
region. Analysis of the 13C NMR with the aid of the HSQC spectrum of 6, and the comparison of
its NMR data with those of related compounds [10,11], revealed the presence of an aryltetralin
lignan glycoside skeleton. This was confirmed by analysis of 2D NMR spectra of 6, where the
methine, methylene, and carboxylate carbon resonances of the aryltetralin lignan framework at
δC 37.6 (C-8), 45.5 (C-8′), 71.9 (C-9), 76.4 (C-7) and 173.9 (C-9′) were assigned. The presence
of a sugar moiety at C-7 was revealed by the cross-peaks of C-7 with the anomeric proton H-1″
at H 4.38 in the HMBC spectrum. The malonate element was connected to C-6′′ via ester bridge
as shown by the HMBC correlations of C-1′′′ at δC 166.6 with the protons CH2-6′′ at δH 4.28
(Figure 5). Proton coupling constants of the sugar protons indicated the presence of β-
glucopyranose (Table 1). The proton H-7 had an anti coupling constant (J = 10.0 Hz), suggesting
a trans-pseudodiaxial relationship between H-7 and H-8. Whereas, proton H-8′ had a gauche (J =
4.5 Hz) and an anti (J = 14.5 Hz) coupling constants. This indicated a pseudoequatorial and a

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pseudoaxial orientations for H-7′ and H-8′, respectively. This observation was clearly confirmed
by interactions of H-7 (δH 5.27) with H-8′ (δH 2.68), and those of H-7′ (δH 4.53) with H-8′ and
one proton of CH2-9 at δH 4.06 in the NOESY spectrum. A positive Cotton effect at 295 nm (Δ
+2.9) was observed in the CD spectrum of 6 revealing the R-configuration for C-7′ [10,19,20].
Base on the relative configuration established above, the R-configuration was thus assigned for
the remaining chiral centers, C-7, C-8 and C-8′. The hydrolyzed sugar had a positive optical
rotation activity and an identical Rf value with the standard D-(+)-glucose, which suggested the
D-configuration for the glucose moiety of cleistonkiside A (6).
Fig. 5. Key HMBC correlations for 6.
30
Compound 7 was obtained as optically active white solids {[α]D -101.2 (c 0.083, MeOH)},
and showed a pseudo molecular ion [M - H]- at m/z 663.1930 in the negative HRESIMS which
1
was in agreement with a molecular formula of C31H36O16. Its H NMR exhibited the resonances
of two aromatic ABX systems which were confirmed by analysis of 13C NMR with the aid of
1
HSQC experiment. The H-1H COSY data indicated the correlations of H-7 (δH 5.21)/H-8 (δH
2.52)/H-8′ (δH 2.88)/CH2-7′ (δH 2.24 and 2.48), and those of H-8′/CH2-9′ (δH 3.93 and 4.28). In
the HMBC spectrum of 7, cross-peaks of the carbonyl carbon C-9 (δC 176.4) with H-7 and CH2-
9′ indicated the formation of the lactone ring. The lignan skeleton was revealed by correlations of
C-7 (δC 75.6) with H-2 (δH 6.94) and H-6 (δH 6.76), and those of C-7′ (δC 38.5) with H-2′ (δH

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6.44) and H-6′ (δH 6.41). Additionally, the positions of the two methylendioxy groups at C-3/C-4
and C-3′/C-4′ were defined by HMBC correlations of C-3 (δC 147.2) and C-4 (δC 146.6) with
methylene protons CH2-10 (δH 5.92), and C-3′ (δC 147.1) and C-4′ (δC 145.5) with protons CH2-
10′ (δH 5.98 and 6.00). The presence of two sugar units was also observed which were assigned
to a glucopyranose and an apiofuranose by analysis of 2D NMR data and proton coupling
constants (Table 1). The bonding of the glucopyranose substructure to C-7 was established from
the HMBC cross-peak of the anomeric carbon at δC 99.1 (C-1″) to the proton at δH 5.21 (H-7),
and the linkage of the apiofuranose to C-6″ of the glucopyranose moiety by the correlation of the
carbon at δC 67.4 (C-6″) with the anomeric proton at δH 4.90 (H-1‴) (Figure 6). The anomeric
proton H-1″ had a coupling constant J = 7.0 Hz, assigning a β-form for the glucopyranose unit
while, the downfield chemical shift of the anomeric carbon C-1′′′ (δC 109.4) suggested a -
apiofuranose conformation in the structure of 7 [11,21]. NOE correlation of H-7 (δH 5.21) with
H-8′ (δH 2.88) indicated that CH-7 and H-8′ were co-facial. This observation demonstrated a
trans relationship for H-8/H-8′ which was supported by the ΔδHa-9′-Hb-9′ value of 0.35 (this value ≥
0.2 for trans, and ≈ 0 for cis) [22,23]. The hydrolyzed glucose and apinose had positive optical
rotation values, assigning the D-configuration for both glucose and apiose moieties of 7. Since,
D-glucose, D-apiose, and trans configuration for H-8/H-8′ were determined, four possible
stereoisomers, 7R,8S,8′R, 7R,8R,8′S, 7S,8R,8′S and 7S,8S,8′R should be attributed for the
aglycone of 7. The ECD spectra of four diastereomers were generated and compared with the
experimental CD spectrum of 7 (Figure 7). The ECD spectrum of the diastereomer 7R,8S,8′R-7
was in accordance with the experimental CD spectrum of 7. Thus, the 7R, 8S and 8′R-
configurations were assigned for 7. This suggestion was in agreement with the negative Cotton
effect at 234 nm in the CD spectrum of 7, indicating the R-configuration for C-8′ [24].

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Fig. 6. Key HMBC correlations for 7.
Fig. 7. Calculated ECD and experimental CD spectra of 7.

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Five known compounds, cleisindoside B (8) [11], cleistantoxin (9) [10], cleisindoside D (10)
[11], neocleistantoxin (11) [10] and polygamain (12) [25], were also isolated from this plant.
Their structures were determined from NMR data and comparison with those previously
reported.
Compounds 1–9 were evaluated for their cytotoxicity against a panel of cancer cell lines,
Hep3B (Human hepatoma cell lines), HeLa (human cervical carcinoma cells), MCF7 (human
breast cancer cells), Pan C1 (pancreas epithelioid carcinoma cell lines) and A549 (human lung
adenocarcinoma cells). The most active compound was cleistantoxin (9) which seems to be less
cytotoxic against HeLa in comparison with A549, Hep3B, Pan C1 and MCF7 (Table 3).
Whereas, neocleistantoxin (11) was more active toward A549, HeLa, Pan C1 and MCF7 in
comparison with Hep3B cells. This is the first report of cytotoxicity of 9 and 11 against A549,
Hep3B and Pan C1 cell lines. Compound 2 and 6 had selective inhibition with moderate
cytotoxicity against Pan C1 and A549 cell lines, respectively. Compound 5 had weak inhibition
against MCF7, Hep3B and Pan C1 cells, and was inactive against A549 and HeLa cell lines
event at concentration of 20 µM. The moderate cytotoxicity of compounds 1‒7 was in agreement
with the SAR study for podophyllotoxin and its derivatives. Accordingly, modification of the
trans-fused γ-lactone ring [26] led to the loss of cytotoxicity. Additionally, compounds with β-
substituents at C-7 exhibited greater inhibitory against DNA topo II than those with α-
configuration [27].
Table 3 In vitro cytotoxic activity of 1‒7, 9 and 11 (IC50 values are expressed in μM)
Compounds
A549
>20
>20
>20
>20
>20
18.1
>20
0.5
HeLa
>20
>20
>20
>20
>20
>20
>20
7.2
MCF7
>20
Hep3B
>20
>20
>20
>20
13.3
>20
>20
0.8
Pan C1
>20
1
2
3
4
5
6
7
9
>20
15.3
>20
>20
>20
>20
17.7
>20
18.5
>20
>20
>20
0.03*
0.7

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11
2.1
1.6
1.2
2.9
2.2
0.2
>20
0.1
5.1
0.9
Camptothecin
*Value taken from ref. 10
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgments
This work was carried out within the framework of an International Associated Laboratory
(LIA) between the Centre National de la Recherche Scientifique (CNRS, France) and the
Vietnam Academy of Science and Technology (VAST, Vietnam). The authors thank Mr. Dao
Dinh Cuong and Dr Nguyen The Cuong (VAST - Vietnam) for plant collection and botanical
determination. The Ministry of Science and Technology of Vietnam is gratefully acknowledged
for financial support (Grant No: ĐTĐLCN.14/16).
Author’s statement
We, the undersigned author(s) of the manuscript entitled “Cytotoxic Lignans from Fruits of
Cleistanthus tonkinensis” hereby declare that the above manuscript which is submitted for
publication in the Fitoterapia is NOT under consideration elsewhere. The manuscript is NOT
published already in part or whole in any journal or magazine for private or public circulation.
Each author has participated sufficiently in the work to take public responsibility for appropriate
portions of the content. The order of authorships on the byline is a joint decision of all the
coauthors.
Supplementary data
Supplementary material
References
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Graphical abstract

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