Chem. Pharm. Bull. Note DNA Topoisomerase Inhibitory

Chemical and Pharmaceutical Bulletin Advance Publication by J-STAGE DOI:10.1248/cpb.c17-00466

Advance Publication September 26, 2017

Chem. Pharm. Bull. Note

DNA Topoisomerase Inhibitory Activity of Constituents from the Fruits of Illicium

verum Tae In Kim,a Bora Shin,d Geum Jin Kim,a Hyukjae Choi,a Chong Soon Lee,b Mi Hee Woo,c

Dong-Chan Oh,d and Jong Keun Son*,a

a College of Pharmacy, Yeungnam University; Gyeongsan 38541, Korea: b Department of

Biochemistry, Yeungnam University; Gyeongsan 38541, Korea: c College of Pharmacy,

Catholic University of Daegu; Gyeongsan 38430, Korea: and d College of Pharmacy, Seoul

National University; Seoul 08826, Korea

Chemical and Pharmaceutical Bulletin Advance Publication

Abstract

Three new compounds, a sesquilignan (1) and two glucosylated phenylpropanoids (2, 3), and

seven known compounds (4-10), were isolated from the fruits of Illicium verum Hook. Fil.

(Illiciaceae). The structures of 1-3 were determined based on one and two dimensional (1D-

and 2D-) NMR data and ECD spectra analyses. Compounds 3, 5, 6, and 8-10 exhibited potent

inhibitory activities against topoisomerase II with IC50 values of 54.6, 25.5, 17.9, 12.1, 0.3

and 1.0 μM, respectively, compared to etoposide, the positive control, with an IC50 of 43.8

Keywords: Illicium verum, topoisomerase I, topoisomerase II, cytotoxicity

Introduction

Star anise (the fruits of I. verum Hook. Fil., Illiciaceae) has been used as a traditional

medicine to treat diverse disease such as stomach aches, insomnia, and skin inflammation in

Asia.1,2) Several sesquiterpenoids, flavonoids, phenylpropanoids and lignans have been

previously isolated from this plant 3-6) and been reported to possess anti-HIV, cancer

chemopreventive and antifungal activities.7-9) The authors previously reported medicinal plant

derived compounds that inhibit topoisomerases I and II.10-15) Topoisomerases are enzymes

that control DNA topology in cells and are targets of anticancer drugs.16-17) Topoisomerase I

cleaves and reseals one DNA strand at a time without requiring ATP to pass the

complementary strand through the enzyme-linked strand break, and thus affect DNA

relaxation. On the other hand, topoisomerase II cleaves both strands of DNA during catalysis.

In a reaction coupled to ATP binding and hydrolysis, these proteins were found to cleave one

DNA duplex, transport a second duplex through the break, and then ligate the cleaved

duplex.18-20) Camptothecin (CPT) and etoposide (VP-16) are well-known inhibitors of

topoisomerases I and II, respectively.16-17) Here, the authors report the isolation of ten

compounds, including a new sesquilignan and two new glycosylated phenylpropanoids, from

the fruits of I. verum, and describe cytotoxicity and their DNA topoisomerases I and II

inhibitory activities.

RESULTS AND DISCUSSION

Three new (1‒3) and seven known compounds (4‒10) were isolated by various

chromatographic separations from the EtOAc extract of the fruits of I. verum (Figure 1). The

molecular formula of 1 was established as C30H36O10 based on a [M-H]- peak at m/z 555.2223

(calcd 555.2230) in its high resolution FAB-MS spectrum. The 13C-NMR and distorsionless

enhancement by polarization transfer (DEPT) spectra showed thirty carbon peaks including

three methyl, five methylene, twelve methine, and ten quaternary carbons. In aliphatic

regions of the 1H-NMR and the 1H-1H correlation spectroscopy (COSY) spectra, 1 exhibited

three spin coupling systems, one among H-9 (δH 3.56), H-10 (δH 1.81) and H-11 (δH 2.62),

another among H-3 (δH 5.49), H-2 (δH 3.42) and C-2-CH2OH (δH 3.77), and a third among

H-7 (δH 4.80), H-6 (δH 4.35) and C-6-CH2OH (δH 3.79) (Figure 2). In their aromatic regions,

signals of two units of 1,3,4-trisubstituted benzene rings, one at δH 6.93 [1H, d, J = 1.8 Hz,

H-2(B), 6.83 [1H, dd, J = 8.3, 1.8 Hz, H-6(B)], and 6.89 [1H, d, J = 8.3 Hz, H-5(B)], another

at δH 6.98 (1H, d, J = 1.8 Hz, H-2'), 6.81 (1H, dd, J = 8.1, 1.8 Hz, H-6') and 6.70 (1H, d, J =

8.1 Hz, H-5'), and one unit of a 1,3,5,6-tetrasubstituted benzene ring based on signals at δH

6.72 [1H, brs, H-4(A)] and 6.71 [1H, brs, H-2(A)] were recognized. Heteronuclear multiple

bond connectivity (HMBC) correlations of H-2/C-1(A), C-2(A) and C-6(A); H-3/C-1(B),

C-2(B) and C-6(B); H-6/C-4(B); H-7/C-9; H-11/C-2(A), C-3(A) and C-4(A) indicated the

presence of a dibenzenacycloundecaphane ring system (Figure 2). Linkage between C-7 and

C-1' was established by HMBC correlations of H-7 with C-1', C-2' and C-6' and positions of

three methoxyl groups observed at δH 3.74 [C-3(B)-OCH3], 3.77 (3'-OCH3) and 3.84

[C-5(A)-OCH3] were determined to be at C-3(B), C-3′ and C-5(A), respectively, based on

HMBC correlations between each methoxy proton and corresponding carbons.

In order to elucidate relative configurations on both C2-C3 and C6-C7, energy-minimized

3D structures of eight possible diastereomers of 1, 2S*,3R*,6R*,7R* (1a), 2R*,3S*,6R*,7R*

(1b), 2S*,3S*,6R*,7R* (1c), 2S*,3R*,6R*,7S* (1d), 2S*,3R*,6S*,7R* (1e), 2S*,3S*,6R*,7S*

(1f), 2S*,3S*,6S*,7R* (1g) and 2S*,3S*,6S*,7S* (1h), were calculated by using Turbomole

6.5 (Figure S39 in supplementary materials) and 1H-1H coupling constants and NOE

correlations of 1 were investigated (Figure 3). In the 1H-NMR spectrum, two sets of 1H-1H

coupling constants between H-2 and H-3, and between H-6 and H-7 were observed to be 6.1

Hz and 5.8 Hz, respectively, and it corresponds to two gauche forms on H-2/H-3 and H-6/H-7

of compound 1. Among energy-minimized structures of eight possible diastereomers, only

three cases, 1a, 1b and 1e agree to two gauche conformations, while 1f and 1g have two

anti-conformations, and 1c, 1d, 1h have one anti and one gauche conformations, on H-2/H-3,

and H-6/H-7 (Figure S39 in supplementary materials). In addition, key NOE correlations

among protons around C-2 and C-3, [H-2/H-2(A), H-2/H-2(B), H-3/C-2-CH2OH,

H-3/H-2(B)], and those around C-6 and C-7, [H-6/H-5(B), H-6/H-6', H-7/H-5(B), H-7/H-2',

H-7/H-6'], were observed in NOESY spectrum (Figure 4). However, in case of 1b,

H-2/H-2(B) and H-3/C-2-CH2OH are positioned in anti-conformations which are inconsistent

to the observed NOE correlations. Two protons, H-6 and H-2', in 1e case are calculated to be

in anti-conformation, but this could not explain the strong NOE correlation between two

protons, H-6 and H-2', of compound 1. The calculated structure of case 1a is consistent to all

the NOE correlations of 1 and dihedral angles of H-2/H-3 and H-6/H-7 of 1a were calculated

to be 52.4˚ and 63.5˚, respectively. Therefore, the relative configuration of compound 1 was

speculated to be 2S*,3R*,6R*,7R*.

The absolute configurations at C-2, C-3, C-6 and C-7 in 1 were determined by comparing

the observed electronic circular dichroism (ECD) spectrum of 1 with calculated ECD spectra

of two enantiomers of 1a [2S,3R,6R,7R and 2R,3S,6S,7S].21) Energy minimized structures of

the two isomers were calculated using Turbomole 6.5. The ECD spectra of the two isomers of

1 were calculated by TD-DFT (time dependent density functional theory) at the

B3LYP/def2-TZVPP//B3LYP/def-SV(P) level for all atoms.21) The experimental ECD

spectrum of 1 exhibited a positive cotton effect at 295 nm and a negative cotton effect at 242

nm and a profile almost identical with the calculated ECD spectrum of the 2S,3R,6R,7R

isomer (Figure 5). Therefore, the structure of 1 was proposed to be

(2S,3R,6R,7R)-7-(4-hydroxy-3-methoxyphenyl)-2,6-bis(hydroxymethyl)-15,43-dimethoxy-

5,8-dioxa-1(1,3),4(1,4)-dibenzenacycloundecaphane-16,3-diol.

The molecular formula of 2 was C22H30O11 based on a [M+Na]+ peak at m/z 493.1683

(calcd : 493.1686) in its HR-FAB-MS spectrum. Chemical shift values of 4-methoxyphenyl

propane and sugar moieties in 1H and 13C NMR spectra of 2 were similar to those of

oligandrumin E (2a) which was previously reported in Illicium oligandrum (Figure 1).22) The

sugar unit of 2 was identified to be D-glucose by acidic hydrolysis followed by methyl

2-(polyhydroxyalkyl)-3-(o-phenylthiocarbamoyl)-thiazolidine-4(R)–carboxylate

derivatization and HPLC.23) The presence of a 1,2-dioxygeneted propyl group was indicated

by COSY correlations between H-7, H-8 and H-9, and linkage between the propyl chain and

the aromatic ring was established by HMBC correlations of H-7 with C-1, C-2, C-6 and C-9

HMBC correlations of H-8/C-1' and H-2'/C-7 revealed the glucosyl moiety was connected to

the propyl chain by two ether linkages (Figure S34 – Figure S35 in supplementary

materials).22) Large coupling constants between H-7/H-8 (9.1 Hz), H-1′/H-2′ (7.7 Hz),

H-2′/H-3′ (9.5 Hz), H-3'/H-4' (9.5 Hz), and H-4′/H-5' (9.5 Hz) and NOE correlations

indicated all these protons were in trans-diaxial positions with respect to adjacent protons and

that the two six-membered rings had chair conformations.22) The presence of

3-hydroxy-3-methylglutaryl (HMG) moiety was confirmed by HMBC correlations of

H-2"/C-4" and 3"-CH3 ; H-4"/C-2", C-3", 3"-CH3 and C-5"; and 3"-CH3/C-2" and the

location of the HMG group was determined by the HMBC correlation from H-6' (δΗ 4.24 and

4.47, each 1H) to C-1". The absolute configuration of C-3" was analyzed to be S by

comparing the ECD spectrum of 2 with the theoretically calculated spectra of the C-3"S and

C-3"R isomers of 2 (Figure 6). ECD absorptions of 2 and its 3"S isomer were almost identical.

In addition, naturally occurring HMG esters were speculated to be biosynthesized by

nucleophilic substitution between a nucleophilic hydroxyl group or RO- and (S)-HMG-CoA,

as this mode of biosynthesis is widely used to produce terpenoids and other natural products.

24-27)

Compound 3 had the molecular formula C16H22O7 as determined by its [M-H]- HR-FAB-MS

peak (m/z 325.1289 calcd 325.1287). Its 13C-NMR and DEPT spectra showed sixteen carbon

peaks of two methyl, one methylene, ten methine, and three quaternary carbons. 1H-NMR

and the 1H-1H COSY spectra revealed three spin coupling systems including, a

trans-propenyl group [H-1' (δH 7.19, d, J = 15.2 Hz), H-2' (δH 6.22, dq, J = 15.2, 6.6 Hz), and

H-3' (δH 1.65, d, J = 6.6 Hz)], a 1, 2, 4-trisubstituted benzene ring [H-3 (δH 7.36, d, J = 2.2

Hz), H-5 (δH ,6.71 dd, J = 8.5, 2.2 Hz) and H-6 (δH 7.56, d, J = 8.5 Hz)], and a glucose moiety

[H-1" (δH 5.66, d, J = 7.0 Hz), H-2" (δH 4.40, m), H-3" (δH 4.12, m), H-4" (δH 4.38, m), H-5"

(δH 4.38, m), H-6a" (δH 4.57, dd, J = 12.0, 2.0 Hz) and H-6b (δH 4.37, dd, J = 12.0, 6.0 Hz)].

The positions of three functional groups on the benzene ring of 3 were determined based on

HMBC correlations (H-2'/C-1, H-1"/C-2 and 4-OCH3/C-4) and NOESY correlations

(4-OCH3/H-3 and H-5; H-1"/H-3; H-2'/H-6) (Figure S36 – Figure S37 in supplementary

materials). A coupling constant of an anomeric proton signal at δH 5.66 (d, J = 7.0 Hz, H-1'')

indicated the presence of β-D-glucopyranoside. The glucose unit of 3 was identified as

D-glucose.23) Therefore, 3 was determined as (E)-2-hydroxyanethole β-D-glucopyranoside.

Compounds 4‒10 were identified by comparing their 1H- and 13C-NMR, FAB-MS spectral

data and optical rotation values with previously reported data as;

(E)-1′-(2-hydroxy-5-methoxyphenyl)propane-β-D-glucopyranoside (4),28) (-)-catechin (5),29)

2-[2-hydroxy-5-(3-hydroxypropyl)-3-methoxyphenyl]-1-(4-hydroxy-3-methoxyphenyl)propa

ne-1,3-diol (6),30) (2S)-2-hydroxy-1-(4-methoxyphenyl)-1-propanone (7),31)

(1R,2R)-1-(4-methoxyphenyl)-1,2-propanediol (8),32)

1-(4'-methoxyphenyl)-(1S,2S)-propan-1-ol-2-O-β-D-glucopyranoside (9)33) and shikimic

acid (10).34)

None of ten compounds showed any topoisomerase I inhibitory activity at concentrations of

less than 100 μM, but compounds 3, 5, 6, and 8-10 inhibited the activity of topoisomerase II

with IC50 values of 54.6, 25.5, 17.9, 12.1, 0.3 and 1.0 μM, respectively (Figure S38 in

supplementary materials). Compounds 5, 6, and 8-10 exhibited more topoisomerase II

inhibitory activity than the positive control, VP-16 (IC50; 43.8 μM). No cytotoxicity of

isolated compounds (1 – 10) at concentrations of less than 100 μM were shown against four

human cancer cell lines, lung carcinoma (A549), ovary adenocarcinoma (SK-OV-3), liver

hepatocellular carcinoma (HepG2) and colon adenocarcinoma (HT-29). In conclusion, ten

compounds including one new sesquilignan (1) and two new glucosylated phenylpropanoids

(2, 3) were isolated from the fruits of Illicium verum. Structures of the new compounds were

determined based on spectroscopic data. Compound 5, 6 and 8-10 showed more potent

inhibitory activities against topoisomerase II than that of VP-16.

Materials and methods General experimental procedures

The NMR spectra were recorded on Bruker 250 MHz (DMX 250, Germany) and Varian

600 MHz (VNS 600, Australia) units. HR-FAB-MS spectra were recorded on a JEOL

JMS-700 (Tokyo, Japan) mass spectrometer at the Daegu center of KBSI, and NCIRF at

Seoul National University in Korea. Stationary phases for column chromatography (silica gel

60, 70-230 and 230-400 mesh, LiChroprep RP-18 gel, 40-63 μm, Sephadex LH-20) and TLC

plates (silica-gel 60 F254 and RP-18 F254, 0.25 mm) were purchased from Merck KGaA

(Darmstadt, Germany). The NMR solvents, chloroform-d, pyridine-d5 and methanol-d4, were

purchased from Aldrich (St. Louis, MO, USA). Optical rotations were measured using a

JASCO DIP-1000 (Tokyo, Japan), and ECD spectra were recorded using an Applied

Photophysics Chirascan-Plus circular dichroism spectrometer (Leatherhead, Surrey, UK).

Plant material

Star anise (the fruit of Illicium verum Hook. Fil., Illiciaceae) was purchased in November

2014 from Pungsan Pharm Co., in Ahndong, South Korea. Materials were confirmed

taxonomically by Professor Gi-Hwan Bae, Chungnam National University, Daejeon, South

Korea. A voucher specimen (YNIV-2015) was deposited at the College of Pharmacy,

Yeungnam University, South Korea.

Extraction and isolation

The dried fruits of I. verum (9 kg) were extracted three times with 4 L of 90% MeOH at 60℃

for 12h, and the MeOH solution was evaporated to dryness (1.3 kg). The dried MeOH extract

was suspended with H2O, and the resulting H2O layer was partitioned with hexane, EtOAc

and n-BuOH, successively. The EtOAc extract (100 g) was loaded on a silica gel column (11

× 22 cm, silica-gel 230‒400 mesh), which was eluted then with hexane-EtOAc (gradient from

100:0 to 0:100) and then EtOAc-MeOH (gradient from 100:0 to 0:100). Eluates were

combined on the basis of TLC analyses, giving 21 fractions (E1‒E21). Fraction E11 (300 mg)

was chromatographed on a reversed-phase column (4.5 × 20 cm) using MeOH-H2O (gradient

from 20:80 to 60:40) to give compound 7 (27 mg). Fraction E16 (2 g) was loaded on a

reversed-phase column (5 × 20 cm) and eluted with gradient solvent CH3CN-H2O (gradient

from 10:90 to 25:75) to afford compounds 5 (156 mg) and 8 (22 mg).

Fraction E17-20 (79 mg) was loaded on a Sephadex LH-20 column (3 × 80 cm) and eluted

with MeOH to give compound 6 (10 mg). Fraction E18 (10 g) was chromatographed on a

reversed-phase column (6 × 25 cm) with MeOH-H2O (gradient from 10:90 to 100:0), to give

9 fractions (E18-1‒E18-9) and pure compounds 10 (6 g) and 3 (701 mg) were obtained from

E18-1 and E18-7, respectively. Fraction E18-5 (500 mg) was chromatographed on a silica gel

column (4.5 × 22 cm, 230-400 mesh) with CH2Cl2- MeOH (gradient from 100:0 to 0:100) to

give compound 4 (91 mg). Fraction E19 (2 g) was chromatographed on a reversed-phase

column (4 × 18 cm) with MeOH-H2O (gradient from 10:90 to 50:50), to give 18 fractions

(E19-1‒E19-18) and E19-7 was pure compound 9 (121 mg). Fraction E19-12 (66 mg) was

separated on a silica-gel column (2.5 × 13.5 cm, 230-400 mesh) by flash column

chromatography using EtOAc-MeOH (gradient from 100:0 to 50:50) as eluent to give

compounds 1 (15 mg) and 2 (11 mg).

Compound 1 White powder; 1H-NMR (MeOH-d4, 600 MHz) δ: 6.98 (1H, d, J = 1.8 Hz, H-2'),

6.93 [1H, d, J = 1.8 Hz, H-2(B)], 6.89 [1H, d, J = 8.3, H-5(B)], 6.83 [1H, dd, J = 8.3, 1.8 Hz,

H-6(B)], 6.81 (1H, dd, J = 8.1, 1.8 Hz, H-6'), 6.72 [1H, s, H-4(A)], 6.71[1H, s, H-2(A)], 6.70

(1H, d, J = 8.1 Hz, H-5'), 5.49 (1H, d, J = 6.1 Hz, H-3), 4.80 (1H, d, J = 5.8 Hz, H-7), 4.35

(1H, ddd, J = 5.8, 5.8, 3.8 Hz, H-6), 3.84 [3H, s, C-5(A)-OCH3], 3.79 (2H, m, C-6-CH2), 3.77

(2H, m, C-2-CH2), 3.77 (3H, s, 3'-OCH3), 3.74 [3H, s, C-3(B)-OCH3), 3.56 (2H, t, J = 7.5 Hz,

H-9), 3.42 (1H, ddd, J = 6.1, 6.1, 6.1 Hz, H-2), 2.62 (2H, t, J = 7.5 Hz, H-11), 1.81 (2H, q, J

= 7.5 Hz, H-10); 13C-NMR (MeOH-d4, 150 MHz) δ: 151.94 [C-3(B)], 148.98 [C-4(B)],

148.65 (C-3'), 147.49 [C-6(A)], 146.99 (C-4'), 145.22 [C-5(A)], 137.57 [C-1(B)], 137.01

[C-3(A)], 134.06 (C-1'), 129.71 [C-1(A)], 121.08 (C-6'), 119.34 [C-6(B)], 118.93 [C-5(B)],

117.94 [C-2(A)], 115.60 (C-5'), 114.15 [C-4(A)], 111.85 (C-2'), 111.14 [C-2(B)], 88.57 (C-3),

86.23 (C-6), 74.12 (C-7), 65.01 (C-2-CH2), 62.29 (C-6-CH2), 62.23 (C-9), 56.77

[C-5(A)-OCH3], 56.47 [C-3(B)-OCH3], 56.34 (C-3'-OCH3), 55.55 (C-2), 35.81 (C-10), 32.90

(C-11). Negative HR-FAB-MS m/z: 555.2223 [M-H]- (calcd for C30H35O10 : 555.2230). [α]D20

-6.6 (c=0.74, MeOH). ECD (c 0.45mM, MeOH) nm (Δε): 295 (+1.40), 242 (-4.81). UV λmax

(MeOH) nm (log ε): 210 (4.85), 230 (4.25), 281 (3.85).

Compound 2 yellow powder; 1H-NMR (MeOH-d4, 600 MHz) δ: 7.29 (2H, d, J = 8.7 Hz, H-2,

H-6), 6.89 (2H, d, J = 8.7 Hz, H-3, H-5), 4.55 (1H, d, J = 7.7 Hz, H-1'), 4.47 (1H, dd, J =

12.0, 2.0 Hz, 6'-Hb), 4.24 (1H, dd, J = 12.0, 5.5 Hz, 6'-Ha), 4.14 (1H, d, J = 9.1 Hz, H-7), 3.84

(1H, dt, J = 9.1, 6.2 Hz, H-8), 3.78 (3H, s, OCH3), 3.66 (1H, ttt, J = 9.5, 5.5, 2.0 Hz, H-5'),

3.57 (1H, t, J = 9.5 Hz, H-3'), 3.44 (1H, t, J = 9.5 Hz, H-4'), 3.14 (1H, dd, J = 9.5, 7.7 Hz,

H-2'), 2.62 (2H, s, H-2''), 2.54 (1H, d, J = 15.3 Hz, 4''-Hb), 2.39 (1H, d, J = 15.3 Hz, 4''-Ha),

2.00 (1H, d, J = 7.5 Hz, 3''-OH), 1.33 (3H, brs, 3''-CH3), 0.97(3H, d, J = 6.2 Hz, H-9);

13C-NMR (MeOH-d4, 150 MHz) δ: 179.65 (C-5''), 172.56 (C-1''), 161.33 (C-4), 131.11 (C-1),

130.14 (C-2, C-6), 114.76 (C-3, C-5), 99.58 (C-1'), 84.96 (C-7), 80.82 (C-2'), 78.05 (C-8),

77.04 (C-5'), 74.87 (C-3'), 71.88 (C-4'), 70.98 (C-3''), 64.36 (C-6'), 55.68 (C-10), 47.68 (C-4''),

47.05 (C-2''), 27.84 (3''-CH3), 17.06 (C-9). Positive HR-FAB MS m/z: 493.1683 [M+Na]+

(calcd for C22H30O11Na : 493.1686). [α]D20 +20.8(c= 0.4, MeOH). ECD (c 0.79mM, MeOH)

nm (Δε): 273 (+1.05). UV λmax (MeOH) nm (log ε): 280 (2.91), 226 (3.88), 204 (3.82)

(E)-2-hydroxyanethole β-D-glucopyranoside (3) yellow powder; 1H-NMR (pyridine-d5, 250

MHz) δ: 7.56 (1H, d, J = 8.5 Hz, H-6), 7.36 (1H, d, J = 2.2 Hz, H-3), 7.19 (1H, d, J = 15.2 Hz,

H-1'), 6.71 (1H, dd, J = 8.5, 2.2 Hz, H-5), 6.22 (1H, dq, J = 15.2, 6.6 Hz, H-2'), 5.66 (1H, d, J

= 7.0 Hz, H-1''), 4.57 (1H, dd, J = 12.0, 2.0 Hz, H-6''a), 4.40 (1H, m, H-2''), 4.38 (1H, m,

H-5''), 4.38 (1H, m, H-4''), 4.37 (1H, dd, J=12.0, 6.0 Hz, H-6''b), 4.12 (1H, m, H-3''), 3.70 (3H,

s, 4'-OCH3), 1.65 (3H, d, J = 6.6 Hz, H-3'); 13C-NMR (pyridine-d5, 62.5 MHz) δ: 160.85

(C-4), 156.63 (C-2), 127.58 (C-6), 126.63 (C-1') 124.19 (C-2'), 121.42 (C-1), 108.80 (C-5),

103.00 (C-3), 102.96 (C-1''), 79.52 (C-3''), 79.21 (C-5''), 75.45 (C-2''), 71.78 (C-4''), 62.81

(C-6''), 55.77 (OCH3), 19.29 (C-3'). Negative HR-FAB-MS m/z 325.1289 [M-H]- (Calcd for

C16H21O7 : 325.1287). [α]D20 -48.5 (c=0.48, MeOH). UV λmax (MeOH) (log ε): 297 (3.56),

258 (3.73), 210 (4.32).

Identification of sugar units in compounds 2 and 3

Compounds 2 and 3 (1 mg each) were independently hydrolyzed by heating in 1 M HCl (0.5

mL) at 90°C for 2 h and neutralizing with 1 M NaOH (0.5 mL). Each reaction mixture was

then washed with EtOAc (1 mL × 3) and the remaining water fraction was filtrated. After

drying the aqueous fraction in vacuo, the residue was dissolved in pyridine (0.5 mL)

containing L-cysteine methyl ester hydrochloride (1.5 mg) and heated at 60°C for 1 h. Phenyl

isothiocyanate (0.1 mL, 99.0%) added to the reaction mixture and then kept at 60°C for 1 h.

The solution was concentrated to dryness with N2 gas. Sugar analysis was performed by

reversed-phase HPLC using the following conditions: column – Agilent eclipse plus C18 (5

μm, 4.6 × 250 mm), detector – UV (250 nm), Flow – 0.8 ml/min, solvent – 25% CH3CN.

Peaks due to derivatives of standard D-glucose and L-glucose were observed at 13.34 and

12.32 min, respectively. For compound 2, the 13.37 min peak was attributed to a D-glucose

derivative.

Computational Analysis

Ground state geometries were optimized using density functional theory (DFT) calculations

and Turbomole 6.5. The basis set was def-SV(P) for all atoms at the DFT level and the

B3LYP/DFT level was used at the functional level. Ground states were confirmed using

harmonic frequency calculations. The calculated ECD data corresponding to optimized

structures were obtained using TD-DFT at the functional B3LYP/DFT level and the TZVPP

basis set. ECD spectra were simulated by overlapping for each transition, where σ is the

width of the band at 1/e height. ΔEi and Ri are the excitation energies and rotatory strengths

for transition i, respectively. The σ value of 0.10 eV, and excitation number of 12 were used

in this calculation.

∆ ( ) = 12.297 × 10 1√2 [ ( ∆ ) /( ) ] Assay of DNA topoisomerase I and II inhibitions in vitro

These inhibition assays were performed by measuring the relaxation of supercoiled pBR

322 plasmid DNA as previoiusly described.14)

Assay for cytotoxicity

A MTT assay (tetrazolium-based colorimetric assay) was used to investigate the cytotoxic

effects of compounds 1–10 on selective cancer cell lines, A549 cells (lung carcinoma),

SK-OV-3 cells (ovary adenocarcinoma), HepG-2 cells (liver hepatoblastoma), and HT-29

cells (colon adenocarcinoma).14) All samples were tested in triplicate.

ACKNOWLEDGEMENTS This research was supported by a Yeungnam University research grant in 2015.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

The online version of this article contains supplementary materials.

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Figure 1. Structures of compounds 1–10 isolated from I. verum

Figure

2. Key 1H,1

3. Key NO

mical and P

OHOCH3

1H COSY (

Pharmaceu

) and HM

) correlatio

utical Bull

MBC(→) co

ons of comp

letin Adva

orrelations o

pound 1

ance Publi

of compound

ication

H-2 - HH-3 - C

H-6 - (

Figure C6-C7 C6-C7 o

H-2/ H-6(B ring)

H-2'/ H-

H-5 (B

H-2/H-6 (B ringCH2OH : gauch

(H-2'/H6') : ga

4. NOESYof 1a (2S*of 1b (2R*,

mical and P

H-2 (A ring

B ring)

g) : gauchehe

Y( ) co,3R*,6R*,7,3S*,6R*,7R

Pharmaceu

g) HOH2C

H-2 - HH-3 - C

orrelations aR*) (A) an

R*) (B) and

utical Bull

H-2'/ H-6'

H-5 (B rin

H-2/ H-6 (B ring)

H-2/H-6 (B ringCH2OH : anti

- (H-2'/H6') : ga

and conformnd conforma

1e (2S*,3R

letin Adva

H-2 (A ring)

g) : anti

mations amoations amon

R*,6S*,7R*)

ance Publi

H-5(B ring)

H-6 - (

H-2 - H-2H-3 - CH2

ong protonsng protons (C), respec

ication

H-2/ H-6 (B ring)

H-2'/ H-6'

(H-2'/H6') : ant

2/H-6 (B ring) :2OH : gauche

s around C2around C2

ctively.

-2 (A ring)

: gauche

2-C3 and -C3 and

Wavelength (nm)

220 240 260 280 300 320 340

Experimental ECD of 1Calculated ECD (2S, 3R, 6R, 7R)Calculated ECD (2R, 3S, 6S, 7S)

Figure 5. Experimental ECD spectrum of 1 (solid line) and calculated ECD spectra of 2S, 3R,6R,7R (long dash line) and 2R,3S,6S,7S (dotted line) isomers of 1

Wavelength (nm)

260 280 300 320 340-2

Experimental ECD of 2Calculated ECD of 2 (3''S)Calculated ECD of 2 (3''R)

Figure 6. Experimental ECD spectrum of 2 (solid line) and calculated ECD spectra of 3''S (long dash line) and 3''R (dotted line) isomers of 2

Chem. Pharm. Bull. Note DNA Topoisomerase Inhibitory

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