A new series of bisbenzimidazole derivatives: Synthesis, antitumor activity and low toxicity on PBMC

Article abstract of DOI:10.3987/COM-12-12521

Herein, a new series of bisbenzimidazole derivatives were designed and synthesized. Most of these new compounds showed significant in vitro anticancer activity when compared to Hoechst 33258 and 5-FU. Among them, the most potent compound 8 had the IC₅₀ values of 0.78 μM for U937 tumor cell line, 5.62 μM for HL60 tumor cell line and 6.97 μM for Hela tumor cell line. Molecular modeling, fluorescence and viscosimetry study showed that compound 8 could bind into the minor groove of DNA. Subsequent toxicity study on human peripheral blood mononuclear cells (PBMC), shows that compound 8 exhibited less toxicity than paclitaxel and 5-FU.

Full text of DOI:10.3987/COM-12-12521

HETEROCYCLES, Vol. 85, No. 8, 2012, pp. -. © 2012 The Japan Institute of Heterocyclic Chemistry
Received, 13th May, 2012, Accepted, 22nd June, 2012, Published online, 25th June, 2012
DOI: 10.3987/COM-12-12505
AURONE CONSTITUENTS FROM THE FLOWERS OF ROSA RUGOSA
AND THEIR BIOLOGICAL ACTIVITIES
Xuemei Gao,1 Liying Yang,1,2 Lidang Shu,1 Yanqiong Shen,1 Yinjie Zhang,1*
and Qiufen Hu1*
1
Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic
Affairs Commission & Ministry of Education, Yunnan University of
2
Nationalities; Key Laboratory of Tobacco Chemistry of Yunnan Province,
Yunnan Academy of Tobacco Science, Kunming 650106, P. R. China. E-mail:
huqiufena@yahoo.com.cn, yingjie@kmust.edu.cn
Abstract – Three new aurones, rugaurones A–C (1-3), together with four known
aurones (4-7) were isolated from the flowers of Rosa rugosa. Their structures
were elucidated by spectroscopic methods, including extensive 1D- and 2D- NMR
techniques. Compounds 1-7 were tested for their anti-HIV-1 activity and
cytotoxicities. The results showed that compound 3 has significant potential
anti-HIV-1 activity, compounds 2 have high cytotoxic abilities, and other
compounds also have moderate biological activities.
INTRODUCTION
The species of Rosa rugosa are one of the most important ornamental plants for it large and attractive
flowers. This species widely distributed in temperate regions of eastern Asia including Japan, Korea and
China,1 and had widely been cultivated in several areas of Yunnan Province because of high commercial
values.2 Meanwhile, the petals and buds of R. rugosa have also been used as food, incense materials, and
in traditional Chinese medicine for treating stomachache, diarrhoea and women’s diseases.3,4 The
previous phytochemical researches on R. rugosa has revealed that tannins, flavonoids, as well as
terpenoids are major components isolated from this plant,5-9 and recent studied reveal that R. rugosa also
have anti-HIV and antitumor activity.8,9
With the aim of multipurpose utilization of R. rugosa and identify bioactive natural products from this
plants, the phytochemical investigation on R. rugosa was carried out. As a result, three new aurones (1-3)
together with four known aurones (4-7), were isolated from this plant. In addition, the anti-HIV-1 activity

and cytotoxicities of compounds 1-7 were evaluated. This article deals with the isolation, structural
elucidation and biological activities of the compounds.
RESULTS AND DISCUSSION
A 70% aq. methanol extract prepared from the flowers of R. rugosa was subjected repeatedly to column
chromatography on silica gel, Sephadex LH-20, RP-18 and preparative HPLC to afford compounds 1-7,
including three new aurones, named rugaurones A–C (1-3), together with four known aurones,
4,6,4-trihydroxy-aurone-4,6-di-O--D-glucopyranoside (4),10 sulfuretin (5),11 hamiltrone (6),12
(E)-3-O--D-glucopyranosyl-4,5,6,4-tetrahydroxy-7,2-dimethoxyaurone (7).13 The structures of the
compounds 1-7 were as shown in Figure 1.
OH
OR2
4′
3′
2′
5′
C
7
B
4
R1O
O
MeO
R1O
6
5
8
9
O
6′
1′
A
10
2
3
O
OR2
O
1 R1 = Me, R2 = H; 2 R1 = H, R2 = H
3 R1 = H, R2 = Me
4 R1 = Glc, R2 = Glc
5 R1 = H, R2 = Rut
OH
OH
OMe
HO
HO
O
MeO
MeO
O
OMe
OGlc
OH
O
OH
O
OMe
6
7
Figure 1. The structures of aurones from the flowers of R. rugosa
Compound 1 was obtained as pale yellow gum. Its molecular formula was determined as C17H14O5 by
HR-ESI-MS m/z 321.0732 [M+Na]+ (calcd 321.0739).
The 1H and 13C NMR spectra of 1 (Table 1) along with
OH
analysis of the DEPT spectra displayed 17 carbon
MeO
MeO
O
signals and 14 proton signals, respectively,
corresponding to an aurone nucleus12,13 (δC 147.0 s,
180.2 s, 113.0 d, 145.8 s, 154.0 s, 102.1 d, 158.3 s,
116.6 s, 110.5 d, 122.5 s, 134.5 d, 115.1 d, 156.7 s,
O
Figure 2 Selected HMBC (
) correlations of 1

115.1 d, 134.5 d), two methoxyl groups (δC 55.8 q, 56.0 q), and one phenolic hydroxyl proton (δH 10.88).
Strong absorption bands accounting for hydroxyl (3421 cm-1), carbonyl group (1667 cm-1) and aromatic
groups (1610, 1492, 1441 cm-1) could also be observed in its IR spectrum. The UV spectrum of 1 showed
absorption maxima at 338, 256 and 210 nm, which confirmed the existence of the aromatic functions. The
HMBC correlations (Figure 2) of H-10 (δH 6.79) with C-2 (δC 147.0), C-3 (δC 180.2), C-1′ (δC 122.5),
C-2′ (δC 134.5), of H-2′ (δH 7.86) with C-10 (δC 110.5), and of H-4 (δC 7.29) with C-3 (δC 180.2), also
supported the aurone nucleus.
Table 1. 1H NMR and 13C NMR data of compounds 1 - 3 in C5ND5(125 and 500 MHz)
Compound 1
H (m, J, Hz)
Compound 2
H (m, J, Hz)
Compound 3
H (m, J, Hz)
No.
C (m)
C (m)
C (m)
2
147.0 s
147.7 s
147.0 s
3
180.2 s
113.0 d
145.8 s
154.0 s
102.1 d
158.3 s
116.6 s
110.5 d
122.5 s
134.5 d
115.1 d
156.7 s
115.1 d
134.5 d
55.8 q
180.2 s
114.9 d
140.0 s
155.5 s
102.8 d
158.4 s
116.9 s
110.7 d
122.2 s
133.9 d
115.7 d
157.0 s
115.7 d
133.9 d
180.6 s
115.0 d
141.0 s
155.1 s
102.3 d
158.2 s
116.7 s
110.2 d
122.0 s
133.0 d
113.5 d
160.6 s
113.5 d
133.0
4
7.29, s
6.69, s
6.79, s
7.30, s
6.62, s
6.80, s
7.31, s
6.62, s
6.82, s
5
6
7
8
9
10
1′
2′
7.86, d, J=8.6
7.02, d, J=8.6
7.85, d, J=8.6
7.02, d, J=8.6
7.92, d, J=8.6
7.05, d, J=8.6
3′
4′
5′
7.02, d, J=8.6
7.86, d, J=8.6
3.76, s
7.02, d, J=8.6
7.85, d, J=8.6
7.05, d, J=8.6
7.92, d, J=8.6
6′
OMe-5
OMe-6
OMe-4′
OH-5
OH-4′
56.0 q
3.80, s
55.6 q
3.80, s
56.0 q
55.7 q
3.80, s
3.82, s
11.10, s
11.10, s
10.83, s
10.88, s
The signals for four coupled aromatic protons at H 7.02 (d, J=8.6 Hz, 2H), and 7.86 (d, J=8.6, 2H),
suggested a 4′-monosubstituted for C ring. The proton signals for two singlets at H 7.29 (s, 1H), and H
6.69 (s, 1H) also revealed that the substituents for B-ring should be located at C-5 and C-6. The HMBC

correlations of the hydroxyl proton signal H 10.88 with C-3 (C 115.1), C-4 (C 156.7), C-5 (C 115.1),
suggested the attachment position of the phenolic hydroxyl group at C-4. The HMBC correlation of two
methoxy proton signals (H 3.76, 3.80) with C-5 (C 145.8) and C-6 (C 154.0) suggested two methoxyl
groups located at C-5 and C-6 respectively. Thus, the structure of 1 was established as
4′-hydroxy-5,6-dimethoxyl-aurone, and given the trivial name of rugaurone A.
Compounds 2 and 3 (rugaurones B and C) were also obtained as pale yellow gum. By comparison of their
1
IR, UV, H, and 13C NMR spectra with those of 1, compounds 2 and 3 were also assigned as
4,5,6-substituted-aurone. The obvious chemical shift differences resulted from the substituents group
variations in the aromatic rings. The NMR spectra of 2, compared to those of 1, displayed an additional
phenolic hydroxyl proton (H 11.10), and the disappearance of a methoxy group signals. This indicated
that a methoxy group in 1 was replaced by a hydroxyl group in 2, and the additional hydroxyl group
located at C-5 was supported by the the HMBC cross-peak between this hydroxyl group (H 11.10) with
1
C-4 (C 114.9), C-5 (C 140.0), and C-6 (C 155.5). The H and 13C NMR spectra of 3 displayed 17
carbon signals and 14 proton signals. The obvious differences are the downfield-shifted signals for C-4
(C 160.4) and the upfield-shifted signal for C-5 (C 141.0) when compared to those of 1. This suggested
that the substituents group variations should be at C-5 and C-4. The hydroxyl group located at C-5 was
supported by the HMBC correlations of hydroxyl group (H 11.10) with C-4 (C 115.0), C-5 (C 141.0),
and C-6 (C 155.1), and the methoxy groups located at C-4′ and C-6 were supported by the HMBC
correlations of methoxy proton signals (H 3.82, 3.80) with C-4′ (C 160.6) and C-6 (C 155.1),
respectively. Thus, the structures of 2 and 3 were established as shown.
Since the configuration of the olefinic bond in aurones can be established on the basis of the chemical
shift of olefic methine resonance as it absorbs at 119.9-121.5 ppm in E-aurones and at 105.9-112.8 ppm
in Z-aurones.14 The configuration of the olefinic bond in 1-3 was defined as Z by the chemical shift value
of carbon atom C-10.
Table 2. Anti-HIV activity of compounds 1 - 7
compounds
CC50 (μg/mL)
EC50 (μg/mL)
TI a
6.38
1
2
3
4
5
6
7
 200
31.34
184
3.25
1.28
4.48
18.2
5.18
2.82
56.70
 200
68.18
186.5
170.3
90.01
156.9
15.22
10.27
32.87
31.92
a TI (therapeutic index) = CC50/ EC50.

For anti-HIV-1 activity assay, the cytotoxicity against C8166 cells (CC50) was assessed using the MTT
method and anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV-1
(EC50), using AZT as a positive control (EC50 = 0.045 g/mL and CC50  200 g/mL).15 The results are
shown in Table 2. The results reveal that compound 3 showed significant potential anti-HIV-1 activity
with therapeutic index (TI) values above 156.9. Compounds 1, 2, 6, and 7 also showed moderate
anti-HIV-1 activitiy with TI values above 30.
The cytotoxicity tests for the isolates were performed using a previously reported procedure.16 All
treatments were performed in triplicate. In the MTT assay, the IC50 was defined as the concentration of
the test compound resulting in a 50% reduction of absorbance compared with untreated cells. The
cytotoxic abilities against HL-60, Hep-G2, KB and MDA-MB-231 tumor cell lines by MTT-assay (with
camptothecin as the positive control) were shown in Table 3. The results showed that compound 2 have
high cytotoxic abilities with IC50 values close to these of the positive control. Other compounds also
showed moderate cytotoxic abilities.
Table 3. Cytotoxicity of compounds 1-7
Cell lines
HepG2
Compound
HL-60
5.46
KB
8.24
3.10
6.23
6.56
16.56
10.35
8.46
1.82
MDA-MB-231
7.61
1
6.12
2
3
4
5
6
7
2.41
6.15
2.37
9.49
12.20
11.11
6.63
3.93
8.26
21.37
12.65
6.12
11.67
9.92
14.87
11.67
1.82
9.94
0.86
9.13
2.20
Camptothecin
Data of IC50 values in μmol/L. For a compound to be deemed effective, an IC50 value < 100
μmol/L is required. Camptothecin was used as a positive control.
HL-60, human acute promyelocytic leukemia; Hep-G2, human hepatocellular carcinoma; KB,
human oropharyngeal epidermoid carcinoma; MDA-MB-231, human breast cancer cells.
EXPERIMENTAL
General. Optical rotation was measured in Horiba SEPA-300 high sensitive polarimeter. IR spectra were
obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ESI-MS were measured on a VG
1
Auto Spec-3000 MS spectrometer. H, 13C and 2D NMR spectra were recorded on Bruker DRX-500
instrument with TMS as internal standard. Column chromatography was performed on silica gel (200-300
mesh), or on silica gel H (10～40 m, Qingdao Marine Chemical Inc., China). Second separate was used
an Agilent 1100 HPLC equipped with ZORBAX-C18 (21.2 mm × 250 mm, 7.0 m) column and DAD
detector.
Plant material. The flowers of R. rugosa were collected in Dali Prefecture, Yunnan Province, People’s

Republic of China, in September 2010. The identification of the plant material was verified by Prof. Chen
Y. J (Yunnan Nationalities University). A voucher specimen (YNNI 10-9-56) has been deposited in our
laboratory.
Extraction and Isolation. The air-dried and powdered flowers of R. rugosa (2.5 kg) were extracted four
times with 70% MeOH (4 × 2.0 L) at room temperature and filtered. The crude extract (92 g) was applied
to silica gel (200–300 mesh) column chromatography, eluting with a CHCl3-acetone gradient system
(20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions A–F. The further separation of fraction C (8:2, 11.5 g)
by silica gel column chromatography, eluted with CHCl3-MeOH (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures
D1–D5. Fraction D1 (9:1, 1.57 g) was subjected to preparative HPLC (35% MeOH, flow rate 12 mL/min)
to give 1 (15.2 mg), 2 (13.8 mg), 3 (8.26 mg), and 6 (13.2 mg). The further separation of fraction F (1:1,
23.6 g) by silica gel column chromatography, and preparative HPLC (15% MeOH, flow rate 12 mL/min)
to give 4 (22.1 mg) 5 (28.4 mg), and 7 (16.3 mg).
Anti-HIV1 Assays. The cytotoxicity assay against C8166 cells (CC50) was assessed using the MTT
method and anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV-1
(EC50).15
Cytotoxicity Assay. The cytotoxicity tests for the isolates were performed by against HL-60, Hep-G2,
KB and MDA-MB-231 tumor cell lines by MTT-assay (with camptothecin as the positive control).16
Rugaurone A (1). Obtained as a pale yellow gum; UV (MeOH), max (log ) 338 (3.69), 256 (3.87), 210
1
(4.18) nm; IR (KBr) max 3421, 2908, 1667, 1610, 1492, 1441, 1269, 1143, 1078, 851, 822, 775 cm-1; H
13
NMR and C NMR data (C5ND5, 500 MHz and 150 MHz, respectively), Table 1; ESIMS (positive ion
mode) m/z 321 [M+Na]+; HRESIMS (positive ion mode) m/z 321.0732 [M+Na]+ (calcd 321.0739 for
C17H14O5Na).
Rugaurone B (2). Obtained as a pale yellow gum; UV (MeOH), max (log ) 336 (3.62), 258 (3.81), 210
1
(4.22) nm; IR (KBr) max 3423, 2905, 1665, 1612, 1490, 1444, 1267, 1143, 1075, 852, 825, 774 cm-1; H
13
NMR and C NMR data (C5ND5, 500 MHz and 150 MHz, respectively), Table 1; ESIMS (positive ion
mode) m/z 307 [M+Na]+; HRESIMS (positive ion mode) m/z 307.0588 [M+Na]+ (calcd 307.0582 for
C16H12O5Na).
Rugaurone C (3). Obtained as a pale yellow gum; UV (MeOH), max (log ) 340 (3.65), 256 (3.88), 210
1
(4.14) nm; IR (KBr) max 3425, 2906, 1662, 1616, 1493, 1440, 1264, 1148, 1076, 850, 822, 776 cm-1; H
13
NMR and C NMR data (C5ND5, 500 MHz and 150 MHz, respectively), Table 1; ESIMS (positive ion
mode) m/z 321 [M+Na]+; HRESIMS (positive ion mode) m/z 321.0746 [M+Na]+ (calcd 321.0739 for
C17H14O5Na).

ACKNOWLEDGMENT
This project was supported financially by the Excellent Scientific and Technological Team of Yunnan
High School (2010CI08), the Yunnan University of Nationalities Green Chemistry and Functional
Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of
Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan University of Nationalities)
(2010XY08).
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