Synthesis and biological evaluation of novel furozan-based nitric oxide-releasing derivatives of oridonin as potential anti-tumor agents

Article abstract of DOI:10.3390/molecules17067556

To search for novel nitric oxide (NO) releasing anti-tumor agents, a series of novel furoxan/oridonin hybrids were designed and synthesized. Firstly, the nitrate/nitrite levels in the cell lysates were tested by a Griess assay and the results showed that these furoxan-based NO-releasing derivatives could produce high levels of NO in vitro. Then the anti-proliferative activity of these hybrids against four human cancer cell lines was also determined, among which, 9h exhibited the most potential anti-tumor activity with IC50 values of 1.82 μM against K562, 1.81 μM against MGC-803 and 0.86 μM against Bel-7402, respectively. Preliminary structure-activity relationship was concluded based on the experimental data obtained. These results suggested that NO-donor/natural product hybrids may provide a promising approach for the discovery of novel anti-tumor agents.

Full text of DOI:10.3390/molecules17067556

Molecules 2012, 17, 7556-7568; doi:10.3390/molecules17067556
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of Novel Furozan-Based
Nitric Oxide-Releasing Derivatives of Oridonin as Potential
Anti-Tumor Agents
Dahong Li 1,2,†, Lei Wang 1,3,†, Hao Cai 1, Yihua Zhang 2,3,* and Jinyi Xu 1,2,
*
1
Department of Medicinal Chemistry, China Pharmaceutical University, 24 Tongjia Xiang,
Nanjing 210009, China
2
3
State Key Laboratory of Natural Medicines, China Pharmaceutical University, 24 Tongjia Xiang,
Nanjing 210009, China
Center of Drug Discovery, China Pharmaceutical University, 24 Tongjia Xiang, Nanjing 210009,
China
†
These authors contributed equally to this work.
* Authors to whom correspondence should be addressed; E-Mails: zyhtgd@sohu.com (Y.Z.);
jinyixu@china.com (J.X.); Tel.: +86-25-8327-1445 (J.X.); Fax: +86-25-8330-2827 (J.X.).
Received: 25 April 2012; in revised form: 8 June 2012 / Accepted: 12 June 2012 /
Published: 18 June 2012
Abstract: To search for novel nitric oxide (NO) releasing anti-tumor agents, a series of
novel furoxan/oridonin hybrids were designed and synthesized. Firstly, the nitrate/nitrite
levels in the cell lysates were tested by a Griess assay and the results showed that these
furoxan-based NO-releasing derivatives could produce high levels of NO in vitro. Then the
anti-proliferative activity of these hybrids against four human cancer cell lines was also
determined, among which, 9h exhibited the most potential anti-tumor activity with IC50
values of 1.82 µM against K562, 1.81 µM against MGC-803 and 0.86 µM against
Bel-7402, respectively. Preliminary structure-activity relationship was concluded based on
the experimental data obtained. These results suggested that NO-donor/natural product
hybrids may provide a promising approach for the discovery of novel anti-tumor agents.
Keywords: NO donor; oridonin; hybrid; anti-tumor agents; SAR

Molecules 2012, 17
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1. Introduction
Nitric oxide, a special gaseous molecular, is a key mediator involved in many physiological and
pathological processes [1,2]. High concentrations of NO and its metabolic derivatives can modify
functional proteins, leading to cell cycle arrest and apoptosis, particularly in tumor cells [3–5]. Indeed,
some synthesized NO-releasing compounds have shown cytotoxic activity against human carcinoma
cells in vitro and inhibit the growth and metastasis of cancers in vivo [6–8]. So, the NO-based
anti-cancer agents have been investigating for cancer therapy at clinic [9,10]. Furoxans represent one
class of NO donors that can produce high levels of NO and exhibit strong anti-cancer activity [11,12].
In the previous work by our group, several classes of NO-releasing compounds have been reported,
which possess strong anti-proliferative activity against human carcinoma cells in vitro, inhibition of
cancer cells growth in vivo and the ability to increase sensitivity of Pgp-mediated multidrug resistance
(MDR) in solid tumors, separately [13–17]. These results motivated us to further design some novel
NO-donor/natural product hybrids.
Oridonin (1, see Figure 1) is a commercially available natural ent-kaurene diterpenoid that has
recently attracted much attention because of its anti-tumor activity with a mechanism of inhibition
effect on nuclear factor κB (NF-κB) activation, induction of G2/M phase arrest and apoptosis [18].
Oridonin has been safely used for the treatment of hepatoma and promyelocytic leukemia in China for
many years. In previous studies, we found that a series of novel 1-O- and 14-O-derivatives of oridonin
exhibited stronger cytotoxicity against six cancer cell lines in vitro and some of them had stronger
anti-tumor activity than the parent compound 1 and the positive control cyclophosphamide in mice
with H22 liver tumor in vivo [19–21]. Hence, it may be a desired lead compound using for further
design of novel furoxan-based NO-releasing derivatives for the development of anti-tumor agents.
Therefore, a series of novel furozan-based nitric oxide-releasing derivatives of oridonin were designed
and synthesized.
Figure 1. The structure and atom numbering of oridonin.
2. Results and Discussion
2.1. Synthesis of Furoxan-Based NO Donor
The substituted furoxans were prepared in five steps in the following way (Scheme 1). The starting
material benzenethiol (2) was converted to 2-(phenylthio)acetic acid (4) by treatment with chloroacetic
acid (3). Then, compound 4 was oxidized by 30% H2O2 aqueous solution to give 2-(phenylsulfonyl)

Molecules 2012, 17
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acetic acid (5) and fuming HNO3 was added to obtain diphenylsulfonylfuroxan (6), which was then
converted to various monophenylsulfonylfuroxans 7a–c by treatment with the corresponding diol.
Finally, anhydrides were added and furoxan-based NO donors 8a–i were obtained.
Scheme 1. Synthesis of compounds 8a–i.
2.2. Synthesis of Furoxan/Oridonin Hybrids
The resulting furoxans 8a–i were treated with oridonin to give the target compounds 9a–i, as shown
in Scheme 2.
Scheme 2. Synthesis of compounds 9a–i.
Treatment of oridonin with 2,2-dimethoxypropane (DMP) in the presence of p-toluenesulfonic acid
(TsOH) in acetone afforded 7,14-(1-methylethylene)-dioxyoridonin derivative 10. Compound 10 upon
reaction with Ac2O/pyridine yielded 1-O-acetyl derivative 11. Deprotection of 11 with AcOH gave
1-O-acetyl-oridonin 12 in quantitative yield. Target compounds 13a–i were prepared by reaction of 12
with furoxan-based NO donors 8a–i in the presence of 4-dimethylaminopyridine/1-ethyl-3-(3-
dimethyllaminopropyl)carbodiimide hydrochloride (DMAP/EDCI) in CH2Cl2, as shown in Scheme 3.

Molecules 2012, 17
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Scheme 3. Synthesis of compounds 13a–i.
2.3. NO-Releasing Test of Hybrids 9a–i and 13a–i in Vitro
The levels of nitrate/nitrite in the lysates of target compounds 9a–i and 13a–i were determined at
100 μM by Griess assay over a duration of 0–60 min. As shown in Figure 2, variable levels of NO
were produced by compounds 9a–i and 13a–i. The concentration of NO increased with time, and at the
time point of 60 min, all tested compounds produced more than 15 μmol/L of NO. The amount of NO
released by compounds 13a–i (13g with the lowest level of 16.88 μmol/L at the 60 min time point) was
less than that of 9a–i (9d with the highest level of 45.44 μmol/L at the 60 min time point).
Figure 2. Variable levels of NO produced by compounds (a) 9a–i and (b) 13a–i.

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2.4. Anti-proliferative Activity in Vitro
The anti-proliferative activity of oridonin and its NO-donor hybrids 9a-i as well as 1-O-derivatives
of oridonin (12) and its NO-donor hybrids 13a–i was evaluated against four human cancer cell lines
(Bel-7402, K562, MGC-803, CaEs-17) by MTT assay. The results are shown in Table 1 [22]. All the
target compounds 9a–i and 13a–i exhibited stronger anti-proliferative activity than their parent
compounds 1 (oridonin) and 12 (1-oxo-oridonin), correspondingly. Among them, 13a–i released less
NO (Figure 2) and showed less potent anti-proliferative activity than 9a–i. For example, 13e with IC50
value of 2.13 µM compared to 9e (1.33 µM) against Bel-7402 cells, 13g with IC50 value of 2.45 µM
compared to 9g (1.33 µM) against MGC803 cells, and so on. These results and our previous
studies [13–17] indicated that the releasing of NO contributed to anti-proliferative activity and higher
levels of NO releasing could produce stronger activity.
Table 1. IC50 values of the furoxan/oridonin hybrids against four human tumor cell lines a.
Compd.
Taxol b
Oridonin
9a
Bel-7402
K562
MGC-803
CaEs-17
1.89 ± 0.09 0.41 ± 0.02 c 0.85 ± 0.06 c 0.43 ± 0.03 d
7.48 ± 0.53
2.37 ± 0.85
1.91 ± 0.09
2.23 ± 0.04
1.89 ± 0.22
1.33 ± 0.15
1.97 ± 0.04
4.76 ± 0.32
4.33 ± 0.14
3.46 ± 0.60
4.02 ± 0.05
3.78 ± 0.19
2.85 ± 0.03
3.72 ± 0.26
5.69 ± 0.39 11.03 ± 1.02
3.22 ± 0.19
2.57 ± 0.07
3.46 ± 0.23
3.08 ± 0.47
2.21 ± 0.16
3.23 ± 0.25
8.46 ± 0.05
6.98 ± 0.20
8.17 ± 1.01
8.04 ± 0.18
6.77 ± 0.32
8.09 ± 0.47
9b
9c
9d
9e
9f
9g
9h
9i
0.95 ± 0.21 c 1.94 ± 0.14
0.86 ± 0.08 c 1.82 ± 0.07
0.97 ± 0.10 c 1.92 ± 0.34
1.98 ± 0.13 4.81 ± 0.10 c
1.81 ± 0.20 4.56 ± 0.32 c
1.90 ± 0.11
4.05 ± 0.04
3.77 ± 0.31
5.24 ± 0.18
7.24 ± 0.41
5.30 ± 0.28
12
3.21 ± 0.25
2.85 ± 0.14
2.19 ± 0.19
2.76 ± 0.42
2.70 ± 0.09
2.13 ± 0.17
2.66 ± 0.30
1.94 ± 0.13
1.72 ± 0.08
1.86 ± 0.15
5.06 ± 0.18
4.65 ± 0.07
3.85 ± 0.06
4.11 ± 0.15
4.08 ± 0.30
3.04 ± 0.21
3.97 ± 0.16
2.22 ± 0.29
2.08 ± 0.34
2.65 ± 0.08
13a
13b
13c
13d
13e
13f
13g
13h
13i
2.90 ± 0.12 4.11 ± 0.07 c
3.65 ± 0.40
3.64 ± 0.12
5.22 ± 0.12
5.38 ± 0.24
2.79 ± 0.10 4.00 ± 0.31 c
3.42 ± 0.27
5.11 ± 0.39
2.45 ± 0.51 3.28 ± 0.06 c
2.22 ± 0.29 3.24 ± 0.23 c
2.41 ± 0.16 3.13 ± 0.21 c
a
b
Results are expressed as IC50 values in µM and the values are means ± SD; n = 3. Taxol was
used as a positive control. c p < 0.05 versus oridonin; d p < 0.01 versus oridonin.
Among the tested compounds, the series 9g–i and 13g–i with a o-C6H4 linker (R1) (compounds g–i)
showed lower IC50 values than the corresponding compounds a–f. Compared the IC50 values of the
compounds of series a–c with d–f in different cell lines, there was a decline with the extension of the
length of R1. In general, when R1 were aromatic groups (compounds g–i), the activity was stronger
than those with alkyl groups. While R1 were alkyl groups, IC50 values decreased with lengthening of
carbon chain. In almost all cases (except 13h), when the length of R2 is three carbons, more potential

Molecules 2012, 17
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anti-proliferative activity was observed than those of two and four carbons, correspondingly (for
instance, 9b > 9a and 9c, 9e > 9d and 9f, 9h > 9g and 9i, 13b > 13a and 13c, 13e > 13d and 13f). This
suggested that the length of the linker group R2 with three carbons would be more suitable. In all the
target synthetic hybrids, compound 9h (R1=o-C6H4; R2=CH2CH2CH2) exhibited the most potential
anti-tumor activity against tested cell lines: IC50 values of 0.86 µM against Bel-7402 (stronger than
parent compound oridonin of 7.48 µM and positive control taxol of 1.89 µM), 1.82 µM against K562,
1.81 µM against MGC-803 (stronger than oridonin of 5.69 µM) and 4.56 µM against CaEs-17
(stronger than oridonin of 11.03 µM). Subsequent design and synthesis of novel NO releasing
anti-tumor agents based on present SAR and more intensive biological studies were undertaking.
3. Experimental
3.1. Chemistry
All commercially available solvents and reagents were used without further purification. Melting
points were taken on XT-4 micro melting point apparatus and are uncorrected. Infrared (IR) spectra
1
(KBr pellets) were recorded on a Nicolet Impact 410 instrument (Madison, WI, USA). H-NMR
spectra were recorded at 300 MHz with a Bruker AV-300 spectrometer (Karlsruhe, Germany) in the
indicated solvents (TMS as internal standard): The values of the chemical shifts are expressed in
δ values (ppm) and the coupling constants (J) in Hz. Mass spectra were obtained using FTMS-2000
(Madison, WI, USA). HR-MS were obtained using an Agilent QTOF 6520 (Palo Alto, CA, USA).
Compounds 2–4 were commercially available. Compounds 5, 6, 7a–c, 10, 11 and 12 were synthesized,
as previously described [13,19,20].
3.1.1. General Procedure for the Preparation of 8a–i
Compound 7a–c (2 mmol) in pyridine (5 mL) was mixed with the corresponding anhydride
(4 mmol) by stirring at room temperature for 6–12 h. The mixture was concentrated in vacuo,
dissolved in H2O (15 mL) and extracted with CH2Cl2 (15 mL × 3). The organic layers were combined,
washed with water and saturated NaCl solution sequentially, dried over anhydrous Na2SO4, and
concentrated in vacuo. The crude products 8a–i used for the next step without further purification.
3.1.2. General Procedure for the Preparation of 9a–i and 13a–i
Compounds 8a–i (0.24 mmol) were dissolved in CH2Cl2 (10 mL) and stirred at room temperature.
Oridonin or its derivative 12 (0.2 mmol), EDCI (93 mg, 0.6 mmol) and DMAP (catalytic amount) were
added. After 8–12 h, the reaction mixture was washed with water and saturated NaCl solution
sequentially, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude products were
purified by column chromatography (MeOH/CH2Cl2 1:200 v/v) to give the title compounds.
ent-1α,6β,7β-Trihydroxy-(14β-O-(4-oxo-butyric
acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-4)
oxyethyl))-15-oxo-7,20-epoxy-16-kaurene (9a). Yield 41%, m.p. 93–95 C; 1H-NMR (CDCl3), δ (ppm)
1.12 (3H, s, –CH3), 1.25 (3H, s, –CH3), 3.16 (1H, d, J = 9.6 Hz, 13-CH), 3.49 (1H, m, 1-CH), 3.76
(1H, m, 6-CH), 4.02(1H, s, 1-OH), 4.06, 4.30 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.51 (2H, t,

Molecules 2012, 17
7562
J = 4.5 Hz, –CH2), 4.62 (2H, t, J = 4.8 Hz, –CH2), 5.53 (1H, s, 17-CH2), 5.92 (1H, s, 14-CH), 6.04
(1H, d, J = 10.8 Hz, 6-OH), 6.15 (1H, s, 17-CH2), 7.64 (2H, t, J = 7.2 Hz, Ar-H), 7.77 (1H, t, J = 7.5 Hz,
Ar-H), 8.07 (2H, d, J = 8.1 Hz, Ar-H); MS(ESI) m/z: 755.4 [M+Na]+; HR-MS (ESI, M+Na) m/z: calcd
for C34H40N2NaO14S: 755.2092, found 755.2095.
ent-1α,6β,7β-Trihydroxy-(14β-O-(4-oxobutyric
acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-4)-
oxypropyl))-15-oxo-7,20-epoxy-16-kaurene (9b). Yield 34%, m.p. 86–88 C; IR υmax 3419, 2955,
1
2025, 1736, 1615, 1554, 1451, 1359, 733, 686 cm−1; H-NMR (CDCl3), δ (ppm) 1.11 (6H, s, –CH3),
3.14 (1H, d, J = 9.6 Hz, 13-CH), 3.49 (1H, m, 1-CH), 3.74 (1H, m, 6-CH), 4.07 (1H, s, 1-OH), 4.28
(2H, m, –CH2), 4.06, 4.30 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.50 (2H, t, J = 6.0 Hz, –CH2),
5.52 (1H, s, 17-CH2), 5.89 (1H, s, 14-CH), 6.07 (1H, d, J = 10.8 Hz, 6-OH), 6.15 (1H, s, 17-CH2), 7.63
(2H, t, J = 7.8 Hz, Ar-H), 7.77 (1H, t, J = 7.2 Hz, Ar-H), 8.06 (2H, d, J = 7.2 Hz, Ar-H); MS(ESI) m/z:
764.3 [M+NH4]+, 747.3 [M+H]+, 781.4 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for
C35H42N2NaO14S: 769.2249, found 769.2254.
ent-1α,6β,7β-Trihydroxy-(14β-O-(4-oxobutyric
acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-4)-
oxybutyl))-15-oxo-7,20-epoxy-16-kaurene (9c). Yield 48%, m.p. 83–85 C; IR υmax 3417, 2955, 2025,
1
1734, 1635, 1554, 1451, 1367, 733, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.11 (6H, s, –CH3), 3.15
(1H, d, J = 9.6 Hz, 13-CH), 3.48 (1H, m, 1-CH), 3.75 (1H, m, 6-CH), 4.07 (1H, s, 1-OH), 4.17 (2H, m,
–CH2), 4.05, 4.29 (each 1H, dd, JA = JB = 11.1 Hz, 20-CH2), 4.45 (2H, t, J = 6.0 Hz, –CH2), 5.52 (1H,
s, 17-CH2), 5.90 (1H, s, 14-CH), 6.06 (1H, d, J = 10.8 Hz, 6–OH), 6.15 (1H, s, 17-CH2), 7.63 (2H, t,
J = 7.5 Hz, Ar-H), 7.77 (1H, t, J = 7.2 Hz, Ar-H), 8.06 (2H, d, J = 7.2 Hz, Ar-H); MS(ESI) m/z: 778.2
[M+NH4]+, 761.1 [M+H]+, 795.2 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for C36H44N2NaO14S:
783.2405, found 783.2411.
ent-1α,6β,7β-Trihydroxy-(14β-O-(5-oxo-pentanoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-
4)-oxyethyl))-15-oxo-7,20-epoxy-16-kaurene (9d). Yield 45%, m.p. 86–89 C; IR υmax 3405, 2952,
1
2025, 1739, 1618, 1554, 1451, 1360, 732, 676 cm–1; H-NMR (CDCl3), δ (ppm) 1.10 (6H, s, –CH3),
3.16 (1H, d, J = 9.9 Hz, 13-CH), 3.48 (1H, m, 1-CH), 3.73 (1H, m, 6-CH), 4.14 (1H, s, 1-OH), 4.05,
4.30 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.47 (2H, m, –CH2), 4.61 (2H, m, –CH2), 5.50 (1H, s,
17-CH2), 5.87 (1H, s, 14-CH), 6.06 (1H, d, J = 10.5 Hz, 6-OH), 6.13 (1H, s, 17-CH2), 7.63 (2H, t,
J = 7.5 Hz, Ar-H), 7.77 (1H, t, J = 7.5 Hz, Ar-H), 8.06 (2H, d, J = 7.8 Hz, Ar-H); MS(ESI) m/z: 764.0
[M+NH4]+, 747.1 [M+H]+, 781.2 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for C35H42N2NaO14S:
769.2249, found 769.225.
ent-1α,6β,7β-Trihydroxy-(14β-O-(5-oxopentanoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-4)-
oxypropyl))-15-oxo-7,20-epoxy-16-kaurene (9e). Yield 42%, m.p. 80–82 C; IR υmax 3439, 2954, 2025,
1
1734, 1615, 1554, 1452, 1383, 733, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.11 (6H, s, –CH3), 3.16
(1H, d, J = 9.6 Hz, 13-CH), 3.50 (1H, m, 1-CH), 3.75 (1H, m, 6-CH), 4.07, 4.30 (each 1H, dd,
JA = JB = 10.8 Hz, 20-CH2), 4.25 (2H, t, J = 6.0 Hz, –CH2), 4.50 (2H, t, J = 6.0 Hz, –CH2), 5.51 (1H, s,
17-CH2), 5.87 (1H, s, 14-CH), 6.05 (1H, d, J = 10.5 Hz, 6–OH), 6.14 (1H, s, 17-CH2), 7.63 (2H, t,
J = 7.5 Hz, Ar-H), 7.77 (1H, t, J = 7.5 Hz, Ar-H), 8.05 (2H, d, J = 7.5 Hz, Ar-H); MS(ESI) m/z: 778.3

Molecules 2012, 17
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[M+NH4]+, 761.3 [M+H]+, 795.4 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for C36H44N2NaO14S:
783.2405, found 783.2419.
ent-1α,6β,7β-Trihydroxy-(14β-O-(5-oxo-pentanoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-
4)-oxybutyl))-15-oxo-7,20-epoxy-16-kaurene (9f). Yield 37%, m.p. 92–94 C; IR υmax 3440, 2955,
1
2025, 1733, 1615, 1554, 1451, 1367, 732, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.11 (6H, s, –CH3),
3.39 (1H, d, J = 9.9 Hz, 13-CH), 3.50 (1H, m, 1-CH), 3.76 (1H, m, 6-CH), 4.06 (1H, s, 1-OH), 4.08,
4.34 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.46 (1H, m, –CH2), 4.57 (1H, m, –CH2), 4.58 (2H, m,
–CH2), 5.56 (1H, s, 17-CH2), 6.05 (1H, d, J = 10.5 Hz, 6-OH), 6.07 (1H, s, 14-CH), 6.17 (1H, s,
17-CH2), 7.52 (2H, m, Ar-H), 7.57 (3H, m, Ar-H), 7.75 (2H, m, Ar-H), 8.06 (2H, d, J = 7.5 Hz, Ar-H);
MS(ESI) m/z: 792.3 [M+NH4]+, 775.5 [M+H]+, 809.6 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for
C37H46N2NaO14S: 797.2562, found 797.2565.
ent-1α,6β,7β-Trihydroxy-(14β-O-(2-formyl benzoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-
4)-oxyethyl))-15-oxo-7,20-epoxy-16-kaurene (9g). Yield 53%, m.p. 123–125 C; IR υmax 3392, 2951,
1
2025, 1714, 1618, 1553, 1451, 1363, 739, 685 cm–1; H-NMR (CDCl3), δ (ppm) 1.09 (6H, s, –CH3),
3.37 (1H, d, J = 9.6 Hz, 13-CH), 3.50 (1H, m, 1-CH), 3.72 (1H, m, 6-CH), 4.05 (1H, s, 1–OH), 4.07,
4.36 (each 1H, dd, JA = JB = 10.2 HZ, 20-CH2), 4.67 (2H, m, –CH2), 4.76 (2H, m, –CH2), 5.54 (1H, s,
17-CH2), 6.04 (1H, d, J = 12.0 Hz, 6-OH), 6.07 (1H, s, 14-CH), 6.14 (1H, s, 17-CH2), 7.46 (2H, t,
J = 7.8 Hz, Ar-H), 7.58 (4H, m, Ar-H), 7.78 (1H, m, Ar-H), 8.01 (2H, d, J = 7.5 Hz, Ar-H); MS(ESI)
m/z: 798.3 [M+NH4]+, 781.2 [M+H]+, 815.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for
C38H40N2NaO14S: 803.2092, found 755.2093.
ent-1α,6β,7β-Trihydroxy-(14β-O-(2-formyl benzoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-
4)-oxypropyl))-15-oxo-7,20-epoxy-16-kaurene (9h). Yield 47%, m.p. 113–115 C; IR υmax 3418, 2954,
1
2025, 1714, 1616, 1554, 1451, 1384, 736, 685 cm–1; H-NMR (CDCl3), δ (ppm) 1.11 (6H, s, –CH3),
3.39 (1H, d, J = 9.9 Hz, 13-CH), 3.50 (1H, m, 1-CH), 3.76 (1H, m, 6-CH), 4.06 (1H, s, 1-OH), 4.08,
4.34 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.46 (1H, m, –CH2), 4.57 (1H, m, –CH2), 4.58 (2H, m,
–CH2), 5.56 (1H, s, 17-CH2), 6.05 (1H, d, J = 10.5 Hz, 6-OH), 6.07 (1H, s, 14-CH), 6.17 (1H, s,
17-CH2), 7.52 (2H, m, Ar-H), 7.57 (3H, m, Ar-H), 7.75 (2H, m, Ar-H), 8.06 (2H, d, J = 7.5 Hz, Ar-H);
MS(ESI) m/z: 812.3 [M+NH4]+, 795.3 [M+H]+, 829.4 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for
C39H42N2NaO14S: 817.2249, found 755.2252.
ent-1α,6β,7β-Trihydroxy-(14β-O-(2-formylbenzoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-oxide-
4)-oxybutyl))-15-oxo-7,20-epoxy-16-kaurene (9i). Yield 50%, m.p. 108–110 C; IR υmax 3384, 2952,
1
2025, 1715, 1615, 1553, 1450, 1368, 734, 685 cm–1; H-NMR (CDCl3), δ (ppm) 1.10 (6H, s, –CH3),
3.32 (1H, d, J = 9.9 Hz, 13-CH), 3.50 (1H, m, 1-CH), 3.76 (1H, m, 6-CH), 4.08, 4.34 (each 1H, dd,
JA = JB = 8.4 HZ, 20-CH2), 4.44 (2H, m, –CH2), 4.50 (2H, t, J = 5.4 Hz, –CH2), 5.30 (1H, s, 1-OH),
5.56 (1H, s, 17-CH2), 6.04 (1H, d, J = 10.5 Hz, 6-OH), 6.09 (1H, s, 14-CH), 6.61 (1H, s, 17-CH2), 7.53
(3H, m, Ar-H), 7.61 (2H, m, Ar-H), 7.76 (2H, m, Ar-H), 8.06 (2H, d, J = 7.8 Hz, Ar-H); MS(ESI) m/z:
826.1 [M+NH4]+, 809.0 [M+H]+, 843.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for C40H44N2NaO14S:
831.2405, found 831.2411.

Molecules 2012, 17
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ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(4-oxobutyric acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-
oxide-4)-oxyethyl))-15-oxo-7,20-epoxy-16-kaurene (13a). Yield 40%, m.p. 105–107 C; IR υmax 3384,
1
2958, 2025, 1739, 1618, 1554, 1452, 1363, 732, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.12 (6H, s,
–CH3), 2.17 (3H, s, –CH3), 3.13 (1H, d, J = 9.6 Hz, 13-CH), 3.76 (1H, m, 6-CH), 4.17, 4.28 (each 1H,
dd, JA = JB = 10, 5 Hz, 20-CH2), 4.51 (2H, m, –CH2), 4.61 (1H, m, 1-CH), 4.62 (2H, m, –CH2), 5.52
(1H, s, 17-CH2), 5.87 (1H, s, 14-CH), 6.09 (1H, d, J = 9.3 Hz, 6–OH), 6.15 (1H, s, 17-CH2), 7.63 (2H,
t, J = 7.2 Hz, Ar-H), 7.91 (1H, t, J = 7.8 Hz, Ar-H), 8.07 (2H, d, J = 7.2 Hz, Ar-H); MS(ESI) m/z:
775.3 [M+H]+, 809.4 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for C36H42N2NaO15S: 797.2198,
found 797.2207.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(4-oxobutyric acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-
oxide-4)-oxypropyl))-15-oxo-7,20-epoxy-16-kaurene (13b). Yield 51%, m.p. 95–97 C; IR υmax 3383,
1
2957, 2025, 1738, 1615, 1554, 1452, 1373, 733, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.12 (6H, s,
–CH3), 1.99 (3H, s, –CH3), 3.12 (1H, d, J = 9.6 Hz, 13-CH), 3.77 (1H, m, 6-CH), 4.31 (2H, m, –CH2),
4.17, 4.34 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.51 (2H, t, J = 6.0 Hz, –CH2), 4.62 (1H, m,
1-CH), 5.52 (1H, s, 17-CH2), 5.85 (1H, s, 14-CH), 6.12 (1H, d, J = 11.1 Hz, 6–OH), 6.15 (1H, s,
17-CH2), 7.64 (2H, t, J = 7.8 Hz, Ar-H), 7.77 (1H, t, J = 7.5 Hz, Ar-H), 8.06 (2H, d, J = 7.2 Hz,
Ar-H); MS(ESI) m/z: 806.3 [M+NH4]+, 789.3 [M+H]+, 823.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z:
calcd for C37H44N2NaO15S: 811.2355, found 811.2362.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(4-oxobutyric acid-(3-phenylsulfonyl-1,2,5-oxadiazole-2-
oxide-4)-oxybutyl))-15-oxo-7,20-epoxy-16-kaurene (13c). Yield 48%, m.p. 109–111 C; IR υmax 3385,
1
2957, 2025, 1737, 1616, 1554, 1451, 1371, 733, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.12 (6H, s,
–CH3), 1.99 (3H, s, CH3), 3.13 (1H, d, J = 9.6 Hz, 13-CH), 3.78 (1H, m, 6-CH), 4.19 (2H, m, –CH2),
4.11, 4.27 (each 1H, dd, JA = JB = 10.5 Hz, 20-CH2), 4.46 (2H, t, J = 7.5 Hz, –CH2), 4.62 (1H, m,
1-CH), 5.53 (1H, s, 17-CH2), 5.88 (1H, s, 14-CH), 6.11 (1H, d, J = 10.5 Hz, 6–OH), 6.16 (1H, s,
17-CH2), 7.64 (2H, t, J = 7.5 Hz, Ar-H), 7.78 (1H, t, J = 7.2 Hz, Ar-H), 8.07 (2H, d, J = 7.8 Hz,
Ar-H); MS(ESI) m/z: 820.4 [M+NH4]+, 803.3 [M+H]+, 837.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z:
calcd for C38H46N2NaO15S: 825.2511, found 825.2525.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(4-oxopentanoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-
2-oxide-4)-oxyethyl))-15-oxo-7,20-epoxy-16-kaurene (13d). Yield 42%, m.p. 92–94 C; IR υmax 3530,
1
3386, 2956, 2025, 1738, 1618, 1553, 1451, 731, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.12 (6H, s,
CH3), 1.99 (3H, s, –CH3), 3.16 (1H, d, J = 9.9 Hz, 13-CH), 3.75 (1H, m, 6-CH), 4.17, 4.27 (each 1H,
dd, JA = JB = 10.5 Hz, 20-CH2), 4.49 (2H, m, –CH2), 4.61 (2H, m, –CH2), 5.50 (1H, s, 17-CH2), 5.83
(1H, s, 14-CH), 6.06 (1H, d, J = 9.6 Hz, 6–OH), 6.13 (1H, s, 17-CH2), 7.63 (2H, t, J = 7.8 Hz, Ar-H),
7.76 (1H, t, J = 7.5 Hz, Ar-H), 8.06 (2H, d, J = 7.5 Hz, Ar-H); MS(ESI) m/z: 806.4 [M+NH4]+, 789.2
[M+H]+, 823.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for C37H44N2NaO15S: 811.2355, found
811.2367.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(5-oxopentanoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-
2-oxide-4)-oxypropyl))-15-oxo-7,20-epoxy-16-kaurene (13e). Yield 36%, m.p. 86–88 C; IR υmax 3421,
1
2958, 2025, 1737, 1616, 1554, 1452, 1374, 732, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.12 (6H, s,

Molecules 2012, 17
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–CH3), 2.19 (3H, s, –CH3), 3.16 (1H, d, J = 10.2 Hz, 13-CH), 3.77 (1H, m, 6-CH), 4.20, 4.38 (each
1H, dd, JA = JB = 10.5 Hz, 20-CH2), 4.26 (2H, t, J = 6.0 Hz, –CH2), 4.50 (2H, t, J = 6.0 Hz, –CH2),
4.62 (1H, m, 1-CH), 5.52 (1H, s, 14-CH), 5.83 (1H, s, 17-CH2), 6.06 (1H, d, J = 10.5 Hz, 6–OH), 6.15
(1H, s, 17-CH2), 7.63 (2H, t, J = 7.5 Hz, Ar-H), 7.77 (1H, t, J = 7.2 Hz, Ar-H), 8.06 (2H, d,
J = 7.5 Hz, Ar-H); MS(ESI) m/z: 803.3 [M+H]+, 837.4 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for
C38H46N2NaO15S: 825.2511, found 825.2523.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(5-oxopentanoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-
2-oxide-4)-oxybutyl))-15-oxo-7,20-epoxy-16-kaurene (13f). Yield 40%, m.p. 98–101 C; IR υmax 3394,
1
2957, 2025, 1737, 1617, 1554, 1451, 1373, 732, 686 cm–1; H-NMR (CDCl3), δ (ppm) 1.12 (6H, s,
–CH3), 2.35 (3H, s, –CH3), 3.17 (1H, d, J = 9.3 Hz, 13-CH), 3.76 (1H, m, 6-CH), 4.20 (2H, t, J = 6.0 Hz,
–CH2), 4.18, 4.27 (each 1H, dd, JA = JB = 10.2 Hz, 20-CH2), 4.46 (2H, t, J = 5.7 Hz, –CH2), 4.61 (1H,
m, 1-CH), 5.52 (1H, s, 17-CH2), 5.83 (1H, s, 14-CH), 6.07 (1H, d, J = 10.2 Hz, 6–OH), 6.15 (1H, s,
17-CH2), 7.63 (2H, t, J = 7.2 Hz, Ar-H), 7.74 (1H, t, J = 7.8 Hz, Ar-H), 8.05 (2H, d, J = 7.2 Hz,
Ar-H); MS(ESI) m/z: 834.4 [M+NH4]+, 817.3 [M+H]+, 851.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z:
calcd for C39H48N2NaO15S: 839.2668, found 839.2679.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(3-formylbenzoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-
2-oxide-4)-oxyethyl))-15-oxo-7,20-epoxy-16-kaurene (13g). Yield 46%, m.p. 154–156 C; IR υmax
3383, 2957, 2025, 1736, 1617, 1554, 1451, 1364, 739, 685 cm–1; 1H-NMR (CDCl3), δ (ppm) 1.12 (6H,
s, –CH3), 2.01 (3H, s, CH3), 3.36 (1H, d, J = 9.6 Hz, 13-CH), 3.75 (1H, m, 6-CH), 4.19, 4.36 (each 1H,
dd, JA = JB = 10.5 Hz, 20-CH2), 4.64 (2H, m, –CH2), 4.79 (2H, m, –CH2), 4.82 (1H, m, 1-CH), 5.54
(1H, s, 17-CH2), 6.02 (1H, s, 14-CH), 6.03 (1H, d, J = 10.5 Hz, 6–OH), 6.15 (1H, s, 17-CH2), 7.44
(2H, t, J = 7.5 Hz, Ar-H), 7.79 (1H, m, Ar-H), 7.58 (4H, m, Ar-H), 8.03 (2H, d, J = 7.8 Hz,
Ar-H); MS(ESI) m/z: 840.2 [M+NH4]+, 823.2 [M+H]+, 857.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z:
calcd for C40H42N2NaO15S: 845.2198, found 845.2208.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(3-formylbenzoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-
2-oxide-4)-oxypropyl))-15-oxo-7,20-epoxy-16-kaurene (13h). Yield 52%, m.p. 136–138 C; IR υmax
3379, 2957, 2025, 1735, 1616, 1554, 1451, 1374, 738, 685 cm–1; 1H-NMR (CDCl3), δ (ppm) 1.12 (6H,
s, –CH3), 2.02 (3H, s, –CH3), 3.33 (1H, d, J = 9.6 Hz, 13-CH), 3.78 (1H, m, 6-CH), 4.22, 4.34 (each
1H, dd, JA = JB = 8.7 Hz, 20-CH2), 4.45 (1H, m, –CH2), 4.57 (1H, m, –CH2), 4.61 (2H, m, –CH2), 4.64
(1H, m, 1-CH), 5.56 (1H, s, 17-CH2), 6.02 (1H, s, 14-CH), 6.10 (1H, d, J = 10.5 Hz, 6–OH), 6.17 (1H,
s, 17-CH2), 7.53 (2H, m, Ar-H), 7.62 (3H, m, Ar-H), 7.72 (2H, m, Ar-H), 8.09 (2H, d, J = 7.8 Hz,
Ar-H); MS(ESI) m/z: 854.3 [M+NH4]+, 837.2 [M+H]+, 871.3 [M+Cl]−; HR-MS (ESI, M+Na) m/z:
calcd for C41H44N2NaO15S: 859.2355, found 859.2368.
ent-(1α-O-Acetyl)-6β,7β-dihydroxy-(14β-O-(2-formylbenzoic acid-(3-phenylsulfonyl-1,2,5-oxadiazole-
2-oxide-4)-oxybutyl))-15-oxo-7,20-epoxy-16-kaurene (13i). Yield 45%, m.p. 116–118 C; IR υmax
3382, 2958, 2025, 1726, 1616, 1553, 1451, 1373, 733, 685 cm–1; 1H-NMR (CDCl3), δ (ppm) 1.12 (6H,
s, –CH3), 1.97 (3H, s, –CH3), 3.45 (1H, d, J = 9.9 Hz, 13-CH), 3.77 (1H, m, 6-CH), 4.21, 4.33 (each
1H, dd, JA = JB = 10.5 Hz, 20-CH2), 4.43 (2H, m, –CH2), 4.50 (2H, t, J = 5.7 Hz, –CH2), 4.64 (1H, m,
1-CH), 5.57 (1H, s, 17-CH2), 6.07 (1H, s, 14-CH), 6.07 (1H, d, J = 10.2 Hz, 6–OH), 6.16 (1H, s,

Molecules 2012, 17
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17-CH2), 7.54 (3H, m, Ar-H), 7.61 (2H, t, J = 7.8 Hz, Ar-H), 7.77 (2H, m, Ar-H), 8.06 (2H, d, J = 7.2 Hz,
Ar-H); MS(ESI) m/z: 868.3 [M+NH4]+, 885.4 [M+Cl]−; HR-MS (ESI, M+Na) m/z: calcd for
C42H46N2NaO15S: 873.2511, found 873.2527.
3.2. In Vitro MTT Assay
The MTT assay was employed as an in vitro cytotoxicity assay, which was performed in 96-well plates.
Test cells at the log phase of their growth cycle (5 × 104 cell/mL) were added to each well (100 µL/well),
then treated in three replicates at various concentrations of the samples (0.39–100 µg/mL), and
incubated for 24 h at 37 °C in a humidified atmosphere of 5% CO2. After 72 h, 20 µL of MTT solution
(5 mg/mL) per well was added to each cultured medium, which was incubated for further 4 h. Then,
DMSO was added to each well (150 µL/well). After 10 min at room temperature, the OD of each well
was measured on a Microplate Reader (BIO-RAD instruments Inc. No. 550, Hercules, CA, USA) at a
wavelength of 490 nm. In these experiments, the negative reference was 0.1% DMSO, and taxol was
used as the positive reference with the concentration of 10 µg/mL.
3.3. NO-Releasing Test: Nitrate/Nitrite Measurement in Vitro
The levels of nitrate/nitrite formed from individual compounds were determined by the colorimetric
assay using the nitrate/nitrite colorimetric assay kit, in triplicate with 100 μM of individual compounds
for 0–60 min according to the manufacturer's instructions (Beyotime, Shanghai, China). The lysates
were mixed with Griess for 40 min and centrifugalized for 10 min, and then followed by measuring at
540 nm, similar as previously reported [13–17].
4. Conclusions
In summary, a series of novel furoxan/oridonin hybrids were synthesized and tested for anti-
proliferative activity against four human cancer cell lines by an in vitro MTT assay. Among them,
compound 9h exhibited the most potential anti-tumor activity against all test cell lines. The
preliminary SAR of the target compounds was discussed based on the experimental data obtained.
Furthermore, more than 15 μmol/L NO were produced by all target compounds at the 60 min time
point, and the results showed that higher levels of NO releasing produced stronger anti-proliferative
activity, so high levels of NO release by these hybrids could play a role in growth inhibition activity.
These results suggested that NO-donor/natural product hybrids may provide a promising approach for
the discovery of novel anti-tumor agents. Further studies on the structure modification of these hybrids
and the mechanism of the derivatives are currently in progress and will be reported in due course.
Acknowledgments
This study was supported by grant from National Natural Science Fund (No. 30973610), Key Fund
of Ministry of Education of China (No. 108069), Specialized Research Fund for the Doctoral Program
of Higher Education (No. 20100096110001), Project for Research and Innovation of Graduates in
Universities of Jiangsu Province (CXZZ11-0800) and the Fundamental Research Funds for the Central
Universities (JKY2011030) for financial assistance.

Molecules 2012, 17
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