Synthesis, biological activity, and apoptotic properties of NO-donor/enmein-type ent-kauranoid hybrids

Article abstract of DOI:10.3390/ijms17060747

Herein, we reported on a series of synthetic nitric oxide-releasing enmein-type diterpenoid hybrids (9a–i). All the target compounds showed potent antibacterial activity against selected Gram-positive bacteria S. aureus and B. subtilis. The antiproliferative activity against human tumor K562, MGC-803, CaEs-17 and Bel-7402 cells, and human normal liver cells L-02 was tested and the structure activity relationships (SARs) were also concluded. Compounds 9b and 9d showed the best activity against S. aureus and B. subtilis with the same minimal inhibitory concentrations (MICs) of 4 and 2 μg/mL, respectively. The derivative 9f displayed IC₅₀ values of 1.68, 1.11, 3.60 and 0.72 μM against the four cancer cell lines above and 18.80 μM against normal liver cells L-02; meanwhile, 9f also released a high level of NO at the time point of 60 min of 22.24 μmol/L. Furthermore, it was also found that 9f induced apoptosis via the mitochondria-related pathway and arrested cell cycle of Bel-7402 cells at S phase. These findings might be important to explore new chemical entities for the main causes of in-hospital mortality of S. aureus infection, combined with a solid tumor.

Full text of DOI:10.3390/ijms17060747

International Journal of
Molecular Sciences
Article
Synthesis, Biological Activity, and Apoptotic
Properties of NO-Donor/Enmein-Type
ent-Kauranoid Hybrids
Dahong Li 1,2,3, Xu Hu 1, Tong Han 1, Shengtao Xu 4, Tingting Zhou 1, Zhenzhong Wang 2,*,
Keguang Cheng 3, Zhanlin Li 1, Huiming Hua 1,*, Wei Xiao 2 and Jinyi Xu 4,
*
1
Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education,
and School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University,
Shenyang 110016, China; lidahong0203@163.com (D.L.); huxu105@163.com (X.H.);
hantong1221@163.com (T.H.); tingtingzhou1118@hotmail.com (T.Z.); lzl1030@hotmail.com (Z.L.)
State Key Laboratory of New-Tech for Chinese Medicine Pharmaceutical Processes,
and National Post-Doctoral Research Workstation, Jiangsu Kanion Pharmaceutical Co., Ltd.,
Lianyungang 222001, China; kanionxw2010@126.com
State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
and School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China;
kgcheng2008@gmail.com
2
3
4
Department of Medicinal Chemistry and State Key Laboratory of Natural Medicines,
China Pharmaceutical University, Nanjing 210009, China; cpuxst@163.com
Correspondence: kanionwzz2016@126.com (Z.W.); huimhua@163.com (H.H.); jinyixu@china.com (J.X.);
Tel.: +86-24-2398-6465 (H.H.); +86-25-8327-1445 (J.X.)
*
Academic Editor: Ge Zhang
Received: 26 February 2016; Accepted: 6 May 2016; Published: 24 May 2016
Abstract: Herein, we reported on a series of synthetic nitric oxide-releasing enmein-type diterpenoid
hybrids (9a–i). All the target compounds showed potent antibacterial activity against selected
Gram-positive bacteria S. aureus and B. subtilis. The antiproliferative activity against human tumor
K562, MGC-803, CaEs-17 and Bel-7402 cells, and human normal liver cells L-02 was tested and the
structure activity relationships (SARs) were also concluded. Compounds 9b and 9d showed the best
activity against S. aureus and B. subtilis with the same minimal inhibitory concentrations (MICs) of 4
and 2
against the four cancer cell lines above and 18.80
also released a high level of NO at the time point of 60 min of 22.24
µ
g/mL, respectively. The derivative 9f displayed IC50 values of 1.68, 1.11, 3.60 and 0.72
M against normal liver cells L-02; meanwhile, 9f
mol/L. Furthermore, it was
µM
µ
µ
also found that 9f induced apoptosis via the mitochondria-related pathway and arrested cell cycle of
Bel-7402 cells at S phase. These findings might be important to explore new chemical entities for the
main causes of in-hospital mortality of S. aureus infection, combined with a solid tumor.
Keywords: NO-donor; enmein-type; ent-kauranoid; antiproliferative; apoptosis
1. Introduction
Bacterial infections, causing deadly diseases, have increased at an alarming rate [1,2]. The development
of drug resistance among the infectious bacterial strains and absence of effective preventive measures
have become the two major driving forces for design and identify suitable active new chemical
entities. On the other hand, cancer, uncontrolled, rapidly proliferating, abnormal cells, is now the
second leading cause of human deaths, and the high mortality rate engages with the rising number
of diverse cancer types [3]. These have triggered research aiming to find new lead structures that
may be beneficial in the design of novel antitumor agents [
4,5
]. Patients with neoplastic disorders
Int. J. Mol. Sci. 2016, 17, 747; doi:10.3390/ijms17060747
www.mdpi.com/journal/ijms

Int. J. Mol. Sci. 2016, 17, 747
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are subjected to chemotherapeutic treatment, and susceptible to microbial infections due to the poor
immunity [ ]. For example, Staphylococcus aureus is a pathogen responsible for the main causes of
in-hospital mortality [ ]. Therefore, the discovery of new novel potent antibacterial and antitumor
6
7
chemical entities is challenging work for medicinal chemists [8–11].
A potential solution is to explore innovative natural scaffolds from natural sources [12,13].
Among numerous exploited medicinal plants, the Isodon species are traditionally used as antitumor
and antibacterial folk medicine and have many complex and bioactive diterpenoids [14 15]. They have
recently received great attention from researchers from many research fields [16 18]. In this context,
,
–
enmein-type ent-kauranoids, one of the important and main classes of diterpenoids from the plants of
Isodon species with antitumor and antibacterial activities, are worthy of intensive investigation.
Nitric oxide (NO) is an endogenous, small and reactive molecule, which has various physiological
and biological properties [19
tumor cell apoptosis, sensitizing drug-resistant tumor cells, and inhibiting tumor metastasis, and
so on [22 23]. NO also exhibits antimicrobial activity and kills bacteria through lipid peroxidation,
DNA cleavage, and protein dysfunction. The multiple bactericidal pathways of NO make it a potent
broad-spectrum antimicrobial agent with low risk [24 26]. Thus, NO-donating groups are good
–21]. High concentration of NO is cytotoxic to tumor cells by inducing
,
–
pharmacophores for both antitumor and antibacterial agents.
On the basis of the above, we developed a series of new enmein-type ent-kauranoid derivatives
containing furozan-based NO-donors. The antibacterial potency, antiproliferative activity and NO
releasing ability of target compounds were tested. Furthermore, the preliminary antitumor mechanisms
were also disclosed.
2. Results and Discussion
2.1. Chemistry
Diphenylsulfonylfuroxan (
), and was then treated with corresponding diol to afford monophenylsulfonylfuroxans (5a
Furoxan-based NO donors intermediate 6a were obtained from the condensation of 5a with
corresponding anhydride. Enmein-type ent-kauranoid derivative was synthesized from oridonin
) by treatment with sodium periodate in water. Target NO donor/enmein-type ent-kauranoid
hybrids 9a were designed and synthesized from with 6a in the presence of DMAP/EDCI in DCM.
4) was obtained in a three-step sequence starting from benzenethiol
(1
–c).
–
i
–c
8
(7
–
i
8
–i
Flash chromatography was used only at the last step of the synthetic route of each target compound
(Scheme 1) [22,27].
2.2. Antimicrobial Activity
The antibacterial activity of NO donor/enmein-type ent-kauranoid hybrids 9a–i against the
Gram-negative bacterium Escherichia coli (ATCC 25922), Gram-positive bacterium Staphylococcus aureus
(ATCC 29213) and Bacillus subtilis (CMCC 63501), and the fungus Monilia albicans (ATCC 10231) was
first disclosed and summarized in Table 1. Parent compound enmein-type 6,7-seco-ent-kauranoid
8
showed the minimum inhibitory concentration (MIC) above 100
exhibited antibacterial activity against gram-positive bacterium S. aureus and B. subtilis to some
extent. In the S. aureus strain, 9a exhibited similar or stronger antibacterial activity than
which meant that structural modifications introduced going in the right direction, improving its
biological properties. In the B. subtilis strain, 9b and 9i were even more potent than positive
µg/mL and derivatives 9a–i
–
i
7,
–
d
control chloromycetin. Thus, the introduction of NO-donor substituent groups at the 14-position
of enmein-type 6,7-seco-ent-kauranoid could improve antimicrobial activity against S. aureus and
B. subtilis. However, almost all of them were inactive (MIC > 100
µg/mL) against E. coli and M. albicans,
and target compounds selectively inhibited Gram-positive bacteria S. aureus and B. Subtilis.

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Scheme 1. Synthesis of NO donor/enmein-type ent-kauranoid hybrids 9a
–
i
. Reagents and conditions:
) fuming HNO3, 90 ˝C, 4 h;
) NaIO4,
(
(
a
d
) ClCH2COOH, NaOH(aq), 140 ˝C, 2 h; (
b
) 30% H2O2, AcOH, rt, 3 h; (
c
) diol, THF, 30% NaOH, 0 ˝C, 4–8 h; (
e
) triethylamine, succinic anhydride, DMAP, rt, 1 h; (f
H2O, rt, 6 h; (g) 6, EDCI, DMAP, rt, 12 h.
Table 1. The antimicrobial activity of the synthesized compounds (MIC µg/mL).
Compound
E. coli
S. aureus
B. subtilis
M. albicans
7
8
9a
9b
9c
9d
9e
9f
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
4
32
>100
16
4
8
4
16
32
16
16
16
4
32
>100
64
2
4
2
16
64
16
16
2
8
NT
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
NT 1
4
9g
9h
9i
Chloromycetin
Fluconazole
NT
NT
1
NT, not test.
The most promising compounds 9b and 9d exhibited the strongest antibacterial activity among
with MICs of 4 and 2 g/mL against S. aureus and B. subtilis. The substituents (R1 and R2)
of 9b were (CH2)2 and (CH2)3, respectively, while the substituents were (CH2)3 and (CH2)2 of 9d
9a
–
i
µ
.
Coincidentally, two of the most active compounds, 9b and 9d, had the same total linkage length of five
carbons (CH2)5. The linkages between NO donor and parent compound always affected the biological
activity. Different lead compounds had their own favorable linker [22,23,28,29].

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2.3. Antiproliferative Activity
The antiproliferative activity of 9a
–
i
against four human cancer cell lines K562 leukemia cell line,
MGC-803 gastric cancer cell line, CaEs-17 esophageal cancer cell line, and Bel-7402 hepatoma cell line,
and human normal liver cells L-02 was evaluated by MTT assay (Table 2). All the target molecules 9a
were stronger than oridonin with IC50 values below 1.33 M and lower than Taxol (1.89 M) against
Bel-7402 cell line. The most potent 9f with IC50 of 0.72 M was 1-fold stronger than Taxol. While, target
compounds were not so sensitive to CaEs-17 cells with IC50 ranging from 3.60 to 5.13 M. IC50 values
against MGC-803 cell line were a little weaker than Taxol, between 1.11 to 1.83 M. For K562 cell line,
–
i
7
µ
µ
µ
µ
µ
the antiproliferative activity of all the derivatives was weaker than Taxol. Most NO donor derivatives
showed cytotoxic selectivity between tumor and normal liver cells to some extent, with IC50 values
ranging from 10.36 to 19.87 µM against L-02 normal liver cells. These results confirmed previous
reports [28,29] that some NO donating derivatives showed less cytotoxicity to normal cells.
Table 2. Antiproliferative activity of the synthesized compounds (IC50 µM).
Compound
K562
MGC-803
CaEs-17
Bel-7402
L-02
SI 2
7
8
9a
9b
9c
9d
9e
9f
9g
9h
9i
4.76 ˘ 0.32
8.11 ˘ 0.76
2.47 ˘ 0.16
2.31 ˘ 0.21
1.93 ˘ 0.10
2.15 ˘ 0.12
1.92 ˘ 0.09
1.68 ˘ 0.12
2.26 ˘ 0.08
2.11 ˘ 0.16
1.86 ˘ 0.18
0.41 ˘ 0.02
5.69 ˘ 0.39
14.21 ˘ 1.22
1.83 ˘ 0.16
1.62 ˘ 0.08
1.34 ˘ 0.13
1.50 ˘ 0.10
1.29 ˘ 0.12
1.11 ˘ 0.05
1.43 ˘ 0.08
1.49 ˘ 0.11
1.25 ˘ 0.10
0.85 ˘ 0.06
11.03 ˘ 1.02
30.84 ˘ 2.09
5.12 ˘ 0.42
4.83 ˘ 0.30
3.76 ˘ 0.37
4.98 ˘ 0.42
4.27 ˘ 0.35
3.60 ˘ 0.12
5.13 ˘ 0.22
4.68 ˘ 0.31
3.82 ˘ 0.19
0.43 ˘ 0.03
7.48 ˘ 0.53
32.96 ˘ 2.19
1.33 ˘ 0.10
1.20 ˘ 0.08
0.83 ˘ 0.06
1.23 ˘ 0.08
0.92 ˘ 0.05
0.72 ˘ 0.04
1.27 ˘ 0.11
1.08 ˘ 0.05
0.78 ˘ 0.04
1.89 ˘ 0.09
18.26 ˘ 0.81
24.37 ˘ 1.59
10.36 ˘ 0.61
16.73 ˘ 0.17
19.87 ˘ 0.18
13.58 ˘ 1.31
17.20 ˘ 0.77
18.80 ˘ 1.25
11.57 ˘ 0.39
12.09 ˘ 1.08
14.57 ˘ 0.86
3.73 ˘ 0.17
2.4
0.7
7.7
13.9
23.9
11.0
18.6
26.1
9.1
11.1
18.6
1.9
Taxol 1
1
Taxol was used a positive control. Data were means
˘
SD of three experiments; 2 SI: selectivity index. It was
calculated as: SI = IC50, L-02/IC50, Bel-7402
.
In most cases against the selected tumor cell lines, among the derivatives 9a
substitution R1 was the same (9a 9d and 9g ) and R2 was changed among (CH2)2, (CH2)3,
and o-C6H4 in each group, compounds (9c 9f and 9i) with R2 of aromatic group of o-C6H4 showed
stronger activity than corresponding ones with alkyl groups (9a 9b 9d 9e 9g and 9h). Between alkyl
substituents (CH2)2 and (CH2)3 in R2, the latter one (9b 9e and 9h) was favorable. For example, 9i with
the same R1 of (CH2)4 as 9g and 9h, and R2 of o-C6H4, showed slightly lower IC50 values of 1.25, 1.86,
3.82 and 0.78 M against K562, MGC-803, CaEs-17 and Bel-7402 tumor cells, respectively. When the
–i, when the
–
c,
–
f
–i
,
,
,
,
,
,
µ
substitution R2 was the same and R1 was changed among (CH2)2, (CH2)3, and (CH2)4, compounds
showed statistically equal IC50 values.
2.4. NO-Releasing Ability
The NO-releasing ability of 9a
the time point of 10, 20, 30, 40, 50 and 60 min (Figure 1). The concentrations of released NO for all
synthetic hybrids increased with time and were more than 20 mol/L (except 9c) at the time point of
60 min. High NO releasing ability might, at least to a certain extent, contribute to antiproliferative and
antibacterial activities [23 26]. The most promising NO-releasing enmein-type 6,7-seco-ent-kauranoid
–i was determined by Griess assay at 100 µM and measured at
µ
,
derivative was 9f with the highest selectivity index (SI) value of 26.1. It was chosen for intensive
mechanism study on hepatoma Bel-7402 cell line.

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Figure 1. NO-releasing ability of compounds 9a–i.
2.5. Influence of 9f on the Bel-7402 Cell Cycle
Cell cycle arrest is an important sign for inhibition of proliferation and the series of events that take
place in a cell leading to its division and replication. Some NO donating hybrids exhibited cell cycle
arrest properties [28]. As the inhibitory effect of 9f on cell proliferation was observed, we next assessed
the effect on the cell cycle distribution of Bel-7402 cells by flow cytometry (Figure 2). Treatment of
Bel-7402 cells with 9f at 0.25, 0.5 and 1 µM resulted in a remarkable increase in the percentage
of cells in S/G2 phase from 25.51% of control group to 31.78%, 45.33%, and 49.78%, respectively.
Compound 9f could influence Bel-7402 cell cycle progression at low micromolar concentrations in a
dose-dependent manner.
Figure 2. Influence of Bel-7402 cell cycle by 9f. Left red part: cells in G1 phase; Right red part: cells in
G2 phase; Oblique line part: cells in S phase; White part: total cells.

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2.6. Induction of Apoptosis by 9f
To examine the potent cancer cell antiproliferative effect of 9f on Bel-7402 cells, the number of
apoptotic cells was monitored using flow cytometry. The total percentage of apoptotic cells (early and
late, Q2 + Q4) was 7.88% treated with a vehicle alone. In comparison with the control group, 2.76-, 4.54-,
and 8.01-fold percentages of apoptotic cells were observed when different concentrations (0.25, 0.5
and 1 µM) of 9f were added to Bel-7402 cells. As shown in Figure 3, compound 9f caused significant
induction of apoptosis in a dose-dependent manner in Bel-7402 cells and resulted in 21.74%, 35.78%
and 63.15% apoptotic cells, respectively.
Figure 3. Induction of apoptosis by 9f in Bel-7402 cells.
2.7. 9f Induced Mitochondrial Depolarization
In the field of cancer, cellular apoptosis plays a very important role. The loss of mitochondrial
membrane potential is a key event during drug-induced apoptosis. In order to investigate the
mitochondria related effect of 9f, the fluorescent probe JC-1 (Keygen, KGA601, Nanjing, China)
was used to detect the changes on mitochondrial membrane potential. Before Bel-7402 cells were
stained with JC-1, cells were incubated with 0, 0.15, 0.3, and 0.6 µM of 9f. Then, the flow cytometry
analysis was carried out to determine the cell numbers with collapsed mitochondria in different
cell groups (Figure 4). The percentage of apoptotic cells increased in a dose-dependent fashion
and showed 4.62%, 8.81%, 21.82% and 25.98%, respectively. These results together with Annexin-V
and PI double stain apoptosis assay confirmed that 9f could induce apoptosis in a Bel-7402 cells
mitochondria-related pathway.

Int. J. Mol. Sci. 2016, 17, 747
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Figure 4. 9f induced mitochondrial depolarization in Bel-7402 cells.
3. Materials and Methods
3.1. Chemistry
3.1.1. General
All commercially available solvents and reagents were purchased from a local commercial supplier
(Yuwang Chemical Industries, Ltd., Shenyang, China) and used directly. Melting points (m.p.) were
tested using micro melting point apparatus XT-4 (Taike, Beijing, China) and not corrected. Infrared (IR)
spectra were recorded on a Nicolet Impact 410 instrument (Thermo Fisher Scientific, Waltham,
MA, USA) using a KBr pellet. 1H and 13C NMR spectra were taken (TMS as internal standard)
on a Bruker AV-300 (Bruker Corp., Karlsruhe, Germany) or ACF 500 spectrometer (Bruker Corp.,
Karlsruhe, Germany) in CDCl3. Low resolution mass spectra (MS) were carried out using FTMS-2000
(Thermo Fisher Scientific, Waltham, MA, USA). High resolution mass spectra (HR-MS) were obtained
with Agilent QTOF 6520 (Agilent Technologies China, Beijing, China).
3.1.2. General Procedure to Synthesize 9a–i
Compound
8 (72 mg, 0.2 mmol) was dissolved in 15 mL of dichloromethane and mixed with 6a–i
(0.24 mmol), EDCI and DMAP, stirring at room temperature for 8 h. When the reaction was finished
(monitored by TLC), the reaction mixture was poured into 10% HCl (about 15 mL), and then extracted
three times (each 10 mL) with dichloromethane. The organic phases were combined together, washed
with water (about 15 mL) and saturated brine (about 15 mL), sequentially, then dried over anhydrous
Na2SO4, and evaporated to dryness in reduced pressure. At the last step, the product was purified by
flash column chromatography (CH2Cl2/MeOH v/v 300:1) to obtain the target molecules.

Int. J. Mol. Sci. 2016, 17, 747
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Compound 9a: white solid, 48% yield: m.p. 92 ˝C–94 ˝C; IR (KBr)
υ
max 3439, 2959, 2025, 1723, 1615,
1553, 1450, 1371, 734, 685 cm´1
;
13C NMR (CDCl3, 100 MHz),
δ
(ppm) 198.07, 172.91, 172.69, 167.22,
158.99, 147.86, 138.18, 135.83, 129.89 (
ˆ
2), 128.68 (
ˆ
2), 120.53, 110.61, 101.90, 76.18, 74.67, 74.09, 68.13,
61.46, 60.34, 53.93, 49.96, 48.60, 40.77, 37.16, 33.12, 33.03, 31.72, 31.13, 29.89, 23.33, 23.16, 19.74; 1H NMR
(CDCl3, 300 MHz),
δ
1(ppm) 8.07 (2H, d, J = 7.5 Hz, 21,61-Ar–H), 7.78 (1H, d, J = 7.5 Hz, 41-Ar–H), 7.64
1
(2H, t, J = 7.5 Hz, 3 ,5 -Ar–H), 6.20 (1H, s, 6-OH), 5.74 (1H, s, 17-CH2), 5.62 (1H, s, 17-CH2), 5.32 (1H, s,
6-CH), 4.47~4.65 (6H, m, overlapped, 1-CH, 14-CH, 25,26-CH2), 4.05, 3.92 (each 1H, dd, JA = JB = 9.6 Hz,
20-CH2), 3.16 (1H, d, J = 9.3 Hz, 13-CH), 2.53~2.90 (4H, m, 22,23-CH2), 2.17 (2H, m, 12-CH2), 1.97 (1H,
m, 5-CH), 1.61~1.74 (5H, m, 3,11-CH2, 9-CH), 1.45 (2H, m, 2-CH2), 1.01 (3H, s, 18-CH3), 0.95 (3H, s,
19-CH3); MS (ESI) m/z: 748.1 [M + NH4]+, 731.0 [M + H]+, 765.3 [M + Cl]´; HR-MS (ESI, M + NH4
m/z: calcd for C34H42N3O14S: 748.2382, found: 748.2388.
)
Compound 9b: white solid, 46% yield: m.p. 88 ˝C–89 ˝C; IR (KBr)
1553, 1452, 1358, 732, 685 cm´1 13C NMR (CDCl3, 100 MHz),
(ppm) 198.14, 172.73, 172.65, 167.27,
158.79, 147.85, 138.12, 135.87, 129.87 ( 2), 128.71 ( 2), 120.54, 110.52, 101.87, 76.17, 74.59, 74.12, 69.04,
61.23, 60.35, 53.90, 49.93, 48.58, 40.73, 37.12, 33.00 (
2), 32.89, 31.10, 29.85, 23.29, 23.13, 19.87, 19.61; 1H
NMR (CDCl3, 500 M Hz), (ppm) 8.00 (2H, d, J = 7.5 Hz, 2 ,6 -Ar–H), 7.89 (1H, d, J = 7.5 Hz, 4 -Ar–H),
υmax 3442, 2954, 2025,1746, 1618,
;
δ
ˆ
ˆ
ˆ
1
1
1
δ
7.74 (2H, t, J = 7.5 Hz, 31,51-Ar–H), 6.18 (1H, s, 6-OH), 5.73 (1H, s, 17-CH2), 5.55 (1H, s, 17-CH2), 5.31
(1H, s, 6-CH), 4.48~4.63 (6H, m, 1-CH, 14-CH, 26,27-CH2), 4.05, 3.92 (each 1H, dd, JA = JB = 9.0 Hz,
20-CH2), 3.14 (1H, d, J = 9.0 Hz, 13-CH), 2.38~2.73 (6H, m, overlapped, 22,23,24-CH2), 2.00 (2H, m,
12-CH2), 1.95 (1H, m, 5-CH), 1.61~1.74 (5H, m, 3,11-CH2, 9-CH), 1.45 (2H, m, 2-CH2), 1.02 (3H, s,
18-CH3), 0.95 (3H, s, 19-CH3); MS (ESI) m/z: 762.3 [M + NH4]+, 745.2 [M + H]+, 779.4 [M + Cl]´;
HR-MS (ESI, M + NH4) m/z: calcd for C35H44N3O14S: 762.2539, found: 762.2532.
˝
˝
Compound 9c: white solid, 45% yield: m.p. 137 C–139 C; IR (KBr)
υ
3443, 2954, 2025, 1757, 1727,
max
1617, 1553, 1450, 735, 685 cm´1
158.87, 147.65, 137.95, 135.74, 131.89, 131.71, 131.19, 130.92, 130.14, 129.76 (
120.72, 110.49, 101.80, 76.24, 75.27, 74.58, 68.88, 62.45, 60.21, 53.87, 50.14, 48.76, 40.74, 37.00, 32.93, 30.97,
;
13C NMR (CDCl3, 100 MHz),
δ
(ppm) 198.21, 167.15, 167.06, 166.58,
2), 128.87, 128.55 ( 2);
ˆ
ˆ
1 1
31.08, 29.86, 23.29, 23.07, 19.76; 1H NMR (CDCl3, 300 M Hz),
δ (ppm) 8.00 (2H, d, J = 7.5 Hz, 2 ,6 -Ar–H),
7.81 (1H, t, J = 4.5 1Hz, 41-Ar–H), 7.69 (1H, t, J = 4.5 Hz, 22-Ar–H), 7.56 (3H, m, 21,23,24-Ar–H), 7.41
1
(2H, t, J = 7.5 Hz, 3 ,5 -Ar–H), 6.24 (1H, s, 6-OH), 5.87 (1H, s, 17-CH2), 5.52 (1H, s, 17-CH2), 5.33 (1H, s,
6-CH), 4.40~4.76 (6H, m, 1-CH, 14-CH, 29,30-CH2), 4.07, 3.96 (each 1H, dd, JA = JB = 9.3 Hz, 20-CH2),
3.29 (1H, d, J = 9.6 Hz, 13-CH), 2.17 (2H, m, 12-CH2), 1.97 (1H, m, 5-CH), 1.52~1.80 (5H, m, 3,11-CH2,
9-CH), 1.41 (2H, m, 2-CH2), 1.02 (3H, s, 18-CH3), 0.98 (3H, s, 19-CH3); MS (ESI) m/z: 801.3 [M + Na]+;
HR-MS (ESI, M + NH4) m/z: calcd for C38H42N3O14S: 796.2382, found: 796.2376.
˝
˝
Compound 9d: white solid, 44% yield: m.p. 110 C–112 C; IR (KBr)
υ
3459, 2955, 2025, 1754, 1615,
max
1554, 1451, 1355, 732, 685 cm´1
158.90, 147.69, 138.01, 135.67, 129.73 (
60.35, 60.16, 53.79, 49.79, 48.47, 40.59, 36.98, 32.88, 30.99, 29.74, 29.05, 28.65, 27.81, 23.18, 23.03, 19.70; 1H
;
13C NMR (CDCl3, 100 MHz),
δ (ppm) 197.92, 172.08, 171.73, 167.13,
ˆ
2), 128.60 ( 2); 120.46, 110.56, 101.76, 76.04, 74.52, 74.18, 67.91,
ˆ
1 1 1
δ (ppm) 8.03 (2H, d, J = 9.0 Hz, 2 ,6 -Ar–H), 7.91 (1H, t, J = 8.1 Hz, 4 -Ar–H),
NMR (CDCl3, 300 M Hz),
7.75 (2H, t, J = 9.0 Hz, 31,51-Ar–H), 6.04 (1H, s, 6-OH), 5.74 (1H, s, 17-CH2), 5.69 (1H, s, 17-CH2), 5.19
(1H, s, 6-CH), 4.68 (2H, m, 1,14-CH), 4.45 (2H, t, J = 6.0 Hz, 25-CH2), 4.14 (2H, t, J = 6.0 Hz, 27-CH2),
3.90, 3.54 (each 1H, dd, JA = JB = 9.0 Hz, 20-CH2), 3.14 (1H, d, J = 5.4 Hz, 13-CH), 2.36~2.63 (6H, m,
22,23,26-CH2), 2.08 (2H, m, 12-CH2), 1.38~1.76 (6H, m, 3,11-CH2, 5,9-CH), 1.43 (2H, m, 2-CH2), 0.93
(3H, s, 18-CH3), 0.85 (3H, s, 19-CH3); MS (ESI) m/z: 767.3 [M + Na]+; HR-MS (ESI, M + NH4) m/z:
calcd for C35H44N3O14S: 762.2539, found: 762.2551.
Compound 9e: white solid, 42% yield: m.p. 82 ˝C–84 ˝C; IR (KBr)
1554, 1451, 1358, 732, 685 cm´1 13C NMR (CDCl3, 100 MHz),
(ppm) 197.96, 172.78, 172.56, 167.11,
158.85, 147.73, 138.02, 135.71, 129.76 ( 2), 128.53 ( 2), 120.40, 110.47, 101.75, 76.05, 74.49, 73.96, 68.00,
61.33, 60.16, 53.79, 49.82, 48.46, 40.63, 37.01, 32.97, 32.89, 31.57, 30.99, 29.74, 27.88, 23.19, 23.03, 19.75,
υmax 3458, 2955, 2025, 1738, 1615,
;
δ
ˆ
ˆ

Int. J. Mol. Sci. 2016, 17, 747
9 of 13
1 1
δ (ppm) 8.02 (2H, d, J = 8.4 Hz, 2 ,6 -Ar–H), 7.90 (1H, t, J = 7.5 Hz,
19.60; 1H NMR (CDCl3, 300 M Hz),
41-Ar–H), 7.74 (2H, t, J = 8.4 Hz, 31,51-Ar–H), 6.25 (1H, d, J = 2.2 Hz, 6-OH), 6.01 (1H, s, 17-CH2), 5.77
(1H, s, 14-CH), 5.68 (1H, s, 17-CH2), 5.19 (1H, d, J = 2.4 Hz, 6-CH), 4.71 (1H, m, 1-CH), 4.55 (2H, t,
J = 6.0 Hz, 26-CH2), 4.12 (2H, t, J = 6.0 Hz, 28-CH2), 3.88, 3.35 (each 1H, dd, JA = JB = 9.0 Hz, 20-CH2),
3.17 (1H, d, J = 9.3 Hz, 13-CH), 2.21~2.51 (8H, m, overlapped, 22,23,24,27-CH2), 2.07 (2H, m, 12-CH2),
1.67~1.78 (6H, m, overlapped, 3,11-CH2, 5,9-CH), 1.24 (2H, m, 2-CH2), 0.94 (3H, s, 18-CH3), 0.88 (3H,
s, 19-CH3); MS (ESI) m/z: 781.4 [M + Na]+; HR-MS (ESI, M + NH4) m/z: calcd for C36H46N3O14S:
776.2695, found: 776.2687.
˝
˝
Compound 9f: white solid, 28% yield: m.p. 128 C–130 C; IR (KBr)
1723, 1616, 1554, 1451, 735, 685 cm´1 13C NMR (CDCl3, 100 MHz),
166.55, 158.86, 147.67, 137.94, 135.77, 131.91, 131.67, 131.18, 130.94, 130.15, 129.76 (
υ
3445, 2956, 2025, 1758,
max
;
δ
(ppm) 198.23, 167.13, 167.02,
2), 128.85, 128.54
ˆ
( 2); 120.75, 110.51, 101.84, 76.27, 75.29, 74.54, 68.91, 62.48, 60.23, 53.91, 50.09, 48.78, 40.72, 37.02,
ˆ
32.97, 30.99,131.07, 29.86, 29.79, 23.30, 23.12, 19.75; 1H NMR (CDCl3, 300 M Hz),
δ
(ppm) 81.07 (2H, d,
1
1
1
J = 7.0 Hz, 2 ,6 -Ar–H), 7.75 (2H, m, 22,4 -Ar–H), 7.64 (3H, m, 21,23,24-Ar–H), 7.57 (2H, m, 3 ,5 -Ar–H),
6.21 (1H, s, 6-OH), 5.92 (1H, s, 17-CH2), 5.56 (1H, s, 17-CH2), 5.32 (1H, s, 6-CH), 4.31~4.60 (6H, m,
overlapped, 1-CH, 14-CH, 29,31-CH2), 4.08, 3.97 (each 1H, dd, JA = JB = 9.0 Hz, 20-CH2), 3.26 (1H, d,
J = 8.7 Hz, 13-CH), 2.57~2.78 (2H, m, 30-CH2), 2.35 (2H, m, 12-CH2), 1.95 (1H, m, 5-CH), 1.42~1.78
(5H, m, 3,11-CH2, 9-CH), 1.24 (2H, m, 2-CH2), 1.03 (3H, s, 18-CH3), 0.96 (3H, s, 19-CH3); MS (ESI) m/z:
815.4 [M + Na]+; HR-MS (ESI, M + NH4) m/z: calcd for C39H44N3O14S: 810.2539, found: 810.2530.
Compound 9g: white solid, 37% yield: m.p. 85 ˝C–87 ˝C; IR (KBr)
1554, 1452, 1360, 733, 685 cm´1 13C NMR (CDCl3, 100 MHz),
(ppm) 197.92, 172.21, 172.00, 167.09,
158.93, 147.69, 138.01, 135.67, 129.72 ( 2), 128.56 ( 2); 120.45, 110.47, 101.76, 76.03, 74.55, 74.16, 70.98,
63.86, 60.14, 53.78, 49.80, 48.46, 40.59, 36.99, 32.88, 30.99, 29.74, 29.09, 28.74, 25.18, 24.95, 23.19, 23.02,
υmax 3442, 2956, 2025, 1755, 1616,
;
δ
ˆ
ˆ
1 1
δ (ppm) 8.06 (2H, d, J = 7.8 Hz, 2 ,6 -Ar–H), 7.90 (1H, t, J = 7.2 Hz,
19.73; 1H NMR (CDCl3, 300 M Hz),
41-Ar–H), 7.65 (2H, t, J = 7.8 Hz, 31,51-Ar–H), 6.25 (1H, s, 6-OH), 6.03 (1H, s, 17-CH2), 5.77 (1H, s,
14-CH), 5.68 (1H, s, 17-CH2), 5.19 (1H, s, 6-CH), 4.62 (1H, m, 1-CH), 4.42 (2H, m, 25-CH2), 4.07 (2H, m,
28-CH2), 3.90, 3.54 (each 1H, dd, JA = JB = 10.5 Hz, 20-CH2), 3.14 (1H, d, J = 9.6 Hz, 13-CH), 2.47~2.54
(8H, m, overlapped, 22,23,26,27-CH2), 1.66~1.81 (8H, m, overlapped, 3,11,12-CH2, 5,9-CH), 1.24 (2H, m,
2-CH2), 0.93 (3H, s, 18-CH3), 0.88 (3H, s, 19-CH3); MS (ESI) m/z: 776.3 [M + NH4]+, 793.2 [M + Cl]´;
HR-MS (ESI, M + NH4) m/z: calcd for C36H46N3O14S: 776.2695, found: 776.2693.
Compound 9h: white solid, 42% yield: m.p. 78 ˝C–80 ˝C; IR (KBr)
1554, 1452, 1365, 732, 685 cm´1 13C NMR (CDCl3, 100 MHz),
(ppm) 198.10, 172.34, 172.11, 167.26,
159.04, 147.69, 138.16, 135.81, 129.85 ( 2), 128.66 ( 2); 120.58, 110.58, 101.85, 76.18, 74.57, 74.30, 71.11,
υmax 3444, 2960, 2025, 1755, 1616,
;
δ
ˆ
ˆ
63.98, 60.28, 53.90, 49.92, 48.57, 40.71, 37.09, 32.98, 31.09, 29.84, 29.20, 28.86, 25.29, 25.05, 23.30, 23.13,
1
22.76, 19.83; H NMR (CDCl3, 300 M Hz),
δ
(ppm) 8.06 (2H, d, J = 7.2 Hz, 21,61-Ar–H), 7.77 (1H, t,
J = 7.2 Hz, 41-Ar–H), 7.63 (2H, t, J = 7.2 Hz, 31,51-Ar–H), 6.21 (1H, s, 6-OH), 5.73 (1H, s, 17-CH2), 5.57
(1H, s, 17-CH2), 5.32 (1H, m, 6-CH), 4.57 (2H, m, 1,14-CH), 4.45 (2H, t, J = 6.3 Hz, 26-CH2), 4.14 (2H,
t, J = 6.0 Hz, 29-CH2), 4.06, 3.93 (each 1H, dd, JA = JB = 9.3 Hz, 20-CH2), 3.13 (1H, d, J = 9.6 Hz,
13-CH), 2.45~2.50 (10H, m, overlapped, 22,23,24,27,28-CH2), 2.35 (2H, m, 12-CH2), 1.58~1.98 (6H, m,
overlapped, 3,11-CH2, 5,9-CH), 1.24 (2H, m, 2-CH2), 0.93 (3H, s, 18-CH3), 0.87 (3H, s, 19-CH3); MS (ESI)
m/z: 795.4 [M + Na]+; HR-MS (ESI, M + NH4) m/z: calcd for C37H48N3O14S: 790.2852, found: 790.2841.
˝
˝
Compound 9i: white solid, 43% yield: m.p. 126 C–128 C; IR (KBr)
υ
3443, 2955, 2025, 1758, 1723,
max
1615, 1553, 1451, 734, 685 cm´1
158.96, 147.64, 137.97, 135.72, 131.71, 131.41, 131.22, 130.89, 130.16, 129.75 (
120.58, 110.49, 101.76, 76.13, 74.98, 74.56, 70.94, 64.72, 60.15, 53.79, 49.97, 48.69, 40.61, 36.97, 32.88, 31.00,
;
13C NMR (CDCl3, 100 MHz),
δ
(ppm) 198.12, 167.09, 166.90, 166.51,
2), 128.64, 128.53 ( 2);
ˆ
ˆ
29.80, 25.26, 24.95, 23.23, 23.02, 19.76; 1H NMR (CDCl3, 300 M Hz),
δ (ppm) 8.07 (2H, d, J = 7.8 Hz,
21,61-Ar–H), 7.73 (3H, m, 23,25,26-Ar–H), 7.60 (2H, m, 24,41-Ar–H), 7.50 (2H, t, J = 7.8 Hz, 31,51-Ar–H),
6.21 (1H, s, 6-OH), 5.93 (1H, s, 17-CH2), 5.56 (1H, s, 17-CH2), 5.34 (1H, m, 6-CH), 4.68 (2H, m, 1,14-CH),
4.40~4.62 (4H, m, 29,32-CH2), 4.08, 3.97 (each 1H, dd, JA = JB = 9.0 Hz, 20-CH2), 3.30 (1H, d, J = 9.0 Hz
,

Int. J. Mol. Sci. 2016, 17, 747
10 of 13
13-CH), 2.79 (2H, m, 30-CH2), 2.62 (2H, m, 31-CH2), 2.42 (2H, m, 12-CH2), 2.03 (1H, m, 5-CH), 1.97 (1H,
m, 9-CH), 1.70~1.81 (4H, m, 3,11-CH2), 1.24(2H, m, 2-CH2), 1.03 (3H, s, 18-CH3), 0.96 (3H, s, 19-CH3);
MS (ESI) m/z: 829.4 [M + Na]+; HR-MS (ESI, M + NH4) m/z: calcd for C40H46N3O14S: 824.2695,
found: 824.2697.
3.2. Biology
3.2.1. Antibacterial Assay
The minimal inhibitory concentrations (MICs) were carried out by microbroth dilution method
described by the Clinical Laboratory Standards Institute [30,31]. A stock solution of all the target
compounds was prepared with DMSO-medium (1:2), and then graded quantities of the test compounds
were diluted with medium. The specified concentration suspension of fungus and bacterium contained
approximately 103, and 105 CFU/mL was added into microtitration plates. The inoculated 96-well
˝
plates were incubated at 35 C for 18 h. MIC was defined as the lowest concentrations that prevented
visible growth of the bacteria.
3.2.2. MTT Assay
MTT assay was developed to monitor mammalian cell survival and proliferation in vitro [32].
CaEs-17 cells (K562, Bel-7402 and MGC-803 cells) from American Type Culture Collection
(ATCC, Manassas, VA, USA) were cultivated in RPMI-1640 medium supplemented with 5% fetal
bovine serum (v/v), 100 U/mL penicillin, and 50 mg/mL streptomycin. Cells (5
log phase of their growth cycle were added to each well of a 96-well plate (100 L/well) and incubated
for 24 h at 37 ˝C in a humidified atmosphere of 5% CO2. Then, cells were treated with or without
test compounds in different concentrations. After 72 h, 5 mg/mL MTT solution (20 L per well) was
added. Cells were incubated at 37 ˝C. After 4 h, MTT solution was removed and DMSO was added
to each well (150 L). Ten minutes later at room temperature, the optical density (OD) values were
measured at the wavelength of 490 nm on a Microplate Reader NO. 550 (BIO-RAD Instruments Inc.,
ˆ
104 cell/mL) at the
µ
µ
µ
Hercules, CA, USA). In this experiment, 10
DMSO was used as the negative reference.
µg/mL of Taxol was used as the positive control and 0.1%
3.2.3. Griess Assay
NO produced by the compounds was determined by assaying the levels of NO2 using the Griess
reagent [33]. The levels of NO released from 100 M of each compound were tested by nitrate/nitrite
µ
in triplicate. The lysates were reacted with Griess reagent (40 min), centrifugalized (10 min), and then
measured at 540 nm. This assay was carried out according to the manufacturer’s instructions for
60 min (Beyotime, Nanjing, China).
3.2.4. Cell Cycle Arrest
Bel-7402 cells (5.0
incubated with different concentrations (0.25, 0.5 and 1
ˆ
103 cells/well) were plated in 6-well plates and incubated for 24 h, and then
˝
M) of 9f at 37 C for 48 h. After 48 h treatment,
µ
cells were treated with 70% ethanol and RNase, sequentially, and stained with PI [34]. Cellular DNA
content was measured by a flow cytometer for cell cycle distribution analysis.
3.2.5. Cellular Apoptosis
Bel-7402 cells were incubated with different concentrations (0.25, 0.5 and 1 µM) of 9f for 72 h.
Then, Annexin-V and PI double staining was used to detect apoptotic cells to analyze apoptosis
according to the manufacturer’s instructions by flow cytometry [35].
3.2.6. Mitochondrial Membrane Potential
Bel-7402 cells were cultured in six-well plates after treatment with 9f or vehicles for 48 h. The cells
were cells were collected by trypsinization, washed with PBS (Sigma Chemical Co., Shanghai, China)

Int. J. Mol. Sci. 2016, 17, 747
11 of 13
and stained according to the manufacturer’s instruction (Keygen, KGA601, Nanjing, China) with the
lipophilic cationic dye JC-1. The flow cytometry analysis was carried out to monitor the percentage of
cells with healthy or collapsed mitochondrial membrane potentials [36].
4. Conclusions
In summary, a series of NO donor/enmein-type diterpenoid derivatives (9a–i) were synthesized
from relevant commercial available resources. All of the target compounds showed antibacterial
activity against Gram-positive bacteria S. aureus and B. subtilis and antiproliferative activity against
the K562 leukemia cell line, the MGC-803 gastric cancer cell line, the CaEs-17 esophageal cancer cell
line, the Bel-7402 hepatoma cell line, and human normal liver cells L-02 to some extent. It was found
that 9b and 9d with the same total linkage length of five carbons were the most active against S. aureus
and B. subtilis with MICs of 4 and 2
µg/mL, respectively. Compound 9f displayed IC50 values of
1.68, 1.11, 3.60 and 0.72 M against four tested cancer cell lines, respectively, and 18.80
µ
µM against
normal liver cells L-02. The SI value was 26.1 between L-02 normal liver and Bel-7402 hepatoma
cell lines. The preliminary SARs were also concluded based on the obtained biological evaluation
data. Furthermore, it was also found that 9f induced apoptosis via the mitochondria-related pathway
and arrested cell cycle of Bel-7402 cells at the S stage. These findings were important to explore
new chemical entities with innovative natural scaffolds for the treatment of infection combined with
solid tumors.
Supplementary Materials: Supplementary materials can be found at http://www.mdpi.com/1422-0067/17/6/
747/s1, including 1H NMR and H-H COSY of 8, 1H and 13C NMR of methyl ester of 6a, and 1H and 13C NMR
of 9f.
Acknowledgments: This work was financially supported by the National Natural Science Foundation of China
(81373280, 21502121), the Project Funded by the China Post Doctoral Science Foundation (2015M570258), General
Scientific Research Projects of the Department of Education in Liaoning Province (L2014382), the Key Laboratory
for the Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal University), the Ministry
of the Education of China (CMEMR2015-B07), the Jiangsu Key Laboratory of New Drug Research and Clinical
Pharmacy (KF-XY-201401) and the Career Development Support Plan for Young and Middle-aged Teachers in
Shenyang Pharmaceutical University.
Author Contributions: Dahong Li, Huiming Hua and Jinyi Xu conceived and designed the experiments;
Dahong Li, Tong Han, Shengtao Xu, Tingting Zhou and Keguang Cheng performed the experiments; Dahong Li,
Keguang Cheng and Zhanlin Li analyzed the data; Dahong Li, Zhenzhong Wang, Wei Xiao and Jinyi Xu
wrote the paper. Xu Hu revised the manuscript and did the corresponding experiments according to the
reviewers’ suggestions.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
NO
Nitric Oxide
SAR
Structure Activity Relationship
4-Dimethylaminopyridine
1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide Hydrochloride
Room Temperature
DMAP
EDCI
rt
THF
Tetrahydrofuran
SI
Selectivity Index
MTT
TLC
FBS
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
Thin Layer Chromatograph
Fetal Bovine Serum
TMS
1H NMR
TMS
ESI MS
HR-MS
DCM
Tetramethylsilane
Proton Nuclear Magnetic Resonance
Tetramethlysilane
Electrospray Ionization-Mass Spectrometry
High Resolution Mass Spectrum
Dichloromethane

Int. J. Mol. Sci. 2016, 17, 747
12 of 13
MIC
PI
Minimal Inhibitory Concentration
Propidium Iodide
NT
Not Test
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