Synthesis and biological activity evaluation of novel peroxo-bridged derivatives as potential anti-hepatitis B virus agents

Article abstract of DOI:10.1039/c6md00344c

Previous studies have demonstrated that natural steroid compounds containing a peroxide bridge exhibited potential anti-hepatitis B virus activity. To continue our research, a simple and regioselective methodology, using Eosin Y as a clean photosensitized oxidation catalyst, was developed for the synthesis of a peroxide bridge in steroids. The method that using Eosin Y as the catalyst was exposed to visible light and furbished in high yields, did not involve tedious work-up or purification, and avoided using environmentally hazardous solvents. It can be regarded as a green protocol. Moreover, a series of cholesterol and β-sitosterol derivatives containing a peroxide bridge were synthesized using this method and screened for their anti-HBV activity. Among the compounds synthesized in this research, 5α,8α-cyclicobioxygen-6-vinyl-3-oxo-cholesterone (1f, 3.13 μg ml^?1) had the most potent activity with inhibition rates of 77.45% ± 6.01% and 58.73% ± 8.64% on the secretion of HBsAg and HBeAg antigens, respectively, after 8 days. Further acute toxicity test showed that the LD₅₀ value of compound 1f was 362.46 mg kg^?1 after an intraperitoneal injection in mice. Moreover, structure-activity relationships of cholesterol and β-sitosterol derivatives were briefly discussed.

Full text of DOI:10.1039/c6md00344c

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ARTICLE
Synthesis and biological activity evaluation of novel peroxo-
bridged derivatives as potential anti-hepatitis B virus agents
Received 00th January 20xx,
Accepted 00th January 20xx
Menglu Jia,‡a Rui Zhao,‡a Bing Xu,a Wenqiang Yan,a Fuhao Chu,a Hongshun Gu,b Tianxin Xie,a
Hongjun Xiang,a Jian Ren,a Dagang Chenc, Penglong Wang*a and Haimin Lei*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
Previous studies have demonstrated that natural steroid compounds containing peroxide bridge exhibited potential anti-
hepatitis B virus activity. To continue our research, a simple and regioselective methodology, by using Eosin Y as a clean
photosensitized oxidation catalyst, is developed for the synthesis of peroxide bridge in steroids. The method was exposed
to visible light and furnished in high yields, does not involve tedious work-up or purification, and avoided using
environmentally hazardous solvents. It could be regarded as a green protocol. Besides, a series of cholesterol and β-
sitosterol derivatives containing a peroxide bridge were synthesized through this method and screened for their anti-HBV
activity. Among compounds synthesized in this research, 5α, 8α-cyclicobioxygen-6-vinyl-3-oxo-cholesterone (1f,
3.13µg/ml) had the most potent activity with inhibition rates of 77.45±6.01% and 58.73±8.64 % on the secretion of HBsAg
and HBeAg antigens after 8 days. Further acute toxicity test showed that the LD50 value of compound 1f was 362.46 mg/kg
after intraperitoneal injection in mice. Moreover, structure-activity relationships of cholesterol and β-sitosterol derivatives
were briefly discussed
Introduction
conventional synthetic methods 14-16. The experimental results from
Steroids,
a widespread class of natural organic compounds
the synthesis parts indicated that Eosin Y could combine with
oxygen in the air and then oxidize dienes into peroxide bridges,
which might be regarded as a specific catalytic to diene ring in the
5,8 position of cholesterol and β-sitosterol. Therefore, we will
continue to focus on its application in other steroids and optimize
9- chemistry technology for synthesizing peroxide bridge.
occurring in animals, plants and fungi, play a highly significant role
in the drug discovery 1-3. These compounds had shown remarkable
biological activities, including antiviral, anti-tumor, antihypertensive,
anticholinergic and so on 4-8. In previous work, a new steroid
compound (alcohol from ascidian, SC) with peroxide bridge was
discovered , which exhibited potent anti-hepatitis B virus activity
11. However, due to the low content of SC in Styela Clava 12, it has
stimulated our interest in synthesizing its structural analogs.
Cholesterol and β-sitosterol have the same parent nucleus with SC
(Figure 1). So they were expected to be anti-HBV drugs by building
peroxide bridges.
In our continuous effort a series of novel cholesterol and β-
sitosterol derivatives with peroxide bridges were synthesized
through the new method. All newly derivatives were fully
1
characterized with H-NMR, 13C-NMR and 2D-NMR spectra (HMQC,
HMBC and 1H-1H COSY) and HRMS. Their inhibitory effects on the
expression of HBsAg and HBeAg in the HepG2.2.15 cells were
evaluated by MTS and ELISA assays. Moreover, acute toxic test, as a
part of safety evaluation of peroxo-bridged derivatives, was carried
out to investigate the potential toxicity by intraperitioneal injection
in ICR mice. In addition, the structure-activity relationships of these
novel compounds were also briefly discussed.
In our present study,
a new method was established to
synthesize peroxide bridge by using Eosin Y as photosensitive
catalytic oxidizer, which had been patented 13. The method was
operated at room temperature and with visible light, which was
simple and environmentally friendly compared with the
Results and Discussion
Chemistry
All designed derivatives were synthesized via the routes outlined in
Scheme 1. Compounds 1a and 2a were prepared via acetylation
reaction of cholesterol and β-sitosterol, and then underwent free
radical and elimination reaction with N-bromosuccinimide (NBS) to
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afford compounds 1b and 2b. Subsequently, products 1c and 2c connected with δC 80.0 (C-8), which were similar with 1b. Besides,
were obtained after deacetylation reaction of 1b and 2b. Then 1b
there were δC 83.4 (C-5) and δC 80.0 (C-8) in the 13C-NMR spectrum
Figure 1 The structures of SC, cholesterol, β-sitosterol and the novel peroxo-bridged derivatives.
Scheme 1 Synthesis of the cholesterol and β-sitosterol derivatives. Reagents and Conditions: (a) Toluene, acetic anhydride, pyridine, reflux, 4 h. (b)
CCl4, N-bromobutanimide (NBS), lighted, reflux, 4 h. (c) Eosin Y, anhydrous ethanol, lighted, r.t., 10 h. (d) Ethanol, 10% sodium hydroxide, 80°C, 30
min. (e) Acetone, chromic acid, ice-bath, 3 h.
of compound 1f, while δC 141.6 (C-5) and δC 138.6 (C-8) in the 13C-
and 2b were further reacted with Eosin Y under visible light to
NMR spectrum of 1b. It suggested that the peroxide bridge of
generate the target compounds 1d and 2d. Compound 1e and 2e
compound 1f was synthesized according to all of the above analysis,
were obtained via two different routes (1): a-b-c-d and (2): a-b-d-c
This inference could also be applied to compounds 1d and 1e.
with different yield rates. Compounds 1f and 2f were synthesized
Within the framework of green chemistry, solvents occupy a
from 1e and 2e with chromic acid solution in acetone eventually. All
strategic place. Green chemistry aims to replace the hazardous
newly derivatives were fully characterized with 1H-NMR, 13C-NMR
and/or harmful solvents with more environmentally friendly
and 2D-NMR spectra (HMQC, HMBC and 1H-1H COSY) and HRMS.
alternatives. In our experiment, Eosin Y, a kind of dye 17was used as
a photosensitive catalytic oxidizer18,19. It could simplify the use of
The molecular formula of 1f was assigned as C27H42O3 through
the basis of the positive high-resolution mass spectrometer (HR-MS)
Eosin Y as a photosensitive catalytic oxidizer simplified the reaction
m/z 437.30087 [M+Na]+. Especially, product 1f was further
and avoided the use of H2SO4, H2O2 and the expensive materials:
identified by HMQC, HMBC and 1H-1H COSY 2D-NMR spectra.
MTO and Re2O7 20, 21, which might be regarded as a relatively green
Combined with the significant HMQC, HMBC and 1H-1H COSY
process. In addition, the mechanism involved of the reaction is
correlations, CH-6 [δH 6.53 (d, 1H, J = 8.5 Hz, =CH-)] was connected
probably that Eosin Y activated the oxygen in the air, and then the
with δC 83.4 (C-5), and CH-7 [δH 6.27 (d, 1H, J = 8.5 Hz, =CH-)] was
structure of conjugated dienes was peroxidized to obtain
corresponding products. What’s more, the result of the catalytic
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reactions appears that Eosin Y has specificity for synthesizing acetyl in β-sitosterol derivatives may play a key role. It thus
peroxide bridge in diene.
appeared that diverse sterol matrixes had different working groups.
The phenomenon could be related to their biogenetic derivation.
Biological activity
The cytotoxicity of all compounds in HepG2.2.15 cells
The cytotoxicity of all compounds was observed by a standard MTS
method. After the incubation with various concentrations of these
synthetics, the inhibition ratio of the HepG2.2.15 cells exposed to
different values of the compounds, as shown in Figure 2. There was
a dose-effect relationship in all derivatives on cytotoxicity. These
results were used to determine the treatment concentration of
these six compounds for the subsequent experiments.
Figure 2 Cytotoxicity effect of 1d-1f and 2d-2f on HepG2.2.15 cells line.
Figure 3 The inhibition ratio of compound 1f on HBsAg and HBeAg in
HepG2.2.15 cells.The inhibition ratio of compound 1f on HBsAg and HBeAg
in HepG2.2.15 cells. HepG2.2.15 cells were treated with four concentrations
(0.39, 0.78, 1.56, and 3.13 µg/ml) of compound 1f for 4d or 8d. The cultural
media of each treatment were collected for viral HBsAg and HBeAg via ELISA
analysis. The data are expressed as the mean and the standard deviation of
the mean. (n = 3, p<0.05).
Inhibition of HBsAg and HBeAg production in HepG2.2.15 cells
As shown in Table 1, all derivatives with peroxide bridge exhibited
higher activity on anti-HBV than the others. Current study proved
that introducing peroxide bridge to SC parent steroid nucleus could
induce the anti-hepatitis B virus activity. Furthermore, it also
proved that various 3-substituents could influence peroxo-bridged
derivatives’ biological activity.
Further study on antigens inhibition of compound 1f was studied
by setting four concentration gradients. As shown in Figure 3, dose-
and time-effect relationship was found, and after 8 days, 1f showed
significant inhibitory effects on HBsAg secretion, with the highest
inhibition at 77.45±6.01%. Similarly, the secretion of HBeAg was
significantly reduced with the highest inhibition at 58.73±8.64%.
Meanwhile, we calculated the therapeutic index of 1f to indicate
its activity of anti-hepatitis B (Table 2).
Table 1 Inhibitory effect of 1d-1f and 2d-2f on HBsAg and HBeAg secretion in
HepG2.2.1 cells at 3.13 µg/mL
4 d
8 d
Compound
HBsAg (%)
HBeAg (%)
HBsAg (%)
HBeAg (%)
Table 2 Inhibitory effect on HBsAg and HBeAg in 2.2.15 cells treated with 1f
1d
1e
1f
12.96±3.45
1.20±4.67
72.28±6.00
26.67±2.98
19.01±2.65
1.91±4.65
5.68±3.78
3.51±2.65
1.35±4.87
54.10±5.06
25.64±3.23
2.88±5.45
3.28±4.98
4.94±4.99
13.77±4.23
1.56±3.12
77.45±6.01
28.79±4.34
20.04±3.12
5.70±4.13
-0.44±3.85
7.56±4.78
2.55±5.01
58.73±8.64
25.78±3.54
10.35±3.56
2.36±5.37
-9.22±6.32
Time
Antigen
IC50
TC50
TC50/IC50
HBeAg
HBsAg
HBeAg
HBsAg
1.78±0.24
1.86±0.09
2.59±0.15
2.95±0.35
5.79±0.52
5.51±0.18
3.40±0.24
3.47±0.37
4d
2d
2e
2f
10.25
8d
3TC
For example, activity of 1f was more remarkable than 1d and 1e,
which suggested 3-carbonyl might promote the anti-HBV activity of
cholesterol derivatives with peroxide bridge. While, compound 2d
exhibited stronger activity than 2e and 2f, which demonstrated 3-
Acute toxicity of compound 1f in vivo
Although, compound 1f exhibited relatively low cytotoxicity in vitro
,
the toxicity in vivo was not clear. To evaluate the safety of
compound 1f, the acute toxicity was tested in ICR mice. At dose of
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56, 178, 350, 422, 750 and 1000 mg/kg administered Notes and references
intraperitoneally (i.p.). Every dose was given 6 mice, compound 1f
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Conclusion
A new method to synthesize peroxo bridge was established by using
Eosin Y as photosensitive catalytic oxidizer. Comparedwith the
conventional synthetic methods, it was in lower consumption and
more convenient. Therefore, our synthesis of peroxide bridges had
been optimized by the special catalyzer. A series of novel steroid
derivatives containing peroxide bridge were synthesized using this
method, and their bioactivity tests showed that most derivatives
had anti-HBV activities. In particularly, compound 1f exhibited
higher inhibitory against HBsAg and HBeAg secretion in HepG2.2.15
cells, while nearly no cytotoxicity on HepG2.2.15 cells at 3.13 µg/mL,
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4
the assembly of virus particles , which was pending further study.
Therefore, these compounds were expected in collaboration with
3TC for the treatment of hepatitis B diseases. In summary, the
steroid derivatives with peroxide bridges demonstrated potential
anti-HBV activity, and it provided us new ideas to discover anti-HBV
leading compounds.
Acknowledgments
This study was financially supported by the National Natural Science
Foundation of China (No. 81173519) and the Innovation Team
Project Foundation of Beijing University of Chinese Medicine (Lead
Compound Discovering and Developing Innovation Team Project
Foundation).
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