Synthesis and biological evaluation of nucleoside analogues than contain silatrane on the basis of the structure of acyclovir (ACV) as novel inhibitors of hepatitis B virus (HBV)

Article abstract of DOI:10.1016/j.bmcl.2012.12.097

Hepatitis B virus (HBV) infection causes major public health problems worldwide. Acyclovir (ACV) is mainly used to inhibit herpes simplex virus (HSV) rather than HBV. In this study, we used the combination principle to design and synthesize nucleoside analogues that contain silatrane on the basis of the structure of ACV. We found that the compounds were effective inhibitors of HBV, both in vitro and in vivo. All of the compounds showed suppressive activity on the expression of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in the HepG2.2.15 cell line with low cytotoxicity. One of compounds was studied in HBV transgenic mice model, and the test results showed its ability to reduce the levels of HBsAg, HBeAg and HBV DNA by ELASE and qPCR. Furthermore, significant improvement of T lymphocyte was observed after treatment, as evaluated by flow cytometry (FCM).

Full text of DOI:10.1016/j.bmcl.2012.12.097

Bioorganic & Medicinal Chemistry Letters 23 (2013) 1310–1314
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of nucleoside analogues than
contain silatrane on the basis of the structure of acyclovir (ACV) as
novel inhibitors of hepatitis B virus (HBV)
Anyue Han a, , Lingna Li a, , Kuiyou Qing a, Xiaolu Qi a, Leping Hou a, Xintong Luo a, Shaohua Shi a,
Faqing Ye a,b,
⇑
a School of Pharmacy, Wenzhou Medical College, Wenzhou 325000, PR China
b Bioorganic & Medicinal Chemistry Research Center, Wenzhou Medical College, Wenzhou 325000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Hepatitis B virus (HBV) infection causes major public health problems worldwide. Acyclovir (ACV) is
mainly used to inhibit herpes simplex virus (HSV) rather than HBV. In this study, we used the combina-
tion principle to design and synthesize nucleoside analogues that contain silatrane on the basis of the
structure of ACV. We found that the compounds were effective inhibitors of HBV, both in vitro and
in vivo. All of the compounds showed suppressive activity on the expression of HBV surface antigen
(HBsAg) and HBV e antigen (HBeAg) in the HepG2.2.15 cell line with low cytotoxicity. One of compounds
was studied in HBV transgenic mice model, and the test results showed its ability to reduce the levels of
HBsAg, HBeAg and HBV DNA by ELASE and qPCR. Furthermore, significant improvement of T lymphocyte
was observed after treatment, as evaluated by flow cytometry (FCM).
Received 13 October 2012
Revised 25 December 2012
Accepted 28 December 2012
Available online 9 January 2013
Keywords:
Hepatitis B virus
Silatrane
ACV
Ó 2013 Elsevier Ltd. All rights reserved.
HepG2.2.15 cell
HBV transgenic mouse
Infection with hepatitis B virus (HBV) leads to a wide spectrum of
clinical presentations ranging from an asymptomatic carrier state to
self-limited acute or fulminant hepatitis to chronic hepatitis with
progression to cirrhosis, hepatocellular carcinoma and other com-
plications.1 The World Health Organization (WHO) estimated that
one-third of the world’s population has been infected by HBV.
Chronic hepatitis B affects more than 350 million people worldwide
and is associated with as many as 1 million deaths per year due to
associated complications.2 Nucleotide analogs, such as lamivudine
(3TC), entecavir and adefovir, and interferon are approved drugs
used for the treatment of HBV infections. However, unresolved crit-
ical issues, such as side effects, dose-dependent side effects, rela-
tively low cure rate, and quick accumulation of drug-resistant
mutants, have limited their use.3 Therefore, alternative strategies
and drugs are being sought to combat this disease, whether for
monotherapy or combination therapy.
Acyclovir (ACV), 9-(2-hydroxyethoxymethyl)guanine, is used
extensively to inhibit several herpes viruses, particularly HSV-1
and HSV-2.4 In cells infected by herpes viruses, acyclovir can be
selectively phosphorylated by virus-encoded thymidine kinase
(TK) to its monophosphate and then transformed by cellular en-
zymes to the triphosphate, which is then used as a substrate by
the viral DNA polymerase. Due to the lack of 30-hydroxyl group,
it functions as a chain terminator, thereby inhibiting the viral
DNA synthesis.5 However, acyclovir has been shown in animal
models to be a modest inhibitor of HBV DNA polymerase for both
in vitro and in vivo activities; however, it has demonstrated disap-
pointing anti-HBV efficacy in human clinical trials.6
Silatrane derivatives are known for their extensive and en-
hanced biological activities, such as immunoregulation, anti-
inflammatory effect, and anti-tumor effect.7 Over the years, re-
search has shown that silatrane derivatives are relatively safe
due to their low toxicity and that they do not have any direct effect
on cancer cells, working instead through the immune system.8 In
addition, it has been reported that 1-chloromethyl silatrane is
effective in the treatment of rheumatoid arthritis due to a higher
activity of T lymphocytes.9
Abbreviations: HBV, hepatitis B virus; ACV, acyclovir; HSV, herpes simplex virus;
HBsAg, hepatitis
B virus surface antigen; HBeAg, hepatitis B e antigen; 3TC,
lamivudine; MTT, methyl thiazolyl tetrazolium; ELISA, enzyme-linked immunosor-
bent assay; qPCR, quantitative polymerase chain reaction; GAPDH, glyceraldehyde-
3-phosphate dehydrogenase; FCM, flow cytometry.
In this study, based on the combination principle, we designed
and synthesized nucleoside analogues that contain silatrane. It was
expected that these analogues could eliminate the virus from the
⇑
Corresponding author. Tel.: +86 0577 86689793; fax: +86 0577 86689983.
E-mail address: yfq664340@163.com (F. Ye).
Both authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.097

A. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1310–1314
1311
Scheme 1. Reagents and conditions: (a) succinic anhydride or glutaric anhydride, pyridine, 120 °C, 10 h; (b) 130 °C, 2 h; (c) EDCꢀHCl, DMF, rt.
Table 1
Primer design for detection of target genes
Genes
Forward primer
Reverse primer
GAPDH
50-GTTGTCTCCTGCGACTTCA-30
50-GGTGGTCCAGGGTTTCTTA-30
IFN-c
50-TGCTCGAGACTTCAAGCTCTACAGC-30
50-TGCTCGAGACACACTGCATCTTGGC-30
50-ATAAAACGCCGCAGACACATC-30
50-CGGGTACCATGCTTTGACAGAAGGC-30
50-CGGGTACCATGAGCTCATTGAATGC-30
50-AACCTCCAATCACTCACCAACC-30
IL-2
HBV-DNA
body by using silatrane to promote the activation and proliferation
of T lymphocytes. Subsequently, we were pleasantly surprised and
encouraged when our biological activity research indicated that
some of our compounds showed very potent anti-HBV activity.
3 days. MTT (5 g/l) reagent was added 4 h before the end of the cul-
ture, and then the cells were dissolved with DMSO, OD values were
read at 570 nm, and the percentage of cell death was calculated.
These same drug concentrations were used in subsequent assays.
The 50% cytotoxic concentration (CC50), defined as the concentra-
tion that caused a 50% reduction in the number of viable cells or
in the optical density, was calculated according to the Berkson logit
method.14 Lamivudine (3TC) and acyclovir (ACV) were used as po-
sitive controls.
Starting from
c-aminopropyltriethoxysilane and triethanola-
mine, we prepared the silatrane in one step and with good yield.10
Subsequently, acyclovir was reacted with anhydride in pyridine for
10 h with excellent yield.11 Pyridine, which is an alkaline solvent,
can promote reaction by the partial dissolution of the compounds.
Compounds L1–L4, combinations of acyclovir derivatives and sila-
trane, were produced definitively by EDCꢀHCl coupling at room
temperature.12 The ultimate products were purified as a light-yel-
low solid by column chromatography and assayed by 1H nuclear
magnetic resonance, mass spectrometry, and infrared spectros-
copy. The general procedure for their synthesis is depicted in
Scheme 1.
The cytotoxicity of each compound, evaluated by using the MTT
assay, was expressed as the concentration of compound required
to kill 50% (CC50) of the HepG2.2.15 cells. In addition, the anti-
HBV activity of each compound was expressed as the concentra-
tion of compound that achieved 50% inhibition (IC50) to the secre-
tion of HBsAg and HBeAg using the ELISA method. As shown in
Table 1, compounds L1–L4 had low toxicity to HepG2.2.15 cells.
According to the results derived from antiviral assays, L2 had a bet-
ter inhibitory effect on the secretion of HBsAg and HBeAg in the
2.2.15 cell line. Therefore, we chosen compound L2 for further ani-
mal experiment to investigate its anti-HBV activities in vivo.
The HBV transgenic mice used in this study were obtained from
Shanghai Research Centre for Model Organisms (Shanghai, China).
The mice were initially produced on a C57BL/6 background and
had 3.2 Kb of the HBV DNA integrated into the mouse genome,
after which it were backcrossed to C57BL/6J for at least seven gen-
erations. A high level of HBsAg in the sera could be detected in the
HBV transgenic mice. In all experiments, the mice were matched
for age (8 weeks) with an equal number of males and females that
had equal hepatitis B surface antigen (HBsAg) levels in their sera
before experimental manipulations. All animals were kept under
standard, pathogen-free conditions in the Laboratory Animal Cen-
tre of Wenzhou Medical College, and all animal experiments were
performed according to the guidelines for the care and use of med-
ical laboratory animals (Ministry of Health PR China, 1998) and the
guidelines of the Laboratory Animal Ethical Commission of Wenz-
hou Medical College.
The HepG2.2.15 cell (clonal cells derived from HepG2cells
transfected with a plasmid containing HBV DNA), which was pro-
vided by Zhejiang University (China), was cultured routinely in
Dulbecco’s Modified Eagle Medium (DMEM, USA) supplemented
with 10% (v/v) fetal bovine serum (Hyclone, USA) at 37 °C in an
incubator at 5% CO2. The cells were seeded in 24-well tissue culture
plates at approximately 5 ꢁ 105/well. After incubation at 37 °C for
24 h, fresh culture media with a variety of concentrations of com-
pounds, lamivudine (3TC) or acyclovir (ACV) was added and re-
placed every 2 days. After the treatment with the drugs for
6 days, the media were collected and used for Measurement of se-
creted HBsAg and HBeAg.13 The expression levels of HBsAg and
HBeAg were measured by enzyme-linked immunosorbent assay
(ELISA) kits (Shanghai KeHua Bio-engineering Corporation, China)
according to the manufacturer’s instructions. The media samples
were collected after 6 days of treatment, centrifuged at 5000 rpm
for 10 min to eliminate cellular debris, and then used immediately
for ELISA.
Cytotoxicity towards HepG2.2.15 was assessed by the MTT as-
say to determine the drug concentrations that did not affect cell
viability. Briefly, confluent cultures of cells in 96-well flat-bot-
tomed tissue culture plates were treated with different doses of
the test compounds (six cultures for each of two dilutions) for
Mice were randomly divided into five group of ten in each
group. The groups included the normal control group, model group,
positive group, and drug-treatment group. The normal control

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A. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1310–1314
Table 2
Liver tissues were dissolved in Trizol Reagent (Invitrogen, Carls-
bad, CA, USA) for mRNA and HBV DNA extraction. Fluorescent
quantitative polymerase chain reaction (qPCR) was performed
using the commercial PCR kit (Fosun High Technology Co. Ltd,
Shanghai, China), following the manufacturer’s directions. Gene-
Cellular toxicity and antigen secretion in HepG2.2.15 cells treated with compounds
and 3TC
a
b
Compounds
n
R
CC50
(
lM)
IC50
(lM)
HBsAg
HBeAg
specific primers for mouse IL-2, IFN-c, and HBV were designed (Ta-
L1
L2
L3
2
2
3
3
—
—
H
CH3
H
CH3
—
—
485 67
467 90
570 68
732 87
>1000
79.7 8.3
34.5 3.7
90.4 3.6
140.9 12.6
405.6 98.4
>1000
125.1 35.7
89.8 7.8
160.2 36.2
485.6 68.3
>1000
ble 2) (synthesized by Sangon Biological Engineering Technology,
Shanghai, China). Fluorescence signals were measured after 40
PCR cycles, and all samples were normalized to GAPDH housekeep-
ing gene levels. All samples were run in duplicate.
L4
3TCc
ACVd
630 40
>1000
To further evaluate the anti-HBV activity of the drug in HBV
transgenic mice, we detected the expression level of HBV DNA in
liver tissue by using qPCR techniques. As shown in Figure 1, the re-
sults demonstrated that the titer of HBV DNA in liver was reduced
markedly in the mice that received the drug compared with those
that received 0.1% DMSO and normal saline (p <0.01). In addition,
the titer of HBV DNA was analogous to the positive group that
was administered 3TC. The results demonstrated that compound
L2 inhibited HBV DNA replication and that it was effective at doing
so than 3TC.
All values are the mean of three independent experiments.
a
CC50, the cytotoxicity concentration of compound that reduced cell viability to
50%, denoted by means SD.
b
IC50, the concentrations of compound needed to inhibit HBsAg and HBeAg
secretions to 50%, denoted by means SD.
c
3TC: lamivudine, an antiviral agent used as positive control.
ACV: acyclovir, an antiviral agent used as positive control.
d
group was given only normal saline with no drugs, and DMSO solu-
tion (0.1%) was used as a model for the animal experiments. Posi-
tive groups were given lamivudine (3TC) and acyclovir (ACV). For
administration to mice, the purified drugs, 3TC and ACV, solubi-
lized by the aid of 0.1% DMSO, were given once daily orally by
intraperitoneally administration with the same dose of 100 mg/
kg for one month, respectively. After 30 days, the mice were eutha-
nized, and their livers, spleens, blood, and sera were collected for
the following detection.
The two mRNA levels of cytokines were assayed by PCR, and the
results were shown in Figure 2. The mRNA levels of cytokines IFN-
and IL-2 were elevated in the drug groups, but they were low in the
control. In addition, liver IFN- mRNA levels were significantly
c
c
higher in the treatment group than in the positive group. In these
assays, mRNA for GAPDH was used as an internal control. The re-
sults indicated that the drug treatment could genuinely and effica-
ciously enhance the expression of IFN-c and IL-2 mRNA.
There were numerous HBsAg and HBeAg in the sera of the HBV
transgenic mice, which are an important animal model for study-
ing the immunotherapeutic effects of antiviral drugs or vaccines.
To investigate the effect of anti-HBV activity elicited by the drugs
in HBV transgenic mice, serum HBsAg and HBeAg levels were de-
tected by ELISA using the HBsAg and HBeAg ELISA kit (Shanghai
KeHua Bio-engineering Corporation, China). As shown in Figure 3,
serum HBsAg and HBeAg levels from the mice that received drug
L2 decreased markedly compared to the normal control group
(normal saline) and the model group (0.1% DMSO) (p <0.01). In
addition, the HBsAg and HBeAg levels of the treatment group
undoubtedly were lower than the levels obtained in the mice with
lamivudine control. These results indicated that the treatment
drug inhibited both HBsAg and HBeAg of HBV effectively and
simultaneously in vivo.
Th1 cells produce IFN-
directing cell-mediated immune responses, which are important
for clearance of intracellular pathogens. Serum levels of IFN- , IL-
c, IL-2, and they play a critical role in
c
2 were detected by ELISA (Shanghai KeHua Bio-engineering Corpo-
ration, China) to assess our drug, and the results are shown in
Figure 4. Our data, which were generated from ELISA analyses,
Figure 1. HBV DNA in liver was extracted and analyzed by the qPCR method using
an HBV DNA PCR kit. Lamivudine (3TC), an antiviral agent, was used as the positive
control. The data were the mean SD from 10 transgenic mice.
Figure 2. The level of IFN-c mRNA and IL-2 mRNA in liver was extracted and analyzed by quantitative PCR method using PCR kit. The 3TC, an antiviral agent, was used as
positive control. The data were the mean SD from 10 transgenic mice.

A. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1310–1314
1313
Figure 3. Serum levels of HBsAg and HBeAg in HBV transgenic mice were analyzed by ELISA. Data are presented as mean values SD from 10 transgenic mice.
Figure 4. The serum levels of cytokine (IFN-c and IL-2) were extracted and examined by the ELISA technique. The results were the mean SD from 10 transgenic mice.
showed elevated serum concentration of IFN-
transgenic mice in comparison to the model group and the normal
c and IL-2 of HBV
Table 3
Peripheral blood T lymphocyte subgroups in transgenic mice treated with com-
pounds, 3TC or ACV
control group (p <0.05).
T lymphocyte and their subpopulations are the key composi-
tions of cellular immune system. CD4+ and CD8+ cells are the major
functional subgroups of T lymphocyte and play an important role
in response to HBV infection, which can reflect the cellular im-
mune function and immunoregulation and usually regarded as a
valuable index.15 Heparinized blood samples were collected, and
erythrocytes of blood were lysed in NH4Cl buffer. All samples were
analyzed with flow cytometry (FCM) by using anti-mouse mono-
clonal antibodies CD3-PE-CY5/CD4-FITC/CD8-PE (Sigma). For each
sample, detection was conducted using CellQuest software (Coul-
ter). The results were expressed as the percentages of CD3+/CD4+
(short for CD4+ below) and CD3+/CD8+ (short for CD8+ below) cells
found to be positive for the marker antigen in the total T-cell pop-
ulation. The handling procedures were performed in strict accor-
dance with the instructions provided on the insert in the reagent
kit package. As shown in Table 3, CD3+, CD4+, and CD8+ subpopula-
tions and the CD4+/CD8+ ratio of peripheral blood increased in the
treatment group in comparison to other groups. The results dem-
onstrated that the impaired balance of T-cell subsets was improved
significantly by our drug.
In this Letter, we examined the anti-HBV activities of our com-
pounds both in vitro and in vivo. For the in vitro study, we took
advantage of the HepG2.2.15 cells, which contain HBV genomes
integrated into the host DNA, and thus produce and secrete HBsAg
and HBeAg. This cell line is widely accepted as a standard model for
the evaluation of anti-HBV activities in vitro.16 Prior to the investi-
gation of anti-HBV activity for these compounds, the cytotoxicity
against HepG2.2.15 cells was determined. The corresponding
CC50 values of [L1–L4] on HepG2.2.15 cells were all similar to that
of lamivudine, which was considered as non-cytotoxic to the cell
line.
Groups
CD3+ (%)
CD4+ (%)
CD8+ (%)
CD4+/CD8+ (%)
Control
Model
L2
3TC
ACV
29.8 1.7
30.1 2.9
48.3 3.4
34.1 2.1
31.2 4.7
14.1 1.4
13.6 2.7
33.0 3.2
19.3 4.8
18.7 2.5
14.7 1.8
14.1 1.4
20.6 2.3
16.7 1.7
15.1 2.0
0.9 0.1
0.9 0.2
1.6 0.3
1.2 0.3
1.2 0.2
Heparinized blood without erythrocyte was analyzed with FCM by suing anti-
mouse monoclonal antibodies. All values are the mean SD from 10 transgenic
mice.
Based on the experimental outcomes discussed above, it could
be suggested that these compounds possess the bioactivity of sup-
pressing the expression of HBsAg and HBeAg in the culture med-
ium. Especially, compound L2 seemed to be more potential than
the other compounds against HBV in HepG2.2.15 cells. This indi-
cated that the compounds may show protective effects via a differ-
ent mechanism to 3TC, and they may have the potential to work
synergistically with other antiviral agents. Therefore, additional
investigation of compound L2 in vivo to clarify its anti-HBV activ-
ities was required.
Although the anti-HBV nucleoside drugs mainly exhibit the
early virological response and target the viral polymerase activity,
the long-term effects of most antiviral drugs are unsatisfactory.17
According to previous reports, in HBV infection, efficient therapy
of anti-HBV drugs results in sustained inhibition of HBV replication
and a significant enhancement of cellular immune responses.18
However, chronic HBV infection, like many other chronic viral
diseases and cancers, is associated with T-cell hyporesponsiveness
or tolerance. The exact molecular mechanisms associated with
this phenomenon are not entirely clear, but negative selection,

1314
A. Han et al. / Bioorg. Med. Chem. Lett. 23 (2013) 1310–1314
peripheral anergy, and imbalances in lymphokine production all
appear to contribute to maintaining the hyporesponsive state of
the host that is exposed chronically to viral or cancer antigens. T-
cell failure was associated significantly with viral replication level,
and viral clearance requires the coordinated action of the immune
response.19 CD4+ cells as T-helpers are essential for the control of
HBV infection by facilitating the induction and maintenance of
the CD8+ T-cell response. Many studies show that lysis of infected
hepatocytes is unlikely to be the only mechanism by which CD8+ T-
cells exert their anti-viral effect and that the control of viral repli-
cation in the transgenic mouse models also may be an important
mechanism.20 The role of antivirals in the setting of chronic infec-
tion is to decrease viral production and also to restore the specific
CD4ꢂ and CD8ꢂ mediated immune response following the decrease
in viral load in the infected host.18a,21
In this study, we observed major changes of T lymphocyte sub-
groups in the peripheral blood from HBV transgenic mice with L2
treatment. Concomitantly with a quantitative reduction in viral
replication, the frequency of CD8+ T-cells increased during effective
therapy. The disappearance of most of the HBV DNA from the liver
was followed by increased expression of T-cell markers, maximal
CD4+ and CD8+ T-cell responses in the blood, and seroconversion
to HBeAg- and HBsAg-specific antibodies. And the impaired bal-
ance of T-lymphocyte subsets was improved significantly.
Reference and notes
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10. Synthesis of silatrane was carried out by placing 0.1 mol of
c-
aminopropyltriethoxysilane and 0.1 mol of triethanolamine into 100-ml
a
reaction flask equipped with a reflux condenser and a dry pipe with anhydrous
calcium chloride. The mixture was stirred magnetically and heated at 130 °C
for 2 h. During the course of the reaction, a large quantity of ethanol was
created, and it was removed by refluxing for 4 h. The remaining liquid was
distilled under reduced pressure, and the fraction that boiled between 164 and
166 °C at 66.6 Pa was collected. After cooling, the resulting yellow solid, that is,
silatrane, was filtered out of the solution and washed. The yield was 81%.
11. A mixture of acyclovir (10 mmol) and anhydride (20 mmol) in pyridine (20 ml)
was stirred and heated in an oil bath at 120 °C for 10 h. The completion of the
reaction was monitored by thin layer chromatography (TLC). After the mixture
was cooled to 0–5 °C, the crystalline solid that separated out was collected by
filtration, thoroughly washed with acetone, and dried in vacuo at 40 °C. An
analytical sample was obtained by crystallization from methanol as a white
solid in 76% yield.
12. A solution of silatrane (10 mmol) in chloroform (15 ml) was added dropwise to
a stirred solution of acyclovir esters (10 mmol) and EDCꢀHCl (20 mmol) in DMF
(15 ml). The resulting mixture was stirred at room temperature for 10 h. After
removing the solvents under reduced pressure, the crude residue was purified
by chromatography on silica (CHCl3/CH3OH 10:1), which produced the
combination of silatrane and acyclovir esters as a light yellow solid (39–58%
yield).
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The production of type 1 cytokines, such as interferon (IFN)-y
and interleukin (IL)-2, favors cellular immune responses and is
associated with protective antiviral immune responses.22 It has
been suggested that the alteration of serum and intrahepatic IFN-
c
and IL-2 cytokine play an important role in viral persistence, host
immune response and liver damage in chronic viral hepatitis. Our
study detected the molecular level of IFN- and IL-2 in serum and
c
their mRNA levels. The results all showed that the compound en-
hanced the secretion of two cytokines, which was due to the acti-
vation of T-cells.
In conclusion, the results of our study indicated that an acyclo-
vir derivative that contains silatrane reduced the production of
HBV-specific antigens, reduced the level of HBV DNA, and activated
immunoregulation. Although further studies are required to eluci-
date the molecular mechanisms, these results suggest that this
kind of compound may play an important role in the therapy asso-
ciated with hepatitis B virus infections.
Acknowledgments
This work was financed in part by Zhejiang Science & Technol-
ogy Funding (Y2080697 to F.Q.Y.; Y204089 to F.Q.Y.). We are grate-
ful to Professor Mifang Liang for the assistance in this work.
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