Discovery of the first series of small molecule H5N1 entry inhibitors

Article abstract of DOI:10.1021/jm900275m

The occurrence of highly pathogenic avian influenza virus H5N1 highlights the urgent need for new classes of antiviral drugs. Inhibition of H5N1 entry into cells may be an effective strategy. We report the first three small molecule inhibitors saponins wi

Full text of DOI:10.1021/jm900275m

7368 J. Med. Chem. 2009, 52, 7368–7371
DOI: 10.1021/jm900275m
Because of the safety concerns in studying highly patho-
genic viral pathogens such as Ebola and Marburg viruses,
SARS-CoV, pseudotyping systems have been established that
alleviate the safety concerns of working with the “live” viruses
and also allow easy manipulation of viral glycoproteins in
receptor binding and viral entry. Pseudotype reporter viruses
have been widely used as a quantitative high-throughput
method for assessing viral entry inhibitors and antibody
neutralization for highly pathogenic enveloped viruses such
as SARS and Ebola viruses.6,7 Influenza A virus surface
glycoprotein HA is the sole surface protein essential for viral
entry. Lentiviral vector pseudotyped with high pathogenic
H5N1 subtypes influenza A viruses hemagglutintins has been
established by us.8 Others have used the same system to
identify antiflu neutralizating antibodies.9 In this report, we
used this efficient HIV-based pseudotyping system to evaluate
potential anti-H5N1 entry inhibitors, while the VSV-G/HIV
pseudovirions7 were used as specificity controls. Using these
pseudotyping systems, we have screened a saponin library
generated from semisynthesis. We found that chlorogenin
3-O-β-chacotrioside (1)10 and chlorogenin 6-R-O-actyl-3-O-
β-chacotrioside (2)10 displayed strong inhibitory activity
against H5N1 entry with IC50 of 7.22 and 9.25 μM, respec-
tively. Notably, the two saponins 1 and 2 beared the same
β-chacotriosyl (R-L-rhamnopyranosyl-(1f2)-[R-L-rhamno-
pyranosyl-(1f4)]-β-D-glucopyranosyl) residue, which was a
typical sugar chain of natural spirostan saponins with the
most cytotoxic activities when compared to the other spiro-
stan saponin members in nature.11 To our knowledge, this is
the first report that these small molecule compounds were
discovered to effectively block H5N1 viral entry (Figure 1).
To develop additional small molecule inhibitors with en-
hanced activity, compound 1 was chosen as the lead com-
pound to design and synthesize a series of analogues 3-9 to
explore the preliminary structure-activity relations of these
designed molecules around the aglycone and sugar chain. We
first investigated the effect of aglycone residue on the inhibi-
tory activity. Dihydrochlorogenin, dehydroisoandrosterone,
methyl oleanolate, methyl ursolate, and stigmasterine12 were
selected as the aglycone moieties, and the resulting five
saponins 3-7 with the same 3-O-β-chacotriosyl residue were
derived (Figure 2). Saponins 8 and 9, simplifing the β-chaco-
triosyl moiety of 1 into R-L-rhamnopyranosyl-(1f4)-β-D-
glucopyranosyl and R-L-rhamnopyranosyl-(1f2)-β-D-gluco-
pyranosyl moiety, were also synthesized in order to understand
the effect of sugar chain on the activity. Here we report the
determination of the saponin inhibitors for H5N1 viral entry
and preliminary structure-activity relations (SARa) of these
designed molecules 3-9.
Discovery of the First Series of Small Molecule
H5N1 Entry Inhibitors
Gaopeng Song,‡,^ Sen Yang,§, ,^ Wei Zhang,‡ Yingli Cao,§
Peng Wang,‡ Ning Ding,‡ Zaihong Zhang,‡ Ying Guo,*,§
and Yingxia Li*,‡
‡Key Laboratory of Marine Drugs, The Ministry of Education of
China, School of Medicine and Pharmacy, Ocean University of
China, Qingdao 266003, China, §Department of Pharmacology,
Institute of Materia Medica, Chinese Academy of Medical Sciences
and Peking Union Medical College, Beijing 100050, China, and
Department of Pharmacognosy, Medical College of Chinese
People’s Armed Police Forces, Tianjin 300162, China.
^ These authors contributed equally to this work
Received March 4, 2009
Abstract: The occurrence of highly pathogenic avian influenza virus
H5N1 highlights the urgent need for new classes of antiviral drugs.
Inhibition of H5N1 entry into cells may be an effective strategy.
We report the first three small molecule inhibitors saponins with
3-O-β-chacotriosyl residue, which showed potent inhibitory activity
with IC50 of 7.22-9.25 μM. The subsequent SAR studies showed the
3-O-β-chacotriosyl residue was essential for the activity, and the
aglycone structure also affected the activity.
Highly pathogenic avian influenza (HPAI) continues to
cause outbreaks in poultry and migratory birds in Asia,
Europe, and Africa. Recently, humans became infected by
this virus with both increasing number of infected individuals
and high mortality rates,1-3 suggesting persisting threat of
human pandemic. In the wake of such a pandemic, the World
Health Organization (WHO) and many individual nations
have developed plans to limit its hazardous consequences.
Tremendous efforts are made already to fully understand the
pandemic of the virus and to develop effective therapies to
control the spread of the virus. To date, only two classes of
anti-influenza drugs have been approved that may be effective
against H5N1 viruses. The first class is the inhibitors of
M2 ion channel such as amantadine and rimantadine. But
use of these inhibitors rapidly leads to the emergence of
resistant variants and thus they are not recommended for a
general and uncontrolled use.4 The second class is the neur-
aminidase inhibitors such as oseltamivir, zanamivir, and
peramivir. However, resistance of H5N1 to oseltamivir has
also been observed recently.5 Therefore, there is an urgent
need for new classes of agents to combat avian H5N1 variants
that are resistant to treatment by targeting at other potential
viral factors.
The first step of influenza virus infection is the attachment
of viral particles to the host cell. This is mediated by viral
envelope glycoprotein, hemagglutinin (HA), which binds to
its receptor on host cell, sialic acid, leading to viral endo-
cytosis. Therefore, it is important to identify and develop
potent entry inhibitors against H5N1 virus as potential anti-
viral treatments.
The preparation of known saponin 3 was performed accord-
ing to our previous procedure.12 The synthesis of target sapo-
nins 4-6 was done by the similar route as that for compounds 1
and 2 reported previously by us.10,12 As shown in Scheme 1,
glycosylation of dehydroisoandrosterone, methyl oleanolate,
a Abbreviations: Lev, levulinoyl; LevOH, acetylpropionic acid;
TMSOTf, trimethylsilyl trifluoromethanesulfonate; EDC HCl, 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide hydrochloride; NIS, N-iodo-
butanimide; AgOTf, silver trifluoromethanesulfonate; SAR, structure-
activity relations.
*To whom correspondence should be addressed. For Y.G.: phone:
þ86-10-63165176; fax: þ86-10-63165176; E-mail: yingguo6@yahoo.
com. For Y.L.: phone: þ86-532-82032150; fax: þ86-532-82033054;
E-mail: liyx417@ouc.edu.cn.
3
r
pubs.acs.org/jmc
Published on Web 06/18/2009
2009 American Chemical Society

Letter
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7369
Scheme 1. Synthesis of Target Compounds 4-6a
Figure 1. Saponin inhibitors for H5N1 viral entry.
aReagents and conditions: (a) TMSOTf, CH2Cl2; (b) MeONa;
(c) 1-BBTZ, Et3N, CH2Cl2; (d) BF3 Et2O, CH2Cl2; (e) MeONa.
3
Scheme 2. Synthesis of Target Compound 7a
Figure 2. Saponins designed for SAR studies.
and methyl ursolate with 2,3,4,6-tetra-O-benzoyl-D-glucopyr-
anosyl trichloroacetimidate (10), respectively, under the action
of TMSOTf afforded the 3-O-β-glucopyranosides 11-13.
Removal of the benzoyl groups with NaOMe in MeOH gave
14-16, which were subjected to 1-BBTZ [1-(benzoyloxy)-
benzotriazole] in the presence of Et3N to protect selectively
the 3,6-OHs of the β-glucopyranosyl residues, leading to inter-
mediates 17-19. Subsequent glycosylation of the 2,4-OHs in
17-19 with 2,3,4-tri-O-acetyl-L-rhamnopyranosyl trichloro-
acetimidate (20) under the “inverse addition conditions” with
aReagents and conditions: (a) LiAlH4, AlCl3; (b) Ac2O, pyridine;
(c) Pd/C; (d) 1-BBTZ, Et3N, CH2Cl2; (e)LevOH, EDC HCl, DMAP;
3
(f) i: NBS, acetone-H2O; ii: DBU, CNCCl3, CH2Cl2; (g) TMSOTf,
CH2Cl2; (h) AcOH-NH2NH2; (i) BF3 Et2O, CH2Cl2; (j) MeONa.
BF3 Et2O as the promoter led to the desired compounds
21-23. Final treatment with NaOMe in MeOH afforded target
saponins 4-6 smoothly.
3
3
corresponding trichloroacetimidate 28. With the acceptor 27
and the donor 28 in hand, their coupling reaction was then
performed under the promotion of TMSOTf to provide the
desired compound 32, and the following deprotection of the
Lev group with hydrazine acetate gave 33. The target saponin 7
was finally obtained from first glycosylation of 33 with 20 under
The synthetic route toward 7 is depicted in Scheme 2.
Glycosyl acceptor 27 was first prepared starting from 3-O-
benzyl-chlorogenin 24.10 Reductive opening of the spiroketal
withLiAlH4/AlCl3 and followedbyprotectionof6, 26-OHsin
25 with acetyl group afforded 26, in which the 3-O-benzyl
group was removed by hydrogenolysis to provide the impor-
tant intermediate 27.
the promotion of BF3 Et2O and then deprotection of the acyl
groups with MeONa.
3
The next step was to elaborate glucopyranosyl donor 28.
Levulinoyl group was chosen to protect the C2-OH and C4-OH
in 28, which was helpful to construct β-glycosidic linkage as the
neighboring ester group and could be selectively removed
without affecting the benzoyl group in 28 and acetyl group in
acceptor 27. Thus treatment of thioglycoside 29 with 1-BBTZ
in the presence of Et3N protected selectively the 3,6-OHs to
afford compound 30, which was then masked the following
2,4-OHs with levulinoyl group to furnish 31. The latter was
hydrolyzed with N-bromosuccinimide (NBS) in acetone-
H2O, followed by treatment with CCl3CN and DBU to give
Target chlorogenin 8 containing R-L-rhamnopyranosyl-
(1f4)-β-D-glucopyranosyl moiety was synthesized as shown
in Scheme 3. Treatment of the compound 24 with BzCl in
the presence of DMAP afforded compound 36, in which the
3-O-benzyl group was removed to give the important inter-
mediate 35. Target compound 8 could be obtained by taking
advantage of a “one-pot” procedure. In this method, trichloro-
acetamidate glycosyl 37 and thioglycoside 38 were used as
sequential glycosyl donors for two glycosidic linkages.
Chlorogenin 9 containing R-L-rhamnopyranosyl-(1f2)-
β-D-glucopyranosylresiduewaspreparedasshowninScheme4.

7370 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23
Scheme 3. Synthesis of Target Compound 8a
Song et al.
Table 1. Effect of Compounds on HA Mediated Viral Entry
HA(Viet)a/
HA(QH)b/
VSVGc/HIV
compd
HIV IC50 (μM)
HIV IC50 (μM)
conc [10 μM]
1
7.84; 36.3% * (10 μM) 7.22; 42.6% (10 μM)
111%
101%
2
9.25; 46.6% (10 μM)
64.6% (10 μM)
106.1% (10 μM)
17.5% (10 μM)
8.54; 43.3% (10 μM)
102.3% (10 μM)
87.4% (10 μM)
57.9% (10 μM)
0.011
7.58; 50.4% (10 μM)
73.6% (10 μM)
108.1% (10 μM)
35.3% (10 μM)
6.00; 37.7% (10 μM)
97.9% (10 μM)
98.2% (10 μM)
61.0% (10 μM)
0.012
3
76.2%
100.2%
48.4%
86%
4
5
6
7
111.1%
116.8%
121.5%
0.015
8
9
AZT
aHA (Viet): HA, H5N1(A/Viet Nam/1203/2004).13 b HA(QH): HA,
H5N1(Goose/Qinghai/59/05).14 c VSV-G: vesicular stomatitis virus
G protein. *The percentages in this table indicated the infectivity
compared to the same amount solvent as 100%.
saponins 3-9 was evaluated by these assays. Among the
saponins 3-7 with the same 3-O-β-chacotriose residue, urso-
latesaponin 6 showed the similarinhibitory activityas1 and 2,
which exhibited much higher inhibitory activity than its
analogue oleanolate saponin 5, suggesting that the subtle
modification of aglycone had the important effect on the
antiviral activity. Compound 6 showed slightly different in-
hibitory activity against HA of the two H5N1 virus like 1 and
2 with IC50 of 8.54 and 6.00 μM, respectively, because HA of
each H5N1 virus has its own special chemical structure. The
reductive ring-opening of spirostan saponin 1, forming furo-
stan saponin 7, led to the disappearance of inhibitory activity.
Two other steroid saponins 3 and 4 did not show inhibitory
activity. These results suggest that the aglycone with more
fused rings is favored for the inhibitory activity. Compounds 8
and 9, two analogues of 1 with reduced sugar chain, showed
no activity. This further demonstrated that the 3-O-β-chaco-
triosyl residue plays a very important role in antiviral activity,
suggesting that attachment of such trisaccharide chain to
some steroids and triterpenes can contribute to enhance
inhibitory activity against H5N1 entry.
aReagents and conditions: (a) BzCl, DMAP; (b) Pd/C; (c) i: TMSOTf,
CH2Cl2; ii: NIS, AgOTf, CH2Cl2; (d) MeONa.
Scheme 4. Synthesis of Target Compound 9a
The potential pandemic of bird flu H5N1 is a global public
health concern because there are no effective vaccines available
for human. Further, the emergence of drug-resistant H5N1
strains in human patients should serve as a wakeup call for
developing new anti-H5N1 (and other flu) therapeutics. Our
strategy for developing new anti-influenza therapeutics is to
target HA, which is involved in multiple steps during viral
entry, including receptor binding, internalization, and fusion.
Theoretically, each of these steps can be a target of antiviral
therapeutics. However, up to date, no entry inhibitor drug is
available for H5N1 or any other influenza viruses. Therefore, it
is imperative to develop potent entry inhibitors against H5N1
and other influenza viruses. Here, we first discovered novel
small molecule inhibitors against HA mediated viral entry
saponins with 3-O-β-chacotriosyl residue. They can be used
as lead compounds for both prophylactic and therapeutic
treatments against bird flu H5N1 and other potential pandemic
influenza viruses. Further structure-activity relationship stu-
dies on these saponins were carried out for additional small
molecule inhibitors with enhanced activity. Such efforts are of
vital importance for the further development of novel and
effective small molecule inhibitors.
aReagents and conditions: (a) TMSOTf, CH2Cl2; (b) AcCl, CH2Cl2,
MeOH; (c) TMSOTf, CH2Cl2; (d) MeONa.
With the acceptor 35 and donor 40 in hand, their coupling
reaction was then carried out under the promotion of
TMSOTf to provide the compound 41, in which the 2-O-
acetyl group was removed from AcCl in MeOH to afford
intermediate 42. The later was glycosylated with 20 promoted
by TMSOTf to provide 43, and final deprotection of all the
acyl groups with MeONa gave the target compound 9.
An HIV-based pseudotyping system was used to screen our
semisynthesized saponin library for the entry inhibitors
against high pathogenic H5N1 (A/Viet Nam/1203/2004) in-
fection, while VSVG/HIV was applied to evaluate the speci-
ficity of H5N1 inhibitors. Potentially effective compounds
were further tested by HA (QH)/HIV model in which the HA
gene was derived from another highly pathogenic H5N1 virus
in migratory birds in Qinghai Lake (Goose/Qinghai/59/05).
The Vietnam strain we used was isolated from a fatally
infected human patient. Sequence alignments shows these
two HAs shares 97% residues similarity. Compounds 1 and
2 showed specific inhibitory effects on both HAs mediated
viral entry (Table 1). Similarly, the antiviral activity of
Acknowledgment. This project was supported by the
national Natural Basic Research Program of China (no.
2003CB716400) and NSFC Grant no. 30701035.

Letter
Supporting Information Available: Procedures for synthesis
and characterization of compounds 3-43, details of the in vitro
biological protocols. This material is available free of charge via
the Internet at http://pubs.acs.org.
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 23 7371
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