Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11

Article abstract of DOI:10.1016/j.bmcl.2011.01.098

In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no antiviral activity against coxsackievirus B₃ (CVB₃), human respiratory syncytial virus (RSV) and influenza virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC₅₀ values of 0.42 ± 0.18 μM and 0.60 ± 0.21 μM, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of action is potent as an anti-HSV clinical trial candidate.

Full text of DOI:10.1016/j.bmcl.2011.01.098

Bioorganic & Medicinal Chemistry Letters 21 (2011) 1675–1677
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro anti-HSV-1 activity of a novel Hsp90 inhibitor BJ-B11
Huai-Qiang Ju a,c, , Yang-Fei Xiang a, , Bao-Juan Xin b, Ying Pei a, Jia-Xin Lu a, Qiao-Li Wang a, Min Xia a,
b,
⇑
Chui-Wen Qian a, Zhe Ren a, Sha-Yan Wang c, Yi-Fei Wang a, , Guo-Wen Xing
⇑
a Guangzhou Jinan Biomedicine Research and Development Center, National Engineering Research Center of Genetic Medicine, Jinan University, No. 601, West Huangpu Road,
Guangzhou 510632, China
b Department of Chemistry, Beijing Normal University, Beijing 100875, China
c Shenzhen People’s Hospital, Clinical Medical College of Jinan University, Shenzhen 518020, China
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a novel Hsp90 inhibitor BJ-B11, was synthesized and evaluated for in vitro antiviral activity
against several viruses. Possible anti-HSV-1 mechanisms were also investigated. BJ-B11 displayed no
antiviral activity against coxsackievirus B3 (CVB3), human respiratory syncytial virus (RSV) and influenza
Received 20 November 2010
Revised 20 January 2011
Accepted 21 January 2011
Available online 27 January 2011
virus (H1N1), but exhibited potent anti-HSV-1 and HSV-2 activity with EC50 values of 0.42 0.18
lM and
0.60 0.21 M, respectively. Additionally, the inhibitory effects of BJ-B11 against HSV-1 were likely to be
l
introduced at early stage of infection. Our results indicate that BJ-B11 with alternative mechanisms of
action is potent as an anti-HSV clinical trial candidate.
Keywords:
Hsp90 inhibitor
Antivirals
Ó 2011 Elsevier Ltd. All rights reserved.
Herpes simplex virus
Herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) are
lipid-enveloped DNA viruses that cause various illnesses ranging
from asymptomatic infection to fulminant disseminated diseases,
such as labials herpes, keratitis, genital herpes, and encephalitis.1
In taxonomy, both HSV-1 and HSV-2 are members of the genera
Simplexvirus from the subfamily Alphaherpesvirinae in Herpesviridae
family,2 which can be distinguished by clinical manifestations and
biochemical as well as serological examinations but possess many
common characteristics, and that currently available anti-HSV-1
medicines are generally active against HSV-2 with analogical
mechanisms.3,4 The major antiviral therapy for the treatment of
HSV infection is to use nucleoside analogues such as acyclovir
(ACV). However, the increasing clinical application of this type of
antiviral agents has been associated with the emergence of drug-
resistant HSV strains.5 Therefore, the development of new anti-
HSV agents with a different mode of action is highly warranted.
Hsp90 is a multifunctional, complex, and highly specialized
chaperone machine that is extremely abundant in most organisms
and cell types.6 It can facilitate the assembly of multiprotein com-
plexes and participate in protein trafficking within the cellular
milieu in response of various types of stress. It was reported that
Hsp90 inhibitors could selectively target and induce apoptosis in
tumor tissues but had modest effects in normal cells.7 Viral
infections have also been shown to associate with Hsp90. For
instance, Hsp90 is required for full activity of the hepatitis B virus
reverse transcriptase. Studies also indicate that HSV-1 employs the
Hsp90 chaperone system during infection and the viral polymerase
may be a client protein of Hsp90.8 The possibility that the
host–pathogen interaction may represent a novel and specific anti-
viral target has led us study novel Hsp90 inhibitors as antiviral
agents.
In previous reports,9 we analyzed the molecular mechanism of
the apoptosis effect of a selective Hsp90 inhibitor, SNX-2112,10,11
on human chronic myeloid leukemia (CML) K562 cells, and a
sensitive and specific reversed-phase high-performance liquid
chromatography method was used for the identification and quan-
tification of SNX-2112 in rat plasma. We also examined the antivi-
ral activities of SNX-2112 and its analogues in order to investigate
the correlation between viral infections and Hsp90. Here, we
report the synthesis and antiviral activity of 2-(4-acetyloxy-
cyclohexylamino)-4-(3,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-
indazol-1-yl)-benzamide (BJ-B11, 1, Scheme 1), a novel analogue of
SNX-2112. Also, the possible anti-HSV-1 mechanisms of BJ-B11
were investigated.
The synthetic route of compound 1 was shown in Scheme 1.
Triketones 3 were conveniently obtained from acetyl chloride
and dimedone 2 in the presence of triethylamine and catalytic
4-dimethylaminopyridine (DMAP) at room temperature. 4-Hydra-
zino-2-(4-hydroxy-cyclohexylamino)-benzonitrile (5) was pre-
pared by treatment of the known compound 410 with hydrazine
Abbreviations: Hsp90, heat shock protein 90; MTT, 3-(4,5-diethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromideassay; Vero, African green monkey kidney cell
line; MOI, multiplicity of infection; PFU, plaque forming unit.
⇑
Corresponding authors. Tel./fax: +86 20 85223426 (Y.F.W.).
E-mail addresses: twang-yf@163.com (Y.-F. Wang), gwxing@bnu.edu.cn
(G.-W. Xing).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.01.098

1676
H.-Q. Ju et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1675–1677
Scheme 1. Chemical structure and general synthesis process of BJ-B11.
at 90 °C for 3 h. Then, indazol-4-one 6 was produced from 4 and 5
in 3:1 ethanol/acetic acid by heated at about 60 °C, followed by
hydration of the resulting benzonitriles to afford 710 in high yield.
Condensation of 7 and acetic acid under DCC/DMAP coupling con-
ditions provided the expected SNX-2112 analogue BJ-B11.12 The
structure of BJ-B11 is different from SNX-2112 in the cyclohexanol
and inazolone moieties. Yields of all compounds are showed in
Scheme 1. Both analytical and spectral data of all target com-
pounds were accordant with the proposed structures.
BJ-B11 was first tested in vitro for antiviral activity against her-
pes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), cox-
sackievirus B3 (CVB3), human respiratory syncytial virus (RSV) and
influenza virus (H1N1) by cytopathic effect (CPE). Results sug-
gested that BJ-B11 only had antiviral activity against HSV-1 and
HSV-2. Possible antiviral mechanisms against HSV-1 were investi-
gated as follows. The cytotoxicity of BJ-B11 and ACV (used as the
positive agent) on Vero cells was determined by MTT assay.13
Based on the results of MTT assay, non-cytotoxic concentrations
of compounds were used for determination of in vitro anti-HSV
activity in Vero cells by plaque reduction assay.14 Results are
shown in Table 1.
HSV entry involves complex ligand–receptor interactions and
has been shown to be an ideal target for antiviral compounds.15 Ef-
fects of BJ-B11 on viral attachment and penetration were individu-
ally investigated based on plaque reduction assay. Results
suggested that the anti-HSV-1 activity of BJ-B11, like that of ACV,
was not attributed to blockage of virus entry (Fig. S1). As shown
in Figure 1, to determine at which stage of HSV-1 (MOI = 1) lifecy-
cle BJ-B11 (1 lM) exerts antiviral activity, time-of-addition (filled
triangle) and time-of-removal (filled square) experiments were
performed.15 For both assays, at 24 hpi (hours post-infection), the
infected cultures were harvested and the virus yield was deter-
mined by plaque assay. As shown in Figure 1, the inhibitory effect
of BJ-B11 declined insignificantly when added at 9 hpi as compared
to that when added at 24 hpi in time-of-addition assay (6.67 vs
7.09 log PFU/mL). Concordantly, virus yield was profoundly af-
fected when BJ-B11 was removed at 6 hpi compared with removal
at 0 hpi in time-of-removal experiment (7.13 vs 6.14 log PFU/mL).
These data suggested that BJ-B11 mainly inhibited HSV-1 replica-
tion before 6 hpi.
Further, real-time PCR was used to explore the effects of
BJ-B11 on HSV-1 DNA synthesis and gene expression.16 Vero cell
Table 1
Cytotoxicity, anti-HSV activity and therapeutic index of BJ-B11
Compound
Cytotoxicitya CC50
(l
M)
Antiviral activityb
HSV-1
HSV-2
EC50
(
l
M)
TIc
84.52
>172.42
EC50
(
l
M)
TIc
59.16
>116.27
BJ-B11
ACV
35.50 2.15
>200.00
0.42 0.18
1.16 1.02
0.60 0.21
1.72 0.44
Values are mean SD of three independent experiments.
a
Cytotoxic effect was determined by MTT assay. CC50 was defined as the concentration reducing 50% cell viability.
b
c
Antiviral activity was determined by plaque reduction assay. EC50 was the concentration that inhibited 50% of HSV replication in Vero cells.
TI (therapeutic index) was defined as the ratio of CC50 to EC50
.

H.-Q. Ju et al. / Bioorg. Med. Chem. Lett. 21 (2011) 1675–1677
1677
expression of UL54, but significantly reduced the expression of
both UL52 and UL27 (P <0.01) (Fig. 2B). ACV was used as a refer-
ence compound here and it showed similar effects. The observation
that E gene expression was reduced correlates with the results in
Figure 1, and suggested that BJ-B11 mainly exerted anti-HSV-1
effects at the early stage of infection.
In this study, a novel Hsp90 inhibitor BJ-B11 was designed, syn-
thesized and evaluated for in vitro antiviral activity. Results sug-
gested that BJ-B11 had antiviral activity against HSV (HSV-1 and
HSV-2). We have focused our study on the anti-HSV-1 mecha-
nisms, which could represent anti-HSV mechanisms of BJ-B11. This
study not only demonstrates that the host-pathogen interface may
represent a novel and specific antiviral target but also provides
information about the basic biology of chaperone-dependent viral
processes and cellular responses to stress. In conclusion, the pres-
ent study describes that BJ-B11 possesses anti-HSV activity, prob-
ably by inhibiting early stage of HSV replication. Further studies
will be required to explore the detailed antiviral mechanism of
BJ-B11, as a possible antiviral therapy.
Figure 1. Effects of delayed addition and early removal of BJ-B11 on virus yields.
(The data shown here represent the mean of three independent experiments.)
Acknowledgments
This work was supported by Grants from the Nation ‘863’ Pro-
gram of China (No. 2007AA02Z142), the Nation ‘211’ project and
the National Natural Science Foundation of China (No. 30873082,
20772013).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.01.098.
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Figure 2. Effects of BJ-B11 on HSV-1 DNA synthesis and HSV-1 gene expression.
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150.10, 170.57, 171.43, 193.41; HRMS (ESI) m/z [M+H]+ calcd for C25H33N4O4:
453.2502. Found: 453.2512.
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