Structure based molecular design, synthesis and biological evaluation of α-pyrone analogs as anti-HSV agent

Article abstract of DOI:10.1016/j.bmcl.2012.07.098

Several options for treating Herpes Simplex Virus type 1 and type 2 are available. However, non-specific inhibition and drug resistance warrants the discovery of new anti-herpetic compounds with better therapeutic profile or different mode of action. The

Full text of DOI:10.1016/j.bmcl.2012.07.098

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6261–6266
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure based molecular design, synthesis and biological evaluation
of a-pyrone analogs as anti-HSV agent
Srinivas Karampuri a, Paromita Bag b, Sabina Yasmin a, Devendra Kumar Chouhan a, Chandralata Bal a,
Debashis Mitra c, Debprasad Chattopadhyay b, Ashoke Sharon a,
⇑
a Department of Applied Chemistry, Birla Institute of Technology, Mesra, Ranchi 835215, India
b ICMR Virus Unit, ID & BG Hospital, Beliaghata, Kolkata 700010, India
c National Centre for Cell Science, NCCS Complex, Pune University Campus, Ganeshkhind, Pune 411007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Several options for treating Herpes Simplex Virus type 1 and type 2 are available. However, non-specific
inhibition and drug resistance warrants the discovery of new anti-herpetic compounds with better ther-
apeutic profile or different mode of action. The non-nucleoside inhibitors of HSV DNA polymerase target
the site that is less important for the binding of a natural nucleoside or nucleoside inhibitors. In the pres-
Received 19 June 2012
Revised 16 July 2012
Accepted 31 July 2012
Available online 4 August 2012
ent study, we have explored the possibility to find a new lead molecule based on
non-nucleoside inhibitors using structure based modeling approach. The designed molecules were syn-
thesized and evaluated for anti-HSV activity using MTT assay. The compound 5h with EC50 7.4 g/ml
and CC50 52.5 g/ml was moderately active against HSV when compared to acyclovir. A plaque reduction
a-pyrone analogs as
Keywords:
l
Molecular docking
In silico drug like properties
Herpes simplex virus
HSV DNA polymerase
l
assay was also carried out and results reveal that 5h is more effective against HSV-1 with better selective
index of 12.8 than against HSV-2 (SI = 3.6). The synthesized compounds were also evaluated for anti-HIV
activity, but none were active.
Ó 2012 Elsevier Ltd. All rights reserved.
Herpes Simplex Virus type 1 and type 2 (HSV-1 and HSV-2) are
members of Herpesviridae family, which causes several symptoms
in humans including cold sores, encephalitis and are responsible
for recurrent orolabial and genital infections.1 Transmission occurs
by contact with secretions from an infected person either by overt
infection or asymptomatic excretion of virus. After primary infec-
tion, HSV establishes long-term latency in the ganglia of sensory
nerves, from which it can reactivate episodically. There is no per-
manent cure for these infections till date. The goals of treatment
are to accelerate lesion healing and to prevent transmission. HSV
infections can be severe in immune-compromised patients, partic-
ularly those with defected cell-mediated immunity, recipients of
solid organ or bone marrow transplants.2–4 DNA replication in
the virus is mediated through its DNA polymerase enzyme. Hence
HSV DNA polymerase can be utilized as a target for designing anti-
HSV drugs. The present treatment involves the use of mainly two
types of HSV DNA polymerase inhibitors as drugs.5–7 The first strat-
egy includes the application of nucleoside analogs (e.g., acyclovir)
or its prodrugs (e.g., valacyclovir). The nucleoside analogs require
phosphorylation by viral thymidine kinase followed by host
kinases to be converted into nucleoside triphosphate which is
the actual inhibitor of viral DNA polymerase. The second category
consists of non nucleoside analogs.8 However, in past few years, a
number of resistant virushave been isolated especially from
immune-compromised patients.9,10 In this era, where the number
of immune-suppressed patients like those suffering from HIV is
continuously increasing, there is an immediate need to find new
drugs to treat HSV infections which have a higher efficacy or have
an alternative mode of action. A number of new anti-viral drugs
against HSV focusing other domains of the viral DNA polymerase
are currently under research and development.11–13 One such no-
vel class of compounds is that of 4-oxo-dihydroquinolines
(4-oxo-DHQs, Fig. 1, I) belonging to the non-nucleoside inhibitor
(NNI) family.14–16 4-oxo-DHQs have shown high specificity index
in inhibiting DNA polymerases of herpesviridae family because
unrelated DNA and RNA viruses were not susceptible to their
inhibitory effect.15,16 Recently, Withaferin A (WA: II; Fig. 1), a
naturally occurring C28-steroidal lactone from Indian ginseng17
has been found to exert inhibitory effect via interaction with a viral
DNA polymerase site that is less important for the binding of
deoxynucleoside triphosphates. Therefore, it is speculated that it
could be active on resistant viruses also.18 Recently, benzylidene
derivatives of bis-(4-hydroxycoumarin) (III) have been reported
for their mild anti-HSV activity.19 The structural inspection of
these three types of molecules reveals; I and III have two close keto
groups as common pharmacophoric motif (red color). Further, the
⇑
Corresponding author.
E-mail address: asharon@bitmesra.ac.in (A. Sharon).
compound II has a steroidal structure and has a a-pyrone ring.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.098

6262
S. Karampuri et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6261–6266
H
O
O
O
H
HO
O
H
O
H
H
N
N
H
OH
CH3
H
O
CH3
CH3
N
Cl
CH3
O
I
R3
II
R2
O
R3 HN
R4
R2
R1
O
O
OH
OH
R1
OO
O
O
III
IV
Figure 1. The chemical structure of anti-HSV compound; 4-oxo-dihydroquinoline analog (I), Withaferin A (WA, II) and benzylidene derivative of bis-(4-hydroxycoumarin)
(III). The possible functional pharmacophore (pyran/di-keto motif) is shown in red color. The prototype molecule IV selected as preliminary designed molecule, consisting of
pyran and di-keto motif.
In continuation to our anti-viral drug discovery program, we in-
tended to merge these specialties together into a single skeleton to
synthesize a-pyrone analogs having two close keto groups. The ini-
the palm region to investigate the binding mode (Fig. 3, prototype
analogs 5h). The surface diagram reveals that the 5h utilizes pyran
and diketo motif to bind into the inside of the palm region cavity.
Most of molecules studied had similar binding mode as that of
5h, however interaction pattern were significantly different which
lead to different G-score (relative binding score by Glide, Table 1).
A recent binding mechanism study of WA (II) through modeling
reports the role of hydrophobic interactions mediated by Phe 718,
Tyr 722, Pro 723 and Tyr 818 as well as electrostatic interaction
mediated by Asn 815 and Phe 718 for stabilization of binding with
target enzyme.18 The ligand-receptor diagram (Fig. 4) of 5h dis-
closes similar major residues (Tyr 818, Tyr 722, Leu 721, Phe
718, Asn 815) as was reported in the case of the binding mode of
anti-HSV compound WA (II). These interactions help the binding
of 5h into central core of HSV DNA polymerase near to palm region
(Fig. 2).
tially selected prototype analogs IV were subjected for HSV DNA
polymerase binding studies. The semi-empirical binding score
and binding mode were evaluated against HSV DNA polymerase
in the palm region and considered as preliminary basis for the
screening of the analogs for synthesis.
A model structure (Fig. 2) was prepared utilizing available PDB
crystal structure of HSV DNA polymerase (PDB ID 2GV9) as starting
structure.20
DNA polymerase is a heterodimer composed of the UL30 and
UL42 gene products. The UL30 gene encodes the catalytic subunit,
while the UL42 gene encodes a phosphoprotein that possesses dou-
ble-stranded DNA-binding activity. The overall structure comprises
of
6 structural domains; Pre –NH2 domain, –NH2 domain,
30-50 exonuclease domain, Palm, Finger and Thumb. All these do-
mains assemble to form a disk like central hole having –NH2 and
–COOH termini at opposite sides of the protein. The catalytic site
is situated in the palm subdomain and contains the catalytic triad
of aspartic acid residues (at positions 717, 886, and 888) essential
for polymerase activity.21 Therefore, prototype analogs (IV) were
docked using docking program Glide22 of Schrodinger Suite23 in
The in silico analysis using QikProp were also conducted to as-
sess the drug like properties of selected molecules (Table 1). None
of the molecules violated Lipinski’s rule24 of five as well as Jorgen-
sen’s rule25,26 of three (Table 1). Thus, the in silico studies
supported the selection of analogs for synthesis to access the
anti-HSV potential.
Ketene dithioacetals are versatile reagents which have been
extensively utilized in literature for the synthesis of diverse class
of molecules.27–32 Appropriate acetophenones were used as an eco-
nomic starting material for the reaction with CS2 to form the
Figure 2. Model structure of HSV DNA polymerase showing 30-50-exonuclease
domain, palm, finger and thumb domain. The docked pose of prototype molecule IV
(5h) was used to show the active site at HSV-polymerase palm region. Overall the
active site is located in the central core of enzyme near to palm surface.
Figure 3. Surface diagram showing active site grooves for the binding of 5h into the
inside of the palm region cavity.

S. Karampuri et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6261–6266
6263
Table 1
Manassas, VA, USA) and was compared with Acyclovir. Compounds
5b, 5d–f and 5i were either not properly soluble in DMSO or
formed crystal with the media. Therefore, their cytopathic effect
could not be determined. The % of inhibition of virus yield was ini-
tially determined for 5a, 5c, 5g and 5h at the concentration of
In silico binding analysis and drug like properties (DLP) evaluation of prototype
analogs (IV)
IV
R1
R2
R3
G-Scorea
DLP violationb
#5c
#3d
20 lg/ml. Our results show that these compounds can reduce the
5a
5b
5c
5d
5e
5f
5g
5h
5i
H
F
C2H4OH
C2H4OH
C6H5
4-Cl-C6H4
4-Br-C6H4
4-F-C6H4
4-Me-C6H4
C2H4OH
C2H4OH
C4H8N
ꢀ5.3
ꢀ4.0
ꢀ2.8
ꢀ2.6
ꢀ2.7
ꢀ2.6
ꢀ2.7
ꢀ3.7
ꢀ4.9
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
0
virus yield in 62–89% range (Table 3). These compounds were also
tested for their ability to inhibit cytopathic effect caused by HSV-1
as well as their toxicity to Vero cells. The EC50 and CC50, respec-
tively, were calculated on the basis of these results (Table 3).
C5H10
N
H
H
H
H
H
H
F
C4H8N
C4H8N
C4H8N
C4H8N
C4H8N
The compound 5h was moderately active with EC50 7.4 0.4
ml compared to 2.1 0.1 g/ml for acyclovir. The CC50 of 5h was
52.5 g/ml and selectivity index (SI) 7.1. Compounds 5c and 5g
lg/
l
C5H10
N
C4H8N
l
a
were not found significant. The compound 5a was found to have
comparable activity with 5h, but with less SI of 3.1 over 7.1. The
specificity of 5h for HSV-1 or HSV-2 was determined by plaque
reduction assay. The test was used to access the antiviral activity
against HSV-1 and HSV-2, using acyclovir and DMSO (0.1%) as po-
sitive and negative control, respectively. The results revealed that
G-Score (Glide Score): The minimized poses are rescored using Schrödinger’s
proprietary GlideScore scoring function and the binding affinity can be estimated by
G-Score.
b
DLP: Drug Like Properties.
#5: Number of violations of Lipinski’s rule of five. The rules are: molecular
c
weight MW <500, QPlogPo/w <5, donorHB 6 5, accptHB 6 10. Compounds that
satisfy these rules are considered drug-like.
d
#3: Number of violations of Jorgensen’s rule of three. The three rules are:
5h at a concentration of 4.1 and 14.5 lg/ml inhibited 50% plaque
QPlogS >ꢀ5.7, QPPCaco > 22 nm/s, Primary Metabolites <7. Compounds with fewer
formation by HSV-1 and HSV-2, (Figs. 5a and 5b). The results also
showed that 5h is more effective against HSV-1 (Table 4), with bet-
ter selective index of 12.8 than against HSV-2 (SI = 3.6).
violations of these rules are more likely to be orally available.
To build a model structure, PDB crystal structure of HSV DNA
polymerase (PDB: 2GV9) was obtained from protein databank.
The structure was optimized and prepared using Protein Prepara-
tion Wizard (PPW) module in Maestro interface of Schrodinger suit
2011.23 As the crystal structure consists two identical polypeptide
chains, one chain was deleted and the other was modeled for fur-
ther studies. The obtained structure was submitted for a short min-
imization through Impref module of PPW followed by
a
MacroModel39 minimization employing OPLS2005 force field with
5000 iterations. A dynamic simulation of 1.2 ns was performed
through Desmond40 in explicit solvent system to observe the con-
formational behavior of structure in presence of water as a solvent.
The average structure obtained was minimized through Macro-
Model.39 The final structure (Fig. 3) thus obtained was considered
to investigate the binding profile with docking studies. Receptor
grid was generated using Glide receptor grid generation utility of
Schrodinger’s suite 2011. The grid was generated around the cata-
lytic traid of aspartic acid residues (717, 886 and 888). All ligands
(IV, Fig. 1) were built in Maestro interface of Schrodinger suite
2011. Finally all compounds were treated in LigPrep module of
Schrodinger suite 2011 using OPLS2005 force field followed by
conformational search analysis through MacroModel using MMFFs
force field to obtain the most favored conformations. The lowest
energy conformer was selected for each ligand to perform docking
studies. The lowest energy conformer of compound 5a–i was sub-
mitted for drug like properties analysis using QikProp module of
Schrodinger. The reference (WA) was also prepared for docking
with the same protocol mentioned above. Glide module of Schro-
dinger suite 2011 was used to perform docking analysis. All ligands
and reference were docked at the predefined receptor grid using XP
mode. Overall, the estimated essential in silico properties of com-
pounds were summarized in Table 1.
Figure 4. Ligand-receptor diagram showing the major hot spot residues (hydro-
phobic: Val 823, Tyr 818, Tyr 722, Leu 721, Phe 718, Pro 723, Ala 719; acidic: Asp
888; hydrophilic: Asn 815, Ser 720, Ser 816, Gln 617, and Thr 887) causing the
binding of 5h into central core of HSV DNA polymerase near to palm region (see
Fig. 2).
ketene dimercapto intermediate in situ followed by addition of
methyl iodide to yield substituted ketene dithioacetals (2a–b,
Scheme 1).33
One-pot synthetic approach was applied to yield the
a-pyrone
derivates (3a–b) having carboxylic acid group at C-3 position from
2a–b using diethyl malonate as a carbanion source in presence of
NaH.34 In brief, the generated diethyl malonate carbanion formed
C–C bond with ketene dithioacetal followed by abstraction of an-
other active methylene proton from the intermediate to facilitate
ring closure to form a-pyrone C-3 ester derivate, which undergone
hydrolysis in situ. 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetra-
methyl uronium hexafluorophosphate (HATU) as a coupling re-
agent35 was used to couple amines with the carboxyl group of
3a–b to generate respective amides (4a–g) in more than 70%
yield.36 The methylthio group at C-4 of 4a–g was replaced with
secondary amines37 by heating in dioxane to yield the designed
molecules 5a–i.38 All the intermediates were characterized by
spectroscopic analysis. The physical and spectroscopic analysis
data for 5a–i are summarized in Table 2.
African green monkey kidney cells (Vero cells, ATCC, Manassas,
VA, USA) were grown and maintained in Eagle’s minimum essen-
tial medium (EMEM), supplemented with 5–10% fetal bovine ser-
um (FBS).41 The standard control strain HSV-2G (ATCC-734) and
HSV-1F (ATCC-733) were purchased from the ATCC. After plaque
purification, the virus was grown and the virus stocks were stored
at ꢀ80 °C for future use42 and whenever required the virus stocks
were grown on Vero cells to determine the titers and used for fur-
ther study.
The antiviral activity of 5a–i against HSV-1 was evaluated by
MTT assay on African green monkey kidney cell (Vero cells, ATCC,
Cell toxicity was monitored by determining the effect of the test
compounds on cell viability.43 For this Vero cells were cultured

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S. Karampuri et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6261–6266
SMe
O
C
O
C
SMe
SMe
COOH
R1
CH3
i)
R1
C
H
C
ii)
O
O
) R1 = H
1a
) R1 = H
3a
3b
) R1 = H
2a
R1
) R1 = F
2b) R1 = F
) R1 = F
1b
iii)
R2
R3 HN
SMe O
R2
iv)
O
N
H
O
O
O
O
R1
R1
5
4
) R1 = H, R2 = C2H4OH, R3 = C4H8N
5a
5b
5c
4a) R1 = H, R2 = C2H4OH
4b) R1 = F, R2 = C2H4OH
4c) R1 = H, R2 = C6H5
) R1 = F, R2 = C2H4OH, R3 = C4H8N
) R1 = H, R2 = C6H5, R3 = C4H8N
5d) R1 = H, R2 = 4-Cl-C6H4, R3 = C4H8N
5e) R1 = H, R2 = 4-Br-C6H4, R3 = C4H8N
) R1 = H, R2 = 4-Cl-C6H4
) R1 = H, R2 = 4-Br-C6H4
4d
4e
1
2
) R = H, R = 4-F-C6H4, R3 = C4H8N
5f
4f) R1 = H, R2 = 4-F-C6H4
4g) R1 = H, R2 = 4-Me-C6H4
) R1 = H, R2 = 4-Me-C6H4, R3 = C4H8N
5g
5h
) R1 = H, R2 = C2H4OH, R3 = C5H10
N
5i) R1 = F, R2 = C2H4OH, R3 = C5H10
N
Scheme 1. Reagents and conditions: (i) NaH, THF, CS2, MeI, 0–5 °C, 4 h; (ii) NaH, diethylmalonate, dioxane, 0–70 °C, 6 h; (iii) R2NH2, HATU, DMF, rt, 6 h; (iv) R3H, dioxane,
70 °C, 4 h.
Table 2
The analytical data of synthesized compounds
Sl.
No.
Yield
(%)
MP (°C)
Spectral data
5a
55
186–187
MS-ESI (m/z): [M+] 329.1; IR: (KBr) ( , cmꢀ1) 3270.2 (NH), 1659.8 and 1525.8 (C@O); 1H NMR (400 MHz, DMSO-d6) d 1.88–1.90 (m, 4H),
m
3.26 (t, J = 6 Hz, 2H), 3.47 (t, J = 6 Hz, 2H), 3.54–3.57 (m, 4H), 4.60–4.63 (m, 1H, OH), 6.80 (s, 1H), 7.51–7.53 (m, 3H), 7.90–7.91 (m, 2H),
8.07 (s, 1H, NH)
5b
5c
46
44
165–166
195–196
MS-ESI (m/z): [M+] 347.1; IR: (KBr) ( , cmꢀ1), 1H NMR (400 MHz, DMSO-d6) d 1.88–1.90 (m, 4H), 3.23 (t, J = 6 Hz, 2H), 3.45 (t, J = 6 Hz,
m
2H), 3.47–3.49 (m,4H), 4.60–4.63 (m,1H, OH), 7.34–7.38 (m, 2H), 7.79 (s, 1H), 7.95–7.99 (m, 2H), 8.08 (s, 1H, NH)
MS-ESI (m/z): [M+] 361.1; IR: (KBr) ( , cmꢀ1) 3275.1 (NH), 1656.8 and 1521.8 (C@O); 1H NMR (400 MHz, DMSO-d6) d 1.88–1.90 (m, 4H),
m
3.48–3.51 (m,4H), 6.85 (s, 1H), 7.03–7.07 (m,1H), 7.53–7.55 (m, 2H) , 7.66–7.7 (m, 3H), 7.92–7.93 (m, 2H), 7.94–8.0 (m, 2H), 10.25 (s,
1H-NH)
5d
5e
5f
53
55
43
56
55
198–199
229–230
208–209
230–231
167–168
MS-ESI (m/z): [M+] 394.8, IR: (KBr) ( , cmꢀ1) 3236.55 (NH), 1658.8 and 1525.7 (C@O); 1H NMR (400 MHz, DMSO-d6) d 1.88–1.91 (m, 4H),
m
3.50–3.53 (m, 4H), 6.86 (s,1H), 7.36–7.40 (m, 2H), 7.53–7.55 (m, 3H), 7.70–7.73 (m, 2H), 7.92–7.95 (m, 2H), 10.42 (s, 1H-NH)
MS-ESI (m/z): [M+] 437.6; IR: (KBr) ( , cmꢀ1) 3234.6 (NH), 1658.8, 1531.5 (C@O); 1H NMR (400 MHz, DMSO-d6) d 1.98–2.01 (m, 4H),
3.60–3.63 (m, 4H), 7.33 (s, 1H), 7.62–7.66 (m, 7H), 8.05–8.12 (m, 2H), 10.79 (s, 1H-NH)
MS-ESI (m/z): [M+] 379.0; IR: (KBr) ( , cmꢀ1) 3255.8 (NH), 1660.7 and 1510.2 (C@O); 1H NMR (400 MHz, DMSO-d6) 1.92–1.95 (m, 4H),
3.70–3.74 (m, 4H) 7.17–7.22 (m, 3H), 7.58–7.62 (m, 3H), 7.67–7.70 (m, 2H), 8.03–8.05 (m, 2H), 10.82 (s, 1H-NH).
MS-ESI (m/z): [M++1] 375.0; IR: (KBr) ( , cmꢀ1) 3333.0 (NH), 1670.3 and 1519.9 (C@O); 1H NMR (400 MHz, DMSO-d6) d 1.80–1.83 (s,
4H), 3.61–3.64 (m,4H), 6.90 (s, 1H), 7.2–7.5 (m, 2H), 7.41–7.60 (m, 5H), 8.01–8.12 (m, 2H), 10.2 (s, 1H-NH).
MS-ESI (m/z): [M++1] 343.2; IR: (KBr) ( , cmꢀ1) 3276.3 (NH), 1654.1 and 1531.3 (C@O); 1H NMR: (400 MHz, DMSO-d6) d 1.60–1.68 (m,
m
m
5g
5h
m
m
6H), 3.25 (t, J = 6 Hz, 2H), 3.47 (t, J = 6 Hz, 2H), 3.48–3.51 (m,4H), 4.60–4.63 (m, 1H, OH), 7.00 (s, 1H), 7.51–7.53 (m, 3H), 7.93–7.94 (m,
2H), 8.19 (s, 1H-NH).
5i
56
193–194
MS-ESI (m/z): [M+] 361.1; IR: (KBr) ( , cmꢀ1) 3270.2 (NH), 1662.1 and 1560.3 (C@O); 1H NMR (400 MHz, DMSO-d6) d 1.49–1.51 (m, 6H),
m
3.51 (m, 4H), 3.37 (t, J = 6 Hz, 2H), 3.47 (t, J = 5.2 Hz, 2H), 4.70–4.71 (m, 1H, OH), 5.92 (s, 1H), 7.30–7.32 (m, 2H), 7.48–7.50 (m, 2H), 8.17
(s, 1H, NH).
onto 96-well plate at 1.0 ꢁ 105 cells/ml. Different concentrations of
test compounds and standard drug acyclovir were added to culture
Table 3
wells at a final volume 100
tration was made in triplicate. DMSO (0.1%) was used as a negative
control. After incubation at 37 °C with 5% CO2 for 2 days, 10 l MTT
ll in each well. Each particular concen-
Antiviral activity of 5a, 5c, 5g and 5h
SIc
3.1
3.0
2.7
7.1
61.9
a
b
Compound
% of inhibition of viral yield
CC50
EC50
l
5a
5c
5g
5h
ACV
70
79
62
89
94
1
2
2
1
1
30.0
37.5
56.5
52.5
130.0
9.7 0.5
12.5 0.6
20.9 1.1
7.4 0.4
2.1 0.1
reagent was added to each well. After 4 h of incubation at 37 °C,
the formazan formed was dissolved by adding 0.04 N HCl in Isopro-
panol, and the absorbance was determined at 570 nm with a refer-
ence wavelength of 690 nm by an ELISA reader. Data were
calculated as the percentage of cell viability using the formula:
[(sample absorbance-blank)/mean media control absorbance)]/
100. The 50% cytotoxic concentration (CC50) for Vero cells was
determined with respect to cell control.42,44
a
The 50% cytotoxic concentration for Vero cells in
l
g/ml.
b
Concentration of compound in
lg/ml producing 50% inhibition of virus induced
cytopathic effect. This experiment was repeated for three times.
c
Selectivity index (SI) = CC50/EC50
.

S. Karampuri et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6261–6266
6265
HSV-2) and incubated at room temperature. After 1–2 h of incuba-
tion, the cells were washed with fresh EMEM and overlaid with
methylcellulose, so that the virus can spread via cell-to-cell route
to form plaques. The plaques that developed after 2–3 days of incu-
bation were stained with crystal violet. The effective concentration
of test compounds that inhibited the number of viral plaques by
50% (EC50) was interpolated from the dose-response curves.46
We have rationally designed novel
a-pyrone analogs using
structure based in silico approach along with drug like properties
estimation for the synthesis and biological evaluation. Compound
5h was found to be moderately active with respect to Acyclovir
against HSV-1 as well as has limited cytotoxicity. Therefore, we se-
lected 5h as our lead for future optimization and detailed structure
activity relationship. The synthesized compounds were also evalu-
ated for anti-HIV activity on MT-4 cell using MTT assay and none of
the compounds were found significant.
Figure 5a. Plaque reduction assay of HSV-1 at different concentrations of 5h and
ACV.
Acknowledgments
This research received funding from Department of Biotechnol-
ogy (DBT), Delhi, India through grant no BT/PR14237/MED/29/196/
2010. SK thanks CSIR, India for the award of Junior Research Fel-
lowship (JRF). D.K.C. is thankful to DBT, India for the JRF. Author
thanks to ILS, Hyderabad and CIF, BIT Mesra for analytical support.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.
07.098.
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Assessment of anti-HSV activity of 5h by plaque reduction assay on vero cell
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5h
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4.1 0.5
1.6 0.2
14.5 0.5
1.8 0.3
12.8
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The antiviral activity against HSV-1 was evaluated by MTT as-
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