Efficient synthesis of 3H,3′H-spiro[benzofuran-2,1′- isobenzofuran]-3,3′-dione as novel skeletons specifically for influenza virus type B inhibition

Article abstract of DOI:10.1016/j.ejmech.2013.01.015

An efficient and novel two step synthetic procedure to prepare various substituted 3H,3′H-spiro[benzofuran-2,1′-isobenzofuran]-3,3′- diones A, was established from very simple and easily available starting materials. The developed method is a robust and general approach for the synthesis of these structures. The prepared compounds were tested against influenza virus type A viz., A/Taiwan/1/86 (H1N1), A/Hong Kong/8/68 (H3N2) and type B viz., B/Panama/45/90, B/Taiwan/2/62, B/Lee/40, B/Brisbane/60/2008. Among 31 compounds tested, some of them showed good activity (selective index values >10) against these influenza viruses preferentially for type B. The most active compound 3b showed activity in 3.0-16.1 μM range with a selectivity index value between 30 and 166 against these type B viruses, in which it was comparable to the antiviral agent favipiravir. Also, 3b is found to be inactive against other enveloped viruses (viz., HIV and HSV) showing its specificity for influenza viruses.

Full text of DOI:10.1016/j.ejmech.2013.01.015

European Journal of Medicinal Chemistry 62 (2013) 534e544
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Efficient synthesis of 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
3,30-dione as novel skeletons specifically for influenza virus
type B inhibition
,
,
Yashwardhan Malpani a b, Raghavendra Achary a b, So Yeon Kim a, Hee Chun Jeong a,
Pilho Kim a b, Soo Bong Han a, Meehyein Kim a , Chong-Kyo Lee b, Jae Nyoung Kim c,
,
,
a,
*
Young-Sik Jung a
,b,
*
a Division of Drug Discovery Research, Korea Research Institute of Chemical Technology, Daejeon 305-606, Republic of Korea
b University of Science and Technology, Daejeon 305-606, Republic of Korea
c Department of Chemistry and Institute of Basic Science, Chonnam National University, Gwangju 500-757, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
An efficient and novel two step synthetic procedure to prepare various substituted 3H,30H-spiro[benzo-
furan-2,10-isobenzofuran]-3,30-diones A, was established from very simple and easily available starting
materials. The developed method is a robust and general approach for the synthesis of these structures. The
prepared compounds were tested against influenza virus type A viz., A/Taiwan/1/86 (H1N1), A/Hong Kong/
8/68 (H3N2) and type B viz., B/Panama/45/90, B/Taiwan/2/62, B/Lee/40, B/Brisbane/60/2008. Among 31
compounds tested, some of them showed good activity (selective index values >10) against these influenza
Article history:
Received 20 July 2012
Received in revised form
10 January 2013
Accepted 11 January 2013
Available online 29 January 2013
viruses preferentially for type B. The most active compound 3b showed activity in 3.0e16.1 mM range with
Keywords:
Spiro
Antiviral activity
Iodine
a selectivity index value between 30 and 166 against these type B viruses, inwhich it was comparable to the
antiviral agent favipiravir. Also, 3b is found to be inactive against other enveloped viruses (viz., HIV and
HSV) showing its specificity for influenza viruses.
Ó 2013 Elsevier Masson SAS. All rights reserved.
Microwave
Anti-influenza virus type B
1. Introduction
with the adamantanes but increasing frequency of oseltamivir-
resistant mutants and their cross-resistance to another NA inhibi-
Influenza virus infection can be prevented by vaccination and
therapeutic treatment available with viral neuraminidase (NA) or
matrix protein (M2) inhibitors. Nevertheless, it has not been
eradicated and has become a major cause of seasonally regional or
pandemic threat to humans [1e6]. Its ability to shelter in various
mammals or birds and to persistently evolve via antigenic drift or
shift results in generation of uncharacterized viruses. It is also
believed that they might have resistance developed to available
treatments. Clinically, a series of adamantanes (M2 inhibitors) are
available for the treatment of influenza type A viruses, but are
ineffective against type B virus or M2-mutant viruses of H1N1 and
H3N2. Also the side effects associated with them in the central
nervous system are severe [7e10]. Well known NA inhibitors have
broad-spectrum activity against various (sub) types of influenza
viruses. Although oseltamivir phosphate (OSV-P) is safe compared
tor, e.g., zanamivir, provoke the need for development of alternative
antiviral drugs with different modes-of-action [7e10]. Thus, the
discovery of novel anti-influenza agents is urgently needed. In this
regard, the anti-influenza ability of various spiro substructures is
worth to investigate, as they are known to be present in many bio-
logically active [11e15] and pharmacologically important com-
pounds [16,17]. In fact, there are well known active compounds
against influenza viruses which contain spiro substructure [18].
Moreover, spiro substructures find their application in not only
in medicinal chemistry but also in many other fields of chemistry
[19,20]. However, despite their application in different areas of
science, general and facile synthetic methods to prepare them still
remain a challenge for organic chemists [21]. It requires special
techniques and reactions to generate these spiro substructures
mainly because of the presence of a quaternary carbon and fused
rings in the structure.
Especially, 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
diones A (Fig. 1) belongs to a class of spiro compounds which have
not received a considerable attention yet. The core structure is
* Corresponding authors. Division of Drug Discovery Research, Korea Research
Institute of Chemical Technology, Daejeon 305-606, Republic of Korea.
E-mail address: ysjung@krict.re.kr (Y.-S. Jung).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.015

Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
535
O
O
O
O
N
O
O
O
O
R
Cl
OH
R
O
O
O
Cl
Me
Spiro lactones A
Fluorescamine
Streptospirole
Fig. 1. Representative compounds with core spiro structure.
known to be present in synthetic compounds used for various ap-
plications such as Fluorogens used for the detection of amino groups
(e.g. Fluorescamine) [22]. Lactum form of this spiro substructure is
also present in Streptospirole derivatives, derived from micro-
organism Streptomyces, which are known to inhibit bacterial
growth [23]. In spite of this, there are only a few methods available
and all of them involve multistep synthesis [24e27].
Scheme 1. General synthetic scheme.
2b from 4b,9b-dihydroxy-7-isopropyl-4bH-indeno[1,2-b]benzo-
furan-10(9bH)-one 2a with iodine (2.0 eq.)/ethanol under reflux for
two days in 51% yield. The product was unambiguously charac-
terized by 1H NMR, 13C NMR and its LC/MS data displayed molec-
ular ion peak at m/z 294.1 (Mþ). Finally, single crystal XRD of this
compound confirmed its formation as shown in Fig. 2 (Table 1,
Supporting information). Similarly, we also obtained 1b from 1a in
69% yield. The lack of in-depth literature precedents for the syn-
thesis of A, led us to optimize the reaction conditions and further
study the scope of the reaction.
Microwave reactions are well known for their high yields and
shorter reaction times in comparison with classical thermal condi-
tions. Since the reaction from a to b took several days under con-
ventional thermal conditions and gave low chemical yields, we
carried out further optimization with microwave irradiation
(Table 1). The reaction with 4b,9b-dihydroxy-7,8-dimethyl-4bH-
indeno[1,2-b]benzofuran-10(9bH)-one 3a was used as a model
system to optimize the reaction condition. Initial trials at 110 ꢀC in
ethanol gave the O-ethyl derivative 3c as a major product even after
2 h of irradiation (Table 1, Entry 1). Increasing the temperature to
150 ꢀC gave the conversion to the desired spiro-derivative 3b in high
yield with 2 h of continuous microwave irradiation (Table 1, Entry 2).
Ethanol proved to be the best solvent for the reaction (Table 1, En-
tries 2, 3 and 4) and higher loadings of iodine shortened the reaction
time (Table 1, Entries 6 and 7). We also found that a minimum of two
equivalents of iodine was required to achieve high yields of 3b as
lesser equivalents showed little conversion to 3b with the formation
of 3c (Table 1, Entry 5). Also, neither 3b nor 3c was obtained when
the reaction was performed in absence of iodine suggesting that
iodine plays an important role to facilitate the reaction (Table 1,
Entry 8). Thus, 2 equivalents of iodine with 2 h of microwave irra-
diation were chosen as the optimum condition to avoid excessive
use of iodine and to maintain appropriate reaction time.
Moreover, the previous reports available for these 3H,30H-spiro
[benzofuran-2,10-isobenzofuran]-3,30-diones A have never attrac-
ted interest among the medicinal chemists for its application.
To the best of our knowledge, there are no earlier reports of
any biological activity of these 3H,30H-spiro[benzofuran-2,10-iso-
benzofuran]-3,30-diones A.
However, based on our experimental data, we found that some
of these novel compounds prepared by our new method are active
particularly against influenza virus type B, including B/Taiwan/2/62
(TWB), B/Lee/40 (LE), B/Panama/45/90 (PNM), and B/Brisbane/60/
2008 (BB): the most potent compound having the 50% effective
concentration (EC50) values ranging from 3.0 to 16.1
these type B viruses.
mM against
Thus, herein we report, the first microwave-assisted and
molecular-iodine mediated efficient oxidative procedure to build
the 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-dione skele-
ton and its application into biological system as the inhibitors of
influenza virus infection.
2. Results and discussion
2.1. Chemistry
A thorough literature survey reveals that, after the first synthesis
of 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-dione 1b [24],
only two reports appeared in the literature for the synthesis of
similar compounds. One report shows the formation of 1b as a side
product in 14% yield during the synthesis of Wrightiadione [25]. And
the other report was by Bullington and Dodd where they prepared
only 30b in a multistep route with low yield [26]. The same authors
also reported a similar conversion of nitrogen analogues of these
spiro compounds from 4b,9b-dihydroxy-4b,5-dihydroindeno[1,2-b]
indol-10(9bH)-ones. Indeed, a few more methods to get the nitro-
gen analogues of these spiro lactones and respective spiro lactams
are available [28,29]. But, to the best of our knowledge, there are no
other reports directly targeting the synthesis of spiro lactones A.
Also, the methods reported earlier for synthesis of A are not general
or have drawbacks such as multistep synthesis, use of harsh re-
agents and conditions, low yields, and reproducibility. Thus, a gen-
eral and efficient method is in need to synthesize these 3H,30H-spiro
[benzofuran-2,10-isobenzofuran]-3,30-diones A.
In order to demonstrate the scope and potential for this mi-
crowave assisted synthesis of A, various substituents on the phe-
nolic and ninhydrin ring were tested under the optimized reaction
condition (Table 2). Thus, compounds with alkyl substituents on
the phenolic ring were applied first under the optimized condition.
Mono and poly-alkyl substituted derivatives were obtained in good
to high yields (Table 2, 1b, 2b, 3b and 4b). The positions of these
alkyl substituents were found to have no effect on the reaction and
the different regioisomers of the methyl substituted derivatives
We started the synthesis by the reaction of the respective nin-
hydrins with various phenols in acidic medium to get the 4b,9b-
dihydroxy-4bH-indeno[1,2-b]benzofuran-10(9bH)-ones (1ae31a)
in good to high yields as shown in Scheme 1 [30,31]. These com-
pounds are known to give the corresponding alkyl ethers (e.g. 3c,
Scheme 1) under iodine/alcohol reflux conditions [32]. When we
continued this reaction for extended time we observed the for-
mation of the 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
diones A as a major byproduct. In particular, we could obtain 6-
isopropyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-dione
Fig. 2. ORTEP diagram of 2b.

536
Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
Table 1
were also obtained in high yields (Table 2, 4b, 5b and 6b). Likewise
the naphthalene ring compounds also gave good yields (Table 2, 7b
and 8b). Interestingly, we observed that the reaction is quite sen-
sitive other type of substituents present on the benzene rings and
their positions. Particularly, in case of electron withdrawing groups
on phenolic ring, we observed that the yields were decreased and
the starting materials were recovered under the optimized condi-
tions (Table 2, 26b and 27b). Nonetheless, with a little change in the
optimized condition, we were able to obtain good yields of the
products (Table 2, 9b, 10b, 11b, 12b, 13b and 14b). In general,
electron withdrawing substituents on any ring slow down the re-
action rate and higher loadings of iodine or longer reaction times
were required to get high yields. Higher iodine loading was not
sufficient and thus the irradiation time also has to be elongated to
get high yields in some cases (Table 2, 12b). The order of these
electronic effects of substitution on phenolic ring was
methyl < phenyl < chloro < fluoro w ester. Transesterified product
was obtained with the ester functionality on the substrate, which is
in accord with the earlier published results (Table 2, 25b) [33].
Strong electron donating substituents such as amino, hydroxy and
Reaction condition optimization for synthesis of 3b.
Entry
Solvent
Iodine
(eq.)
Temp
(ꢀC)
Time
(h)
Yielda
(3b)
Yielda
(3c)
1
2
3
4
5
6
7
8
EtOH
EtOH
iPrOH
tBuOHb
EtOH
EtOH
EtOH
EtOH
2.0
2.0
2.0
2.0
1.0
4.0
10.0
0.0
110
150
150
150
150
150
150
150
2
2
2
2
2
1
0.25
2
e
92%
e
90%
42%
e
e
e
30%
84%
80%
e
28%
e
e
e
a
Isolated yield.
The reaction gives a very messy TLC pattern.
b
Table 2
Scope for the microwave assisted synthesis of the spiro derivatives of A.

Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
537
methoxy groups at position 2 of the phenolic ring (Scheme 1),
resulted in either iodinated O-alkyl derivatives at lower tempera-
ture through electrophilic substitution reaction of iodine or tends
to get decompose at higher temperature (see Supporting
information). But, the methoxy group at position 1 on the phe-
nolic ring gave the desired product in moderate yield (Table 2, 24b).
Substitution on the ninhydrin ring has a negative effect on the re-
action (Table 2, 28b and 29b). As we obtained the initial results of
SAR for these compounds and dimethyl group on the phenolic ring
showed good activity with low toxicity (Table 3), we decided to
prepare more derivatives with alkyl substitution. In these cases we
found that in general using four equivalents of iodine with 2 h of
irradiation gave reasonable yields with the completion of the re-
action in almost every case (Table 2, 15be23b).
speculated that the same intermediate could have been formed in
our case. To prove whether the 8-membered ring compound is the
reactive intermediate of the reaction, we prepared two 8-
membered ring compounds (Scheme 3, 1d and 2d) with the pre-
viously known method [26] and applied the intermediate under the
microwave condition in ethanol without iodine. As a result, we
were able to obtain the desired spiro products (30b and 31b),
suggesting that the spiro compounds could be formed through 8-
membered ring intermediates.
Further, this reactive 8-membered ring intermediate may un-
dergo two possible pathways (Scheme 2). In path A, the interme-
diate could be hydrolyzed to give the ester compound 5 under the
reaction condition. Subsequently, the attack of phenolic hydroxyl
group to the electron deficient carbonyl group may generate the 2-
phenyl hydroxy furanone 6. The hydroxy group later could cyclize
with the ortho ester on the phenyl substituent and generate the
lactone ring. In fact, the ester compound 1e (Scheme 2) was isolated
(in <5% yield) in our reaction conditions, when the reaction of 3a to
3b was quenched just after 15 min of microwave irradiation (see
the Supporting information). 1e [34] has also been converted to 3b
2.2. Mechanistic insight
Formation of the 8-membered ring intermediate (Scheme 2)
was reported earlier in the case of nitrogen analogues either
through air oxidation or sodium periodate oxidation [26]. We also
Table 3
The CC50 and EC50 values of the spiro compounds against influenza A and B virus.
Compounda
CC50
(
mM)
EC50 (m
M)b
TWAc
HKd
PNMe
TWBf
LEg
BBh
1b
2b
7b
8b
>500
>500
>500
88.1 ꢁ 32.7
44.9 ꢁ 3.7
<6.2
N.A.i
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
314.4 ꢁ 29.7
40.4 ꢁ 4.2
>500
147.9 ꢁ 20.7
129.3 ꢁ 26.7
8.9 ꢁ 0.6
185.7 ꢁ 11.7
>36.3
>500
>500
63.8 ꢁ 23.9
17.3 ꢁ 15.7
110.0 ꢁ 4.5
>500
<6.2
205.6 ꢁ 15.0
7.6 ꢁ 1.9
>500
96.1 ꢁ 57.2
10b
13b
25b
26b
27b
28b
29b
30b
31b
3b
36.3 ꢁ 4.1
<6.2
>500
>500
>500
>500
>500
>500
100.1 ꢁ 0.5
25.5 ꢁ 19.2
321.4 ꢁ 46.2
>500
8.2 ꢁ 2.0
18.4 ꢁ 4.0
>500
33.3 ꢁ 10.1
27.4 ꢁ 0.8
>500
3.0 ꢁ 0.1
24.7 þ 2.9
25.7 ꢁ 1.2
7.6 ꢁ 0.4
96.1 ꢁ 57.2
4.3 ꢁ 2.1
6.6 ꢁ 0.0
21.7 ꢁ 2.3
7.8 ꢁ 0.1
11.2 ꢁ 1.7
7.5 ꢁ 0.2
9.8 ꢁ 1.3
9.9 ꢁ 0.9
>500
86.0 ꢁ 3.7
>500
>500
305.3 ꢁ 7.0
49.0 ꢁ 36.0
30.0 ꢁ 10.9
>500
>500
>500
N.A.
N.A
N.A
>500
>500
23.4 ꢁ 0.6
83.2 þ 1.8
27.8 ꢁ 151.6
32.9 ꢁ 2.4
205.6 ꢁ 15.0
12.9 ꢁ 0.6
>30.3
173.8 ꢁ 3.5
25.2 ꢁ 0.0
34.6 ꢁ 7.7
28.1 ꢁ 4.7
30.8 ꢁ 0.5
7.1 ꢁ 0.3
11.4 ꢁ 7.3
>186.7
19.1 ꢁ 0.9
335.3 ꢁ 151.6
100.1 ꢁ 19.6
125.3 ꢁ 6.0
185.7 ꢁ 11.7
>29.2
3.6 ꢁ 0.3
53.0 þ 0.0
31.2 ꢁ 5.1
24.8 ꢁ 0.1
39.4 ꢁ 5.1
8.3 ꢁ 1.1
10.8 ꢁ 1.7
61.4 ꢁ 4.4
21.7 ꢁ 0.2
12.1 ꢁ 0.3
9.3 ꢁ 0.4
25.0 ꢁ 1.5
28.9 ꢁ 0.5
>500
16.1 ꢁ 0.2
198.7 þ 27.4
59.9 ꢁ 28.6
32.8 ꢁ 0.9
159.3 ꢁ 0.3
12.2 ꢁ 2.9
14.1 ꢁ 3.7
226.6 ꢁ 2.9
38.8 ꢁ 3.5
34.4 ꢁ 2.5
31.2 ꢁ 2.4
33.7 ꢁ 2.2
32.0 ꢁ 3.8
>500
4.8 ꢁ 0.0
103.9 þ 1.8
46.9 ꢁ 7.3
28.0 ꢁ 0.0
132.1 ꢁ 4.6
11.2 ꢁ 3.3
12.1 ꢁ 1.0
96.3 ꢁ 6.4
28.6 ꢁ 1.9
32.2 ꢁ 3.7
25.4 ꢁ 2.4
27.6 ꢁ 0.9
23.5 ꢁ 3.7
>500
4b
5b
6b
8b
376.1 ꢁ 41.7
292.9 ꢁ 81.9
>500
29.2 ꢁ 0.3
30.3 ꢁ 0.4
>500
152.6 ꢁ 14.3
103.3 ꢁ 12.6
111.6 ꢁ 2.8
100.3 ꢁ 0.9
370.2 ꢁ 183.6
>500
186.7 ꢁ 24.8
80.4 ꢁ 2.7
>500
9b
11b
12b
14b
15b
16b
17b
18b
19b
20b
21b
22b
23b
24b
AMTj
OSV-Ck
T-705l
>30.3
92.3 ꢁ 0.0
36.4 ꢁ 7.1
>103.3
41.6 ꢁ 9.8
>100.3
16.3 ꢁ 3.2
>500
>186.7
>186.7
66.6 ꢁ 9.2
5.7 ꢁ 9.2
23.2 ꢁ 4.1
>500
>186.7
10.6 ꢁ 0.1
254.5 ꢁ 48.4
>500
42.7 ꢁ 4.2
4.8 ꢁ 0.1
103.2 ꢁ 27.8
>500
9.8 ꢁ 0.3
>80.4
2.1 ꢁ 0.0
11.0 ꢁ 1.0
>500
>500
296.5 ꢁ 3.6
289.8 ꢁ 5.3
40.4 ꢁ 1.0
1.9 ꢁ 0.4
<0.01
>500
6.3 ꢁ 0.1
126.4 ꢁ 17.6
0.6 ꢁ 0.0
0.05 ꢁ 0.04
6.95 ꢁ 1.75
253.2 ꢁ 1.6
23.6 ꢁ 4.9
42.3 ꢁ 2.0
58.2 ꢁ 9.9
81.1 ꢁ 10.0
>100
>100
>100
>100
>100
>100
>100
0.06 ꢁ 0.01
1.94 ꢁ 0.22
0.05 ꢁ 0.00
2.84 ꢁ 0.93
0.82 ꢁ 0.14
1.93 ꢁ 0.88
13.63 ꢁ 0.53
1.32 ꢁ 0.23
4.71 ꢁ 0.98
a
Concentration of chemicals required for reducing 50% of viability of normal MDCK cells.
b
c
d
e
f
Concentration of chemicals required for improving 50% of viability of influenza virus-infected cells.
A/Taiwan/1/86 (H1N1).
A/Hong Kong/8/68 (H3N2).
B/Panama/45/90.
B/Taiwan/2/62.
B/Lee/40.
B/Brisbane/60/2008.
Not applicable.
Amantadine.
Oseltamivir carboxylate.
Favipiravir.
g
h
i
j
k
l

538
Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
MDCK cells, but was not as potent as OSV-C or RBV. In the com-
parison analysis with 1b, the presence of a methyl group on the
phenolic ring as seen in 4b, 5b and 6b showed considerably
improved antiviral effect against not only PNM but alsoTWB, LE and
BB, but cytotoxicity was exhibited at the maximum concentration of
5b and 6b (CC50 values, 376.1 and 292.9 mM, respectively). It sug-
gests that the methyl group at position 1, 2, or 3 does not damage the
function of 1b required for inhibiting the influenza B virus infection,
but substitution at position 1 is most desirable to maintain both
antiviral efficacy and cell viability. The antiviral and cytotoxic pro-
files for the other two chloride derivatives (12b and 14b) were very
similar to methyl substituents. Again, chloride at position 1 of the
phenolic ring has improved cell viability. Notably, upon treatment of
12b, the cell viability even at its maximum concentration was
comparable to mock cells viability. However, substitution with
methoxy at the same position (24b) showed again cytopathicity but
still was accompanied with the antiviral activity against the type B
viruses. Although the compound 21b has strong anti-flu B activity
Scheme 2. Proposed mechanism.
in ethanol under microwave irradiation (see the Supporting
information) without iodine. These results were supportive to
corroborate our proposed mechanism. Further, in path B, which
was reported by Bullington and Dodd [26], the intermediate may
undergo ethanol-induced nucleophilic attack to the electron defi-
cient carbonyl, which could result in formation of the 5-membered
lactone 7 after intramolecular transesterification. The oxonium ion
8, generated by liberating ethoxide anion, could be converted to the
spiro derivative by the attack of the phenolic hydroxyl group.
(EC50, between 2.1 and 10.6 mM) its toxicity to host cells made it to
have a narrow therapeutic window (data not shown). Other
dimethyl substitution at positions 1 and 2, 2 and 4, or 1 and 4 (18b,
19b and 20b) was not as efficient as 3b and trimethyl substitutions
(as in 22b and 23b) also did not improve the antiviral property
compare to 3b. The cyclopentyl derivative (21b) structurally very
similar to 3b maintained the activity but induced toxicity. In accord
with the initial test results (10b), the presence of phenyl sub-
stituents on any position of the phenolic ring (9b and 11b) was
a supplement to the high toxicity with CC50 values between 30 and
2.3. Biology
Present therapeutic treatment of influenza virus is mostly based
on viral NA inhibitors such as oseltamivir, zanamivir, peramivir and
laninamivir, which are all sialic acid derivatives. Clinically, ada-
mantanes such as amantadine and rimantadine as M2 protein ion
channel inhibitors are also available. As a continuation of ongoing
research in search of novel anti-influenza agents with entirely
different modes-of-action, our in-house chemical library contain-
ing about 7000 diverse compounds was screened. The compounds
1b and 2b (Table 3), the initial products obtained by us under
thermal conditions were identified to have some activity. Based on
the structure of 1b diverse spiro compounds (2b, 3b, 7b, 8b, 10b,
13b and 25be31b) were then synthesized by the chemical route
developed by us, which were subjected to test for their antiviral
activity against the influenza A and B viruses. They were treated to
MDCK cells infected with three different strains of influenza virus
such as A/Taiwan/1/86 (H1N1) (TWA), A/Hong Kong/8/68 (H3N2)
(HK) and PNM, and then reduction of the virus-induced cytotoxicity
was measured by the fluorescein diacetate-based (FDA) cytopathic
effect assay [35,36]. The initial activity results of these 14 com-
pounds showed that 3b, 26b and 29b have significant antiviral
activity particularly against the PNM strain, with the 50% cytotoxic
40 mM, thus resulting in reduction of their selectivity index (the ratio
of CC50 to EC50) less than 5, which is lower than that of the 1b having
about 20 for PNM. The other prepared derivatives having a higher
alkyl substitution on the phenolic ring at 3 positions (16b and 17b)
still maintained the activity but harboured cellular toxicity (CC50,
about 100 mM). We also found that any substitution at position 2 on
the phenolic ring with isopropyl, ethyl or fluoro group 2b, 15b and
26b) still maintained the antiviral activity against the type B viruses
but induced cytotoxicity of the MDCK cells.
Additionally, we could observe from Table 3 that the naphthyl
rings (7b and 8b) are responsible for inducing toxicity or loss of
activity. The ester substitution (25b) showed neither antiviral ac-
tivity nor toxicity. Dichloride substitution at positions 1 and 4 (27b)
was also highly toxic. It should be mentioned here that although
26b has CC50 value above 500 mM, it displayed above 20% normal
cell death at the maximum concentration (data not shown). In
addition, more complex substitutions at both rings 1 and 2 lost the
type B-specific antiviral activity (28b) or showed toxicity causing
concentration (CC50) values above 500
mM and selectivity index
(the ratio of CC50 to EC50) greater than 15. 3b being most active and
without any considerable cytotoxicity (Fig. A, Supporting
information) was used as a reference for a further systematic SAR
study performed with more abundant repertoire of A. Also, as the
most predominant inhibitory effect of 3b was against influenza
virus type B (PNM) infection thus the three additional influenza B
viruses (TWB, LE and BB) as well as the primarily used viral strains
were tested simultaneously. Thus our further antiviral study with
these spiro compounds gets focused on inhibition of the type B
influenza virus infection.
cell death of about 47% at 500 mM (29b). Amino group at position 3
(30b) of the phenolic ring showed activity accompanied with
toxicity whereas the methoxy group (31b) was neither active nor
toxic.
As 3b was the most active compounds without cytotoxicity
throughout the assay, we carried out the plaque inhibition assay to
confirm its efficacy against the various strains of influenza B viruses
as shown (Fig. 3). Notably, it was not active against other enveloped
viruses, such as herpes simplex virus (HSV) or human immuno-
deficiency virus (HIV) (Tables 4 and 5). Thus it was confirmed that
the antiviral activity of 3b is specific for influenza virus only.
Overall the data suggest that for spiro compounds A to be active
and relatively safe antiviral agents for targeting the influenza B
virus, it should be incorporated with the dimethyl group at posi-
tions 2 and 3 on the phenolic ring (3b). As an alternative and
simplified approach, it can be substituted at the position 1 either
with chloride (12b) or methyl (4b). In this report it can be stressed
that although the activity of 3b was less potent than the effect of
OSV-C, it inhibited influenza B virus with nearly comparable
Based on the antiviral test, the core compound 1a was active
against PNM but not against TWA or HK. It was not toxic to host
Scheme 3. Preparation of 8-membered intermediates and respective spiro
compounds.

Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
539
Fig. 3. A) Plaque inhibition assay of compound 3b with MDCK cells. B) Plaque numbers were counted from crystal violet-stained plates and the percentage number relative to mock-
treated, virus-infected cells (100%) was calculated. n.d., not detected. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
being robust and more general over the earlier reported procedures
with a direct one pot conversion of a to the spiro derivatives b in
good to high yields (Table 2). This novel short and efficient syn-
thetic methodology can be usefully applied in medicinal and
organic chemistry. The prepared compounds show preferential
inhibition of influenza virus type B over type A in general. The SAR
analysis showed that the presence of chloride or methyl sub-
stitutions at position 1 of the phenolic ring gave a potent antiviral
activity without any cytotoxicity. In particular, the presence of
dimethyl group at positions 2 and 3 on the ring exhibited the most
enhanced activity with 3b showing EC50 values between 3.0 and
Table 4
Anti-HSV activity of 3b measured by an MTT-based CPE assay.
Compound
CC50
(
m
m)a
EC50 (m
M)b
HSV-1c
HSV-2d
3b
311.7 ꢁ 9.1
>50.0
>311.7
1.7 ꢁ 0.3
>311.7
58.6 ꢁ 3.3
ACVe
a
Concentration of chemicals required for reducing 50% of viability of normal Vera
cells.
b
Concentration of chemicals required for improving 50% of viability of herpes
simplex virus (HSV)-infected cells.
c
HSV type 1 strain F.
HSV type 2 strain MS.
Acyclovir.
d
16.1 mM, without any cytotoxicity at 500 mM. In addition 3b was
e
found to be active against only influenza viruses and showed no
activity against other enveloped viruses such as HIV and HSV.
Further studies to establish in-depth SAR and modes-of-action are
in progress. It is expected that 3b could be further optimized to be
a candidate for development of a novel and specific therapeutics
inhibiting influenza B viruses which are not sensitive to ada-
mantanes, as well as influenza A virus to some extent.
Table 5
Anti-HIV activity of 3b measured by an MTT-based CPE assay.
Compound
CC50
(
m
m)a
EC50 (m
M)b
HIV-1c
HIV-2d
3b
158.1 ꢁ 5.5
75.2 ꢁ 7.9
>500.0
>158.1
>158.1
ddCe
ddIf
0.4 ꢁ 0.1
0.4 ꢁ 0.1
4. Experimental section
7.8 ꢁ 0.6
34.7 ꢁ 4.2
a
Concentration of chemicals required for reducing 50% of viability of normal MT4
4.1. Materials and methods in chemistry
cells.
b
Concentration of chemicals required for improving 50% of viability of human
immunodeficiency virus (HIV)-infected cells.
Unless otherwise stated, all commercially available solvents and
reagents were used without further purification. Melting points
were recorded on Mettler Toledo MP50 apparatus and are uncor-
rected. IR spectra were recorded on an FT-IR Smiths IdentifyIR and
the values are mentioned in cmꢂ1. The 1H NMR and 13C NMR was
recorded on Varian 300 and Bruker 300 NMR spectrophotometer
operating at 300 MHz, using commercial NMR solvents obtained
from Aldrich with TMS as internal standard and chemical shifts are
c
HIV type 1 strain III-B infected in HUT78 cells.
HIV-2 strain ROD.
Zalcitabine.
Didanosine.
d
e
f
antiviral activity to that of T-705 and with higher selectivity than
influenza A virus. Other enveloped viruses, HSV and HIV, were not
sensitive to 3b. Thus, it is expected that the compound 3b could be
a novel specific inhibitor of influenza B viruses which are resistant
to spiro adamantanes. Potentially, it may also be used as a hit
compound for the discovery of novel inhibitors against influenza
virus infection for which efforts are ongoing. Also, further studies to
elucidate the mode-of-action of 3b and its target molecule(s) are
under progress.
mentioned in d ppm scale. Single crystal X-ray was performed with
Bruker SMART Apex II X-ray Diffractometer. The Mass spectra were
obtained over Varian 1200L quadrupole MS (EI) spectrophotometer
and LCMS were recorded over Waters Acquity UPLC coupled with
Micromass Quattro micro spectrophotometer. Elemental Analysis
was done on Thermopack instruments. The microwave irradiation
was carried on Anton Paar Synthos 3000 Microwave. The reaction
progress and purity of the products was checked by Thin-Layer
Chromatography (TLC) on Merck TLC Silica gel 60 F254 TLC plates.
KMnO4 and PMA stains were used as detecting agent.
3. Conclusion
In summary, we report a short and efficient synthesis of a series
of 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-diones A, by
microwave-assisted and molecular iodine-mediated novel reaction
pathway. This provides a new general route to prepare spiro com-
pounds A in two simple steps from easily available starting mate-
rials. In addition, our synthetic methodology is advantageous as
4.1.1. Synthesis of 4b,9b-dihydroxy-4bH-indeno[1,2-b]benzofuran-
10(9bH)-ones 1ae31a
4b,9b-Dihydroxy-4bH-indeno[1,2-b]benzofuran-10(9bH)-ones
(1ae31a) were synthesized according to previously reported
approach [30,31].

540
Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
4.1.2. Preparation of 3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
3,30-dione (1b) under thermal condition
3H, CH3), 7.01 (s, 1H, ArH), 7.34e7.37 (m, 1H, ArH), 7.51 (s, 1H, ArH),
7.68e7.70 (m, 2H, ArH), 7.97e7.99 (m, 1H, ArH); 13C NMR (75 MHz,
4b,9b-Dihydroxy-4bH-benzo[d]indeno[1,2-b]furan-10(9bH)-
one 1a (0.50 g, 1.96 mmol) was taken in ethanol (5 mL) with iodine
(0.99 g, 3.93 mmol) at room temperature. This mixture was
refluxed for 72 h. Then ethanol was evaporated completely and the
residue was dissolved in DCM (100 mL). This was washed with 10e
15% aqueous sodium thiosulfate solution (50 mL ꢃ 2), water
(50 mL) and brine (50 mL). Then it was dried over anhydrous so-
dium sulphate and concentrated. The crude mass obtained was
then purified over silica gel column chromatography (10% ethyl
acetate in hexane) to get the pure product as white solid. Yield:
0.34 g (69%); mp: 170e175 ꢀC; IR (cmꢂ1): 1786, 1732; 1H NMR
CDCl3) d 19.5, 21.8, 104.8, 114.2, 116.5, 122.6, 125.5, 126.2, 127.2,
131.9,133.1,135.2,142.8,151.6,167.2,170.4,192.0 (C]O); HRMS (EI):
mass calculated for C17H12O4 (Mþ), 280.0736; m/z found, 280.0737.
4.1.4.4. 7-Methyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (4b). Yield: 35 mg (70%); mp: 155e157 ꢀC; IR (cmꢂ1): 1798,
1728; 1H NMR (300 MHz, CDCl3)
d 2.35 (s, 3H, CH3), 7.15 (t,
J ¼ 7.5 Hz, 1H, ArH), 7.39e7.41 (m, 1H, ArH), 7.59e7.62 (m, 2H, ArH),
7.68e7.76 (m, 2H, ArH), 7.98e8.01 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3)
d 14.3, 104.4, 118.3, 122.7, 123.1, 123.8, 124.0, 126.2, 127.3,
132.0, 135.2, 140.6, 142.7, 167.1, 170.1, 193.1 (C]O); HRMS (EI): mass
(300 MHz, CDCl3)
7.70e7.80 (m, 4H, ArH), 7.99e8.01 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3) 104.2, 113.6, 118.6, 122.5, 123.8, 125.8, 126.1, 127.1, 131.9,
d 7.22e7.29 (m, 2H, ArH), 7.38e7.41 (m, 1H, ArH),
calculated for C16H10O4 (Mþ), 266.0579; m/z found, 266.0581.
d
4.1.4.5. 6-Methyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
135.1, 139.7, 142.3, 166.8, 171.2, 192.4 (C]O); HRMS (EI): mass cal-
culated for C15H8O4 (Mþ), 252.0423; m/z found, 252.0435.
dione (5b). Yield: 36 mg (72%); mp: 185e187 ꢀC; IR (cmꢂ1): 1787,
1724; 1H NMR (300 MHz, CDCl3)
d 2.41 (s, 3H, CH3), 7.13 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.37e7.39 (m, 1H, ArH), 7.56e7.59 (m, 2H, ArH),
4.1.3. Preparation of 6-isopropyl-3H,30H-spiro[benzofuran-2,10-
isobenzofuran]-3,30-dione (2b) under thermal condition
7.66e7.74 (m, 2H, ArH), 7.97e7.99 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3)
d 23.0, 104.8, 113.9, 116.4, 122.7, 125.4, 125.5, 126.2, 127.2,
4b,9b-Dihydroxy-7-isopropyl-4bH-benzo[d]indeno[1,2-b]furan-
10(9bH)-one 2a (0.50 g, 1.68 mmol) was taken in ethanol (10 mL)
with Iodine (0.85 g, 3.37 mmol) at room temperature. This mixture
was refluxed for 48 h. Then ethanol was evaporated completely and
the residue was dissolved in DCM (100 mL). This was washed with
10e15% aqueous sodium thiosulfate solution (50 mL ꢃ 2), water
(50 mL) and brine (50 mL). Then it was dried over anhydrous sodium
sulphate and concentrated. The crude mass obtained was then pu-
rified over silica gel column chromatography (10% ethyl acetate in
hexane) to get the pure product as white solid. Yield 250 mg (51%);
mp: 132e135 ꢀC; IR (cmꢂ1): 1775, 1730; 1H NMR (300 MHz, CDCl3)
132.0, 135.2, 142.7, 152.6, 167.1, 171.8, 191.8 (C]O); HRMS (EI): mass
calculated for C16H10O4 (Mþ), 266.0579; m/z found, 266.0580.
4.1.4.6. 5-Methyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (6b). Yield: 40 mg (80%); mp: 166e168 ꢀC; IR (cmꢂ1): 1794,
1724; 1H NMR (300 MHz, CDCl3)
d 2.41 (s, 3H, CH3), 7.13 (d,
J ¼ 8.4 Hz, 1H, ArH), 7.37e7.39 (m, 1H, ArH), 7.56e7.59 (m, 2H, ArH),
7.66e7.74 (m, 2H, ArH), 7.97e7.99 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3)
d 20.8, 104.7, 113.4, 118.7, 122.7, 125.3, 126.2, 127.2, 132.0,
133.9, 135.2, 141.0, 142.6, 167.1, 169.8, 192.7 (C]O); HRMS (EI): mass
calculated for C16H10O4 (Mþ), 266.0579; m/z found, 266.0580.
d
1.32 (d, J ¼ 6.9 Hz, 6H, CH3), 3.05 (septet, J ¼ 6.9 Hz, 1H, CH), 7.07
(s,1H, ArH), 7.13 (d, J ¼ 7.8 Hz,1H, ArH), 7.38e7.41 (m,1H, ArH), 7.68e
4.1.4.7. 3H,30H-Spiro[isobenzofuran-1,20-naphtho[1,2-b]furan]-3,30-
7.74 (m, 3H, ArH), 7.98e8.00 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
dione (7b). Yield: 21 mg (20%); mp: 187e189 ꢀC; IR (cmꢂ1): 1794,
d
23.3, 35.3, 104.6, 111.1, 116.5, 122.5, 123.0, 125.6, 126.1, 127.1, 131.8,
1713; 1H NMR (300 MHz, CDCl3)
d 7.39e7.42 (m, 1H, ArH), 7.64e
135.0, 142.5, 163.2, 166.9, 171.9, 191.7 (C]O); Anal. Calcd for
C18H16O4: C, 72.96; H, 5.44; O, 21.6. Found C, 73.37; H, 4.80; O, 21.58;
HRMS (EI): mass calculated for C18H14O4 (Mþ), 294.0892; m/z found,
294.0893.
7.68 (m, 3H, ArH), 7.71e7.74 (m, 2H, ArH), 7.77e7.82 (m, 1H, ArH),
7.97 (d, J ¼ 8.1 Hz, 1H, ArH), 8.04e8.06 (m, 1H, ArH), 8.23 (d,
J ¼ 8.7 Hz, 1H, ArH); 13C NMR (75 MHz, CDCl3)
d 105.2, 113.7, 119.3,
121.1, 122.7, 122.8, 124.4,126.4, 127.2, 127.6, 128.8, 132.0, 132.1,135.3,
139.6, 142.7, 167.1, 172.6, 191.9 (C]O); HRMS (EI): mass calculated
for C19H10O4 (Mþ), 302.0579; m/z found, 302.0580.
4.1.4. General method for the preparation of spiro compounds
under microwave condition 1be29b
4b,9b-Dihydroxy-4bH-indeno[1,2-b]benzofuran-10(9bH)-ones
(2aeac) (50 mg, 1.0 eq.) was taken in ethanol (1 mL) and to these
specified equivalents of iodine was added. The reaction mass was
then irradiated in microwave for specified time at 150 ꢀC. Then the
reaction mass was concentrated and then dissolved in DCM
(100 mL). This was washed with 20% aqueous sodium thiosulfate
solution (30 mL), then with water (30 mL) and brine (30 mL). This
was dried with anhydrous sodium sulphate and concentrated to get
the crude product which was purified over silica gel column
chromatography (ethyl acetate/hexanes) to afford the spiro com-
pounds (1be29b).
4.1.4.8. 3H,30H-Spiro[isobenzofuran-1,20-naphtho[2,3-b]furan]-3,30-
dione (8b). Yield: 40 mg (80%); mp: 223e225 ꢀC; IR (cmꢂ1): 1786,
1710; 1H NMR (300 MHz, CDCl3)
d
7.36 (d, J ¼ 9.0 Hz,1H, ArH), 7.40e
7.43 (m, 1H, ArH), 7.54e7.59 (m, 1H, ArH), 7.68e7.73 (m, 3H, ArH),
7.92 (d, J ¼ 7.8 Hz,1H, ArH), 7.96e8.03 (m,1H, ArH), 8.25 (d, J ¼ 9.0 Hz,
1H, ArH), 8.67 (d, J ¼ 8.4 Hz, 1H, ArH); 13C NMR (75 MHz, CDCl3)
d
104.8,111.4,113.5,122.7,123.5,126.3,126.5,127.3,129.1,129.3,130.0,
130.9, 132.0, 135.3, 142.1, 142.6, 167.2, 174.4, 191.9 (C]O); HRMS (EI):
mass calculated for C19H10O4 (Mþ), 302.0579; m/z found, 302.0580.
4.1.4.9. 7-Phenyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (9b). Yield: 45 mg (90%); mp: 199e201 ꢀC; IR (cmꢂ1): 1798,
4.1.4.1. 3H,30H-Spiro[benzofuran-2,10-isobenzofuran]-3,30-dione (1b).
Yield: 35 mg (70%). Analytical data matched as mentioned above.
1728; 1H NMR (300 MHz, CDCl3)
d
7.31e7.46 (m, 5H, ArH), 7.66e
7.77 (m, 5H, ArH), 7.87e7.89 (m, 1H, ArH), 7.96e7.99 (m, 1H,
ArH); 13C NMR (75 MHz, CDCl3)
104.3, 119.4, 122.8, 124.4, 124.7,
d
4.1.4.2. 6-Isopropyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
3,30-dione (2b). Yield: 38 mg (76%). Analytical data matched as
mentioned above.
126.3, 127.3, 127.7, 128.61, 128.63, 128.9, 132.0, 134.1, 135.3, 139.2,
142.5, 167.0, 168.4, 193.0 (C]O); HRMS (EI): mass calculated for
C21H12O4 (Mþ), 328.0736; m/z found, 328.0735.
4.1.4.3. 5,6-Dimethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
4.1.4.10. 6-Phenyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
3,30-dione (3b). Yield: 45 mg (90%); mp: 199e201 ꢀC; IR (cmꢂ1):
dione (10b). Yield: 30 mg (60%); mp: 168e170 ꢀC; IR (cmꢂ1): 1799,
1784, 1725; 1H NMR (300 MHz, CDCl3)
d
2.31 (s, 3H, CH3), 2.41 (s,
1720; 1H NMR (300 MHz, CDCl3)
d 7.40e7.54 (m, 6H, ArH), 7.65e

Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
541
7.77 (m, 4H, ArH), 7.84 (d, J ¼ 7.8 Hz, 1H, ArH), 7.99e8.02 (m, 1H,
ArH); 13C NMR (75 MHz, CDCl3)
104.9, 111.8, 117.4, 122.7, 123.4,
126.2, 126.3, 127.2, 127.6, 129.4, 129.6, 132.1, 135.3, 139.2, 142.6,
153.4, 167.1, 171.8, 192.0 (C]O); HRMS (EI): mass calculated for
C21H12O4 (Mþ), 328.0736; m/z found, 328.0736.
ArH), 7.38e7.40 (m, 1H, ArH), 7.53e7.60 (m, 2H, ArH), 7.66e7.74 (m,
2H, ArH), 7.97e8.00 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3) 13.7,
24.6, 37.2, 104.7, 113.4, 118.7, 122.7, 124.8, 126.3, 127.3, 132.0, 135.2,
138.8, 140.6, 142.6, 167.1, 170.0, 192.8 (C]O); HRMS (EI): mass
calculated for C18H14O4 (Mþ), 294.0892; m/z found, 294.0894.
d
4.1.4.11. 5-Phenyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
4.1.4.18. 6,7-Dimethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
dione (11b). Yield: 35 mg (70%); mp: 202e204 ꢀC; IR (cmꢂ1): 1791,
3,30-dione (18b). Yield: 41 mg (82%); mp: 178e180 ꢀC; IR (cmꢂ1):
1734; 1H NMR (300 MHz, CDCl3)
d
7.31 (d, J ¼ 8.4 Hz,1H, ArH), 7.38e
7.51 (m, 4H, ArH), 7.58 (d, J ¼ 7.5 Hz, 2H, ArH), 7.69e7.76 (m, 2H,
ArH), 7.97e8.02 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
104.9,
1797, 1722; 1H NMR (300 MHz, CDCl3)
d 2.24 (s, 3H, CH3), 2.43 (s,
3H, CH3), 7.06 (d, J ¼ 7.8 Hz, 1H, ArH), 7.37e7.40 (m, 1H, ArH), 7.51
(d, J ¼ 7.8 Hz, 1H, ArH), 7.67e7.74 (m, 2H, ArH), 7.96e8.00 (m, 1H,
ArH); 13C NMR (75 MHz, CDCl3) 10.9, 20.7, 104.9, 116.2, 122.2, 122.5,
122.7, 125.9, 126.2, 127.3, 131.9, 135.2, 142.9, 150.7, 167.2, 170.2, 192.6
(C]O); HRMS (EI): mass calculated for C17H12O4 (Mþ), 280.0736;
m/z found, 280.0737.
d
114.0, 119.2, 122.7, 123.8, 126.3, 127.1, 127.2, 128.1, 129.3, 132.1, 135.3,
137.8, 139.0, 142.5, 167.0, 170.7, 192.7 (C]O); HRMS (EI): mass cal-
culated for C21H12O4 (Mþ), 328.0736; m/z found, 328.0735.
4.1.4.12. 7-Chloro-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (12b). Yield: 36 mg (72%); mp: 200e202 ꢀC; IR (cmꢂ1): 1789,
4.1.4.19. 5,7-Dimethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
1735; 1H NMR (300 MHz, CDCl3)
d
7.20e7.25 (m,1H, ArH), 7.41e7.44
(m, 1H, ArH), 7.69e7.79 (m, 4H, ArH), 7.99e8.02 (m, 1H, ArH); 13C
NMR (75 MHz, CDCl3) 104.7, 119.4, 120.5, 122.9, 124.1, 124.7, 126.4,
3,30-dione (19b). Yield: 35 mg (70%); mp: 196e198 ꢀC; IR (cmꢂ1):
1795, 1717; 1H NMR (300 MHz, CDCl3)
d
2.44 (s, 3H, CH3), 2.57 (s,
3H, CH3), 6.82 (s, 2H, ArH), 7.38e7.40 (m, 1H, ArH), 7.65e7.73 (m,
2H, ArH), 7.96e8.00 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
17.9,
d
127.2, 132.4, 135.4, 139.5, 141.9, 166.6, 166.8, 191.9 (C]O); HRMS
(EI): mass calculated for C15H7ClO4 (Mþ), 286.0033; m/z found,
286.0032.
d
22.8, 104.6, 111.0, 114.8, 122.7, 126.1, 126.6, 127.3, 131.9, 135.1, 141.1,
142.9, 151.8, 167.2, 172.0, 192.1 (C]O); HRMS (EI): mass calculated
for C17H12O4 (Mþ), 280.0736; m/z found, 280.0737.
4.1.4.13. 6-Chloro-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (13b). Yield: 28 mg (56%); mp: 215e217 ꢀC; IR (cmꢂ1): 1793,
4.1.4.20. 4,7-Dimethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
1728; 1H NMR (300 MHz, CDCl3)
d
7.23e7.26 (m, 2H, ArH), 7.37e
7.40 (m, 1H, ArH), 7.69e7.76 (m, 3H, ArH), 7.99e8.02 (m, 1H,
ArH); 13C NMR (75 MHz, CDCl3)
104.8, 114.4, 117.4, 122.7, 125.0,
3,30-dione (20b). Yield: 34 mg (68%); mp: 136e138 ꢀC; IR (cmꢂ1):
1791,1722; 1H NMR (300 MHz, CDCl3)
d 2.29 (s, 3H, CH3), 2.57 (s, 3H,
d
CH3), 6.90 (d, J ¼ 7.5 Hz, 1H, ArH), 7.40e7.42 (m, 2H, ArH), 7.67e7.75
126.4, 126.7, 127.1, 132.3, 135.4, 142.1, 146.4, 166.7, 171.4, 191.1 (C]
O); HRMS (EI): mass calculated for C15H7ClO4 (Mþ), 286.0033; m/z
found, 286.0034.
(m, 2H, ArH), 7.98e8.00 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
d
14.0, 17.6, 104.2, 116.5, 120.6, 122.8, 125.0, 126.2, 127.4, 131.9, 135.1,
138.5, 140.1, 142.9, 167.3, 170.1, 193.5 (C]O); HRMS (EI): mass cal-
culated for C17H12O4 (Mþ), 280.0736; m/z found, 280.0737.
4.1.4.14. 5-Chloro-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (14b). Yield: 38 mg (76%); mp: 162e165 ꢀC; IR (cmꢂ1): 1790,
4.1.4.21. 6,7-Dihydro-30H-spiro[indeno[5,6-b]furan-2,10-iso-
1736; 1H NMR (300 MHz, CDCl3)
d
7.20 (d, J ¼ 8.4 Hz,1H, ArH), 7.37e
7.39 (m, 1H, ArH), 7.71e7.74 (m, 1H, ArH), 7.98e8.00 (m, 1H, ArH);
13C NMR (75 MHz, CDCl3)
104.9, 115.2, 119.9, 122.7, 125.2, 126.4,
benzofuran]-3,30(5H)-dione (21b). Yield: 35 mg (70%); mp: 202e
205 ꢀC; IR (cmꢂ1): 1792, 1722; 1H NMR (300 MHz, CDCl3)
d 2.13e
d
2.26 (m, 2H, CH2), 2.94 (t, J ¼ 7.5 Hz, 2H, CH2), 3.02 (t, J ¼ 7.5 Hz,
2H, CH2), 7.06 (s, 1H, ArH), 7.36e7.39 (m, 1H, ArH), 7.57 (s, 1H, ArH),
7.68e7.73 (m, 2H, ArH), 7.96e7.99 (m, 1H, ArH); 13C NMR (75 MHz,
127.1, 129.6, 132.3, 135.4, 139.7, 142.0, 166.7, 169.6, 191.6 (C]O);
HRMS (EI): mass calculated for C15H7ClO4 (Mþ), 286.0033; m/z
found, 286.0032.
CDCl3) d 26.0, 31.7, 34.2, 105.2, 109.5, 117.3, 120.4, 122.6, 126.2, 127.2,
131.9,135.25,140.7,142.9,159.6,167.2,171.4,192.0 (C]O); HRMS (EI):
4.1.4.15. 6-Ethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
mass calculated for C18H12O4 (Mþ), 292.0736; m/z found, 292.0735.
dione (15b). Yield: 36 mg (72%); mp: 173e175 ꢀC; IR (cmꢂ1): 1787,
1726; 1H NMR (300 MHz, CDCl3)
d
1.32 (t, J ¼ 7.5 Hz, 3H, CH3), 2.79
4.1.4.22. 4,5,6-Trimethyl-3H,30H-spiro[benzofuran-2,10-iso-
(q, J ¼ 7.5 Hz, 2H, CH3), 7.05 (s, 1H, ArH), 7.09 (d, J ¼ 7.8 Hz, 1H, ArH),
7.38e7.41 (m, 1H, ArH), 7.63e7.74 (m, 3H, ArH), 7.97e8.00 (m, 1H,
ArH); 13C NMR (75 MHz, CDCl3) 15.0, 30.1, 104.8, 112.7, 116.6, 122.7,
124.4, 125.7, 126.2, 127.3, 132.0, 135.2, 142.7, 158.8, 167.1, 172.0, 191.9
(C]O); HRMS (EI): mass calculated for C17H12O4 (Mþ), 280.0736;
m/z found, 280.0736.
benzofuran]-3,30-dione (22b). Yield: 84 mg (50%); mp: 164e166 ꢀC;
IR (cmꢂ1): 1785, 1720; 1H NMR (300 MHz, CDCl3)
d 2.19 (s, 3H, CH3),
2.32 (s, 3H, CH3), 2.57 (s, 3H, CH3), 6.86 (s, 1H, ArH), 7.35e7.38 (m,
1H, ArH), 7.64e7.72 (m, 2H, ArH), 7.95e8.00 (m, 1H, ArH); 13C NMR
(75 MHz, CDCl3)
d 14.2, 14.6, 22.6, 104.6, 111.6, 115.0, 122.6, 126.1,
127.4, 131.6, 131.8, 135.1, 139.0, 143.1, 150.5, 167.3, 169.8, 192.7 (C]
O); HRMS (EI): mass calculated for C18H14O4 (Mþ), 294.0892; m/z
found, 294.0889.
4.1.4.16. 5-Ethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (16b). Yield: 33 mg (66%); mp: 158e162 ꢀC; IR (cmꢂ1): 1791,
1726; 1H NMR (300 MHz, CDCl3)
d
1.28 (t, J ¼ 7.5 Hz, 3H, CH3), 2.72 (q,
4.1.4.23. 4,5,7-Trimethyl-3H,30H-spiro[benzofuran-2,10-iso-
J ¼ 7.5Hz, 2H, CH3), 7.15 (d, J ¼ 7.8Hz,1H, ArH), 7.37e7.40(m,1H, ArH),
7.56e7.74 (m, 4H, ArH), 7.98e8.00 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3) 15.6, 28.2, 104.7, 113.5, 118.7, 122.7, 124.2, 126.3, 127.3, 132.0,
135.2, 140.2, 140.3, 142.7, 167.1, 170.0, 192.8 (C]O); HRMS (EI): mass
calculated for C17H12O4 (Mþ), 280.0736; m/z found, 280.0736.
benzofuran]-3,30-dione (23b). Yield:36mg(72%); mp:171e173 ꢀC;IR
(cmꢂ1): 1790,1720; 1H NMR (300 MHz, CDCl3)
d 2.18 (s, 3H, CH3), 2.36
(s, 3H, CH3), 2.53 (s, 3H, CH3), 6.81 (s,1H, ArH), 7.38e7.40 (m,1H, ArH),
7.65e7.73 (m, 2H, ArH), 7.97e8.00 (m, 1H, ArH); 13C NMR (75 MHz,
CDCl3)
d 10.5, 17.6, 20.6, 104.7, 114.6, 119.0, 122.8, 126.1, 127.0, 127.4,
131.8, 135.1, 137.7, 143.1, 150.0, 167.4, 170.1, 192.9 (C]O); HRMS (EI):
4.1.4.17. 5-Propyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
mass calculated for C18H14O4 (Mþ), 294.0892; m/z found, 294.0889.
dione (17b). Yield: 40 mg (80%); mp: 156e160 ꢀC; IR (cmꢂ1): 1784,
1735; 1H NMR (300 MHz, CDCl3)
d
0.97 (t, J ¼ 7.5 Hz, 3H, CH3), 1.61e
4.1.4.24. 7-Methoxy-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
1.74 (m, 2H, CH2), 2.64 (q, J ¼ 7.5 Hz, 2H, CH2), 7.14 (d, J ¼ 8.4 Hz, 1H,
3,30-dione (24b). Yield: 25 mg (50%); mp: 173e175 ꢀC; IR (cmꢂ1):

542
Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
1805,1727; 1H NMR (300 MHz, CDCl3)
d
3.98 (m, 3H, OCH3), 7.17e7.43
concentrated and then directly purified over silica gel column
(m, 4H, ArH), 7.66e7.74 (m, 2H, ArH), 7.97e8.00 (m,1H, ArH);13CNMR
chromatography (50% ethyl acetate in hexanes) to get the pure
(75 MHz, CDCl3)
d
55.6, 104.5, 116.8, 119.9, 121.0, 122.9, 124.5, 126.3,
product as solid. Yield: 15 mg (38%); mp: 258e260 ꢀC; 1H NMR
127.2, 132.1, 135.3, 142.3, 146.5, 161.1, 167.0, 192.7 (C]O); HRMS (EI):
(300 MHz, DMSO)
d
6.29 (s, 1H, ArH), 6.50 (d, J ¼ 8.4 Hz, 1H, ArH),
mass calculated for C16H10O5 (Mþ), 282.0528; m/z found, 282.0525.
7.24 (br, 2H, NH2), 7.44 (d, J ¼ 8.4 Hz,1H, ArH), 7.63 (d, J ¼ 7.5 Hz,1H,
ArH), 7.78e7.87 (m, 2H, ArH), 8.03 (d, J ¼ 7.2 Hz, 1H, ArH); 13C NMR
4.1.4.25. Ethyl 3,30-dioxo-3H,30H-spiro[benzofuran-2,10-isobenzofur-
(75 MHz, CDCl3) d 95.0, 105.5, 107.6, 111.6, 122.5, 125.7, 127.0, 127.4,
an]-5-carboxylate (25b). Yield: 13 mg (26%); mp: 205e208 ꢀC; IR
131.5, 134.9, 143.3, 158.7, 167.3, 173.5, 187.5 (C]O); HRMS (EI): mass
(cmꢂ1): 1807, 1736, 1703; 1H NMR (300 MHz, CDCl3)
d 1.43 (t,
calculated for C15H9NO4 (Mþ), 267.0532; m/z found, 267.0533.
J ¼ 7.2 Hz, 3H, CH3), 4.43 (q, J ¼ 7.2 Hz, 2H, CH2), 7.27e7.31 (m, 1H,
ArH), 7.38e7.40 (m, 1H, ArH), 7.74e7.53 (m, 2H, ArH), 8.02e8.04 (m,
4.1.7. Preparation of 3-methoxy-6H-dibenzo[b,f]oxocine-6,11,12-
trione (2d)
1H, ArH), 8.47e8.50 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3)
d 14.4,
61.8, 105.0, 113.8, 118.8, 122.7, 126.5, 126.9, 127.1, 128.1, 132.4, 135.4,
141.1, 141.9, 164.8, 166.6, 173.6, 191.8 (C]O); HRMS (EI): mass cal-
culated for C17H10O6 (Mþ), 324.0634; m/z found, 324.0632.
Compound 31a (410 mg, 1.44 mmol) was taken in ACN:water
(1:1) (10 mL) and to this NaOI4 (618 mg, 2.89 mmol) was added at
r.t. This was then monitored by TLC. After 10 min of vigorous stir-
ring the precipitated solid was filtered off and then washed with
cold water and dried under vacuum to get the pure product as solid.
Yield: 210 mg (51%); IR (cmꢂ1): 1754, 1686, 1656; mp: 193e195 ꢀC;
4.1.4.26. 6-Fluoro-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-3,30-
dione (26b). Yield: 20 mg (20%); mp: 225e227 ꢀC; IR (cmꢂ1): 1795,
1727; 1H NMR (300 MHz, CDCl3)
d
6.95e7.02 (m, 2H, ArH), 7.35e
7.45 (m, 1H, ArH), 7.65e7.90 (m, 3H, ArH), 7.95e8.03 (m, 1H,
ArH); 13C NMR (75 MHz, CDCl3)
101.6, 102.0, 105.1, 112.6, 112.9,
1H NMR (300 MHz, CDCl3)
ArH), 7.68e7.80 (m, 2H, ArH), 7.83e7.90 (m, 2H, ArH), 8.00e8.03
(m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
56.4, 108.4, 114.6, 121.9,
d 3.96 (s, 3H, OCH3), 6.95e6.98 (m, 2H,
d
d
115.4, 122.7, 126.4, 127.1, 128.0, 128.1, 132.3, 135.4, 142.1, 166.7, 168.6,
172.1, 172.8, 172.9, 190.6 (C]O); HRMS (EI): mass calculated for
C15H7FO4 (Mþ), 270.0328; m/z found, 270.0328.
127.7, 128.8, 131.0, 131.5, 132.5, 135.8, 139.8, 155.8, 165.1, 167.1, 191.4
(C]O), 198.6 (C]O); MS/EI: m/z (rel. intensity) 282.2 (Mþ, 8.1),
254.2 (11.2), 76.1 (100).
4.1.4.27. 4,7-Dichloro-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
4.1.8. Preparation of 6-methoxy-3H,30H-spiro[benzofuran-2,10-
isobenzofuran]-3,30-dione (31b)
3,30-dione (27b). Yield: 20 mg (19%); mp: 219e221 ꢀC; IR (cmꢂ1):
1798, 1737; 1H NMR (300 MHz, CDCl3)
d
7.17 (d, J ¼ 8.4 Hz, 1H, ArH),
7.42e7.45 (m, 1H, ArH), 7.676 (d, J ¼ 8.7 Hz, 1H, ArH), 7.71e7.76 (m,
2H, ArH), 8.00e8.02 (m,1H, ArH); 13C NMR (75 MHz, CDCl3)
104.6,
Compound 2d (50 mg, 0.18 mmol) was taken in ethanol (1 mL)
and then irradiated with microwaves at 110 ꢀC for 15 min. This was
concentrated and then directly purified over silica gel column
chromatography (20% ethyl acetate in hexanes) to get the pure
product as solid. Yield: 22 mg (44%); mp: 185e187 ꢀC; IR (cmꢂ1):
d
117.6, 117.7, 123.0, 125.7, 126.5, 127.1, 131.8, 132.5, 135.5, 139.3, 141.5,
166.4, 167.0, 189.1 (C]O); HRMS (EI): mass calculated for
C15H6Cl2O4 (Mþ), 320.9721; m/z found, 320.9718.
1787, 1719; 1H NMR (300 MHz, CDCl3)
d 3.95 (s, 3H, OCH3), 6.65 (s,
1H, ArH), 6.78 (d, J ¼ 8.1 Hz, 1H, ArH), 7.39 (m, 1H, ArH), 7.66e7.69
4.1.4.28. 6-Isopropyl-50,60-dimethyl-3H,30H-spiro[benzofuran-2,10-
(m, 3H, ArH), 7.97e7.99 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
isobenzofuran]-3,30-dione (28b). Yield: 15 mg (29%); mp: 200e
d 56.4, 97.1, 105.4, 111.7, 113.1, 122.7, 126.2, 127.16, 127.22, 132.0,
202 ꢀC; IR (cmꢂ1): 1772, 1732; 1H NMR (300 MHz, CDCl3)
d
1.33
135.2, 142.8, 167.2, 169.8, 173.9, 189 (C]O); HRMS (EI): mass cal-
(d, J ¼ 6.9 Hz, 6H, CH3), 2.35 (s, 3H, CH3), 2.40 (s, 3H, CH3), 3.06
(sept, J ¼ 6.9 Hz, 1H, CH), 7.07 (s, 1H, ArH), 7.13 (d, J ¼ 8.1 Hz, 2H,
ArH), 7.16 (d, J ¼ 7.8 Hz,1H, ArH), 7.70 (d, J ¼ 7.8 Hz,1H, ArH), 7.75 (s,
culated for C16H10O5 (Mþ), 282.0528; m/z found, 282.0527.
4.1.9. 4b-Ethoxy-9b-hydroxy-7,8-dimethyl-4bH-indeno[1,2-b]
benzofuran-10(9bH)-one (3c)
1H, ArH); 13C NMR (75 MHz, CDCl3)
d 20.4, 20.9, 35.4, 104.8, 111.2,
116.8, 123.0, 123.3, 125.1,125.7, 126.6, 140.7, 141.6, 145.7, 163.2, 167.5,
172.0, 192.4 (C]O); HRMS (EI): mass calculated for C20H18O4 (Mþ),
322.1205; m/z found, 322.1203.
4b,9b-Dihydroxy-7,8-dimethyl-4bH-indeno[1,2-b]benzofuran-
10(9bH)-one 3a (100 mg, 0.34 mmol) was taken in ethanol (2 mL)
and to this iodine (180 mg, 0.70 mmol) was added. The reaction
mass was then irradiated with microwaves for 2 h at 110 ꢀC. Then
the reaction mass was concentrated in a r.b flask and then dissolved
in ethyl acetate (100 mL). This was washed with 20% Na2S2O3 so-
lution (30 mL) and then with water (30 mL) and brine (30 mL). This
was dried with anhydrous sodium sulphate and concentrated to get
the crude which was purified over silica gel column chromatogra-
phy (10% ethyl acetate in hexane) to get the pure product as solid.
Yield: 100 mg (92%); mp: 165e168 ꢀC; IR (cmꢂ1): 1702; 1H NMR
4.1.4.29. 6-Isopropyl-50,60-dimethoxy-3H,30H-spiro[benzofuran-2,10-
isobenzofuran]-3,30-dione (29b). Yield: 14 mg (14%); mp: 135e
137 ꢀC; IR (cmꢂ1): 1788, 1737; 1H NMR (300 MHz, CDCl3)
d 1.33
(d, J ¼ 6.9 Hz, 6H, CH3), 3.06 (sept, J ¼ 6.9 Hz, 1H, CH), 3.91 (s, 3H,
OCH3), 3.997 (s, 3H, OCH3), 6.75 (s, 1H, ArH), 7.09 (s, 1H, ArH), 7.14
(d, J ¼ 7.8 Hz, 1H, ArH), 7.37 (s, 1H, ArH), 7.71 (d, J ¼ 8.1 Hz, 1H, ArH);
13C NMR (75 MHz, CDCl3)
d 23.6, 35.4, 56.65, 56.74, 104.1, 106.6,
111.3, 116.8, 119.8, 123.1, 125.8, 136.7, 152.7, 155.7, 163.3, 167.3, 172.0,
192.3 (C]O); HRMS (EI): mass calculated for C20H18O6 (Mþ),
354.1103; m/z found, 354.1104.
(300 MHz, CDCl3)
d
1.31 (t, J ¼ 6.3 Hz, 3H, CH3), 2.17 (s, 3H, CH3), 2.18
(s, 3H, CH3), 3.44 (s, 1H, OH), 3.94e4.14 (m, 2H, OCH2), 6.65 (s, 1H,
ArH), 7.28 (s, 1H, ArH), 7.50e7.56 (m, 1H, ArH), 7.74e7.79 (m, 2H,
ArH), 7.90e7.93 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3)
d 15.9, 19.4,
4.1.5. Preparation of 3-amino-6H-dibenzo[b,f]oxocine-6,11,12-
trione (1d)
The same procedure is followed as previously reported and
analytical data matched [26].
20.6, 62.1, 84.0, 111.8, 112.0, 122.3, 124.1, 125.5, 125.8, 130.7, 131.2,
134.7, 136.5, 141.3, 148.2, 155.6, 199.4 (C]O); MS/EI: m/z (rel. in-
tensity) 310.1 (Mþ, 56.9), 264.1 (100).
4.1.10. Ethyl 2-(2-(2-hydroxy-4,5-dimethylphenyl)-2-oxoacetyl)
benzoate (1e)
4.1.6. Preparation of 6-amino-3H,30H-spiro[benzofuran-2,10-
isobenzofuran]-3,30-dione (30b)
5,6-Dimethyl-3H,30H-spiro[benzofuran-2,10-isobenzofuran]-
3,30-dione, 3b (500 mg, 1.60 mmol) was taken in ethanol (20 mL)
and to this sodium ethoxide (0.138 g, 2.04 mmol) was added. After
Compound 1d (40 mg, 0.15 mmol) was taken in ethanol (1 mL)
and then irradiated with microwaves at 110 ꢀC for 15 min. This was

Y. Malpani et al. / European Journal of Medicinal Chemistry 62 (2013) 534e544
543
10 min of stirring the reaction mass was concentrated to remove
the solvent and then dissolved in ethyl acetate (100 mL) and
washed with water (50 mL) and brine (20 mL). This was dried over
sodium sulphate and concentrated to get the crude which was
purified over silica gel column chromatography (5% ethyl acetate in
hexanes) to get the pure compound as solid. Yield: 120 mg (21%);
mp: 159e162 ꢀC; IR (cmꢂ1): 1733, 1698, 1628; 1H NMR (300 MHz,
and the percentage number relative to mock-treated, virus-infec-
ted cells (100%) was calculated. The values are presented as
means ꢁ standard deviations and are representative of at least two
independent experiments.
Acknowledgements
CDCl3)
d
1.23e1.29 (m, 6H þ 3H, CH3), 2.93 (sept, J ¼ 6.9 Hz, 1H, CH),
This work was supported by Transgovernmental Enterprise for
Pandemic Influenza in Korea (TEPIK) (Grant A103001). The authors
like to thank Korea Research Institute of Chemical Technology for
providing financial support and facility for this work and also Korea
Chemical Bank in KRICT for supplying the chemical library. We also
thank University of Science and Technology for providing fellow-
ships to the student authors. Finally, we thank Dr. I.Y. Lee at KRICT
for generously providing the known compound favipiravir syn-
thesized at his lab.
4.22 (q, J ¼ 7.2 Hz, 3H, OCH2), 6.88e6.91 (br, 2H, ArH), 7.61e7.71 (m,
3H, ArH), 8.05 (d, J ¼ 7.5 Hz, 1H, ArH), 8.18 (d, J ¼ 8.4 Hz, 1H, ArH),
11.29 (br, 1H, OH); 13C NMR (75 MHz, CDCl3)
d 14.2, 23.3, 23.5, 34.7,
62.2, 114.7, 115.6, 118.7, 129.6, 130.41, 130.42, 131.8, 132.9, 133.4,
138.5, 160.0, 164.3, 166.7, 192.4 (C]O), 193.0 (C]O); MS (EI) m/z
(rel. intensity) 340.4 (Mþ, 73), 323.3 (84), 295.2 (100), 163 (67).
4.2. Antiviral assay
4.2.1. Materials and methods
Appendix A. Supplementary data
MadineDarby canine kidney (MDCK) cells (ATCC; Manassas, VA)
were grown in minimum essential medium (MEM; Gibco/Invi-
trogen, Carlsbad, CA) supplemented with 10% fetal bovine serum
(FBS; Invitrogen) at 37 ꢀC. Influenza virus HK were obtained from
ATCC and propagated in 10-day-old chicken embryos at 37 ꢀC.
Influenza virus strain BB was obtained from the Center for Disease
Control and Prevention (CDC), Atlanta, GA via Korea CDC at
Cheongwon, South Korea, while TWA and PNM were obtained from
the Korean CDC. The rest influenza B viruses, TWB and LE, were
purchased from ATCC. All viral strains except HK were propagated
via the infection of MDCK cells in serum-free medium containing
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2013.01.015.
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