Synthesis and anti-norovirus activity of pyranobenzopyrone compounds

Article abstract of DOI:10.1016/j.bmcl.2012.03.084

During the last decade, noroviruses have gained media attention as the cause of large scale outbreaks of gastroenteritis on cruise ships, dormitories, nursing homes, etc. Although noroviruses do not multiply in food or water, they can cause large outbreaks because approximately 10-100 virions are sufficient to cause illness in a healthy adult. Recently, it was shown that the activity of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT1) enzyme may be important in norovirus infection. In search of anti-noroviral agents based on the inhibition of ACAT1, we synthesized and evaluated the inhibitory activities of a class of pyranobenzopyrone molecules containing amino, pyridine, substituted quinolines, or 7,8-benzoquinoline nucleus. Three of the sixteen evaluated compounds possess ED₅₀ values in the low micrometer range. 2-Quinolylmethyl derivative 3A and 4-quinolylmethyl derivative 4A showed ED₅₀ values of 3.4 and 2.4 μM and TD₅₀ values of >200 and 96.4 μM, respectively. The identified active compounds are suitable for further modification for the development of anti-norovirus agents.

Full text of DOI:10.1016/j.bmcl.2012.03.084

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3480–3484
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and anti-norovirus activity of pyranobenzopyrone compounds
Laxman Pokhrel a, Yunjeong Kim b, Thi D.T. Nguyen a, Allan M. Prior a, Jianyu Lu a,
Kyeong-Ok Chang b, Duy H. Hua a,
⇑
a Department of Chemistry, Kansas State University, Manhattan, KS 66506, USA
b Department of Diagnostic Medicine and Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
During the last decade, noroviruses have gained media attention as the cause of large scale outbreaks of
gastroenteritis on cruise ships, dormitories, nursing homes, etc. Although noroviruses do not multiply in
food or water, they can cause large outbreaks because approximately 10–100 virions are sufficient to
cause illness in a healthy adult. Recently, it was shown that the activity of acyl-coenzyme A:cholesterol
acyltransferase-1 (ACAT1) enzyme may be important in norovirus infection. In search of anti-noroviral
agents based on the inhibition of ACAT1, we synthesized and evaluated the inhibitory activities of a class
of pyranobenzopyrone molecules containing amino, pyridine, substituted quinolines, or 7,8-benzoquin-
oline nucleus. Three of the sixteen evaluated compounds possess ED50 values in the low micrometer
range. 2-Quinolylmethyl derivative 3A and 4-quinolylmethyl derivative 4A showed ED50 values of 3.4
Received 24 February 2012
Revised 15 March 2012
Accepted 22 March 2012
Available online 29 March 2012
Keywords:
Acyl-coenzyme A:cholesterol
acyltransferase (ACAT)
Caliciviruses
and 2.4
lM and TD50 values of >200 and 96.4 lM, respectively. The identified active compounds are suit-
Norovirus
able for further modification for the development of anti-norovirus agents.
Anti-noroviruses
Pyranobenzopyrones
Ó 2012 Elsevier Ltd. All rights reserved.
Noroviruses, category B bioterrorism agents, are the leading
cause of food- or water-borne gastroenteritis outbreaks. Studies
have shown that noroviruses are responsible for approximate
90% of epidemic non-bacterial food–water-borne gastroenteritis
outbreaks with an estimated 23 million cases annually in the US
causing 50,000 hospitalizations and 300 deaths.1–3 Norovirus
belongs to the Caliciviridae family, and may infect an individual
with as few as 10 viral particles. Outbreaks often occur in closed
environments such as dormitories, cruise ships, hospitals, and
care facilities. Caliciviruses (Family Caliciviridae) are small, non-
enveloped RNA viruses of 27–35 nm in diameter. They possess a
single-stranded, plus-sense genomic RNA of 7–8 kb, which encodes
a nonstructural polyprotein, a major structural capsid protein of
58–80 kDa (VP1), and a small basic protein (VP2).4 Currently, there
are no specific drugs for norovirus infection. Vaccine development
for human noroviruses have faced challenges because noroviruses
do not grow in cell culture, show high diversity, and immunity
from heterologous strains do not seem to confer protection.5
Furthermore, repeat infections in adults indicate that long-term
immunity may be absent. Consequently there is an urgent need
for the development of effective anti-noroviral drugs. The recent
development of replicon-harboring cells for norovirus6,7 has made
possible the study of norovirus replication in cells. DNA microarray
analysis of norovirus replicon-harboring cells (HG23) by an
Affymetrix Gene Chip showed the up regulations of acyl-coenzyme
A:cholesterol acyltransferase (ACAT) and other cholesterol modu-
lating genes (> 1.5-fold).8 Moreover, commercially available ACAT
inhibitors were shown to inhibit norovirus replication.8 These
findings suggest that cellular ACAT may be a potential therapeutic
target for norovirus infection. Previously, we reported a class of
pyranobenzopyrones that possesses ACAT inhibitory activity.9
Hence, anti-norovirus activities of pyranobenzopyrones were
investigated. Herein, we report the synthesis and anti-norovirus
activity of a small library of sixteen pyranobenzopyrones contain-
ing amino, pyridine, quinoline, and 7,8-benzoquinoline nucleus
(Fig. 1). Among the pyranobenzopyrones, several compounds
possess anti-norovirus activity in low micromolar concentrations
in vitro with therapeutic index values of P40.
The synthesis of pyranobenzopyrones 1–7 stems from a reduc-
tive amination reaction10 of amine 10 and various aldehydes,
11–17 as depicted in Scheme 1. Initially, amine 10 was prepared
via a four-step sequence of reactions starting from pyranobenzopy-
rones 9 by hydroboration–hydroxylation reaction followed by
mesylation, displacement with sodium azide, and reduction with
H2/Pd.9 The synthesis was simplified by a one-pot hydrobora-
tion–amination reaction of 9 with BH3ÁTHF followed by hydroxyl-
amine-O-sulfonic acid11 in 50% yield (Scheme 1). A mixture of
two diastereomers in a ratio of 1:1 resulted at the newly created
carbon center C12 from the hydroboration reaction indicated by
its 1H and 13C NMR spectra. The diastereomers are separable by
HPLC but not silica gel column chromatograph. Alkylation of amine
10 with 1 equivalent each of aldehydes 11–17 separately in meth-
anol followed by sodium cyanoborohydride afforded amines 1–7,
⇑
Corresponding author. Tel.: +1 785 532 6699; fax: +1 785 532 6666.
E-mail address: duy@ksu.edu (D.H. Hua).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.084

L. Pokhrel et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3480–3484
3481
O
O
O
CH3
O
O
O
CH3
O
O
O
CH3
O
O
O
CH3
H
H
H
H
R1
R2
H
4
6
H
N
H
N
N
H
N
2
3
R2
N
N
4
6
1
2N
3A : R1 = R2 = H
4
R1
3B : R1 = Me, R2 = H
3C : R1 = H, R2 = Me
N
4A : R1 = R2 = H
2
4B : R1 = Me, R2 = H
4C : R1 = CH2OAc, R2 = H
4D : R1 = CH2OH, R2 = H
O
O
4E : R1
=
, R2 = H
O
4F : R1 = H, R2 = Me
O
O
CH3
O
O
CH3
O
O
O
CH3
O
O
O
CH3
H
O
H
H
H
O
N
H
N
N
H
N
H
N
O
4
H
N
6
8
8
7
4
N
5
6
7
N
8
Figure 1. Synthesized and bioevaluated pyranobenzopyran compounds 1–8.
O
O
O
CH3
O
O
O
CH3
O
O
O
O
CH3
H
Ar
BH3•THF;
H
H2NOSO3H
H
(11 - 17)
H
(50% yield)
MeOH;
AcOH, NaCNBH3
H
N
NH2
Ar
12
9
10
1 - 7
Me
16 : Ar =
6 (53% yield)
4B (60% yield)
4C (66% yield)
8
13B: Ar =
3B (53% yield)
4
2
11 : Ar =
12 : Ar =
13A: Ar =
3
1 (60% yield)
2 (64% yield)
3A (60% yield)
N
N
N
N
4
14B: Ar =
4
Me
N
Me
14F: Ar =
13C: Ar =
17 : Ar =
2
4F (62% yield)
14C: Ar =
N
N
N
OAc
Me
4
14A: Ar =
15 : Ar =
4A (65% yield)
5 (47% yield)
2
3C (56% yield)
7 (62% yield)
14D: Ar =
14E: Ar =
N
N
4D (61% yield)
4E (47% yield)
N
OH
O
6
4
N
N
N
O
O
Scheme 1. Synthesis of compounds 1–7.
respectively. Yields of compounds 1–7 range from 47–66%.
Pyridinecarboxaldehydes, various substituted quinolinecarboxal-
dehydes, and 7,8-benzoquinoline-4-carboxaldehyde (17 or
4-azaphenanthrene-1-carboxaldehyde) were used in the reductive
amination reaction, and functional groups such as primary alcohol,
ester, and trioxane are stable under the reaction conditions.

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L. Pokhrel et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3480–3484
Amide 8 was synthesized from the coupling reaction of quino-
(15) was obtained from 6-methylquinoline (20) in 54% yield after
the treatment with SeO2 in refluxing xylene. To our surprise, oxida-
tion of 8-methylquinoline (21) under similar reaction conditions
provided only a trace amount of 8-quinoline-carboxaldehyde
(16). Apparently, methyl group appended on ring A of quinoline
is activated toward oxidation, but methyl group on ring B is not,
and a sluggish oxidation resulted. To overcome the problem,
benzylic bromination of 21 was carried out. Treatment of 21 with
N-bromosuccinimide (NBS) and a catalytic amount of azobisisobu-
tyronitrile (AIBN) followed by aqueous hydrolysis accompanying
air oxidation gave aldehyde 16 (37% yield)13 along with the hydro-
lyzed product, 6-hydroxymethylquinoline (22) (53% yield). Oxida-
tion of alcohol 22 with o-iodoxybenzoic acid (IBX) and DMSO16
furnished 16 in a 79% yield.
line-4-carboxylic acid (18), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC), and amine 10 in 65% yield (Scheme 2).
Aldehydes 11, 12, and 13A and carboxylic acid 18 along with var-
ious methylquinolines were obtained from commercial sources.
Aldehydes 14A, 15, and 16 were prepared by benzylic oxidation
with selenium dioxide12 or bromination followed by hydroly-
sis/oxidation13 of methylquinolines, and aldehydes 13B, 13C,
14B–14F, and 17 were achieved from free-radical heteroaromatic
trioxanylation14,15 with trioxane-t-BuOOH–ferrous sulfate
(Schemes 2–4). Hence, oxidation of 4-methylquinoline (19) with
SeO2 under refluxing toluene gave quinoline-4-carboxaldehyde
(14A) in 73% yield along with a small amount of over oxidized car-
boxylic acid 18 (Scheme 2). Similarly, quinoline-6-carboxaldehyde
CO2H
EDC, DMAP
CH2Cl2
4
+
10
8
(65% yield)
N
18
Me
1) NBS, AIBN
CH2Cl2
SeO2
toluene
4
16
+
8
8
14A
18
+
N
2) H2O
reflux
N
reflux, 24 h
N
(37% yield)
(73% yield)
(7% yield)
Me
OH
(53% yield)
22
19
20
21
SeO2
Me
xylene
6
IBX
15
DMSO
reflux, 24 h
(54% yield)
N
22
16
(79% yield)
Scheme 2. Syntheses of compound 8 and quinolinecarboxaldehydes 14A, 15, and 16.
O
O
O
FeSO4, TFA
-BuOOH
10% H2SO4
reflux
t
2
14B
trioxane
2
Me
N
(79% yield)
(51% yield)
Me
N
23
24
O
O
O
H
SeO2
toluene
2 N HCl
O
24
H
80oC
N
2
H
O
N
26
O
(68% overall yield)
25
HO
NaBH4
HO
CH2Cl2/EtOH
14D
+
+
26
H
O
N
(32% yield)
N
OH
28
(16% yield)
27
(35% yield)
Ac2O
ZnO
14D
14C
(85% yield)
Scheme 3. Syntheses of quinolinecarboxaldehydes 14B–14D.

L. Pokhrel et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3480–3484
3483
1) FeSO4, TFA
t
-BuOOH, trioxane
Me
N
19
13B
FeSO4, TFA
-BuOOH
2) 10% H2SO4
(50% yield)
t
19
O
+
13B
trioxane
SeO2
O
O
toluene
(3% yield)
14E
29
29
(43% yield)
reflux
(35% yield)
1) FeSO4, TFA
-BuOOH, trioxane
t
13C
14F
(17% yield)
+
20
2) 10% H2SO4
reflux
(16% yield)
1) FeSO4, TFA
-BuOOH, trioxane
7
t
17
N
8
2) 2 N HCl
(18% yield)
30
Scheme 4. Syntheses of quinolinecarboxaldehydes 13B, 13C, 14E, 14F and 7,8-benzoquinolinecarboxaldehyde (17).
Substituted quinolinecarboxaldehydes 13B, 13C, 14B–14F, and
7,8-benzoquinolinecarboxaldehyde (17) were obtained from
heteroaromatic trioxanylation reactions (Schemes 3 and 4).14,15
Treatment of 2-methylquinoline (23) with trioxane, t-butyl hydro-
peroxide and trifluoroacetic acid (TFA) in the presence of a cata-
lytic amount of ferrous sulfate afforded trioxanylquinoline 24,
which underwent acidic hydrolysis to give aldehyde 14B. 2-
Hydroxymethyl-4-quinolinecarboxaldehyde (14D) was obtained
from the benzylic oxidation of 24 with selenium dioxide followed
by the hydrolysis of the resulting trioxanyl aldehyde 25 with 2 N
HCl to give dialdehyde 26. Subsequent reduction of 26 with sodium
borohydride afforded 14D. In the reduction process, regioisomer,
4-hydroxymethyl-2-quinolinecarboxaldehyde (27) and 2,4-
di(hydroxymethyl)quinoline (28) were also isolated. Acetylation
of the hydroxyl function of 14D with acetic anhydride and zinc
oxide17 produced acetate 14C (Scheme 3).
methylquinolines can either be oxidized to the corresponding
quinolinecarboxaldehydes or formylated to methylquinolinecarb-
oxaldehydes.
From our initial screening of the effects of pyranobenzopyrone
compounds on the reduction of NV replicon-harboring cells
(HG23 cells), 3-pyridyl analog 1 showed promising results with
ED50 value (effective dosage at reducing NV genome levels by
50% at 24 h post-treatment) and TD50 value (cytotoxic dosage in
killing 50% HG23 cells at 48 h post-treatment determined by cyto-
toxicity assay at 48 h of treatment)7,8 of 4 and >200
lM, respec-
tively. Hence, a small library of pyridylmethyl, quinolylmethyl,
quinolylcarbonyl, and 4-azaphenanthrenylmethyl derivatives
along with their synthetic precursor, amine 10, was evaluated for
their anti-norovirus activities. Results of the inhibition of NV
RNA replication are summarized in Table 1. To our surprise, amine
10, 4-pyridylmethyl 2, 8-quinolylmethyl 6, and 2-acetoxymethyl-
and 2-hydroxymethyl-4-quinolylmethyls 4C and 4D have ED50
Similar trioxanylation of 4-methylquinoline (19) furnished 2-
trioxanequinoline 29 in 43% yield along with a small amount of
4-methyl-2-quinolinecarboxaldehyde (13B) (3% yield), which de-
rived from the hydrolysis of the trioxane moiety of 29 with TFA
and water in the reaction mixture (Scheme 4). Alternatively, alde-
hyde 13B can be achieved in 50% yield from a two-step trioxanyla-
tion of 19 followed by acidic hydrolysis with 10% aqueous sulfuric
acid of the resulting trioxane without purification. 2-Trioxanyl-4-
carboxaldehyde (14E) was obtained from the benzylic oxidation
of 29 with selenium dioxide. Similar to the selenylation reaction
of 8-methylquinoline (21), trioxanylation of 6-methylquinoline
(20) appeared to be sluggish. Hence, the reaction of 20 with triox-
ane, t-butyl hydroperoxide, TFA and ferrous sulfate followed by
acidic hydrolysis furnished a mixture of regioisomers, 6-methyl-
4-carboxaldehyde (14F) (17% yield) and 6-methyl-2-carboxalde-
hyde (13C) (16% yield), which were separated by silica gel column
chromatography. The spectral data of compound 13C are similar to
that reported,18 and the assignment of regiochemistry of 14F is
based on its 1H NMR spectral data. The chemical shift of C2-H of
14F appears at d 9.14 ppm as a doublet with coupling constant J
value of 4.3 Hz, and that of C3-H at d 7.77 ppm as a doublet with
J value of 4.3 Hz, which are similar to that of 4-quinolinecarboxal-
dehyde (14A). Formylation of 4-azaphenanthrene (30) under
similar reaction conditions followed by hydrolysis with 2 N
HCl afforded 7,8-benzoquinoline-4-carboxaldehyde (17). Hence,
values >10 lM. Due to high ED50 values, TD50 values of these
compounds were not determined except compound 10. Other
Table 1
Effects of pyranobenzopyrone compounds on the reduction of NV RNA replication
(ED50
) and toxicity (TD50) in HG23 cells (NV replicon-haboring cells) and their
therapeutic indexes (TI)
Compound ED50 value in
l
M
TD50 value in
lM
Therapeutic index (TI)
1
2
3A
4A
5
4.1
9.6
3.4
2.4
8.1
>10
8.2
9.7
9.5
8.4
5.3
3.4
8.1
5.3
5.5
>10
>200
NDa
>200
96.4
ND
>160
ND
ND
>50
—
>58.8
40.2
—
>16
—
—
6
4B
4C
4D
4E
3B
4F
3C
7
ND
ND
—
—
>200
61.2
83.7
>200
103.5
>200
>40
18.0
10.3
>40
18.8
—
8
10
a
ND: not determined due to high ED50 values. Each value is the average of at
least two independent tests.

3484
L. Pokhrel et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3480–3484
quinolylmethyl, substituted quinolylmethyl, and 4-aza-
phenanthrenylmethyl derivatives along with 4-quinolyl amide 8
be found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.03.084.
possess ED50 values ranging from 2–8
lM and TD50 values of 61
to >200 M. In particular, 2-quinolylmethyl 3A, 4-quinolylmethyl
l
References and notes
4A, and 6-methyl-4-quinolylmethyl 4F possess the strongest
anti-norovirus activities with respective ED50 values of 3.4, 2.4,
1. Lindesmith, L.; Moe, C.; Marionneau, S.; Ruvoen, N.; Jiang, X.; Lindblad, L.;
Stewart, P.; LePendu, J.; Baric, R. Nat. Med. 2003, 9, 548.
and 3.4
ues of 58.8, 40.2, and 18.0, respectively. 6-Methyl-2-quinolylmeth-
yl 3C on the other hand showed a lower ED50 value of 8.1 M.
lM and TI (therapeutic index; derived from TD50/ED50) val-
2. Widdowson, M. A.; Sulka, A.; Bulens, S. N.; Beard, R. S.; Chaves, S. S.; Hammond,
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l
Hence, addition of substituent at C6 or ring B of 4-quinolylmethyl
analog appears to retain its antiviral activity and substituent on
ring A diminishes its activity.
In conclusion, various pyranobenzopyrone molecules contain-
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2-quinolylmethyl analog 3A possess the strongest anti-norovirus
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This work has been supported by the National Institutes of
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Supplementary data
Supplementary data (synthetic procedure, analytical data, and
protocols for antiviral evaluation) associated with this article can