Synthesis and evaluation of quinoxaline derivatives as potential influenza NS1A protein inhibitors

Article abstract of DOI:10.1016/j.bmcl.2011.03.042

A library of quinoxaline derivatives were prepared to target non-structural protein 1 of influenza A (NS1A) as a means to develop anti-influenza drug leads. An in vitro fluorescence polarization assay demonstrated that these compounds disrupted the dsRNA-NS1A interaction to varying extents. Changes of substituent at positions 2, 3 and 6 on the quinoxaline ring led to variance in responses. The most active compounds (35 and 44) had IC₅₀ values in the range of low micromolar concentration without exhibiting significant dsRNA intercalation. Compound 44 was able to inhibit influenza A/Udorn/72 virus growth.

Full text of DOI:10.1016/j.bmcl.2011.03.042

Bioorganic & Medicinal Chemistry Letters 21 (2011) 3007–3011
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of quinoxaline derivatives as potential influenza
NS1A protein inhibitors
Lei You a, Eun Jeong Cho b, John Leavitt c, Li-Chung Ma d, Gaetano T. Montelione d,e, Eric V. Anslyn a,
,
⇑
a
a
Robert M. Krug c, , Andrew Ellington , Jon D. Robertus
⇑
a Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, TX 78712, USA
b The Institute for Drug and Diagnostic Development, University of Texas at Austin, Austin, TX 78712, USA
c Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712, USA
d Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers The State University of New Jersey, Piscataway, NJ 08854, USA
e Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A library of quinoxaline derivatives were prepared to target non-structural protein 1 of influenza A
(NS1A) as a means to develop anti-influenza drug leads. An in vitro fluorescence polarization assay dem-
onstrated that these compounds disrupted the dsRNA–NS1A interaction to varying extents. Changes of
substituent at positions 2, 3 and 6 on the quinoxaline ring led to variance in responses. The most active
compounds (35 and 44) had IC50 values in the range of low micromolar concentration without exhibiting
significant dsRNA intercalation. Compound 44 was able to inhibit influenza A/Udorn/72 virus growth.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 18 January 2011
Revised 10 March 2011
Accepted 11 March 2011
Available online 17 March 2011
Keywords:
Quinoxaline derivatives
NS1A protein
Influenza A virus
Fluorescence polarization
Influenza viruses cause a highly contagious respiratory disease
in humans. They are RNA viruses composed of three general types:
influenza A, influenza B, and influenza C.1 The type A viruses cause
the most severe diseases, and as a result, are the most serious
threat to human health.2 The influenza A virus can be further di-
vided into different serotypes. H1N1 caused the 2009 flu pan-
demic,3 and H5N1 is a current pandemic threat.4 Therefore, the
development of small molecule based anti-influenza therapeutics
continues to capture significant attention.5,6
can fit into this cavity, it can block dsRNA binding and thus inacti-
vate the NS1 protein.
(ꢀ)-Epigallocatechin-3-gallate (EGCG)9 was identified to inhibit
NS1A through high-throughput screening. EGCG and its deriva-
tives10 display a broad range of biological activities.11 In an effort
to design and synthesize structurally simple molecules targeting
NS1A protein,
OH
HO
The NS1 protein,7 a highly conserved influenza virus encoded
protein, has been identified as a potential target for antiviral devel-
opment.8 Specifically, the double-stranded RNA (dsRNA) binding
domain, comprising residues 1–73, is crucial for virus replication,
and is the primary target of our work. Detailed biophysical and
structural studies by high-resolution NMR and X-ray analysis re-
vealed that the N-terminal domain of the NS1A protein forms a
homodimer with a unique six-helical chain fold.7 There is a deep
cavity at the center of dsRNA-binding surface. If a small molecule
O
OH
HO
4
5
8
3
2
6
X
Ar
Ar
N
O
HO
HO
OH
7
O
N
1
OH
(-) Epigallocatechin-3-Gallate
Quinoxaline
derivative
we turned our attention into the quinoxaline scaffold, which can be
rapidly constructed. Quinoxalines, an important class of heterocy-
cles, are components of several biologically active compounds.12
Quinoxaline and EGCG share structural similarities: a bicyclic ring
and the potential for substitution with polar groups on the ring.
⇑
Corresponding authors. Tel.: +1 512 471 0068; fax: +1 512 471 7791 (E.V.A.);
tel.: +1 512 232 5563; fax: +1 512 232 5565 (R.M.K.).
E-mail addresses: anslyn@austin.utexas.edu (E.V. Anslyn), rkrug@mail.utexas.
edu (R.M. Krug).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.03.042

3008
L. You et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3007–3011
O
Here, we report a structure–activity relationship (SAR) study with
quinoxaline analogs targeting the NS1A protein.
PyBOP or TBTU,
DIPEA, DMF
N
N
COOH
+
N
N
O
O
N
R
R
H
NH2
A library of 46 compounds were designed and synthesized. While
keeping the quinoxaline core, various aromatic residues, such as
4-methoxyphenyl, 4-hydroxyphenyl, 2-furyl, and 2-pyridyl, were
incorporated into positions 2 and 3, and different substituents were
also placed in position 6. In general, 2,3-disubstituted quinoxalines
were prepared by condensation of 1,2-diketones and o-phenylene-
diamine derivatives in refluxing EtOH or HOAc/NaOAc (Eq. 1).12
O
O
ð2Þ
In order to examine whether the quinoxaline analogues can dis-
rupt the dsRNA binding to NS1A protein, an in vitro fluorescence
polarization-based binding assay (FP assay)14 was employed. In
this assay, a carboxyfluorescein-labeled dsRNA (FAM-dsRNA) was
used as a signaling probe. In detail, when FAM-dsRNA binds to
the NS1A protein, the mobility of the fluorophore (FAM) decreases
and as a result, the fluorescence polarization increases. The addi-
tion of potential NS1A inhibitors targeting the dsRNA-binding do-
main will displace the FAM-dsRNA from NS1A and lead to a
decrease of fluorescence polarization. The data were reported as
Ar
Ar
O
O
H2N
H2N
X
HOAc, NaOAc, reflux
or, EtOH, reflux
Ar
N
X
+
Ar
N
ð1Þ
For demethylation of the methoxyphenyl substituted deriva-
tives, many conditions were tested, including HBr/HOAc, BBr3/
CH2Cl2, and EtSNa/DMF. For 3-methoxyphenyl and 4-methoxy-
phenyl substituted quinoxalines, treatment with EtSNa in refluxing
DMF afforded the corresponding 3-hydroxyphenyl and 4-hydroxy-
phenyl derivatives when either H or OMe was in position 6. When
electron-withdrawing groups, such as COOH and NO2, were in po-
sition 6 of quinoxalines, demethylation of 3,30-dimethoxybenzil or
4,40-dimethoxybenzil was achieved utilizing 48% HBr in HOAc un-
der refluxing conditions, prior to condensation with o-phenylene-
diamine derivatives (Scheme 1).
% binding at 50 lM, where a higher percentage represents stronger
activity in breaking the dsRNA–NS1A interaction. A similar FP
based assay to probe dsRNA intercalation of the quinoxaline deriv-
atives was utilized as a control experiment, because targeting
NS1A instead of dsRNA was desired. The data were reported as %
intercalation at 50 lM, and (+) sign means intercalating to the
dsRNA while (ꢀ) sign means denaturation of the dsRNA to ssRNAs.
All assays were run in duplicates, and data were averaged. The
compounds with high % binding at 50
lM and low % intercalation
at 50 M were subjected to further studies.
l
Several of the 1,2-diketones we used in (Eq. 1) are not readily
available. For example, 2,20-dimethoxybenzil was prepared from
o-anisaldehyde using Pinacol coupling followed by oxidation.13
Benzoin condensation of piperonal followed by oxidation afforded
3,4,30,40-bis(methylenedioxy)-benzil (Scheme 2). Condensation
with these 1,2-phenylenediamines was done as described above.
However, attempts to deprotect the catechol using either BBr3/
CH2Cl2 or EtSNa/DMF afforded a complicated mixture.
We first set out to explore SARs of 2,3,6-substituted quinoxaline
derivatives, and the results are shown in Table 1. Substitution at
positions 2 and 3 on the quinoxaline core had the most significant
impact on the activity. Compounds with bis-2-furyl substitutions
(27–30) were the most potent. Replacements of the furyl residue
with methoxyphenyl (1–12), phenol (13–22), 3,4-(methylenedi-
oxy)phenyl (23–25), or 2-pyridyl groups (31–33), reduced the
activity. Compounds with phenol substitution (13–22) generally
showed improved activity compared to the corresponding com-
pounds with methoxyphenyl substitution (1–12). For bis-2-furyl
substituted quinoxaline derivatives, substitution at position 6 also
played an important role in the biological activity. Compounds 27,
In addition, 2,3-furyl-quinoxaline-6-carboxylic acid was cou-
pled with various amines using PyBOP or TBTU as a coupling re-
agent and DIPEA as
a base to afford a library of amide
substituted quinoxaline analogs (Eq. 2).
N
N
R
EtSNa. DMF,
reflux
N
N
R
MeO
MeO
HO
HO
R = H (48%), OH (64%)
OH
R = H, OMe
N
COOH
EtOH, reflux
79%
N
OMe
OH
OH
OH
48% HBr,
O
HOAc,
reflux
92%
O
O
O
HOAc, NaOAc,
reflux
71%
OMe
OH
N
N
NO2
OH
Scheme 1.

L. You et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3007–3011
3009
MeO
O
O
1, Al, KOH, MeOH
H
2, DMSO, 40% HBr
O
OMe
46%
MeO
O
O
O
O
OH
O
O
NH4NO3, Cu(OAc)2
H
KCN
EtOH, H2O
28%
HOAc, H2O
55%
O
O
O
O
O
O
O
O
Scheme 2.
Table 1
Activities of 2,3,6-substituted quinoxaline derivatives
Ar
Ar
N
N
X
Compound
Ar
X
% Binding at 50
l
M
% Intercalation at 50
lM
1
2
3
4
5
6
7
8
9
2-OMe-Ph–
2-OMe-Ph–
2-OMe-Ph–
2-OMe-Ph–
3-OMe-Ph–
3-OMe-Ph–
3-OMe-Ph–
3-OMe-Ph–
4-OMe-Ph–
4-OMe-Ph–
4-OMe-Ph–
4-OMe-Ph–
2-OH-Ph–
2-OH-Ph–
3-OH-Ph–
3-OH-Ph–
3-OH-Ph–
3-OH-Ph–
4-OH-Ph–
4-OH-Ph–
4-OH-Ph–
4-OH-Ph–
3,4-O,O-CH2-Ph–
3,4-O,O-CH2-Ph–
3,4-O,O-CH2-Ph–
2-Furyl-
H
5.0
4.4
2.8
6.7
1.4
7.2
9.1
1.5
0.5
ꢀ6.7
ꢀ5.0
ꢀ2.5
ꢀ3.7
ꢀ6.0
ꢀ8.4
ꢀ4.4
1.9
OMe
COOH
NO2
H
OMe
COOH
NO2
H
OMe
COOH
NO2
H
OH
H
OH
COOH
NO2
H
1.4
5.7
10
11
12
13
14
15
16
17
18
19
20
21
22
2315
24
25
26
27
28
29
30
31
32
33
1.8
4.5
ꢀ4.8
112.7
ꢀ1.0
0.9
ꢀ4.8
18.2
ꢀ3.4
ꢀ15.8
8.5
29.5
ꢀ7.7
ꢀ32.0
7.9
ꢀ4.8
24.4
ꢀ23.7
ꢀ3.2
5.8
ꢀ19.7
ꢀ0.9
10.9
10.9
25.0
28.7
42.8
10.2
38.8
13.3
19.5
1.5
23.2
56.6
7.3
54.3
60.9
76.0
25.5
-3.1
15.7
7.8
OH
COOH
NO2
H
COOH
NO2
H
OMe
COOH
COOMe
NO2
H
2-Furyl-
2-Furyl-
2-Furyl-
2-Furyl-
2-Pyridyl-
2-Pyridyl-
2-Pyridyl-
ꢀ12.3
25.9
2.3
2.9
ꢀ5.6
COOH
NO2
28 and 29 exhibited significantly higher activity than 26, which has
a hydrogen atom at position 6.
Next, we prepared a library based on compound 28, and the
results are listed in Table 2. Keeping 2-furyl at positions 2 and 3,
phenyl groups with various substitution or heterocycles were
2-Pyridyl (43) substitution also decreased the biological activity.
Replacement of 2-furyl with 2-thenyl (45) was not tolerated. An
aliphatic 2-furyl mimic, glycine methyl ester derivative (46),
showed decreased activity. The four compounds with the highest
percentage binding at 50
lM were further studied in dose depen-
incorporated into position
6
through an amide linker. The
dent FP assay. They have the following IC50 (lM): 74.8 (29), 6.2
compounds showing the most potency were 35 and 44, with
3-methoxyphenyl and 2-furyl at position 6, respectively. Their per-
(35), 43.3 (43) and 3.5 (44). None of these four compounds showed
significant dsRNA interference.
centage binding at 50
35, respectively, which is comparable to that of EGCG
(89.0 11.4 M). Replacement of 3-methoxyphenyl with other
methoxy substituted phenyl groups (34, 36–38), fluorophenyl
(39–41), or phenyl 4-methyl ester (42) resulted in reduced activity.
l
M is 79.5 1.5 and 74.0 1.4
l
M for 44 and
We then set out to explore the biological activity of the most
potent compound (44) identified from the in vitro assay described
above. Viral growth was assayed using MDCK cells infected with
influenza A/Udorn/72 virus. Figure 1 shows the single cycle growth
curve at MOI (multiplicity of infection) 5 (top panel) and multiple
l

3010
L. You et al. / Bioorg. Med. Chem. Lett. 21 (2011) 3007–3011
Table 2
effectiveness of inhibition after 37 h (bottom panel). Although
these are qualitative experiments, they demonstrate the antiviral
potential of those quinoxaline derivatives.
Activities of amide derivatives of compound 28
O
In conclusion, we have explored preliminary structure–activity
relationships (SARs) for a library of quinoxaline derivatives target-
ing the NS1A protein for the development of anti-influenza thera-
peutics. Those quinoxaline derivatives were designed to mimic
(ꢀ)-epigallocatechin-3-gallate, a hit identified by high-throughput
screening. In vitro fluorescence polarization experiments showed
that these compounds can bind to NS1A to disrupt the NS1A–
dsRNA interaction. Substitution at positions 2 and 3 on the quinox-
aline core played an important role, with 2-furyl being the best
among those investigated. Substitution at position 6 was also cru-
cial. Compounds 35 and 44 were the most potent, with an IC50 of
N
O
N
R
H
N
O
Compound
R
% Binding at
50
% Intercalation at
50
lM
lM
34
35
36
37
38
2-OMe-Ph–
3-OMe-Ph–
4-OMe-Ph–
3,4-(OMe)2-Ph–
3,4,5-(OMe)3-
Ph–
29.5
74.0
44.0
39.5
53.6
ꢀ17.7
4.5
7.6
5.6
ꢀ4.7
6.2 and 3.5 lM, respectively. The dsRNA intercalation experiments
indicated that both 35 and 44 do not inhibit NS1A–dsRNA interac-
tions by interfering with dsRNA, but likely function by binding to
the NS1A dsRNA-binding domain itself. Results from a cell assay
demonstrated that compound 44 was able to inhibit influenza A
virus growth. Detailed structural analysis and optimization are
currently ongoing.
39
40
41
42
43
44
45
46
2-F-Ph–
3-F-Ph–
4-F-Ph–
11.6
13.6
49.0
47.9
64.7
79.5
8.2
ꢀ11.3
ꢀ11.6
6.3
4-COOMe-Ph–
2-Pyridyl-
2-Furyl-
8.8
ꢀ9.6
5.9
2-Thenyl-
COOMe
ꢀ10.5
ꢀ27.9
26.7
Acknowledgments
We thank the National Institutes of Health (U01 AI074497) and
The Texas Institute for Drug and Diagnostic Development for sup-
port of this work.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.03.042.
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