3-Hydroxyquinolin-2(1H)-ones as inhibitors of influenza A endonuclease

Article abstract of DOI:10.1021/ml4001112

Several 3-hydroxyquinolin-2(1H)-ones derivatives were synthesized and evaluated as inhibitors of 2009 pandemic H1N1 influenza A endonuclease. All five of the monobrominated 3-hydroxyquinolin(1H)-2-ones derivatives were synthesized. Suzuki-coupling of p-fluorophenylboronic acid with each of these brominated derivatives provided the respective p-fluorophenyl 3-hydroxyquinolin(1H)-2-ones. In addition to 3-hydroxyquinolin-2(1H)-one, its 4-methyl, 4-phenyl, 4-methyl-7-(p-fluorophenyl), and 4-phenyl-7-(p-fluorophenyl) derivatives were also synthesized. Comparative studies on their relative activity revealed that both 6- and 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)- one are among the more potent inhibitors of H1N1 influenza A endonuclease. An X-ray crystal structure of 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one complexed to the influenza endonuclease revealed that this molecule chelates to two metal ions at the active site of the enzyme.

Full text of DOI:10.1021/ml4001112

Letter
pubs.acs.org/acsmedchemlett
3‑Hydroxyquinolin-2(1H)‑ones As Inhibitors of Influenza A
Endonuclease
Hye Yeon Sagong,† Ajit Parhi,† Joseph D. Bauman,‡ Disha Patel,† R. S. K. Vijayan,‡ Kalyan Das,‡
Eddy Arnold,*,‡ and Edmond J. LaVoie*,†
†Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway,
New Jersey 08854-8020, United States
‡Center for Advanced Biotechnology and Medicine and Department of Chemistry and Chemical Biology, Rutgers University,
Piscataway, New Jersey, 08854-5627, United States
S
* Supporting Information
ABSTRACT: Several 3-hydroxyquinolin-2(1H)-ones derivatives were synthesized and evaluated
as inhibitors of 2009 pandemic H1N1 influenza A endonuclease. All five of the monobrominated 3-
hydroxyquinolin(1H)-2-ones derivatives were synthesized. Suzuki-coupling of p-fluorophenylbor-
onic acid with each of these brominated derivatives provided the respective p-fluorophenyl 3-
hydroxyquinolin(1H)-2-ones. In addition to 3-hydroxyquinolin-2(1H)-one, its 4-methyl, 4-phenyl,
4-methyl-7-(p-fluorophenyl), and 4-phenyl-7-(p-fluorophenyl) derivatives were also synthesized.
Comparative studies on their relative activity revealed that both 6- and 7-(p-fluorophenyl)-3-
hydroxyquinolin-2(1H)-one are among the more potent inhibitors of H1N1 influenza A
endonuclease. An X-ray crystal structure of 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one
complexed to the influenza endonuclease revealed that this molecule chelates to two metal ions at
the active site of the enzyme.
KEYWORDS: antiviral, influenza A, quinolinones, 3-hydroxyquinolin-2-ones, endonuclease
domains, PAN (∼25 kDa N-terminal domain; residues 1−197)
ntiviral agents are used for both prophylactic and
Atherapeutic treatments of influenza infection. The antiviral and PAC (∼55 kDa C-terminal domain; residues 239−716).
Crystal structures of PAC have been determined in complexes
with N-terminal fragments of PB1.5 The structure of PAN has
been solved in several crystal forms both unliganded and with
various ligands.6−8
agents in clinical use against influenza infection target the M2
ion-channel protein (adamantanes) and neuraminidase (zana-
mivir and oseltamivir). The adamantane drugs, amantadine and
rimantadine, are ineffective due to emergence of resistance
(predominantly through a M2 mutation, S31N) that limit their
clinical use. The neuraminidase (NA)-inhibiting oral drug,
oseltamivir (Tamiflu) is widely used for treating flu.
Oseltamivir-resistant seasonal influenza A strains have been
circulating for several years.1 The mutant viruses predominantly
contain the NA H274Y mutation. When accompanied by
compensatory mutations, the mutant viruses exhibit fitness
comparable to wild-type influenza A and remain resistant to
oseltamivir.2 These mutations can emerge in almost all
influenza A subtypes/strains, including the pandemic 2009
H1N1 virus, resulting in a major concern for an effective
treatment of flu.3 Therefore, new drugs are essential for treating
drug-resistant and future pandemic flu strains.
The RdRP of influenza A is responsible for the replication
and transcription of the viral RNA genes. Viral mRNA
transcription involves a cap-snatching mechanism in which
the polymerase binds to cellular mRNA via the 5′-cap and
cleaves the mRNA 12−13 nucleotides downstream. The
cleaved RNA fragment containing the 5′ cap acts as a primer
for viral mRNA synthesis.9 Cap-snatching is an important event
in the life cycle of all members of the Orthomyxoviridae family
including influenza A, B, and C viruses, and the host cell has no
analogous activity. Inhibitors of cap-snatching have the
potential to selectively act against all influenza subtypes and
strains, including oseltamivir-resistant influenza A viruses
without interfering with host cell activities.
Several small molecules have been identified that have the
ability to inhibit viral endonuclease. These include 2,4-
dioxobutanoic acid derivatives,8−11 5-hydroxy-1,6-dihydropyr-
imidine-4-carboxylic acid derivatives,9 flutimide and its
Influenza A contains eight negative-stranded RNA genomic
segments. The three largest genomic RNA segments encode
the viral RNA-dependent RNA polymerase (RdRP) proteins
consisting of the polymerase acidic protein (PA) and
polymerase basic protein 1 (PB1) and 2 (PB2) subunits. The
PA subunit (i) has endonuclease activity, (ii) is involved in viral
RNA (vRNA)/complementary RNA (cRNA) promoter bind-
ing, and (iii) interacts with the PB1 subunit.4 PA has two
Received: March 15, 2013
Accepted: May 7, 2013
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
derivatives,8,11−13 and tetramic acid derivatives.14 As correctly
hypothesized, the endonuclease activity of influenza polymerase
belongs to the two metal ion group of phosphate-processing
enzymes.14 Fragment screening of influenza A endonuclease
enzyme using X-ray crystallography, performed using similar
methods as we previously reported,15 identified the compound
5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating
ligand at the active site of the enzyme. Using this information,
we developed a 3-hydroxyquinolin-2(1H)-ones series. Here we
describe the synthesis and structure−activity relationships
associated with various 3-hydroxyquinolin-2(1H)-ones with
regard to their ability to inhibit the endonuclease activity as
measured by a high-throughput fluorescence assay.
Scheme 2. Formation of Compounds 2 and 14 and Use in
Suzuki-Coupling Reactions
a
3-Hydroxyquinolin(1H)-2-one, 1, was prepared as outlined
in Scheme 1 by ring expansion of isatin with (TMS)-
Scheme 1. Synthesis of 3-Hydroxyquinolin-2(1H)-one, 5-, 6-,
7-, and 8-Bromo 3-Hydroxyquinolin-2(1H)-ones, and 5-, 6-,
7-, and 8-(p-Fluorophenyl) 3-Hydroxyquinolin-2(1H)-ones
a
a
Reagents and conditions: (a) NBS, DMF, under Ar; (b) phenyl-
boronic acid (for 12 and 15), Pd(PPh3)4, Na2CO3, dioxane/H2O
(2:1), trimethylboroxine (for 13 and 16), TMSCl, NEt3; Pd(PPh3)4,
Na2CO3, dioxane/H2O (2:1).
4-phenyl-3-hydroxyquinolin-2(1H)-one, 12, and 4-methyl-3-
hydroxyquinolin-2(1H)-one, 13, respectively.
Treatment of 9 with N-bromosuccinimide in DMF gave
exclusively the 4-bromo derivative, 14 (Scheme 2), which
underwent Suzuki-coupling with either phenylboronic acid or
trimethylboroxine to yield 4-phenyl-7-(p-fluorophenyl)-3-hy-
droxyquinolin-2(1H)-one, 15, or 4-methyl-7-(p-fluorophenyl)-
3-hydroxyquinolin-2(1H)-one, 16, respectively.
Each of these 3-hydroxyquinolin-2-(1H)ones were evaluated
as inhibitors of influenza A endonuclease. A high-throughput
96-well plate based assay, similar to those developed by
Kowalinski et al.19 as well as Noble et al.,20 was used to
demonstrate the inhibition of endonuclease cleavage by PAN. A
TaqMan-like oligonucleotide contains a 6-carboxy-fluorescein
(FAM) fluorophore at the 5′-end followed by 19 nucleotides
and a minor groove binding nonfluorescent quencher
(MGBNFQ, Applied Biosystems) at the 3′-end (Figure 1).
a
Reagents and conditions: (a) TMSCHN2 (1.0 mol equiv), EtOH,
TEA under Ar; (b) TMSCHN2 (2.0 mol equiv), EtOH, TEA under
Ar; (c) p-fluorophenylboronic acid, Pd(PPh3)4, Na2CO3, dioxane/
H2O (2:1); (d) BBr3 in CH2Cl2, 0° to r.t.
diazomethane, followed by treatment of the resulting 3-
methoxyquinolin-2(1H)-one with BBr3 as previously de-
scribed.16 Employing this methodology, several bromo 3-
hydroxyquinolin(1H)-2-one derivatives, 3−6, were prepared
using the appropriately substituted bromo isatin and with
(TMS)diazomethane. As illustrated in Scheme 1, treatment of
the 5-, 6-, 7-, and 8-bromo-3-methoxyquinolin-2(1H)-one
intermediates with excess BBr3 in dichloromethane provided
3−6 (Scheme 1). Suzuki-coupling of each of the brominated 3-
methoxyquinolin-2-(1H)-ones with p-fluorophenylboronic acid
as outlined in Scheme 1 provided the p-fluorophenyl derivatives
7−10. 4-Phenyl-3-hydroxyquinolin-2(1H)-one, 12, and 4-
methyl-3-hydroxyquinolin-2(1H)-one, 13, have been previously
synthesized.16,17 In this study, we synthesized 4-bromo-3-
hydroxyquinolin-2(1H)-one, 2, from 1 as previously de-
scribed.18 and used this as an intermediated for the preparation
of the 4-substituted 3-hydroxyquinolin-2(1H)-ones, 11−13.
Treatment of 2 with p-fluorophenylboronic acid as illustrated
in Scheme 2 provided 4-(p-fluorophenyl)-3-hydroxy-quinolin-
2(1H)-one, 11. Under similar reaction conditions, treatment of
2 with either phenylboronic acid or trimethylboroxine provided
Figure 1. (A) Diagram showing the cleavage of nucleic acid probe by
PAN. After cleavage the FAM fluorophore fluoresces when excited by
light with a wavelength of 488 nm. (B) Change in fluorescence after 1
h measured for compound 9 during a titration, IC50 of 0.5 μM.
When excited, MGBNFQ quenches the fluorescence of FAM
via fluorescence resonance energy transfer. Upon cleavage of
the oligonucleotide, the quencher is no longer coupled to the
fluorophore, and therefore, FAM fluoresces. The Z′ score for
this assay as described in the Supporting Information was a very
B
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ACS Medicinal Chemistry Letters
Letter
acceptable 0.87. This assay was used to determine the
inhibitory activity of the compounds.
The results of these assays are summarized in Table 1. These
data indicate that the presence of a p-fluorophenyl substitutent
Table 1. Inhibition Assay of Influenza A Endonuclease
compound
IC50 (μM)
1
3-OH-quinolin-2-one (3HQ)
4-Br-(3HQ)
24
2
53
3
5-Br-(3HQ)
12
4
6-Br-(3HQ)
7.4
7.6
11
5
7-Br-(3HQ)
6
8-Br-(3HQ)
7
5-(p-FC6H4)-(3HQ)
6-(p-FC6H4)-(3HQ)
7-(p-FC6H4)-(3HQ)
8-(p-FC6H4)-(3HQ)
4-(p-FC6H4)-(3HQ)
4-C6H5-(3HQ)
3.3
0.5
0.5
4.7
11
8
9
10
11
12
13
14
15
16
>20
>100
1.1
2.0
13
4-CH3-(3HQ)
4-Br-7-(p-FC6H4)-(3HQ)
4-Phenyl-7-(p-FC6H4)-(3HQ)
4-CH3-7-(p-FC6H4)-(3HQ)
at either the 6- or 7-position of 3-hydroxyquinolin-2(1H)-one is
associated with a significant enhancement in enzyme inhibition
relative to other positional isomers, as well as the unsubstituted
parent compound, 1.
Substitution at the 4- and 8-positions was frequently
associated with reduced activity. Among the bromo-substituted
3-hydroxyquinolin-2(1H)-ones, the 6- and 7-positional isomers
were the more active. A similar trend was observed for the p-
fluorophenyl substituted 3-hydroxyquinolin-2(1H)-ones. The
presence of either a 4-methyl or a 4-phenyl substituent attached
to 3-hydroxyquinolin-2(1H)-one did not enhance enzyme
inhibition relative to the unsubstituted derivative. In the case
of 7-(p-fluorophenyl)-3-hydroxyquinolin-2(1H)-one, the pres-
ence of either a 4-methyl or 4-phenyl substituent was
detrimental to its ability to inhibit endonuclease activity.
The X-ray crystal structure of influenza A endonuclease with
9 was obtained by soaking unliganded endonuclease crystals
with 10 mM 9 (Figure 2). The crystal structure clearly shows 9
chelating to the two active site metals. Additionally, the core
scaffold is coordinated by a hydrogen bond between the
hydroxyl at the 3-position and the ε nitrogen of Lys134. The
protonated nitrogen of the core scaffold also coordinates to a
water molecule chelating to Mn2. Importantly, unlike what has
been determined for known endonuclease inhibitors, the ring
system binds at 50° tilt toward His41.9,19 The binding angle
increases π−π stacking interactions with His41 and allows for
development of additional interactions as the molecule is
extended into the surrounding pockets. The 7-p-fluorophenyl
extends the molecule into a pocket formed by Ala20, Met21,
Tyr24, Asp26, Lys34, and Ile38. A hydrophobic network is
formed between the fluorophenyl and Ala20, Tyr24, and Ile38.
These data are consistent with the structure−activity data
associated with the inhibition of influenza A endonuclease. The
data indicate that these compounds bind through bimetal
chelation at the active site. The presence of substituents at
either the 4- or 8-position could interfere with the establish-
ment of a favorable interaction with these two metals. Several 3-
hydroquinolin-2(1H)ones were reported as inhibitors of D-
Figure 2. Binding of 9 at the endonuclease active site. Ligand is shown
in yellow, while the receptor is purple. Chelation is depicted as black
dashes, hydrogen bonds are depicted as blue dashes, and strong
hydrophobic interactions are gray dashes. The blue mesh around the
ligand is calculated from a 4.5σ omit map. The figure was generated
using PyMol (www.pymol.org). PDB code: 4KIL.
amino acid oxidase (DAAO).16 As the binding modes for
DAAO inhibitors do not involve metal chelation, it is not
surprising that the structure−activity relationships for DAAO
inhibition is unique.
A series of 3-hydroxyquinolin-2-(1H)-ones was also recently
reported as selective inhibitors of HIV-1 reverse transcriptase
associated RNase H activity.21 This series of compounds
consisted of 4-carboxylic acids, 4-ethylcarboxylates, and 4-
carboxamides. The formation of magnesium chelation was
examined in this study. The authors noted that the ability of
their three oxygen pharmacophore to chelate both metal
cofactors within the active site of the enzyme was consistent
with their results. The N-hexylamide and various N-
benzylamides were among the more active compounds. The
authors reported that, with these 4-substituted 3-hydroxyqui-
nolin-2(1H)-ones, significant cytotoxicity was observed in cell
culture. In light of these results, we did evaluate the relative
cytotoxic activity of 3−6 toward Madin−Darby canine kidney
(MDCK) cells and human embryonic kidney 293 (HEK 293)
cells using the MTT-microtiter plate tetrazolium cytotoxicity
assay. The assays were performed as previously described.22
The IC50 values for all of these compounds were >10 μM,
which was the highest concentration tested.
The results suggest that 6- and 7-substituted 3-hydroxyqui-
nolin-2(1H)-ones could provide a useful scaffold for the
development of endonuclease inhibitors that could block the
cap snatching associated with influenza A replication. Studies
C
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ACS Medicinal Chemistry Letters
Letter
an Avian Influenza Polymerase PAN Reveals an Endonuclease Active
are in progress to design and discover more potent
endonuclease inhibitors using soakable endonuclease crystals
with varied 6- and 7- substituted 3-hydroxyquinolin-(1H)-ones.
Site. Nature 2009, 458, 909−914.
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(7) Dias, A.; Bouvier, D.; Crepin, T.; McCarthy, A. A.; Hart, D. J.;
Baudin, F.; Cusack, S.; Ruigrok, R. W. H. The Cap-Snatching
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ASSOCIATED CONTENT
* Supporting Information
■
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Basis for Development of Influenza Virus Inhibitors Targeting the PA
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Synthetic methods and spectral characterization of compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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Cap(m7GpppXm)-Dependent Influenza Virion Endonuclease Cleaves
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AUTHOR INFORMATION
Corresponding Author
*(E.J.L.) Tel: (848)-445-2674. E-mail: elavoie@pharmacy.
rutgers.edu. (E.A.) Tel: (732)-235-5323. E-mail: arnold@
cabm.rutgers.edu.
■
(10) Tomassini, J.; Selnick, H.; Davies, M. E.; Armstrong, M. E.;
Bladwin, J.; Bourgeois, M.; Hastings, J.; Hazuda, D.; Lewis, J.;
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A.; Wolfe, A. Inhibition of Cap (m7GpppXm)-Dependent Endonu-
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Notes
The authors J.D.B., K.D., E.A., and E.J.L. are cofounders of
Prodaptics Pharmaceuticals, Inc., which has licensed the
technology associated with these compounds from Rutgers
University
The authors declare the following competing financial
interest(s): Dr. Joseph Bauman, Dr. Kalayan Das, Dr. Eddy
Arnold, and Dr. Edmond LaVoie are co-founders of Prodaptics
Pharmaceuticals, Inc. Using a soakable crystal developed at
CABM, we are working on developing small molecule
inhibitors of influenza endonuclease A. Successful development
of a clinically useful agent would be of financial benefit to the
founders.
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Inhibitor of Influenza Virus. Tetrahedron Lett. 1995, 36, 2009−2012.
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ACKNOWLEDGMENTS
■
We thank Angela Liu from the Department of Pharmacology at
UMDNJ-Robert Wood John Medical School for performing
the cytotoxicity studies. The Bruker Avance III 400 MHz NMR
spectrometer used for this study was purchased with funds from
NCRR Grant No. 1S10RR23698-1A1. Mass spectrometry was
provided by the Washington University Mass Spectrometry
Resource with support from the NIH National Center for
Research Resources Grant No. P41RR0954. We thank the
laboratories of Ann Stock and Gaetano Montelione for access
to equipment used in this study. X-ray data collection was
conducted at the Cornell High Energy Synchrotron Source
(CHESS). CHESS is supported by the NSF & NIH/NIGMS
via NSF award DMR-0225180, and the MacCHESS resource is
supported by NIH/NCRR award RR-01646.
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