Fragment-Based Identification of Influenza Endonuclease Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00628

The influenza virus is responsible for millions of cases of severe illness annually. Yearly variance in the effectiveness of vaccination, coupled with emerging drug resistance, necessitates the development of new drugs to treat influenza infections. One a

Full text of DOI:10.1021/acs.jmedchem.6b00628

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Article
Fragment-based Identification of Influenza Endonuclease Inhibitors
Cy V. Credille, Yao Chen, and Seth M. Cohen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00628 • Publication Date (Web): 11 Jun 2016
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Fragmentꢀbased Identification of Influenza
Endonuclease Inhibitors
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Cy V. Credille,1 Yao Chen,1 and Seth M. Cohen1*
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA
92093, United States
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Abstract: The influenza virus is responsible for millions of cases of severe illness annually.
Yearly variance in the effectiveness of vaccination, coupled with emerging drugꢀresistance,
necessitates the development of new drugs to treat influenza infections. One attractive target is
the RNAꢀdependent RNA polymerase PA subunit. Herein we report the development of
inhibitors of influenza PA endonuclease derived from lead compounds identified from a metalꢀ
binding pharmacophore (MBP) library screen. Pyromeconic acid and derivatives thereof were
found to be potent inhibitors of endonuclease. Guided by modeling and previously reported
structural data, several sublibraries of molecules were elaborated from the MBP hits. Structureꢀ
activity relationships were established, and more potent molecules were designed and
synthesized using fragment growth and fragment merging strategies. This approach ultimately
resulted in the development of a lead compound with an IC50 value of 14 nM, which displayed an
EC50 value of 2.1 µM against H1N1 influenza virus in MDCK cells.
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Introduction
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The influenza virus is responsible for annual seasonal epidemics, resulting in between 3 ꢀ 5
million yearly cases of severe illness and an estimated 250,000 to 500,000 deaths worldwide.1
The last century alone has seen the advent of four influenza pandemics, each resulting in millions
of deaths.2 While vaccinations are a reasonable prophylactic for healthy adults, they must be reꢀ
administered annually and are markedly less effective for individuals with compromised
immunity or similar highꢀrisk medical conditions. In addition, efficacy of these vaccines is
heavily dependent on correctly predicting the predominant infectious strains for any given year,
and incorrect predictions can render vaccination less than 25% effective.3 Existing drugs, such
as zanamivir (GlaxoSmithKline) and oseltamivir (Roche), which target viral neuraminidase, can
be useful in treating influenza infections, but must be administered within 1ꢀ2 days of infection
to be effective. These therapeutics also suffer from undesirable side effects, including unusual
neurologic or psychiatric events such as delirium, hallucinations, confusion, and abnormal
behavior, primarily in children.4,5,6 M2 ion channel blockers such as rimantadine (Sun Pharma)
and amantadine (Endo) were previously effective at inhibiting viral replication; however, 100%
of seasonal H3N2 and 2009 pandemic H1N1 influenza strains now show resistance to these
drugs.7,8 Considering this, there is an urgent need for the development of new drugs to prevent
and treat influenza infection.
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The influenza virus is a lipidꢀenveloped, negativeꢀsense, singleꢀstrand RNA virus. The
viral genome is divided into 8 distinct genomic segments, each encoding one or two of the 11
total viral proteins.9 This segmented genome allows for the exchange of segments between
different viruses in infected host cells and, coupled with the low fidelity of the viral RNA
polymerase, explains the high rates of drugꢀresistance and antigenic shift seen in influenza
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viruses.4,10 Each RNA segment is packaged in complex with a single heterotrimeric RNA
dependent RNA polymerase.11 The polymerase complex is comprised of three distinct subunits
(PA, PB1, PB2) and is responsible for both transcription and replication of the viral genome.
However, the complex is unable to synthesize the 5′ꢀmRNA cap necessary for translation by
eukaryotic hostꢀcell translation machinery. To overcome this limitation, the polymerase hijacks
a mature 5′ꢀcap from host cell preꢀmRNA. This ‘capꢀsnatching’ mechanism is accomplished by
the polymerase B2 subunit tightly binding the modified 5′ꢀguanine nucleotide with subsequent
cleavage 10ꢀ13 nucleotides downstream by the Nꢀterminal endonuclease portion of the PA
subunit.12,13 The sequestered, capped RNA segment is then used as a primer for viral mRNA
synthesis, and the resulting hybrid RNA is translated by the host cell.14
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The viral polymerase complex is an attractive target for new antiviral therapies. It is
highly conserved across all influenza strains and subtypes, and inhibitors should therefore have
broad efficacy against multiple serotypes. Of particular interest, the capꢀsnatching mechanism is
essential to the virus lifecycle, is conserved in all members of the influenza virus family, and has
no human analogue.4 Capꢀsnatching, and subsequent viral replication, has been shown to be
inhibited by inactivating one of two different domains of the viral polymerase complex: either
inhibiting the 5′ꢀmRNA cap binding site on the PB2 subunit,15,16,17 or inhibiting the Nꢀterminal
endonuclease portion of the PA subunit.4,18,19
The Nꢀterminal domain of the PA subunit contains the endonuclease active site.
Crystallographic and biochemical studies have shown the endonuclease to contain a dinuclear
metal active site, employing either two Mn2+ or Mg2+ cations (Figure 1).13 The structure of the
endonuclease domain closely resembles type II restriction endonucleases,13 and it has been
shown that coordinating the metal centers of the endonuclease active site effectively inhibits
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endonuclease activity. There are currently no FDA approved endonuclease inhibitors, but
several classes of inhibitors have been reported in recent literature. These include diketo
acids,20,21 hydroxamic acids,22 flutimide derivitives,23 green tea catechins,24,25
dihydroxypyrimidinones,26 3,2ꢀhydroxypyridinones,27 and others. Most of these were identified
by experimental or computational highꢀthroughput screening (HTS) of large libraries, and
produced only modest inhibitors in spite of great synthetic effort. More recently, some very
potent in vitro inhibitors have been identified.21,28,29
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Figure 1. Structural model of the influenza RNAꢀdependent RNA polymerase PA subunit
(PDB: 4M5Q). The endonuclease active site employs two divalent metal cations to facilitate the
hydrolytic cleavage of the phosphodiester backbone of nucleic acids. Key active site residues
and binding pockets are highlighted. Pocket 2 is obscured by residue Tyr24. Divalent metal
cations are shown as orange spheres with the bridging hydroxide anion shown as a red sphere.
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In an effort to efficiently identify novel, potent inhibitors of influenza PA endonuclease,
we carried out a fragmentꢀbased drug discovery (FBDD) campaign, using a designer metalꢀ
binding pharmacophore (MBP) library. This library is an expansion of our prior report,30 and
consists of ~300 fragment molecules designed to interact with metal ions found in
metalloenzyme active sites. From this screening, pyromeconic acid was identified as a ligandꢀ
efficient scaffold for inhibitor development. Guided by modeling and SAR identified in the
initial library screen, a modest sublibrary of molecules was elaborated from the MBP hits. Our
metalloenzymeꢀfocused FBDD approach ultimately leads to molecules with potent in vitro
inhibitory activity (IC50 <50 nM) against viral PA endonuclease. The most potent of these, 1ꢀ(4ꢀ
(1Hꢀtetrazolꢀ5ꢀyl)phenyl)ꢀ2ꢀ(((4ꢀchlorophenyl)(methyl)amino)methyl)ꢀ5ꢀhydroxypyridinꢀ4(1H)ꢀ
one (71), was found to inhibit endonuclease activity with an IC50 of 14 nM in enzymatic assays.
Compound 71 further showed good antiviral activity against H1N1 influenza A virus in Marvinꢀ
Darby canine kidney (MDCK) cells with an EC50 value of 2.1 µM and a CC50 value of 280 µM.
These findings demonstrate that a metalꢀcentric FBDD approach can result in a rapid and
efficient identification of leadꢀlike molecules, especially when compared to FBDD using
unbiased libraries or highꢀthroughput screening.
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Results
MBP Library Screening. Fragments in the MBP library were initially screened for
endonuclease inhibition against the PA subunit of the influenza polymerase complex. The screen
was performed using a FRETꢀlabeled DNA oligonucleotide substrate based on previously
reported protocols with some minor modifications (Supporting Information).13,28 Compounds
that exhibited >80% inhibition at a concentration of 200 µM were reꢀevaluated at a concentration
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of 50 µM. Doseꢀresponse curves were compiled and IC50 values determined for fragments
exhibiting >50% inhibition at 50 µM. This screen identified pyromeconic acid (1) as a moderate
inhibitor of endonuclease activity (IC50 = 22.5 µM; LE = 0.79), and unveiled important structureꢀ
activity trends associated with simple modifications to the hydroxypyrone ring (Table 1). A
proposed mode of metal coordination for 1 is shown in Figure 2. This model is based on
superposition of the donorꢀatoms in 1 with crystallographic water molecules present in Xꢀray
structures of uninhibited influenza PA endonuclease and superposition of the pyrone ring with
other similar cyclic metalꢀbinding inhibitors that have been crystallographically validated against
the endonuclease.28
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It was found that the addition of a methyl group to the 6ꢀposition of the pyromeconic acid
ring (2, allomaltol) had little effect on the potency of the fragment, while the addition of a methyl
group to the 2ꢀposition (3, maltol) effectively abolished activity. This is consistent with other
endonuclease inhibitors that employ a similar MBP motif, which demonstrate that addition of
substituents alpha to the hydroxyl donor atom reduces potency.26,27 Addition of hydrophilic
substituents in the 6ꢀposition (4, 5) were well tolerated. The addition of a carboxylic acid moiety
to the 5ꢀposition, as in compound 6, afforded an increase in potency, as did a methyl amide
derivative. Other derivatives of 2 (6ꢀposition substituent) gave rise to modest improvements in
potency (8, 9).
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Table 1. Select MBP fragments that displayed potent inhibition against influenza PA
endonuclease. pIC50 is defined as pIC50 = ꢀlog(IC50) and is included to allow a linear comparison
between IC50 values. Ligand efficiency (LE) provides a measure of binding energy per nonꢀ
hydrogen atom in the fragment molecule.31
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Figure 2. Proposed binding of pyromeconic acid (1) to the PA subunit active site. Ring
positions 5ꢀ and 6ꢀ were deemed amenable to further derivatization for targeting hydrophobic
pocket 2 and key residues Tyr24 and Arg84. Derivatization at the 1ꢀposition was proposed to
probe interactions with pocket 4.
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Conversion of the pyrone ring to an Nꢀmethylpyridinone resulted in a marked increase in
potency, as seen in 9 and 10. Pyridinones, as compared to pyrones, have more aromatic
character and greater electron density on the donating oxygen atoms.32 This change in the
electronic structure of the ligand typically results in greater ligand basicity, which can lead to
better interactions with hard Lewis acidic metal centers, such as the Mg2+ or Mn2+ found in the
endonuclease active site.32 Based on this preliminary SAR, synthetically feasible elaborations
were designed using pyromeconic acid derivatives as an MBP core. Substitutions at the 1ꢀ, 5ꢀ,
and 6ꢀpositions were deemed most accessible, and were evaluated independently.
5-Position Derivatives. Based on the SAR outlined in Table 1, initial efforts focused on
exploring amide derivatives of compound 6/7. Guided by modeling and prior crystallographic
evidence,18,28 it was proposed that aryl substituents at the 5ꢀposition could favorably interact with
residues Tyr24 or Arg84, or make favorable hydrophobic interactions with pocket 2 (Figure 1).
A sublibrary of 11 aryl amides derived from 6 were synthesized and evaluated for inhibitory
activity (Table 2). While the methyl amide 7 was equipotent to the parent fragment 6, all aryl
amide derivatives were found to be less active. This trend persisted regardless of additional
functionalization of the aryl ring or the introduction of methylene spacers between the amide and
aryl ring. The only exception to this trend was compound 20, a tryptamine amide, but this
derivative was not viewed as an attractive lead based on the substantial increase in molecular
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weight for little gain in activity. The resulting SAR suggested that 5ꢀposition amide
functionalization was not a productive route to more active inhibitors.
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6-Position Derivatives. Initial SAR (8, 9) indicated that hydrophilic substituents at the 6ꢀ
position were well tolerated. To continue probing interactions with pocket 2 (Figure 2) a series
of 6ꢀaminomethyl derivatives were synthesized. Starting with commercially available kojic acid,
derivatives of the 6ꢀposition could be readily prepared with amine, amide, and sulfonamide
linkages. Amine, amide, and sulfonamide derivatives were between 3ꢀ to 18ꢀfold more active
than kojic acid (8), with compound 31 being the most potent of the set. Aryl amine and ether, as
well as benzyl amine derivatives displayed similar activity, with tertiary amine derivatives being
slightly more potent than secondary amine derivatives (compound 26 vs. 27; 32 vs. 34).
Functionalization of the aryl ring substituent in some cases provided added potency; however,
the introduction of a pyridine substituent drastically decreased activity. Ultimately, the SAR at
the 6ꢀposition was relatively flat among aryl derivatives, but did indicate some gains could be
made, with compound 35 identified as the most potent derivative of this set with an IC50 value of
0.94 ± 0.08 µM.
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Table 2. Inhibition values of various 5ꢀ and 6ꢀposition derivatives of pyromeconic acid.
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Pyridinone Derivatives. Conversion of the pyrone ring to a pyridinone ring not only
increases ligand basicity (and hence metal binding), but also affords a chemical handle for facile
exploration of the large pocket located above the metal center active site of endonuclease (Figure
2). To probe this pocket, a sublibrary of Nꢀsubstituted pyridinones was synthesized and screened
against endonuclease (Table 3). It was found that both Nꢀaryl and Nꢀcycloalkyl pyridinones
(compounds 44 and 47) were significantly more active than the parent fragment compound 10.
Simple extension of the aryl substituent (compounds 46 and 50) did not improve activity.
Interestingly, compounds 45 and 48 showed a significant loss in activity, consistent with the
previously observed negative effect of substituents at the 2ꢀposition (compound 3, Table 1).
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In addition to elongation of the substituent, a series of more polar, saturated, heterocyclic
derivatives of 47 were prepared. As detailed in Table 3, all of these derivatives showed reduced
activity, with modifications to the cyclohexyl moiety not being well tolerated. The simple
phenyl ring in compound 44 was deemed more suitable for further synthetic elaboration.
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Table 3. Inhibition values of various Nꢀfunctionalized hydroxypyridinones. (a) A sublibrary of
Nꢀfunctionalized pyridinones designed to probe shape and lipophilicity of endonuclease binding
pocket. (b) Saturated Nꢀfunctionalized hydroxypyridinones. Hydrophilic modifications of this
ring system showed detrimental effects on activity.
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A series of derivatives were synthesized exploring substituents at either the 3′ or 4′
positions of the Nꢀinserted phenyl ring (Table 4). In general, 3′ modifications yielded a flat
SAR, with only small changes in activity. The SAR around the 4′ derivatives was more varied,
and revealed that the addition of an aryl ether (compound 60) or carboxylic acid (compound 62)
both afforded increases in activity. While several other polar 4′ derivatives resulted in a reduced
potency (64, 65, 66), the addition of a tetrazole group to the 4′ position (63) resulted in a
substantial increase in potency. This finding is consistent with previous reports that phenyl
tetrazoles make very favorable interactions with pocket 4.26ꢀ28 Compound 63 was identified as
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the most potent derivative of this series with an IC50 value of 36 ± 7 nM against PA
endonuclease.
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Table 4. Inhibition values and SAR of 3′ and 4′ derivatives of 5ꢀhydroxyꢀ2ꢀmethylꢀ1ꢀ
phenylpyridinꢀ4(1H)ꢀone. (a) Derivatives of the 3′ position yielded flat SAR with effectively no
gains in activity. (b) Derivatives of the 4′ position revealed phenyl ether and carboxylic acid
groups as active derivatives. Other carboxylic acid isosteres were examined, and a tetrazole
moiety (63) was identified as the optimal derivative at this position.
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Fragment Merging. In an effort to maximize potency from derivatives of pyromeconic
acid (1), a fragmentꢀmerging strategy was pursued. Computational modeling suggested that
derivatives at the 5ꢀ and 6ꢀposition were likely interacting with pocket 2 in the endonuclease
active site, and that Nꢀsubstituted derivatives at the 1ꢀposition were interacting with pocket 4. It
was decided to merge the most potent 5ꢀ or 6ꢀposition derivatives with the most potent Nꢀaryl
derivative of the 1ꢀposition to interact with both pockets simultaneously. Because amide
derivatization of compound 6 showed effectively no improvement to potency, incorporation of a
5ꢀposition carboxylic acid to Nꢀsubstituted compounds 44, 60, and 63 was pursued. Ethyl esters
67, 68, and 69 surprisingly showed markedly decreased potency after the addition of the
carboxylate group at the 5ꢀposition. Base catalyzed hydrolysis of these compounds to produce
the free carboxylic acid resulted in complete degradation of these molecules; however, 70 was
synthesized via an alternate route. Disappointingly, 70 showed no improvement over the parent
compound 44. Due to the poor inhibition shown by 70, further efforts towards 5ꢀposition
carboxylic acids were abandoned.
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Extensive SAR of the 6ꢀposition revealed that aryl amine and sulfonamide derivatives
were the most potent elaborations at this position, likely due to favorable hydrophobic
interactions with pocket 2 and possible πꢀstacking interactions with Tyr24. As detailed in Table
5, the addition of a 4ꢀchloroꢀNꢀmethyl aniline substituent to the already potent 63 resulted in a
oneꢀhalf log increase in potency from an IC50 value of 36±7 nM to 14±3 nM for compound 71.
While fragment merging did result in an overall more potent molecule, the fact that the observed
potency increase was not more additive suggests either a conformational change in binding mode
as compared to the original fragments or a possible overlap in binding pockets between the two
substituents.
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Table 5. Inhibition values of fragment merged inhibitors.
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Docking. In lieu of a coꢀcrystal structure of 71 with endonuclease (efforts are currently
underway), docking studies were performed to gain insight about the probable binding mode of
71. Previously reported Xꢀray crystal structures of inhibitors with similar MBPs bound to the
endonuclease active site were used as a starting point for these docking experiments.28 Using the
Molecular Operating Environment (MOE) software, constrained docking and minimization
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studies were performed starting from a reported endonuclease crystal structure with a bound
hydroxypyridinoneꢀbased inhibitor (PDB: 4M4Q, Figure 3). The bound inhibitor was removed
from the structure and replaced with compound 71, utilizing the position of the metalꢀ
coordinating donor atoms of the coꢀcrystallized inhibitor as an initial guide for the binding mode
of 71. Constrained minimizations were then performed on this model in which the metalꢀbinding
oxygen atoms of 71 were fixed in optimal geometries based on several parameters, primarily
previous crystal structures of inhibitors with similar ligand donor atom identity and
geometry.26,27 These constrained simulations ensure that a reasonable metal binding geometry is
maintained, as MMꢀdocking simulations do not appropriately account for metalꢀligand
interactions.33,34 Flexible receptor modeling (induced fit) was employed in which active site
residue side chains proximal to the inhibitor molecule were allowed to move, while residue
backbones were more constrained. In the simulation the coordinating phenolic oxygen atom on
inhibitor 71 was deprotonated, and the charge on each metal center was assigned as 2+ (i.e.
Mn2+). Two water molecules coordinated to one of the Mn2+ centers in the template structure
(PDB: 4M4Q, Mn302) were retained in the simulation and their positions were fixed relative to
the metal center.
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Analysis showed that the MBP chelates one of the activeꢀsite metal ions, with the
carbonyl oxygen atom replacing the bridging water molecule, and the hydroxyl oxygen atom
binding one metal center and forming a hydrogen bond with Lys134. The phenylaminomethyl
moiety at the 6ꢀposition was found to be too short to allow for πꢀstacking with Tyr24, but
allowed for hydrophobic interactions with pocket 2. The 4ꢀchloro substituent is also positioned
to allow for halogen bonding with the backbone carbonyl of Arg82. The Nꢀphenyltetrazole
moiety at the 1ꢀposition fits well into pocket 4, with the aryl ring making hydrophobic
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interactions with the hydrophobic wall of the pocket and the tetrazole making favorable contacts
with the basic residues found at the end of the pocket (Figure 3). It was found that the 4'
tetrazole was able to hydrogen bond with two nitrogen atoms from the guanidine moiety of
Arg124 and simultaneously hydrogen bond with the terminal nitrogen of Lys34. The larger size
and greater number of available hydrogen bond acceptors in the tetrazole ring, when compared to
a carboxylic acid group, may account for the observed difference in activity between the
compounds containing these isosteres (62 vs. 63).
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Figure 3. Docking analysis of 71 in influenza PA endonuclease (PDB: 4M4Q). (a) Docked
structure of 71 bound to endonuclease. Strong hydrophobic interactions were found between the
6ꢀposition phenylaminomethyl moiety and hydrophobic pocket 2, as well as halogen bonding
with Arg82. The Nꢀphenyltetrazole moiety was found to hydrogen bond simultaneously to
Arg124 and Lys34. (b) Ligand interaction diagram detailing ligand/protein/solvent interactions
in rendered in 2ꢀdimensions. Interactions between the ligand and protein active site are
displayed as colored dotted lines; coordination bonds in purple, hydrogen bonds and πꢀπ
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interactions in green, and halogen bonds in blue. Blue halos indicate a measure of ligand
solventꢀexposure, with larger halos indicating greater exposure.
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Cytotoxicity and Antiviral Activity. Cytotoxicity and antiviral activity studies were
performed in MDCK cells (Supporting Information). MDCK cells were incubated for a period
of 48 hours in the presence of varying concentrations of compound 71 or the commercially
available diketo acid compound 89 (Lꢀ742,001; Supporting Information), as a control.
Compound 89 is one of the most potent inhibitors of influenza PA endonuclease reported, both in
cellular and protein based assays.19, 35 Cell viability was then determined using CellTiterꢀGlo
luminescent assay (Promega). Antiviral activity was determined by exposing MDCK cells to a
lethal challenge of influenza virus, in the presence of varying concentrations of inhibitor.
Influenza A virus obtained from the American Tissue Culture Collection (H1N1, ATCC VRꢀ
1737) was used to infect healthy MDCK cells, and inhibitor molecules were coꢀadministered in
varying concentrations with the viral challenge. The cells were then incubated for 48 hours and
analyzed for viability via luminescence assay. Compound 71 was found to have an EC50 value
of 2.1 µM and a CC50 value of 280 µM (Figure 4). Both of these values were comparable to the
positive control (89), with 71 being 4ꢀ to 5ꢀfold more potent than the control in the viral
challenge assay.
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Figure 4. Potency and cytotoxicity analysis of 71 and the reported diketo acid inhibitor 89 in
MDCK cell lines. Cytotoxicity was determined by incubating cells in the presence of inhibitors
for 48 h, followed by evaluating cell viability. Potency was determined by coꢀadministration of
inhibitor and a lethal challenge of virus particles, followed by a 48 h incubation and subsequent
analysis of cell viability.
Discussion and Conclusions
Of the reported binding pockets in the influenza PA endonuclease active site, many
inhibitors that have been structurally characterized in the active site interact with pocket 4. This
common interaction space is due to the open nature of pocket 4 (Figure 1, Figure 3), coupled
with its proximity to the catalytic metal ions that many inhibitors also interact with.18,25,28
A
number of reported inhibitors interact with pocket 3 for similar reasons.26,36 Fewer reported
inhibitors have been shown to interact with pockets 1 or 2, with the only examples being
dioxobutanoic acid derivatives.18 Examples of inhibitors with good interactions with pockets 3
and 4 include compounds containing a phenyl tetrazole moiety for pocket 4 (Figure 5).26,28
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Arnold and LaVoie initially identified a 5ꢀchloroꢀ3,2ꢀhydroxypyridinone as a fragment hit
against endonuclease using a crystallographyꢀbased fragment screening of 775 compounds.27
Fragment elaboration resulted in the development of Parhi-6 (Figure 5), which contains the
aforementioned phenyl tetrazole moiety and displays a reported IC50 value of 368±41 nM. In an
effort to develop even more potent inhibitors of endonuclease, additional efforts lead to
additional MBP heterocycle analogues in combination with a phenyl tetrazole substituent (Figure
5, Figure 6).
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Figure 5. Chemical structure and inhibitory activity of several reported influenza endonuclease
inhibitors that contain a phenyl tetrazole moiety. Reported IC50 values for each compound are
provided. The MBP portion of each molecule is highlighted in blue. Compounds were initially
reported by Parhi et al.27 and Sagong et al.26
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Figure 6. Structural comparison of several phenyl tetrazoleꢀcontaining influenza endonuclease
inhibitors. Left: Crystal structure of Sagongꢀ11 (PDB: 4W9S). Middle: Crystal structure of a
more elaborated molecule based on Parhi-6 (PDB: 4M4Q). Right: Docking analysis of 71
(PDB: 4M4Q). Crystal structures of phenyl tetrazoleꢀcontaining inhibitors show the binding
orientation the phenyl tetrazole moiety to be conserved. Docking analysis of 71 predicts a very
similar binding mode for this moiety to that observed in crystallographically validated inhibitors.
The MPB scaffolds and elaborated inhibitors shown in Figure 5 were the result of a
‘scaffold hopping’ campaign, which is common in inhibitor development.37 Successful scaffold
hopping requires the identification of more potent, yet functionalizable, core molecules. As
such, the FBDD approach reported here was able to further advance this strategy, using new
MBP fragments, resulting in compound 63 that exhibits improved activity over all of these other
derivatives shown in Figure 5. Therefore, the MBPꢀbased FBDD approach facilitates the
identification of potent core molecules and can allow for more effective and rapid scaffold
hopping, which also serves to further validate the pharmacophore model and SAR observed in
these prior studies.26,28 The MBP library used here helps compliment the previous screening
efforts by exploring a broader chemical space of metalꢀbinding functionality, rapidly identifying
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novel MBPs, and providing significant improvements in activity. Furthermore, by examining
other fragment sublibraries, a ‘fragment growth’ strategy was applied here, as demonstrated by
compound 71, which led to even greater enhancements in activity by exploiting lessꢀtargeted
active site pockets (pocket 2).
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Employing a FBDD strategy, using a metalloenzymeꢀtargeted MBP library, pyromeconic
acid (1) and similar fragments were identified as active inhibitors against the influenza PA
endonuclease. Prior work in the field, along with modeling, allowed for the generation of a
SAR, leading to the synthesis of several pyrone and pyridinone derivatives based on the initial
fragment hits. Ultimately, five small molecules with in vitro IC50 values of <200 nM, and two
with IC50 values of <50 nM, were obtained. The most active compound (71) was found to inhibit
endonuclease activity with an IC50 of 14 nM. Compound 71 showed minimal cytotoxicity with a
CC50 of 280 µM and could rescue cells from a lethal challenge of H1N1 influenza virus with a
viral EC50 of 2.1 µM. As opposed to other reported screening methods, which generally involve
much larger libraries and more costly methods (e.g. crystallography), the FBDD approach
described here readily produced ligand efficient fragments that targeted the metal active site.
This targeted library screening method shows the potential to be a more timeꢀ and costꢀeffective
route than those employed by traditional FBDD and HTS campaigns, yielding a similar number
of active hits, and ultimately resulting in the development of effective lead molecules.
Experimental Section
General Experimental Details
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All reagents and solvents were obtained from commercial sources and used without
further purification. All reactions, unless otherwise stated, were performed under a nitrogen
atmosphere. Reactions were monitored using either aluminum or glassꢀbacked silica TLC plates
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impregnated with a fluorescent indicator, absorbing at 254 nm.
Silica gel column
chromatography was performed on a CombiFlash Rf Teledyne ISCO system using hexane, ethyl
acetate, methylene chloride, or methanol as eluent. Reverse phase column chromatography (C18
column) was performed on the same instrument using 0.1% formic acid in methanol, acetonitrile,
or water as eluent. Separations were monitored by mass spectrometry via a Teledyne ISCO RF+
PurIon ESIꢀMS or APCIꢀMS detector with 1 Da resolution. The purity of all compounds used in
1
assays was determined to be ≥95% by H NMR spectroscopy and confirmed by highꢀresolution
mass spectrometry (HRMS) experiments using an Agilent 6230 AccurateꢀMass LCꢀTOFMS
located in the U.C. San Diego Molecular Mass Spectrometry Facility (MMSF). Standard
resolution MS was performed either at the aforementioned MMSF or Teledyne ISCO RF+ PurIon
MS. Microwave reactions were performed using a CEM Discover series Sꢀclass microwave
reactor in pressureꢀsealed vessels. Docking simulations were performed using MOE version
2014.0901. Flexible receptor modeling (induced fit) was employed in these simulations, and
metalꢀbinding atoms were fixed.
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Scheme 1a
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aReagents and conditions: (a) triethyl orthoformate, H2SO4 (catalytic), rt, 24 h; (b) 4ꢀnitrophenyl
trifluoroacetate, pyridine, rt, 18 h; (c) ethyl or tertꢀbutyl acetoacetate, NaH, reflux in dry THF, 4ꢀ
6 h; (d) TFA, CH2Cl2, rt, 2ꢀ4 h; (e) HATU, triethyl amine, DMF, 60 ºC, o/n; (f) 1:1
HCOOH:H2O, 80 ºC, 2ꢀ6 h.
Synthesis of 5ꢀhydroxyꢀ2ꢀmethylꢀ4ꢀoxoꢀ4Hꢀpyranꢀ3ꢀcarboxamide derivatives is outlined
in Scheme 1. Bromopyruvic acid was transformed to bromodiethoxypropanoic acid by treatment
with triethyl orthoformate in the presence of catalytic sulfuric acid. The acetalꢀprotected
propanoic acid was then activated as the pꢀnitrophenyl ester 74. Pyrone ring formation was
achieved over two steps by the slow addition of 74 to a solution of ethyl or tertꢀbutyl
acetoacetate was previously deprotonated by sodium hydride. After the initial nucleophilic
attack of the activated ester by the acetoacetate, ring closing was accomplished via nucleophilic
addition by heating the reaction mixture to reflux for 4ꢀ6 h. The pyroneꢀacetal 75a was
deesterified by stirring briefly with trifluoroacetic acid (TFA) in CH2Cl2. The tertꢀbutyl ester
was removed selectively by controlling the time of the reaction, as the ester is more labile in the
absence of water than the acetal. Key intermediate 76 was used to prepare various amides using
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analogous conditions; specifically, 76 was activated with HATU and triethyl amine in DMF.
Addition of the amine was followed by heating and stirring for ~18 h at 60 ºC. After isolation of
the formed amide the acetal was deprotected in water and acid to reveal the 3ꢀketo intermediate,
which rapidly tautomerizes to form the desired 3ꢀhydroxide species.
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Scheme 2a
aReagents and conditions: (a) benzyl bromide, K2CO3, DMF, 80 ºC, 8ꢀ12 h; (b) thionyl chloride,
CH2Cl2, rt, 8 h; (c) R1R2NH, triethyl amine, DMF, 75 ºC, o/n; (d) 5:5:1 HOAc:HCl:TFA, rtꢀ40
ºC, 24ꢀ48 h; (e) sodium azide, DMF, rt, o/n; (f) triphenylphosphine, THF, rt, 30ꢀ60 min; (g) acid
chloride or sulfonyl chloride, CH2Cl2, rt, o/n; (h) BCl3, CH2Cl2, 0 ºC, 30 min, rt, 30 min.
To acquire SAR at the 6ꢀposition, a library of amine derivatives was prepared from
commercially available kojic acid (Scheme 2). The phenolic oxygen of 8 was selectively
protected as a benzyl ether by treatment with benzyl bromide in the presence of potassium
carbonate in DMF at 80 ºC. Compound 78 was prepared quantitatively by reacting 77 with
thionyl chloride. Nucleophilic addition of various primary and secondary amines, followed by
selective hydrolysis of the benzyl ether in a mixture of TFA, concentrated HCl, and glacial acetic
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acid afforded aminomethyl pyrones in good yields. Amide and sulfonamide derivatives were
generated by the nucleophilic addition of sodium azide to 78, following by reduction of 79 with
triphenylphosphine to afford 80 as a key intermediate. Compound 80 was reacted with various
acid chlorides and sulfonyl chlorides to generate 28ꢀ31, after deprotection of the phenol by boron
trichloride.
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Scheme 3a
aReagents and conditions: (a) thionyl chloride, CH2Cl2, rt, 4ꢀ6 h; (b) Zinc dust, HCl, water, 70
ºC, 4ꢀ6 h; (c) benzyl bromide, K2CO3, DMF, 80 ºC, 8ꢀ12 h; (d) aryl or alkyl amine, HOAc, 3:1
EtOH:H2O, MW 125 ºC, 90ꢀ120 min; (e) BCl3, CH2Cl2, 0 ºC, 30 min, rt, 30 min; (f) 5:5:1
HOAc:HCl:TFA, rtꢀ40 ºC, 24ꢀ48 h.
Preparation of pyridinone derivatives of allomaltol (2) possessing Nꢀaryl and ꢀalkyl
substituents is described generally in Scheme 3. Kojic chloride (81) was derived from 8, and
reduced to 2 using metallic zinc and strong acid. While dehydration of the hydroxypyrone ring
to afford hydroxypyridinone derivatives was shown to be possible, benzyl protection of the
hydroxyl group prior to dehydration greatly improved yields and suppressed formation of sideꢀ
products. Key intermediate 82 was irradiated in a microwave reactor in the presence of excess
amine and acetic acid to produce a wide variety of pyridinone derivatives. Microwave heating
under increased pressure greatly accelerated reaction rates; dehydration using conventional
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heating could be accomplished, but required refluxing reactants in ethanol for a period of days.
Removal of the benzyl ether after dehydration was shown to be accomplished quickly and
efficiently employing boron trichloride as a dealkylating agent, or less efficiently using a 5:5:1
mixture of concentrated hydrochloric acid, glacial acetic acid, and TFA—as a milder alternative
to boron trihalides (Supporting Information).
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Scheme 4a
aReagents and conditions: (a) 1:1 HCOOH:H2O, 80 ºC, 2ꢀ6 h; (b) benzyl bromide, K2CO3, DMF,
80 ºC, 8ꢀ12 h; (c) aryl amine, HOAc, 3:1 EtOH:H2O, MW 130 ºC, 3ꢀ5 h; (d) 5:5:1
HOAc:HCl:TFA, rtꢀ40 ºC, 24ꢀ48 h; (e) 4% KOH, 1:2:1 THF:MeOH:water, rt, o/n; (f) RCH3NH,
triethyl amine, DMF, 75 ºC, o/n.
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Fragment merging was accomplished as shown in Scheme 4. Merging of Nꢀaryl
pyridinones with 5ꢀposition carboxylates began with advanced intermediate 76a. Hydrolysis of
the acetal followed by tautomerization yielded 83. After benzyl protection of the phenol,
dehydration was accomplished with microwave heating using dry ethanol as a solvent.
Hydrolysis of only the benzyl ether using strong acid afforded compounds 67ꢀ69 in moderate
yields; hydrolysis of both ester and ether was not observed. Further base catalyzed hydrolysis of
the ethyl ester proved difficult, and ultimately resulted in the decomposition of the ring system.
As an alternative route, compound 86 was hydrolyzed to the free carboxylic acid by stirring in
4% KOH and methanol for several hours. This reaction proceeded almost quantitatively with no
apparent decomposition of the starting material. Compound 86 was found to be stable to
strongly acidic conditions, and was hydrolyzed to 70 using a mixture of strong acids with good
yields. Synthesis of compound 71 began with chloride compound 78. Nucleophilic substitution
with 4ꢀchloroꢀNꢀmethylaniline afforded pyrone 87. Dehydration, as previously described,
yielded 88 in moderate to low yields. Hydrolysis in the presence of strong acid afforded 71.
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Ancillary and Supporting Information
Details of experimental methods and additional information as indicated in the text. This
material is available free of charge via the Internet at http://pubs.acs.org.
Abbreviations Used
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