Discovery of a highly selective PLD2 inhibitor (ML395): A new probe with improved physiochemical properties and broad-spectrum antiviral activity against influenza strains

Article abstract of DOI:10.1002/cmdc.201402333

Further chemical optimization of the halopemide-derived family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded ML395 (VU0468809), a potent, >80-fold PLD2 selective allosteric inhibitor (cellular PLD1, IC₅₀>30 000 nm; cellular PLD2, IC₅₀=360 nm). Moreover, ML395 possesses an attractive in vitro DMPK profile, improved physiochemical properties, ancillary pharmacology (Eurofins Panel) cleaner than any other reported PLD inhibitor, and has been found to possess interesting activity as an antiviral agent in cellular assays against a range of influenza strains (H1, H3, H5 and H7).

Full text of DOI:10.1002/cmdc.201402333

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DOI: 10.1002/cmdc.201402333
Discovery of a Highly Selective PLD2 Inhibitor (ML395): A
New Probe with Improved Physiochemical Properties and
Broad-Spectrum Antiviral Activity against Influenza Strains
[
a]
[b]
[a]
[b]
Matthew C. O’Reilly, Thomas H. Oguin, III, Sarah A. Scott, Paul G. Thomas,
[
a]
[a]
[a]
[a]
Charles W. Locuson, Ryan D. Morrison, J. Scott Daniels, H. Alex Brown, and
[a]
Craig W. Lindsley*
Further chemical optimization of the halopemide-derived
family of dual phospholipase D1/2 (PLD1/2) inhibitors afforded
ML395 (VU0468809), a potent, >80-fold PLD2 selective alloste-
ric inhibitor (cellular PLD1, IC >30000 nm; cellular PLD2,
50
IC =360 nm). Moreover, ML395 possesses an attractive in
50
vitro DMPK profile, improved physiochemical properties, ancil-
lary pharmacology (Eurofins Panel) cleaner than any other re-
ported PLD inhibitor, and has been found to possess interest-
ing activity as an antiviral agent in cellular assays against
a range of influenza strains (H1, H3, H5 and H7).
Phospholipase D (PLD) is a phospholipase that catalyzes the
production of phosphatidic acid, an important lipid second
messenger involved in a myriad of critical signaling and meta-
[
1–3]
bolic pathways.
In mammals, there are two isoforms of PLD,
coined PLD1 and PLD2, which are differentially regulated and
perform distinct physiological roles. Data from biochemical
and genetic studies have implicated aberrant PLD function
and/or overexpression in cancer, viral infection and central
nervous systems (CNS) disorders; however, due to a lack of
highly isoform-selective small-molecule PLD inhibitors, the
therapeutic potential of modulating PLD function has re-
[
1–4]
mained elusive.
In 2007, halopemide 1, an atypical antipsychotic agent, was
[
5]
Figure 1. Structures of recently reported PLD inhibitors (1–5). Halopemide
1), an atypical antipsychotic agent was shown to be a dual PLD1/2 inhibitor,
which spawned optimization campaigns that afforded isoform-selective
either PLD1 (2) or PLD2 (3 and 4)) inhibitors, as well as a highly potent dual
reported to inhibit PLD. This report triggered a resurgence of
interest in the therapeutic relevance of PLD inhibition since
(
1
) no potent chemical inhibitors of PLD were previously known
(
and 2) 1 had been involved in clinical trials where PLD1 and
PLD1/2 inhibitor (5).
[
6]
PLD2 were inhibited without adverse events. Therefore, our
lab quickly initiated a diversity-oriented synthesis campaign
around 1 (Figure 1), and developed the first direct, isoform-se-
lective PLD inhibitors represented by 2 (1700-fold PLD1 selec-
tion tools were important in defining the individual contribu-
tions of PLD1 and PLD2 in various systems and diseases, nei-
ther 2 nor 3 possessed ideal physiochemical or DMPK proper-
[
2]
[7–9]
tive) and 3 (75-fold PLD2 selective). While these first genera-
ties for rigorous in vivo evaluation.
Subsequent
optimization efforts provided the PLD2-selective probe ML298
4) and the potent dual PLD1/2 inhibitor ML299 (5), with signif-
icantly improved ancillary pharmacology; however, the DMPK
[
a] M. C. O’Reilly, Dr. S. A. Scott, Dr. C. W. Locuson, R. D. Morrison,
Prof. J. S. Daniels, Prof. H. A. Brown, Prof. C. W. Lindsley
Department of Pharmacology
Vanderbilt Center for Neuroscience Drug Discovery
Vanderbilt Specialized Chemistry Center (MLPCN)
Vanderbilt University Medical Center, Nashville, TN 37232-6600 (USA)
E-mail: craig.lindsley@vanderbilt.edu
(
[10]
profiles and physiochemical properties were still lacking.
Therefore, we launched an optimization effort aimed at devel-
oping a third generation PLD2-selective inhibitor devoid of cy-
totoxicity, that displays improved DMPK and ancillary pharma-
cology profiles coupled with good CNS exposure.
[b] T. H. Oguin, III, Prof. P. G. Thomas
Department of Immunology
Thus far, all triazaspirone-based PLD inhibitors, such as 3–5,
bore an N-aryl moiety (only naked phenyl or halogen-substitut-
ed phenyl were evaluated), which left a great deal to explore
St. Jude Children’s Hospital, Memphis, TN 38105 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402333.
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in terms of structure–activity relationship (SAR) for PLD2 inhibi-
tion as well as in enhancement of physiochemical and DMPK
inhibitors with IC50 values of 440 nm and 320 nm, respectively;
however, both were modestly selective versus PLD1 (7–12-
fold). Benzamide congeners, 6d–f, were uniformly inactive at
both PLD1 and PLD2, while certain benzyl amines proved to
be potent PLD2 inhibitors, such as the 2-fluorobenzyl analogue
[
8–10]
properties.
Further probe optimization efforts were focused
on surveying alternative, non-N-aryl moieties in the triazaspir-
one core (Figure 2) and to evaluate diverse analogues 6. To
6
h, displaying 22-fold selectivity
over PLD1 and a PLD2 IC50 of
05 nm. The most interesting
2
SAR concerned regioisomeric
methylene-linked pyridyl conge-
ners.
In this case, the methylene-
linked 2-pyridyl analogue 6k
was 30-fold selective versus
PLD1, the 3-pyridyl analogue 6l
was >80-fold selective and very
potent (PLD2 IC =360 nm), and
Figure 2. Optimization plan for 3–5 to produce analogues 6 with non-N-aryl diversity to improve activity, physio-
chemical properties and DMPK profile.
50
enable this effort, a synthetic route was devised to access ad-
the 4-pyridyl methyl analogue 6m was 28-fold selective versus
PLD1 (PLD2 IC =850 nm). Thus, the 3-pyridyl methyl would
[
11]
vanced intermediate 11 (Scheme 1).
Beginning with N-
50
benzyl-protected piperidinone 7, a Strecker reaction with am-
be retained in future analogues 12 while surveying alternative
amides (Table 2), employing a variation of the route depicted
in Scheme 1. In this instance, SAR was shallow, with functional-
ized benzamides and heteroaryl amides affording inactive
compounds. However, indole-based amides, 12a–e, were the
exception from this library, though SAR was still shallow. Nota-
bly, 12a, a 2-indolyl amide was a potent PLD2 inhibitor (PLD2
IC =52 nm) with ~69-fold selectivity versus PLD1 (PLD1 IC =
50
50
3,600 nm). The 5-fluoro congener 8b, suffered a moderate loss
in potency (PLD2 IC =120 nm) and selectivity (26-fold), but
50
other substitution patterns were inactive. The N-methyl ana-
logue of 12a, 12c, was uniformly inactive as were aza deriva-
tives, such as 12d. Lastly, the 3-indolyl amide 8e was also
devoid of PLD activity inhibition.
Scheme 1. Preparation of analogues 6. Reagents and conditions: a) 1. NaCN,
NH
SO
mine (DIEA), 0.5 equiv 4-dimethylaminopyridine (DMAP), tetrahydrofuran,
. H , 10% Pd/C, MeOH, RT, 3. tert-butyl (2-bromoethyl)carbamate, K CO
Cl
4
Cl, 7m NH
3
/MeOH, RT, 4 h, 2. H
2
SO
4
, CH
2
Cl
2
, 62%; b) 1. formamide,
H
2
4
, 1758C, 16 h 2. NaBH
4
, MeOH, 25% c) 1. Boc
2
O, N,N-diisopropylethyla-
Since the methylene-linked 3-pyridyl moiety was still opti-
mal, this group was attached to the nitrogen of the triazaspir-
2
2
2
3
,
DMF, RT, 62% over three steps; d) 1. HCl, dioxanes, RT, 2. RCOCl, CH
DIEA, RT, 51–84%.
2
2
,
one and N-pyridyl congeners evaluated. S Ar chemistry afford-
N
ed rapid access to a set of 12 analogues with either a 3-pyridyl
(13) or 3-pyridyl-5-fluoro ring (14), the latter of which to com-
bine the optimal moieties in 3–5 and the 3-pyridyl moiety
(Figure 3). Interestingly, these analogues were all uniformly in-
monia followed by sulfuric acid mediated hydrolysis, delivered
carboxamide 8. Condensation with formamide followed by re-
duction with sodium borohydride provided the triazaspirone
core 9 in 25% yield. Subsequent Boc protection of the secon-
dary amine, deprotection of the primary benzyl amine, and re-
ductive amination with tert-butyl (2-oxoethyl)carbamate fur-
nished the bis-Boc 10 in 62% yield. Finally, global deprotection
with HCl and selective acylation of the primary amine afforded
advanced intermediate 11, with the free secondary amine to
participate in alkylations, reductive aminations, and acylations
to survey non-N-aryl moieties in analogues 6.
From 11, multiple iterative libraries were synthesized (in
total 80 novel analogues) that were evaluated for inhibitory ac-
tivity against PLD1 and PLD2 in our standard cell-based
Figure 3. Direct N-pyridyl analogue libraries 13 and 14 were all uniformly in-
active at both PLD1 and PLD2 (IC50 values >30 mm).
[
2]
assay. In the first iteration, the 2-naphthylamide moiety was
held constant (a preferred group) and alterative N-substituents
were surveyed in analogues 6. Table 1 highlights selected SAR
for this series. Robust SAR was noted. The unsubstituted sec-
ondary amine 6a was inactive, while simple cycloalkyl amides,
such as cyclopropyl 6b and cyclobutyl 7c were potent PLD2
active. Thus, 3-pyridyl analogue 6l, a potent, direct inhibitor of
PLD2 (cellular PLD2 IC =360 nm, exogenous biochemical
50
assay with purified PLD2 IC =8.7 mm) with no measureable
50
activity at PLD1 up to 30 mm (>80-fold selective versus PLD1)
was designated ML395, an Molecular Libraries Probe Produc-
tion Centers Network (MLPCN) probe molecule to be further
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À1
À1
ance (rat CL : 82.1 mLmin kg , human CL :
int
À1
int
Table 1. Structures and PLD inhibitory activities of analogues 6.
À1
4
3 mLmin kg ) with predicted hepatic clearance
À1
À1
values (rat CLhep: 64.3 mLmin kg , human CLhep:
À1
À1
1
7 mLmin kg ) near the respective rates of hepatic
blood flow in each species (Table 3). In order to de-
termine the relevant biotransformation pathway(s)
contributing to the modest metabolic stability ob-
served in vitro (CLint and CLhep), metabolite identifica-
tion (Met ID) experiments were performed using the
rat hepatic S9 fraction (Figure 4). This analysis re-
vealed NADPH-dependent oxidation of the triazaspir-
one core consistent with common biotransformation
pathways of piperidines where an initial oxygenation
is followed by subsequent alcohol oxidation or dehy-
dration. Another NADPH-dependent mono-oxidation
pathway identified in rat S9 was N-dealkylation at the
Compd
R
PLD1
IC50 [nM]
PLD2
IC50 [nM]
Fold PLD2
selectivity
[
a]
[a]
6
6
a
H
>30000
>30000
440Æ5
–
7
b
3100Æ80
4000Æ40
6
6
c
320Æ60
12.5
9
d
19000Æ1100
8760Æ100
2050Æ40
[11]
amide (M1; Figure 4).
6
6
e
f
2750Æ80
2540Æ70
~3
In order to gauge distribution to the central nerv-
ous system (CNS), concentrations of ML395 in whole
brain and plasma at a single time point (0.25 h) were
measured following a single intravenous (IV) adminis-
>20000
~10
À1
tration (0.2 mgkg ) to male, Sprague Dawley rats
[11]
6
6
g
h
8260Æ50
4590Æ90
890Æ10
205Æ10
9.2
22
(n=2). This study revealed a brain:plasma partition
coefficient (K ) of 1.48, indicating excellent distribu-
p
tion to the CNS (Table 1), and in a bidirectional
MDCK–MDR1 transwell assay, ML395 (5 mm) exhibited
an efflux ratio (ER) of 1.4, suggesting an absence of
P-glycoprotein (P-gp)-mediated active efflux liabilities
at the blood–brain barrier. Moreover, ML395 was
screened in a Eurofins radioligand binding panel of
68 G protein-coupled receptors (GPCRs), ion channels
6
6
6
i
8100Æ150
3500Æ110
2650Æ60
1780Æ80
7200Æ370
80Æ10
4.5
–
j
k
30
[12]
and transporters at a concentration of 10 mm, and
6
6
l
>30000
24000Æ400
360Æ10
850Æ29
>80
no significant activity was noted (no inhibition
>
50% at 10 mm) including hERG. Thus, in addition to
m
28
unprecedented selectivity versus PLD1, ML395 dis-
played clean ancillary pharmacology against a diverse
array of discrete molecular targets, and notably elimi-
[a] IC50 values ÆSEM were determined in triplicate.
characterized. To further evaluate ML395 (6l), the physiochemi-
cal and DMPK profile of this highly PLD2-selective inhibitor
[
11]
was assessed. ML395 conforms to Lipinski’s rules (MW=443,
H-bond donors=2) with favorable lipophilicity (cLogP=2.74)
and an acceptable topological polar surface area (TPSA) of 77.
Moreover, ML395 was found be both soluble in phosphate-buf-
fered saline (PBS, 95 mm at pH 7.4 at 238C) and stable (~100%
parent remaining at 48 h in PBS at 238C). Moreover, ML395 dis-
played a favorable cytochrome P450 profile (CYP3A4 IC =
50
3
.9 mm, CYP2D6 IC =16.4 mm, CYP1A2 IC >30 mm, CYP2C9
50 50
IC >30 mm). In plasma protein binding experiments, the com-
50
pound exhibited exceptionally high fraction unbound (fuplasma
)
in rat (26.3%) and moderately high fuplasma in human (8.8%),
which prompted investigation into the compound’s stability
due to the presence of amides. ML395 was stable over time in
plasma from both species (4 h, 378C), revealing that the ob-
served fuplasma values were accurate. In rat and human hepatic
microsomes, ML395 exhibited moderate to high intrinsic clear-
Figure 4. Metabolite identification for 6l, ML395 (VU0468809), in rat S9 with
and without NADPH. Three routes of metabolism (M1–M3) were identified in
the presence of NADPH, and none in the absence of NADPH.
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[13]
fection. By decreasing PLD2 activity using chemical
inhibition, using 3, or through RNA interference, de-
layed viral entry and reduced viral titers in vitro were
observed. In vivo, PLD2 inhibition with 3 reduced
viral titer, increased survival, and correlated with sig-
nificant increases in transcription of innate antiviral
Table 2. Structures and PLD inhibitory activities of analogues 12.
[13]
effectors.
However, due to the cytotoxicity of 3,
Compd
R
PLD1
PLD2
Fold PLD2
selectivity
[
a]
[a]
and the modest activity toward PLD1 (IC =1.5 mm),
IC50 [nM]
IC50 [nM]
50
evaluation of the antiviral activity of ML395 (6l) due
to its improved properties and lack of both cytotoxic-
ity and activity at PLD1 was essential.
1
1
1
1
2a
2b
2c
2d
3600Æ100
3100Æ90
52Æ6
69
26
–
Employing the traditional tissue culture infective
dose (TCID ) assay to assess viral reproduction in
120Æ8
50
[13]
vitro,
A549 cells were treated with 10 mm 3 or
>20000
>20000
ML395 (6l) for 1 h before infection (Figure 6), and the
cells were then infected with one multiplicity of in-
fection (MOI) with four distinct strains of influenza
>20000
>20000
>20000
>20000
–
(
(
A/California/04/2009 (H1N1), A/Brisbane/10/2007
H3N2), rg-A/Vietnam/1203/2004 (H5N1) and A/
Anhui/01/2013 (H7N9)). At 24 h post infection, infec-
tious supernatant was removed from the A549 cells
1
2e
–
and titrated on MDCK cells to measure viral repro-
[a] IC50 values ÆSEM were determined in triplicate.
[11,13]
duction.
Across all four strains, both 3 and
ML395 significantly lowered viral reproduction. The
improved physiochemical properties of ML395 trans-
Table 3. DMPK profile of ML395 (6l).
lated into comparable efficacy to 3, despite an ~20-fold reduc-
tion in PLD2 potency. Additionally, since this assay was per-
formed at 10 mm, the effect of ML395 can be directly linked to
PLD2 inhibition while a 10 mm concentration of 3 undoubtedly
inhibits both PLD1 and PLD2. Notably, the PLD inhibitors dis-
played pan-anti-influenza activity across seasonal influenza
(H1N1), a low pathogenicity strain of influenza (H3N2), a highly
pathogenic avian influenza (H5N1), and a recently emergent
virus with pandemic potential (H7N9). Importantly, the appar-
ent ease of resistance to both adamantane drugs and oseltami-
vir (Tamiflu) with H7N9 viruses is concerning, and thus small-
molecule PLD inhibition represents an exciting new mecha-
nism with which to combat pathogenic influenza and provide
new tools to assist in overcoming evolution of antiviral drug
[
a]
Species
Property
Value
À1
À1
À1
Hepatic Microsomal CLint
Predicted CLhep
82.1 mLmin kg
64.3 mLmin kg
0.26Æ0.03; 0.12Æ0.03
[b]
À1
Sprague Dawley rat
fuplasma; fubrain
C
brain:Cplasma (K
p
)
1.48
À1
À1
À1
Hepatic Microsomal CLint
Predicted CLhep
fuplasma
43 mLmin kg
17 mLmin kg
À1
0.088Æ0.01
Human
P450 1A2 IC50
P450 2C9 IC50
P450 2D6 IC50
P450 3A4 IC50
>30 mm
>30 mm
16.3Æ0.04 mm
3.9Æ0.4 mm
[
a] Values represent means ÆSEM of at least two replicates with similar
À1
p
results. [b] K determined at 0.25 h following a 0.2 mgkg IV dose (n=2).
fu=fraction unbound.
[14]
resistance.
In summary, a potent (PLD2 IC =360 nm) and >80-fold se-
50
lective (PLD1 IC >30000 nm) PLD2 inhibitor was developed
50
[
3–10]
nated biogenic amine activity that persisted with 1–5.
To-
(6l, also known as ML395 or VU0468809), with improved physi-
ochemical properties, no cytotoxicity, high CNS penetration
and a favorable DMPK profile. SAR was steep for this series,
with subtle modifications leading to complete loss of PLD in-
hibitory activity. In cell-based antiviral assays, ML395 was
shown to dramatically inhibit infection of A549 cells by multi-
ple strains of influenza. Importantly, inhibition of PLD2 has
now emerged as a powerful new mechanism to treat not only
classical H1N1, but also the highly virulent and treatment-re-
sistant H5N1 and H7N9 strains. Studies are underway to further
explore the breadth of antiviral activity of PLD2 inhibition, as
well as in vivo survival studies with ML395. ML395 is an
MLPCN probe and is freely available upon request.
gether, these findings suggest that ML395 possesses accepta-
ble CNS compound exposure properties for pharmacodynamic
studies in rodent species.
Based on the pronounced cytotoxicity of our standard selec-
tive PLD2 tool compound 3, we needed to assess if ML395
provided an improvement prior to advancing into key studies.
As shown in Figure 5, ML395 was devoid of cytotoxicity at con-
centrations up to 50 mm, as opposed to 3, which displays sig-
[
11]
nificant cyototoxicity at concentrations above 10 mm. There-
fore, ML395 emerged as a preferred tool compound to assess
selective PLD2 inhibition in cell-based assays, and represented
a major advance over 3.
Recently, we reported that influenza virus stimulates host-
cell PLD activity, and PLD co-localizes with influenza during in-
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Acknowledgements
This work was generously sup-
ported by the US National Insti-
tutes of Health (NIH) / Molecular
Libraries Probe Production Centers
Network (MLPCN) grant U54
MH084659 (C.W.L.). Dr. Lindsley
acknowledges the Warren Family
and Foundation for funding the
William K. Warren, Jr. Chair in
Medicine. M.C.O. acknowledges
funding from a Predoctoral ACS
Medicinal Chemistry Fellowship
Figure 5. Assessing the cytotoxicity of PLD2 inhibitor 3 versus ML395 (6l). A) microscopic image of A549 cells
treated with ML395 (6l) showing no cellular toxicity after 24 h, bar=50 mm. B) microscopic image of A549 cells
treated with 3 showing significant cellular toxicity after 24 h, bar=50 mm. C) A dose response of 3 and ML395 in
a 24 h cytotoxicity in A549 cells. PLD2 inhibitor 3 displays significant toxicity at concentrations above 10 mm,
whereas ML395 is comparable to DMSO at concentrations up to 50 mm.
(
2011–2012).
Keywords: antivirals
inhibitors
phospholipase D · PLD2
·
·
·
lipids
[
[
1] P. E. Selvy, R. Lavieri, C. W. Lindsley, H. A. Brown, Chem. Rev.
011, 111, 6064–6119.
2] S. A. Scott, P. E. Selvy, J. R. Buck, H. P. Cho, T. L. Criswell, A. L.
Thomas, M. D. Armstrong, C. L. Arteaga, C. W. Lindsley, H. A.
Brown, Nat. Chem. Biol. 2009, 5, 108–117.
2
[
[
[
3] H. A. Brown, L. G. Henage, A. M. Preininger, Y. Xiang, J. H.
Exton, Methods Enzymol. 2007, 434, 49–87.
4] S. A. Scott, T. P. Matthews, P. T. Ivanova, C. W. Lindsley, H. A.
Brown, Biochim. Biophys. Acta 2014, 1841, 1060–1084.
5] L. Monovich, B. Mugrage, E. Quadros, K. Toscano, R. Tomma-
si, S. LaVoie, E. Liu, Z. M. Du, D. LaSala, W. Boyar, P. Steed,
Bioorg. Med. Chem. Lett. 2007, 17, 2310–2311.
[
[
6] H. De Cuyper, H. M. van Praag, D. Verstraeten, Neuropsy-
chobiology 1984, 12, 211–223.
7] J. A. Lewis, S. A. Scott, R. Lavieri, J. R. Buck, P. E. Selvy, S. L.
Stoops, M. D. Armstrong, H. A. Brown, C. W. Lindsley, Bioorg.
Med. Chem. Lett. 2009, 19, 1916–1919.
[
[
8] R. Lavieri, J. A. Lewis, S. A. Scott, P. E. Selvy, J. Buck, M. D.
Armstrong, H. A. Brown, C. W. Lindsley, Bioorg. Med. Chem.
Lett. 2009, 19, 2240–2244.
9] R. Lavieri, S. A. Scott, P. E. Selvy, H. A. Brown, C. W. Lindsley,
J. Med. Chem. 2010, 53, 6706–6719.
Figure 6. Influenza replication is significantly reduced when PLD2 is inhibited by either
VU034739 (3) or ML395 (6l). A549 cells were pretreated with either DMSO or 10 mm
VU0364739 or 10 mmML395 (6l) for 1 h, then infected with 1 MOI of either A) H1N1 influ-
enza, B) H3N2 influenza, C) H5N1 influenza, D) H7N9 influenza. After 24 h post-infection,
the infectious supernatant containing the virus was removed and titrated on MDCK cells
to assess viral production. Under PLD2 inhibitor treatment, poor viral replication was ob-
served in all influenza strains tested by 24 h post-infection, and the viral output defect
was noted as early as 12 h post-infection in the case of H3N2 and H7N9. Differences
were assessed using a two-way ANOVA and Bonferroni’s post-test, where *p<0.05,
[
10] M. C. O’Reilly, S. A. Scott, K. A. Brown, T. H. Oguin III, P. G.
Thomas, J. S. Daniels, R. Morrison, H. A. Brown, C. W. Linds-
ley, J. Med. Chem. 2013, 56, 2695–2699.
11] For experimental details of the molecular pharmacology,
DMPK, antiviral assays, chemical synthesis and characteriza-
tion, see the Supporting Information.
12] For informaiton on the Eurofins Lead Profiling Screen, see:
http://www.eurofins.com.
13] T. Oguin III, S. Sharma, A. D. Stuart, C. K. Jones, J. S. Daniels,
C. W. Lindsley, P. G. Thomas, H. A. Brown, J. Biol. Chem. 2014,
DOI: 10.1074/jbc.M114.558817.
[
[
[
**p<0.01, ***p<0.001, ****p<0.0001.
Experimental Section
[
14] Y. Hu, S. Lu, W. Wang, P. Hao, J. Li, H-L. Yen, B. Shi, T. Li, W. Guan, L. Zu,
Y. Liu, X. Zhang, D. Tian, Z. Zhu, J. He, K. Huang, H. Chen, L. Zheng, X.
Li, J. Ping, B. Kang, X. Xi, L. Zha, Z. Zhang, M. Peiris, Z. Yuan, Lancet
2
013, 381, 2273–2279.
Experimental procedures for medicinal chemistry, pharmacology
and drug metabolism as well as characterization of compounds are
provided in the Supporting Information.
Received: August 4, 2014
Published online on September 10, 2014
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