Expeditious Lead Optimization of Isoxazole-Containing Influenza A Virus M2-S31N Inhibitors Using the Suzuki-Miyaura Cross-Coupling Reaction

Article abstract of DOI:10.1021/acs.jmedchem.6b01852

The existence of multidrug-resistant influenza viruses, coupled with the continuously antigenic shift and antigenic drift of influenza viruses, necessitates the development of the next-generation of influenza antivirals. As the AM2-S31N mutant persists in more than 95% of current circulating influenza A viruses, targeting the AM2-S31N proton channel appears to be a logical and valid approach to combating drug resistance. Starting from compound 1, an isoxazole compound with potent AM2-S31N channel blockage and antiviral activity, in this study we report an expeditious synthetic strategy that allows us to promptly explore the structure-activity relationships of isoxazole-containing AM2-S31N inhibitors. Propelled by the convenient synthesis, the lead optimization effort yielded a number of potent antivirals with submicromolar efficacy against several human clinical isolates of influenza A viruses, including both oseltamivir-sensitive and -resistant strains.

Full text of DOI:10.1021/acs.jmedchem.6b01852

Article
pubs.acs.org/jmc
Expeditious Lead Optimization of Isoxazole-Containing Influenza A
Virus M2-S31N Inhibitors Using the Suzuki−Miyaura Cross-Coupling
Reaction
Fang Li,† Yanmei Hu,† Yuanxiang Wang,† Chunlong Ma,‡ and Jun Wang*,†,‡
†Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, United
States
‡BIO5 Institute, The University of Arizona, Tucson, Arizona 85721, United States
S
* Supporting Information
ABSTRACT: The existence of multidrug-resistant influenza
viruses, coupled with the continuously antigenic shift and
antigenic drift of influenza viruses, necessitates the develop-
ment of the next-generation of influenza antivirals. As the
AM2-S31N mutant persists in more than 95% of current
circulating influenza A viruses, targeting the AM2-S31N
proton channel appears to be a logical and valid approach to
combating drug resistance. Starting from compound 1, an
isoxazole compound with potent AM2-S31N channel blockage
and antiviral activity, in this study we report an expeditious
synthetic strategy that allows us to promptly explore the
structure−activity relationships of isoxazole-containing AM2-S31N inhibitors. Propelled by the convenient synthesis, the lead
optimization effort yielded a number of potent antivirals with submicromolar efficacy against several human clinical isolates of
influenza A viruses, including both oseltamivir-sensitive and -resistant strains.
target the conserved viral proteins such as the AM2-S31N are
highly desired.2,12,13
INTRODUCTION
■
Influenza is a contagious respiratory illness that affects an
estimated 10−15% of the population in the United States
during the annual influenza season.1 For the immune-
competent person, infection with seasonal influenza virus is
usually self-confined and non-life-threating. However, the
symptoms and disease outcomes are exacerbated in seniors,
children, and immunocompromised individuals.2 Furthermore,
various influenza strains differ significantly in terms of their
lethality for the general population.3 Mortality rates are
disproportionally high for emerging and re-emerging pandemic
influenza viruses as well as highly pathogenic avian influenza
viruses (HPAIVs).4 For example, the 1918 Spanish H1N1
influenza pandemic claimed an estimated 50 million lives
worldwide in a matter of months.5 The recent 2009 H1N1
influenza pandemic led to 284,000 deaths globally in the first 12
months of outbreak, as estimated by the CDC.6 The mortality
rate for HPAIVs such as H5N1 is more than 50%;7 therefore,
effective vaccines and antiviral drugs are clearly needed to
confine and limit the impact of influenza viruses.8
The AM2-S31N mutant is one of the most conserved viral
proteins among currently circulating influenza A viruses, and
more than 95% of the viruses carry this mutation.14 Although
this mutation confers amantadine resistance, this is not a result
of the amantadine drug selection pressure.15 Instead, the AM2-
S31N mutant is a natural mutation that happens to maintain
nearly identical channel function as the wild-type AM2 (AM2-
WT) but is resistant to amantadine.16 Our goal is to design
potent channel blockers of the AM2-S31N mutant and develop
them as the next-generation of influenza antivirals. Propelled by
mechanistic studies of AM2-S31N drug inhibition and proton
conductance using X-ray crystallography,17−23 NMR,19−21 and
molecular dynamics simulations,24 we were able to design
inhibitors that target the AM2-S31N mutant,14 which was
previously deemed undruggable, as no progress had been made
for decades.14 Briefly, at the initial stage of drug design, there
was no AM2-S31N structure available, so molecular dynamics
simulations were applied to generate homology models of
AM2-S31N.20,24 In the drug-free form, AM2 adapts two
conformations, Openout-Closedin and Closedout-Openin. In the
Openout-Closedin conformation, the AM2 channel opens to the
viral exterior to allow protons to diffuse into the channel, while
Vaccines are generally effective in preventing influenza
infection, with an overall effectiveness of approximately 60%.9
Due to the six-months delay between antigen identification and
vaccine production, viruses might mutate during this period,
which would render vaccines less effective.10,11 Moreover,
vaccines are not always available at the beginning of an
influenza outbreak. Thus, small-molecule antiviral drugs that
Received: December 20, 2016
© XXXX American Chemical Society
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Figure 1. AM2-S31N inhibitors. (A) Pharmacophore of AM2-S31N inhibitors. (B) Representative isoxazole-containing AM2-S31N inhibitor. (C)
Synthesis of potent AM2-S31N inhibitors by microwave-assisted Suzuki−Miyaura cross-coupling.
Scheme 1. Previously Reported and Newly Discovered Synthesis Routes to Isoxazole-Containing AM2-S31N Inhibitors
in the Closedout-Openin conformation, the AM2 channel opens
to the viral interior such that the protonated histidines can
release protons to the viral cytoplasm. We have designed
inhibitors that intend to target either the Openout-Closedin or
the Closedout-Openin conformation of AM2-S31N, and so far,
we were only able to successfully target the Openout-Closedin
conformation.14,25 With one of the AM2-S31N inhibitors,
M2WJ332, in hand, we were able to solve the solution NMR
structure of AM2-S31N (PDB: 2LY0), and the structure indeed
showed that the drug M2WJ332 bound to the AM2-S31N
channel when it was in the Openout-Closedin conformation.21 In
terms of the biological activity, the designed AM2-S31N
inhibitors were found to have potent antiviral activity against
multidrug-resistant influenza A viruses.26,27 More importantly,
the AM2-S31N inhibitors have a higher genetic barrier to drug
resistance than amantadine, and resistant mutants that emerge
under AM2-S31N inhibitors drug selection pressure in cell
culture are rare mutations.26 In light of these promising results,
we are interested in continuously developing AM2-S31N
inhibitors and providing lead compounds for in vivo mouse
studies.
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Table 1. Optimization of the Suzuki−Miyaura Cross-coupling Reaction for Isoxazole
a
b
Entry
Temp (°C)
Catalyst
Solvent
Time
Base
Yield (%)
c
1
120
100
100
100
90
Pd(OAc)2
Toluene/H2O
DME
30 min
60 min
60 min
60 min
60 min
60 min
60 min
60 min
60 min
30 min
60 min
K3PO4
15
47
37
51
55
70
78
68
57
77
70
c
2
Pd2dba3
K3PO4
c
3
Pd2dba3
Dioxane/H2O
Dioxane
K3PO4
c
4
Pd2dba3
K3PO4
5
Pd(dppf)Cl2
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
Pd(PPh3)4
DME
K2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
Na2CO3
6
150
150
150
150
150
Dioxane/H2O
Dioxane
Toluene
7
8
9
DME
10
11
Dioxane
120
Dioxane
a
b
c
Microwave irradiation. Isolated yield. Tricyclohexylphosphine was used as a ligand.
Initial structure−activity relationship (SAR) studies of the
it takes five steps to synthesize every single compound. This is
less than ideal, as it poses a rate-limiting step in the iterative
cycles of design, synthesis, and biological characterization. In
searching for an alternative synthesis route to speed up the lead
optimization process, we devised a late-step diversification
strategy (Scheme 1B). In the newly proposed synthesis
strategy, the diversity was introduced in the last step using
Suzuki−Miyaura cross-coupling. The success of the proposed
route relies on not only the synthesis of the key intermediate
14, which has not been reported before, but also the last-step
Suzuki−Miyaura cross-coupling reaction. As an initial trial, a
condensation−cyclization reaction between diethyl oxalacetate
sodium 10 and hydroxylamine in the presence of sodium
bicarbonate gave the intermediate 11 with a 40% yield, which
was then converted to the key intermediate ethyl 5-
bromoisoxazole-3-carboxylate 14 using phosphoryl bromide.
Although we were able to obtain this intermediate 14, the yield
for the second-step bromination was only 23%. We then
switched to another strategy which started from 3 + 2
cycloaddition between a nitrile oxide generated from 2-
chloro-2-(hydroxyimino)acetate 12 and ethynyltributylstan-
nane to give the intermediate 13 in a 75% yield.30 Replacement
of the tributylstannane group in 13 by bromide using bromine
proceeded smoothly with a decent yield of 90%. With the key
intermediate 14 in hand, we then proceeded to the reduction of
14 with diisobutylaluminum hydride at 0 °C to give the alcohol
intermediate 15, which was then oxidized by Dess−Martin
periodinane to give the aldehyde 16. The yields for these two
steps were 85% and 64%, respectively. Reductive amination of
the isoxazole aldehyde 16 with adamantane analogues 8a−b
using previously optimized conditions gave intermediate 17a−b
in high yields: 79% for 17a and 82% for 17b.
AM2-S31N inhibitors revealed that potent channel blockers
contain the following structural features: a hydrophobic
template such as adamantane or hydroxyl adamantane, an
ammonium linker, and an aryl headgroup with a hydrophobic
substitution (Figure 1A).21,28,29 It has been found that the aryl
headgroup prefers to be isoxazole, and a representative example
is compound 1 (Figure 1B). As previous studies suggested that
the hydrophobic substitution at the 5-position of isoxazole is
critical for the channel blockage and antiviral activity,29 we are
interested in exploring various aryls and heteroaryls at this
substitution. To facilitate iterative cycles of design, synthesis,
and biological characterization, we aim to develop an
expeditious synthetic strategy that allows us to install diverse
aryls and heteroaryls at the last step of the synthesis (Figure
1C). In this study, we report the optimized synthesis route as
well as the channel blockage and antiviral activity of the
synthesized compounds. These efforts resulted in several
promising lead compounds with improved potency and a
high selectivity index against multidrug-resistant influenza A
viruses.
RESULTS AND DISCUSSION
■
Chemistry. Previous syntheses of isoxazole-containing
AM2-S31N inhibitors involved a five-step synthesis procedure
(Scheme 1A).29 Specifically, condensation of methyl ketone 2
and dimethyl oxalate 3 using potassium tert-butoxide as the
base gave the intermediate 4, which was cyclized to isoxazole
ester 5 upon treatment with hydroxylamine hydrochloride
under mild heating at 50 °C in methanol. The ester 5 was
subsequently reduced to alcohol 6 by sodium borohydride and
was then converted to bromide 7 by triphenyl phosphine and
carbon tetrabromide in dichloromethane. The last step
alkylation of bromide 7 with the amantadine analog 8 furnished
the isoxazole-containing AM2-S31N inhibitor 9. Although this
is a highly reliable synthesis route with overall yields ranging
from 26% to 62% (depending on substrates), one apparent
limitation of this strategy from the medicinal chemistry
perspective is that the diversity at the 5-position of isoxazole
was carried from the first step to the last step, which means that
The next critical step was to establish a cross-coupling
condition to install a variety of aryls and heteroaryls at the 5-
position of isoxazole, which has not been widely explored.31,32
To this end, we chose N-[(5-bromo-1,2-oxazol-3-yl)methyl]-
adamantan-1-amine 17a and phenylboronic acid as a model
reaction for reaction condition optimization (Table 1). Using
microwave irradiation, a series of Suzuki coupling conditions
with variations in catalyst, solvent, base, and reaction time were
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Scheme 2. Synthesis of a Library of Isoxazole-Containing AM2-S31N Inhibitors Using the Optimized Suzuki−Miyaura Cross-
coupling Reaction Conditions
explored (Table 1). It was found that Pd(PPh3)4 was a
preferred catalyst over Pd(OAc)2, Pd2dba3, and Pd(dppf)Cl2.
Solvent and temperature also played important roles in this
reaction, and the highest yield (78%) was achieved when
dioxane was used as the solvent and the reaction was heated at
150 °C (Table 1, entry 7). A reaction performed at a lower
temperature, 120 °C, had a lower yield (Table 1, entry 11).
Reduction of the reaction time from 60 to 30 min had little to
no effect on the yield (Table 1, entry 7 versus entry 10).
Nevertheless, considering the yield might be substrate-depend-
ent, a 60 min reaction time was employed for the following
cross-coupling reactions. In summary, the optimized reaction
conditions were 5% Pd(PPh3)4, 1.2 equiv of boronic acid, 1.4
equiv of sodium bicarbonate, and heating at 150 °C for 60 min
in dioxane under microwave irradiation.
With the optimized cross-coupling conditions, we then
synthesized a library of isoxazole-containing compounds
(Scheme 2) with the substations at the 5-position of isoxazole
being substituted benzene (9a, 9d−9n), pyridine (9b−9c),
1,1′-diphenyl (9o), naphthalene (9p), and substituted thio-
phene (9q−9v). It was found that this cross-coupling
conditions were not very sensitive to the substrate, and the
reaction proceeded smoothly in the presence of functional
groups such as amine, alcohol, and phenol, with yields ranging
from 60% to 76%. Overall, this synthesis strategy proved to be
robust and amenable for parallel synthesis.
compound was added at 100 μM concentration, and the
channel conductance current at the 2 min time point was
recorded. The activity of the compound was expressed as a
percentage of current inhibition, which was calculated by
dividing the current at the 2 min time point by the maximum
conductance at pH 5.5. For AM2-S31N proton channel
inhibition, it was found that benzene and thiophene are the
preferred aryl substitutions at the 5-position of isoxazole (Table
2), while pyridines (9b and 9c) were not tolerated. The
channel blockage was also sensitive to the substitutions on the
benzene and thiophene rings. For example, when R equals
benzene, compounds with chloride (9d) and thiomethyl (9f)
substitutions at the ortho position had similar channel blockage
as compound 9a, while compounds with methoxyl (9e) and
hydroxyl (9h) had reduced channel blockage activity. When a
mesyl group was present at the ortho position, compound 9g
was nearly completely inactive. Compounds with substitutions
at the meta position of benzene (9i and 9j) were also less
potent in blocking the AM2-S31N channel than 9a, suggesting
substitution at the meta position is not tolerated. For
compounds with substitutions at the para position of benzene,
dimethylamine (9k), cyano (9l), and methyl ester (9n) were
tolerated, while amide (9m) was not compatible (47.4 0.5%).
Compounds with either 1,1′-diphenyl (9o) or naphthalene
(9p) substitution had decreased channel blockage activity,
probably due to steric hindrance. In contrast, when R equals
thiophene, compounds 9q−9v all showed potent channel
blockage (>78% at 100 μM). We also tested a few selected
compounds (9a, 9d, 9f, 9h, 9q, 9s, 9t, and 9v) against the wild-
type AM2 channel (AM2-WT). It was found that none of the
compounds had inhibition greater than 30% at 100 μM, except
compound 9v, which showed moderate inhibition (38.2
1.6%). This result was not a surprising finding, as this class of
compounds were designed to specifically target the amantadine-
resistant AM2-S31N mutant channel. In the next step, all
compounds were tested for their antiviral activity in plaque
assays at 10 μM against the amantadine-resistant influenza A
virus A/California/07/2009 (H1N1), which contains the AM2-
S31N mutation. Several compounds were able to reduce the
CHANNEL INHIBITION, ANTIVIRAL ACTIVITY, AND
CYTOTOXICITY OF ISOXAZOLE-CONTAINING
AM2-S31N INHIBITORS
■
All synthesized compounds were tested for channel blockage
against the AM2-S31N proton channel and for antiviral activity
against the A/California/07/2009 (H1N1) virus using the two-
electrode voltage clamp (TEVC) assay and plaque assay,
respectively (Table 2). TEVC is one of the standard assays used
to measure AM2 ion channel blockage by small molecules.
Briefly, the AM2-S31N channel was expressed in the oocyte cell
membrane and was activated by acidic pH (pH = 5.5),
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Table 2. Channel Blockage, Antiviral Activity, and Cytotoxicity of Isoxazole-Containing AM2-S31N Inhibitors
*
Values represent the mean of three independent measurements. The data are presented as percent inhibition at 100 μM at the 2 min time point.
N.T. = not determined.
plaque formation to less than 20% compared to that of the
DMSO control (9a, 9d, 9f, 9h, 9q, 9r, 9s, 9t, 9u, and 9v). All of
these compounds (9a, 9d, 9f, 9h, 9q, 9r, 9s, 9t, 9u, and 9v) also
had potent channel blockage against the AM2-S31N channel
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(>70% at 100 μM), which is consistent with their potent
antiviral activity. To further dissect the correlation between
channel blockage efficacy and antiviral activity of all the
synthesized AM2-S31N inhibitors, we plotted the percentage of
channel blockage against the percentage of plaque formation
and calculated the correlation coefficient (r) (Figure 2). The
MDCK cells and human epithelial A549 cells using the neutral
red method (Table 2).33 Compounds 9r, 9t, and 9u were found
to have moderate cytotoxicity against both MDCK and A549
cells, with CC50 values less than 65 μM; thus, these three
compounds were excluded from further studies. Compounds
9d and 9f also showed moderate cytotoxicity against the
MDCK cells (CC50 < 65 μM) but were less cytotoxic to the
A549 cells (CC50 > 100 μM). Compounds 9h, 9q, 9s, and 9v
appear to be well-tolerated for both MDCK and A549 cells, and
the CC50 values were greater than 70 μM.
Next, potent lead compounds with low cytotoxicity (9a, 9d,
9f, 9h, 9q, 9s, and 9v) were titrated against human clinical
isolates of influenza A viruses in the plaque assay (Figure 3).
According to the CC50 values (Table 2), the highest drug
concentration applied in the plaque assay was 30 μM, which
conferred minimal cellular cytotoxicity. It was found that all
Figure 2. Correlation of channel blockage and antiviral activity for
isoxazole-containing AM2-S31N inhibitors. P < 0.0001.
correlation efficient r was calculated to be −0.81, suggesting
that there was a strong correlation between the compound’s
channel blockage and antiviral activity. Specifically, 12 out of
the twenty-two compounds (9a, 9d, 9e, 9f, 9g, 9l, 9m, 9n, 9r,
9t, 9u, and 9v) fall on the curve, meaning that their channel
blockage correlated well with their antiviral activity. However,
there were compounds that fell both above and under the
curve. Four compounds (9b, 9i, 9j, and 9k) fell to the northeast
side of the curve (above the curve), meaning that their actual
antiviral activity was less potent than that predicted by their
AM2-S31N channel blockage efficacy. Likewise, six compounds
(9c, 9h, 9o, 9p, 9q, and 9s) fell to the southwest side of the
curve (below the curve), meaning that their actual antiviral
activity was more potent than that predicted by their AM2-
S31N channel blockage efficacy. The lack of a straight linear
correlation between the results from the in vitro target-specific
electrophysiological assay and the cellular antiviral assay might
be due to many factors, such as the compound’s membrane
permeability, off-target effects, binding kinetics, etc. Specifically,
in the antiviral plaque assay, compounds were incubated with
the virus-infected cells for 2 days; thus, the percentage of
plaque formation represents the true potency of the compound.
In contrast, the electrophysiological assay is under kinetic
control: the percentage of current inhibition was recorded at
the 2 min time point after applying the compounds in which
the drug-binding equilibrium may or may not be achieved.26
Therefore, the electrophysiological percentage inhibition might
underestimated the compound’s true potency if the compound
showed slow binding kinetics. In summary, the electro-
physiological assay results should be viewed as a means to
confirm the compound’s mechanism of action but not a
predictor for the compound’s cellular antiviral potency.
Understanding this concept is critical for the lead optimization,
as it suggests that the structure−activity relationship studies
should not only focus on improving the channel blockage
activity, but also on improving the cellular antiviral potency.
Another important factor that needs to be taken into
consideration during lead optimization is the cellular toxicity.
For this reason, the cytotoxicity of the potent lead compounds
(9d, 9f, 9h, 9q, 9r, 9s, 9t, 9u, and 9v) was tested against both
Figure 3. Antiviral efficacy of potent AM2-S31N inhibitors in
inhibiting clinically relevant human influenza A viruses. The antiviral
efficacy was determined using plaque reduction assays. (A) Dose
response curves of compounds 9a, 9d, 9f, and 9h in inhibiting the A/
California/07/2009 (H1N1) virus. (A) Dose response curves of
compounds 9q, 9s, and 9v in inhibiting the A/California/07/2009
(H1N1) virus. (B) Dose response curves of compound 9q in
inhibiting the A/California/07/2009 (H1N1), A/Washington/29/
2009 (H1N1), A/Switzerland/9715293/2013 (H3N2), and A/Den-
mark/528/2009 (H1N1) viruses. Each data point represents duplicate
experiments.
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Figure 4. Molecular docking of compound 9q in the AM2-S31N channel. (A) X-ray crystal structure of the drug-free AM2-S31N structure (PDB:
5C02).22 (B) Overlay of the drug-free AM2-S31N structure (orange) and the amantadine-bound AM2-WT structure (cyan) (PDB: 3C9J).18 (C)
Docking model of compound 9q in the AM2-S31N channel. The solution NMR structure (PDB: 2LY0) of AM2-S31N was used for the docking.21
Docking was performed using Autodock Vina.35 (D) Overlay of the docking model (gray) and drug-free AM2-S31N structure (orange) (PDB:
5C02).22
compounds had potent antiviral activity against the 2009
pandemic H1N1 virus A/California/07/2009 (H1N1) in a
dose-dependent manner except compound 9v, which showed
incomplete inhibition at the highest drug concentration of 30
μM (Figure 3A and 3B). Testing of compound 9v at a higher
drug concentration was not attempted due to cellular
cytotoxicity. The EC50 values for compounds 9a, 9d, 9f, 9h,
9q, and 9s range from 0.1 μM to 1.2 μM, with the most potent
compound being 9q (EC50 = 0.1 0.03 μM). Compound 9q
was also noncytotoxic, and the CC50 values for MDCK cells
and A549 cells were 190.5 14.2 μM and 208.6 20.9 μM,
respectively, resulting in selectivity indexes of 1905 and 2086
for MDCK and A549 cells, respectively. To further profile its
antiviral spectrum, compound 9q was tested against three
additional influenza A strains, A/Washington/29/2009
(H1N1), A/Denmark/528/2009 (H1N1), and A/Switzer-
land/9715293/2013 (H3N2). The A/Washington/29/2009
(H1N1) and A/Denmark/528/2009 (H1N1) viruses were
chosen because they are representative examples of multidrug-
resistant influenza A viruses, and these two strains are resistant
to both amantadine and oseltamivir due to AM2-S31N and
H275Y mutations in their AM2 and neuraminidase genes,
respectively. The A/Switzerland/9715293/2013 (H3N2) virus
was chosen because it is an emerging predominant strain that
was circulating in the 2014−2015 influenza season in North
America.34 This strain is resistant to amantadine due to AM2-
S31N mutation but is sensitive to oseltamivir. It was found that
compound 9q had potent antiviral activity against all three
drug-resistant influenza A strains, with EC50 values ranging
from 0.1 μM to 0.2 μM. Overall, these results suggest that
AM2-S31N inhibitors are potent antivirals against influenza A
viruses with diverse genetic backgrounds.
contrast, when AM2-S31N inhibitors such as M2WJ332 bind to
the AM2-S31N channel, the channel adapts the Openout-
Closedin conformation as shown by the solution NMR structure
(PDB: 2LY0).21 For this reason, the Openout-Closedin
conformation of the AM2-S31N channel (PDB: 2LY0) was
chosen for the docking of the most potent compound 9q. It
was found that compound 9q binds to the AM2-S31N channel
with its aryl group facing toward the N-terminus of the channel
(Figure 4C). The adamantane ring of 9q is surrounded by the
G34 residues, and the positively charged ammonium from 9q
forms a hydrogen bond with one of the N31 side chain
carbonyl groups. The distal thiophene ring from 9q is
surrounded by the V27 side chain methyl groups, and this
might be driven by hydrophobic interactions. It can be seen
clearly from the overlay structures of the docking model and
the drug-free AM2-S31N structure (Figure 4D) that when
compound 9q binds to the AM2-S31N channel, the channel
undergoes a conformation change, leading to the opening of
the N-terminus of the channel to accommodate the bulky drug
9q. In summary, the docking model shows that compound 9q
binds to the Openout-Closedin conformation of the AM2-S31N
channel, which is consistent with the previously solved solution
NMR structure (PDB: 2LY0).21
CONCLUSIONS
■
Despite the urgent need of influenza antivirals to combat not
only existing drug-resistant strains but also emerging or re-
emerging strains that will undoubtedly appear in the near
future, the drug discovery process, however, is unfortunately a
lengthy process. With our continuous efforts in developing
antivirals to target the predominant drug-resistant AM2-S31N
mutant, we aimed to develop an expeditious synthesis strategy
in order to speed up the iterative cycles of design, synthesis, and
biological characterization. The synthesis route we devised
allowed us to introduce a diverse set of substitutions at the 5-
position of isoxazole at the last step, and the optimized Suzuki−
Miyaura cross-coupling reaction condition was compatible with
functional groups such as amine, hydroxyl, and phenol. All
synthesized compounds were tested for their channel blockage
in electrophysiological assays, and potent lead compounds were
further validated in antiviral assays. Potent channel blockers
To gain further insights into how the most potent compound
9q binds to the AM2-S31N channel, molecular docking was
performed (Figure 4). In the drug-free form, AM2-S31N adapts
the Closedout-Openin conformation, as shown by the X-ray
crystal structure (PDB: 5C02) in Figure 4A.22 The AM2-WT
channel similarly adapts the Closedout-Openin conformation
when it binds to amantadine.18 The overlay of the drug-free
AM2-S31N structure (PDB: 5C02) with the amantadine-bound
AM2-WT structure (PDB: 3C9J) was shown in Figure 4B. In
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7.1 Hz, 2H), 3.71 (s, 2H), 1.43 (t, J = 7.1 Hz, 3H). 13C NMR (101
MHz, CDCl3) δ 172.83, 158.11, 157.51, 63.24, 33.25, 14.00.
C6H7NO4 EI-MS: m/z (M+H+): 158.1 (calculated), 158.0 (found).
Yield: 40%.
were generally found to have greater antiviral efficacy; however,
the correlation was not straightly linear (r = −0.81). It is worth
highlighting that this was the first attempt to systematically
correlate the channel blockage activity with antiviral activity for
AM2-S31N inhibitors. The important message is that the
TEVC assay results can be used to confirm the compound’s
mechanism of action, but caution should be taken to use them
to predict the antiviral activity. To corroborate with this
conclusion, the most potent compound emerging from this
study was compound 9q, which had single- to submicromolar
efficacy against all four AM2-S31N-containing influenza A
viruses (A/California/07/2009 (H1N1), A/Washington/29/
2009 (H1N1), A/Denmark/528/2009 (H1N1), and A/
Switzerland/9715293/2013 (H3N2)). However, this com-
pound did not necessarily have the most potent channel
blockage (78.8 3.0% at 100 μM) when compared with other
compounds, such as 9a and 9f (>90% inhibition at 100 μM).
Overall, these results suggest that lead optimization should not
focus solely on improving the channel blockage potency, and
optimizing antiviral activity should also be taken into
consideration.
Ethyl 5-(Tributylstannyl)isoxazole-3-carboxylate (13). To a
solution of ethyl 2-chloro-2-(hydroxyimino)acetate (1.2 g, 7.9
mmol) in anhydrous dichloromethane (20 mL) was added
ethynyltributylstannane (2.5 g, 7.9 mmol) and potassium carbonate
(1.2 g, 8.7 mmol) at room temperature. The mixture was stirred for 24
h and was then quenched with water. The resulting solution was
extracted with dichloromethane. The organic layer was separated,
dried over anhydrous magnesium sulfate, filtered and concentrated
under reduced pressure. The mixture was then purified by silica gel
flash column chromatography (0−5% EtOAc/Hexane) to give the
final product as a colorless oil. 1H NMR (400 MHz, CDCl3) δ 6.82 (s,
1H), 4.47 (q, J = 7.1 Hz, 2H), 1.74−1.56 (m, 8H), 1.45 (t, J = 7.1 Hz,
3H), 1.40−1.20(m, 16H), 0.98−0.89 (m, 12H). C18H33NO3Sn EI-
MS: m/z (M+H+): 432.1 (calculated), 432.0 (found). Yield: 75%.
Ethyl 5-Bromoisoxazole-3-carboxylate (14). To a mixture of 11
(550 mg, 3.5 mmol) in toluene (50 mL) was added phosphoryl
bromide (5.5 g, 17.5 mmol) and triethyl amine (2 mL) at 0 °C. The
mixture was stirred at room temperature for 1 h and then heated at 80
°C for 48 h. The resulting residue was cooled to room temperature,
quenched with water, and extracted with dichloromethane. The
organic layer was separated, dried over anhydrous magnesium sulfate,
filtered, and concentrated under reduced pressure. The mixture was
then purified by silica gel flash column chromatography (0−10%
In summary, the potent antiviral efficacy, coupled with the
high selectivity index of compound 9q, warrants further
developing it as a next-generation of antiviral drug. The
major therapeutic value of AM2-S31N inhibitors is that they are
active against both oseltamivir-sensitive and -resistant strains;
therefore, they can either be used alone to combat oseltamivir-
resistant strains or be used in combination with oseltamivir to
delay the evolution of resistant strains.
1
EtOAc/Hexane) to give the final product as a colorless oil. H NMR
(400 MHz, CDCl3) δ 6.70 (s, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.47−
1.35 (t, J = 7.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 158.75,
157.95, 143.16, 107.10, 62.58, 14.09. C6H7BrNO3 EI-MS: m/z (M
+H+): 220.0 (calculated), 220.1 (found). Yield: 23%.
Ethyl 5-Bromoisoxazole-3-carboxylate (14). To a mixture of 13
(0.9 g, 2.1 mmol) and sodium carbonate (250 mg, 2.3 mmol) in
dichloromethane (15 mL) was added bromine (82 uL, 3.1 mmol) at
room temperature. The mixture was stirred for 24 h and then
quenched with sodium thiosuflate solution (10%, 10 mL). The
resulting residue was extracted with dichloromethane. The organic
layer was separated, dried over anhydrous magnesium sulfate, filtered
and concentrated under reduced pressure. The mixture was then
purified by silica gel flash column chromatography (0−10% EtOAc/
Hexane) to give the final product as a colorless oil (415 mg). Yield:
90%.
EXPERIMENTAL SECTION
■
Chemistry. All chemicals that were commercially available were
used without further purification. Detailed synthesis procedures can be
found below for reactions described in Scheme 1, Table 1, and Scheme
2. All final compounds were purified by flash column chromatography.
1H and 13C NMR spectra were recorded on a Bruker-400 NMR
spectrometer. Chemical shifts are reported in parts per million
referenced with respect to residual solvent (CD3OD) 3.31 ppm and
(Chloroform-d) 7.24 ppm or from internal standard tetramethylsilane
(TMS) 0.00 ppm. The following abbreviations were used in reporting
spectra: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; dd,
doublet of doublets; ddd, doublet of doublet of doublets. All reactions
were carried out under N2 atmosphere, unless otherwise stated.
HPLC-grade solvents were used for all reactions. Flash column
chromatography was performed using silica gel (230−400 mesh,
Merck). Low-resolution mass spectra were obtained using an ESI
technique on a 3200 Q Trap LC/MS/MS system (Applied
Biosystems). The purity was assessed by using Shimadzu LC-MS
with Waters XTerra MS C-18 column (part #186000538), 50 × 2.1
mm, at a flow rate of 0.3 mL/min; λ = 250 and 220 nm; mobile phase
A, 0.1% formic acid in H2O, and mobile phase B′, 0.1% formic in 60%
isopropanol, 30% CH3CN and 9.9% H2O. All compounds submitted
for testing in TEVC assay and plaque reduction assay were confirmed
to be >95.0% purity by LC-MS traces. All compounds were
characterized by proton and carbon NMR and MS.
(5-Bromoisoxazol-3-yl)methanol (15). To a solution of 14 (160
mg, 0.73 mmol) in tetrahydrofuran (10 mL) at 0 °C was added
diisobutylaluminum hydride (4 mL, 1.1 M, 4.4 mmol) and the
resulting solution was stirred for 2 h. Then the reaction was quenched
by adding 1 M HCl (10 mL) and the solution was stirred at room
temperature for 2 h. The resulting solution was extracted with
dichloromethane. The organic layer was separated, dried over
anhydrous magnesium sulfate, filtered, and concentrated under
reduced pressure. The mixture was clean on NMR and was used
1
directly for the next step oxidation without further purification. H
NMR (400 MHz, CDCl3) δ 6.37 (d, J = 1.1 Hz, 1H), 4.72 (d, J = 5.0
Hz, 2H), 3.21 (br, 1H). 13C NMR (101 MHz, CDCl3) δ 165.59,
141.80, 105.48, 56.81. C4H5BrNO2 EI-MS: m/z (M+H+): 178.0
(calculated), 178.1 (found). Yield: 85%.
5-Bromoisoxazole-3-carbaldehyde (16). To a mixture of alcohol
15 (200 mg, 1.12 mmol) in dichloromethane was added Dess-Martin
periodinane (712 mg, 1.68 mmol) and the solution was stirred for 1 h.
The reaction was quenched with sodium thiosulfate (10%, 5 mL) and
saturated sodium bicarbonate (5 mL) solution. The resulting mixture
was extracted with ethyl ether (2 × 10 mL) and ethyl acetate (2 × 10
mL). The organic layer was separated, dried over anhydrous
magnesium sulfate, filtered, and concentrated under reduced pressure.
The mixture was then purified by silica gel flash column
chromatography (0−20% EtOAc/Hexane) to give 16 as pale yellow
Synthesis Procedures. Ethyl 5-Oxo-4,5-dihydroisoxazole-3-
carboxylate (11). To a suspension of diethyl oxalacetate 10 (3 g,
15.94 mmol) in water (20 mL) was added sodium bicarbonate (1.2 g,
17.1 mmol) and hydroxylamine hydrochloride (1.2 g, 34.5 mmol) at
room temperature. The mixture was stirred for 18 h. Then HCl (1M,
20 mL) was added and the resulting solution was extracted with
dichloromethane. The combined organic layers were then dried over
MgSO4, filtered and concentrated under reduced pressure. The
resulting mixture was then purified by silica gel flash column
chromatography (0−10% EtOAc/Hexane) to give the final product
solid. 1H NMR (400 MHz, CDCl3) δ 10.07 (s, 1H), 6.68 (s, 1H). 13
C
1
as a white solid (1.01 g) H NMR (400 MHz, CDCl3) δ 4.45 (q, J =
NMR (101 MHz, CDCl3) δ 183.28, 163.12, 143.97, 104.07.
H
DOI: 10.1021/acs.jmedchem.6b01852
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
C4H3BrNO2 EI-MS: m/z (M+H+): 176.0 (calculated), 176.1
(found). Yield: 64%.
13C NMR (100 MHz, CDCl3 + CD3OD) δ 169.23, 156.74, 137.92,
130.33, 128.63, 126.06, 124.35, 104.26, 68.30, 60.35, 45.81, 42.86,
36.60, 35.61, 33.58, 30.01, 15.83. C21H26N2O2S EI-MS: m/z (M
+H+): 371.5 (calculated), 371.0(found). Yield: 75%.
N-[(5-Bromo-1,2-oxazol-3-yl)methyl]adamantan-1-amine (17a).
Adamantane 8a (147 mg, 0.97 mmol) and 16 (170 mg, 0.97 mmol)
were mixed with 1.5 mL of titanium(IV) isopropoxide. The resulting
slurry was heated in microwave at 120 °C for 30 min. Then the
solution was cooled down to room temperature and CH3OH (10 mL)
was added. The resulting solution was cooled to 0 °C and NaBH4 (147
mg, 3.88 mmol) was added portionwise in 10 min. The solution was
then warmed to room temperature and stirred for another hour. The
reaction was quenched with 1 M NaOH and filtered through Celite.
The filtrate was extracted with dichloromethane and was purified by
silica gel flash column chromatography (5−10% CH3OH/CH2Cl2) to
give the final product as a white solid. 1H NMR (400 MHz, CDCl3) δ
6.25 (t, J = 0.9 Hz, 1H), 3.90 (s, 2H), 2.07 (s, 3H), 1.74−1.53 (m,
12H). C14H20BrN2O EI-MS: m/z (M+H+): 311.0 (calculated),
311.1 (found). Yield: 79%.
N-((5-(2-(Methylsulfonyl)phenyl)isoxazol-3-yl)methyl)-
1
adamantan-1-amine (9g). H NMR (400 MHz, CD3OD) δ 7.70−
7.60 (m, 1H), 7.40 (td, J = 7.4, 1.3 Hz, 1H), 7.35−7.29 (m, 1H), 7.23
(d, J = 7.3 Hz, 1H), 6.52 (s, 1H), 4.18 (s, 2H), 2.83 (s, 3H), 1.93 (q, J
= 3.0 Hz, 4H), 1.68 (d, J = 2.9 Hz, 7H), 1.55−1.40 (m, 5H).
C21H27N2O3S EI-MS: m/z (M+H+): 387.5 (calculated), 387.0
(found). Yield: 69%.
2-(3-(((Adamantan-1-yl)amino)methyl)isoxazol-5-yl)phenol (9h).
1H NMR (400 MHz, CDCl3) δ 7.96−7.90 (m, 1H), 7.53−7.47 (m,
1H), 7.46−7.36 (m, 2H), 6.98 (s, 1H), 3.96 (s, 2H), 2.13 (s, 3H),
1.75−1.63 (m, 12H). C20H24N2O2 EI-MS: m/z (M+H+): 325.4
(calculated), 325.0 (found). Yield: 68%.
N-((5-(3-(Methylthio)phenyl)isoxazol-3-yl)methyl)adamantan-1-
1
3-{[(5-Bbromo-1,2-oxazol-3-yl)methyl]amino}adamantan-1-ol
amine (9i). H NMR (400 MHz, CD3OD) δ 7.56−7.35 (m, 2H),
(17b). Compound 17b was synthesized using the same procedure as
7.32−7.24 (m, 2H), 6.82 (d, J = 0.7 Hz, 1H), 4.49 (d, J = 0.6 Hz, 2H),
2.46 (s, 3H), 2.28−2.21 (m, 3H), 1.98 (m, 6H), 1.88−1.66 (m, 6H).
C21H27N2OS EI-MS: m/z (M+H+): 355.5 (calculated), 355.1
(found). Yield: 77%.
1
described for 17a. H NMR (400 MHz, CDCl3) δ 7.23 (s, 1H), 6.24
(s, 1H), 3.89 (s, 2H), 2.26 (br, 3H), 1.75−1.40 (m, 14H). 13C NMR
(101 MHz, CDCl3) δ 174.88, 140.35, 105.30, 69.59, 54.12, 50.09,
44.31, 41.29, 37.56, 37.35, 37.15, 35.00, 30.85, 30.83. C14H20BrN2O2
EI-MS: m/z (M+H+): 327.1 (calculated), 327.0 (found). Yield: 82%.
General Procedure of Suzuki Coupling Reaction (Table 1).
To a solution of admantane isoxazole bromide (17a-b) (1 equiv) in
dioxane (2 mL) was added boronic acid or boronic ester (1.2 equiv)
and sodium carbonate (1.4 equiv). The mixture was purged with dry
nitrogen for 5 min. Then tetrakis(triphenylphosphine)palladium(0)
(0.05 equiv) was added and the solution was heated in microwave at
150 °C for 30 min. The solvent was removed under reduced pressure,
and the resulting residue was extracted with dichloromethane and
water. The organic layer was separated, dried over anhydrous
magnesium sulfate, filtered, and concentrated under reduced pressure.
The mixture was then purified by silica gel flash column
chromatography (0−10% CH3OH/CH2Cl2) to give the final product.
N-[(5-Phenyl-1,2-oxazol-3-yl)methyl]adamantan-1-amine (9a).
Characterization of 9a was reported before.21
N-((5-(3-(Trifluoromethoxy)phenyl)isoxazol-3-yl)methyl)-
adamantan-1-amine (9j). 1H NMR (400 MHz, CD3OD) δ 7.64 (d, J
= 7.4 Hz, 1H), 7.51 (s, 1H), 7.46−7.41 (m, 1H), 7.28 (ddt, J = 8.2, 2.4,
1.1 Hz, 1H), 6.81 (s, 1H), 4.49 (d, J = 0.6 Hz, 2H), 2.25 (s, 3H), 1.98
(d, J = 3.0 Hz, 6H), 1.90−1.59 (m, 6H). C21H24F3N2O2 EI-MS: m/
z (M+H+): 393.4 (calculated), 393.1 (found). Yield: 70%.
N-((5-(4-(Dimethylamino)phenyl)isoxazol-3-yl)methyl)-
adamantan-1-amine (9k). 1H NMR (400 MHz, CDCl3) δ 7.96−7.90
(m, 1H), 7.53−7.47 (m, 1H), 7.46−7.36 (m, 2H), 6.98 (s, 1H), 3.96
(s, 2H), 2.13 (s, 3H), 1.75−1.63 (m, 12H). C22H29N3O EI-MS: m/z
(M+H+): 352.5 (calculated), 352.0 (found). Yield: 76%.
4-(3-(((Adamantan-1-yl)amino)methyl)isoxazol-5-yl)benzonitrile
(9l). 1H NMR (400 MHz, CDCl3) δ 7.88−7.71 (m, 2H), 7.28 (dt, J =
7.9, 1.0 Hz, 2H), 6.56 (s, 1H), 3.89 (s, 2H), 2.09 (s, 3H), 1.72−1.47
(m, 12H). C21H23N3O EI-MS: m/z (M+H+): 334.4 (calculated),
334.0 (found). Yield: 71%.
N-((5-(2-(Methylsulfonyl)phenyl)isoxazol-3-yl)methyl)-
adamantan-1-amine (9m). 1H NMR (400 MHz, CDCl3) δ 9.63 (br,
2H), 7.64 (ddd, J = 6.8, 3.5, 1.7 Hz, 2H), 7.07 (dd, J = 5.6, 2.9 Hz,
2H), 6.87−6.74 (m, 1H), 4.19 (d, J = 4.5 Hz, 2H), 2.09 (s, 3H), 1.91
(d, J = 4.1 Hz, 6H), 1.75−1.49 (m, 6H). C21H26N3O2 EI-MS: m/z
(M+H+): 352.5 (calculated), 352.1 (found). Yield: 73%.
N-((5-(Pyridin-3-yl)isoxazol-3-yl)methyl)adamantan-1-amine
1
(9b). H NMR (400 MHz, CDCl3) δ 9.02 (dd, J = 2.3, 0.9 Hz, 1H),
8.69 (dd, J = 4.8, 1.7 Hz, 1H), 8.15 (ddd, J = 7.9, 2.3, 1.7 Hz, 1H), 7.41
(ddd, J = 7.9, 4.8, 0.9 Hz, 1H), 6.58 (t, J = 0.8 Hz, 1H), 4.01 (d, J = 0.9
Hz, 2H), 2.14 (s, 3H), 1.84−1.59 (m, 12H). C19H24N3O EI-MS: m/
z (M+H+): 310.4 (calculated), 310.0 (found). Yield: 61%.
Methyl 4-(3-(((Adamantan-1-yl)amino)methyl)isoxazol-5-yl)-
N-((5-(6-Methoxypyridin-3-yl)isoxazol-3-yl)methyl)adamantan-
1
1
benzoate (9n). H NMR (400 MHz, CDCl3) δ 7.72 (d, J = 9.0 Hz,
1-amine (9c). H NMR (400 MHz, CD3OD) δ 8.19 (dd, J = 5.2, 2.3
Hz, 1H), 8.05 (ddd, J = 7.2, 5.1, 2.1 Hz, 1H), 7.14−7.00 (m, 1H), 6.84
(d, J = 1.7 Hz, 1H), 4.53 (s, 2H), 3.31 (s, 3H), 2.31−2.18 (m, 3H),
2.01−1.96 (m, 6H), 1.86−1.72(m, 6H). C20H26N3O2 EI-MS: m/z
(M+H+): 340.4 (calculated), 340.0 (found). Yield: 70%.
2H), 7.06−6.96 (m, 2H), 6.46 (s, 1H), 3.91 (d, J = 0.3 Hz, 2H), 3.88
(s, 3H), 2.13 (s, 3H), 1.84−1.60 (m, 12H). C22H27N2O3 EI-MS: m/
z (M+H+): 367.5 (calculated), 367.0 (found). Yield: 72%.
N-((5-([1,1′-Biphenyl]-4-yl)isoxazol-3-yl)methyl)adamantan-1-
1
amine (9o). H NMR (400 MHz, CDCl3) δ 7.57−7.52 (m, 5H),
3-({[5-(2-Chlorophenyl)-1,2-oxazol-3-yl]methyl}amino)-
1
adamantan-1-ol (9d). HNMR (400 MHz, CDCl3 + CD3OD): δ
7.48−7.40 (m, 4H), 7.38−7.32 (m, 1H), 6.25 (t, J = 0.9 Hz, 1H), 3.90
(d, J = 0.8 Hz, 2H), 2.08 (s, 3H), 1.78−1.50 (m, 12H). C26H28N2O
EI-MS: m/z (M+H+): 385.5 (calculated), 385.0 (found). Yield: 76%.
N-((5-(Naphthalen-1-yl)isoxazol-3-yl)methyl)adamantan-1-
amine (9p). 1H NMR (400 MHz, CD3OD) δ 7.85 (dt, J = 7.8, 1.0 Hz,
2H), 7.76 (d, J = 7.6 Hz, 1H), 7.58−7.38 (m, 4H), 6.80 (d, J = 0.6 Hz,
1H), 4.47 (d, J = 0.5 Hz, 2H), 2.22 (s, 3H), 1.95 (d, J = 3.0 Hz, 6H),
1.84−1.69 (m, 6H). C24H27N2O EI-MS: m/z (M+H+): 359.5
(calculated), 359.0 (found). Yield: 70%.
7.80−7.78 (m, 1H), 7.51−7.48 (m, 1H), 7.44−7.41 (m, 1H), 7.34−
7.30 (m, 2H), 4.21 (s, 2H), 2.31 (s, 2H), 1.96 (s, 2H), 1.91−1.85 (m,
4H), 1.65−1.62 (m, 4H), 1.51 (s, 2H). 13CNMR (100 MHz, CDCl3 +
CD3OD) δ 171.97, 160.83, 135.98, 135.40, 134.86, 133.31, 131.17,
129.43, 108.82, 72.22, 64.48, 49.42, 46.66, 40.79, 39.29, 38.02, 34.10.
C20H23ClN2O2 EI-MS: m/z (M+H+): 359.9 (calculated), 360.0
(found). Yield: 72%.
N-((5-(2-Methoxyphenyl)isoxazol-3-yl)methyl)adamantan-1-
amine (9e). 1H NMR (400 MHz, CDCl3) δ 7.76 (ddd, J = 7.8, 1.6, 0.5
Hz, 1H), 7.37 (ddd, J = 8.2, 7.3, 1.5 Hz, 1H), 7.30 (dd, J = 8.1, 1.3 Hz,
1H), 7.24−7.20 (m, 1H), 6.82 (s, 1H), 3.92 (s, 2H), 2.49 (s, 3H), 2.08
(s, 3H), 1.72−1.59 (m, 12H). C21H26N2O2 EI-MS: m/z (M+H+):
339.5 (calculated), 339.0 (found). Yield: 74%.
N-((5-(5-Chlorothiophen-2-yl)isoxazol-3-yl)methyl)adamantan-
1
1-amine (9q). H NMR (400 MHz, CDCl3) δ 7.33 (d, J = 6.0 Hz,
1H), 7.11 (d, J = 5.9 Hz, 1H), 6.22 (s, 1H), 3.89 (s, 2H), 2.07 (s, 3H),
1.74−1.55 (m, 13H). C18H22ClN2OS EI-MS: m/z (M+H+): 349.9
(calculated), 350.0 (found). Yield: 65%.
N-((5-(5-Chlorothiophen-3-yl)isoxazol-3-yl)methyl)adamantan-
3-[({5-[2-(Methylsulfanyl)phenyl]-1,2-oxazol-3-yl}methyl)amino]-
1
1
1-amine (9r). H NMR (400 MHz, CD3OD) δ 7.54 (s, 1H), 6.93 (s,
adamantan-1-ol (9f). H NMR (400 MHz, CDCl3 + CD3OD): δ
7.68−7.65 (m, 1H), 7.38 (s, 1H), 7.37−7.34 (m, 1H), 7.28−7.26 (m,
1H), 7.20−7.16 (m, 1H), 4.23 (s, 2H), 3.64 (s, 1H), 2.45 (s, 3H), 2.34
(s, 2H), 2.03 (s, 2H), 1.95−1.87 (m, 4H), 1.68 (m, 4H), 1.58 (s, 2H).
1H), 6.61−6.46 (s, 1H), 3.95 (s, 2H), 2.12−2.01 (m, 3H), 1.74−1.59
(m, 12H). C18H22ClN2OS EI-MS: m/z (M+H+): 349.9 (calculated),
350.0 (found). Yield: 60%.
I
DOI: 10.1021/acs.jmedchem.6b01852
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3-({[5-(3-Methoxythiophen-2-yl)-1,2-oxazol-3-yl]methyl}amino)-
adamantan-1-ol (9s). Characterization of compound 9s was reported
before.29
N-((5-(2-Methoxythiophen-3-yl)isoxazol-3-yl)methyl)-
adamantan-1-amine (9t). 1H NMR (400 MHz, CDCl3) δ 7.39−7.22
(m, 1H), 6.54 (d, J = 5.3 Hz, 1H), 6.28 (d, J = 1.0 Hz, 1H), 4.77 (br,
1H), 3.90 (d, J = 0.9 Hz, 2H), 3.83 (s, 3H), 2.09 (d, J = 3.8 Hz, 3H),
1.79−1.56 (m, 12H). C19H25N2O2S EI-MS: m/z (M+H+): 345.5
(calculated), 345.0 (found). Yield: 68%.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b01852.
■
S
SMILES data, electrophysiological assay results, antiviral
efficacy, and cytotoxicity results (CSV)
N-((5-(2,5-Dichlorothiophen-3-yl)isoxazol-3-yl)methyl)-
1
adamantan-1-amine (9u). H NMR (400 MHz, CDCl3) δ 7.05 (s,
AUTHOR INFORMATION
Corresponding Author
*Jun Wang, Tel: 520-626-1366, Fax: 520-626-0749, e-mail:
junwang@pharmacy.arizona.edu.
ORCID
Jun Wang: 0000-0002-4845-4621
Notes
The authors declare no competing financial interest.
■
1H), 6.82 (s, 1H), 3.91 (d, J = 1.0 Hz, 2H), 2.07 (s, 3H), 1.74−1.57
(m, 12H). C18H21Cl2N2OS EI-MS: m/z (M+H+): 384.3 (calcu-
lated), 384.3 (found). Yield: 67%.
3-({[5-(2,5-Dichlorothiophen-3-yl)-1,2-oxazol-3-yl]methyl}-
amino)adamantan-1-ol (9v). 1H NMR (400 MHz, CDCl3) δ 7.24 (s,
1H), 6.25 (s, 1H), 3.90 (d, J = 0.9 Hz, 2H), 2.32−2.22 (m, 2H), 1.70−
1.42 (m, 13H). C18H21Cl2N2O2S EI-MS: m/z (M+H+): 400.3
(calculated), 400.0 (found). Yield: 60%.
Two-Electrode Voltage Clamp (TEVC) Assay. The compounds
were tested in a two-electrode voltage clamp assay using Xenopus laevis
frog oocytes microinjected with RNA expressing either the AM2-WT
or the AM2-S31N mutant of the AM2 protein, as previously
reported.36 The potency of the inhibitors was expressed as percentage
inhibition of A/M2 current observed after 2 min of incubation with
100 μM of compounds at pH 5.5. All measurements were repeated
three times with different oocytes.
ACKNOWLEDGMENTS
■
This research is supported by startup funding from the
University of Arizona, the 2015 PhRMA Foundation Research
Starter Grant in Pharmacology and Toxicology, and NIH grant
AI119187 to J.W. We thank David Bishop for proofreading and
editing the manuscript.
Plaque Reduction Assay. The plaque reduction assay was
performed as previously reported,21 except MDCK cells expressing
ST6Gal I were used instead of regular MDCK cells.37 Briefly, confluent
monolayer of ST6Gal MDCK cells were incubated with ∼100 pfu
virus samples in DMEM with 0.5% BSA for 1 h at 4 °C, then 37 °C for
1 h. The inoculums were removed, and the cells were washed with
phosphate buffered saline (PBS). The cells were then overlaid with
DMEM containing 1.2% Avicel microcrystalline cellulose (FMC
BioPolymer, Philadelphia, PA) and NAT (2.0 μg/mL). To examine
the effect of the compounds on plaque formation, the overlay media
was supplemented compounds at testing concentrations. At 2 days
after infection, the monolayers were fixed and stained with crystal
violet dye solution (0.2% crystal violet, 20% methanol). The viruses
used for this assay were A/California/07/2009 (H1N1), A/
Washington/29/2009 (H1N1), A/Denmark/528/2009 (H1N1) and
A/Switzerland/9715293/2013 (H3N2), all of which contain the AM2-
S31N mutant in their AM2 genes. Influenza A viruses A/Switzerland/
9715293/2013 X-247 (H3N2), FR-1366, A/Washington/29/2009
(H1N1), FR-460, and A/California/07/2009 (H1N1), FR-201, were
obtained through the Influenza Reagent Resource, Influenza Division,
WHO Collaborating Center for Surveillance, Epidemiology and
Control of Influenza, Centers for Disease Control and Prevention,
Atlanta, GA, USA. Influenza virus A/Denmark/528/2009 (H1N1) was
obtained from Dr. Elena Govorkova at St. Jude Children’s Research
Hospital.
ABBREVIATIONS USED
■
WT, wild-type; DMEM, Dulbecco’s modified eagle medium;
MDCK, Madin−Darby Canine Kidney; TEVC, two-electrode
voltage clamps
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