Design and expeditious synthesis of organosilanes as potent antivirals targeting multidrug-resistant influenza A viruses

Article abstract of DOI:10.1016/j.ejmech.2017.04.038

The efficacy of current influenza vaccines and small molecule antiviral drugs is curtailed by the emerging of multidrug-resistant influenza viruses. As resistance to the only FDA-approved oral influenza antiviral, oseltamivir (Tamiflu), continues to rise, there is a clear need to develop the next-generation of antiviral drugs. Since more than 95% of current circulating influenza A viruses carry the S31N mutation in their M2 genes, the AM2-S31N mutant proton channel represents an attractive target for the development of broad-spectrum antivirals. In this study we report the design and synthesis of the first class of organosilanes that have potent antiviral activity against a panel of human clinical isolates of influenza A viruses, including viruses that are resistant to amantadine, oseltamivir, or both.

Full text of DOI:10.1016/j.ejmech.2017.04.038

European Journal of Medicinal Chemistry 135 (2017) 70e76
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design and expeditious synthesis of organosilanes as potent antivirals
targeting multidrug-resistant influenza A viruses
, b, *
Yanmei Hu a, Yuanxiang Wang a, Fang Li a, Chunlong Ma b, Jun Wang a
a Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, AZ 85721, United States
b BIO5 Institute, The University of Arizona, Tucson, AZ, 85721, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
The efficacy of current influenza vaccines and small molecule antiviral drugs is curtailed by the emerging
of multidrug-resistant influenza viruses. As resistance to the only FDA-approved oral influenza antiviral,
oseltamivir (Tamiflu), continues to rise, there is a clear need to develop the next-generation of antiviral
drugs. Since more than 95% of current circulating influenza A viruses carry the S31N mutation in their M2
genes, the AM2-S31N mutant proton channel represents an attractive target for the development of
broad-spectrum antivirals. In this study we report the design and synthesis of the first class of orga-
nosilanes that have potent antiviral activity against a panel of human clinical isolates of influenza A
viruses, including viruses that are resistant to amantadine, oseltamivir, or both.
Received 4 February 2017
Received in revised form
12 April 2017
Accepted 14 April 2017
Keywords:
Influenza A virus
AM2 proton channel
AM2-S31N inhibitor
Organosilane
© 2017 Elsevier Masson SAS. All rights reserved.
Antiviral
1. Introduction
the prevention and treatment of influenza virus infection (Fig. 1):
M2 inhibitors, e.g., amantadine and rimantadine, that inhibit virus
The current countermeasures in preventing and treating influ-
enza virus infection have limited efficacy [1]: despite the existence
of vaccines and antiviral drugs, influenza virus infection accounts
for approximately 36,000 deaths and millions of hospitalizations in
the United States during the annual influenza epidemic [2e4]. In
addition, influenza pandemics caused by emerging or re-emerging
influenza viruses have more catastrophic impact as demonstrated
by the 1918 Spanish influenza and the recent 2009 swine influenza
[5,6]. Influenza vaccine remains the mainstay in prophylaxis of
influenza virus infection [7]. However, it has to be reformulated
each year to match the antigens of influenza viruses in the coming
influenza season. As the manufacturing of influenza vaccines takes
at least six months, influenza viruses might continue to mutate
during this period, resulting in vaccine mismatch [8e10]. Moreover,
influenza vaccines have little to no efficacy in young children, the
elderly, and immunocompromised persons [11]. In addition to
vaccines, there are two classes of anti-influenza drugs approved for
uncoating, and neuraminidase inhibitors, e.g., oseltamivir, zana-
mivir, and peramivir, that inhibit virus release [1]. Resistance to
both classes of drugs, however, now necessitates the development
of the next generation of anti-influenza drugs [12]. Amantadine and
rimantadine are no longer recommended due to widespread drug
resistance. Resistance to the only orally available drug, oseltamivir,
continues to rise, and the 2008e2009 seasonal H1N1 strain was
completely resistant to oseltamivir due to a H275Y mutation [12].
Influenza strains that are resistant to both classes of drugs have
been reported [13,14]. Moreover, certain highly pathogenic avian
influenza viruses such as H7N9 and H5N1, which have the potential
to lead to the next influenza pandemic, are also resistant to osel-
tamivir [15,16]. Thus the next generation of antiviral drugs with
broad-spectrum antiviral activity against both drug-sensitive and
drug-resistant influenza strains is clearly needed [17,18].
In pursuing the next generation of influenza antivirals, we chose
the AM2-S31N mutant as the drug target. AM2 forms a proton-
selective channel in the viral membrane and plays important
roles during the viral replication cycle [19]: in the early stage AM2
facilitates viral uncoating by acidifying the viral interior, which
leads to the dissociation of viral ribonucleoproteins from the matrix
protein M1; in the late stage of viral replication AM2 equilibrates
the pH across the Golgi apparatus and prevents the premature
Abbreviations used: WT, wild type; DMEM, Dulbecco's modified eagle medium;
MDCK, MadineDarby Canine Kidney; TEVC, two-electrode voltage clamps.
* Corresponding author. Department of Pharmacology and Toxicology, College of
Pharmacy, The University of Arizona, Tucson, AZ 85721, United States.
E-mail address: junwang@pharmacy.arizona.edu (J. Wang).
http://dx.doi.org/10.1016/j.ejmech.2017.04.038
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

Y. Hu et al. / European Journal of Medicinal Chemistry 135 (2017) 70e76
71
Fig. 1. FDA-approved influenza antivirals.
conformational change of hemagglutinin. More than 95% of
currently circulating influenza A viruses carry the S31N mutant in
their M2 genes, which renders them resistant to adamantanes [20].
The prevalence of this mutation thus makes AM2-S31N a desired
target for drug design [21]. Propelled by structural and mechanistic
understandings of AM2 proton conductance and drug inhibition
[21,22], we successfully designed the first-in-class AM2-S31N in-
hibitors with both potent channel blockage and antiviral activity
[21,23e26]. Encouraged by this progress, in this study we report the
design and expeditious synthesis of organosilane-based AM2-S31N
inhibitors. Silicon is a bioisostere of carbon, and the design of
organosilane-based bioactive molecules has been hotly pursued
[27,28]. Unique features of organosilanes include but are not
limited to improved binding affinity, higher metabolism stability,
and reduced cytotoxicity. More importantly, from the synthesis
perspective, organosilanes are generally easier to synthesize than
their carbon analogues. Furthermore, organosilanes have several
unique properties that cannot be mimicked by their carbon ana-
logues: (1) silicon can form stable silanediol to mimic the amide
bond hydrolysis intermediate, and silanediols have been reported
to be potent protease inhibitors (Fig. 2, compound 1) [29]. (2)
organosilanes can go beyond tetrahedral coordination to octahe-
dral coordination (Fig. 2, compound 2) and such compounds can be
used as DNA chelators [30]. (3) Incorporation of silicone into
rhodamine results in Si-rhodamine, which emits in the near
infrared region (650e900 nm) (Fig. 2, compound 3) [31]. Such
fluorescence probes have been successfully applied for live cell and
in vivo imaging [32].
consists of a hydrophobic scaffold such as adamantane, a positively
charged ammonium linker, and an aromatic head group with a
hydrophobic substitution (Fig. 3, compound 4) [24e26]. (2) The
designed organosilanes should be easy to synthesize by late-stage
diversification. (3) Introduction of silicon to a drug molecule in-
creases its hydrophobicity, which might lead to enhanced cellular
cytotoxicity; thus a hydroxyl group should be added to the 3-
position of adamantane to reduce the overall hydrophobicity and
thus cellular cytotoxicity [24e26]. Taking these factors into
consideration, the designed organosilanes 5 contain an ada-
mantane cage, an ammonium methylene linker, and a para-
substituted aromatic/heterocyclic head group. Such a design al-
lows the introduction of diverse silicon substitutions starting from
a common intermediate.
3. Chemistry
We then proceed to synthesize the designed organosilanes 5aef
(Fig. 4). Reductive amination of amantadine 6a or 3-amino-1-
hydroxyadamantane 6b with 4-bromobenzaldehyde 7a or 5-
bromo-2-pyridinecarbaldehyde 7b gave the intermediates 8a-8c.
The yields range from 75% to 82%. In the next step, five equivalents
of n-butyl lithium were used to generate the aryl lithium in the
presence of a hydroxyl and an amine group. The resulting aryl
lithium was then reacted with a variety of silyl chlorides to furnish
the final products 5aef. The yields range from 62% to 78%.
4. Results and discussion
2. Design rationale
4.1. Channel blockage, antiviral efficacy, and cytotoxicity of
organosilane-based AM2-S31N inhibitors
In light of advantages of exploring organosilanes as bioactive
molecules, we are interested in designing organosilanes as AM2-
S31N inhibitors. Three criteria were taken into consideration
when designing AM2-S31N inhibitors: (1) the designed organo-
silanes should meet the pharmacophore requirements of AM2-
S31N inhibitors. The pharmacophore of AM2-S31N inhibitors
To validate the design hypothesis, the synthesized organosilanes
were tested for their AM2-S31N channel blockage, antiviral activity,
and cellular cytotoxicity by electrophysiological two-electrode
voltage clamp (TEVC) assay, plaque assay, and neutral red assay,
respectively (Table 1). Two compounds, 9 and 10, which were the
Fig. 2. Representative bioactive organosilanes. Compound 1 is a silanediol peptidomimetic and was designed as a serine protease inhibitor [29]. Compound 2 is an octahedral silicon
complex and was designed as a DNA intercalator [30]. Compound 3 is a Si-rhodamine that emits in the near infrared region [31].

72
Y. Hu et al. / European Journal of Medicinal Chemistry 135 (2017) 70e76
Fig. 3. Rational design of organosilanes as AM2-S31N inhibitors. Left is a cartoon representation of the pharmacophore of AM2-S31N inhibitors and the binding mode of AM2-S31N
inhibitors in the AM2-S31N channel. Right is the designed AM2-S31N inhibitors.
Fig. 4. Expeditious synthesis of organosilanes as AM2-S31N inhibitors.
carbon analogues of compounds 5a and 5b, were also synthesized
and used for comparison. It was found that organosilane 5a had
similar channel blockage and antiviral activity as its carbon
analogue 9. However, the difference between compounds 5b and
10 was more obvious, with the organosilane 5b being more potent
than its carbon analogue 10 in blocking the AM2-S31N channel
(86.7 0.7% versus 75.4 2.3%). Consistent with the electrophys-
iological assay results, the antiviral activity of organosilane 5b was
AM2-S31N channel, not the WT-AM2. In summary, the most potent
compound was 5b, which not only had potent channel blockage
and antiviral activity, but also had the highest selectivity index (SI)
of 101.8.
4.2. Broad-spectrum antiviral activity of organosilane 5b
To further characterize the therapeutic potential of organosilane
5b, its cytotoxicity against the human epithelial cell line A549 and
antiviral activity against multidrug-resistant influenza A viruses
were further evaluated (Table 2). Compound 5b was found to be
less cytotoxic to A549 cells than to MDCK cells (CC50 ¼ 73.2 6.2 vs
6-fold more potent than its carbon analogue 10 (0.4 0.2
mM versus
2.5 1.1 M). The increased potency might be a result of the
m
favorable hydrophobic interaction between the hydrophobic tri-
methylsilyl group from 5b and the valine side chain methyls located
at the N-terminus of the AM2-S31N channel (Fig. 3). Substituting
benzene with pyridine led to a slight increase in AM2-S31N channel
inhibition (5c vs 5a). Further increase in the size of the silyl sub-
stitution led to compounds 5def. Compounds 5d and 5e both had
slightly reduced percentage channel blockage against the AM2-
S31N channel when compared with compound 5b, and both were
also less potent than 5b in terms of their antiviral activity. Com-
pound 5f with bulky triisopropylsilyl substitution was inactive in
both electrophysiological and antiviral assays. All three compounds
(5def) were more cytotoxic than 5b, probably due to increased
clogP (Table 1). None of the compounds showed significant inhi-
bition (>35%) against the AM2-WT channel, which was expected
since these compounds were designed to target the drug-resistant
40.7 8.2 mM), resulting in an SI of 183. The antiviral efficacy of 5b
against human clinical isolates of influenza A viruses was also
profiled. The viruses tested include multidrug-resistant strains such
as the A/Denmark/528/2009 (H1N1), A/Washington/29/2009
(H1N1), and A/Texas/04/2009 (H1N1), all of which are resistant to
both amantadine and oseltamivir due to AM2-S31N and H275Y
mutations in their M2 and neuraminidase genes, respectively.
Encouragingly, compound 5b showed potent antiviral activity with
single to submicromolar efficacy against all three multidrug-
resistant strains. In addition, organosilane 5b also showed potent
antiviral activity against oseltamivir-sensitive strains such as A/
California/07/2009 (H1N1), A/Switzerland/9715293/2013 (H3N2),
and A/Denmark/524/2009 (H1N1) with EC50 values ranging from

Y. Hu et al. / European Journal of Medicinal Chemistry 135 (2017) 70e76
73
Table 1
Channel blockage, antiviral activity, cytotoxicity, and hydrophobicity of organosilane AM2-S31N inhibitors.
Compound structure and ID
a% AM2-S31N inhibition
77.5 0.1
a% AM2-WT inhibition
21.8 1.6
A/WSN/33 EC50
4.8 1.8
(
m
M)b
CC50 (m
M) MDCKc and cLogPd
CC50 ¼ 8.0 0.6
clogP ¼ 5.6
SI ¼ 1.7
5a
5b
5c
86.7 0.7
81.3 2.1
81.4 0.8
9.5 0.5
5.9 1.6
18.2 1.4
0.4 0.2
3.4 0.5
5.5 0.4
CC50 ¼ 40.7 8.2
clogP ¼ 4.9
SI ¼ 101.8
CC50 ¼ 10.3 0.2
clogP ¼ 5.0
SI ¼ 3.0
CC50 ¼ 21.8 0.5
clogP ¼ 5.2
SI ¼ 4.0
5d
5e
84.9 1.5
33.6 2.0
33.6 2.3
17.8 1.8
5.9 2.2
CC50 ¼ 13.3 1.0
clogP ¼ 6.2
SI ¼ 2.3
>10
CC50 ¼ 11.4 1.0
clogP ¼ 6.5
5f
9
72.6 1.4
75.4 2.3
9.9 0.5
3.5 1.2
2.5 1.1
CC50 ¼ 12.8 0.6
clogP ¼ 5.3
SI ¼ 3.7
22.3 0.7
CC50 ¼ 32.0 1.21
clogP ¼ 4.6
SI ¼ 12.8
10
a
Values represent the mean of three independent measurements. Compounds were tested at 100
m
M concentration.
b
c
Antiviral activity was tested with the A/WSN/33 (H1N1) virus, which contains the AM2-S31N mutant, in plaque assay. The experiments were done in duplicate.
Cytotoxicity was tested by incubating MDCK cells with compounds for 48 h and the cells were stained with neutral red [33].
clogP values were calculated using Schrodinger Glide QikProp.
d
Table 2
Antiviral activity of organosilane 5b against drug-resistant influenza A viruses.
Viruses
Resistance to oseltamivir
5b EC50
(m
M)
SI (A549)
A/California/07/2009 (H1N1)
A/Switzerland/9715293/2013 (H3N2)
A/Denmark/524/2009 (H1N1)
A/Denmark/528/2009 (H1N1)
A/Washington/29/2009 (H1N1)
A/Texas/04/2009 (H1N1)
NO
NO
NO
1.2 0.1
0.7 0.1
0.6 0.1
3.0 1.5
1.4 0.3
0.6 0.1
61.0
104.6
122.0
24.4
52.3
122.0
YES (H275Y)
YES (H275Y)
YES (H275Y)
0.6 to 1.2
m
M. The common feature among these influenza A strains
minimized pose (Fig. 5), organosilane 5b binds to the AM2-S31N
channel with its trimethylsilyl-substituted benzene ring facing to-
wards the N-terminus of the channel. The adamantane cage from
5b fits in a space created by G34, and the positively charged
ammonium from 5b forms a hydrogen bond with one of the N31
side chain carbonyl. The trimethylsilyl is surrounded by a ring of
methyls from V27. Based on this docking model, it is expected that
compounds having a sterically bulky substitution at the 4-position
of the benzyl ring, such as 5f, will experience steric clash with the
V27 side chain methyls, thus will not be able to fit in the channel.
This prediction is validated by the antiviral assay result which
showed that compound 5f was not active in inhibiting the AM2-
is that they all contain the AM2-S31N mutation in their M2 genes,
which explains their drug sensitivity to organosilane 5b. The
selectivity index of 5b ranges from 24.4 to 122.0, which suggests
that compound 5b might be a suitable lead compound to be further
optimized.
4.3. Molecular docking of organosilane 5b in the AM2-S31N
channel
To gain further insights how organosilane 5b binds to the AM2-
S31N channel, molecular docking was performed. The solution
NMR structure of AM2-S31N channel (PDB: 2LY0) was used for the
docking [23]. Consistent with the design principle, in the energy
S31N-containing A/WSN/33 (H1N1) virus (EC50 > 10
mM, Table 1).
Overall, the docking model is consistent with the experimental

74
Y. Hu et al. / European Journal of Medicinal Chemistry 135 (2017) 70e76
Fig. 5. Docking model of organosilane 5b in the AM2-S31N channel. The solution NMR structurer of AM2-S31N (2LY0) was used for the docking [23]. Docking was performed using
AutoDock Vina [34]. (A) Side view of the docking pose of organosilane 5b in AM2-S31N channel. The front helix was removed for clarity. (B) Top view of the docking pose of
organosilane 5b in AM2-S31N channel.
results and could serve as a reference for further lead optimization.
unless otherwise stated. HPLC-grade solvents were used for all
reactions. Flash column chromatography was performed using sil-
ica gel (230e400 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),
5. Conclusions
In summary, guided by the pharmacophore of AM2-S31N in-
hibitors, we were able to design organosilanes as potent AM2-S31N
channel blockers. Compared with its carbon analogue 10, the most
potent organosilane, 5b, had improved antiviral activity in inhib-
iting the A/WSN/33 (H1N1) virus. Significantly, compound 5b was
also highly active against both oseltamivir-sensitive and -resistant
influenza A viruses, highlighting its promise for further develop-
ment. Moreover, the synthesis of organosilanes is straightforward,
and a diverse silyl groups can be installed in a one-step SN2 reac-
tion, which would greatly facilitate the iterative cycles of design,
synthesis, and biological characterization. In contrary, similar
chemistry is not feasible for the corresponding carbon analogues.
The major therapeutic importance of AM2-S31N inhibitors such as
organosilane 5b is that they are active against both oseltamivir-
sensitive and -resistant influenza viruses. Thus they can be used
either alone to combat oseltamivir-resistant strains or used in
combination with oseltamivir to delay the evolution of resistance.
Overall, the results from this study reaffirm the advantages of
exploring organosilanes as bioactive molecules, and organosilane
therapeutics might become a reality in the near future.
50 ꢀ 2.1 mm, at a flow rate of 0.3 mL/min;
l
¼ 250 and 220 nm;
mobile phase A, 0.1% formic acid in H2O, and mobile phase B0, 0.1%
formic in 60% isopropanol, 30% CH3CN and 9.9% H2O. All com-
pounds 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.
6.1.2. General procedures for the synthesis of organosilanes
Compounds 8a-8c, 9 and 10 were synthesized according to the
following general procedure.
General procedure of reductive amination (Fig. 4):
Adamantane or 3-amino-1-adamantanol (1 equiv.) and alde-
hyde (1 equiv.) were mixed with 2 mL of titanium (IV) isopropoxide.
The resulting slurry was heated in microwave at 120 ꢁC for 30 min.
Then the solution was cooled down and CH3OH (10 mL) was added.
The mixture was cooled down to 0 ꢁC using ice bath, then NaBH4 (4
equiv.) was added portionwise in 10 min. The solution was subse-
quently warmed to room temperature and stirred for 4 h. The re-
action was quenched with 1M NaOH and filtered through celite.
The filtrate was concentrated under reduced pressure and was
purified by silica gel flash column chromatography (5e10% CH3OH/
CH2Cl2) to give the final product.
6. Experimental section
6.1. Chemistry
6.1.2.1. N-[(4-bromophenyl)methyl]adamantan-1-amine
Yield: 82.4%. 1HNMR (400 MHz, CDCl3):
7.41 (d, J ¼ 8.0 Hz, 2H),
7.22 (d, J ¼ 8.0 Hz, 2H), 3.70 (s, 2H), 2.08 (s, 3H), 1.68e1.60 (m, 12 H).
(8a).
6.1.1. General chemical methods
d
All chemicals were purchased from commercial vendors and
used without further purification unless otherwise noted. 1H and
13C NMR spectra were recorded on a Bruker-400 NMR spectrom-
eter. Chemical shifts are reported in parts per million referenced
with respect to residual solvent (CD3OD) 3.31 ppm and (Chloro-
form-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,
13C NMR (100 MHz, CDCl3)
d 140.43, 131.40, 129.82, 120.41, 50.09,
44.50, 42.64, 36.76, 29.71. EI-MS: m/z (MþHþ): 320.1 (calculated),
320.0 (found).
Compounds 8b and 8c were reported before [25].
6.1.2.2. N-[(4-tert-butylphenyl)methyl]adamantan-1-amine
Yield: 78.0%. 1HNMR (400 MHz, CD3Cl):
7.33 (d, J ¼ 8.0 Hz, 2H),
7.27 (d, J ¼ 8.0 Hz, 2H), 3.76 (s, 2H), 2.10 (m, 3H), 1.79e1.78 (m, 6H),
(9).
d

Y. Hu et al. / European Journal of Medicinal Chemistry 135 (2017) 70e76
75
1.68e1.66 (m, 6H), 1.63 (s, 1H), 1.36 (s, 9H). 13NMR (100 MHz,
(m, 2H), 1.41 (s, 2H), 0.46 (s, 6H).$13C NMR (100 MHz, CDCl3)
139.86, 137.52,134.59, 134.04,131.46,130.30, 129.24,127.87, 69.24,
CDCl3):
d
149.58, 137.97, 128.04, 125.32, 50.94, 44.69, 42.84, 36.80,
d
34.43, 31.38, 31.52, 29.68. EI-MS: m/z (MþHþ): 298.2 (calculated),
61.55, 46.93, 44.99, 42.92, 36.86, 34.13, 30.25, ꢂ2.66. EI-MS: m/z
298.0 (found).
(MþHþ): 392.6 (calculated), 393.0 (found).
6.1.2.3. 3-{[(4-tert-butylphenyl)methyl]amino}1adamantan-1-ol
6.1.2.9. 3-[({4-[tris(propan-2-yl)silyl]phenylmethyl)amino]ada-
(10). Yield: 75.3%. 1HNMR (400 MHz, CD3Cl):
d
7.33 (d, J ¼ 8.0 Hz,
mantan-1-ol (5f). Yield: 62.3%. 1HNMR (400 MHz, CDCl3þ CD3OD):
2H), 7.27 (d, J ¼ 8.0 Hz, 2H), 3.75 (s, 2H), 2.29e2.28 (m, 2H), 1.77 (s,
d 7.58e7.47 (m, 4H), 3.83 (s, 2H), 3.88 (s, 2H), 2.32 (s, 2H), 1.88e1.87
2H), 1.68e1.67 (m, 6H), 1.66 (m, 4H), 1.54 (m, 2H), 1.43 (s, 9H).
(m, 2H),1.83e1.85 (m, 4H),1.70e1.63 (m, 4H),1.52 (s, 2H),1.37e1.30
13NMR (100 MHz, CDCl3):
d 149.91, 137.55, 128.11, 125.39, 69.77,
(m, 3H), 1.00e0.97 (m, 18H). 13NMR (100 MHz, CDCl3þ CD3OD):
54.44, 50.22, 45.03, 44.48, 41.34, 35.24, 34.45, 31.38, 30.83. EI-MS:
d 136.82, 135.97, 131.42, 128.99, 67.95, 60.00, 45.81, 43.85, 42.87,
m/z (MþHþ): 314.2 (calculated), 314.0 (found).
36.26, 33.93, 30.34, 18.40, 10.85. EI-MS: m/z (MþHþ): 414.7
Compounds 5a-5f were synthesized according to the following
general procedure [35].
(calculated), 415.0 (found).
General procedure of silylation (Fig. 4):
6.2. Biological evaluation
n-Butyllithium (1.6 M in hexanes; 5 equiv.) was added dropwise
to a stirred solution of intermediate 8 (1 equiv.) in THF (15 mL)
at ꢂ78 ꢁC. The reaction was stirred at ꢂ78 ꢁC for 1 h, then silyl
chloride (5 equiv.) was added dropwise, and the mixture was
allowed to gradually warm to room temperature (4 h) and stirred
for another 12 h. The reaction was quenched with water (10 mL),
and the aqueous phase was separated and extracted with ethyl
acetate (3 ꢀ 30 mL). The combined organic portions were com-
bined and dried over anhydrous magnesium sulfate, filtered and
concentrated under reduced pressure. The mixture was then pu-
rified by silica gel flash column chromatography (0e10% CH3OH/
CH2Cl2) to give the final products 5a-5f.
6.2.1. Cell lines, viruses, and viral infection
Madin-Darby Canine Kidney (MDCK) cells were grown at 37 ꢁ
C
-
in 5% CO2 atmosphere in DMEM media (high glucose, with
L
glutamine) supplemented with 10% fetal bovine serum (FBS), 100
IU/mL penicillin and 100 g/mL streptomycin. MDCK cells over-
m
expressing ST6Gal I were obtained from Dr. Yoshihiro Kawaoka at
the University of Wisconsin at Madison through material transfer
agreement and were maintained in the presence of 7.5 mg/mL of
puromycin, except when they were used for viral infection. Influ-
enza A virus strains A/California/07/2009 (H1N1) and A/Texas/04/
2009 (H1N1) were obtained from Dr. James Noah at the Southern
Research Institute; influenza A virus strains A/Denmark/524/2009
(H1N1) and A/Denmark/528/2009 (H1N1) were obtained from Dr.
Elena Govorkova at St. Jude Children's Research Hospital; influenza
A virus strains A/Switzerland/9715293/2013 X-247 (H3N2), FR-
1366, and A/Washington/29/2009 (H1N1), FR-460, 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. Virus stocks were amplified in MDCK cells in the presence of
6.1.2.4. N-{[4-(trimethylsilyl)phenyl]methyladamantan-1-amine
(5a). Yield: 78.3%. 1HNMR (400 MHz, CD3Cl):
d
7.47 (d, J ¼ 8.0 Hz,
2H), 7.34 (d, J ¼ 8.0 Hz, 2H), 3.75 (s, 2H), 2.10 (m, 3H), 1.73e1.72 (m,
6H), 1.68e1.66 (m, 6H), 1.63 (s, 1H), 0.25 (s, 9H). 13NMR (100 MHz,
CDCl3):
d 138.47, 133.37, 127.66, 50.90, 44.98, 42.78, 36.68,
29.57, ꢂ1.18. EI-MS: m/z (MþHþ): 314.6 (calculated), 314.0 (found).
6.1.2.5. 3-({[4-(trimethylsilyl)phenyl]methyl}amino)adamantan-1-ol
(5b). Yield: 70.4%. 1HNMR (400 MHz, CD3Cl):
d
7.47 (d, J ¼ 8.0 Hz,
2 mg/ml N-acetyl trypsin. Two days post infection, the culture media
2H), 7.33 (d, J ¼ 8.0 Hz, 2H), 3.75 (s, 2H), 2.28 (m, 2H), 1.71e1.64 (m,
were harvested and cell debris was removed by centrifugation at
3000 rpm for 30 min. Virus titers were determined by plaque
reduction assay using MDCK cells expressing ST6Gal I.
12H), 1.55e1.53 (m, 2H), 0.25 (s, 9H). 13NMR (100 MHz, CDCl3):
d
141.65, 138.87, 133.62, 127.74, 69.78, 54.28, 50.34, 45.43, 44.51,
41.49, 35.62, 30.84, ꢂ1.08. EI-MS: m/z (MþHþ): 330.6 (calculated),
331.0 (found).
6.2.2. Plaque reduction assay
Plaque reduction assay were carried out as previously described
[36,37], except MDCK cells expressing ST6Gal I were used instead of
regular MDCK cells.
6.1.2.6. N-{[5-(trimethylsilyl)pyridine-2-yl]methyladamantan-1-
amine (5c). Yield: 80.4%. 1HNMR (400 MHz, CD3Cl):
d 8.56 (s, 1H),
7.89 (d, J ¼ 8.0 Hz, 1H), 7.47 (d, J ¼ 8.0 Hz, 1H), 4.38 (s, 2H), 2.20 (m,
3H), 2.01 (s, 6H), 1.75e1.71 (m, 6H), 1.70e1.67 (m, 1H), 0.31 (s, 9H).
6.2.3. Cytotoxicity assay
13NMR (100 MHz, CDCl3):
d
151.46, 150.37, 143.92, 122.95, 58.03,
Evaluation of the cytotoxicity of compounds was carried out
using neutral red uptake assay [33]. Briefly, 80,000 cells/mL MDCK
or A549 cells in DMEM medium supplemented with 10% FBS and
100 U/mL Penicillin-Streptomycin were dispensed into 96-well cell
41.91, 38.88, 35.50, 28.00, ꢂ1.46. EI-MS: m/z (MþHþ): 315.5
(calculated), 316.0 (found).
6.1.2.7. 3-({[4-(ethyldimethylsilyl)phenyl]methyl}amino)adamantan-
culture plates at 100
medium was removed and washed with 100
cytotoxicity assay, 200 L fresh DMEM (No FBS) medium contains
m
L/well. Twenty-four hours later, the growth
1-ol (5d). Yield: 67.5%. 1HNMR (400 MHz, CD3Cl):
d
7.49 (d,
mL PBS buffer; then for
J ¼ 8.0 Hz, 2H), 7.342 (d, J ¼ 8.0 Hz, 2H), 3.76 (s, 2H), 2.25 (m, 2H),
2.01 (s, 2H) 1.88e1.58 (m, 10H), 1.47 (m, 2H), 0.87 (t, J ¼ 8.0 Hz, 2H),
0.61 (q, J ¼ 8.0 Hz, 3H), 0.12 (s, 6H). 13NMR (100 MHz, CDCl3):
m
serial diluted compounds was added to each well. After incubating
for 48 h at 37 ꢁC with 5% CO2 in a CO2 incubator, the medium was
d
139.58, 133.34, 129.60, 69.49, 48.21, 45.73, 43.20, 34.78, 30.06,
removed and replaced with 100 mL DMEM medium contains 40 mg/
29.60, 7.33, 7.24, ꢂ3.43. EI-MS: m/z (MþHþ): 344.6 (calculated),
mL neutral red for 4 h 37 ꢁC. The amount of uptaken neutral red was
determined at absorbance 540 nm using a Multiskan FC Microplate
Photometer (Fisher Scientific). The CC50 values were calculated
from best-fit dose response curves with variable slope in Prism 5.
345.0 (found).
6.1.2.8. 3-[({4-[dimethyl(phenyl)silyl]phenyl}methyl)amino]ada-
mantan-1-ol (5e). Yield: 68.7%. 1HNMR (400 MHz, CD3Cl):
d 8.06 (s,
1H), 7.63 (d, J ¼ 8.0 Hz, 2H), 7.48 (d, J ¼ 8.0 Hz, 2H), 7.42e7.37 (m,
2H), 7.34e7.27 (m, 3H), 3.83 (s, 2H), 3.38 (s, 1H), 2.22 (m, 4H),
1.99e1.96 (m, 2H), 1.83e1.80 (m, 2H), 1.67e1.64 (m, 2H), 1.55e1.52
6.2.4. Electrophysiological TEVC assay
The compounds were tested in a two-electrode voltage clamp
assay using Xenopus laevis frog oocytes microinjected with RNA

76
Y. Hu et al. / European Journal of Medicinal Chemistry 135 (2017) 70e76
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6.3. Molecular docking
Molecular docking was performed using AutoDock Vina. The
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docking. The center of the grid box was set as the following:
center_x ¼ 0, center_y ¼ 0, center_z ¼ 11. The size of the grid box
was set as the following: size_x ¼ 22, size_y ¼ 20, size_z ¼ 18.
Acknowledgements
This research is supported by startup funding from the Univer-
sity of Arizona, the 2015 PhRMA Foundation Research Starter Grant
in Pharmacology and Toxicology, and NIH grant AI119187 to J.W. We
thank Dr. David Bishop for proofreading and editing the
manuscript.
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the M2 ion channel of influenza A virus, Proc. Natl. Acad. Sci. U. S. A. 110
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Appendix A. Supplementary data
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targeting the predominant drug-resistant S31N mutant of the influenza a
virus M2 proton channel, J. Med. Chem. 59 (2016) 1207e1216.
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Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2017.04.038.
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