Aminoadamantanes with persistent in vitro efficacy against H1N1 (2009) influenza A

Article abstract of DOI:10.1021/jm500598u

A series of 2-adamantanamines with alkyl adducts of various lengths were examined for efficacy against strains of influenza A including those having an S31N mutation in M2 proton channel that confer resistance to amantadine and rimantadine. The addition of as little as one CH₂ group to the methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-aminoadamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1). Solid state NMR of the transmembrane domain (TMD) with a site mutation corresponding to S31N shows evidence of drug binding. But electrophysiology using the full length S31N M2 protein in HEK cells showed no blockade. A wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to representative drugs within one passage with mutations in M2 TMD, but A/Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and the resistant virus had no mutations in M2 TMD. The results indicate that 2-alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus entry to the host cell.

Full text of DOI:10.1021/jm500598u

Article
pubs.acs.org/jmc
Aminoadamantanes with Persistent in Vitro Efficacy against H1N1
(2009) Influenza A
Antonios Kolocouris,*,† Christina Tzitzoglaki,† F. Brent Johnson,‡ Roland Zell,§ Anna K. Wright,∥
Timothy A. Cross,∥ Ian Tietjen,⊥ David Fedida,⊥ and David D. Busath*,#
†Department of Pharmaceutical Chemistry, Faculty of Pharmacy, National and Kapodistrian University of Athens, Athens 15771,
Greece
‡Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 84602, United States
§Department of Virology and Antiviral Therapy, Jena University Hospital, CMB Building, R. 443, Hans Knoell Strasse 2, D-07745
Jena, Germany
∥Department of Chemistry and Biochemistry and National High Magnetic Field Laboratory, Florida State University, Tallahassee,
Florida 32306, United States
⊥Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, British Columbia, V6T
1Z3, Canada
#Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah 84602, United States
S
* Supporting Information
ABSTRACT: A series of 2-adamantanamines with alkyl adducts of various
lengths were examined for efficacy against strains of influenza A including those
having an S31N mutation in M2 proton channel that confer resistance to
amantadine and rimantadine. The addition of as little as one CH2 group to the
methyl adduct of the amantadine/rimantadine analogue, 2-methyl-2-amino-
adamantane, led to activity in vitro against two M2 S31N viruses A/Calif/07/
2009 (H1N1) and A/PR/8/34 (H1N1) but not to a third A/WS/33 (H1N1).
Solid state NMR of the transmembrane domain (TMD) with a site mutation
corresponding to S31N shows evidence of drug binding. But electrophysiology
using the full length S31N M2 protein in HEK cells showed no blockade. A
wild type strain, A/Hong Kong/1/68 (H3N2) developed resistance to
representative drugs within one passage with mutations in M2 TMD, but A/
Calif/07/2009 S31N was slow (>8 passages) to develop resistance in vitro, and
the resistant virus had no mutations in M2 TMD. The results indicate that 2-
alkyl-2-aminoadamantane derivatives with sufficient adducts can persistently block p2009 influenza A in vitro through an
alternative mechanism. The observations of an HA1 mutation, N160D, near the sialic acid binding site in both 6-resistant A/
Calif/07/2009(H1N1) and the broadly resistant A/WS/33(H1N1) and of an HA1 mutation, I325S, in the 6-resistant virus at a
cell-culture stable site suggest that the drugs tested here may block infection by direct binding near these critical sites for virus
entry to the host cell.
successful adamantane- and pinanamine-based M2 S31N
blockers have appeared.6,10 The design of these molecules
was not based on the enlargement of the amantadine WT-M2
binding site, and the structural analysis of one active
compound, comprising an amantadine linked through a
methylene bridge to an isoxazole having an aryl substituent,
showed that its heterocyclic ring may be trapped by the V27
side chains at the mouth of the channel.6 Triggered by previous
efforts aimed at adequately filling the empty expanded pore
region due to the S31N mutation and to determine
progressively the minimal variation of amantadine required to
block influenza A (H1N1, M2 S31N), we evaluated drug
INTRODUCTION
■
Since 2005,1 the amantadine/rimantadine-insensitive S31N
mutation has become prevalent globally,2 abrogating clinical
usefulness of amantadine 1 and rimantadine 23 and possibly
previously developed M2 blocking compounds.4 If the
replacement of Ser31 with the larger Asn in M2 S31N splays
the helix bundle at the drug binding site,5 as suggested by
solution state NMR studies,5d,6 then drugs larger than
rimantadine might be expected to be effective blockers.
However, initial attempts to identify larger adamantane-based
compounds that block amantadine-resistant viruses were
unsuccessful.7 Further efforts identified spiranamine analogues
based on BL-17438 that were effective against V27A and L26F
mutants9a but not against S31N, while other large templates
could inhibit V27A9b−d but not S31N. Subsequently, reports of
Received: January 16, 2014
Published: May 4, 2014
© 2014 American Chemical Society
4629
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
efficacy and mechanism for variations of amantadine 1 with
alkyl adducts ranging from small to moderate and larger sizes
(Scheme 1) as represented by the 2-alkyl-2-aminoadamantane
The amines 4, 5, 7−11 were synthesized according to
Scheme 3. Tertiary alcohols 16−22 were obtained by treating
Scheme 3. Preparation of 2-Alkyl-2-aminoadamantane
Derivatives 4, 5, 7−11
a
Scheme 1. Amantadine 1, Rimantadine 2, and 2-Alkyl-2-
aminoadamantane Derivatives 3−11
a
Reagents and conditions: (a) RLi, Ar, ether, THF, 0 °C, 2 h rt for
17−20 or RMgCl, ether, THF, 2 h rt for 16, 20, 21, then NH4Cl/H2O
(85−96%); (b) NaN3, TFA, CH2Cl2, 0 °C, then rt (50−96%); (c)
LiAlH4, ether, rt, 5 h (23−65%).
derivatives 3−11, which are simpler than previously reported
aminoadamantane derivatives active against S31N viruses10e
and the larger of which have increased volume compared to
amantadine.
2-adamantanone 12 with an oganolithium (R = Et, n-Bu, i-Bu,
n-hexyl) or organomagnesium reagent (R = Me,4b Ph, or
PhCH2) (Scheme 3). While 2-methyl-2-adamantanol 16 was
obtained after treating 2-adamantanone 12 with CH3MgI, this
is not an efficient method for the preparation of alcohols 17−
22 because of the bulky 2-adamantanone 12 and the soft
carbanion character of the Grignard reagent making the β-
hydride transfer a competitive reaction to the alkyl addition and
leading to a mixture of the desired tertiary alcohol with 2-
adamantanol. The conversion of tertiary alcohols 16−22 to the
corresponding azides 23−29 was accomplished efficiently
through treatment with NaN3/TFA 1 M in dichloromethane
or dichloroethane for 24 h at room temperature. The primary
tert-alkylamines 4, 5, 7−11 were prepared by means of LiAlH4
reduction of the azides 23−29 in refluxing ether for 5 h.
Solid State NMR of the M2 TMD Tetramer. PISA wheel
analysis gives a direct readout of helix tilt relative to the
membrane normal for membrane proteins in uniformly
oriented lipid bilayer preparations from solid state NMR
PISEMA experiments.13 15N anisotropic chemical shifts and
15N−1H dipolar interactions observed in these spectra are very
sensitive to the orientation of the peptide planes relative to the
bilayer normal. Binding of compound 6 shifts the signals for
three pertinent backbone amides that were isotopically labeled
(Figure 1).
We found with 5 that the addition of as little as one CH2
group to the methyl adduct of the amantadine/rimantadine
analogue, 2-methyl-2-aminoadamantane 4 (Scheme 1), recov-
ers activity in vitro against the amantadine-resistant A/Calif/
07/2009. However, the mechanism of action is not M2-block
but a second aminoadamantane target.
RESULTS AND DISCUSSION
■
Chemistry. Compounds 3−11 belong to the class of 2-
alkyl-2-aminoadamantanes, which thus bear a substitution at
adamantane C2 carbon. Compounds 3−64b and 1011 were
previously synthesized but resynthesized with slightly modified
procedures in this work. Tertiary alcohol 13 was obtained by
treating 2-adamantanone 12 with allylmagnesium bromide
(Scheme 2). The unsaturated alcohol 13 was converted to the
a
Scheme 2. Preparation of 2-n-Propyl-2-aminoadamantane 6
Binding of amantadine 1 to WT M2 TMD (A/Udorn/307/
72 sequence) produces an 11° kink near G34 in each helix of
the tetramer.5e When drug-bound, the helix tilt for the N-
terminal half (residues 22−34) is 31° and in the C-terminal half
(residues 35−46) just 20°.14 Here, the S31N M2 TMD is
labeled at two sites in the N-terminal half (residues V28 and
A30) and one site in the C-terminal half (residue I42) of the
TMD helix. The S31N data without drug suggest a helical tilt of
approximately 36°, similar to that seen in the WT structure.5
The shifts in the anisotropic spin interactions upon drug
binding demonstrate a significant change in the structure of the
tetrameric complex. With compound 6, there is a uniform tilt of
∼33°. Thus, the 6-induced changes in the resonance
frequencies of these three sites indicate that the tilt angle for
the entire TMD helix is decreased by 3° while maintaining a
similar rotational orientation for the helices. Unlike the
response of the WT to amantadine 1, with 6 the S31N TMD
helices do not appear to have kinked the helix at G34. Instead,
a
Reagents and conditions: (a) CH2CHCH2MgBr, ether, THF, rt, 2
h, then NH4Cl/H2O (quant); (b) H2/PtO2 (quant); (c) NaN3, TFA,
CH2Cl2, 0 °C, then rt (quant); (c) LiAlH4, ether, rt, 5 h (74%).
n-propyl derivative 14 through catalytic hydrogenation over
PtO2. After experiments with tertiary alcohols in the
adamantane series and an acyclic series (unpublished data),
we concluded that the conversion of tertiary alcohols to the
corresponding azides through NaN3/H2SO4 (various concen-
4b,12
11
trations)/CHCl3
or NaN3/TFA/CHCl3 can result in
unreacted alcohol and found that the transformation proceeds
efficiently using NaN3/TFA 1 M in CH2Cl2. The amine 6 was
prepared by means of LiAlH4 reduction of the azide 15 in
refluxing ether.
4630
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
Biological Evaluation. (a) Antiviral Evaluation. EC50
values from dose−response tests against five strains of influenza
A in Madin−Darby canine kidney (MDCK) cells were
measured using a primary infection assay (Table 2). This
assay detects block at the early stages of viral replication, from
endocytotic uptake to protein synthesis.
All compounds except 1−5 display potent antiviral activity
against the pandemic 2009 strain (arbitrarily designated as <24
μM based on the amantadine insensitivities observed here). It is
striking that the addition of as little as one CH2 group to the
methyl adduct of the amantadine/rimantadine analogue, 2-
methyl-2-aminoadamantane 4, essentially recovers activity in
vitro against this amantadine-resistant form of influenza A.
Likewise, the amantadine-resistant H1N1 strain from 1934
(second column), containing a double mutant M2 (T27 +
N31), is highly sensitive to these compounds. But the drugs do
not block all M2(S31N)-bearing or all H1N1 strains10e as
shown by the third column, which shows that the 1933 Wilson
Smith H1N1 isolate is insensitive to most of these compounds.
Furthermore, the M2 pore region (residues 22−46) of A/WS/
33 and A/Calif/2009 are identical except for the L43T variation
at the C-terminus (Supporting Information Table 1). These
observations are consistent with the negative electrophysiology
results for the A/Calif/2009/M2, further suggesting that the
antiviral effects observed against the A/Calif/2009 strain are
independent of M2 and that instead they attack a second
target.7,10g This second target is most likely present in A/Calif/
07/2009 and probably in A/PR/8/34 but not in A/WS/33.
Strains with WT M2 are very sensitive to these compounds
(fourth and fifth columns), suggesting that, like amantadine,
these drugs also block M2. However, from the structure−
activity point of view, differences between the sequence and
optimal efficacies vary, suggesting that nonbinding site residue
differences in the M2 may alter efficacy. For instance 6 is the
one of the most potent in the set against A2/Taiwan/1/64
H2N2 but the least potent of the set against A/Victoria/3/75
H3N2, even though both have WT-M2 amantadine-binding
sites. They differ in only two residues, 13 and 56, neither of
which is in the pore region (Supporting Information Table 1).
This suggests that extra-pore residues may affect M2 block. On
the other hand, 9 is the most effective from the set against all of
the strains tested except A2/Taiwan/1/64, where it is among
the least effective, which may suggest that the relative impacts
of M2 block and any alternative mechanisms of action are also
dependent on drug structure.
Figure 1. Superimposed PISEMA spectra of the S31N M2
transmembrane domain (residues 22−46), 15N labeled at residues
V28, A30, and I42, in dimyristoylphosphatidylcholine bilayers
uniformly aligned on glass slides with (red) and without (black)
compound 6. Assignments were made based on the known structure
and spectra of WT M2 TMD.15 The assignments with drug follow
based on the rotational orientation of the helices.
the entire helix−helix interface changes with the ∼3° reduction
in tilt of the four helices. Similar results from ssNMR
experiments and proteoliposome assays were obtained with
two related aminoadamantanes that are not included in Scheme
1.
Electrophysiology Results Using Full-Length M2.
Representative compounds 3 and 6 were subsequently tested
for block of proton currents through full-length M2 having the
same amino acid sequence as A/California/07/2009 (viz.,
S31N) using transiently transfected, voltage-clamped HEK
cells16 and found not to block inward proton currents on the 3
min time scale with any improvement over amantadine 1
(Table 1, Figure S1 in Supporting Information). Prolonged
exposure (30 min) yielded but little increase in net block over 3
min exposure for the two drugs. When the M2 protein was
reverted to the S31 WT sequence (through an N31S
mutation), inward proton currents in M2-transfected HEK
cells were well blocked by 1, 3, and 6. This electrophysiology
result suggests (a) that these two drugs do not block the M2
S31N channel in full length A/Calif/07/2009 and must have a
different target and (b) that biophysical models for the M2
protein should be based on the whole protein rather than
segments. This conclusion supports other studies suggesting
another target of aminoadamantane compounds.7,10e,g Similar
results were also obtained with a few other related amino-
adamantanes not included in this work.
(b) Resistance Experiments: Sequencing of Resistant
Strains. Resistance testing with semiweekly passages in
MDCK cell cultures was performed for amantadine 1 against
an amantadine-sensitive H3N2 virus and for compound 6
against amantadine-resistant H1N1 (2009) (Table 3).
In the amantadine−H3N2 system, drug resistance appeared
after one passage in the presence of drug, with no detectable
a
Table 1. Proton Channel Block Measured in Transfected HEK Cells for Compounds Tested
b
A/England/195/2009 (H1N1), M2/N31
A/England/195/2009 (H1N1), M2/S31
compd
% block after 3 min
% block after 30 min
% block after 3 min
IC50, μM
1
3
6
14 2 (100 μM; 26)
13 3 (100 μM; 2)
(N/A)
75 9 (10 μM; 4)
95 8 (10 μM; 2)
63 5 (10 μM; 2)
1.6 2.7 (3)
2.5 0.5 (2)
16 (100 μM; 1)
9.4 10 (100 μM, 3)
0
5 (100 μM; 2)
7
2 (2)
a
For each compound, % block of pH-dependent M2 current at 10 or 100 μM ( SEM) or the IC50 (μM) is shown. Number of replicates is shown in
b
parentheses. The M2 sequence for this strain is identical to that of A/Calif/07/2009 M2.
4631
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
a
Table 2. In Vitro Efficacy (EC50, μM) of Scheme 1 Compounds against Initial MDCK Cell Infection
compd
A/Calif/07/09 (H1N1)
A/PR/8/34 (H1N1)
A/WS/33 (H1N1)
A2/Taiwan/1/64 (H2N2)
A/Victoria/3/75 (H3N2)
M2
1
S31N
V27T/S31N
S31N
WT
WT
240 90 (13)
110 40 (13)
150 30 (20)
54 2 (20)
25 3 (21)
4.7 0.9 (20)
8.5 0.6 (20)
8.0 0.3 (21)
0.13 0.02 (2)
21 2 (21)
24 3.5 (21)
3.3 0.5 (2)
3.8 1.0 (2)
0.4 0.4 (2)
1.8 0.9 (2)
0.5 0.2 (2)
0.3 0.3 (2)
0.3 0.5 (2)
0.07 0.09 (2)
<0.3 0.5 (2)
1.2 1.1 (2)
24 1.1 (21)
310 140 (2)
110 15 (2)
19 4 (2)
0.34 0.01 (21)
1.6 0.3 (2)
0.8 0.3 (2)
0.5 0.5 (2)
0.8 0.3 (2)
<0.24 (2)
2.8 0.3 (16)
0.53 0.07 (18)
3.3 0.9 (2)
2.0 0.4 (2)
2.0 0.4 (2)
23 8 (2)
2
3
4
5
23 3 (2)
6
390 8 (2)
355 4 (2)
210 40 (2)
13.0 3.6 (2)
86 20 (2)
280 150 (2)
7
1.5 0.3 (2)
0.4 0.1 (2)
1.5 0.3 (2)
0.2 0.2 (2)
0.2 0.3 (2)
4
1 (2)
8
13 2 (2)
9
1.1 0.1 (2)
10
11
8
1 (21)
8.6 0.8 (21)
18 2 (21)
a
EC50 its standard error (N) from miniplaque testing for dose−response or single-dose screens, using cultured MDCK cells, based on least-
squares fitting of single-site binding curves. N is the number of assay counts fitted. Experiments with N = 2 are based on replicate 50 μM screens
(except for 9, which were based on replicate 5 μM screens), with a single control (N = 4) for each virus. Row M2 gives variations from the WT
amantadine-binding site (i.e., L26, V27, A30, S31, and G34) for the specific strain listed, WT if none. (See Tables S3, S4, and S5 for the M2
sequences of the isolates used here.) No microscopic evidence of cytotoxicity to MDCK cells was detected after an 18 h exposure at 50 μM except
with compound 9, where a 5 μM dose was used instead. The EC50 values of amantadine 1 and rimantadine 2, known to be inactive against H1N1
(2009), and other cases where EC50 ≥ 24 μM are highlighted.
S187P, and I325S (numbering started after the 13-residue HA
signal sequence).
Table 3. Resistance Testing of Amantadine 1 and 2-n-Propyl-
2-aminoadamantane 6
a
To evaluate whether these mutations were merely due to
adaptation to MDCK cell culture growth, we also sequenced
the M (Tables S2 and S3) and HA segments (Table S1) from
the parent virus after 30 passages in MDCK culture without
drug. For the M segment of these drug-free controls, no
changes were found in M1, while 2 of 5 plaques had an E14G
EC50 SE (μM)
passage
no.
1 (5 μM), A/Victoria/3/75
6 (5 μM), A/Calif/07/2009
(H3N2, M2 WT)
(H1N1, M2 S31N)
0
2.77 0.29
inactive
inactive
ND
4.71 0.92
5.4 1.4
3.7 0.5
2.1 1.6
18.5 1.0
1
2
5
8
ND
10
12
ND
76
9
ND
149 115
a
EC50
SE (μM) (N = 21) after designated passage (incubation)
stages. Drug concentration in medium as specified except that for 6,
passages 1 and 2 were done in 10 μM. Inactive: no miniplaque
reduction by 50 μM amantadine. ND: not done.
activity of amantadine 1 against the progeny from passage 1 or
passage 2 at 50 μM but normal amantadine 1 activity against
the original virus post hoc (EC50 = 3.0 0.5 μM; N = 9). In
contrast, in the 6−H1N1 system, virus progeny produced in the
presence of drug at passages 1−5 maintained full drug
sensitivity (EC50 = 2.1−5.4 μM). Resistance to 6 developed
steadily between passage 6 and passage 12, becoming significant
after passage 10. Without any drug in the medium, the
development of viral resistance to compound 6 was negligible;
i.e., the EC50 retested at passage 0 was 4.7 0.7 μM, at passage
10 was 3.0
0.3, and at passage 30 was 7.7
0.6 μM.
Resistance to amantadine develops rapidly in vitro,17 in mice,18
and in the clinical setting19 through a small set of mutations,
primarily L26F, V27A, V27T, A30T, S31N, and G34E.20 These
are residues whose side chains are near the 4-fold symmetric
amantadine binding site.5 No changes from the parent A/
California/07/2009 were observed for the amino acid trans-
lation of the M-segment of the passage-12 6-resistant strain for
residues sequenced, 10−73. Hence, resistance did not develop
by selection of additional amantadine-resistance mutations in
M2. Sequencing of segment 4 (HA gene), however, revealed
three amino acid substitutions (Figure 2, Table S1) compared
to the parental A/California/07/2009 sequence, i.e., N160D,
Figure 2. CPT structure of the HA trimer, produced by Gamblin et al.
(1RVX, A/Puerto Rico/8/1934)21 with a bound NAG-GAL-SIA
ligand as ball-and-stick (red) and with the two nearby 6-resistance sites
highlighted in blue, residues 159 (above ligand) and 186 (left of
ligand). The third 6-resistance site, 324, also in blue, is near the
bottom of the structure. Because of a common insertion after residue
133 found in A/Calif/07/2009 (H1N1), these correspond to N160,
S187, and I325, respectively, in the A/Calif/07/2009 6-resistant
mutants.
4632
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
a
substitution in M2, which is outside the transmembrane
domain.
Table 4. Mutations Developing in Influenza A (M2 WT)
b
after Passaging in Aminoadamantane Derivatives 4 and 6
Substitution S187P, located near the N-terminus of the 190
sialic acid binding helix, is frequent in pandemic H1N1 and was
observed previously by Torres et al. in resistance development
using a related set of aminoadamantanes.10g In the drug-free
controls, 2 out of 5 plaques showed this mutation, as well as 1
of 4 previously sequenced isolates of A/California/07/2009
(KF00954, Table S1).
c
compd
d
passage no.
2
plaque no.
4, 1 μg/mL
6, 5 μg/mL
1
2
3
1
2
3
WT
A30T
A30T
A30T
A30T
A30T
A30T
WT
WT
5
A30T
A30V
A30T
N160 is located on the tip of a nearby loop that is very close
to the 190 helix, the region where sialic acid residues of the host
cell receptor bind. Among 1750 HA sequences of pandemic
H1N1 deposited in the GenBank only four sequences with
D160 were observed. Substitution of N160 by an aspartic acid
residue would modify the local charges and may thus affect
receptor interactions. Interestingly, D160 is also observed in A/
WS/33, which may account for the insensitivity of this strain to
the compounds tested here (Table 1). However, in the drug-
free controls, 1 of 5 plaques tested had this mutation (Table
S1). Three other plaques had the G159E mutation, suggesting
that an acidic group in that neighborhood is advantageous for
the pandemic virus replication in MDCK culture. On the other
hand that mutation is also present in our sample of the highly
6-sensitive A/PR/8/34 strain (Table S1), suggesting that if
D160 is critical for drug inhibition, an acid group at position
159 is not sufficient for drug resistance. The possibility that
N160D is important to escape from 6 cannot be ruled out.
S325 is close to the HA0 processing site at R331 and the
corresponding residue, 324, was also found to be modified in
the aminoadamantane resistance development study by Torres
et al.10g None of the 1750 HA sequences of pandemic H1N1 in
GenBank has a serine at position 325. This substitution may
affect maturation cleavage or pH stability of HA. Although it
was pointed out10g that a mutation to T at this site is found in
one sequence of A/Puerto Rico/8/1934 and that this site may
be polymorphic, we found seven sequences for that strain
without the mutation and no I325T substitution was observed
in our GenBank set of 1750 pandemic H1N1 strains.
Furthermore, no instances of an I325 mutation were observed
in the drug-free control virus plaques (Table S1). This suggests
that drugs inhibit an important function at this site such as
enzyme binding or cleavage.
To examine the resistance development pathways of the
H3N2 M2 WT virus to these compounds in more detail,
passaging experiments were carried out with plaque sequencing
analysis in the presence of active compound 4 or 6 (Table 4).
The WT virus rapidly develops resistance to both compounds
through mutation at Ala30, especially to Thr, suggesting that
these drugs block the M2 WT but do not block A30T.
Conversely, the lack of sequence changes for M2(S31N)-
bearing virus in the presence of compound 6 mentioned above
indicates that M2(S31N)-bearing virus has a different escape
route than M2(WT)-bearing virus. In the latter case changes
inside the M2 pore confer resistance, while in the former no
mutations were observed in the M2 channel amantadine
binding site; therefore, some other change in the virus is
implicated.
c
compd
d
passage no.
10
plaque no.
4, 2 μg/mL
6, 5 μg/mL
1
2
3
A30T
A30T
A30T
A30T
A30T
A30T
b
a
Parent strain: A/Hong Kong/1/1968 (H3N2M2 WT). Sequences
of resistant progeny of WT induced by compounds in the top row.
MDCK cells were bathed in medium containing the concentrations
specified. Three separate plaques were sampled and sequenced at
c
d
passages 2, 5, and 10. Compound number from Scheme 1. Passage
number.
The apparent simplicity of the synthetic schemes is a virtue of
2-alkyl-2-aminoadamantane derivatives. Resistance develop-
ment in cell culture is markedly reduced for one representative
compound 6 (R = n-Pr) compared to amantadine 1. These
compounds found to be active against two of three S31N
strains (A/Calif/07/009 and A/PR/8/34 but not A/WS/33)
did not block M2, judging by the lack of transfected HEK cell
current block and the lack of M2 changes in the 6-resistant A/
Calif/07/2009, and therefore must have acted on a second
target. The ssNMR study that confirmed that drugs with large
alkyl adducts were sterically suited to fit in the amantadine
binding site in M2 were done at effectively high drug
concentrations and using truncated M2 protein (22−46) and
do not indicate the potential of drugs to block the S31N variant
of M2.
A few alternative candidate mechanisms of action for these
drugs include pH buffering of the endosome, pH buffering of
the viral interior, stabilization of hemagglutinin against acid
activation, and mechanical stabilization at lipid−water interfaces
against envelope−endosomal membrane fusion. The observa-
tions of an HA1 mutation, N160D, near the sialic acid binding
site in both 6-resistant A/Calif/07/2009(H1N1) and the
broadly resistant A/WS/33(H1N1) and of an HA1 mutation
I325S in the 6-resistant virus at a cell-culture stable site suggest
that the drugs tested here may block infection by direct binding
near these critical sites. The region near residue 160 is critical
for binding virus to the cell surface, and the region near residue
325 is critical for HA activation by proteolytic cleavage, both
necessary for the virus entry into the host cell. It is also possible
that the drugs neutralize the endosome and that these sites,
individually or in combination, affect pH sensitivity of HA, as
has been suggested in similar situations previously.7,10g
However, miniplaque assays with compounds 3−6 against
influenza B/Russia/69 in MDCK cells and compound 6 against
bovine parvovirus in bovine embryonic cells, respectively, both
of which are chloroquine sensitive,22,23 showed no effect of 3−6
or 6, respectively, on virus growth with 50 μM drug in the
medium (data not shown), suggesting that these compounds
are less potent endosome neutralizers than chloroquine.
CONCLUSION
■
The addition of as little as one CH2 group to the methyl adduct
of the amantadine/rimantadine analogue, 2-methyl-2-amino-
adamantane 4, has been discovered to largely recover activity in
vitro against the amantadine-resistant 2009 H1N1 influenza A.
4633
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
Anal. Calcd for C12H22NCl: C, 66.80; H, 10.28; N, 6.49. Found: C,
66.93; H, 10.42; N, 6.87.
Further experiments are needed to explore these and other
possibilities.
2-n-Propyltricyclo[3.3.1.13,7]decan-2-amine (6). Tertiary alco-
hol 13 was obtained after treating adamantanone 12 (1.0 g, 6.67
mmol) with CH2CHCH2MgBr in 1:2 ratio (obtained from
CH2CHCH2Br (1.61 g, 13.3 mmol), 1.5 molar excess of Mg (486
mg, 20.01 mmol) in 20 mL of dry ether/g bromobenzene): yield 89%;
1H NMR δ 1.52 (d, J = 12 Hz, 2H, 4′eq, 9′eq-H), 1.53−1.90 (m, 10H,
adamantane-H), 2.15 (d, J = 12 Hz, 1H, 4′ax, 9′ax-H), 2.40 (d, J = 6
Hz, 2H, CH2CHCH2), 5.05−5.15 (m, 2H, CH2CHCH2), 5.75−
6.0 (m, 1H, CH2CHCH2). The unsaturated alcohol 13 (890 mg,
4.64 mmol) was hydrogenated under PtO2 (45 mg) (catalyst was used
Continued outbreaks of amantadine-resistant viruses like
H7N9 merit the urgency to develop new antivirals with
persistent efficacy in global preparations for pandemic threats.24
The new observation of persistent efficacy of these amantadine-
like drugs via second targets, while retaining potency (albeit
resistance vulnerable) to WT M2, makes this family of
compounds intriguing starting points for further studies on
resistance and mechanism of action against influenza A.
1
in /20 percentage to the weight of the unsaturated compound) to
1
EXPERIMENTAL SECTION
afford the n-propyl analogue 14: yield, quant; H NMR (CDCl3, 400
■
MHz) δ 0.92 (t, J = 7 Hz, 3H, CH3), 1.30−1.40 (m, 2H,
CH2CH2CH3), 1.52 (d, J = 12 Hz, 2H, 4′eq, 9′eq-H), 1.58−1.61
(m, 2H, CH2CH2CH3), 1.68 (d, J = 12 Hz, 2H, 8′eq, 10′eq-H), 1.67
(br s, 2H, 6′-H), 1.68 (br s, 2H, 1′, 3′-H), 1.79 (m, 2H, 5′, 7′-H), 1.83
(d, J = 12 Hz, 2H, 8′ax, 10′ax-H), 2.16 (d, J = 12 Hz, 2H, 4′ax, 9′ax-
H); 13C NMR (CDCl3, 50 MHz) δ 14.9 (CH3), 15.4 (CH2CH2CH3),
27.4, 27.6 (5, 7-C), 33.1 (CH2CH2CH3) 34.7 (4, 9-C), 37.1 (8, 10-C),
38.5 (1, 3-C), 40.9 (6-C), 75.2 (2-C).
(A) Chemistry. Melting points were determined using a Buchi
capillary apparatus and are uncorrected. IR spectra were recorded on a
Perkin-Elmer 833 spectrometer. 1H and 13C NMR spectra were
recorded on a Bruker DRX 400 and AC 200 spectrometer at 400 and
50 MHz, respectively, using CDCl3 as solvent and TMS as internal
standard. Carbon multiplicities were established by DEPT experi-
ments. The 2D NMR techniques (HMQC and COSY) were used for
the elucidation of the structures of intermediates and final products.
Microanalyses were carried out by the Service Central de
Microanalyse (CNRS), France, and by the Microanalyses lab of the
National Center for Scientific Research, Demokritos, Athens, Greece,
and the results obtained had a maximum deviation of 0.4% from the
theoretical value. All tested synthesized compounds possess a purity
above 95% as determined through elemental C, H, N analysis.
Full experimental details that were not given previously for
compounds 4 and 54b are included in this paper.
The alcohol 14 (700 mg, 4.22 mmol) was added to a stirred mixture
of H2SO4 70% w/w (10 mL) and chloroform (25 mL) at 0 °C.
Sodium azide was added in small portions at 0 °C, and the mixture was
stirred for 48 h at ambient temperature. The mixture was poured into
an ice−water mixture and was extracted with dichloromethane. The
organic phase was washed with water, saturated NaHCO3, and brine,
dried (Na2SO4), and evaporated under vacuum at room temperature.
The oily residue (650 mg) was flash-chromatographed on silical gel
(35−70 μm) with hexane−AcOEt 5/1 as an eluent to give the pure
azide 15: yield 530 mg (66%). The azide 15 was found to form
quantitatively using TFA/CH2Cl2/NaN3 system (1 mmol of alcohol
14 was treated with 10 mmol of TFA and 4 mmol of NaN3 in 40 mL
of CH2Cl2; see experimental procedure for ethyl- or 2-n-butyltricyclo-
2-Ethyltricyclo[3.3.1.13,7]decan-2-amine (5). 2-Ethyl-2-adaman-
tanol 17 was obtained after treating a solution of 2-adamantanone 12
(500 mg, 3.34 mmol) in dry THF (10 mL, 30% solution w/v) with n-
ethyllithium at 0 °C in a 3.7 molar excess (25 mL, 12.5 mmol, 0.5 M in
1
benzene) and stirring the mixture overnight: yield 94%; H NMR
1
[3.3.1.13,7]decan-2-azide 24 or 25): H NMR (CDCl3, 400 MHz) δ
(CDCl3, 400 MHz) δ 0.86 (t, J = 7 Hz, CH2CH3), 1.40−1.70 (m, 10
H, 1′,3′-H, 4′eq, 9′eq-H, 6′-H, 8′eq,10′eq-H, CH2CH3), 1.75−1.83
(m, 2H, 5′,7′-H), 1.94 (d, J = 12 Hz, 2H, 8′ax, 10′ax-H), 2.07 (d, J =
12 Hz, 2H, 4′ax, 9′ax-H); 13C NMR (CDCl3, 50 MHz) δ 6.4
(CH2CH3), 27.4, 27.5 (5′,7′-C), 30.6 (CH2CH3), 33.0 (8′,10′-C),
34.6 (4′,9′-C), 36.6 (1′,3′-C), 38.5 (6′-C), 74.9 (2′-C).
0.96 (t, J = 7 Hz, 3H, CH3), 1.42 (m, 2H, CH2CH2CH3), 1.59 (d, J =
12 Hz, 2H, 4′eq, 9′eq-H), 1.68−2.03 (m, 12H, CH2CH2CH3,
adamantane-H), 2.10 (d, J = 12 Hz, 1H, 4′ax, 9′ax-H); 13C NMR
(CDCl3, 50 MHz) δ 14.7 (CH3), 16.4 (CH2CH2CH3), 27.2, 27.4 (5,
7-C), 33.8 (CH2CH2CH3) 34.4 (4, 9-C), 37.9 (8, 10-C), 38.5 (1, 3-C),
40.0 (6-C), 69.7 (2-C).
To a stirred mixture of NaN3 (0.195 g, 3.0 mmol) and dry
dichloromethane (5 mL) at 0 °C, TFA (1.14 g, 10.0 mmol) was added.
To the stirred mixture, a solution of 2-ethyl-2-adamantanol 17 (0.180
g, 1.0 mmol) in dry dichloromethane (5 mL) was added, and stirring
was maintained at 0 °C for 4 h. The mixture was stirred at ambient
temperature for 24 h and then was treated with NH3 12% (30 mL) at
0 °C. The organic phase was separated, and the aqueous phase was
extracted twice with an equal volume of dichloromethane. The
combined organic phase was washed with water and brine, dried
(Na2SO4), and evaporated to afford oily 2-ethyl-2-adamantylazide 24:
IR (Nujol) ν(N3) 2100 cm−1; yield 0.160 g (80%).
To a stirred suspension of LiAlH4 (0.120 g, 0.78 mmol) in dry ether
(10 mL) was added, dropwise at 0 °C, a solution of the 2-ethyl-2-
adamantylazide 24 (0.160 g, 3.12 mmol) in dry ether (5 mL). The
reaction mixture was refluxed for 5 h (TLC monitoring) and then
hydrolyzed with water and NaOH (15%) and water under ice cooling.
The inorganic precipitate was filtered off and washed with ether, and
the filtrate was extracted with HCl (6%). The aqueous layer was made
alkaline with solid Na2CO3, and the mixture was extracted with ether.
The combined ether extracts were washed with water and brine and
dried (Na2SO4). After evaporation of the solvent the oily amine 5 was
obtained: yield 100 mg (71%); 1H NMR (CDCl3, 400 MHz) δ 0.85 (t,
J = 7 Hz, 3H, CH3), 1.55 (br s, 2H, 1′,3′-H), 1.58−1.68 (m, 6H, 4′eq,
9′eq-H, 8′eq 6′-H), 1.78 (br s, 1H, 5′-H), 1.81 (br s, 1H, 7′-H), 1.93
(d, J = 12 Hz, 2H, 8′ax, 10′ax-H), 2.06 (d, J ≈ 12 Hz, 2H, 4′ax, 9′ax-
H); 13C NMR (CDCl3, 50 MHz) δ 6.5 (CH2CH3), 27.2, 27.6 (5′,7′-
C), 30.7 (CH2CH3), 33.0 (4′,9′-C), 33.8 (8′,10′-C),36.6 (1′,3′-C),
38.5 (6′-C), 74.9 (2′-C). Hydrochloride: mp >250 °C (EtOH−Et2O).
To a stirred suspension of LiAlH4 (390 mg, 10.3 mmol) in dry ether
(20 mL) was added, dropwise at 0 °C, a solution of the azide 15 (490
mg, 2.57 mmol) in dry ether (10 mL). The reaction mixture was
refluxed for 5 h (TLC monitoring) and then hydrolyzed with water
and NaOH (15%) and water under ice cooling. The inorganic
precipitate was filtered off and washed with ether, and the filtrate was
extracted with HCl (6%). The aqueous layer was made alkaline with
solid Na2CO3, and the mixture was extracted with ether. The
combined ether extracts were washed with water and brine and dried
(Na2SO4). After evaporation of the solvent the oily amine 6 was
obtained: yield 350 mg (74%); 1H NMR (CDCl3, 400 MHz) δ 0.92 (t,
J = 7 Hz, 3H, CH3), 1.29−1.40 (m, 2H, CH2CH2CH3), 1.52 (d, J = 12
Hz, 2H, 4′eq, 9′eq-H), 1.58−1.61 (m, 2H, CH2CH2CH3), 1.67 (d, J =
12 Hz, 2H, 8′eq, 10′eq-H), 1.66 (br s, 2H, 6′-H), 1.68 (br s, 2H, 1′, 3′-
H), 1.78 (br s, 2H, 5′, 7′-H), 1.83 (d, J = 12 Hz, 2H, 8′ax, 10′ax-H),
2.16 (d, J = 12 Hz, 1H, 4′ax, 9′ax-H). Hydrochloride: mp > 250 °C
(EtOH−Et2O). Anal. Calcd for C13H24NCl: C, 67.95; H, 10.53; N,
6.10. Found: C, 68.02; H, 10.63; N, 5.95.
2-n-Butyltricyclo[3.3.1.13,7]decan-2-amine (7). Tertiary alcohol
18 was obtained after treating a solution of adamantanone 12 (500
mg, 3.34 mmol) in dry THF (30% solution w/v) with 3 molar excess
of n-butyllithium (6 mL, 10.02 mmol, 1.6 M in hexanes) at 0 °C and
stirring the mixture overnight: yield 96%; 1H NMR (CDCl3, 400
MHz) δ 0.91 (t, J = 7 Hz, 3H, CH3), 1.25−1.38 (m, 4H,
CH3CH2CH2CH2), 1.54 (d, J = 12 Hz, 2H, 4′eq, 9′eq-H), 1.58−
1.72 (m, 8H, 1′, 3′, 5′, 7′, 8′eq, 10′eq-H, CH3CH2CH2CH2), 1.78−
1.90 (m, 4H, 8′ax, 10′ax-H, 5′,7′-H), 2.16 (d, J = 12 Hz, 1H, 4′ax, 9′ax-
H); 13C NMR (CDCl3, 50 MHz)
δ 14.3 (CH3), 23.5
4634
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
(CH2CH2CH2CH3), 24.4 (CH2CH2CH2CH3), 27.4, 27.6 (5′,7′-C),
34.7 (4′,9′-C), 33.1 (8′,10′-C), 37.1 (1′,3′-C), 38.2
(CH2CH2CH2CH3), 38.5 (6′-C), 75.2 (2′-C).
2-n-Hexyltricyclo[3.3.1.13,7]decan-2-amine (9). Tertiary alco-
hol 20 was obtained after the reaction of n-hexyllithium with 2-
adamantanone 12 (500 mg, 3.34 mmol) in dry THF (5 mL) with n-
hexyllithium (4 mL, 10.02 mmol, 2.47 M in hexanes) at 0 °C in a 1:3
To a stirred mixture of NaN3 (280 mg, 4.32 mmol) and dry
dichloromethane (20 mL) at 0 °C, TFA (1.6 mg, 14.4 mmol) was
added. To the stirred mixture, a solution of tertiary alcohol 18 (300
mg, 1.44 mmol) in dry dichloromethane (10 mL) was added, and
stirring was maintained at 0 °C for 4 h. The mixture was stirred at
ambient temperature for 24 h and then was treated with NH3 12% (30
mL) at 0 °C. The organic phase was separated, and the aqueous phase
was extracted twice with an equal volume of dichloromethane. The
combined organic phase was washed with water and brine, dried
(Na2SO4), and evaporated to afford oily azide 25: yield 96%; IR
(Nujol) ν(N3) 2088 cm−1; 1H NMR (CDCl3, 400 MHz) δ 0.96 (t, J =
7 Hz, 3H, CH3), 1.32−1.42 (m, 4H, CH3CH2CH2 CH2), 1.62 (d, J =
12 Hz, 2H, 4′eq, 9′eq-H), 1.70−1.93 (m, 12H, adamantane-H,
CH3CH2CH2CH2), 2.14 (d, J = 12 Hz, 2H, 4′ax, 9′ax-H); 13C NMR
(CDCl3, 50 MHz) δ 14.2 (CH3), 23.3 (CH2CH2CH2CH3), 24.9
(CH2CH2CH2CH3), 27.2, 27.4 (5′,7′-C), 33.8 (4′,9′-C),33.7 (8′,10′-
C), 34.4 (1′,3′-C), 35.2 (CH2CH2CH2CH3), 38.5 (6′-C), 69.7 (2′-C).
To a stirred suspension of LiAlH4 (163 mg, 4.29 mmol) in dry ether
(15 mL) was added, dropwise at 0 °C, a solution of the azide 25 (250
mg, 1.07 mmol) in dry ether (10 mL). The reaction mixture was
refluxed for 5 h (TLC monitoring) and then hydrolyzed with water
and NaOH (15%) and water under ice cooling. The inorganic
precipitate was filtered off and washed with ether, and the filtrate was
extracted with HCl (6%). The aqueous layer was made alkaline with
solid Na2CO3, and the mixture was extracted with ether. The
combined ether extracts were washed with water and brine and dried
(Na2SO4). After evaporation of the solvent the oily amine 7 was
obtained: yield 50 mg (23%); 1H NMR (CDCl3, 400 MHz) δ 0.88 (t, J
= 7 Hz, 3H, CH3), 1.18−1.32 (m, 4H, CH3CH2CH2CH2), 1.45−165
(m, 10H, adamantane-H, CH3CH2CH2CH2), 1.77 (br s, 2H, 5′,7′-H),
1.93 (d, J = 12 Hz, 2H, 8′ax, 10′ax-H), 2.03 (d, J = 12 Hz, 2H, 4′ax,
9′ax-H), 2,13 (br s, 2H, NH2); 13C NMR (CDCl3, 50 MHz) δ 14.3
(CH3), 23.7 (CH2CH2CH2CH3), 24.6 (CH2CH2CH2CH3), 27.5, 27.8
(5′,7′-C), 34.1 (4′,9′-C), 33.2 (8′,10′-C), 37.5 (1′,3′-C), 38.6 (6′-C),
39.1 (CH2CH2CH2CH3), 54.5 (2′-C). Fumarate: mp 220 °C (EtOH−
Et2O). Anal. Calcd for C18H29NO4: C, 66.86; H, 9.26; N, 4.32. Found:
C, 66.91; H, 9.30; N, 4.29.
1
ratio as before: yield 97%; IR (Nujol) ν(OH) 3391 cm−1; H NMR
(CDCl3, 400 MHz) δ 0.87 (t, J = 7 Hz, 3H, CH3), 1.24−1.33 (m, 8H,
CH2(CH2)4CH3), 1.51−154 (d, J = 12 Hz, 2H, 4′eq, 9′eq-H), 1.60−
1.64 (m, 2H CH2(CH2)4CH3), 1.66−1.69 (m, 6H, 1′,3′,6′, 5′,7′-H),
1.78−1.81(d, J ≈ 11 Hz, 2H, 8′ax, 10′ax-H), 2.14−2.17 (d, J = 12 Hz,
2H, 4′ax, 9′ax-H); 13C NMR (CDCl3, 50 MHz) δ 14.2 ((CH2)5CH3),
22.1 ((CH2)4CH2CH3), 22.7 ((CH2)3CH2CH2CH3), 27.4−27.6
( 5 ′ , 7 ′ - C ) , 3 0 . 1 ( C H 2 C H 2 C H 2 ( C H 2 ) 2 C H 3 ) , 3 2 . 0
(CH2CH2(CH2)3CH3), 33.1 (4′,9′-C), 34.7 (8′, 10′-C), 37.1 (1′, 3′-
C), 38.4 (CH2(CH2)4CH3), 38.5 (6′-C), 75.1 (2′-C).
The corresponding azide 27 was prepared from the alcohol 20 (400
mg, 1.69 mmol) according to the same procedure followed for azide
25 using CH2Cl2 (30 mL)/NaN3 (330 mg, 5.07 mmol)/TFA (1.9 mg,
16.9 mmol): yield 91%; IR (Nujol) ν(N3) 2088 cm−1; 13C NMR
(CDCl3, 50 MHz) δ 14.2 ((CH2)5CH3), 22.6 ((CH2)4CH2CH3), 22.7
( C H 2 ) 3 C H 2 C H 2 C H 3 ) , 2 7 . 2 − 2 7 . 4 ( 5 ′ , 7 ′ - C ) , 2 9 . 9
(CH2CH2CH2(CH2)2 CH3), 31.9 (CH2CH2(CH2)3 CH3), 33.7
(4′,9′-C), 33.8 (8′,10′-C), 34.4 (1′,3′-C), 35.4 (CH2(CH2)4 CH3),
38.5 (6′-C), 69.7 (2′-C).
The corresponding oily amine 9 was prepared through LiAlH4 (233
mg, 6.13 mmol) reduction of azide 27 (400 mg, 1.53 mmol) in
refluxing ether for 5 h according to the same procedure followed for
amine 7: yield 97%; 1H NMR (CDCl3, 400 MHz) δ 0.87 (t, J = 7 Hz,
3H, CH3), 1.24−1.30 (m, 8H, CH2(CH2)4CH3), 1.51−156 (m, 4H,
4′eq, 9′eq-H, CH2(CH2)4CH3), 1.57−1.67 (m, 6H, 1′, 3′, 6′, 8′eq,
10′eq-H), 1.79 (br s, 2H, 5′,7′-H), 1.93 (d, J = 12 Hz, 2H, 8′ax, 10′ax-
H), 2.04 (d, J = 12 Hz, 2H, 4′ax, 9′ax-H); 13C NMR (CDCl3, 50
MHz)
δ 14.2 (CH3 ), 22.3 ((CH2 )4 CH2 CH3 ), 22.8
( ( C H 2 ) 3 C H 2 C H 2 C H 3 ) , 2 7 . 4 − 2 7 . 8 ( 5 ′ , 7 ′ - C ) , 3 0 . 3
(CH2CH2CH2(CH2)2CH3), 32.0 (CH2CH2(CH2)3CH3), 33.1 (4′,9′-
C), 34.1 (8′, 10′-C), 37.4 (1′, 3′-C), 38.8 (CH2(CH2)4 CH3), 39.1 (6′-
C), 54.6 (2′-C). Fumarate: mp 225 °C (EtOH−Et2O). Anal. Calcd for
C20H33NO4: C, 68.94 H; H, 9.46; N, 3.99. Found: C, 68.59; H, 9.55;
N, 3.79.
2-Phenyltricyclo[3.3.1.13,7]decan-2-amine (10). Tertiary alco-
hol 21 was obtained after treating a solution of adamantanone 12 (500
mg, 3.34 mmol) in dry THF (30% solution w/v) with 2 molar excess
PhMgBr (obtained from bromobenzene (1.05 g, 6.68 mmol) and 1.5
molar excess of Mg (240 mg, 10.02 mmol) in 20 mL of dry ether/g
bromobenzene) and stirring the mixture overnight: yield 95%; 1H
NMR (CDCl3, 400 MHz) δ 1.67−1.77 (m, 8H, adamantane-H), 1.89
(br s, 2H, 5′,7′-H), 2.14 (s, 1H, OH), 2.40 (d, J = 12 Hz, 1H, 4′ax,
9′ax-H), 2.56 (br s, 2H, 1′,3′-H), 7.20−7.60 (m, 5H, phenyl-H); 13C
NMR (CDCl3, 50 MHz) δ 27.0, 27.5 (5′,7′-C), 33.1 (4′,9′-C), 34.9
(8′,10′-C), 35.7 (1′,3′-C), 37.8 (6′-C), 75.8 (2′-C), 125.5, 127.1,
127.2, 128.8, 143.0 (Ph).
2-Isobutyltricyclo[3.3.1.13,7]decan-2-amine (8). Tertiary alco-
hol 19 was obtained after treating a solution of 2-adamantanone 12
(500 mg, 3.34 mmol) in dry THF (5 mL) with isobutyllithium (8 mL,
10.02 mmol, 1.6 M in hexanes) at 0 °C in a 1:3 ratio as before: yield
85%; 1H NMR (CDCl3, 400 MHz) δ 0.96 (d, J = 7 Hz, 6H, 2 x CH3),
1.52 (d, J = 12 Hz, 2H, 4′eq, 9′eq-H), 1.57 (d, J = 6 Hz, 2H,
CH2CHMe2), 1.66 (1′,3′,6′-H), 1.68−1.74 (m, 2H, 8′eq,10′eq-H),
1.78 (br s, 2H, 5′,7′-H), 1.76−1.87 (m, 1H, CH2CHMe2), 1.82 (d, J =
12 Hz, 2H, 8′ax,10′ax-H), 2.16 (d, J = 12 Hz, 2H, 4′ax,9′ax-H); 13C
NMR (CDCl3, 50 MHz) δ 23.2 (2 × CH3), 25.3 (CH2CHMe2), 27.5
(5′,7′-C), 35.1 (4′,9′-C),33.1 (8′,10′-C), 37.6 (1′,3′-C),38.5 (6′-C),
46.5 (CH2CHMe2), 75.9 (2′-C). The corresponding azide 26 was
prepared from the alcohol 19 (300 mg, 1.44 mmol) according to the
same procedure followed for azide 25 using CH2Cl2 (30 mL)/NaN3
(280 mg, 4.32 mmol)/TFA (1.6 mg, 14.4 mmol): yield 95%; IR
(Nujol) ν(N3) 2095 cm−1; 13C NMR (CDCl3, 50 MHz) 23.4 (2 ×
CH3), 24.5 (CH2CHMe2), 27.3 (5′,7′-C), 33.9 (4′,9′-C), 33.6 (8′,10′-
C), 34.7 (1′,3′-C), 38.5 (6′-C), 43.0 (CH2CHMe2), 69.7 (2′-C).
The corresponding oily amine 8 was prepared through LiAlH4 (183
mg, 4.80 mmol) reduction of azide 25 (280 mg, 1.20 mmol) in
refluxing ether for 5 h according to the same procedure followed for
amine 7: yield 65%; 1H NMR (CDCl3, 400 MHz) δ 0.94 (d, J = 7 Hz,
6H, 2 x CH3), 1.49 (d, J = 6 Hz, 2H, CH2CHMe2), 1.52−1.65 (m, 2H,
1′,3′,6′,4′eq,9′eq-H), 1.73−1.83 (m, 1H, CH2CHMe2), 1.75 (br s, 2H,
5′,7′-H), 1.95 (d, J = 12 Hz, 2H, 8′ax, 10′ax-H), 2.05 (d, J = 12 Hz,
2H, 4′ax, 9′ax-H); 13C NMR (CDCl3, 50 MHz) δ 23.4 (2 × CH3),
25.7 (CH2CHMe2), 27.6 (5′,7′-C), 34.3 (4′,9′-C), 33.1 (8′,10′-C),
38.0 (1′,3′-C), 39.1 (6′-C), 47.4 (CH2CHMe2), 55.4 (2′-C).
Fumarate: mp 225 °C (EtOH−Et2O). Anal. Calcd for C18H29NO4:
C, 66.86; H, 9.26; N, 4.32. Found: C, 66.91; H, 9.30; N, 4.29.
The corresponding azide 28 was prepared from alcohol 21 (300 mg,
1.31 mmol) according to the same procedure followed for azide 25
using CH2Cl2 (30 mL)/NaN3 (256 mg, 3.94 mmol)/TFA (1.49 mg,
13.1 mmol): yield 95%; IR (Nujol) ν(N3) 2098 cm−1; 13C NMR
(CDCl3, 50 MHz) δ 26.8, 27.4 (5′,7′-C), 33.1 (4′,9′-C), 33.4 (8′,10′-
C), 34.1 (1′,3′-C), 37.7 (6′-C), 70.3 (2′-C), 125.6, 127.3, 127.8, 128.9,
140.3 (Ph).
The corresponding oily amine 10 was prepared through LiAlH4
(175 mg, 4.58 mmol) reduction of azide 28 (290 mg, 1.15 mmol) in
refluxing ether for 5 h according to the same procedure followed for
amine 7: yield 55%; 1H NMR (CDCl3, 400 MHz) δ 1.53 (br s, 2H, 6′-
H), 1.61−1.80 (m, 6H, adamantane-H), 1.90 (br s, 2H, 5′,7′-H), 2.33
(d, J = 12 Hz, 1H, 4′ax, 9′ax-H), 2.45 (br s, 2H, 1′,3′-H), 7.18−7.25
(m, 5H, phenyl-H); 13C NMR (CDCl3, 50 MHz) δ 27.2, 27.6 (5′,7′-
C), 32.9 (4′,9′-C), 34.6 (8′,10′-C), 35.8 (1′,3′-C), 38.2 (6′-C), 57.8
(2′-C), 125.2, 126.2, 128.8, 148.7 (Ph). Hydrochloride: mp > 265 °C
(EtOH−Et2O). Anal. Calcd for C16H22NCl: C, 72.85; H, 8.41; N,
5.31. Found: C, 72.81; H, 8.63; N, 5.29.
2-Benzyltricyclo[3.3.1.13,7]decan-2-amine (11). Tertiary alco-
hol 22 was obtained after treating a solution of adamantanone 12 (500
4635
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
mg, 3.34 mmol) in dry THF (30% solution w/v) with 2-molar excess
PhCH2MgCl (obtained from PhCH2Cl (846 mg, 6.68 mmol) and 1.5
molar excess of Mg (243 mg, 10.02 mmol) in 20 mL of dry ether/g
bromobenzene) and stirring the mixture overnight: yield 95%; 1H
NMR (CDCl3, 400 MHz) δ 1.51 (d, J = 12 Hz, 2H, 4′eq, 9′eq-H),
1.65 (br s, 1H, 6′-H), 1.69 (br s, 1H, 5′,7′-H), 1.77 (d, J = 12 Hz, 2H,
8′eq, 10′eq-H), 1.78 (br s, 1H, 3′-H), 1.90 (br s, 1H, 1′-H), 2.07 (d, J
= 12 Hz, 1H, 8′ax, 10′ax-H), 2.12 (d, J = 12 Hz, 1H, 4′ax, 9′ax-H),
2.97 (s, 2H, CH2Ph), 7.10−7.32 (m, 5H, phenyl-H); 13C NMR
(CDCl3, 50 MHz) δ 27.4, 27.5 (5′,7′-C), 33.1 (4′,9′-C), 34.7 (8′,10′-
C), 36.9 (1′,3′-C), 38.5 (6′-C), 43.9 (CH2Ph), 74.7 (2′-C), 126.5,
128.3, 130.7, 137.4 (Ph).
The corresponding azide 29 was prepared from alcohol 22 (300 mg,
1.24 mmol) according to the same procedure followed for azide 25
using CH2Cl2 (30 mL)/NaN3 (241 mg, 3.71 mmol)/TFA (1.41 mg,
12.4 mmol): yield 50%; IR (Nujol) ν(N3) 2096 cm−1; 13C NMR
(CDCl3, 50 MHz) δ 27.1, 27.4 (5′,7′-C), 33.7 (4′,9′-C), 33.8 (8′,10′-
C), 34.1 (1′,3′-C), 38.4 (6′-C), 41.4 (CH2Ph), 69.8 (2′-C), 126.7,
128.2, 130.3, 136.6 (Ph).
100 μg/mL streptomycin, and the assay compound at concentrations
corresponding to 5−10× EC50 was determined using the CPE assay.25
Miniplaque Assay. In cell culture, miniplaques consist of single
infected cells, double or multiple infected cells contiguously linked,
that are observed microscopically and identified by immunofluor-
escence using FITC-labeled monoclonal antibody against viral protein.
Antiviral activity of test drugs was detected in cultures exposed to drug
by assessing inhibition of viral protein synthesis (virus replication) as
measured by reduction in number of miniplaques. The tests were
performed in MDCK cells. Cells were grown on 12 mm glass
coverslips in shell vials (Sarstadt) to a cell density of 80−99%
confluency in 1 mL of DMEM growth medium per vial. Prior to
infection the cultures were washed with serumless media. The
serumless medium was replaced with 1 mL per vial of DMEM
containing BSA at a concentration of 0.125%. Test drugs at
appropriate concentrations were added to the cultures and allowed
to equilibrate with the media. Stock virus was thawed, and appropriate
concentrations of virus (contained in BSA media) were then exposed
to 1.0 μg/mL trypsin for 30 min at room temperature, then added to
the cultures. Replicate cultures were included at each dilution step of
test chemical. Control cultures containing no antiviral drug were
included in each assay. The cultures were then incubated at 33 °C
overnight. Cultures were washed with phosphate buffered saline (PBS)
within the shell vials, fixed in −80 °C acetone, then stained with anti-
influenza A, FITC-labeled monoclonal antibody (Millipore, Billerica,
MA, USA). Possible drug toxicity in culture was assessed by
microscopic observation of cytologic changes and cell multiplication
rates. EC50 determinations were carried out with a fluorescence
microscope by counting miniplaques (clusters of infected cells) in
confluent MDCK monolayers on a coverslip at drug concentrations of
50, 20, 10, 5 μM, and if necessary, 2 μM. From two to four replicate
cultures were included at each drug concentration step. Plaque counts,
C(D) (including controls and weighted by the standard error of the
count for each concentration), were fitted, using the Levenberg−
Marquardt algorithm (in KaleidaGraph from Synergy Software,
Reading, PA, USA), to the sigmoidal function:
The corresponding oily amine 11 was prepared through LiAlH4
(130 mg, 3.45 mmol) reduction of azide 29 (230 mg, 0.861 mmol) in
refluxing ether for 5 h according to the same procedure followed for
1
amine 7: yield 45%; H NMR (CDCl3, 400 MHz) δ 1.61 (d, J = 12
Hz, 2H, 4′eq, 9′eq-H), 1.61 (br s, 1H, 6′-H), 1.73 (br s, 1H, 5′,7′-H),
1.78 (d, J = 12 Hz, 2H, 8′eq, 10′eq-H), 1.87 (br s, 1H, 3′-H), 1.97 (br
s, 1H, 1′-H), 2.09 (d, J = 12 Hz, 1H, 8′ax, 10′ax-H), 2.29 (d, J = 12 Hz,
1H, 4′ax, 9′ax-H), 2.97 (s, 2H, CH2Ph), 7.10−7.32 (m, 5H, phenyl-
H); 13C NMR (CDCl3, 50 MHz) δ 27.6, 27.8 (5′,7′-C), 33.2 (4′,9′-
C), 34.3 (8′,10′-C), 37.3 (1′,3′-C), 39.2 (6′-C), 44.2 (CH2Ph), 55.1
(2′-C), 126.3, 128.1, 130.7, 138.4 (Ph). Fumarate: mp 205 °C
(EtOH−Et2O). Anal. Calcd for C20H33NO4: C, 70.56; N, 3.92. Found:
C, 70.99; N, 3.89.
(B) Biological Testing Methods. Cells and Media. Tissue used
for preparation of virus stock cultures, virus infectivity titrations, and
miniplaque drug assays were Madin−Darby canine kidney (MDCK)
cells (ATCC CCL 34). The cell culture growth medium used was
Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich)
supplemented with 0.11% sodium bicarbonate, 5% Cosmic calf
serum (Hyclone), 10 mM HEPES buffer, and 50 μg/mL gentamycin.
For culture of virus stocks and virus infectivity assays, 0.125% bovine
serum albumin (BSA, Sigma-Aldrich) was substituted for the Cosmic
calf serum.
Virus. Influenza A virus, the 2009 pandemic strain (A/California/
07/2009), was provided by Dr. Don Smee, Utah State University.
Trypsin added to BSA-supplemented medium for virus activation was
TPCK-treated bovine pancreas trypsin (Sigma-Aldrich). A virus stock
culture (passage 1) was prepared in MDCK cells in a 150 cm2 culture
flask. The cells were planted in growth medium and incubated until
the cell monolayer was at 90% confluency. The monolayer was washed
with medium containing no serum, then renewed with BSA medium
containing 2.5 μg/mL trypsin. The culture was infected with 1 mL of
the virus inoculum obtained from Dr. Smee, then incubated at 33 °C.
At 2 days postinfection the culture had reached complete cytopathic
effect. Detached cells and cell debris were removed by low speed
centrifugation (600g for 5 min). The supernate was aliquoted in 1 mL
quantities, then frozen at −80 °C for storage. For virus titration,
aliquots of the stock were thawed and dilution series were inoculated
in MDCK cultures in shell vials and virus-infected cells were detected
by immunofluorescence. Other virus strains were obtained from
American Type Culture Collection (ATCC): influenza A (H3N2)
Victoria/3/75 (ATCC VR-822), influenza A (H1N1) A/PR/8/34
(ATCC VR-95), influenza A (H1N1) A/WS/33 (ATCC VR-1520),
and influenza A (H2N2) A2/Taiwan/1/64 (ATCC VR-480). Virus
stock cultures were prepared in MDCK cells grown in BSA-
supplemented media, processed, and stored as described above.
For resistance studies with A/Hong Kong/1/1968 (H3N2) (Table
4), Petri dishes with MDCK cells (Federal Research Institute for
Animal Health, Greifswald-Insel Riems, catalog no. RIE328) were
preincubated overnight in Eagle minimum essential medium (EMEM)
supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and
C0
C(D) =
D
1 +
EC50
with D being the drug concentration and C0 and EC50 being free
parameters. The standard error of the EC50, used as reported by the
software, reflects the uncertainties due to variances in the counts at all
concentrations, including the controls. The value of C0 was
constrained by the four independent controls. For the replicate
screens, where the value of EC50 was based only on the four controls
and a pair of tests at a fixed concentration, the formal standard errors
of the parameters may not adequately represent the uncertainty
associated with extrapolating or interpolating the 50% reduction dose
from the miniplaque reduction at the assay dose, which would
probably be greater the greater the difference is between the assay dose
and the EC50. Nevertheless, in spite of this limitation, we found
reproducibility of EC50 values to be high (i.e., within factors of ∼2) on
several occasions where experiments were repeated, either screens
repeated by screens or screens compared to complete dose−response
curves.
Resistance Testing. For Table 4, cultured MDCK cells bathed in
a concentration corresponding to approximately the EC50 concen-
tration were exposed to the usual quantities of virus for 3−4 days (5−7
virus replication cycles). After that time, the cultures developed
cytopathic effects, and the cultures were terminated. The medium,
containing virus, was then collected by low speed centrifugation.
Dose−response tests utilizing the miniplaque technique were
performed on the recovered virus for determination of the EC50
against the potentially mutated virus. An increase in the EC50 above
the original value represents resistance development. A crude sequence
on the passage-12 virus developed in 6 (see text) was carried out by
extracting the virus directly with the RNaqueous kit (Life
Technologies), transcribed with the Superscript III first-strand
4636
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
synthesis kit (Life Technologies), amplified by PCR, and sequenced
with an Applied Biosystems 3730xl DNA analyzer.
(C) Peptide Synthesis and Sample Preparation for Solid
State NMR. S31N M2 TM (22−46) (A/Udorn/307/72) with 15N
labeled V28, A30, and I42 was synthesized using Fmoc (9-
fluorenylmethoxycarbonyl) chemistry. Fmoc-[15N]Val, fmoc-
[15N]Ala, and fmoc-[15N]Ile were purchased from Cambridge Isotope
Laboratory (Andover, MA). Solid-phase 0.25 mmol syntheses of M2
TMD were performed on an Applied Biosystems 430A peptide
synthesizer as previously described.28 The peptide was cleaved from
the resin by the treatment with ice cold 95% TFA, 2.5% H2O, 1.25%
ethanedithiol, 1.25% thioanisole and precipitated from TFA using ice
cold ether. Following centrifugation, the supernatant was discarded
and the pellet was washed with cold ether again. The precipitated
peptide was dried under vacuum. Peptide purity and identity were
confirmed using ESI mass spectrometry (positive ion mode).
Resistance Test Plaque Sequencing. For the more detailed
sequencing in (Supporting Information Table 1), MDCK cells were
washed and incubated with influenza virus (multiplicity of infection is
1) for 1 h to allow virus adsorption. Then excessive virus was washed
off and cells were incubated with EMEM supplemented with the assay
compound for 3−4 days. If no cytopathic effect was visible, 0.5 mL of
supernatant was centrifuged (2000 rpm) to remove cell detritus and
transferred to Petri dishes with confluent MDCK monolayers (blind
passage). Cells were incubated again up to 4 days in EMEM
supplemented with the assay compound. If CPE was visible, 1 mL of
supernatant was stored at −80 °C and 0.5 mL was passaged. Up to 10
passages were executed. For sequencing of resistant viruses, serial
dilution (10-fold) of the stocks of the first, fourth, and ninth passages
were used for plaque assays. Three to five arbitrarily selected plaques
of each tested passage and compound were picked, amplified in
MDCK cells (yielding second, fifth, and tenth passage virus) and used
for RNA preparation as described.26 Briefly, total RNA was prepared
from virus-infected MDCK cells using the RNeasy Mini kit and
Qiashredder kit (Qiagen, Hilden, Germany). Reverse transcription was
conducted with a primer specific to the 3′-end of genomic RNA (5′-
RGCRAAAGCAGG-3′), 20 units reverse transcriptase (Fermentas, St.
Leon-Rot, Germany), and 5 μg of RNA in a final reaction volume of
20 μL. Specific oligonucleotide primers Bm-M-1 and Bm-M-1027R
and Bm-HA-1 and Bm-HA-rev27 were used for the amplification of the
M and HA segments from cDNA. Amplified DNA fragments were
analyzed by agarose gel electrophoresis and gel-extracted employing
the QIAquick gel extraction kit (Qiagen, Hilden, Germany). Purified
DNA fragments were sequenced by cycle sequencing using the CEQ
DTCS quick start kit (Beckman Coulter, Krefeld, Germany) and
analyzed on a CEQ8000 sequencer (Beckman Coulter, Krefeld,
Germany).
15N-V28A30I42 S31N M2 TMD was co-dissolved in trifluoroethanol
(TFE) with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in
a 1:30 molar ratio. The solvent was removed under a stream of
nitrogen gas to yield a lipid film and then dried to remove residual
organic solvent under vacuum for 12 h. Thoroughly dried lipid film
was hydrated with 8 mL of 10 mM HEPES buffer at pH 7.5 to form
multilamellar vesicles containing M2 TMD in tetrameric state. This
suspension was bath sonicated, dialyzed against 2 L of HEPES 10 mM,
pH 7.5, buffer for 1 day and centrifuged at 196000g to harvest
unilamellar proteoliposomes. The pellet was resuspended in a 1 mL
aliquot of the decanted supernatant containing compound 6, resulting
in a 1:6 molar ratio of the M2 TMD tetramer to drug. Following
overnight incubation at 37 °C, the pellet was deposited on 5.7 × 10
mm glass strips (Matsunami Trading, Osaka, Japan). The bulk of the
water from the sample was removed during a 2-day period in a 98%
relative humidity environment at 298 K. Rehydration of the slides,
before stacking and sealing into a rectangular sample cell, generated
40−50% by weight water in the sample. The final sample composition
is 1 mg of drug/60 mg of lipid/8 mg of peptide (mole ratio 1:20:0.7)
with 40−50% hydration.
Electrophysiology Methods. cDNA sequences encoding the full-
length A/California/04/09 M2 protein containing an N-terminal
FLAG-tag plus 3(Gly) repeat linker and either N31 or an S31
mutation were cloned into pcDNA3 and transiently co-transfected
with a pcDNA3 vector encoding eGFP into TSA-201 cells using
standard transfection protocols (Lipofectamine 2000, Life Technolo-
gies). Single GFP-positive transfected cells were then used for
electrophysiological experiments.
Solid State NMR Experiments. PISEMA spectra were acquired at
720 MHz utilizing a low-E 1H/15N double resonance probe.28,29
Acquisition took place at 303 K, above the gel to liquid crystalline
phase transition temperature of DMPC lipids. Experimental
parameters included a 90° pulse of 5 μs and cross-polarization contact
time of 1 ms, a 4 s recycle delay, and a SPINAL decoupling
sequence;30 Sixteen t1 increments were obtained for the spectrum of
15N-V28A30I42 S31N M2 TMD with compound 6, and nine t1
Macroscopic ionic currents were recorded in the whole-cell
configuration 24−48 h after transfection. Cells were perfused
continuously at 3−5 mL/min with external (bath) solution containing
the following (in mM): 150 NMG, 10 HEPES, 10 D-glucose, 2 CaCl2,
1 MgCl2 buffered at pH 7.4 with HCl. For low pH (5.5) solution,
HEPES was replaced by MES. Solutions containing either K+ or Na+
were prepared by replacing NMG with the corresponding ion. Patch
electrodes were pulled from thin-walled borosilicate glass (World
Precision Instruments, Fl) and fire-polished before filling with
standard pipet solution containing the following (in mM): 140
NMG, 10 EGTA, 10 MES, and 1 MgCl2 buffered at pH 6.0 with HCl.
Pipettes typically had a resistance of 3−5 MΩ. Voltage-clamp
experiments were performed with an Axopatch 200B amplifier
(Molecular Devices, CA) connected to a Digidata 1322A 16-bit
digitizer. Data were acquired with the pCLAMP8.0 software
(Molecular Devices, CA) sampled at 10 kHz and low-pass-filtered at
5 kHz. Cells were held at −40 mV. The standard voltage protocol
consisted of a 100 ms pulse to −80 mV followed by a 300 ms ramp to
+40 mV and a 200 ms step to 0 mV before stepping back to −40 mV
and repeated every 4 s. All experiments were performed at room
temperature (20−22 °C). All drugs were prepared as DMSO stocks
(50 or 100 mM) and diluted with external solution to the desired
concentration. To measure block of M2 currents by compounds, cells
were recurrently treated with pH 7.4 and pH 5.5 solutions until stable,
pH-dependent inward currents were reproducibly observed, followed
by treatment with compound and concentration of interest at pH 5.5
for 2−30 min. At the end of each experiment, cells were then treated
with 100 μM amantadine.
increments were obtained for the sample without compound. Spectral
processing was done with NMRPIPE31 and plotting with SPARKY.
15N chemical shifts were referenced to a concentrated solution of
N2H8SO4, defined as 26.8 ppm relative to liquid ammonia.
ASSOCIATED CONTENT
■
S
* Supporting Information
Plaque hemagglutinin-sequencing results, plaque M1-sequenc-
ing results, plaque M2-sequencing results, and representative
diary plots from tSA-201 HEK cells. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*A.K.: phone, 30-210-727-4834; e-mail, ankol@pharm.uoa.gr.
*D.D.B.: phone, 801-422-8753; e-mail, david_busath@byu.edu.
Author Contributions
A.K. and C.T. synthesized compounds. F.B.J. and R.Z. did
dose−response and resistance tissue-culture testing. T.A.C. and
A.K.W. did solid state NMR. I.T. and D.F. did electro-
physiology tests. A.K. and D.D.B. conceived and supervised the
project and wrote the paper with input from the coauthors.
Notes
The authors declare no competing financial interest.
4637
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
(7) Scholtissek, C.; Quack, G.; Klenk, H. D.; Webster, R. G. How to
overcome resistance of influenza A viruses against adamantane
derivatives. Antiviral Res. 1998, 37, 83−95.
(8) Kurtz, S.; Luo, G.; Hahnenberger, K. M.; Brooks, C.; Gecha, O.;
Ingalls, K.; Numata, K.; Krystal, M. Growth impairment resulting from
expression of influenza virus M2 protein in Saccharomyces cerevisiae:
identification of a novel inhibitor of influenza virus. Antimicrob. Agents
Chemother. 1995, 39, 2204−2209.
ACKNOWLEDGMENTS
■
The authors thank Dr. Donald Smee for providing influenza A/
California/07/2009 (H1N1) and Dr. Michaela Schmidtke for
her collaboration and her valuable suggestions. D.D.B. thanks
Curtis Evans, Steven Kearnes, Nathan Gay, Ben Nielsen,
Trevor Anderson, Robert Childs, and Joseph Moulton. R.Z.
thanks Birgit Jahn and Martina Muller, and D.F. thanks Daniel
̈
(9) (a) Balannik, V.; Wang, J.; Ohigashi, Y.; Jing, X.; Magavern, E.;
Lamb, R. A.; Degrado, W. F.; Pinto, L. H. Design and pharmacological
characterization of inhibitors of amantadine-resistant mutants of the
M2 ion channel of influenza A virus. Biochemistry 2009, 49, 696−708.
(b) Duque, M. D.; Ma, C.; Torres, E.; Wang, J.; Naesens, L.; Juarez-
Jimenez, J.; Camps, P.; Luque, F. J.; DeGrado, W. F.; Lamb, R. A.;
Pinto, L. H.; Vazquez, S. Exploring the size limit of templates for
inhibitors of the M2 ion channel of influenza A virus. J. Med. Chem.
2011, 54, 2646−2457. (c) Wang, J.; Ma, C.; Fiorin, G.; Carnevale, V.;
Wang, T.; Hu, F.; Lamb, R. A.; Pinto, L. H.; Hong, M.; Klein, M. L.;
DeGrado, W. F. Molecular dynamics simulation directed rational
design of inhibitors targeting drug-resistant mutants of influenza A
virus M2. J. Am. Chem. Soc. 2011, 133, 12834−12841. (d) Rey-Carrizo,
M.; Torres, E.; Ma, M.; Barniol-Xicota, M.; Wang, J.; Wu, Y.; Naesens,
Kwan for technical assistance. The work is part of C.T.’s
Master’s thesis and was supported by grants from Chiesi Hellas
(Project 10354, Special Account for Research Grants), the
German Ministry for Education and Science (FluResearchNet,
Grant 01 KI1006J), and NIH (Grants AI 23007 and AI
074805).
REFERENCES
■
(1) Bright, R. A.; Medina, M. J.; Xu, X.; Perez-Oronoz, G.; Wallis, T.
R.; Davis, X. M.; Povinelli, L.; Cox, N. J.; Klimov, A. I. Incidence of
adamantane resistance among influenza A (H3N2) viruses isolated
worldwide from 1994 to 2005: a cause for concern. Lancet 2005, 366,
1175−1181.
́
L.; DeGrado, W. F.; Lamb, R. A.; Pinto, L. H.; Vazquez, S. 3-
Azatetracyclo[5.2.1.15,8.01,5]undecane derivatives: from wild-type in-
hibitors of the M2 ion channel of influenza A virus to derivatives with
potent activity against the V27A mutant. J. Med. Chem. 2013, 56,
9265−9274.
(2) (a) Bright, R. A.; Shay, D. K.; Shu, B.; Cox, N. J.; Klimov, A. I.
Adamantane resistance among influenza A viruses isolated early during
the 2005−2006 influenza season in the United States. JAMA, J. Am.
Med. Assoc. 2006, 295, 891−894. (b) Lan, Y.; Zhang, Y.; Dong, L.;
Wang, D.; Huang, W.; Xin, L.; Yang, L.; Zhao, X.; Li, Z.; Wang, W.; Li,
X.; Xu, C.; Guo, J.; Wang, M.; Peng, Y.; Gao, Y.; Guo, Y.; Wen, L.;
Jiang, T.; Shu, Y. A comprehensive surveillance of adamantane
resistance among human influenza A virus isolated from mainland
China between 1956 and 2009. Antiviral Ther. 2010, 15, 853−859.
(3) High levels of adamantane resistance among influenza A (H3N2)
viruses and interim guidelines for use of antiviral agentsUnited
States, 2005−06 influenza season. Morbidity Mortality Wkly. Rep. 2006,
55, 44−46.
(4) See, for examples, the following: (a) Kolocouris, N.; Foscolos, G.
B.; Kolocouris, A.; Marakos, P.; Pouli, N.; Fytas, G.; Ikeda, S.; De
Clercq, E. Synthesis and antiviral activity evaluation of some
aminoadamantane derivatives. J. Med. Chem. 1994, 37, 2896−2902.
(b) Kolocouris, A.; Spearpoint, P.; Martin, S. R.; Hay, A. J.; Lopez-
Querol, M.; Sureda, F. X.; Padalko, E.; Neyts, J.; De Clercq, E.
Comparisons of the influenza virus A M2 channel binding affinities,
anti-influenza virus potencies and NMDA antagonistic activities of 2-
alkyl-2-aminoadamantanes and analogues. Bioorg. Med. Chem. Lett.
2008, 18, 6156−6160.
(5) (a) Stouffer, A. L.; Acharya, R.; Salom, D.; Levine, A. S.; Di
Costanzo, L.; Soto, C. S.; Tereshko, V.; Nanda, V.; Stayrook, S.;
DeGrado, W. F. Structural basis for the function and inhibition of an
influenza virus proton channel. Nature 2008, 451, 596−599. (b) Cady,
S. D.; Schmidt-Rohr, K.; Wang, J.; Soto, C. S.; Degrado, W. F.; Hong,
M. Structure of the amantadine binding site of influenza M2 proton
channels in lipid bilayers. Nature 2010, 463, 689−692. (c) Cady, S. D.;
Wang, J.; Wu, Y.; DeGrado, W. F.; Hong, M. Specific binding of
adamantane drugs and direction of their polar amines in the pore of
the influenza M2 transmembrane domain in lipid bilayers and
dodecylphosphocholine micelles determined by NMR spectroscopy.
J. Am. Chem. Soc. 2011, 133, 4274−4284. (d) Pielak, R. M.; Oxenoid,
K.; Chou, J. J. Structural investigation of rimantadine inhibition of the
AM2-BM2 chimera channel of influenza viruses. Structure 2011, 19,
1655−1663. (e) Hu, J.; Fu, R.; Cross, T. A. The chemical and
dynamical influence of the anti-viral drug amantadine on the M2
proton channel transmembrane domain. Biophys. J. 2007, 93, 276−
283.
(10) (a) Zhang, W. Heterodimers of histidine and amantadine as
inhibitors for wild type and mutant M2 channels of influenza A. Chin.
J. Chem. 2010, 28, 1417−1423. (b) Shibnev, V. A.; Garayev, T. M.;
Finogenova, M. P.; Shevchenko, E. S.; Burtseva, E. I. New adamantane
derivatives and ways of overcoming the resistance of influenza A
viruses to rimantadine and amantadine. Vopr. Virusol. 2011, 56, 36−39.
(c) Zhao, X.; Jie, Y.; Rosenberg, M. R.; Wan, J.; Zeng, S.; Cui, W.;
Xiao, Y.; Li, Z.; Tu, Z.; Casarotto, M. G.; Hu, W. Design and synthesis
of pinanamine derivatives as anti-influenza A M2 ion channel
inhibitors. Antiviral Res. 2012, 96, 91−99. (d) Shibnev, V. A.;
Garaev, T. M.; Finogenova, M. P.; Shevchenko, E. S.; Burtseva, E. I.
New adamantane derivatives can overcome resistance of influenza
A(H1N1)pdm2009 and A(H3N2) viruses to rimantadine. Bull. Exp.
Biol. Med. 2012, 153, 233−235. (e) Torres, E.; Fernan
́
dez, R.; Miquet,
quez, S.
S. P.; Font-Bardia, M.; Vanderlinden, E.; Naesens, L.; Vaz
́
Synthesis and anti-influenza A virus activity of 2,2-dialkylamantadines
and related compounds. ACS Med. Chem. Lett. 2012, 3, 1065−1069.
(f) Wang, J.; Ma, C.; Jo, H.; Canturk, B.; Fiorin, G.; Pinto, L. H.;
Lamb, R. A.; Klein, M. L.; Degrado, W. F. Discovery of novel dual
inhibitors of wild-type and the most prevalent drug-resistant mutant,
S31N, of M2 proton channel from influenza A virus. J. Med. Chem.
2013, 56, 2804−2812. (g) Torres, E.; Duque, M. D.; Vanderlinden, E.;
Ma, C.; Pinto, L. H.; Camps, P.; Froeyen, M.; Vazquez, S.; Naesens, L.
Role of the viral hemagglutinin in the anti-influenza virus activity of
newly synthesized polycyclic amine compounds. Antiviral Res. 2013,
99, 281−291.
(11) Kalir, A.; Balderman, D. Org. Synth. 1981, 60, 104−107.
(12) Sasaki, T.; Eguchi, S.; Toi, N. Synthesis of adamantane
derivatives. 47. Photochemical synthesis of 4-azahomoadamant-4-enes
and further studies on their reactivity in some cycloadditions. J. Org.
Chem. 1979, 44, 3711−3715.
(13) Wang, J.; Denny, J.; Tian, C.; Kim, S.; Mo, Y.; Kovacs, F.; Song,
Z.; Nishimura, K.; Gan, Z.; Fu, R.; Quine, J. R.; Cross, T. A. Imaging
membrane protein helical wheels. J. Magn. Reson. 2000, 144, 162−167.
(14) Hu, J.; Asbury, T.; Achuthan, S.; Li, C.; Bertram, R.; Quine, J.
R.; Fu, R.; Cross, T. A. Backbone structure of the amantadine-blocked
trans-membrane domain M2 proton channel from influenza A virus.
Biophys. J. 2007, 92, 4335−4343.
(6) Wang, J.; Wu, Y.; Ma, C.; Fiorin, G.; Pinto, L. H.; Lamb, R. A.;
Klein, M. L.; Degrado, W. F. Structure and inhibition of the drug-
resistant S31N mutant of the M2 ion channel of influenza A virus.
Proc. Natl. Acad. Sci. U.S.A. 2013, 110, 1315−1320.
(15) Li, C.; Qin, H.; Gao, F. P.; Cross, T. A. Solid-state NMR
characterization of conformational plasticity within the transmembrane
domain of the influenza A M2 proton channel. Biochim. Biophys. Acta
2007, 1768, 3162−3170.
4638
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639

Journal of Medicinal Chemistry
Article
(16) (a) Chizhmakov, I. V.; Geraghty, F. M.; Ogden, D. C.;
Hayhurst, A.; Antoniou, M.; Hay, A. J. Selective proton permeability
and pH regulation of the influenza virus M2 channel expressed in
mouse erythroleukaemia cells. J. Physiol. 1996, 494, 329−336.
(b) Balannik, V.; Carnevale, V.; Fiorin, G.; Levine, B.. G.; Lamb, R.
A.; Klein, M. L.; DeGrado, W. F.; L Pinto, L. H. Functional studies and
modeling of pore-lining residue mutants of the influenza A virus M2
ion channel. Biochemistry 2010, 49, 696−708.
(17) Appleyard, G. Amantadine-resistance as a genetic marker for
influenza viruses. J. Gen. Virol. 1977, 36, 249−255.
(18) Oxford, J. S.; Potter, C. W.; Logan, I. S. Passage of influenza
strains in the presence of aminoadamantane. Ann. N.Y. Acad. Sci. 1970,
173, 300−313.
(19) Hayden, F. G.; Sperber, S. J.; Belshe, R. B.; Clover, R. D.; Hay,
A. J.; Pyke, S. Recovery of drug-resistant influenza A virus during
therapeutic use of rimantadine. Antimicrob. Agents Chemother. 1991,
35, 1741−1747.
(20) Hay, A. J.; Wolstenholme, A. J.; Skehel, J. J.; Smith, M. H. The
molecular basis of the specific anti-influenza action of amantadine.
EMBO J. 1985, 4, 3021−3024.
(21) Gamblin, S. J.; Haire, L. F.; Russell, R. J.; Stevens, D. J.; Xiao, B.;
Ha, Y.; Vasisht, N.; Steinhauer, D. A.; Daniels, R. S.; Elliot, A.; Wiley,
D. C.; Skehel, J. J. The structure and receptor binding properties of the
1918 influenza hemagglutinin. Science 2004, 303, 1838−1842.
(22) Shibata, M.; Aoki, H.; Tsurumi, T.; Sugiura, Y.; Nishiyama, Y.;
Suzuki, S.; Maeno, K. Mechanism of uncoating of influenza B virus in
MDCK cells: action of chloroquine. J. Gen. Virol. 1983, 64, 1149−
1156.
(23) Dudleenamjil, E.; Lin, C. Y.; Dredge, D.; Murray, B. K.;
Robison, R. A.; Johnson, F. B. Bovine parvovirus uses clathrin-
mediated endocytosis for cell entry. J. Gen. Virol. 2010, 91, 3032−
3041.
(24) Kuehn, B. M. CDC: Use antivirals early, aggressively for H7N9
flu. JAMA, J. Am. Med. Assoc. 2013, 309, 2086.
(25) Schmidtke, M.; Schnittler, U.; Jahn, B.; Dahse, H. M.; Stelzner,
A. A rapid assay for evaluation of antiviral activity against
coxsackievirus B3, influenza virus A, and herpes simplex virus type 1.
J. Virol. Methods 2001, 95, 133−143.
(26) Krumbholz, A.; Schmidtke, M.; Bergmann, S.; Motzke, S.;
Bauer, K.; Stech, J.; Durrwald, R.; Wutzler, P.; Zell, R. High prevalence
̈
of amantadine resistance among circulating European porcine
influenza A viruses. J. Gen. Virol. 2009, 90, 900−908.
(27) Hoffmann, E.; Stech, J.; Guan, Y.; Webster, R. G.; Perez, D. R.
Universal primer set for the full-length amplification of all influenza A
viruses. Arch. Virol. 2001, 146, 2275−2289.
(28) Kovacs, F. A.; Cross, T. A. Transmembrane four-helix bundle of
influenza A M2 protein channel: structural implications from helix tilt
and orientation. Biophys. J. 1997, 73, 2511−2517.
(29) Wu, C. H.; Ramamoorthy, A.; Opella, S. J. High-resolution
heteronuclear dipolar solid-state NMR spectroscopy. J. Magn. Reson.
1994, 109, 270−272.
(30) Fung, B. M.; Khitrin, A. K.; Ermolaev, K. An improved
broadband decoupling sequence for liquid crystals and solids. J. Magn.
Reson. 2000, 142, 97−101.
(31) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.;
Bax, A. NMRPipe: a multidimensional spectral processing system
based on UNIX pipes. J. Biomol. NMR 1995, 6, 277−293.
4639
dx.doi.org/10.1021/jm500598u | J. Med. Chem. 2014, 57, 4629−4639