Discovery of novel dual inhibitors of the wild-type and the most prevalent drug-resistant mutant, S31N, of the M2 proton channel from influenza A virus

Article abstract of DOI:10.1021/jm301538e

Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009 H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation M2 inhibitors targeting the S31N mutant. However, the S31N mutant presents a huge challenge to drug discovery, and it has been considered undruggable for several decades. Using structural information, classical medicinal chemistry approaches, and M2-specific biological testing, we discovered benzyl-substituted amantadine derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-1,3-diol (44) is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza A infections.

Full text of DOI:10.1021/jm301538e

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Article
Discovery of Novel Dual Inhibitors of Wild-type and the Most Prevalent
Drug-Resistant Mutant, S31N, of M2 Proton Channel from Influenza A Virus
Jizhou Wang, Chunlong Ma, Jun Wang, Hyunil Jo, Belgin Canturk, Giacomo Fiorin,
Lawrence H. Pinto, Robert A. Lamb, Michael L. Klein, and William F. DeGrado
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm301538e • Publication Date (Web): 25 Feb 2013
Downloaded from http://pubs.acs.org on March 25, 2013
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Journal of Medicinal Chemistry
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Discovery of Novel Dual Inhibitors of Wild-type and the Most Prevalent Drug-
Resistant Mutant, S31N, of M2 Proton Channel from Influenza A Virus
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Jizhou Wang*,a, Chunlong Mab, Jun Wangc, Hyunil Joc, Belgin Canturkd, Giacomo Fiorine,
Lawrence H. Pintob, Robert A. Lambf, g, Michael L. Kleine, and William F. DeGrado*,c
a Influmedix Inc., 170 North Radnor-Chester Road, Suite 300, Radnor, PA 19087
bDepartment of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208
cDepartment of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158
dDepartment of Chemistry, University of Pennsylvania, 231 South 34th St., Philadelphia, PA 19104
eInstitute for Computational Molecular Science and Department of Chemistry, Temple University, Philadelphia,
Pennsylvania 19122-6078
fDepartment of Molecular Biosciences, and gHoward Hughes Medical Institute, Northwestern University,
Evanston, Illinois 60208-3500
Abstract: Anti-influenza drugs, amantadine and rimantadine, targeting the M2 channel from influenza A
virus are no longer effective because of widespread drug resistance. S31N is the predominant and amantadine-
resistant M2 mutant, present in almost all of the circulating influenza A strains as well as in the pandemic 2009
H1N1 and the highly pathogenic H5N1 flu strains. Thus, there is an urgent need to develop second-generation
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M2 inhibitors targeting S31N mutant. However, S31N mutant presents a huge challenge to drug discovery, and
it has been considered undruggable for several decades. Using structural information, classical medicinal
chemistry approaches and M2-specific biological testing, we discovered benzyl-substituted amantadine
derivatives with activity against both S31N and WT, among which 4-(adamantan-1-ylaminomethyl)-benzene-
1,3-diol is the most potent dual inhibitor. These inhibitors demonstrate that S31N is a druggable target and
provide a new starting point to design novel M2 inhibitors that address the problem of drug-resistant influenza
A infections.
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Figure 1. Structures of four approved antiviral drugs for influenza A
INTRODUCTION
Seasonal influenza infections as well as the emergence of life-threatening strains of influenza are a major
worldwide health concern. Every year, influenza epidemics cause numerous deaths and millions of
hospitalizations.1,2 Besides the yearly epidemic outbreaks, influenza viruses are even more threatening
pathogens due to their potency to cause pandemics, as occurred in 2009 with the emergence and worldwide
spread of pandemic H1N1. Available prophylactic vaccines are not completely effective against emerging flu
strains and thus effective anti-viral therapy is an essential component in the fight against influenza A infections.
Currently, there are only four antivirals approved for use against influenza virus infections in humans, two
targeting the M2 proton channel, amantadine (oral) and rimantadine (oral) and two targeting the neuraminidase,
oseltamivir (oral), and zanimivir (inhaled) (Fig.1). Resistance to both classes is a problem: resistance to the
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adamantane class of drugs is now so pervasive that the Centers for Disease Control and Prevention (CDC) has
advised against its continued use2,3 and widespread resistance to oseltamivir has been increasingly reported
since 2007-2008.4-8 Even more concerning are the reports of the emergence of flu strains with dual resistance to
both adamantane and neuraminidase classes of antivirals.9
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Resistance to M2 proton channel drugs is associated with mutations in the transmembrane domain of the M2
protein. The homotetrameric structure of the M2 channel places constraints on the types of drug-resistant
mutations that can be accommodated. A single mutation to the pore-lining residues of the protein results in four
changes to the residues lining the pore, where the drug binds. While many mutations can be tolerated with
retention of function in vitro,10-12 only a few mutants appear to be tolerated in highly transmissible viruses seen
in the wild. V27A, L26F, and S31N have consistently comprised more than 99% of transmissible M2 mutants;
among which S31N is now predominant in 98-100% of the transmissible amantadine-resistant H1N1, H5N1,
and H3N2 strains isolated from humans, birds and swine in the last decade.1 As a result, there is an urgent need
to develop second-generation novel M2 inhibitors targeting the most prevalent S31N mutant. However, this
mutation presents significant challenges to drug discovery, because the side chain of Asn31 partially fills the
amantadine-binding site and also changes its physical properties markedly.
M2 inhibitors generally consist of a hydrophobic portion (adamantane in the drugs amantadine and rimantadine)
connected to a polar group, which is generally a positively charged substituent such as an amine in amantadine
or an aminoethyl group in rimantadine (Fig. 1). A variety of scaffolds can be substituted for the hydrophobic
adamantane group, including fused and spiro-fused multi-cyclic alkanes, acyclic branched alkanes, and
silanes.13, 14 These compounds showed excellent activity for WT, and some were highly active against V27A
and L26F.14, 15 However, none showed significantly better activity vs. S31N than had already been seen for
amantadine. The polar group has also been the subject of extensive structure activity relations (SAR). Again, a
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variety of substituents were tolerated when the target was WT, but only the simple primary amine found in
amantadine gave significant activity against S31N. Thus, after several decades of medicinal chemistry
investigations, no progress had been made towards inhibiting this predominant mutant form, suggesting that this
variant might be essentially undruggable.16-20
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Our journey to S31N inhibitors discovery started with modification of amantadine (1) whose potency against
S31N drops approximately 12-fold from 16 µM against WT21 to 200 µM22 in a TEVC (two electrode voltage
clamp) electrophysiological assay. Amantadine (1) is, however, more active than rimantadine (2) against S31N
(199.9 µM vs > 2 mM)23 (Fig. 2A). We envisioned that the S31N inhibitory activity could be restored if we
captured additional interactions within the channel. Recent crystallographic and NMR studies have provided
major advances in our understanding of the mode of inhibition of M2 by amantadine and related drugs (Fig. 2B
and 2C).15,21,24-33
A.
B.
C.
Figure 2. (A) Activity of amantadine and rimantadine against WT (S31) and S31N mutant in the TEVC assay.
(B) The M2 channel-lining residues found in transmissible drug-resistant mutants are shown in orange letters.
Amantadine (orange bound drug) causes a break in the aqueous path (water molecules, red balls). One helix has
been removed for clarity. (C) Snapshot from a simulation of amantadine with WT15. Water molecules (red)
associate with carbonyl groups (green/red sticks) in a square planar array. This array of water molecules can
stabilize the bound ammonium group of amantadine (green and blue bound drug) or a centrally located water
molecule (magenta).
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The location of the pharmacologically relevant drug binding site in M2 was extensively debated until recently.
Our earlier crystallographic structures suggested a pore-binding model,24 while Chou and co-workers proposed
a more peripheral site on the surface of the protein based on NMR studies.25 However, last year, Chou and co-
workers solved a structure, showing that drug binds to the site we had originally identified, ending the
controversy, and validating our earlier crystallographic structures.31 Specifically, the drug binds in a pocket
within the channel lumen, and is surrounded by the apolar side chain of Val27, Ala30, the C-beta of Ser31, and
the C-alpha of Gly34. The ammonium group projects downward towards the invariant His37 residue, but does
not actually interact with this residue. Instead it is stabilized by water molecules, which are in turn stabilized by
interactions with backbone carbonyl groups lining the pore.15 The mutation S31N disrupts the size and polarity
of the pore in precisely the region that accommodates the adamantane group of amantadine, explaining the
decrease in potency of amantadine from 16 µM against WT to 200 µM in a TEVC electrophysiological assay.
Moreover, NMR studies showed that S31N was more dynamic and possibly also more hydrated in the pore than
the corresponding WT protein.34 This led us to consider strategies in which we either increased the polarity or
dimensions of the adamantane core to capture more interactions with the backbone of the channel (Fig. 3,
approach I). We synthesized over 300 amantadine analogues and other diverse cores, some of which have been
published.14,15,22,35-38 While we succeeded in inhibiting V27A and L26F, none of these compounds inhibited
S31N.15
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Figure 3. Two strategies to overcome drug-resistance
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We turned our attention to the strategy of introducing additional groups (warhead groups) to the amine, in order
to enhance affinity for S31N as well as WT by introducing direct/water mediated hydrogen binding
/electrostatic interactions with backbone or the groups lining the pore of the M2 channel (Fig. 3, approach II).
Examination of the structure of M2’s channel showed there was sufficient space in the channel to accommodate
additional substituents to mediate these interactions. However, this line of modification of amantadine (1) was
not obvious; primary amines tended to be more potent than secondary amines and many early substitutions of
the primary amines decreased activity towards the WT channel.14 Nevertheless, two reports had shown that
aromatic groups could be appended to the amines of 1-adamantanemethylamine (a rimantadine (2) derivative)39
and pinanamine (4)40 and showed activity against M2 channels. We therefore synthesized the most potent
inhibitors in these two series, compounds 339 and 440, and found that they were active against WT (21% and
91% inhibition for 3 and 4, respectively) (Table 1). However, they showed no activity against S31N (0%
inhibition for both) in our electrophysiological TEVC assay. Since amantadine (1) is more potent against S31N
(35% inhibition) than rimantadine (2) (13% inhibition) and pinanamine (0% inhibition23) in a TEVC assay, we
envisioned that installation of aromatic group to the amine group of amantadine (5-49) might improve activity
against S31N as well as WT. This led to the discovery of benzyl-substituted amantadine derivatives as dual WT
and S31N inhibitors, which is described in the present manuscript. These compounds serve as starting points
for design of novel compounds that address the problem of drug-resistant influenza A infections. At the same
time of writing this manuscript, a pinanamine bearing imidazole derivative was reported to show weak
inhibitory activity against the S31N M2 channel.41
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Table 1. M2 inhibitors with an appended aromatic group1
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Structure
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Rimantadine-based Pinanamine-based
Amantadine-based
(this work)
S31N
0%
0%
See the text
% inhibition (TEVC)
WT
27 ± 2%
91 ± 2%
% inhibition (TEVC)
1 The activity of the inhibitors was measured using the TEVC technique with full length AM2 protein in
the Xenopus oocytes membrane; average of three measurements was reported (see Experimental Section
in the Supporting Information).
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A significant obstacle to developing inhibitors of M2 is to devise assays that are specific to inhibition of M2. In
establishing an assay for S31N, we found it important to verify that the assay identified true M2 inhibitors, as
we found both vesicle proton flux assays and viral inhibition assays are complicated by the fact that many
amantadine-like compounds are proton carriers. Proton carriers such as chloroquine are good inhibitors of virus
in vitro, but lack efficacy in animal models and human trials.42-44 However, by conducting electrophysiological
assays in parallel with antiviral assays it is possible to eliminate many false positives from compounds that
acted as proton carriers and delay acidification of the endosome. We report here the first examples of
compounds with specificity for both S31N and WT. Such compounds will be particularly important as
mechanistic probes as well as serving as starting points for design of novel compounds that address the problem
of drug-resistant influenza A infections.
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Chemistry
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The synthetic routes of the compounds 5, 8, 9, 11, 12, 15, 17-23, 29-49 are shown in Scheme 1. A variety of
benzyl groups were installed to N-terminal of 1 either through reductive amination (procedure a or b)45-47 or
amide bond formation followed by lithium aluminium hydride (LAH) reduction (procedure c).48-50 Subsequent
reactions included deprotection, oxidation of a thioether,51 tetrazole formation,52or N-methylation, yielding
compounds 6, 10, 13, 16, and 27, respectively. Due to a low yield in the reductive animation with 2,4-di-
hydroxybenzaldehyde, compound 44 was synthesized by reductive amination with 2-hydroxy-4-methoxy-
benzaldehyde followed by demethylation with BBr3.53 Compounds 14, 24, 25, 31, 33 were prepared via similar
routes (Scheme 2). The syntheses of the imine analogue, 2640 and the ether analogue, 23, are shown in Scheme
3.
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Scheme 1
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Scheme 2
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Scheme 3
Structure-Activity Relationship (SAR) Study
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The activity of the inhibitors was measured using the TEVC technique with full length M2 protein expressed in
Xenopus oocytes.21,54-56 All inhibitors were initially tested at 100 µM; those that inhibited the AM2 channel
activity more than amantadine (Amt) (1) (by 35% inhibition) were chosen for measurement of their IC50.
Inhibitors of M2 enter the channel from the outside of the virus through a narrow opening near Val27. Thus,
M2-blockers show slow on/off inhibition, with association occurring on the min. to hour time scale (at 1 µM)
and dissociation hour to day time scale. Due to the limited stability of oocytes at low pH, we measure the
percent inhibition after only 2 min, before equilibrium is reached. Due to the kinetics of inhibition, this
provides an apparent kinetic IC50 up to 100-fold higher than the true Kdiss measured with much longer times.57
Nevertheless, the relative potency of the compounds tracks well with their relative potencies measured in other
methods. Thus, the low µM IC50 values of compounds seen in our primary electrophysiologic assays are
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numerically larger than their antiviral potency in plaque assays. While the TEVC assay underestimates the true
potencies of the compounds, it has the advantage of being remarkably reproducible, allowing us to reliably
interpret even small degrees of inhibition. All measurements were made in triplicate, with a typical standard
error being between 1% and 3%. Moreover, we observed less than 5% error when making repeat measurements
over time and with different batches of oocytes.
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Since the reported nonamantadine-based aryl-containing M2 inhibitors (Table 1)39,40 showed essentially no
inhibition against S31N, we started to explore amantadine-based derivatives with an aromatic group appended
to the primary amine group, which led to the discovery of a new series of substituted N-benzyl amantadines as
promising M2 inhibitors against S31N as well as WT. Beginning with the unsubsituted N-benzyl analogue of
amantadine we examined the effects of substitution at various positions in the ring. Because unsubstituted
amantadine is already a relatively hydrophobic drug, we focused the present studies primarily on polar
substituents to capture additional hydrogen bonding or electrostatic interactions as well as to maintain desirable
drug-like properties. Although the unsubstituted derivative 5 was inactive towards S31N (1% inhibition), it did
show significant inhibition against the WT (77% inhibition) at 100 µM drug concentration. We therefore
focused on phenyl substituents with hydrogen-bonding donor/acceptor at 4-position (Table 2), and were
gratified to observe that the S31N inhibitory activity did increase gradually for 4-substituted series.
Introduction of a 4-amino substituent (compound 6) increased S31N activity from 1% to 10% inhibition, but
decreased activity against WT from 77% to 31% inhibition. Interestingly, a Boc derivative of the aniline of 6
improved S31N activity to 20% inhibition (compound 7), and acetylation of 6, further boosted S31N activity to
32% inhibition (compound 8), which is similar to the activity of 1 (35% inhibition). However, the closely
related benzamide 9 and benzylamine 10 lost considerable potency against both S31N and WT.
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Next, heteroaryl substituents at 4-position were examined. Introduction of a pyrazole 11, or imidazole 12 gave
compounds with some activity against S31N (26% and 25% inhibition, respectively), but still less potent than
unsubstituted amantadine (1). A tetrazole derivative was even less active than the pyrazole and imidazole
derivatives (8% inhibition) therefore, we examined other substituents at the 4-position. Replacement of aniline
of 6 with its bioisomer, 4-pyridine 14 slightly increased S31N and WT activity. Other substituents at the 4-
position such as sulfone 15, sulfoxide 16, nitro 17, cyano 18, and fluorine 19 showed some improvement in
S31N activity compared to the parent compound 5, but did not reach the same activity as amantadine (1).
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The 4-phenolic derivative, 4-(adamantan-1-ylaminomethyl)-phenol (20) was the first compound that showed
slightly higher inhibitory activity against S31N (IC50 = 166 µM) than amantadine (1) (IC50 = 199.9 µM).
Methylation of the 4-hydroxyl group of 20, reduced S31N inhibitory activity from 42% to 31% inhibition
(compound 21), which indicated the importance of the 4-hydroxy group. Methyl ketone 23 and methyl ether 21
showed similar S31N activity (31% inhibition). One carbon homologated derivative of 20 (compound 22),
markedly decreased S31N inhibitory activity from 42% to 17% inhibition. Interestingly, 4-cyclohexanol 24, a
saturated version of 20, dramatically dropped S31N activity from 42% to 19% inhibition, which demonstrated
that the aromaticity is crucial for S31N activity. We also found that the 4-hydroxy of 20 could not be replaced
by its bioisomer, 3-furan 25, as this modification led to a marked loss of S31N inhibitory activity from 42% to
8%, respectively.
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Table 2. SAR of 4-substituted adamantan-1-yl-benzyl-amines 1
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R1
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HN
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-H
R1=
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Amt
/ ID #
S31N
% inh/
IC50
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5
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14
35 ± 2/
1 ± 1
10 ± 1
20 ± 2
32 ± 2
3 ± 1
8 ± 1
26 ± 3
25 ± 2
8 ± 1
15 ± 1
199.9 µM
WT
% inh/
IC50
91 ± 3/
77 ± 1
31 ± 2
13 ± 2
3 ± 2
7 ± 2
3 ± 2
4 ± 3
4 ± 2
6 ± 1
6 ± 1
16.1 µM
R1=
/ ID #
S31N
% inh/
IC50
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23
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25
12 ± 1
21 ± 2
14 ± 1
8 ± 2
8 ± 2
42 ± 2/
31 ± 2
17 ± 2
31 ± 2
19 ± 2
8 ± 2
166 µM
WT
% inh/
IC50
2 ± 2
45 ± 2
49 ± 2
36 ± 2
77 ± 1
67 ± 2
43 ± 1
26 ± 2
2 ± 2
16 ± 1
73 ± 2
1 The activity of the inhibitors was measured using the TEVC technique with full length AM2 protein in the
Xenopus oocytes membrane; average of three measurements was reported (see Experimental Section in the
Supporting Information).
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We also explored the SAR of the linkers (Table 3). A secondary amine is preferred over an imine analogue for
S31N activity (42% and 21% inhibition for compounds 20 and 26, respectively). Secondary amine linker also
showed better S31N activity than either a tertiary amine or an ether linkage (31%, 20%, and 13% inhibition for
compounds 21, 27, and 28, respectively). Accordingly, in the following SAR studies, we kept the secondary
amine linker constant.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. The SAR of the linkers
Structure
20
26
21
27
28
S31N % inh
WT % inh
42 ± 2%
21 ± 1
%
%
31 ± 2%
20 ± 3%
13 ± 1%
67 ± 2%
81 ± 2
43 ± 1%
34 ± 3%
51 ± 2%
We further examined SAR of 2- and 3-substituted benzyl amine analogues (Table 4). Two phenol regioisomers
of 20, 3-hydroxy 34 and 2-hydroxy 29, displayed much lower S31N inhibitory activity (6% and 18% inhibition,
respectively), but improved WT inhibitory activity (88% and 81% inhibition, respectively). As was the case for
4-methyl ether 21 (Table 2), 2-methyl ether 30 also decreased rather than enhanced the S31N inhibitory activity.
Similarly, replacement of the 2-hydroxy in 29 with a 2-furan 31, 2-nitro-phenyl 32, or 2-pyridyl 33 group led to
the further decrease in activity.
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Table 4. SAR of 2 and 3-substituted adamantan-1-yl-benzyl-amines
1
2
3
4
5
R1
HN
6
7
8
9
-H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
Amt
R1=
/ ID #
1
5
20
29
30
31
32
33
34
S31N
% inh/
IC50
35 ± 2/
1 ± 1
42 ± 2/
18 ± 2
14 ± 1
6 ± 2
1 ± 1
0%
6 ± 2
199.9 µM
166 µM
WT
% inh/
IC50
91 ± 3/
77 ± 1
67 ± 2
81 ± 2
22 ± 2
66 ± 3
48 ± 1
12 ± 1
88 ± 1
16.1 µM
Attention was turned to effects of 3-substituents in the context of the 4-hydroxy 20, which had significant
activity against S31N and WT (Table 5). Both electron withdrawing groups (3-Cl, 3-F, 3-NO2) (compounds 37,
38, and 39, respectively) and electron donating groups (3-OH, 3-OMe) (compounds 35 and 36, respectively)
reduced potency, with 3-fluoro derivative 38 showing the smallest decrease in potency (31% inhibition). 3-
Methoxy analogue 36 is less effective than 3-hydroxy derivative 35, which may be due to steric hindrance. This
was supported by a more potent inhibitor 41, in which the methylene at 3-position was constrained into a 5-
membered ring. Methylation of the 4-hydroxyl group of 35 (compound 40) maintained both WT and S31N
activity and displayed slightly decreased activity than the more constrained 41. We also found that 4-hydroxy
cannot be replaced with two cyclic bioisomers, 42 and 43, which displayed much weaker activity against S31N
(10% and 12% inhibition, respectively).
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Table 5. SAR of 3,4-disubstituted adamantan-1-yl-benzyl-amines
1
2
3
4
5
6
7
8
9
10
R1
HN
H
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
R1=
Amt
/ ID #
1
20
35
36
37
38
39
40
41
42
43
S31N
% inh/
IC50
35 ± 2/
42 ± 2/
23 ± 1
3 ± 1
16 ± 1
31 ± 3
13 ± 2
20 ± 1
29 ± 1
10 ± 1
12 ± 1
199.9 µM 166 µM
WT
% inh/
IC50
91 ± 3/
67± 2
44 ± 3
76 ± 2
78 ± 1
65 ± 1
66 ± 1
46 ± 3
54 ± 2
26 ± 1
79 ± 1
16.1 µM
Because 2- and 4-hydroxyl and methoxy analogues provided better inhibition compared to the 3-hydroxy
analogue (Table 4), we further examined whether their beneficial effects were additive. Toward this end, we
synthesized 2,4-disubstituted benzyl amine derivatives (Table 6). Pleasingly, 2,4-dihydroxy substituted analog,
44, markedly enhanced S31N inhibitory activity (67% inhibition at 100 µM, IC50 = 35.2 µM). Compound 44
was approximately 6-fold more potent against S31N than amantadine (1) (IC50 = 199.9 µM), and only 2-fold
less potent than amantadine (1) for WT (IC50 = 16 µM) in the TEVC assay. Compound 44 was also active
against the WT with an IC50 of 59 µM, approximately 3.7-fold less potent than amantadine (1). The antiviral
activity of 44 was also profiled in viral plaque reduction assays (Fig. 4). The plaque assays showed that the
compounds were more active than in the electrophysiological TEVC assays, which underestimated the affinities
due to the brief period of inhibition. Compound 44 had an EC50 of 3.2 µM against S31N and was 7-fold more
potent than amantadine (1) (EC50 = 22.5 µM) (Fig. 3A). In the plaque assay, compound 44 was also active
against the WT virus with complete inhibition of plaque formation at l µM (Fig. 3B).
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1
2
3
4
5
6
7
8
9
We also found that methylation of the 4-hydroxy 45 maintained the potency for both WT and S31N with IC50
values of 79 µM and 41.3 µM, respectively. The importance of the 2-hydroxy was observed in other 4-
substituted analogues. For example, installation of 2-hydroxy to 4-fluoro analogue 19 markedly improved S31N
inhibitory activity from 8% to 37% inhibition (compound 46). However, methylation of 2-hydroxy in 44 and
45 (compounds 47 and 48, respectively) dramatically reduced both S31N and WT activities, which might be
due to the steric hindrance at this position. 2-Chloro analogue 49 also showed much lower S31N activity than
44, which may be attributed to the fact that chlorine is a weaker hydrogen acceptor than a hydroxyl group.
These data might also indicate that a hydrogen bond donating hydroxyl is essential at this position of the ring.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 6. SAR of 2,4-disubstituted adamantan-1-yl-benzyl-amine of selective compounds
R1
HN
-H
Amt
R1=
/ID #
1
20
29
44
45
46
37 ± 2
NA
47
12 ± 2
NA
48
11 ± 1
NA
49
14 ± 1
NA
% inh
35 ± 2
42 ± 2
18 ± 2
67 ± 1
66 ± 1
S31N
WT
IC50 (µM)
199.9 µM 166 µM
35.2 µM
43.1 µM
% inh
91 ± 3
67 ± 2
NA
81 ± 2
87 ± 2
NA
0
16 ± 2
NA
78 ± 1
NA
64 ± 1
56 ± 1
IC50 (µM)
16.1 µM
NA
59 µM
79 µM
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1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. (A) Plaque reduction assay with A/WSN/33-S31N influenza virus. (B) Plaque reduction assay with
A/WSN/33-WT virus. The inhibitory effect of compound 44 and amantadine on influenza A virus replication
was evaluated by plaque formation in MDCK cells as described in the Experimental Section.
Conclusions:
Using a combination of structural information of M2 proton channel and medicinal chemistry approaches, we
report the discovery of promising dual M2 inhibitors for S31N and WT M2 channels, demonstrating that M2
S31N is a druggable target. Based on this work, several more potent series of S31N inhibitors have been
discovered, one series has already been published58 and others will be the subject of future reports. Such
compounds will be also important as mechanistic probes to address the problem of drug-resistant influenza A
infections.
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1
2
3
4
5
AUTHORSHIP INFORMATION
Corresponding Authors:
6
7
8
Jizhou Wang, Tel: (484) 598-2376; Fax: (484) 598-2401; E-mail: jwang@influmedix.com
9
William F. DeGrado, Tel: (415) 476-9679; Fax: (415)-502-4690; E-mail: William.DeGrado@ucsf.edu
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACKNOWLEDGEMENT
We acknowledge support of grant AI74571 from the NIH.
SUPPORTING INFORMATION
Details of the Two-Electrode Voltage Clamp (TEVC) Assay, Plaque Reduction Assay, and Synthesis of
Compounds, 5-49, are available free of charge via the Internet at http://pubs.acs.org.
LIST OF ABBREVIATIONS
Amantadine (Amt), Serine 31 (S31), Serine 31 Arganine (S31N), Wild type (WT), Compound (Cmpd), Two-
Electrode Voltage Clamp (TEVC), % inhibition (% inh), The half maximal inhibitory concentration (IC50),
Madin-Darby Canine Kidney Cells (MDCK), Lithium aluminium hydride (LAH), 1-Hydroxy-7-
azabenzotriazole (HOAT), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), Nuclear Magnetic
Resonance (NMR), N,N-Diisopropylethylamine (DIPEA), Dimethylformamide (DMF), Room temperature
(RT), meta-Chloroperoxybenzoic acid (m-MCPA).
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