Discovery of Highly Potent Inhibitors Targeting the Predominant Drug-Resistant S31N Mutant of the Influenza A Virus M2 Proton Channel

Article abstract of DOI:10.1021/acs.jmedchem.5b01910

With the emergence of highly pathogenic avian influenza (HPAI) H7N9 and H5N1 strains, there is a pressing need to develop direct-acting antivirals (DAAs) to combat such deadly viruses. The M2-S31N proton channel of the influenza A virus (A/M2) is one of t

Full text of DOI:10.1021/acs.jmedchem.5b01910

Subscriber access provided by Albany College of Pharmacy and Health Sciences
Article
Discovery of highly potent inhibitors targeting the predominant drug-
resistant S31N mutant of the influenza A virus M2 proton channel
Fang Li, Chunlong Ma, William F. DeGrado, and Jun Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01910 • Publication Date (Web): 15 Jan 2016
Downloaded from http://pubs.acs.org on January 19, 2016
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 29
Journal of Medicinal Chemistry
1
1
2
3
4
5
6
7
8
9
Discovery of highly potent inhibitors targeting the predominant
drug-resistant S31N mutant of the influenza A virus M2 proton
channel
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
Fang Li,†,‡ Chunlong Ma,†,‡ William F. DeGrado,§ Jun Wang*,†,‡
†Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona,
Tucson, Arizona 85721, United States
‡BIO5 Institute, The University of Arizona, Tucson, Arizona, 85721, United States
§Department of Pharmaceutical Chemistry, University of California, San Francisco, California
94158, United States
* Corresponding author:
Jun Wang, Tel: 520ꢀ626ꢀ1366, Fax: 520ꢀ626ꢀ0749, email: junwang@pharmacy.arizona.edu
KEYWORDS. Influenza A virus, M2 proton channel, M2ꢀS31N inhibitor
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 29
2
1
2
3
4
ABSTRACT
5
6
7
8
9
With the emergence of highly pathogenic avian influenza (HPAI) H7N9 and H5N1 strains, there
is a pressing need to develop directꢀacting antivirals (DAAs) to combat such deadly viruses. The
M2ꢀS31N proton channel of the influenza A virus (A/M2) is one of the validated and most
conserved proteins encoded by the current circulating influenza A viruses, thus it represents a
highꢀprofile drug target for therapeutic intervention. We recently discovered a series of S31N
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
+
inhibitors with the general structure of adamantylꢀ1ꢀNH2 CH2ꢀaryl, but they generally had poor
physical properties and some showed toxicity in vitro. In this study, we sought to optimize both
the adamantyl as well as the aryl/heteroaryl group. Several compounds from this study exhibited
submicromolar EC50 values against S31Nꢀcontaining A/WSN/33 influenza viruses in antiviral
plaque reduction assays with a selectivity index greater than 100, indicating that these
compounds are promising candidates for in depth preclinical pharmacology.
ACS Paragon Plus Environment

Page 3 of 29
Journal of Medicinal Chemistry
3
1
2
3
4
INTRODUCTION
5
6
7
8
9
Influenza virus infections are a major global health threat. Despite the availability of influenza
vaccines and small molecule antivirals, an estimated 10–15% of the population is infected
annually in the United States,1 leading to approximately 36,000 deaths and 200,000
hospitalizations.2, 3 Currently, vaccination remains the most effective way to prevent influenza
virus infection; however, it is only partially effective and offers 65% protection in the best
scenario.4 Moreover, due to the antigenic shift and drift of influenza viruses, influenza vaccines
have to be regenerated every year.5, 6 Even though more effective broadꢀneutralizing antibodies
are still in development,7ꢀ9 there is an immediate need for small molecule drugs, particularly for
combating emerging highly pathogenic influenza strains, such as H5N1 and H7N9 for which
vaccines were not immediately available in the first few months of influenza outbreak. These
highly pathogenic avian influenza strains might become humanꢀtoꢀhuman transmissible with just
a few additional mutations.10 Thus, small molecule antivirals that target the most conserved viral
proteins, such as the A/M2 proton channel, are highly desired.11, 12
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
A/M2 is a viral membrane protein that forms a homotetrameric protonꢀselective channel in the
viral envelope.13, 14 The recognized function of A/M2 includes acidifying the viral interior after
endocytosis, thereby initiating viral uncoating. In certain strains of influenza A viruses, A/M2
also functions to equilibrate the pH across the lumen of the late Golgi apparatus, thus preventing
premature conformational change of the viral fusogenic proteinꢀhemagglutinin.15
Interest in understanding the proton conductance and drug inhibition mechanism of A/M2 has
been strongly motivated by its involvement in influenza virus infections.14 A/M2 was first
discovered as the protein target of the antiꢀinfluenza drug amantadine.16, 17 However, the use of
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 29
4
1
2
amantadine was discontinued due to the prevalence of drugꢀresistant mutants.18 Among the large
number of drugꢀresistant mutants identified in cell culture and amantadineꢀtreated patients, only
three major mutants, namely V27A, L26F, and S31N, have been found in transmissible viruses.19,
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
20
The stringency of sequence conservation in M2 reflects tight functional constraints on the
poreꢀlining residues, where a single mutation to a monomer in M2 causes four changes within
the highly constricted pore.13 This small set of transmissible mutants suggests that M2 is a highly
conserved drug target compared with other viral proteins, rendering it an ideal drug target for the
development of antiꢀinfluenza drugs.21 However, drug discovery targeting M2 has been
hampered by the lack of a reliable highꢀthroughput screening assay and highꢀresolution
structures. Nevertheless, guided by information gathered from molecular dynamics simulations,22,
23
Xꢀray crystallography,24, 25 solutionꢀ and solidꢀstate NMR spectroscopy,22, 26, 27 recent years
have witnessed significant progress in this area, and several classes of compounds have been
shown to inhibit all three major drug resistant mutants: V27A, L26F, and S31N.22, 27ꢀ31
Subsequently, through iterative cycles of modeling, medicinal chemistry, electrophysiological
testing, and antiviral assaying, the potencies of several lead compounds were further improved to
the point where their IC50 values against drugꢀresistant M2 mutants were better than that of
amantadine in inhibiting the wildꢀtype M2 channel (Figure 1). The general structure of an S31N
inhibitor contains adamantylꢀ1ꢀNH2 CH2ꢀAryl.27, 31 In this study, we systematically explored
+
various hydrophobic scaffolds and substitutedꢀadamantanes as replacements of adamantane and
examined several other heterocycles as the aryl headgroup. This study resulted in several
compounds with improved potency and selectivity.
ACS Paragon Plus Environment

Page 5 of 29
Journal of Medicinal Chemistry
5
1
2
3
4
5
6
7
Previous
Work
1
3
8
2
4
5
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
A/M2
WT
16.1
0.3
S31N
199.9
22.5
WT
0.9
WT
18.7
N.T.
V27A
0.3
L26F
0.6
S31N
16.0
0.4
WT
59.0
N.T.
S31N
35.2
3.2
IC50 (µM)
EC50 (µM)
N.T.
N.T.
N.T.
This Work
Figure 1. Chemical structures of inhibitors targeting the drugꢀresistant influenza A virus M2
proton channels, V27A, L26F, and S31N, and the SAR of S31N inhibitors explored in this study.
IC50 values were determined in twoꢀelectrode voltage clamp assays. EC50 values were
determined from plaque reduction assays. A/Udorn/72, A/Udorn/72ꢀV27A, and A/WSN/33
influenza strains were used in plaque reduction assays for determining drug sensitivity to WT,
V27A, and S31N inhibitors, respectively. N.T. = not tested.
RESULTS AND DISCUSSION
CHEMISTRY
The synthetic routes to compounds with various hydrophobic scaffolds, 6a–c and 8a–d, are
shown in Scheme 1. The synthesis of the adamantylꢀbenzyl series of compounds was carried out
by reductive amination of amines with 2,4ꢀdihydroxybenzaldehyde (Scheme 1A).31 The
isoxazole series of compounds was synthesized by direct alkylation (Scheme 1B).27
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 29
6
1
2
3
4
Scheme 1. Synthetic routes to M2 inhibitors bearing various hydrophobic scaffolds.
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
Synthesis of compounds 14 with various substitutions at the 5 position of isoxazole is shown in
Scheme 2A.32 The synthesis started with a condensation reaction of methyl ketone 9 and
dimethyl oxalate using potassium tertꢀbutoxide as the base (Scheme 2A). Subsequent cyclization
with hydroxylamine hydrochloride under mild heating at 50 °C in CH3OH gave the isoxazole
ester intermediate 11. Yields for these two steps range from 61% to 82%. The ester was next
reduced to the alcohol 12 by addition of sodium borohydride and converted to bromide 13 by
triphenyl phosphine and carbon tetrabromide in dichloromethane. Overall yields for these two
steps are 68–83%. Bromination using PPh3 and CBr4 significantly improved the yield compared
to phosphorous tribromide.27 The final Nꢀalkylation of amantadine was performed under
refluxing condition in isopropanol with cesium iodide as the catalyst and triethyl amine as the
base in yields ranging from 62% to 91%.
The synthesis of compound 18 bearing 1,2,4ꢀoxadiazole is shown in Scheme 2B.33 Condensation
of carboxylic acid 15 with 2ꢀchloroꢀNꢀhydroxyethanimidamide gave the hydroxylamine ester 16,
which was cyclized under heating in DMF to give the oxadiazole methylene chloride 17.
Subsequent Nꢀalkylation gave oxadiazole analogs 18 with 30–65% overall yields. 1,2,4ꢀ
oxadiazoles (20a–c) with the adamantylamino methylene at the 5 position were synthesized by
alkylation (Scheme 2C).
ACS Paragon Plus Environment

Page 7 of 29
Journal of Medicinal Chemistry
7
1
2
3
4
5
6
Scheme 2. Synthetic routes to adamantane derivatives bearing an isoxazole or oxadiazole
headgroup.
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
The synthesis of 1,3,4ꢀoxadiazole and 1,3,4ꢀthiadiazole compounds 26a,b is shown in Scheme
3.34 Treatment of the 2ꢀthiophenecarboxylic acid hydrazide 21 with methyl oxalyl chloride gave
methyl 2ꢀoxoꢀ2ꢀ(thiophenꢀ2ꢀylformohydrazido)acetate 22 in nearly quantitative yield. This
intermediate (22) was cyclized upon treatment with pꢀtoluenesulfonyl chloride (TsCl) or
Lawesson’s reagent to give 1,3,4ꢀoxadiazole ester 23a and 1,3,4ꢀthiadiazole ester 23b,
respectively.34 The yields for 23a and 23b were 80% and 75%, respectively. The esters were
subsequently reduced to hydroxyl 24, followed by bromination and alkylation to give the final
compounds 26a,b. Other 1,3,4ꢀoxadiazole analogs 26c,d and thiazoles 26e-j were synthesized by
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 29
8
1
2
3
4
5
6
reductive amination or alkylation using commercially available aldehydes and halides as shown
in Scheme 1.27
7
8
9
Scheme 3. Synthetic route to adamantane derivatives bearing 1,3,4-oxadiazole or 1,3,4-
thiadiazole headgroup.
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
Compounds with substitutedꢀadamantanes or adamantane analogs as the hydrophobic scaffolds
were synthesized from a common precursor, 4ꢀoxoadamantaneꢀ1ꢀcarboxylic acid (27) (Scheme
4A). Oneꢀpot Curtius rearrangement followed by treatment with tertꢀbutanol gave tertꢀbutyl Nꢀ
(4ꢀoxoadamantanꢀ1ꢀyl)carbamate (28) in 78% yield.35 Subsequent deprotection with
hexafluoroisopropanol (HFIP) under microwave irradiation gave 5ꢀaminoadamantanꢀ2ꢀone (29).
Alkylation of 29 with 3ꢀ(bromomethyl)ꢀ5ꢀ(thiophenꢀ2ꢀyl)ꢀ1,2ꢀoxazole gave the key intermediate
30a in 75% yield. The ketone in 30a was next either reduced by NaBH4 or alkylated by MeMgBr,
or oxidized by the in situ–generated CoreyꢀChaykovsky reagent36 to give 30b, 30c, and 30d,
respectively.
The ringꢀexpanded 4ꢀoxatricyclo [4.3.1.13,8]undecanꢀ1ꢀamine (32) was synthesized by Baeyerꢀ
Villiger oxidation of 28 with metaꢀchloroperoxybenzonic acid (mCPBA), followed by reduction
with indium bromide and triethylsilane and deprotection with HFIP. Finally, installation of the 5ꢀ
ACS Paragon Plus Environment

Page 9 of 29
Journal of Medicinal Chemistry
9
1
2
3
4
5
6
7
8
9
(thiophenꢀ2ꢀyl)ꢀ1,2ꢀoxazole was achieved through alkylation using CsI and DIEA under reflux
condition in isopropanol to give 30e in 27% overall yield. Compounds 30f,g were synthesized
through the same alkylation procedure starting from 3ꢀaminoadamantanꢀ1ꢀol (33). Compound
30h with 3,5ꢀdihydroxyl adamantane was synthesized from 3ꢀbromoꢀ1ꢀadamantanecarboxylic
acid (34). Oxidation of 34 using KMnO4 gave 3,5ꢀdihydroxylꢀ1ꢀadamantanecarboxylic acid (35).
Subsequent Curtius rearrangement followed by treatment with tertꢀbutanol gave 36. Next
deprotection of 36 followed by alkylation afforded the final compound 30h. The overall yield
was 17%.
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
Scheme 4. Synthetic route to M2-S31N inhibitors bearing adamantane analogs
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 29
10
1
2
3
4
STRUCTURE–ACTIVITY RELATIONSHIP (SAR) STUDY
5
6
7
Exploring the hydrophobic scaffold as a replacement for adamantane in S31N inhibitors.
8
9
Our previous studies of the S31N inhibitors revealed two classes of potent S31N inhibitors (4
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
+
and 5), both of which share the same general structure of adamantylꢀ1ꢀNH2 CH2ꢀaryl (Figure
1).27, 31 The adamantylꢀbenzyl series of compounds, a representative example of which is
compound 5, are active against both the WT and the S31N mutant of M2,31 while the adamantylꢀ
isoxazole series, such as 4, show selective inhibitory against S31N.27 SAR studies of the
adamantylꢀbenzyl series revealed that the –NH2 CH2– linker is essential for this activity.31
+
However, the adamantyl scaffold has not been explored. It has been shown that inhibitors with a
wide variety of hydrophobic scaffolds, such as linear alkyl, monoꢀcyclic alkyl, biꢀcyclic alkyl,
and spiroꢀalkyl, are potent channel blockers of the WT M2.28, 37ꢀ41 Thus we sought to explore
various hydrophobic scaffolds in both series of S31N inhibitors and examine how this
modification would affect their inhibition against both WT and S31N. The hydrophobic scaffolds
include adamantane analogs such as ringꢀcontracted adamantane (6a, 8b), ringꢀexpanded
adamantane (6b, 8c), and spiroamine (6c) (Table 1). All of the hydrophobic scaffolds examined
(without aryl headgroup) are potent inhibitors against WTꢀM2.37
The synthesized compounds were first tested in twoꢀelectrode voltage clamp (TEVC)
electrophysiological assays, in which M2 was expressed in Xenopus laevis oocytes and current
conductance across cell membrane was recorded in a whole cell format.29 All compounds were
initially tested at 100 ꢁM drug concentration against both WT and S31N. The antiviral activity of
potent compounds with greater than 80% M2 channel inhibition at 100 µM in TEVC assays were
further confirmed in plaque reduction assays. For the 2,4ꢀdihydroxyl benzyl series of compounds,
replacing the adamantane scaffold in 5 with noradamantane (6a), azaꢀadamantane (6b) or
ACS Paragon Plus Environment

Page 11 of 29
Journal of Medicinal Chemistry
11
1
2
3
4
5
6
7
8
9
spiropiperidine substituent (6c) decreased the M2ꢀS31N channel blockage (Table 1). However,
the resulting compounds (6a–c) all displayed similar or higher inhibitory activity against WTꢀM2.
Similar SAR was also observed for the isoxazole series of compounds: replacing the adamantane
in 8a with other hydrophobic scaffolds ameliorated activity against S31N, and all compounds in
this series (8a–d) remained inactive against WT. Insertion of a methylene linker between the
adamantyl and ammonium gave 8d, which is significantly less active than the parent compound
8a in inhibiting the S31N channel. Collectively, adamantane (5 and 8a) and ringꢀexpanded
adamantane (8c) appeared to be the optimal hydrophobic scaffolds for S31N inhibition, while a
wide variety of hydrophobic scaffolds are tolerated for WTꢀM2 inhibition. Noradamantane
scaffold was also tolerated for S31N inhibition, although corresponding compounds (6a and 8b)
were less active than their adamantane counterparts. A secondary amine group appeared to be
essential for S31N inhibition as compounds with a tertiary amine (6b and 6c) were completely
inactive.
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 1. Exploring various hydrophobic scaffolds in S31N inhibitors
%S31N
%WT
R
Compound ID
AMT (1)
5
inhibition*
inhibition*
36
67
91
64
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 29
12
1
2
3
4
5
6
7
8
40
0
64
74
65
5
6a
6b
6c
8a
8b
8c
8d
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
3
79
71
75
34
19
11
14
*Values represent the mean of three independent measurements. We typically see no more than 5%
variation in the percent inhibition on a given day, or 10% error for measurements made on
different days with different batches of oocytes. Data listed is percent inhibition when tested at
100 ꢁM drug concentration. N.T. = Not tested.
Exploring hydrophobic substitutions of isoxazole and 1,2,4-oxadiazole. Solution NMR
structure of 4 bound to M2ꢀS31N (19–49) shows the distal thienyl ring of the drug forms
ACS Paragon Plus Environment

Page 13 of 29
Journal of Medicinal Chemistry
13
1
2
hydrophobic interactions with V27 side chain methyl groups (Figure 2),27 suggesting that other
hydrophobic alkane substituents can be similarly accommodated at the same position.
3
4
5
6
7
8
9
A
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 2. Drug binding mode of the A/M2ꢀS31N mutant. (a) Solution NMR structure of A/M2ꢀ
S31N bound with the isoxazole inhibitorꢀM2WJ332 (4) (PDB: 2LY0). (b) Representation of
drug–protein interactions.
Replacing a small methyl substituent at the 5 position of isoxazole or 1,2,4ꢀoxadiazole (14a and
18a) with a bulkier propyl (14b and 18b), cyclopropyl (14c and 18c), isopropyl (14f and 18d),
cyclobutyl (14d), or 2ꢀmethylꢀ2ꢀ(methylsulfanyl)propyl (14g) group dramatically increased the
activities against S31N. Compound 14e with a terminal methoxy group was much less active
than its methylene analog 14b, highlighting the importance of hydrophobic interactions with the
V27 side chains in highꢀaffinity binding of drugs. Increasing the size of the substituent from
isopropyl (18d) to isobutyl (18e), however, led to a slight decrease in S31N inhibition. The most
active compound against S31N in this series is 14h, showing 90% inhibition at 100 ꢁM. In
general, isoxazoleꢀcontaining compounds are more active than 1,2,4ꢀoxadiazoleꢀcontaining
analogs. It was also noticed that increasing the size of the substituents at the 5 position of
isoxazole and oxadiazole decreased their inhibition against WT M2. The active compounds were
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 29
14
1
2
3
4
5
6
7
8
9
further tested in plaque reduction assays against S31Nꢀcontaining A/WNS/33 viruses, and two
compounds, 14c and 14d, were found to decrease the plaque number more than 50% at 1 µM,
indicating their EC50 values are less than 1 µM.
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 2. Exploring hydrophobic substitutions of isoxazole and 1,2,4-oxadiazole
% plaque formation
% S31N
% WT
R
Z
Compound ID
at 1 µM
(A/WSN/33)**
N.T.
inhibition*
inhibition*
49/N.T.
60/N.T.
81/49
55
41
16
19
19
18
C
N
C
N
C
N
14a a
18a a
14b
N.T.
73±7
77/19
N.T.
18b
14c a
18c a
85/65
42±3
81/60
82±5
R
O
84/N.T.
30
42±2
C
14d
N
Z
HN
39/N.T.
80/55
18
18
10
N.T.
66±2
N.T.
C
C
N
14e
14f
18d a
70/N.T.
62/N.T.
70/N.T.
12
14
N.T.
N.T.
N
C
18e
14g
90/N.T.
13
57±2
C
14h
*Values represent the mean of three independent measurements. We typically see no more than 5%
variation in the percent inhibition on a given day, or 10% error for measurements made on
different days with different batches of oocytes. All compounds were initially tested at 100 µM.
Compounds that showed greater than 80% inhibition at 100 µM were further tested at 30 µM.
The data are presented as percent inhibition at 100 µM/percent inhibition at 30 µM. N.T. = Not
ACS Paragon Plus Environment

Page 15 of 29
Journal of Medicinal Chemistry
15
1
2
a
**
3
tested. Percent inhibition in TEVC assays was previously reported. Plaque reduction assays
were performed against the M2ꢀS31N containing A/WSN/33 virus in duplicates; percent of
plaque formation is defined as the ratio of plaque numbers in the presence of compounds divided
by the plaque numbers in the absence of compounds.
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
Exploring substitutions in the distal aromatic ring of isoxazole and 1,2,4-oxadiazole
It has been found that S31N inhibition is relatively insensitive to different regioisomers of the
distal ring at the 5 position of isoxazole and 1,2,4ꢀoxadiaole: compounds 14i and 18g showed
similar activities as 4 and 18f, respectively. This finding suggests that the sulfur atom in the
distal thienyl ring is not involved in specific interactions with the M2 channel, which is
consistent with the solution NMR structure showing that the thienyl group of 4 forms
hydrophobic interactions with the Val27 side chain methyls.27 Compounds with a furanyl
substitution, 14j and 18h, were slightly less active, which might due to decreased hydrophobicity
(the clogP for 4 and 14j is 3.74 and 2.37, respectively, as calculated from ChemBioDraw 14).
Compound 14k with a methoxyl at the 3 position of the distal thienyl ring was highly active
against S31N, showing 87% inhibition at 30 ꢁM. When a phenyl ring was placed at the 5
position of isoxazole and 1,2,4ꢀoxadiazole, the S31N inhibition of the resulting compounds, 14l
and 18i, was retained. Halogen substitution in the distal phenyl ring was also examined, and the
S31N inhibition was found to be insensitive to these structural modifications. All chloroꢀ
substituted (14m and 18j), bromoꢀsubstituted (14n), and difluoroꢀsubstituted (14o and 14p)
compounds showed similar potency as 14l and 18i. Monoꢀmethoxyl substitution at all three
positions of the phenyl ring, ortho (18l), meta (14r), and para (14q and 18k), did not affect the
S31N inhibition. Similarly, compounds with monoꢀmethylsulfanyl substitutions at the ortho (14s)
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 16 of 29
16
1
2
3
4
5
6
7
8
9
and para (14a) of the distal phenyl ring retained potent inhibition against S31N. Doubleꢀ
methoxyl substitutions decreased the S31N inhibition of the isoxazoleꢀcontaining compound 14t,
but not the 1,2,4ꢀoxadiazoleꢀcontaining compound 18m. Consistent with previous SAR
conclusions, compounds with 1ꢀ(1ꢀadamantylamino)ꢀmethylene substitution were more active
when placed at the 3 position of isoxazole and 1,2,4ꢀoxadiazole than at the 5 position (20b versus
18l, 20c versus 14h).27 In plaque reduction assays, several compounds, 14k, 14l, and 14r,
showed comparable or even better antiviral activity than the parent compound 4 in inhibiting
A/WSN/33 virus replication.
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. Exploring aromatic substitutions of isoxazole and 1,2,4-oxadiazole
% S31N
inhibition*
90/63
% WT
% plaque formation at 1
R
Z
Compound ID
inhibition*
µM (A/WSN/33)**
4 a
18f a
14i
11
11
28
3
32±2
66±3
47±5
74±10
47±1
N.T.
C
N
C
N
C
N
83/N.T.
90/N.T.
81/N.T.
84/N.T.
73/N.T.
18g
14j
12
24
18h
90/87
27
39±3
C
14k
91/N.T.
75/N.T.
87/65
20
8
26±3
N.T.
84±6
C
N
C
14l a
18i a
14m
14
86/72
16
12
88±1
N.T.
N
C
18j
77/N.T.
14n
78/N.T.
23
N.T.
C
14o
ACS Paragon Plus Environment

Page 17 of 29
Journal of Medicinal Chemistry
17
1
2
3
4
85/68
8
56±7
C
14p
5
6
7
8
9
14q a
18k a
87/N.T.
86/N.T.
13
18
59±9
92±7
C
N
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
87/73
12
39±1
C
14r
79/N.T.
79/N.T.
90/77
8
5
7
N.T.
N.T.
66±3
C
C
N
14s
8a
18l
62/N.T.
82/N.T.
10
18
N.T.
C
N
14t
76±5
18m
39/N.T.
18
N.T.
N
20a
58/N.T.
67/N.T.
13
27
N.T.
N.T.
N
C
20b
20c
*Values represent the mean of three independent measurements. We typically see no more than 5%
variation in the percent inhibition on a given day, or 10% error for measurements made on
different days with different batches of oocytes. The data are presented as percent inhibition at
100 µM/percent inhibition at 30 µM. N.T. = Not tested. a Percent inhibition in TEVC assays was
**
previously reported. Plaque reduction assays were performed against the M2ꢀS31N containing
A/WSN/33 virus in duplicates. The percent of plaque formation is defined as the ratio of plaque
numbers in the presence of compounds divided by the plaque numbers in the absence of
compounds.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 18 of 29
18
1
2
3
4
Exploring 1,3,4-oxadiazole-, 1,3,4-thiadiazole-, and thiazole-containing M2 inhibitors
5
6
7
8
9
To investigate whether isoxazole and 1,2,4ꢀoxadiazole can be replaced by other heterocycles,
several 1,3,4ꢀoxadiazoleꢀcontaining compounds (26a, 26c, and 26d) were synthesized. All three
1,3,4ꢀoxadiazoleꢀcontating compounds were less active than their isoxazole and 1,2,4ꢀoxadiazole
counterparts (26a versus 4 and 18f; 26c versus 14l and 18i; 26d versus 14q and 18k).
Encouragingly, a 1,3,4ꢀthiadiazoleꢀcontaining compound, 26b, was similarly active as the
isoxazole analog (4) in both TEVC and plaque reduction assays. 26b also showed no apparent
toxicity up to 200 µM. Compound 26e with a 1ꢀ(1ꢀadamantylamino)ꢀmethylene substitution at
the 5 position of thiazole was also potent, showing 75% inhibition at 100 ꢁM concentration.
Replacement of the distal thienyl ring at the 2 position of thiazole in 26e with methyl (26g) and
tertꢀbutyl (26f) dramatically decreased S31N inhibition. Compounds containing another thiazole
regioisomer with 1ꢀ(1ꢀadamantylamino)ꢀmethylene at the 4 position of thiazole were much less
active. All compounds were found to have minimal inhibition against WT M2, except 26j (57%
inhibition at 100 ꢁM).
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 4. Exploring 1,3,4-oxadiazole, 1,3,4-thiadiazole, and thiazole
% plaque
% S31N
% WT
CC50
(ꢁM)
R
Compound ID
formation at 1 µM
(A/WSN/33)**
inhibition* inhibition*
52/N.T.
83/N.T.
16
21
N.T.
N.T.
26a
26b
39±2
>200
61/N.T.
58/N.T.
18
3
N.T.
N.T.
N.T.
N.T.
26c
26d
ACS Paragon Plus Environment

Page 19 of 29
Journal of Medicinal Chemistry
19
1
2
3
4
5
6
7
8
9
75/N.T.
38/N.T.
28/N.T.
13
5
N.T.
N.T.
N.T.
113
N.T.
N.T.
26e
26f a
26g a
22
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
37/N.T.
24/N.T.
36/N.T.
7
0
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
26h
26i
26j a
57
*Values represent the mean of three independent measurements. We typically see no more than 5%
variation in the percent inhibition on a given day, or 10% error for measurements made on
different days with different batches of oocytes. The data are presented as percent inhibition at
100 µM/percent inhibition at 30 µM. N.T. = Not tested. a Percent inhibition in TEVC assays was
**
previously reported. Plaque reduction assays were performed against the M2ꢀS31N containing
A/WSN/33 virus in duplicates. The percent of plaque formation is defined as the ratio of plaque
numbers in the presence of compounds divided by the plaque numbers in the absence of
compounds.
Exploring adamantane analogs as potent M2 inhibitors
Having identified the favorable aryl auxiliary substitutions on M2ꢀS31N inhibitors, we decided
to revisit the hydrophobic scaffolds. Our preliminary data suggested adamantane is the optimal
scaffold for S31N inhibition; we therefore focused on substituted adamantanes, such as 4ꢀ
hydroxyl adamantane (30b), 3ꢀhydroxyl adamantane (30f,g), 3,ꢀ5ꢀdihydroxyl adamantane (30h),
4ꢀhydroxylꢀ4ꢀmethyl adamantane (30c), spiro[adamantaneꢀ2,2'ꢀoxirane] (30d), and 4ꢀ
oxatricyclo[4.3.1.13,8]undecane (30e). 5ꢀ(thiophenꢀ2ꢀyl)ꢀ1,2ꢀoxazole and 5ꢀ(3ꢀmethoxythiophenꢀ
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 20 of 29
20
1
2
3
4
5
6
7
8
9
2ꢀyl)ꢀ1,2ꢀoxazole were chosen as the aryl auxiliary substitutions as they are among the most
favorable aryl substitutions for S31N inhibition. Compared with the parent compound 4, all
modifications except 30d retained potent channel blockage against M2ꢀS31N, showing more
than 70% inhibition at 100 µM. In plaque reduction assays, compounds with hydroxylation on
the adamantane ring, 30b, 30f, and 30g, were similarly potent as the parent compound 4, all
showing EC50s less than 1 µM (Figure 3). However, further increasing the hydrophilicity by
adding an additional hydroxyl group led to a significant loss of activity (compound 30h versus
compound 30f). Notably, hydroxylation of the adamantane ring greatly decreased the cellular
cytotoxicity of the parent compound (compound 30f versus compound 4).
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 5. Exploring adamantane analogs as the hydrophobic scaffolds
% plaque
formation
at 1 µM**
Plaque
% S31N
CC50
X, R
Compound ID
Reduction
EC50 (ꢁM)**
clogPꢂ
inhibition*
(ꢁM)***
90
32±2
31±1
0.353
N.T.
100
4.2
4
78/N.T.
N.T.
2.6
2.1
2.6
30a
30b
30c
91/56
87/48
10±3
27±2
0.727
N.T.
116
N.T.
ACS Paragon Plus Environment

Page 21 of 29
Journal of Medicinal Chemistry
21
1
2
3
4
5
61/N.T.
72±3
N.T.
N.T.
2.5
30d
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
72/N.T.
82/N.T.
86/N.T.
58±1
40±1
33±2
N.T.
0.510
0.404
N.T.
>200
N.T.
2.8
2.7
2.2
30e
30f
30g
88/45
69±4
2.5
>200
2.0
30h
*Values represent the mean of three independent measurements. We typically see no more than 5%
variation in the percent inhibition on a given day, or 10% error for measurements made on
different days with different batches of oocytes. The data are presented as percent inhibition at
100 µM/percent inhibition at 30 µM. N.T. = Not tested. **Plaque reduction assays were
performed against the M2ꢀS31N containing A/WSN/33 virus in duplicates. The percent plaque
formation is defined as the ratio of plaque numbers in the presence of compounds divided by the
plaque numbers in the absence of compounds. ***CC50 values were determined using MDCK
cells in the MTT assay. ꢂ clogP was calculated in ChemBioDraw Ultra 14.0.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 22 of 29
22
1
2
3
4
5
6
7
8
9
120
100
80
60
40
20
0
30g EC50 = 404 nM
30f EC50 = 510 nM
30b EC50 =727 nM
30h EC50 = 2.5 µM
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
-4
-2
0
2
4
6
log(X nM)
Figure 3. Antiviral activity of potent S31N inhibitors. Compounds are tested in duplicate with
serial dilutions against A/WSN/33 (M2ꢀS31N).
CONCLUSIONS
M2 belongs to a family of proteins called viroporins, which are virusꢀencoded ion channels.42
Viroporins are involved in diverse steps during viral replication, including viral entry, assembly,
and release. They are made of small (normally fewer than 100 amino acids), hydrophobic
peptides by selfꢀoligomerization and reside in the viral or cellular compartment membranes.
Representative examples of viroporins include the hepatitis C virus p7 ion channel, the HIV vPu
ion channel, M2 from the influenza virus, and the 6K proteins from alphavirus. Compounds that
block cation flux through viroporins inhibit virus replication, corroborating viroporins as
validated antiviral drug targets. The advantage of targeting viroporins compared to other viral
proteins is that viroporins are relatively conserved, as one mutation causes multiple changes
simultaneously in the constrained homoꢀoligomeric channel. For A/M2, only three predominant
ACS Paragon Plus Environment

Page 23 of 29
Journal of Medicinal Chemistry
23
1
2
3
4
5
6
drugꢀresistant mutations have been identified in transmissible viruses, namely V27A, L26F, and
S31N,19, 20 all of which have similar levels of specific activity compared to the WT M2.43
7
8
9
Starting from the lead compounds we identified earlier for S31N inhibition,27, 31 we hereby
disclose the SAR studies of the S31N inhibitor with the generic structure of adamantylꢀ1ꢀ
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
+
NH2 CH2ꢀAryl. The adamantane framework was found to be the optimal hydrophobic core,
suggesting that it fits snuggly in the S31N channel. We next explored adding polar substitutions
to the adamantane framework to increase the drugꢀlike properties of the compounds. Monoꢀ
hydroxylation at the adamantane scaffold was well tolerated (compounds 30b, 30f, and 30g).
Systematic substitutions of the heterocycle immediately attached to adamantane revealed that 1ꢀ
(1ꢀadamantylamino)ꢀmethylene prefers to be placed at the 3 position of isoxazole and 1,3,4ꢀ
oxadiazole,27 and the remaining variable 5 position favors hydrophobic substitutions, such as
alkyl, thienyl, and substituted phenyl. A wide variety of substitutions are tolerated at the orthoꢀ,
metaꢀ, and paraꢀposition of the distal ring when it is a phenyl group. Significantly, we have
identified several compounds (26b and 30f) with much a higher selectivity index (relative to
toxicity towards mammalian cells) than the parent compound 4. Collectively, the SAR agrees
with the mode of inhibitor binding inside the S31N channel,27, 44 in which the adamantane ring is
surrounded by hydrophobic G34, the positively charged amine along with the heteroatom from
the first aryl ring forms bidentate hydrogen bonds with the N31 side chain amide, and the distal
aryl ring fits in the hydrophobic pocket formed by the V27 gate. The lead compounds identified
here have improved physical properties, and should provide excellent tools for in depth
pharmacological examinations.
EXPERIMENTAL SECTION
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 24 of 29
24
1
2
3
4
5
6
7
8
9
Chemistry. All chemicals are commercially available and were used without further purification.
Detailed synthesis procedures can be found in the Supporting Information for reactions described
in Schemes 1–4. All final compounds were purified by flash column chromatography, and the
purify of all compounds tested was ≥ 95% as determined by LCꢀMS.
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
Two-electrode voltage clamp (TEVC) assay. The inhibitors were tested in a twoꢀelectrode
voltage clamp assay using Xenopus laevis frog oocytes microinjected with RNA expressing
either the WT or the S31N mutant of the A/M2 protein as previously reported.29 The potency of
the inhibitors was expressed as percentage inhibition of A/M2 current observed after 2 min of
incubation with either 100 or 30 ꢁM of compounds.
Plaque reduction assay. Plaque reduction assay was performed as previously reported.27 The
viruses used for this assay were A/Udorn/72 and A/WSN/33, which contain WT and the S31N
mutant of M2, respectively.
Cytotoxicity assay. The cytotoxicity of compounds was determined using the CellTiterꢀGlo
Luminescent Cell Viability Assay kit from Promega. Briefly, MadinꢀDarby canine kidney
(MDCK) cells in DMEM + 10% FBS + P/S medium at 50,000 cells/mL were dispensed into
opaque 96ꢀwell plates (Cat #: CLS3362) at 100 µL/well. Twentyꢀfour hours later, the growth
medium was replaced with fresh DMEM and compounds were added at twoꢀfold series dilution
starting from 300 µM. After incubating for 72 h at 37 °C with 5% CO2 in a CO2 incubator, the
plates were equilibrated to room temperature before 100 µL of the CellTiterꢀGlo solution was
added. The plates were shaken for 5 s on a plate reader to allow even mixing. Luminescence
signals were recorded on a multimode SpectraMax M5 plate reader, and the signals were
ACS Paragon Plus Environment

Page 25 of 29
Journal of Medicinal Chemistry
25
1
2
3
4
5
6
normalized against the wells containing cells only. The CC50 values were calculated from bestꢀfit
dose response curves with variable slope.
7
8
9
ASSOCIATED CONTENT
Supporting information
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
13
1HNMR, CNMR, and mass spectrometry of intermediates and final products. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Jun Wang, Tel: 520ꢀ626ꢀ1366, Fax: 520ꢀ626ꢀ0749, email: junwang@pharmacy.arizona.edu
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENTS
This research is supported by NIH Grant AI119187 and the PhRMA foundation 2015 research
starter grant in pharmacology and toxicology to J.W. and NIH Grants GM056423 and AI74571
to W.F.D.
ABBREVIATIONS USED
WT, wild type; SAR, structure–activity relationship; DMEM, Dulbecco’s modified eagle
medium; MDCK, MadinꢀDarby Canine Kidney; TEV, twoꢀelectrode voltage clamps; HPAI,
highly pathogenic avian influenza; DAA, directꢀacting antiviral.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 26 of 29
26
1
2
3
4
REFERENCES
5
6
7
8
1.
Cox, N.; Subbarao, K. Global epidemiology of influenza: Past and present. Annu. Rev.
Med. 2000, 51, 407ꢀ421.
9
2.
Thompson, W.; Shay, D.; Weintraub, E.; Brammer, L.; Cox, N.; Anderson, L.; Fukuda, K.
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
Mortality associated with influenza and respiratory syncytial virus in the United States. JAMA
2003, 289, 179ꢀ186.
3.
Influenzaꢀassociated hospitalizations in the United States. JAMA 2004, 292, 1333ꢀ1340.
4. Osterholm, M.; Kelley, N.; Sommer, A.; Belongia, E. Efficacy and effectiveness of
influenza vaccines: a systematic review and metaꢀanalysis. Lancet Infect. Dis. 2012, 12, 36ꢀ44.
5. Lambert, L.; Fauci, A. Influenza Vaccines for the Future. N. Engl. J. Med. 2010, 363,
2036ꢀ2044.
Thompson, W.; Shay, D.; Weintraub, E.; Brammer, I.; Bridges, C.; Cox, N.; Fukuda, K.
6.
Wong, S.; Webby, R. Traditional and New Influenza Vaccines. Clin. Microbiol. Rev.
2013, 26, 476ꢀ492.
7.
Laursen, N. S.; Wilson, I. A. Broadly neutralizing antibodies against influenza viruses.
Antiviral Res. 2013, 98, 476ꢀ483.
8.
and Challenges. Annu. Rev. Med. 2013, 64, 189ꢀ202.
9. Jin, H.; Chen, Z. Production of live attenuated influenza vaccines against seasonal and
potential pandemic influenza viruses. Curr. Opin. Virol. 2014, 6, 34ꢀ39.
10. Qi, X.; Qian, Y. H.; Bao, C. J.; Guo, X. L.; Cui, L. B.; Tang, F. Y.; Ji, H.; Huang, Y.; Cai,
Pica, N.; Palese, P.; Caskey, C. Toward a Universal Influenza Virus Vaccine: Prospects
P. Q.; Lu, B.; Xu, K.; Shi, C.; Zhu, F. C.; Zhou, M. H.; Wang, H. Probable person to person
transmission of novel avian influenza A (H7N9) virus in Eastern China, 2013: epidemiological
investigation. BMJ 2013, 347, f4752.
11.
Opin. Pharmacother. 2011, 12, 1523ꢀ1549.
12. Webster, R. G.; Govorkova, E. A. Continuing challenges in influenza. Annu. N. Y. Acad.
Sci. 2014, 1323, 115ꢀ139.
Preziosi, P. Influenza pharmacotherapy: present situation, strategies and hopes. Expert
13.
Wang, J.; Qiu, J. X.; Soto, C.; DeGrado, W. F. Structural and dynamic mechanisms for
the function and inhibition of the M2 proton channel from influenza A virus. Curr. Opin. Struct.
Biol. 2011, 21, 68ꢀ80.
14.
De Clercq, E. Antiviral agents active against influenza A viruses. Nat. Rev. Drug Discov.
2006, 5, 1015ꢀ1025.
15.
Hong, M.; DeGrado, W. F. Structural basis for proton conduction and inhibition by the
influenza M2 protein. Protein Sci. 2012, 21, 1620ꢀ1633.
16.
Davies, W. L.; Grunert, R. R.; Haff, R. F.; McGahen, J. W.; Neumayer, E. M.; Paulshock,
M.; Watts, J. C.; Wood, T. R.; Hermann, E. C.; Hoffmann, C. E. Antiviral Activity of 1ꢀ
Adamantanamine (Amantadine). Science 1964, 144, 862ꢀ863.
17.
Activity. Cell 1992, 69, 517ꢀ528.
18. Dong, G.; Peng, C.; Luo, J.; Wang, C.; Han, L.; Wu, B.; Ji, G.; He, H. Adamantaneꢀ
Pinto, L. H.; Holsinger, L. J.; Lamb, R. A. InfluenzaꢀVirus M2 Protein Has Ion Channel
Resistant Influenza A Viruses in the World (1902–2013): Frequency and Distribution of M2
Gene Mutations. PLoS ONE 2015, 10, e0119115.
19.
Furuse, Y.; Suzuki, A.; Kamigaki, T.; Oshitani, H. Evolution of the M gene of the
influenza A virus in different host species: largeꢀscale sequence analysis. Virol. J. 2009, 6, 67.
ACS Paragon Plus Environment

Page 27 of 29
Journal of Medicinal Chemistry
27
1
2
3
4
5
6
20.
Furuse, Y.; Suzuki, A.; Oshitani, H. Largeꢀscale sequence analysis of M gene of
influenza A viruses from different species: mechanisms for emergence and spread of amantadine
resistance. Antimicrob. Agents Chemother. 2009, 53, 4457ꢀ4463.
7
8
9
21.
resistant M2 proton channels from the influenza A viruses. Biopolymers 2015, 104, 291ꢀ309.
22. Wang, J.; Ma, C.; Fiorin, G.; Carnevale, V.; Wang, T.; Hu, F.; Lamb, R. A.; Pinto, L. H.;
Wang, J.; Li, F.; Ma, C. Recent progress in designing inhibitors that target the drugꢀ
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
Hong, M.; Kein, M. L.; DeGrado, W. F. Molecular Dynamics Simulation Directed Rational
Design of Inhibitors Targeting DrugꢀResistant Mutants of Influenza A Virus M2. J. Am. Chem.
Soc. 2011, 133, 12834ꢀ12841.
23.
Khurana, E.; Dal Peraro, M.; DeVane, R.; Vemparala, S.; DeGrado, W.; Klein, M.
Molecular dynamics calculations suggest a conduction mechanism for the M2 proton channel
from influenza A virus. Proc. Natl. Acad. Sci. U S A 2009, 106, 1069ꢀ1074.
24.
Acharya, R.; Carnevale, V.; Fiorin, G.; Levine, B. G.; Polishchuk, A. L.; Balannik, V.;
Samish, I.; Lamb, R. A.; Pinto, L. H.; DeGrado, W. F.; Klein, M. L. Structure and mechanism of
proton transport through the transmembrane tetrameric M2 protein bundle of the influenza A
virus. Proc. Natl. Acad. Sci. U S A 2010, 107, 15075ꢀ15080.
25.
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, 452, 596ꢀ599.
26.
Wang, J.; Cady, S. D.; Balannik, V.; Pinto, L. H.; DeGrado, W. F.; Hong, M. Discovery
of SpiroꢀPiperidine Inhibitors and Their Modulation of the Dynamics of the M2 Proton Channel
from Influenza A Virus. J. Am. Chem. Soc. 2009, 131, 8066ꢀ8076.
27.
Wang, J.; Wu, Y.; Ma, C.; Fiorin, G.; Wang, J.; Pinto, L. H.; Lamb, R. A.; Klein, M. L.;
DeGrado, W. F. Structure and inhibition of the drugꢀresistant S31N mutant of the M2 ion
channel of influenza A virus. Proc. Natl. Acad. Sci. U S A 2013, 110, 1315ꢀ1320.
28.
Wang, J.; Ma, C.; Wu, Y.; Lamb, R. A.; Pinto, L. H.; DeGrado, W. F. Exploring
organosilane amines as potent inhibitors and structural probes of influenza a virus M2 proton
channel. J. Am. Chem. Soc. 2011, 133, 13844ꢀ13847.
29.
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 amantadineꢀ
resistant mutants of the M2 ion channel of influenza A virus. Biochemistry 2009, 48, 11872ꢀ
11882.
30.
Wu, Y.; Canturk, B.; Jo, H.; Ma, C.; Gianti, E.; Klein, M. L.; Pinto, L. H.; Lamb, R. A.;
Fiorin, G.; Wang, J.; DeGrado, W. F. Flipping in the Pore: Discovery of Dual Inhibitors that
Bind in Different Orientations to the WildꢀType versus the AmantadineꢀResistant S31N Mutant
of the Influenza A Virus M2 Proton Channel. J. Am. Chem. Soc. 2014, 136, 17987ꢀ17995.
31.
Wang, J.; Ma, C.; Wang, J.; Jo, H.; Canturk, B.; Fiorin, G.; Pinto, L. H.; Lamb, R. A.;
Klein, M. L.; DeGrado, W. F. Discovery of Novel Dual Inhibitors of the WildꢀType and the
Most Prevalent DrugꢀResistant Mutant, S31N, of the M2 Proton Channel from Influenza A Virus.
J. Med. Chem. 2013, 56, 2804ꢀ2812.
32.
E.; Hsieh, J. Smallꢀmolecule activation of neuronal cell fate. Nat. Chem. Biol. 2008, 4, 408ꢀ410.
33. McBriar, M. D.; Clader, J. W.; Chu, I.; Del Vecchio, R. A.; Favreau, L.; Greenlee, W. J.;
Schneider, J. W.; Gao, Z.; Li, S.; Farooqi, M.; Tang, T. S.; Bezprozvanny, I.; Frantz, D.
Hyde, L. A.; Nomeir, A. A.; Parker, E. M.; Pissarnitski, D. A.; Song, L. X.; Zhang, L. L.; Zhao,
Z. Q. Discovery of amide and heteroaryl isosteres as carbamate replacements in a series of orally
active gammaꢀsecretase inhibitors. Bioorg. Med. Chem. Lett. 2008, 18, 215ꢀ219.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 28 of 29
28
1
2
3
4
5
6
34.
Garfunkle, J.; Ezzili, C.; Rayl, T. J.; Hochstatter, D. G.; Hwang, I.; Boger, D. L.
Optimization of the central heterocycle of alphaꢀketoheterocycle inhibitors of fatty acid amide
hydrolase. J. Med. Chem. 2008, 51, 4392ꢀ4403.
7
8
9
35.
Cady, S. D.; SchmidtꢀRohr, 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, 689ꢀ692.
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
36.
Corey, E. J.; Chaykovsky, M. Dimethyloxosulfonium Methylide ((CH3)2SOCH2) and
Dimethylsulfonium Methylide ((CH3)2SCH2). Formation and Application to Organic Synthesis.
J. Am. Chem. Soc. 1965, 87, 1353ꢀ1364.
37.
Requirements for the Hydrophobic Scaffold and Polar Amine in inhibitors of M2 from Influenza
A Virus. ACS Med. Chem. Lett. 2011, 2, 307ꢀ312.
38.
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,
2646ꢀ2657.
Wang, J.; Ma, C.; Balannik, V.; Pinto, L. H.; Lamb, R. A.; Degrado, W. F. Exploring the
Duque, M. D.; Ma, C.; Torres, E.; Wang, J.; Naesens, L.; JuarezꢀJimenez, J.; Camps, P.;
39.
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 antiꢀinfluenza A M2
ion channel inhibitors. Antiviral Res. 2012, 96, 91ꢀ99.
40.
virus. Eur. J. Med. Chem. 2011, 46, 52ꢀ57.
41. Hu, W. H.; Zeng, S. G.; Li, C. F.; Jie, Y. L.; Li, Z. Y.; Chen, L. Identification of Hits as
Matrixꢀ2 Protein Inhibitors through the Focused Screening of a Small Primary Amine Library. J.
Med. Chem. 2010, 53, 3831ꢀ3834.
42.
Rev. Micro. 2012, 10, 563ꢀ574.
43. Balannik, V.; Carnevale, V.; Fiorin, G.; Levine, B.; Lamb, R.; Klein, M.; DeGrado, W.;
Pinto, L. Functional Studies and Modeling of PoreꢀLining Residue Mutants of the Influenza A
Virus M2 Ion Channel. Biochemistry 2010, 49, 696ꢀ708.
44.
Conformational and Dynamical Changes of the S31N Mutant of the Influenza M2 Proton
Channel Investigated by SolidꢀState NMR. J. Am. Chem. Soc. 2013, 135, 9885ꢀ9897.
Zhao, X.; Li, C.; Zeng, S.; Hu, W. Discovery of highly potent agents against influenza A
Nieva, J. L.; Madan, V.; Carrasco, L. Viroporins: structure and biological functions. Nat.
Williams, J. K.; Tietze, D.; Wang, J.; Wu, Y.; DeGrado, W. F.; Hong, M. DrugꢀInduced
ACS Paragon Plus Environment

Page 29 of 29
Journal of Medicinal Chemistry
29
1
2
3
4
TOC
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
ACS Paragon Plus Environment