Structure-Property Relationship Studies of Influenza A Virus AM2-S31N Proton Channel Blockers

Article abstract of DOI:10.1021/acsmedchemlett.8b00336

Majority of current circulating influenza A viruses carry the S31N mutation in their M2 genes, rendering AM2-S31N as a high profile antiviral drug target. With our continuous interest in developing AM2-S31N channel blockers as novel antivirals targeting both oseltamivir-sensitive and -resistant influenza A viruses, we report herein the structure-property relationship studies of AM2-S31N inhibitors. The goal was to identify lead compounds with improved microsomal stability and membrane permeability. Two lead compounds, 10d and 10e, were found to have high mouse and human liver microsomal stability (T_1/2 > 145 min) and membrane permeability (>200 nm/s). Both compounds also inhibit both currently circulating oseltamivir-sensitive and -resistant human influenza A viruses (H1N1 and H3N2) with EC₅₀ values ranging from 0.4 to 2.8 μM and a selectivity index of >100. We also showed for the first time that AM2-S31N channel blockers such as 10e inhibited influenza virus replication at both low and high multiply of infection (10²-10⁶ pfu/mL) and the inhibition was not cell-type dependent. Overall, these studies have identified two promising lead candidates for further development as antiviral drugs against drug-resistant influenza A viruses.

Full text of DOI:10.1021/acsmedchemlett.8b00336

Subscriber access provided by UNIV OF NEWCASTLE
Letter
Structure-property relationship studies of influenza
A virus AM2-S31N proton channel blockers
Yanmei Hu, Raymond Kin Hau, Yuanxiang Wang, Peter Tuohy,
Yongtao Zhang, Shuting Xu, Chunlong Ma, and Jun Wang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00336 • Publication Date (Web): 03 Oct 2018
Downloaded from http://pubs.acs.org on October 5, 2018
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 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 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.
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 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
9
Structure-property relationship studies of influenza A virus AM2-
S31N proton channel blockers
Yanmei Hu,†,# Raymond Kin Hau,†,# Yuanxiang Wang,† Peter Tuohy,‡ Yongtao Zhang,‡ Shuting Xu,‡
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
Chunlong Ma,† Jun Wang†,*
†Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721,
United States
‡Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721, United States
#Yanmei Hu and Raymond Kin Hau contributed equally to this work
Keywords: Influenza A virus, AM2 proton channel, AM2-S31N inhibitor, microsomal stability, antiviral.
ABSTRACT: Majority of current circulating influenza A viruses carry the S31N mutation in their M2 genes, rendering AM2ꢀ
S31N as a high profile antiviral drug target. With our continuous interest in developing AM2ꢀS31N channel blockers as novel antiꢀ
virals targeting both oseltamivirꢀsensitive and –resistant influenza A viruses, we report herein the structureꢀproperty relationship
studies of AM2ꢀS31N inhibitors. The goal was to identify lead compounds with improved microsomal stability and membrane
permeability. Two lead compounds, 10d and 10e, were found to have high mouse and human liver microsomal stability (T1/2 > 145
min) and membrane permeability (>200 nm/s). Both compounds also inhibit both currently circulating oseltamivirꢀsensitive and –
resistant human influenza A viruses (H1N1 and H3N2) with EC50 values ranging from 0.4 to 2.8 µM and a selectivity index of
>100. We also showed for the first time that AM2ꢀS31N channel blockers such as 10e inhibited influenza virus replication at both
low and high multiply of infection (102 –106 pfu/ml) and the inhibition was not cell type dependent. Overall, these studies have
identified two promising lead candidates for further development as antiviral drugs against drugꢀresistant influenza A viruses.
Influenza viruses are negative sense, segmented RNA viꢀ
ruses that are the causative agents for annual influenza epiꢀ
demic and sporadic influenza pandemics.1 Despite the availaꢀ
bility of small molecule antivirals and influenza vaccines,
there is an influenza season every year. More concerning is the
emerging of influenza pandemic outbreaks that normally occur
every 10 to 20 years.2 Part of explanation for the reoccurring
influenza virus infection might be because influenza virus not
only infects human, but also many animals such as swine,
migrating birds, chicken, horse, sea lions, etc. As such, there
are multiple sources where human can contract the virus.
When healthy immunocompetent adults are infected with seaꢀ
sonal influenza viruses, the symptoms are normally mild and it
is rarely fatal.3 Therefore, it might be somewhat surprising to
learn that influenza virus infection is currently listed among
the topꢀten leading causes of deaths in the United Sates.4 The
number of influenza virusꢀrelated mortality actually surpasses
that of breast cancer. There are several factors that might conꢀ
tribute to the surprising death toll of influenza virus infection:
(1) influenza virus is transmissible through the airways and
can be quickly spread among humans. In each seasonal influꢀ
enza epidemic, an estimate of 10ꢀ20% of the population are
infected; (2) Mortality rate of influenza virus infection among
people in highꢀrisk groups is high.5 They include seniors 65
years or older, people with chronic diseases such as cardiovasꢀ
cular diseases, diabetes, and high blood pressure, and people
with compromised immune system. In such cases, influenza
virus infection normally serves as a trigger of these preꢀ
existing conditions. Overall influenza virus infection is a perꢀ
sistent public health concern that cannot be possibly ignored.
Currently there are two classes of FDAꢀapproved small
molecule influenza antivirals6: (1) adamantanes such as amanꢀ
tadine and rimantadine. They are channel blockers of the inꢀ
fluenza virus AM2 proton channel and inhibit the early stage
of viral replication by blocking the virus uncoating. (2) neuꢀ
raminidase (NA) inhibitors such as oseltamivir, zanamivir, and
peramivir. They are mimics of sialic acid and inhibit the late
stage of viral replication by blocking the virus egress. Reꢀ
sistance to both classes of drugs now necessitates the develꢀ
opment of newer influenza antivirals.7 Majority of influenza A
viruses (>95%) are now resistant to adamantanes due to the
AM2ꢀS31N mutation in their M2 genes, and CDC no longer
recommends the use of adamantanes in the prophylaxis and
treatment of influenza virus infection. Resistance to oseltamiꢀ
vir has been continuously reported, and more alarmingly, the
2007ꢀ2009 seasonal influenza virus circulating in North Amerꢀ
ican and Japan was completely resistant to oseltamivir due to
the H275Y mutation in it NA gene.7ꢀ8 To tackle these drugꢀ
resistant viruses, several drug candidates are currently in deꢀ
6, 9
velopment
which include both directꢀacting antivirals such
as the PA (polymerase acidic protein) endonuclease inhibitor
baloxavir marboxil (approved in Japan and in late stage cliniꢀ
cal trial in U.S.), polymerase inhibitor Tꢀ705 and PB2 inhibiꢀ
tor JNJꢀ63623872, as well as hostꢀtargeting antivirals such as
nitazoxanide and DAS181. In addition, a large number of othꢀ
er drug targets are also actively pursued in the earlyꢀstage of
development.10
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
To design novel antivirals that are active against both
Page 2 of 7
microsomal stability in mouse liver microsomes. Surprisingly,
all compounds except compound 4 had low microsomal stabilꢀ
ity (T1/2 < 30 min) (Table 1), precluding them from further
progression. Encouragingly, compound 4 showed good microꢀ
somal stability with a T1/2 > 145 min, and the predicted liver
clearance is less than 38.0 ml/min/kg. Therefore, compound 4
was selected as a lead compound for further optimization.
Given the promising results of compound 4, a library of
thiophene containing AM2ꢀS31N inhibitors were synthesized
and tested for channel blockage, antiviral activity, cellular
cytotoxicity and microsomal stability. Specifically, two series
of new adamantane analogs, the monoꢀaryl adamantanes (10aꢀ
10i) and the bisꢀaryl adamantanes (12aꢀ12i), were designed
and synthesized (Figure 1).
1
2
3
4
5
6
7
8
oseltamivirꢀsensitive and –resistant influenza A viruses, we
focus on targeting the influenza AM2ꢀS31N proton channel.14ꢀ
16
AM2ꢀS31N is a high profile antiviral drug target and more
than 95% of current circulating influenza A viruses carry this
mutation.17 Therefore AM2ꢀS31N channel blockers are exꢀ
pected to inhibit both oseltamivirꢀsensitive and –resistant inꢀ
fluenza A viruses. As a proofꢀofꢀconcept, we have shown that
our rationally designed AM2ꢀS31N inhibitors not only have
potent channel blockage, but also effective antiviral activity
against multiple human influenza A viruses that are in circulaꢀ
tion in recent years, including both H1N1 and H3N2 viruses
that are resistant to either amantadine, oseltamivir, or both.14ꢀ16
Importantly, the newly developed AM2ꢀS31N inhibitors
showed a higher genetic barrier to drug resistance than amanꢀ
tadine, and drug resistance only emerged under high drug seꢀ
lection pressure after several passages.18ꢀ19 To further advance
these promising lead compounds to in vivo mice model studies
and prove their in vivo antiviral efficacy, we report herein our
progress in profiling the metabolism stability of previous reꢀ
ported AM2ꢀS31N inhibitors. One candidate, compound 4,
was found to have good metabolic stability. Subsequent strucꢀ
tureꢀactivity and ꢀproperty studies led to the discovery of two
lead compounds, 10d and 10e, that have a long halfꢀlife in
mouse and human liver microsomes (T1/2 > 145 min) as well
as a high membrane permeability (Pe > 200 nm/s). Both comꢀ
pounds 10d and 10e showed improved selectivity index than
compound 4. Importantly, compounds 10d and 10e retain poꢀ
tent antiviral activity against both oseltamivirꢀsensitive and –
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
resistant human H1N1 and H3N2 influenza A viruses (EC50
=
0.4–2.8 µM), rendering them as promising candidates for the
next step in vivo metabolic and efficacy studies in mice.
We have discovered several classes of adamantlyꢀcontaining
AM2ꢀS31N inhibitors based on structureꢀguided design and
14ꢀ16, 20
medicinal chemistry optimization.11ꢀ12,
Representative
examples of most potent AM2ꢀS31N inhibitors were shown in
Table 1. They had single to submicromolar efficacy in inhibitꢀ
ing AM2ꢀS31Nꢀcontaining human influenza A viruses, includꢀ
ing current circulating H1N1 and H3N2 strains. As a first step
to profile the in vitro absorption, distribution, metabolism,
excretion, and toxicity (ADMET) properties, we first tested
their Table 1. Microsomal stability profiling of previously
reported AM2-S31N inhibitors.
Figure 1. Design and synthesis of AM2ꢀS31N inhibitors. (A)
Pharmacophore of AM2ꢀS31N inhibitors. Adamantane cage fits in
the hydrophobic cavity formed by Gly34, the ammonium linker
forms a hydrogen bond with the Asn31 side chain carbonyl, and
the substitution on the aryl group forms hydrophobic interactions
with the Val27 side chain. (B) Solution NMR structure of monoꢀ
aryl AM2ꢀS31N inhibitor 4 in complex with AM2ꢀS31N (19ꢀ49)
(PDB: 2MUV).12 (C) Solution NMR structure of bisꢀaryl AM2ꢀ
S31N inhibitor 5 in complex with AM2ꢀS31N (19ꢀ49) (PDB:
2LY0).13 (D) Synthesis of monoꢀaryl adamantanes by reductive
amination. (E) Synthesis of biꢀaryl adamantanes by Suzukiꢀ
Miyaura crossꢀcoupling reaction.
MLM 0.5
Compound structure and ID
T1/2
CLint(liver)
(min)
(ml/min/kg)
19.4
0.8
282.7
6762.7
248.3
1 (ref11)
2 (ref11)
The design was based on the AM2ꢀS31N inhibitor pharꢀ
macophore (Figure 1A), which was derived from previous
structureꢀactivity relationship studies.16 Both series of adamanꢀ
tane analogs (10aꢀ10i and 12aꢀ12i) meet the requirements of
the AM2ꢀS31N pharmacophore. Therefore, it is likely that
they will have potent channel blockage and antiviral activity.
Representative examples of monoꢀaryl and bisꢀaryl AM2ꢀ
S31N inhibitors are compounds 4 and 5, respectively, and
their binding modes to the AM2ꢀS31N channel were deterꢀ
mined by solution NMR as shown in Figures 1B and 1C, reꢀ
spectively.12ꢀ13 The monoꢀaryl adamantanes were synthesized
using our previously optimized reduction amination condition
(Figure 1D),21 and the yields range from 62% to 85%. The bisꢀ
aryl adamantanes were synthesized using the expeditious miꢀ
H
N
N
N
N
22.1
3 (ref11)
4 (ref12)
> 145
1.0
< 38.0
5290.9
5 (ref13)
6 (ref14)
H
N
Si
4.4
566
HO
ACS Paragon Plus Environment

Page 3 of 7
ACS Medicinal Chemistry Letters
crowaveꢀmediated SuzukiꢀMiyaura crossꢀcoupling reaction
with Pe greater than 200 nm/s in the EggꢀPAMPA assay, indiꢀ
cating they can passively diffuse through the transcellular
membrane. Both compounds 10d and 10e also didn’t violate
the Lipinski rule of five. The microsomal stability of comꢀ
pounds 10d and 10e was further confirmed in human liver
microsomal stability test and both compounds 10d and 10e
were found to have a long halfꢀlife with T1/2 > 145 mins. Takꢀ
en together, compounds 10d and 10e appear to be promising
leads for further development.
One of the major therapeutic advantages of AM2ꢀS31N
inhibitors is that they have no crossꢀresistance with neuramꢀ
inidase inhibitors such as oseltamivir.18 Therefore they are
expected to have potent antiviral activity against both oseltaꢀ
mivirꢀsensitive and –resistant influenza A viruses since more
than 95% of current circulating influenza A viruses have
AM2ꢀS31N mutation in their M2 genes. To prove this hypothꢀ
esis, we tested the antiviral activity of compounds 10d and 10e
against several human influenza A viruses, including both
H1N1 and H3N2 strains (Table 4). These viruses were chosen
because of their clinical relevance, and similar viruses are
circulating among humans in recent years. All viruses contain
the AM2ꢀS31N mutation in their M2 genes and are resistant to
amantadine. In addition, the four oseltamivirꢀresistant strains
encode the H275Y mutant in their neuraminidase gene which
confer to their resistance to oseltamivir. It was found that
compounds 10d and 10e inhibited all seven influenza A virusꢀ
es with EC50 values ranging from 0.4 µM to 2.8 µM.
The antiviral activity of compound 10e was further valiꢀ
dated when MDCK cells were infected with the
A/California/07/2009 (H1N1) or the A/Wisconsin/67/2005
(H3N2) virus at high MOIs, a condition which mimics the late
stage of treatment when the virus was already amplified in the
host. It was found that compound 10e significantly suppressed
the viral replication when MOI was as high as 106 pfu/ml for
both influenza strains (Figure 2). In contrast, oseltamivir carꢀ
boxylate was only effect at low MOIs (101 to 103 pfu/ml) and
was not effective at high MOIs (104ꢀ106 pfu/ml). Overall comꢀ
pound 10e showed potent inhibition against human H1N1 and
H3N2 influenza A strains at both low and high MOIs.
1
2
3
4
5
6
7
8
(Figure 1E), and the yields range from 75% to 92%.
The challenge of structureꢀproperty relationship studies is
that the ADMET properties need to be optimized without negꢀ
atively affecting the channel blockage and antiviral efficacy.
In the first step, we focus on optimizing the mouse liver miꢀ
crosomal stability because mouse is a commonly used animal
model for the evaluation of the in vivo antiviral efficacy of
influenza antivirals.22ꢀ23 Changing the bromide in compound 4
with iodide resulted in compound 10a, which had improved
antiviral activity (EC50 = 1.0 ± 0.1 µM vs 2.9 ± 0.8 µM), but
drastically reduced microsomal stability (T1/2 = 24.5 min vs
>145 min) (Table 2). The selenium analog 10b had similar
antiviral activity as the thiophene 4, but it was less metaboliꢀ
cally stable (T1/2 = 32.1 min vs >145 min). Replacing the adaꢀ
mantane in 4 with ringꢀexpanded adamantane gave compound
10c, which had increased antiviral activity (EC50 = 0.6 ± 0.1
µM vs 2.9 ± 0.8 µM), but drastically reduced microsomal
stability (T1/2 = 9.1 min vs >145 min). In contrast, replacing
adamantane in compound 4 with hydroxylꢀadamantane yielded
compound 10d that not only had improved antiviral activity
(EC50 = 1.1 ± 0.2 µM vs 2.9 ± 0.8 µM), but also high microꢀ
somal stability (T1/2 >145 min). Encouraged by this result, we
then synthesized the iodide analog 10e. It was found that the
iodide analog 10e retained the potent antiviral activity and
high microsomal stability (EC50 = 0.9 ± 0.1 µM, T1/2 >145
min). Both compounds 10d and 10e also had an improved
selectivity index than compound 4. Two additional bromothiꢀ
ophene analogs 10f and 10g had reduced antiviral activity
compared to compound 10d, and compound 10f was also less
metabolically stable than 10d (T1/2 = 100.8 min vs >145 min).
The bromothiazole analogs 10h and 10i had drastically reꢀ
duced channel blockage and antiviral activity, showing less
than 40% channel inhibition at 100 µM. For the bisꢀaryl adaꢀ
mantane analogs 12aꢀ12i, only compounds 12b and 12f had
comparable antiviral activity as compound 10d. The antiviral
EC50 values of 12b and 12f were 2.1 ± 0.2 µM, and 2.5 ± 0.8
µM respectively. However both 12b and 12f had reduced seꢀ
lectivity index than compound 10d, therefore they were not
further pursued. All other bisꢀaryl compounds (12a, 12c, 12d,
12e, 12g, 12h, and 12i) had significantly reduced antiviral
activity as shown by the values of percentage plaque forꢀ
mation at 10 µM. All compounds (10aꢀ10i and 12aꢀ12i) were
also tested against the wildꢀtype (WT) AM2 channel, and
compounds 4, 10a, and 10c were found to have potent channel
blockage against AM2ꢀWT (>77% inhibition at 100 µM).
Compound 10b had moderate activity (56.1 ± 0.8% inhibition
at 100 µM), and all other compounds were inactive. In sumꢀ
mary, through the structureꢀproperty relationship studies, we
identified two lead compounds 10d and 10e that had potent
channel blockage, antiviral activity, a high selectivity index,
and optimal microsomal stability.
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 membrane permeability of the two lead compounds
10d and 10e was further profiled (Table 3). First, the memꢀ
brane permeability and oral absorption of compounds 10d and
10e were predicted by the Schrödinger Glide QikProp proꢀ
gram, and both compounds were predicted to have high memꢀ
brane permeability and oral absorption (Table 3). Next, to
experimentally determine their membrane permeability, comꢀ
pounds 10d and 10e were tested in the parallel artificial memꢀ
brane permeability assay (PAMPA), which is commonly used
as an in vitro model of passive, transcellular permeation. Both
compounds 10d and 10e showed high membrane permeability
Figure 2. Antiviral activity of compound 10e in inhibiting
A/California/07/2009 (H1N1) and A/Wisconsin/67/2005 (H3N2)
viruses at different MOIs (101ꢀ106 pfu/ml). Confluent MDCK
cells were infected with either A/California/07/2009 (H1N1) virus
o
(A) or the A/Wisconsin/67/2005 (H3N2) virus (B) for 1h at 4 C
follow by 1h at 37 oC. Avicel overlay containing 10 µM of comꢀ
pound 10e was added to the cell and the plate was incubated for
46 h and 56 h for A and B, respectively. Cells were stains with
crystal violet dye. The images shown were representative results
from
two
repeats.
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 4 of 7
1
2
Table 2. Channel blockage, antiviral activity, cytotoxicity, and mouse liver microsomal stability of AM2-S31N inhibitors.
3
4
TEVC assaya
(% S31N channel
inhibition)
TEVC assaya
(% WT channel
inhibition)
EC50 (µM)
A/California/07/2009
(H1N1)b
% plaque forꢀ
CC50 (µM)
T 1/2
(min)
CLint(liver)
(ml/min/kg)
Structure/Compound ID
mation at 10 µMb
MDCK cellsc
5
6
7
8
76.3 ± 2.5
78.5 ± 2.4
70.6 ± 2.2
72.6 ± 1.4
77.2 ± 4.3
91 ± 1.6
3.2 ± 0.5
2.9 ± 0.8
1.0 ± 0.1
2.2 ± 1.9
0.6 ± 0.1
123.2 ± 8.6
70.2 ± 3.6
82.1 ± 5.2
43.1 ± 2.8
>145
24.5
32.1
9.1
<38.0
223.6
170.7
603.6
4
1.8 ± 0.3
10a
10b
10c
9
56.1 ± 0.8
86.5 ± 0.3
0
0
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
64.7 ± 3.5
75.6 ± 1.2
56.6 ± 1.3
48.3 ± 0.5
11.4 ± 1.1
3.2 ± 1.5
6.5 ± 1.4
1.9 ± 0.9
3.9 ± 1.2
2.5 ± 1.9
31.4 ± 0.5
31.3 ± 0.5
1.1 ± 0.2
0.9 ± 0.1
3.1 ± 0.3
5.1 ± 0.6
238.1 ± 2.1
317.4 ± 12.0
380.7 ± 24.9
363.7 ± 18.3
>145
>145
100.8
N.T.
<38.0
<38.0
54.5
10d
10e
10f
N.T.
10g
10h
36.0 ± 0.4
3.9 ± 0.0
108.0 ± 1.8
N.T.
N.T.
N.T.
N.T.
33.5 ± 1.2
86.1 ± 1.2
84.6 ± 1.2
2.7 ± 0.9
17.9 ± 3.2
0.3 ± 0.2
102.1 ± 0.4
58.5 ± 3.2
27.9 ± 0.7
N.T.
N.T.
N.T.
2.8
N.T.
1939.0
N.T.
10i
12.16 ± 1.11
2.1 ± 0.2
19.5 ± 2.0
80.5 ± 5.5
12a
N.T.
12b
12c
37.6 ± 0.6
74.5 ± 1.1
0
0
67.5 ± 1.9
44.1 ± 1.1
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
12d
12e
73.4 ± 1.8
78.7 ± 1.3
0
0
47.7 ± 1.2
20.4 ± 1.4
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
2.5 ± 0.8
65.2 ± 5.7
12f
12g
12h
12i
4.4 ± 2.4
0
0
74.0 ± 0.5
40.5 ± 1.0
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
N.T.
68.3 ± 1.2
64.3 ± 2.4
90.3 ± 3.4
0
52.0 ± 0.7
0
N.T.
N.T.
100
N.T.
1.0
N.T.
O
N
H
N
11.2 ± 1.3
0.3 ± 0.1
5290.9
S
5
aValues represent the mean of three independent measurements ± standard deviation. Compounds were tested at 100 µM concentraꢀ
tion. Antiviral activity was tested with the A/California/07/2009 (H1N1) virus, which contains the AM2ꢀS31N mutant, in plaque
b
c
assay. Values represent the mean of two independent measurements ± standard deviation. Cytotoxicity was tested by incubating
MDCK cells with compounds for 48 h and the cells were stained with neutral red.24 N.T. = not tested.
ACS Paragon Plus Environment

Page 5 of 7
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
7
8
Table 3. PK predictions, membrane permeability, physiochemical properties, and mouse microsomal stability of lead com-
pounds 10d and 10e.
EggꢀPAMPA
Permeability
(nm/s)
Human liver microꢀ
some
stability T1/2 (min)
Compound strucꢀ
ture
Predicted values by Glide
QikPropa
Lipinski rule of Mouse liver microsome
fivea
stability T1/2 (min)
Cacoꢀ2: 892 nm/s
MDCK: 2163 nm/s
(<25 poor, >500 great)
% human oral absorption in
GI: 100 (<25% is poor)
M.W. = 342.3
HBD = 2
HBA = 3
252.814
>145
>145
cLogP = 3.69
10d
Cacoꢀ2: 892 nm/s
MDCK: 2374 nm/sec
(<25 poor, >500 great)
% human oral absorption in
GI: 100 (<25% is poor)
9
M.W. = 389.3
HBD = 2
HBA = 3
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
204.422
>145
>145
cLogP = 3.80
10e
aMembrane permeability and cLogP were calculated by the Schrödinger Glide QikProp program.
Table 4. Antiviral activity of compounds 10d and 10e in inhibiting oseltamivir-sensitive and –resistant influenza A viruses.
A/California/
07/
2009 (H1N1)
EC50
A/Switzerland/9
715293/2013
(H3N2)
A/North Caroꢀ
lina/29/2009
(H1N1)
A/Wisconsin/67/
2005 (H3N2)
EC50
A/Washington/
29/2009 (H1N1)
EC50
A/Texas/04/20
09 (H1N1)
EC50
A/Denmark/52
8/2009 (H1N1)
EC50
CC50
MDCK
cells
Compound strucꢀ
tures
EC50
EC50
Amantadineꢀresistant (AM2ꢀS31N)
Oseltamivirꢀsensitive
Amantadineꢀresistant (AM2ꢀS31N)
Oseltamivirꢀresistant (H275Y)
1.1 ± 0.2
µM
0.5 ± 0.1
µM
1.4 ± 0.1
µM
0.7 ± 0.1
µM
0.6 ± 0.1
µM
2.8 ± 0.2
µM
2.0 ± 0.3
µM
238.1 ± 2.1
µM
10d
10e
0.9 ± 0.1
µM
0.7 ± 0.1
µM
0.8 ± 0.1
µM
0.4 ± 0.1
µM
0.6 ± 0.1
µM
1.1 ± 0.2
µM
0.7 ± 0.1
µM
317.4 ±
12.0 µM
Oseltamivir Carꢀ
boxylate
2.5 ± 0.5
nM
2.4 ± 0.3 nM
12.3 ± 5.2 nM
> 200 nM
> 200 nM
>200 nM
> 200 nM
> 20 µM
To test whether the antiviral efficacy of compound 10e is
cellꢀtype dependent, we performed viral titer reduction assay
using the A/California/07/2009 (H1N1) virus and the A549
cells. In this assay, confluent A549 cells were infected with
the A/California/07/2009 (H1N1) virus at MOI of 0.001, and
the cell culture supernatant was collected at 24, 48, and 72
hours post infection and the viral titer was quantified by
plaque assay. Compared with the DMSO control, treatment
with 10 µM of compound 10e significantly reduced the viral
titers of 1.8, 2.1, and 2.9 log10 unites at 24, 48, and 72 hrs,
respectively (Figure 3). In comparison, oseltamivir carboxꢀ
ylate only delayed viral replication and showed no effect at 72
hpi under similar drug selection pressure (10ꢀfold of EC50 valꢀ
ue).
phene group facing towards the Nꢀterminal of the channel. The
hydroxyl group from 10e forms a hydrogen bond with the
backbone amide carbonyl from one of the helixes, and the
ammonium from 10e forms another hydrogen bond with the
side chain amide carbonyl from the neighboring helix. The
iodothiophene substitution forms hydrophobic interactions
with the V27 side chain methyls. Overall, the docking pose of
compound 10e is similar to that of compound 10e as shown in
the solution NMR structure (PDB: 2MUV) (Figure 1b).
Figure 4. Docking model of compound 10e in the transmembrane
domain of AM2ꢀS31N (PDB: 2LY0) 13. The transparency of the
front helix was set as 0.7 for clarity. Docking was performed usꢀ
ing Schrödinger Glide standard precision.
Drug discovery is a lengthy and expansive endeavor,25
and lead compounds can fail in any step during the early preꢀ
clinical development phase or the later human clinical trials.
Therefore, it is essential to provide additional backup comꢀ
pounds with favorable PK properties for the following in vivo
animal and human studies. Herein we report our progress of
optimizing the mouse microsomal stability and cell membrane
permeability of thiopheneꢀcontaining AM2ꢀS31N inhibitors.
Starting from a promising lead compound 4, we were able to
identify two compounds 10d and 10e with improved antiviral
efficacy and selectivity index. The optimized lead compounds
Figure 3. Antiviral activity of compound 10e in inhibiting
A/California/07/2009 (H1N1) virus replication in A549 cells.
Confluent A549 cells were infected with A/California/07/2009
(H1N1) virus at MOI of 0.001. Supernatant was collected at 24,
48, and 72 hours post infection and the viral titer was quantified
by plaque assay. The results shown were from two repeats.
The binding of compound 10e in the AM2ꢀS31N channel
was modeled by the Schrödinger Glide standard precision
docking program. In the energy minimized docking pose (Figꢀ
ure 4), compound 10e fitted inside the channel with the thioꢀ
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
Page 6 of 7
Obuchi, M.; Odagiri, T.; Tashiro, M., A twoꢀyear survey of the
10d and 10e retained high mouse liver microsomal stability
(T1/2 > 145 mins), had favorable membrane permeability in the
PAMPA assay (Pe > 200 nm/s) as well as a high selectivity
index (SI > 100). As a demonstration of the therapeutic value
of AM2ꢀS31N inhibitors, compounds 10d and 10e were found
to have potent antiviral potency against several oseltamivirꢀ
sensitive and ꢀresistant human influenza A viruses. Compound
10e was also able to inhibit A/California/07/2009 (H1N1) and
A/Wisconsin/67/2005 (H3N2) at MOIs ranging from 102 to
106 pfu/ml, and the antiviral activity of compound 10e was
further confirmed in human A549 cell line. Taken together,
the potent antiviral efficacy, a high selectivity index, a long
halfꢀlife in mouse liver microsomes, and a high membrane
permeability of the identified lead compounds 10d and 10e
warrant their further development as orally bioavailable influꢀ
enza antivirals. Indeed, several FDAꢀapproved oral drugs such
as rivaroxaban, chlorothen, brotizolam, and lornoxicam simiꢀ
larly contain halogenꢀdeactivated thiophene,26 which reassures
continuous development of this series of compounds.
oseltamivirꢀresistant influenza A(H1N1) virus in Yamagata, Japan and the
clinical effectiveness of oseltamivir and zanamivir. Virol J 2010, 7, 53.
9. Koszalka, P.; Tilmanis, D.; Hurt, A. C., Influenza antivirals currently in
lateꢀphase clinical trial. Influenza Other Respir Viruses 2017, 11 (3), 240ꢀ
246.
10. Loregian, A.; Mercorelli, B.; Nannetti, G.; Compagnin, C.; Palu, G.,
Antiviral strategies against influenza virus: towards new therapeutic
approaches. Cell Mol Life Sci 2014, 71 (19), 3659ꢀ3683.
11. Li, F.; Ma, C.; Hu, Y.; Wang, Y.; Wang, J., Discovery of Potent
Antivirals against AmantadineꢀResistant Influenza A Viruses by Targeting
the M2ꢀS31N Proton Channel. ACS Infect Dis 2016, 2 (10), 726ꢀ733.
12. 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 (52), 17987ꢀ17995.
13. 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 drugꢀ
resistant S31N mutant of the M2 ion channel of influenza A virus. Proc.
Natl. Acad. Sci. U. S. A. 2013, 110 (4), 1315ꢀ1320.
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
14. Hu, Y.; Wang, Y.; Li, F.; Ma, C.; Wang, J., Design and expeditious
synthesis of organosilanes as potent antivirals targeting multidrugꢀresistant
influenza A viruses. Eur J Med Chem 2017, 135, 70ꢀ76.
ASSOCIATED CONTENT
Supporting Information
15. Li, F.; Hu, Y.; Wang, Y.; Ma, C.; Wang, J., Expeditious Lead
Optimization of IsoxazoleꢀContaining Influenza
A Virus M2ꢀS31N
Inhibitors Using the SuzukiꢀMiyaura CrossꢀCoupling Reaction. J Med
Chem 2017, 60 (4), 1580ꢀ1590.
16. Li, F.; Ma, C.; DeGrado, W. F.; Wang, J., Discovery of Highly Potent
Inhibitors Targeting the Predominant DrugꢀResistant S31N Mutant of the
Influenza A Virus M2 Proton Channel. J Med Chem 2016, 59 (3), 1207ꢀ
1216.
17. Dong, G.; Peng, C.; Luo, J.; Wang, C.; Han, L.; Wu, B.; Ji, G.; He, H.,
Adamantaneꢀresistant influenza a viruses in the world (1902ꢀ2013):
frequency and distribution of M2 gene mutations. PLoS One 2015, 10 (3),
e0119115.
18. Ma, C.; Zhang, J.; Wang, J., Pharmacological Characterization of the
Spectrum of Antiviral Activity and Genetic Barrier to Drug Resistance of
M2ꢀS31N Channel Blockers. Mol Pharmacol 2016, 90 (3), 188ꢀ198.
19. Musharrafieh, R.; Ma, C. L.; Wang, J., Profiling the in vitro drugꢀ
resistance mechanism of influenza A viruses towards the AM2ꢀS31N
proton channel blockers. Antiviral Res 2018, 153, 10ꢀ22.
20. Wang, Y.; Hu, Y.; Xu, S.; Zhang, Y.; Musharrafieh, R.; Hau, R. K.;
Ma, C.; Wang, J., In Vitro Pharmacokinetic Optimizations of AM2ꢀS31N
Channel Blockers Led to the Discovery of SlowꢀBinding Inhibitors with
Potent Antiviral Activity against DrugꢀResistant Influenza A Viruses. J
Med Chem 2018, 61 (3), 1074ꢀ1085.
21. 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 (7), 2804ꢀ2812.
22. Hu, Y.; Musharrafieh, R.; Ma, C.; Zhang, J.; Smee, D. F.; DeGrado,
W. F.; Wang, J., An M2ꢀV27A channel blocker demonstrates potent in
vitro and in vivo antiviral activities against amantadineꢀsensitive and ꢀ
resistant influenza A viruses. Antiviral Res 2017, 140, 45ꢀ54.
23. Smee, D.; Barnard, D., Methods for Evaluation of Antiviral Efficacy
Against Influenza Virus Infections in Animal Models. In Antiviral
Methods and Protocols, Gong, E. Y., Ed. Humana Press: 2013; Vol. 1030,
pp 407ꢀ425.
The Supporting Information is available free of charge on the
ACS Publications website.
Synthesis procedures, characterization of compounds; antiviral
and cytotoxicity assay; electrophysiological assay; mouse microꢀ
somal stability assay; membrane permeability assay.
AUTHOR INFORMATION
Corresponding Author
* Email: junwang@pharmacy.arizona.edu. Tel: +1 (520)ꢀ626ꢀ
1366.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script. ‡These authors contributed equally.
Funding Sources
This research is supported by the startup funding from the Univerꢀ
sity of Arizona and the NIH grant AI119187 to J. W.
ABBREVIATIONS
WT, wild type; DMEM, Dulbecco’s modified eagle medium;
MDCK, Madin–Darby Canine Kidney; TEVC, twoꢀelectrode
voltage clamps.
REFERENCES
1. Webster, R. G.; Monto, A. S.; Braciale, T. J.; Lamb, R. A., Textbook of
Influenza. Wiley: 2013.
2. http://www.flu.gov/pandemic/history/. Accessed on 09/28/2018.
3.https://www.cdc.gov/flu/professionals/acip/clinical.htm. Accessed on
09/28/2018.
4.https://www.cdc.gov/nchs/fastats/leadingꢀcausesꢀofꢀdeath.htm. Accessed
on 09/28/2018.
5.https://www.cdc.gov/flu/about/disease/high_risk.htm. Accessed on
09/28/2018.
6. Shaw, M. L., The Next Wave of Influenza Drugs. ACS Infect Dis 2017,
3, 691ꢀ694.
7. Hurt, A. C., The epidemiology and spread of drug resistant human
influenza viruses. Curr Opin Virol 2014, 8, 22ꢀ29.
8. Matsuzaki, Y.; Mizuta, K.; Aoki, Y.; Suto, A.; Abiko, C.; Sanjoh, K.;
Sugawara, K.; Takashita, E.; Itagaki, T.; Katsushima, Y.; Ujike, M.;
24. Repetto, G.; del Peso, A.; Zurita, J. L., Neutral red uptake assay for the
estimation of cell viability/cytotoxicity. Nat Protoc 2008, 3 (7), 1125ꢀ
1131.
25. DiMasi, J. A.; Grabowski, H. G.; Hansen, R. W., Innovation in the
pharmaceutical industry: New estimates of R&D costs. J Health Econ
2016, 47, 20ꢀ33.
26. Scott, K. A.; Njardarson, J. T., Analysis of US FDAꢀApproved Drugs
Containing Sulfur Atoms. Top Curr Chem (Cham) 2018, 376 (1), 5.
6
ACS Paragon Plus Environment

Page 7 of 7
ACS Medicinal Chemistry Letters
TOC
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
7
ACS Paragon Plus Environment