Discovery of novel 1-phenyl-cycloalkane carbamides as potent and selective influenza fusion inhibitors

Article abstract of DOI:10.1016/j.bmcl.2010.04.141

A novel class of HA inhibitors (4a) was identified based on ligand similarity search of known HA inhibitors. Parallel synthesis and further structural modifications resulted in 1-phenyl-cyclopentanecarboxylic acid (4-cyano-phenyl)-methyl-amide 4t as a potent and selective inhibitor to phylogenetic H1 influenza viruses with an EC₅₀ of 98 nM against H1N1 A/Weiss/43 strain and over 1000-fold selectivity against host MDCK cells.

Full text of DOI:10.1016/j.bmcl.2010.04.141

Bioorganic & Medicinal Chemistry Letters 20 (2010) 3507–3510
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of novel 1-phenyl-cycloalkane carbamides as potent and selective
influenza fusion inhibitors
a
a
a
a
b
b
Guozhi Tang a, , Zongxing Qiu , Xianfeng Lin , Wentao Li , Lei Zhu , Shaohua Li , Haodong Li ,
*
Lisha Wang a, Li Chen a, Jim Z. Wu a, Wengang Yang a
a Roche R&D Center China, Shanghai 201203, China
b WuXi Apptec Co., Ltd, Shanghai 200131, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel class of HA inhibitors (4a) was identified based on ligand similarity search of known HA inhib-
itors. Parallel synthesis and further structural modifications resulted in 1-phenyl-cyclopentanecarboxylic
acid (4-cyano-phenyl)-methyl-amide 4t as a potent and selective inhibitor to phylogenetic H1 influenza
viruses with an EC50 of 98 nM against H1N1 A/Weiss/43 strain and over 1000-fold selectivity against host
MDCK cells.
Received 2 April 2010
Revised 28 April 2010
Accepted 30 April 2010
Available online 5 May 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Influenza A
Fusion inhibitor
Hemagglutinin
Influenza or flu is a highly contagious respiratory illness caused
by a group of enveloped RNA viruses, whose genomes are com-
posed of seven or eight single-stranded RNA segments.1 There
are three types of influenza viruses, named A, B, and C, that cause
seasonal flu and account for significant morbidity, mortality, and
economic loss worldwide.2 Among them, influenza A viruses infect
many species including Homo sapiens, evolve rapidly through ge-
netic shift and drift, and cause periodical epidemics and global pan-
demics.3 Recently, a pandemic influenza A H1N1 swine flu strain
emerged from Mexico in 2009, arguably as a result of the genetic
reassortment of human, avian, and swine influenza viruses before
overcoming transmission barriers and adapting fitness in human.4
So far it has infected millions of people around the world and caused
many human deaths. In the face of the persistent threat of human
influenza infections and the potential of highly pathogenic avian
influenza H5N1 to unleash another pandemic once inter-human
transmission becomes possible, there is an urgent medical demand
to come up with new and effective anti-influenza agents.5
zanamivir, which target viral neuraminidase, and proton channel
M2 blockers amantadine and rimantadine.7 These four drugs are
effective upon prompt administration after infection or through
prophylactic use, but possess various degree of undesirable CNS
side effects. More importantly, the drug-resistance issues have
emerged in the clinic.8
The membrane fusion process is an early event in the influenza
virus life cycle.9 Hemagglutinin (HA) is a trimeric protein that an-
chors to the viral membrane and mediates host cell recognition,
endocytosis, and the fusion of virus envelope with the host cell
endosome membrane. Each HA monomer is expressed as a single
polypeptide precursor HA0 and proteolytically cleaved during viral
maturation to HA1 and HA2, which are linked by a disulfide bond.
Highly conserved among all 16 HA subtypes, the HA2 section con-
stitutes the core fusion machinery in the stem region which under-
goes significant conformational rearrangements upon acidification
in endosomal compartments. Several small molecule HA inhibitors
such as 1–3 have been identified as potent anti-influenza agents
that bind to HA and prevent low-pH triggered conformational
changes (Fig. 1).10–12 Recently, two independent studies reported
Besides activities to develop anti-influenza antibodies and new
vaccines, there have been significant efforts devoted to identifying
small molecule drugs for the prophylaxis and treatment of influ-
enza infections.6 Of the 11 influenza A viral proteins, many are
essential for viral replication and are potential targets for the
development of anti-influenza drugs. Examples of anti-influenza
small molecules that are in clinical use include oseltamivir and
O
OH
O
O
OH
N
H
N
O
OH
Cl
O
1
2 (LY-180299)
3 (Triperiden)
* Corresponding author. Tel.: +86 21 38954910x3350; fax: +86 21 50790291.
E-mail address: gordon.tang@roche.com (G. Tang).
Figure 1. The structures of HA inhibitors 1–3.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.04.141

3508
G. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3507–3510
that HA2 stem-targeting antibodies can effectively inhibit influ-
enza membrane fusion and neutralize the H5N1 avian virus as well
as other seasonal flu strains.13,14 It is anticipated that small mole-
cule HA inhibitors that bind to the conserved stem region of HA2
would have a similar anti-influenza effect.
4a negatively affected RBC hemolysis by binding to HA and inter-
fering with crucial conformational changes of HA normally trig-
gered by a low-pH environment. This assumption was further
verified in an HA trypsin sensitivity assay. In this assay, HA protein
was isolated from viral preparations after cleavage with bromelain.
The purified HA, in the presence or absence of compounds, was
subjected to a brief low-pH treatment followed by trypsin diges-
tion.20 It has been well-demonstrated that the low-pH triggered
conformational changes of HA expose its internal trypsin cleavage
sites and render HA sensitive to trypsin. Compound 4a, like other
known HA stabilizers, protected isolated HA from trypsin-induced
degradation (Fig. 2, lane 8). Taken together, the CPE assay data and
mechanism of action studies indicate that 4a inhibits the virus fu-
sion step by binding to HA and stabilizing the pre-fusion HA
structure.
Due to its promising anti-influenza potency, compound 4a was
chosen for further medicinal chemistry optimizations. A fast paral-
lel synthesis route via coupling of sterically hindered acyl chlorides
with amines was used for structure–activity relationship (SAR)
studies on scaffold 4 (Scheme 1). In a few instances, N-alkylation
reaction was carried out for some of the compound synthesis.
Early modifications indicated that fusing additional rings onto
the piperidine of 4a was generally not tolerated. For instance, com-
pound 4c with the 1,2,3,4-tetrahydro-quinolinyl group was inac-
tive in the CPE assay (Table 2). Attempts to replace the
piperidine with other versatile primary amines also gave inactive
compounds, such as anilide derivative 4d. Surprisingly, when the
amide NH was alkylated, two of the compounds (4e and 4f)
showed submicromolar potency in the CPE assay with an EC50 of
Besides compounds 1–3, a number of structurally versatile HA
inhibitors have been reported. However, many of these compounds
turned out to be promiscuous and may not be true fusion inhibi-
tors.15 Herein we report the identification of a novel class of HA
inhibitors through ligand similarity-based virtual screening. In this
study, nine known HA inhibitors including 1–3 were selected as
the basis for an MOS (maximum overlapping set)16 similarity
search against the Roche collection of about 1 million compounds.
The top scored hits were identified, clustered, and filtered through
in silico profiling and analyses. Nearly 2000 compounds arising
from this search were evaluated for their prevention of viral cyto-
pathic effect (CPE) to Madin–Darby canine kidney (MDCK) cells,
which were infected with the influenza A/Weiss/43 strain.17
A
few compounds showed positive cell protection in the primary
screening, and their anti-viral activities were confirmed by titrat-
ing multiple doses of the compounds in the CPE assays. The mech-
anism of action of the positive hits was further analyzed and
confirmed using a red blood cell (RBC)-based hemolysis assay
and an in vitro trypsin-catalyzed HA proteolysis assay.18
Based on the results of a primary single dose CPE screening and
the secondary multiple dose confirmation assays, two closely re-
lated compounds 4a and 4b were found to effectively protect
MDCK cells from cytopathic effects caused by influenza virus rep-
lication with an EC50 of 2.1 and 27.9 lM, respectively (Table 1). As
a fact, they are among the many hits of MOS similarity search
based on 3-(1-phenyl-cyclohexyl)-propionic acid, which was re-
ported as an anti-influenza fusion inhibitor.19 Compound 4a, an
amide of piperidine was over 10 times more potent than its mor-
pholine analog 4b. Notably, 4a was not significantly cytotoxic to-
0.24 and 0.52 lM, respectively. While the exact role of the N-
methyl group is not clear, N-methyl substitution was predicted
to change the dihedral angle between the amide bond (O@CAN)
and the aniline moiety (NAC@C) by about 26°, whereas in the case
of 4d, the amide and phenyl ring are almost coplanar. Additional
changes on N-substitution with both hydrophobic and hydrophilic
groups confirmed that N-substitution is important to control the
orientation of the phenyl group. Among many N-substituted deriv-
atives synthesized, the N-ethyl analog 4g and the N-hydroxyethyl
analog 4h showed similar anti-viral activities with that of 4e.
Given the preliminary SAR we obtained and the promising po-
tency of 4e, we decided to carry out systemic structural modifica-
tions to further improve the anti-viral potency and cytotoxicity
profile of this class of compounds. In the studies, the R1 phenyl
ward MDCK cells (CC50 > 100 lM), indicating a good selectivity
window between its anti-viral activity and general cytotoxicity.
To confirm that these compounds indeed target influenza hem-
agglutinin protein and are able to block virus fusion as we ex-
pected, their inhibitory effects on the hemolysis of chicken red
blood cells (RBCs) were tested. In this assay, fresh chicken RBCs
were collected and incubated with influenza A/Weiss/43 viruses.
After the binding of HA to host receptors on the surface of chicken
RBC, a brief acidification (pH ꢀ5) triggered an irreversible confor-
mational change of HA protein, and resulted in pore formations
on the RBC membrane and subsequent release of hemoglobin from
RBCs to the supernatant. As summarized in Table 1, 4a inhibited
1
2 3 4 5 6 7 8 9
the hemolysis of RBCs with an IC50 of 2.2 lM, but did not have
an effect on hemagglutination, in which only viral HA-receptor
binding is involved (data not shown). This result suggested that
HA1
HA2
Table 1
Biological activities of compounds 1, 2, and 4a–b
O
N
N
O
O
4a
4b
Compound
EC50
(l
M)/CPEa
CC50
(l
M)b
IC50 (l
M)/hemolysisc
1
2
4a
4b
0.06
0.89
2.1
>50
67
>100
>100
0.53
3.2
2.2
Figure 2. SDS–PAGE analysis of trypsin sensitivity assay showing 1, 4a, and 4t
protected purified HA from trypsin digestion. Lane 1, purified HA; lane 2, HA treated
with trypsin without a prior acidification step; lane 3, acidified HA without trypin
treatment; lane 4, trypsin only (overloaded); lane 5, DMSO control; lane 6, ribavirin
27.9
NA
a
Compound concentration to inhibit 50% of cytopathic effect. Values are the
mean of triplicate experiments.
(10
compound 4t (10
trypsin sensitivity assay. Positions of HA1 and HA2 are marked.
l
M); lane 7, compound 1 (10
lM); lane 8, compound 4a (10 lM); lane 9,
b
Compound concentration to inhibit 50% of MDCK cell growth relative to control.
l
M). Lanes 5–9 included all steps and components in a typical
c
Half maximal inhibition concentration to prevent hemolysis of chicken RBCs.

G. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3507–3510
3509
Y
R3
Y
N
R3
N
Y
Cl
Amine
MeI, NaH
R2
R2
R1
R1
R1
when R3 = H
O
O
O
4a-n
Scheme 1. A general synthetic route for HA inhibitors 4a–v.
4o-v (R3 = Me)
Table 2
Influenza inhibitory activities of compounds 4c–u against A/Weiss/43 strain
R3
N
Y
R2
R1
O
4c-u
a
b
Compound
Y
R1
R2
R3
EC50
(l
M)
CC50
24
(lM)
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–CH2–
–(CH2)2–
–(CH2)4–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
–(CH2)3–
H
H
H
H
H
H
1,2,3,4-Tetrahydroquinolinyl
>100
>100
0.24
0.45
0.26
0.58
2.26
0.19
0.75
1.86
0.27
>10
0.18
0.46
0.73
16.4
0.080
0.098
2.12
Ph
Ph
c-Hexyl
Ph
Ph
Ph
Ph
Ph
Ph
Ph
H
Me
Me
Et
–(CH2)2OH
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
>100
52
21
56
79
20
21
69
49
24
3-CF3
4-Cl
4-OMe
4-Cl
4-Cl
4-Cl
H
H
H
H
H
Ph
7.4
23
17
2-Cl-Ph
3-Cl-Ph
3-CN-Ph
3-Pyridinyl
4-Cl-Ph
4-CN-Ph
4-CF3-Ph
56
>100
20
>100
21
H
H
4u
Me
a
Values are mean of at least duplicate experiments.
Cytotoxicity in MDCK cells.
b
substitution was generally not well tolerated except for small sub-
stituents at the para-position (4j–k). The cyclopentane group was
found to be very important for anti-viral activity, as none of the
ring-opened analogs we synthesized was active. We also looked
into the relationship between the anti-viral activity and the size
of aliphatic ring. Except for the cyclobutane derivative 4m, which
exhibited an EC50 equivalent to that of 4e, all other ring expan-
sion/contraction analogs were much less active in the CPE assay
(4l and 4n in Table 2). These results indicate that the orientation
of the two phenyl rings and the cyclopentane moiety is crucial
for effective interaction with the HA protein. To further improve
the anti-viral potency, structural elaborations of 4e on the aniline
phenyl group were carried out. The SAR indicates that 2,4-substitu-
tion of the phenyl group is preferred for better activity in the CPE
assay, while meta-substitution is detrimental to the activity. Struc-
tural modifications at the para-position yielded highly potent HA
inhibitors, such as 4s and 4t21, with an EC50 value below 100 nM
against the A/Weiss/43 strain. In addition, viral specificity studies
showed that 4s and 4t had similar anti-viral potency against an-
other group 1 influenza strain A/PR/8/34 (H1N1) (data not shown),
suggesting that they may be broadly active against other group 1
influenza viruses. The cytotoxicity studies showed that 4t with a
fully optimize the anti-viral activity and DMPK properties of this 1-
phenyl-cycloalkane carbamide scaffold.
In line with other previously reported HA inhibitors including 1
and 2, all compounds reported in Table 2 were specific for group 1
influenza virus based on our limited viral specificity tests. For
example, they were inactive against influenza A/Hongkong/8/68
virus, a H3N2 strain that belongs to the group 2 influenza viruses.
This selectivity between group 1 and 2 viruses is probably due to
the structural difference at the putative binding site in the HA stem
region between the two groups of viruses, as revealed by the co-
crystal studies of tert-butyl hydroquinone (TBHQ) and H3 HA pro-
tein complex.22 TBHQ inhibits the H3 and H14 strains specifically
by binding to a site located at the stem region which is not found
in the similar range of HAs of group 1 influenza viruses. It is clear
that co-crystal structural studies of HA with the current leads will
shed light on our understanding of the anti-flu specificity and ben-
efit future structural optimizations that could broaden the activity
of this series of compounds. Nevertheless, considering that both
current pandemic H1N1 swine flu and highly pathogenic H5N1
avian influenza are group 1 influenza viruses and that the vast
majority of influenza infections are caused by group 1 influenza
viruses, highly potent and effective HA group 1 specific inhibitors
like the ones described here should be a valuable addition to our
arsenal in battling with the vicious influenza infections.
4-cyano substitution had over 1000-fold selectivity (CC50/EC50
)
against MDCK cells. Overall, compound 4t is about 20-fold more
potent than the preliminary hit 4a in the cell-based anti-viral as-
says. In the subsequent trypsin sensitivity test, 4t was able to sta-
bilize the HA protein and prevented trypsin mediated HA digestion
more effectively than 4a, which is constant with its improved anti-
viral activity in the CPE assay (Fig. 2, lane 9). Efforts are ongoing to
In summary, we report a ligand-based similarity approach to
identify a novel class of anti-influenza inhibitors that bind to the
HA protein and prevent its low-pH triggered conformational
change and thus inhibit the membrane fusion process. Structural
modifications of an initial hit resulted in a lead molecule, 1-phe-

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G. Tang et al. / Bioorg. Med. Chem. Lett. 20 (2010) 3507–3510
13. Sui, J.; Hwang, W.; Perez, S.; Wei, G.; Aird, D.; Chen, L.; Santelli, E.; Stec, B.;
Cadwell, G.; Ali, M.; Wan, H.; Murakami, A.; Yammanuru, A.; Han, T.; Cox, N. J.;
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nyl-cyclopentanecarboxylic acid (4-cyano-phenyl)-methyl-amide
4t, that exhibits high activity against influenza A/Weiss/43 strain
(H1N1) with an EC50 of 98 nM in the CPE assay and greater than
1000-fold selectivity against host MDCK cells. The identification
of this series of molecules provides a good starting point for further
optimization and development of influenza fusion inhibitors for
treatment of flu.
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Acknowledgments
19. Calacino, J. M.; Hatch, S. D.; Hornback, W. J.; Muesing, M. A.; Munroe, J. E.;
Staschke, K. A.; Tang, J. C. PCT Int. Appl. WO9728802, 1997.
The authors acknowledge Xihan Wu, Jason C. Wong, Hongying
Yun, and Lu Gao for many helpful discussions. The authors also
thank Yongguo Li, Wenzhi Chen, and Qingshan Gao for their ana-
lytical assistance.
20. In
a trypsin sensitivity assay, 6 lg of purified HA was incubated with
compounds at 31 °C for 15 min. Then the pH of the solution was adjusted to
ꢀ5.0 to trigger HA conformational changes. After neutralization with Tris
buffer, the solution was treated with 4 lg of trypsin at 37 °C for 30 min. The
extent of trypsin digestion of HA was revealed on a 10% SDS–PAGE gel that was
stained with Coomassie blue G-250.
References and notes
21. As an example, synthesis of 4t: To a solution of 1-phenyl-cyclopentanecarb-
oxylic acid (190 mg, 1.0 mmol) in 5 mL of CH2Cl2 was added 0.5 mL of SOCl2.
The mixture was heated to reflux for 1 h before the solvent and excess SOCl2
were removed under reduced pressure. The residue was dissolved in 10 mL of
ClCH2CH2Cl. To this solution was added 4-aminobenzonitrile (130 mg,
1.1 mmol) and N-methylmorpholine (0.2 mL). After refluxed for 2 h, the
reaction mixture was cooled down and washed with 10 mL of HCl (1 N) and
saline. The organic layer was dried over anhydrous Na2SO4 and concentrated.
The residue was dissolved in 10 mL of DMF, to which was added NaH (60 mg,
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iodomethane (2.0 mmol) was added. After stirred at rt for 4 h, the reaction
mixture was evaporated and worked up with water and ethyl acetate. From the
concentrated organic phase, 4t was isolated by preparative RP-HPLC (column:
C18), using water and acetonitrile as eluents. 1H NMR (MeOD, 400 MHz), 7.59
(d, 2H, J = 8.4 Hz), 7.32–7.22 (m, 3H), 7.12–7.06 (m, 4H), 3.08 (s, 3H), 2.44–2.37
(m, 2H), 1.98–1.96 (m, 2H), 1.77–1.62 (m, 4H).
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