Optimization of N-Substituted Oseltamivir Derivatives as Potent Inhibitors of Group-1 and -2 Influenza A Neuraminidases, Including a Drug-Resistant Variant

Article abstract of DOI:10.1021/acs.jmedchem.8b00929

On the basis of our earlier discovery of N1-selective inhibitors, the 150-cavity of influenza virus neuraminidases (NAs) could be further exploited to yield more potent oseltamivir derivatives. Among the synthesized compounds, 15b and 15c were exceptionally active against both group-1 and -2 NAs. Especially for 09N1, N2, N6, and N9 subtypes, they showed 6.80-12.47 and 1.20-3.94 times greater activity than oseltamivir carboxylate (OSC). They also showed greater inhibitory activity than OSC toward H274Y and E119V variant. In cellular assays, they exhibited greater potency than OSC toward H5N1, H5N2, H5N6, and H5N8 viruses. 15b demonstrated high metabolic stability, low cytotoxicity in vitro, and low acute toxicity in mice. Computational modeling and molecular dynamics studies provided insights into the role of R group of 15b in improving potency toward group-1 and -2 NAs. We believe the successful exploitation of the 150-cavity of NAs represents an important breakthrough in the development of more potent anti-influenza agents.

Full text of DOI:10.1021/acs.jmedchem.8b00929

Subscriber access provided by Technical University of Munich University Library
Article
Optimization of N-Substituted Oseltamivir Derivatives
as Potent Inhibitors of Group-1 and -2 Influenza A
Neuraminidases, Including a Drug-Resistant Variant
Jian Zhang, Vasanthanathan Poongavanam, Dongwei Kang, Chiara Bertagnin, Huamei
Lu, Xiujie Kong, Han Ju, Xueyi Lu, Ping Gao, Ye Tian, haiyong Jia, Samuel Desta, Xiao
Ding, Lin Sun, Zengjun Fang, Boshi Huang, Xuewu Liang, Ruifang Jia, Xiuli Ma, Wenfang
Xu, Natarajan Arul Murugan, Arianna Loregian, bing huang, Peng Zhan, and Xinyong Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00929 • Publication Date (Web): 02 Jul 2018
Downloaded from http://pubs.acs.org on July 2, 2018
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of their duties.

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Sciences, Shandong University
Ma, Xiuli; Institute of Poultry Science, Shandong Academy of Agricultural
Sciences
Xu, Wenfang; Shandong Univ, School of Pharmaceutical Sciences,
Shandong University, Department of Medicinal Chemistry
Murugan, Natarajan Arul; Division of Theoretical Chemistry and Biology,
School of Biotechnology, KTH Royal Institute of Technology
Loregian, Arianna; University of Padova,
huang, bing; Shandong Academy of Agricultural Sciences, Institute of
Poultry Science
Zhan, Peng; Shandong Univ,
Liu, Xinyong; School of Pharmaceutical Sciences, Shandong University,
Department of Medicinal Chemistry
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Optimization of N-Substituted Oseltamivir Derivatives as Potent
Inhibitors of Group-1 and -2 Influenza A Neuraminidases, Including
a Drug-Resistant Variant
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†
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&
Jian Zhang, Vasanthanathan Poongavanam, Dongwei Kang, Chiara Bertagnin,
‡
†
†
†
†
†
†
Huamei Lu, Xiujie Kong, Han Ju, Xueyi Lu, Ping Gao, Ye Tian, Haiyong Jia,
†
†
†
†,#
†
†
Samuel Desta, Xiao Ding, Lin Sun, Zengjun Fang, Boshi Huang, Xuewu Liang,
†
‡
†
≠
&
Ruifang Jia, Xiuli Ma, Wenfang Xu, Natarajan Arul Murugan, Arianna Loregian,
‡
,
†,
†,
Bing Huang, * Peng Zhan, * and Xinyong Liu *
†
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmaceutical Sciences, Shandong University, 44 West
Culture Road, 250012 Jinan, Shandong, PR China.
‡
Institute of Poultry Science, Shandong Academy of Agricultural Sciences, 1,
Jiaoxiao Road, Jinan, Shandong 250023, P. R. China.
&
Department of Molecular Medicine, University of Padova, via Gabelli 63, 35121
Padova, Italy
|
Department of Physics, Chemistry, and Pharmacy, University of Southern Denmark,
DKꢀ5230 Odense M. Denmark.
≠
Division of Theoretical Chemistry and Biology, School of Biotechnology, KTH Royal
Institute of Technology, Sꢀ106 91 Stockholm, Sweden.
#
The Second Hospital of Shandong University, No. 247 Beiyuan Avenue, Jinan
250033, China.
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ABSTRACT: Based on our earlier discovery of N1ꢀselective inhibitors, the
150ꢀcavity of influenza virus neuraminidases (NAs) could be further exploited to
yield more potent oseltamivir derivatives. Among the synthesized compounds, 15b
and 15c were exceptionally active against both groupꢀ1 and ꢀ2 NAs. Especially for
1
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09N1, N2, N6 and N9 subtypes, they showed 6.80ꢀ12.47 and 1.20ꢀ3.94 times greater
activity than oseltamivir carboxylate (OSC). They also showed greater inhibitory
activity than OSC towards H274Y and E119V variant. In cellular assays, they
exhibited greater potency than OSC towards H5N1, H5N2, H5N6, and H5N8 viruses.
15b demonstrated high metabolic stability, low cytotoxicity in vitro, and low acute
toxicity in mice. Computational modeling and molecular dynamics studies provided
insights into the role of R group of 15b in improving potency towards groupꢀ1 and ꢀ2
NAs. We believe the successful exploitation of the 150ꢀcavity of NAs represents an
important breakthrough in the development of more potent antiꢀinfluenza agents.
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INTRODUCTION
Influenza A virus is the pathogenic agent of seasonal, occasional, and pandemic
influenza in both humans and animals, causing acute contagious respiratory infections
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ꢀ3
that may be lifeꢀthreatening or even lethal.
According to the World Health
Organization (WHO, 2018), the annual incidence of severe illness is about 3 to 5
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million cases, with about 290,000 to 650,000 deaths. Currently, two subtypes
influenza A virus (H1N1pdm09 and H3N2) are the predominant circulating strains
4
responsible for annual/seasonal influenza epidemics. The influenza A virus has been
the causative agent of five major pandemics in the last century and the beginning of
this century: the 1918 Spanish flu (H1N1), 1957 Asian flu (H2N2), 1968 Hong Kong
flu (H3N2), 1977 Russian flu (H1N1), and 2009 swineꢀorigin pandemic flu
5ꢀ7
(H1N1pdm09).
Avian influenza virus (AIV), which belongs to the influenza A virus family, has
caused substantial economic losses throughout the world due to high bird mortality
8
and the cost of control measures to prevent its spread in poultry. In general, AIV
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1
strains have strong hostꢀspecies barriers, but nevertheless, human and mammals are
often infected with various AIV subtypes, including H5N1, H5N2, H5N6, H6N1,
1
2ꢀ15
H7N9, and H9N2 strains.
In particular, H5N1 and H7N9 (first identified in China
in March 2013) showed mortality rates of 60% and 40%, respectively, in humans.
Though their mode of transmission is not yet fully established, and there is no clear
14,16
evidence of efficient humanꢀtoꢀhuman transmission,
the worrying possibility
remains that further adaptation of AIV for personꢀtoꢀperson transmission could give
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rise to a worldwide pandemic.
Influenza A viruses contain two important surface antigenic glycoproteins,
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hemagglutinin (HA) and neuraminidase (NA). Different subtypes are named
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according to the antigenic properties of the HA and NA molecules. In terms of NA,
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influenza A viruses can be classified into N1ꢀN11 subtypes. NAs play an essential
role in the release of newly formed virions from infected cells and facilitate
1
9,20
propagation of the virus,
therefore, NAs are promising targets for antiꢀinfluenza
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1
drugs. Indeed, several neuraminidases inhibitors (NAIs) have been approved for
®
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® 23
clinical use, including oseltamivir (1 Tamiflu ), zanamivir (2 Relenza ),
®
24
® 25
laninamivir octanoate (3 Inavir ) and peramivir (4 Rapivab ) (Figure 1). Among
the approved drugs, only oseltamivir (a prodrug of oseltamivir carboxylate) (OSC) is
orally available, and it has been a firstꢀline therapy since its approval in 1999. Other
neuraminidase inhibitors are mainly administered by inhalation or intravenously due
25
to their high polarity. However, various mutants with resistance to these four drugs
2
6ꢀ29
have appeared;
H274Y) and H3N2 (E119V) subtypes confer resistance to oseltamivir without
altering susceptibility to zanamivir, which have severely limited Tamiflu’s clinical
such as the most frequent NA substitutions reported in H5N1
(
26,28,29
27
application.
For NA of H5N1ꢀH274Y mutant, Collins et al. revealed the
molecular basis of the oseltamivir resistance of His274Y, suggesting that the mutation
of histidine to a bulkier tyrosine residue could disorder the hydrophobic pocket
formed by the methylene of Glu276, thus preventing OSC from making hydrophobic
contact with the ligandꢀbinding pocket. Thus, there is an urgent need to develop
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improved NAIs for effective influenza therapy.
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Figure 1. Structures of the licensed NA inhibitors 1-4
Typically, the classic NA inhibitors were designed based on the crystal structures
of NAs. Based on the phylogenetic tree, the N1ꢀN9 subtypes can be divided into two
groups: groupꢀ1 (N1, N4, N5, and N8), and groupꢀ2 (N2, N3, N6, N7, and N9) (see
30
Supporting Information, Figure S1). For groupꢀ1 NAs (except pH1N1 (09N1)), the
crystal structures reveal that the 150ꢀloop, containing residues 147ꢀ152, adopts an
open conformation, forming the 150ꢀcavity adjacent to the active site. In contrast, no
groupꢀ2 NA has yet been crystallographically shown to have the 150ꢀloop in a
completely open conformation. As exemplified by N2, there is a key salt bridge
between the conserved residues Asp147 and His150, which was considered to lock the
150ꢀloop in the closed conformation and to control the formation of the
31,32
150ꢀcavity.
However, in contrast to the other characterized N1 subtypes, 09N1
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does not have an open 150ꢀcavity, and this surprising finding suggested that the
9N1 150ꢀloop closely resembles that of groupꢀ2 rather than groupꢀ1 NAs. Therefore,
except for 09N1, the 150ꢀcavity is an unique feature of groupꢀ1 NAs. Various
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compounds, such as Cꢀ5 aminoꢀsubstituted oseltamivir derivatives 5, 6, and 7,
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(N1ꢀselective, more potent than OSC), oseltamivir analogues 8, (N1ꢀselective, less
3
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potent than zanamivir), sialic acid derivative 9, (N1ꢀselective, less potent than
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zanamivir and sialic acid), zanamivir derivative 10, (less potent than zanamivir) and
3
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Cꢀ6 substituted oseltamivir derivatives 11 and 12 (H3N2ꢀselective) have been
discovered to target the 150ꢀcavity of NAs (Figure 2).
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Figure 2. Structures of the groupꢀ1ꢀspecific influenza NA inhibitors 5-10 and H3N2ꢀselective
inhibitors 11 and 12.
Recently, molecular dynamics (MD) simulation studies indicated that the
conformation of the 150ꢀloop in groupꢀ1 NAs could generate an even more extended
31,40,41
1
50ꢀcavity than that observed in the crystal structures.
Similarly, the flexibility
of the 150ꢀloop of 09N1 is very high, and biophysical simulations showed that the
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9N1 favors an open 150ꢀcavity conformation overall. In addition, the amino acid
sequences of the groupꢀ1 and groupꢀ2 NA 150ꢀloops show a high degree of
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similarity. The recent crystal structure of a halfꢀopen 150ꢀloop in a complex of N2
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with OSC, and several computational studies further suggested that the conformation
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of the 150ꢀloop in NAs of both groups could be easily influenced by an introduced
ligand, which might interact with the closed 150ꢀloop and lock it in an open
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conformation.
Thus, the open 150ꢀcavity may exist not only in groupꢀ1 NAs, but
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also in groupꢀ2 NAs in the physiological situation. In other words, intrinsic protein
flexibility and ligandꢀinduced protein flexibility could provide a basis for further
antiꢀinfluenza drug discovery via a structureꢀbased approach.
DESIGN OF OSELTAMIVIR DERIVATIVES
The main objective of this research was to discover potent groupꢀ1 and groupꢀ2
NAIs as candidate antiꢀinfluenza agents by exploiting the 150ꢀcavity adjacent to the
active site of influenza A viral NAs. As shown in Figure 3, the Cꢀ5 amino group and
Cꢀ6 of OSC in the NAs active site lie very close to the open 150ꢀcavity of groupꢀ1
NAs (1918H1N1, H5N1, and H5N8) and the closed 150ꢀloop of 09N1 and groupꢀ2
NAs (H3N2, H5N2, H5N6, and H7N9). The Cꢀ6 substituted oseltamivir derivatives
11 and 12 were designed to target both the active site and the 150ꢀcavity of groupꢀ1
and ꢀ2 NAs, but the results were unsatisfactory, even though they showed potent
39
H3N2ꢀselective inhibitory activity. On the other hand, Cꢀ5 aminoꢀsubstituted
oseltamivir derivatives (5, 6, and 7) were potent N1ꢀselective inhibitors, being more
34ꢀ36
potent than OSC against N1.
Moreover, at high concentration, the deprotonated
Cꢀ5 amino group of OSC exerts a significant effect on the rigid closed N2 150ꢀloop
32
and can induce halfꢀopening. So, we set out to modify the Cꢀ5 amino group of
oseltamivir, aiming to probe the groupꢀ1 NAs 150ꢀcavity and to force the unexplored
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closed 150ꢀcavity of 09N1 and groupꢀ2 NAs into open conformation.
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Figure 3. Comparison of the crystal structures of the NA subtypes used in our NA inhibition assay,
bound with OSC. Structures of N1 (1918H1N1, PDB ID: 3BEQ), 09N1 (H1N1pdm09, PDB ID:
3TI6), N2 (H3N2, PDB ID: 4GZP), N1 (H5N1, PDB ID: 2HU0), N2 (H5N2, PDB ID: 5HUK),
N6 (H5N6 PDB ID: 5HUM), N8 (H5N8, PDB ID: 2HT7) and N9 (H7N9, PDB ID: 5L15) are in
surface representation. It can be seen that 09N1 (H1N1pdm09), along with N2, N6, and N9 have a
closed 150ꢀloop, but N1 and N8 have an open 150ꢀcavity.
Our previous report revealed that structureꢀbased design of specific N1 inhibitors
targeting the activity site and 150ꢀcavity was a successful strategy for discovering
antiꢀinfluenza agents, such as 5 and 6 (lead compounds, originally numbered 20I and
32, J. Med. Chem. 2014, 57, 8445ꢀ8458), which contain hydrophobic pꢀphenyl and
pꢀ(thiophenꢀ2ꢀyl)ꢀbenzyl groups, respectively, and exhibit more potent N1ꢀselective
34
inhibition than OSC. It turned out that the substituted benzyl group is an excellent
34
privileged moiety for the 150ꢀcavity. Recent computational models of N1 have
revealed that Nꢀcontaining polar groups (including hydrazine, imineamide and amino
derivatives) and bulky moieties (including aromatic rings, heterocyclic group and
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4
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5
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5
5
6
alkene moieties) are available for hydrogen bond formation with Gly147, Val149, and
Asp151 and for van der Waals (vdW) interactions with Gln136, Asp151, and Thr439
in the 150ꢀcavity, respectively. Consequently, an aromatic ring with polar moieties
0
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43
may be sterically and electronically complementary to the 150ꢀcavity. In addition,
39
MD studies indicated that the triazoleꢀcontaining oseltamivir analogs (11 and 12,
which were designed to adapt to the 150ꢀcavity of N1 and N2, and showed higher
efficacy against H3N2 strain than H1N1 strain) could alternatively occupy the
150ꢀcavity and the catalytic site of N1 or N2, because of the short triazole group and
the rigid and large angle between the central ring and the triazole group. Even so, it
was clear that the 150ꢀcavity of groupꢀ2 could be induced to open by oseltamivir
derivatives, and Nꢀcontaining groups (triazole group) play an important role in this
process.
Our strategy here was to explore and optimize the substituted benzyl structure of
lead compound 5 or 6 and to identify structureꢀactivity relationships (SARs) that
could be utilized to improve the potency against both groupꢀ1 and ꢀ2 NAs while also
showing good potency against H5N1ꢀ274Y mutant, by introducing phenyl and
alkylꢀsubstituted Nꢀcontaining groups or Nꢀcontaining aryl groups at the paraꢀposition
of the benzyl group of 5 or 6. Thus, we designed and synthesized a series of Cꢀ5
aminoꢀsubstituted oseltamivir derivatives (Figure 4), anticipating that 1) phenyl and
alkylꢀsubstituted Nꢀcontaining groups or Nꢀcontaining aryl groups at the paraꢀposition
of the benzyl group would have sufficiently large size, sufficient length, relative
flexibility, and appropriate hydrophobicity to bind effectively to the 150ꢀcavity of
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groupꢀ1 NAs, 2) the 150ꢀcavity of 09N1 and groupꢀ2 NAs is likely to be opened and
bound by Cꢀ5 amino and substituted benzyl groups, and 3) the substituted benzyl
group may form strong interactions with the 150ꢀcavity of NAs of both groups to
increase the affinity between the compounds and the targets and thus improve the
antiꢀresistance profile. Given the importance of interactions between the substituted
benzyl groups and 150ꢀcavity, we were optimistic that these design and optimization
strategies would be fruitful.
1
1
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0
Figure 4. Further structural optimization of oseltamivir derivatives 5 and 6.
CHEMISTRY
The synthetic protocols for the new oseltamivir derivatives are outlined in
Schemes 1ꢀ6. All derivatives were synthesized by wellꢀestablished methods using
commercially available oseltamivir phosphate as the primary starting material. In
Scheme 1, oseltamivir phosphate was treated with a series of different aldehydes in
the presence of NaBH
4a-14d were hydrolyzed in the presence of NaOH aqueous solution and acidified
with hydrochloric acid aqueous solution to give the corresponding target compounds
5a-15d.
3
CN to afford the key intermediates 14a-14d. Subsequently,
1
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6
Compound 19 was synthesized according to the synthetic protocols in Scheme 2.
4ꢀThiomorpholinobenzaldehyde (17) was prepared by reaction of thiomorpholine with
4ꢀfluorobenzaldehyde (16) in the presence of potassium carbonate. The target
0
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0
compound 19 was prepared via similar procedures from 15a-15d. Compound 22 was
obtained according to the method shown in Scheme 3.
A BuchwaldꢀHartwig crossꢀcoupling reaction of the 4ꢀbromobenzaldehyde with
aniline, Nꢀmethyl or ethyl substituted aniline afforded another three aryl aldehydes
2
4a-24c, which were transformed to the corresponding target compounds 26a-26c
Scheme 4).
Compounds 29a-29b were also prepared via similar procedures from 15a-15d, as
(
depicted in Scheme 5. Lead compound 6 (originally numbered 32, J. Med. Chem.
34
2014, 57, 8445ꢀ8458) was obtained according to the method we previously reported.
All novel derivatives were fully characterized by electrospray ionization mass
spectrometry (ESIꢀMS), highꢀresolution mass spectroscopy (HRMS), proton nuclear
1
magnetic resonance ( H NMR) spectroscopy, and carbon nuclear magnetic resonance
1
3
(
C NMR) spectroscopy. The purity of all target compounds was >95% as determined
by analytical HPLC.
a
Scheme 1. Synthesis of Compounds 15a-15d
a
(
3 2
i) NaBH CN, EtOH, MeOH, rt, 60ꢀ70%; (ii) NaOH, MeOH, H O, rt, then HCl (2 mol/L),
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0ꢀ75%.
a
Scheme 2. Synthesis of Compound 19
1
1
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1
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a
(
i) DMSO, K CO , 100ꢀ120°C, 4h, 68%; (ii) NaBH CN, EtOH, MeOH, rt, 63%; (iii) NaOH,
2 3 3
2
MeOH, H O, rt, then HCl (2 mol/L), 60%.
a
Scheme 3. Synthesis of Compound 22
a
(
i) NaBH
3
2
CN, EtOH, MeOH, rt, 76%; (ii) NaOH, MeOH, H O, rt, then HCl (2 mol/L), 74%.
a
Scheme 4. Synthesis of Compounds 26a-26c
O
O
HO
O
O
R1
Br
oseltamivir
phosphate
NH
O
N
O
N
O
O
H
H
ii
R1
HN
O
iii
R1
HN
i
N
N
N
R1
2
3
24
25
26
2
3a, 24a, 25a 26a R = H; 23b, 24b, 25b, 26b R = Me; 23c, 24c, 25c, 26c R = Et.
1 1 1
a
(
2 3 2 3
i) toluene, Cs CO , Pd(OAc) , BINAP, reflux, 60ꢀ70%; (ii) NaBH CN, EtOH, MeOH, rt,
60ꢀ70%; (iii) NaOH, MeOH, H
2
O, rt, then HCl (2 mol/L), 58ꢀ70%.
a
Scheme 5. Synthesis of Compounds 29a-29b
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a
(
i) NaBH
3
2
CN, EtOH, MeOH, rt, 55ꢀ65%; (ii) NaOH, MeOH, H O, rt, then HCl (2 mol/L),
6
0ꢀ70%.
Scheme 6. Synthesis of Lead Compound 6 (originally numbered 32, J. Med.
a
Chem. 2014, 57, 8445-8458)
a
(
2 3 3 4 3
i) DMSO, K CO , Pd(PPh ) , reflux, 68%; (ii) NaBH CN, EtOH, MeOH, rt, 68%; (iii) NaOH,
MeOH, H O, rt, then HCl (2 mol/L), 74%.
2
RESULTS AND DISCUSSION
Inhibition of Influenza Neuraminidases
All the newly designed oseltamivir derivatives were screened for NAꢀinhibitory
activity using chemiluminescenceꢀbased assay with
′ꢀ(4ꢀmethylumbelliferyl)ꢀαꢀDꢀNꢀacetylneuraminic acid (MUNANA) as the
a
2
3
4
substrate. For NA inhibition screening, we chose five NAs in each group (10
influenza subtypes in total): the endemic human strains seasonal H1N1, pandemic
H1N1 (H1N1pdm09), and H3N2, which caused significant morbidity and mortality in
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humans, and avian influenza viruses (H5N1, H5N2, H5N6, H5N8, H7N9, H9N2, and
oseltamivirꢀresistant H5N1ꢀH274Y) that pose a significant threat to birds, poultry,
5ꢀ7, 12ꢀ16
mammals and humans.
In addition to the reference compounds oseltamivir
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
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5
6
7
8
9
0
carboxylate (OSC) and zanamivir, we also employed compound 6 as a reference,
34
since it has been reported to show selectivity for N1 over N2. Table 1 summarizes
the measured inhibition potencies of the synthesized compounds towards the above
NAs. It is clear that OSC showed greater inhibitory potency towards wildꢀtype
groupꢀ1 NAs (IC50 = 4.45ꢀ33.56 nM) and groupꢀ2 NAs (IC50 = 4.17ꢀ15.72 nM).
However, OSC showed weaker activity towards the H5N1ꢀH274Y mutant (IC =
50
34,44
1630 nM), as previously reported.
Impressively, several of the designed compounds had greater inhibitory potency
than the reference compounds, and in particular, 15b showed singleꢀdigit nanomolar
activity against NAs of the H1N1, H1N1pdm09, H3N2, H5N2, H5N8, H9N2 and
H7N9 strains. However, compound 15a exhibited weaker activity against NAs of
H5N1 (IC50 = 120.93 nM) and H5N6 (IC50 = 33.85 nM), showing approximately 3ꢀ
and 2ꢀfold lower potency than OSC, respectively.
The inhibitory effects of 15b and 15c bearing N,Nꢀdiethylamino and
1ꢀpyrrolidine groups, respectively, toward the nine wild NA subtypes lay within a
narrow concentration range (IC values ranging from 0.35 nM to 33.72 nM and 1.81
5
0
nM to 14.31 nM, respectively). In the case of H5N1 NA, the IC50 value of 15b was
3.72 nM, which is similar to that of OSC (IC50 = 33.56 nM). As for the other eight
wild NA subtypes, 15b turned out to be a highly potent inhibitor of both groupꢀ1 NAs
3
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5
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5
5
6
(
H1N1, IC50 = 1.16 nM; H1N1pdm09, IC50 = 0.55 nM; H5N8, IC50 = 5.34 nM) and
groupꢀ2 NAs (H3N2, IC50 = 1.05 nM; H5N2, IC50 = 0.49 nM; H9N2, IC50 = 0.35 nM;
H5N6, IC50 = 1.26 nM; H7N9, IC50 = 1.79 nM), with greater potency than OSC or
zanamivir. In addition, compound 15c exhibited better inhibition of all wildꢀtype NAs
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of groupꢀ1 (H1N1, IC50 = 2.65 nM; H1N1pdm09, IC50 = 2.31 nM; H5N1, IC50
=
=
1
4.31 nM; H5N8, IC50 = 4.06 nM) and groupꢀ2 (H3N2, IC50 = 1.81 nM; H5N2, IC50
4
.50 nM; H9N2, IC50 = 2.13 nM; H5N6, IC50 = 10.85 nM; H7N9, IC50 = 6.51 nM),
being 1.2 to 3.94ꢀfold more potent than OSC. It is worth noting that the IC50 values of
1
5b were much lower against the five NAs of groupꢀ2 (especially H7N9 NA) and
0
9N1 than against the three NAs of groupꢀ1. Indeed, the IC50 values of 15b against
NAs of H1N1pdm09, H3N2, H5N2, H5N6, H9N2 and H7N9 were approximately 8, 7,
1, 13, 13, and 6 times, and 8, 6, 27, 18, 26, and 10 times lower than those of OSC
1
and zanamivir for the same NAs, respectively. In general, 15b showed comparable or
much better inhibitory activities relative to 15c against both groupꢀ1 and ꢀ2 wildꢀtype
NAs, suggesting that the flexible N,Nꢀdiethylamino group at the Rꢀposition was more
advantageous for NAs inhibitory activity than the rigid 1ꢀpyrrolidine group.
The presence of the 1ꢀpiperidine group (15d) and 4ꢀthiomorpholine group (19)
resulted in decreased activities towards groupꢀ1 and ꢀ2 NAs (15d, IC50
=
1
6.64ꢀ165.90 nM; 19, IC = 48.38ꢀ557.77 nM) in comparison with OSC. Moreover,
5
0
neither compound displayed selectivity for groupꢀ1 or ꢀ2 NAs. Compared with 15d,
compound 19 was less potent against both groupꢀ1 and ꢀ2 wildꢀtype NAs,
demonstrating that replacing the 1ꢀpiperidine group with a 4ꢀthiomorpholine group
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resulted in reduced NAꢀinhibitory activity. Compound 22 (IC50 = 10.91ꢀ581.03 nM)
with an Nꢀacetamide group at the Rꢀposition showed significantly reduced activity
against both groupꢀ1 and ꢀ2 wildꢀtype strains, especially H5N1 (IC50 =581.03 nM),
H5N2 (IC = 31.73 nM), H5N6 (IC = 181.17 nM), H5N8 (IC = 216.53 nM) and
1
1
1
1
1
1
1
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1
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2
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2
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2
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2
2
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0
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5
0
50
50
H7N9 (IC50 = 31.47 nM) relative to 15a-15c, OSC and zanamivir. Therefore, we did
not further explore amide groups as the R group of oseltamivir derivatives.
Based on the above information and our previously established structureꢀactivity
34,43
relationships (SAR) results,
we concluded that N,Nꢀdialkylꢀsubstituted aniline
derivatives preferentially bind to the 150ꢀcavity of both groupꢀ1 and ꢀ2 NAs, and the
key factors influencing the activities are the size, flexibility and hydrophobicity of the
N,Nꢀdialkyl substituted group. Compounds with N,Nꢀdiethylamino (15b) and
1ꢀpyrrolidinyl groups (15c), which possess moderate size, flexibility, and
hydrophobicity, at the Rꢀposition enhanced the activities against both groupꢀ1 and ꢀ2
wildꢀtype NAs, while relatively bulky, rigid (15d and 19) or small (15a) substituent
groups appeared to have a negative effect on the potency, possibly because their
conformational effects impaired the binding of other structural parts of oseltamivir
derivatives to the corresponding pockets of the active site. In addition, an amide
substituent group reduced the activity, as exemplified by compound 22.
To further examine the effects of Nꢀsubstituted fragments, one of the two methyl
groups of 15a or ethyl groups of 15b was replaced with a phenyl group or a hydrogen
atom (cf. compounds 26aꢀ26c). Among them, compound 26b (Nꢀmethylaniline
derivative) exhibited the most potent activity against NAs of H3N2 and H9N2 (IC50
=
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6
5
.65 nM and 2.29 nM, respectively), being more potent than the reference compounds.
It also exhibited similar potency towards NAs of H1N1 (IC50 = 9.62 nM),
H1N1pdm09 (IC50 = 29.82 nM), H5N2 (IC50 = 62.84 nM), H5N8 (IC50 = 29.00 nM),
and H7N9 (IC = 11.37 nM) relative to OSC and zanamivir, but was less potent than
0
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9
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5
6
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9
0
50
OSC and zanamivir against NAs of H5N1 and H5N6. Nevertheless, 26a (IC50
=
27.39ꢀ599.17 nM) and 26c (IC50 = 275.3ꢀ2586 nM) showed significantly reduced
NAꢀinhibitory activity towards both groupꢀ1 and ꢀ2 wildꢀtype NAs. These results
suggest that either too large (phenyl or ethyl), or too small (hydrogen atom) or too
rigid (phenyl) Nꢀsubstituted groups are unfavorable for NAꢀinhibitory activity, in
accordance with the aboveꢀestablished SARs (i.e., relatively bulky, rigid or small
substituent groups appeared to have a negative effect on the potency).
In addition, we replaced the N,Nꢀdiethylamino group of 15b with 1Hꢀpyrrole and
1Hꢀpyrazole groups (cf. 29a and 29b). Like reference compound 6, 29a and 29b
turned out to be selective inhibitors of groupꢀ1 NAs (cf. Table 1). As per our
34
established SARs for this class of compound, 29a and 29b showed significantly
decreased inhibitory activity for groupꢀ2 NAs (H3N2, H5N2, H5N6, H9N2, and
H7N9). It is noteworthy that 29a was a much more potent inhibitor of both groupꢀ1
and 2 wildꢀtype NAs (1.17ꢀ to 15.22ꢀfold) than 29b, indicating that increasing the
number of N atoms at the Rꢀposition is not effective to improve the activity of
oseltamivir derivatives.
Collectively, apart from the identified groupꢀ1 NAsꢀselective inhibitors (29a and
29b), the NAꢀinhibitory activities of the novel oseltamivir derivatives are very
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sensitive to the size, flexibility, and hydrophobicity of the R group. Indeed, 15b and
15c with R groups of moderate size, flexibility, and hydrophobicity proved to be the
most potent NA inhibitors and exhibited increased inhibitory activities towards the
whole panel of wildꢀtype NAs, especially the groupꢀ2 NAs. Our results indicate that
N,Nꢀdiethylamino and 1ꢀpyrrolidine groups bound well with the 150ꢀcavity of
groupꢀ1 NAs, and that they are likely to induce opening of the 150ꢀloop of 09N1 and
groupꢀ2 NAs, while other structural parts interact with the active site in a similar
manner to the case of OSC.
1
1
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0
As for NA of the H5N1ꢀH274Y mutant virus, which has been reported to be
34,44,45
oseltamivirꢀresistant,
OSC showed sharply reduced potency (IC50 = 1630 nM),
which was approximately 50ꢀfold lower than that towards the wildꢀtype NA of H5N1
34,44
(IC50 = 33.56 nM); this result is in agreement with the finding of Schade and Xie.
Among these oseltamivir derivatives, nine were found to be less active or to lack
activity at the highest tested concentration of 100 ꢁM against the mutant
H5N1ꢀH274Y NA. However, it is noteworthy that compounds 15b and 15c displayed
superior activity (IC50 = 387.07 and 663.9 nM, respectively), being 4ꢀ and 2ꢀfold more
potent than OSC, respectively. Besides, two representative compounds 15b and 15c
were also performed an enzymatic assay against NA of H3N2ꢀE119V, and the results
are shown in Table 2. The NA of H3N2ꢀE119V confer resistance to oseltamivir
46
without altering susceptibility to zanamivir, which is consistent with reported data.
5b and 15c displayed robust potencies against NA of H3N2ꢀE119V, with IC50 values
of 603.83 and 288.5 nM, respectively, which were more potent than OSC. These
1
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compounds could serve as suitable lead compounds for further optimization studies
0
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Table 1. Neuraminidase (NA) Inhibition of Oseltamivir Derivatives in Chemiluminescenceꢀbased Assay
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
a
NA Enzyme-Inhibitory Assay, IC50 (nM)
Compound
R
b
c
d
e
f
g
h
i
j
k
H1N1
H1N1pdm09
group-1
H3N2
H5N1
H5N2
H5N6
H5N8
H9N2
H7N9
H5N1-H274Y
group-1
group-1
group-2
group-1
group-2
group-2
group-1
group-2
group-2
1
5a
5.06±0.04
4.09±0.13
0.55±0.03
2.31±0.03
18.61±0.68
48.38±1.54
17.31±0.17
66.72±0.83
4.11±0.13
120.93±14.59
8.89±0.43
33.85±4.48
9.53±0.56
5.74±0.61
8.41±0.74
3365.67±181.95
387.07±16.46
663.9±98.31
15b
1.16±0.01
2.65±0.09
1.05±0.01
1.81±0.07
17.15±0.83
62.59±4.55
10.91±0.47
31.12±0.83
33.72±2.95
14.31±1.80
0.49±0.06
4.50±0.09
1.26±0.08
10.85±0.73
5.34±0.67
4.06±0.38
0.35±0.003
2.13±0.08
1.79±0.17
6.51±0.12
1
5c
15d
24.92±0.20
65.93±1.82
17.49±1.96
73.48±13.87
165.90±16.09
557.77±60.10
581.03±34.54
599.17±20.71
16.64±0.77
127.63±10.79
31.73±6.92
138.23±13.11
73.82±18.22
363.57±37.51
181.17±21.68
539.03±54.17
57.46±3.01
20.74±0.12
74.18±3.22
13.66±0.30
27.39±1.47
40.46±3.83
155.90±14.72
31.47±0.18
94.20±6.08
10028.33±679.75
l
1
2
9
2
288.37±24.70
216.53±23.56
368.70±42.86
>100000
9387±717.29
2
6a
12540±1126.41
2
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6b
9.62±0.98
29.82±0.34
5.65±0.29
454.67±57.07
275.30±20.54
62.84±8.02
224.87±12.47
29.00±3.41
2.29±0.04
11.37±0.54
2586±0.29
1485.67±168.84
0
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9
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6c
361.67±24.80
697.77±57.33
473.73±31.78
1558.33±198.56
24803.33±5028.49
817.37±21.47
559.63±24.45
38083.33±2.46.62
2
9a
72.64±4.08
374.43±24.66
22.63±1.27
59.34±1.96
297±27.34
21.27±0.28
327.23±37.86
869.30±115.28
353.50±35.68
38.11±3.33
266.97±39.42
9.79±0.09
192.6±52.08
2923.67±499.44
389.10±28.76
1038.8±222.30
1322±156.69
6870±1438.31
43.89±5.57
360.70±30.71
17.87±2.85
504.10±31.13
592.80±59.80
163.57±9.58
1820.67±95.52
9123.33±968.92
1463.33±112.33
5007±242.19
29b
46686.67±7085.37
n
m
6
160±20.
OSC
5.39±0.47
5.31±0.19
4.45±0.12
4.32±0.09
7.14±0.29
6.82±0.25
33.56±3.40
9.81±0.56
5.40±0.49
15.72±2.69
22.80±1.08
7.77±1.46
7.39±0.92
4.17±0.15
9.15±0.45
8.42±0.68
1630±41.87
6.71±1.17
o
zanamivir
13.01±0.59
17.61±0.43
a
Concentration required to reduce NA activity to 50% of control NA activity (IC ). Values are the mean of three experiments, presented as the mean ± standard
50
b
c
d
e
f
g
deviation (SD).
A/PuertoRico/8/1934.
A/California/04/2009.
A/Babol/36/2005.
A/goose/Guangdong/SH7/2013.
A/Chicken/Hebei/LZF/2014.
h
i
j
k
l
m
A/duck/Guangdong/674/2014. A/goose/Jiangsu/1306/2014. A/chicken/china/415/2013. A/Anhui/1/2013. A/Anhui/1/2005. Highest tested concentration. Xie
n
o
Y. J. Med. Chem. 2014, 57, 8445–8458. Lead compound (originally numbered 32, J. Med. Chem. 2014, 57, 8445ꢀ8458). Zanamivir was found to be more potent
towards groupꢀ1 NAs (with IC50 values of 4.32ꢀ9.81 nM) than towards groupꢀ2 NAs (with IC50 values of 6.82ꢀ22.80 nM). In agreement with the results of a
47
previous study, zanamivir is more potent than OSC towards groupꢀ1 NAs but less potent than OSC towards groupꢀ2 NAs. The inhibitory effect of zanamivir on the
2
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NA (H5N1ꢀH274Y) (IC50 = 6.71 nM) is still at a very effective level.
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a
Table 2. N2 (H3N2-E119V) Inhibitory Activities of Compounds 15b and 15c
compounds
15b
15c
OSC
zanamivir
b
N2 (H3N2ꢀE119V) (nM)
603.83±32.39
288.5±26.18
817.43±98.61
33.55±1.99
a
b
Values are the mean of three independent experiments. A/Babol/36/2005 (H3N2)
Anti-Influenza-Virus Activity in Cell Culture
0
1
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5
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8
9
0
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2
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8
9
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9
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8
9
0
1
2
3
4
5
6
7
8
9
0
In order to investigate the efficacy of target compounds against influenza virus
infection, we examined all the synthesized NAIs in cellꢀbased assays that address the
cytopathic effect (CPE) of AIV infection, using A/goose/Guangdong/SH7/2013
(
H5N1) and A/goose/Jiangsu/1306/2014 (H5N8), as models for groupꢀ1
NAsꢀcontaining influenza strains, and A/Chicken/Hebei/LZF/2014 (H5N2),
A/duck/Guangdong/674/2014 (H5N6) as models for groupꢀ2 NAsꢀcontaining
influenza strains. As in the enzyme inhibition assay, OSC, zanamivir, and 6 were used
as reference compounds in parallel. The values of EC50 (antiꢀavian influenza A virus
potency) and CC50 (cytotoxicity) of the synthesized compounds are summarized in
Table 3. Notably, all the tested compounds exhibited no appreciable cytotoxicity at the
highest tested concentrations (CC50 > 200 ꢁM) in chicken embryo fibroblasts (CEFs).
In the case of the H5N1 virus, the activities of 15a (EC50 = 0.53 ꢁM), 15b (EC50
=
0.47 ꢁM) and 15c (EC50 = 0.49 ꢁM) were superior to that of OSC. In contrast, 15d,
19, 22, 26a-26c, 29a, and 29b displayed sharply reduced antiviral activity. In
particular, 19 bearing 4ꢀthiomorpholine (EC50 = 49.72 ꢁM) and 22 bearing acetamide
substituents (EC50 > 100 ꢁM) almost lacked antiꢀinfluenza potency. Overall, the
antiꢀH5N1 activities of the novel oseltamivir derivatives were consistent with the
results of the N1 (H5N1)ꢀinhibitory activities, with the exception of 15a.
As for H5N2 virus, the antiviral activities of 15b (EC = 0.012 ꢁM) and 15c
50
(EC50 = 0.063 ꢁM) were consistent with the N2 (H5N2)ꢀinhibitory activities, and
these compounds showed significantly greater activity than the reference compounds.
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In addition, 15a, 15d, 22, 29a, and 26a-26c exhibited EC50 values in the
submicromolar range, being inferior to OSC, in accordance with their less potent
inhibitory activities for NA of H5N2. Similarly, 29b and 19 showed reduced antiviral
activities (EC50 = 1.25 and 1.54 ꢁM, respectively), in accordance with their N2
1
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0
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0
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(H5N2)ꢀinhibitory activities.
Against H5N6 virus, 15b (EC50 = 0.17 ꢁM) and 15c (EC50 = 0.38 ꢁM) turned out
to be the most potent inhibitors, being 6ꢀ and 3ꢀfold more potent than OSC,
respectively. This is in line with their N6 (H5N6)ꢀinhibitory activities. Moreover, 19
also showed more potent antiviral activity (EC = 0.49 ꢁM) than OSC. The activities
5
0
of 15a (EC = 0.86 ꢁM), 15d (EC = 1.60 ꢁM), 26a (EC = 1.07 ꢁM), 26b (EC =
50
50
50
50
1.78 ꢁM) and 26c (EC50 = 0.74 ꢁM) were equivalent to that of OSC. Compound 22
(EC50 = 5.69 ꢁM), in accordance with its lack of N6 (H5N6)ꢀinhibitory activity, was
ineffective against H5N6 virus. Like 6, which showed selective antiꢀH5N1 and H5N8
virus activity, 29a and 29b showed 5ꢀ14 times lower antiꢀH5N6 potency than OSC.
More than half of the new oseltamivir derivatives in this study were found to
show improved antiꢀH5N8 virus activities in comparison with OSC and zanamivir.
Among them, 15a, 15b, 15c and 29a were highly potent antiꢀH5N8 virus agents, with
EC50 values ranging from 0.61 to 0.95 ꢁM, which are lower than those of OSC and
zanamivir. Compounds 29b and 26a showed comparable antiꢀH5N8 virus activities
relative to OSC. Interestingly, 26b and 26c exhibited the most potent activities, with
EC50 values of 0.29 and 0.31 ꢁM, respectively, being greatly superior to the reference
compounds. Although 15d and 22 displayed moderate activity in N8 (H5N8)
inhibition assay, they showed attenuated antiꢀH5N8 virus activities with EC50 = 6.78
ꢁM and 16.27 ꢁM, respectively, comparable to the reference compounds.
Unsurprisingly, 19, which displayed a considerable decrease in N8 (H5N8)ꢀinhibitory
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6
activity, was inactive toward H5N8 virus even at the highest tested concentration of
100 ꢁM.
Among the novel oseltamivir derivatives examined, 15b and 15c showed robust
antiviral activity against the viral panel, being more effective than OSC in the same
cellular assays; the EC50 values of 15b, 15c, and OSC were in the ranges of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0.012ꢀ0.61 ꢁM, 0.063ꢀ0.75 ꢁM, and 0.07ꢀ1.22 ꢁM, respectively. More importantly,
15b and 15c exhibited more potent activity towards H5N2 and H5N6 than towards
H5N1 and H5N8 viruses. This is consistent with their N1 (H5N1), N2 (H5N2), N6
(H5N6), and N8 (H5N8) inhibitory activities. It is also noteworthy that 26b and 26c
showed remarkably improved antiꢀH5N8 virus activity in the cellular system,
although they displayed only moderate N8 (H5N8)ꢀinhibitory activities. The
phenomenon that the potency of compounds can increase in a cellular environment
48
may be due to multiple factors, including synergistic inhibition, cellular metabolism,
49
proteinꢀprotein interactions, and asymmetric intracellular localization.
Table 3. AntiꢀInfluenza Virus Activity and Cytotoxicity of Oseltamivir Derivatives (CEFs)
a
EC50 values (µM) towards influenza viruses
b
Compound
15a
R
CC50
H5N1
H5N2
H5N6
H5N8
group-1
group-2
group-2
group-1
c
0.53±0.28
0.47±0.09
0.17±0.05
0.86±0.11
0.17±0.03
0.95±0.64
0.61±0.15
>200
1
5b
0.012±0.002
>200
1
5c
0.49±0.02
3.02±0.77
0.063±0.02
0.86±0.38
1.25±0.27
0.38±0.13
1.60±0.79
0.49±0.07
0.75±0.21
6.78±1.62
>200
>200
>200
1
5d
c
1
9
49.72±18.33
>100
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c
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2
>100
0.299±0.14
0.17±0.06
0.44±0.18
5.69±2.37
1.07±0.16
1.78±0.79
16.27±4.84
1.16±0.06
0.29±0.13
>200
>200
>200
2
6a
5.11±1.43
13.13±1.12
26b
26c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
23.54±6.88
0.57±0.12
0.74±0.14
0.31±0.13
>200
2
9a
2.32±0.52
10.94±2.67
0.34±0.06
0.36±0.17
1.54±0.26
2.19±0.54
14.17±5.53
9.26±2.19
9.67±1.98
0.63±0.02
1.99±0.44
0.85±0.07
>200
>200
>200
29b
d
6
OSC
0.63±0.07
0.23±0.11
0.07±0.03
0.51±0.24
1.05±0.18
0.58±0.08
1.22±0.20
3.76±1.41
>200
>200
e
Zanamivir
a
EC50: concentration of compound required to achieve 50% protection of CEF cell cultures
against influenza virusꢀinduced cytotoxicity, presented as the mean ± standard deviation (SD) and
determined by the CCKꢀ8 method.
b
CC50: concentration required to reduce the viability of mockꢀinfected cell cultures by 50%, as
determined by the CCKꢀ8 method.
c
d
Highest tested concentration. Lead compound 6 (originally numbered 32, J. Med. Chem. 2014,
5
7, 8445ꢀ8458).
e
Zanamivir displayed antiviral activity against H5N1, H5N2 and H5N6 virus with EC50 values in
the submicromolar range and against H5N8 with EC50 values in the micromolar range.
Then, we tested compounds 15b, 15c, 19, 26b, and 29a in plaque reduction
assays (PRA) with both the A/PR/8/34 (H1N1) and the A/Wisconsin/67/05 (H3N2)
strain in MDCK cells. OSC and ZAN were used as positive controls for inhibition. We
also tested the cytotoxicity of the test compounds, as well as OSC and ZAN as a
reference, in MDCK cells by MTT assays. No cytotoxic effect was observed at the
tested concentrations (250 ꢁM) for any of the compounds. In the inhibitory activity
evaluation against H1N1 and H3N2 strains, 15b exhibited the greatest inhibition, with
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EC50 values of 0.065 and 1.70 ꢁM, respectively, which are similar to those of OSC
and ZA. 15c had slightly decreased activity against H1N1 and H3N2 relative to OSC.
However, other compounds (19, 26b, and 29a) showed remarkably reduced antiviral
activity than OSC and ZA toward the two strains.
0
1
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. AntiꢀInfluenza Virus Activity and Cytotoxicity of Oseltamivir Derivatives (MDCK)
a
EC50 values (µM) towards influenza viruses
b
Compound
R
CC50
c
d
H1N1
H3N2
group-1
group-2
e
15b
0.07 ± 0.01
1.7 ± 0.6
>250
1
5c
0.22
±
±
0.01
2.35
±
±
0.2
>250
>250
1
9
4.4
1.95
0.69
1.0
19.0
25.55
29.0
1.0
6.7
8.9
26b
±
±
0.35
0.06
±
>250
>250
29a
±
OSC
0.04
0.05
±
±
0.02
0.03
0.90
0.27
±
0.14
0.06
>250
>250
Zanamivir
±
a
EC : concentration of compound required to achieve 50% protection of MDCK cell cultures
50
against influenza virusꢀplaque formation, presented as the mean ± standard deviation (SD) and
determined by the PRA method.
b
CC50: concentration required to reduce the viability of mockꢀinfected cell cultures by 50%, as
determined by the MTT method.
c
d
e
A/PR/8/34. A/Wisconsin/67/05. Highest tested concentration.
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Page 29 of 71
Journal of Medicinal Chemistry
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MOLECULAR MODELING
To further understand and characterize the interaction pattern of the synthesized
compounds with the various NAs, the most promising ligand 15b and OSC were
subjected to computational docking into an open 150ꢀloop conformation of the N1,
N2, N6, N8, N9 and 09N1 strains, followed by explicit solvent MD simulations.
Initially, both compounds were docked into the openꢀform structure of each NA using
the Glide program and the best proteinꢀligand complex (pose) was chosen as the
starting configuration for MD simulations; i.e., 12 simulations were performed in total.
Of the 20 ns production run, only trajectories from the last 5 ns (500 snapshots) were
retrieved for analysis (see the method section for more information). For each
complex, the 500 snapshots were analyzed with respect to energy convergence in
order to ensure that the proteinꢀligand complex is energetically stable (Supporting
information, Figure S2).
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