Design and synthesis of constrained bicyclic molecules as candidate inhibitors of influenza A neuraminidase

Article abstract of DOI:10.1371/journal.pone.0193623

The rise of drug-resistant influenza A virus strains motivates the development of new antiviral drugs, with different structural motifs and substitution. Recently, we explored the use of a bicyclic (bicyclo[3.1.0]hexane) analogue of sialic acid that was designed to mimic the conformation adopted during enzymatic cleavage within the neuraminidase (NA; siali-dase) active site. Given that our first series of compounds were at least four orders of magnitude less active than available drugs, we hypothesized that the new carbon skeleton did not elicit the same interactions as the cyclohexene frameworks used previously. Herein, we tried to address this critical point with the aid of molecular modeling and we proposed new structures with different functionalization, such as the introduction of free ammonium and guanidinium groups and ether side chains other than the 3-pentyl side chain, the characteristic side chain in Oseltamivir. A highly simplified synthetic route was developed, starting from the cyclopropanation of cyclopentenone and followed by an aziridination and further functionalization of the five-member ring. This allowed the efficient preparation of a small library of new bicyclic ligands that were characterized by enzyme inhibition assays against influenza A neuraminidases N1, its H274Y mutant, and N2. The results show that none of the new structural variants synthesized, including those containing guanidinium groups rather than free ammonium ions, displayed activity against influenza A neuraminidases at concentrations less than 2 mM. We conclude that the choice and positioning of functional groups on the bicyclo[3.1.0]hexyl system still need to be properly tuned for producing complementary interactions within the catalytic site.

Full text of DOI:10.1371/journal.pone.0193623

RESEARCH ARTICLE
Design and synthesis of constrained bicyclic
molecules as candidate inhibitors of influenza
A neuraminidase
1
,2
3
1
4
Cinzia Colombo *, Črtomir Podlipnik , Leonardo Lo Presti , Masahiro Niikura , Andrew
2
1
J. Bennet , Anna Bernardi
1
Università degli Studi di Milano, Dipartimento di Chimica, Milano, Italy, 2 Department of Chemistry, Simon
Fraser University, Burnaby, British Columbia, Canada, 3 University of Ljubljana, Faculty of Chemistry and
Chemical Technology, Ljubljana, Slovenia, 4 Faculty of Health Sciences, Simon Fraser University, Burnaby,
British Columbia, Canada
a1111111111
a1111111111
a1111111111
a1111111111
a1111111111
* cinzia.colombo@unimi.it
Abstract
The rise of drug-resistant influenza A virus strains motivates the development of new anti-
viral drugs, with different structural motifs and substitution. Recently, we explored the use
of a bicyclic (bicyclo[3.1.0]hexane) analogue of sialic acid that was designed to mimic the
conformation adopted during enzymatic cleavage within the neuraminidase (NA; siali-
dase) active site. Given that our first series of compounds were at least four orders of
magnitude less active than available drugs, we hypothesized that the new carbon skele-
ton did not elicit the same interactions as the cyclohexene frameworks used previously.
Herein, we tried to address this critical point with the aid of molecular modeling and we
proposed new structures with different functionalization, such as the introduction of free
ammonium and guanidinium groups and ether side chains other than the 3-pentyl side
chain, the characteristic side chain in Oseltamivir. A highly simplified synthetic route was
developed, starting from the cyclopropanation of cyclopentenone and followed by an azir-
idination and further functionalization of the five-member ring. This allowed the efficient
preparation of a small library of new bicyclic ligands that were characterized by enzyme
inhibition assays against influenza A neuraminidases N1, its H274Y mutant, and N2. The
results show that none of the new structural variants synthesized, including those contain-
ing guanidinium groups rather than free ammonium ions, displayed activity against influ-
enza A neuraminidases at concentrations less than 2 mM. We conclude that the choice
and positioning of functional groups on the bicyclo[3.1.0]hexyl system still need to be
properly tuned for producing complementary interactions within the catalytic site.
OPEN ACCESS
Citation: Colombo C, Podlipnik Č, Lo Presti L,
Niikura M, Bennet AJ, Bernardi A (2018) Design
and synthesis of constrained bicyclic molecules as
candidate inhibitors of influenza A neuraminidase.
PLoS ONE 13(2): e0193623. https://doi.org/
1
0.1371/journal.pone.0193623
Editor: Joseph J Barchi, National Cancer Institute
at Frederick, UNITED STATES
Received: December 18, 2017
Accepted: February 14, 2018
Published: February 28, 2018
Copyright: © 2018 Colombo et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The research leading to these results
received funding from the People Programme of
the European Union’s Seventh Framework
Programme by a Marie Curie Outgoing Fellowship
to Dr. Cinzia Colombo under REA grant agreement
PIOF-GA-2012-327579. Dr. Črtomir Podlipnik
acknowledges for support of Slovenian Research
Agency via research program P1-0201. The
Introduction
Influenza A viruses are the most virulent human pathogens among the three influenza types
A, B, C. The virus uses its neuraminidases (sialidases, NA), expressed on the surface of viral
envelope, for mobility through the mucus in the respiratory tract and for spreading the
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Molecules as candidate inhibitors of influenza A neuraminidase
funders had no role in study design, data collection
and analysis, decision to publish, or preparation of
the manuscript.
infection.[1–2] After invasion and replication through the host cell machinery, the budded
virions are anchored to sialic acid (NeuAc) residues on the host cell membrane via interac-
tion with viral hemagglutinin (HA). The viral NA, at this point, cleaves the sialic acid resi-
dues from the anchored glycoconjugates and releases new virus particles. Antiviral drugs,
like oseltamivir 1,[3] zanamivir 2,[4] and peramivir 3[5] (Fig 1) have been developed based
on an understanding of the neuraminidase mechanism of action, by mimicking sialic acid
undergoing cleavage in the binding site. For retaining sialidases, the glycosylated enzyme
intermediate generated in the catalytic pocket is subjected to both glycosylation and degly-
cosylation via transition states (TS) that have an oxacarbenium ion character and feature a
distorted six-membered ring (Fig 2).[6–9] Oseltamivir (1, Fig 1) uses a cyclohexene ring in
place of the sugar pyran to mimic this distortion. The ring is substituted at both C4 and C6
with an amino group, replacing NeuAc hydroxyl groups, and at C-5 with a 3- pentyl ether
chain in place of NeuAc glycerol side chain. Zanamivir (2, Fig 1) conserves both the NeuAc
pyran ring and glycerol side chain at C6, but is modified at C4, where the hydroxyl group is
replaced with a guanidino group. Peramivir (3, Fig 1), built on a cyclopentane skeleton,
maintains the guanidino group and other key elements essential for NA binding.
Competing interests: The authors have declared
that no competing interests exist.
Molecular modeling studies suggest that the Michaelis complex between influenza NA and
4
its substrate forces the pyran ring into a B2 or a S conformation (Fig 2, B boat shown),
,5
2
2,5
6
[
10] whereas a S skew-boat conformation has been proposed for bacterial sialidases.[8] The
2
introduction of a double bond into the six-membered ring of 1 and 2 has been used as a gen-
eral strategy to mimic the flattened geometry of the enzymatic TS. Recently, we synthesized
bicyclo[3.1.0]hexane analogues 4 (Fig 1)[11] based on the hypothesis that these molecules
should also provide the ring distortion required to mimic the TS structure (Fig 2).
The synthetic approach to access these derivatives involved a photochemical pyridine ring
contraction followed by a Johnson-Corey-Chaykovsky cyclopropanation, allowing systematic
variation of the relative stereochemistry of the scaffold’s stereocenters.[11] The compounds
displayed ’slow-binding’ time-dependent inhibition of N1 and N2 sialidases with IC50 values
in the micromolar range, i.e. four orders of magnitude less efficient than 1–3. The strongest
inhibition exhibited by these compounds (IC 10 μM) was observed with compound 4a
50
(R = 4-phenylbenzyl, Fig 1).[11] Despite the low activity observed, the results provided proof
of principle for the potential of the bicyclo[3.1.0]hexane scaffold in NA inhibition and offered
some insight into the requirements for the configuration and substitution patterns of con-
strained cyclopropyl sialic acid analogues.
To further develop the structure activity relationship of this new scaffold, we synthesized a
second set of compounds in order to analyze the effect of replacing the amino group of 4 with a
larger guanidium moiety and probe the role of the ether side chain (Fig 3, compounds of gen-
eral formula 5). Indeed, the introduction of a guanidium group as a substituent of the dihydro-
pyran ring was a critical feature in the design of zanamivir.[4] The guanidino unit in zanamivir
and peramivir engages in hydrogen-bonding interactions that do not occur in the oseltamivir-
,
neuraminidase complex, as revealed by available cocrystal structures.[4] [5] In particular, the
guanidino group in zanamivir and peramivir is involved in charge-based interactions and salt
bridges with residues Glu 119 and Glu 227 (N2 numbering), through the replacement of a water
molecule. These additional interactions allowed speculation about the lack of resistance of some
oseltamivir resistant strains toward inhibitors containing the guanidino function [12] and led to
the synthesis and evaluation of NA inhibitors with mixed features from oseltamivir and zanami-
,
,
vir.[13] [14] [15]
It is now well known that the 3-pentyl ether side chain of oseltamivir is a major cause for
resistance: neuraminidases must undergo a significant rearrangement to form a pocket for
oseltamivir binding and this movement can be affected by mutations, such as H274Y.[16]
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 1. Sialic acid (α configuration), oseltamivir 1, zanamivir 2, peramivir 3 and the bicyclo[3.1.0]hexane scaffold 4.
https://doi.org/10.1371/journal.pone.0193623.g001
Resistance to oseltamivir is a growing concern,[17] also considering that permissive secondary
mutations have enabled the evolution of resistant strains.[18] The 3-pentyl ether moiety is a
key element in the design of oseltamivir, but there is no reason to assume that it would be opti-
mal on the bicyclo[3.1.0]hexane framework. Preliminary modeling data on the complex betw-
een N1 and compound 4 (R = H, Fig 1) had suggested that simultaneous binding of isopentyl
group and amine cannot be achieved from this scaffold.
In the current study, to carry out the synthesis of new derivatives 5, a new, much simplified
synthetic route for the bicyclo[3.1.0]hexane framework was adopted, starting from cyclopropa-
nation of cyclopentenone followed by aziridination and further functionalization of the five-
membered ring. This strategy allowed the efficient preparation of a small library of 10 bicyclic
ligands (5, Fig 3), densely functionalized. These compounds were tested against various influ-
enza A neuraminidases.
Results and discussion
The results obtained in our previous campaign highlighted 4a (R = 4-phenylbenzyl, Fig 1) as
the most active stereoisomer of the series.[11] Docking using Schr o¨ dinger’s Induced Fit Dock-
ing (IFD) protocol[19] suggested that compound 4a could make all the relevant contacts in the
carboxylate binding pocket of N1 and that its phenyl-benzyl moiety extended into the 150 cav-
ity, contributing non-polar interactions to the stability of the complex (Fig 4). On the other
hand, the distance between the charged amino moiety of 4a and the side chain of Glu119 is too
large (about 5 Å) to establish a solid salt bridge.
As observed during the development of zanamivir, [4] some of these issues could be
solved by increasing the size and surface of the basic group from an ammonium ion to a
guanidinium group. According to IFD calculations, introduction of a guanidinium group
þ
in the scaffold (compounds 5, R’ ¼ NHCðNH Þ ) is expected to provide better orientation
2
2
of the ligand within the binding site. As can be seen from Fig 5, the guanidino group
should be optimally exposed toward the side chains of Glu119 and Asp151 forming salt
Fig 2. Sialic acid ring distortion during catalysis and mimic 4 in its predicted conformation.
https://doi.org/10.1371/journal.pone.0193623.g002
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 3. Compounds of general formula 5, described in this paper.
https://doi.org/10.1371/journal.pone.0193623.g003
þ
bridge interactions. We also observe that 3-pentyl moiety of 5A (R’ ¼ NHCðNH Þ
2
2
R = CH(CH CH ) ) fits nicely into the pocket surrounded by Ser246, Arg224, Glu277,
2
3 2
Glu278 and Asn294 (Fig 5A). On the other hand, the substitution of the 3-pentyl moiety
þ
with an ethyl group (Fig 5B, compound 5B with R’ ¼ NHCðNH Þ and R = CH CH ) does
2
2
2
3
not give maximal stabilization for hydrophobic interactions within the pocket mentioned
above. The introduction of a dihydroxypropoxy ether moiety, similar to the glycerol side
chain of NeuAc and zanamivir, is expected to produce beneficial interactions within the
þ
binding pocket of N1 (Fig 5C, compound 5C with R’ ¼ NHCðNH Þ and R = CH CH
2
2
2
(
OH)(CH OH)). Compound 5C is interesting because it contains all of zanamivir’s phar-
2
macophoric groups attached to the bicyclic scaffold. Indeed, the interaction diagrams of
þ
compound 5C (R’ ¼ NHCðNH Þ and R = CH CH(OH)(CH OH)) and zanamivir are very
2
2
2
2
similar. The major difference is in the contacts of the guanidino moiety which in the case
of zanamivir is calculated to engage in an additional contact with Glu227.
With these docking hypotheses in hand, we focused our efforts on developing a direct pro-
tocol for the synthesis of new derivatives 5, with the aim of comparing the effect of different
substitutions of the bicyclo[3.1.0]hexane framework on NA inhibitory activity. The synthetic
strategy was based on the cyclopropanation of cyclopentenone 6 with ethyl-(dimethylsulfura-
nylidene)-acetate (EDSA), generated in situ from the corresponding sulfonium bromide 7 and
,
DBU in CHCI at r.t. (Fig 6).[20] [21] The cyclopropane 8 was obtained as a racemic mixture
3
in 85% yield. The second double bond was introduced in the five-membered ring in 78% yield
by transforming the ketone in the corresponding silylenolether (LHMDS, TMSCl), which was
Fig 4. Induced fit docking of ligand 4a (R = 4-phenylbenzyl) with N1 neuraminidase (source: pdb 2HU0).
https://doi.org/10.1371/journal.pone.0193623.g004
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 5. Docking of ligand 5 with various substitutions in N1 neuraminidases (sources: pdb 2HU0 and 2HU4). (A) Docking of 5A
þ
þ
); (C) Docking of 5C R’ ¼ NHCðNH2Þ2þ
(
R’ ¼ NHCðNH Þ R = CH(CH
2
CH
3
)
2
); (B) Docking of 5B (R’ ¼ NHCðNH Þ and R = CH
2 3
CH
2
2
2
2
2 2
and R = CH CH(OH)(CH OH).
https://doi.org/10.1371/journal.pone.0193623.g005
then oxidized to the enone 9 with Pd(OAc) .[22] The aziridination of 9 was inspired by asym-
2
,
metric catalytic aziridination protocols[23] [24] that make use of N-Boc-sulfonylamide 10[25]
and chiral bisamines, but was actually performed with achiral bis-amino ligand 11, to afford
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 6. Synthesis of the key aziridine intermediate 12 and aziridine opening reaction.
https://doi.org/10.1371/journal.pone.0193623.g006
12 in 73% yield. Compared to our previous route,[11] this approach afforded the key aziridine
intermediate in high yields and in only three synthetic steps, avoiding the use of photochemical
reactions that were difficult to scale-up to the required levels.
Opening of the aziridine catalyzed by Copper (II) salts[26] with a variety of alcohols allowed
us to introduce variability at the ether function in the final molecules (Fig 6). Primary alcohols
reacted in good yields giving one major diastereoisomer (13a-d) in good to acceptable yields.
Reaction with furfuryl alcohol generated many impurities, mainly due to cross-reactivity of the
furan ring, and product 13e was not isolated. Isopropanol did not react with 12, even after pro-
longed heating and addition of more copper triflate. The reaction with 3-pentanol produced a
complex mixture of products: opening of the aziridine took place from both faces of the ring
and at both positions 3 and 4 (Figure A in S3 File). Elimination of 3-pentyl ether from the pri-
mary aziridine opening product was also observed, as described previously for this type of
molecules.[27] Different Lewis acids (BF ÁEt O, Sn(OTf) , Zn(OTf) , Ti(OEt) , MgCl ) and
3
2
2
2
4
2
reaction conditions (increasing temperature and concentration) did not improve the desired
reactivity. Attempts to convert the N-Boc aziridine 12 to N-acetyl aziridine, in order to reduce
the steric hindrance of the substrate, failed. Despite all efforts, the reaction with 3-pentanol
never afforded yields above 10% and was not scalable to an adequate level to complete the syn-
thesis of final compounds bearing the 3-pentyl ether chain.
Compounds 13a-d were then subjected to removal of N-Boc protection and nitrogen acety-
lation to give 14a-d (Fig 7). The ketone in position 2 was converted to the corresponding
amines through reductive amination (ammonia, Ti(OEt) , NaBH )[28] followed by N-Boc
4
4
protection, to afford the differentially protected bis-ammino esters 15a-d. Removal of the pro-
tecting groups from 15a-d (NaOH, then TFA; Fig 7) afforded amino acids 16a-d in quantita-
tive yields.
Compound 15c was crystallized (acetone, 4˚C) allowing the determination of the relative
configuration of the products (Fig 8) and the final targets. The compound is chiral and crystal-
lizes in the centric space group P21/c as a racemate, with one molecule per asymmetric unit
(for details see Sup. Info). Fig 8 shows the absolute configuration of the stereocentres for the
arbitrary choice of one enantiomer. The configurational descriptors of the stereogenic centres
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 7. Synthesis of amino acids 16a-d.
https://doi.org/10.1371/journal.pone.0193623.g007
are either C1 (R), C2 (S), C3 (R), C4 (R), C5 (S) and C6 (S) or C1 (S), C2 (R), C3 (S), C4 (S),
C5 (R) and C6 (R) (see Fig 8 for the atom numbering).
Two significant hydrogen bond (HB) donors are present, namely the amide N1–H1 and
N2–H2 groups (Fig 8). These functions are employed to set up supposedly strong H-bonded
pillars along the monoclinic b axis, which involve the carbamate and the acetamide carbonyl
groups (Fig 8, Figure A and B in S1 File, Table A in S1 File). Cumbersome substituents, on the
other hand, are allocated in the free space between adjacent pillars and extend mainly in the
(a,c) plane (Fig 8).
Removal of the t-butylcarbamate from 15a-d (TFA; Fig 9) also set the stage for installation
of the guanidino group on the bicyclic framework. To this end, the resulting amines were
treated with N,N'-bis(tert-butoxycarbonyl)thiourea 17 and Mukayiama’s reagent, following
Lipton’s guanylation protocol,[29] to afford the corresponding N,N'-bis(tert-butoxycarbonyl)
guanidines 18a-d in satisfactory yields. Removal of the protecting groups (NaOH, then TFA;
Fig 9) afforded the target guanidinium derivatives 19a-d.
To introduce the dihydroxypropoxy ether side-chain modification, the allyl ether in 15c
and 18c was dihydroxylated (OsCl , Me NO, acetone/water) to afford glycerol 15e and 18e, as
a diastereomeric mixture at the newly generated stereogenic center (Fig 10). The use of Sharp-
3
3
less methodology with AD-mix α and β did not result in better stereoselectivity, as revealed by
Fig 8. X-ray structure of 15c at room temperature, crystallized from acetone (4˚C). Right: crystal packing viewed along a is
shown, with hydrogen atoms not involved in hydrogen bonds (red dashed lines) omitted for clarity. Left: asymmetric unit of 15c,
with the atom numbering scheme. Thermal ellipsoids are drawn at the 30% probability level.
https://doi.org/10.1371/journal.pone.0193623.g008
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 9. Synthesis of guanidines 19a-d.
https://doi.org/10.1371/journal.pone.0193623.g009
1
3
C-NMR of final compounds obtained with the three different methods. A derivative with a
hydroxy group in position 2 was also prepared (Fig 10, compound 16f), to systematically com-
pare the effect of ammonium and guanidinium substitution on the compounds activities. By
adding 4 equivalents of Ti(OEt) in the reduction of the ketone with ammonia in EtOH, alco-
4
hol 15f formed preferentially (y = 93%), as revealed by NMR analysis (Supp. Info) and was
then subjected to dihydroxylation to afford compound 15g. Protecting group removal afforded
final compounds 16e, 19e and 16f (Fig 10).
Enzyme inhibition and cell culture assays
Initially, we measured K values of the three NAs (A/Anhui/1/2005 H5N1 wild-type and
m
H274Y mutant, and A/Chicken/HongKong/G9/1997 H9N2) for the MUNANA substrate
in MES buffer (32.5 mM 2-(N-morpholino)ethanesulfonic acid, 4 mM CaCl2, pH 6.5).
The K values were 12.8 ± 1.9 μM for N1 (H5N1), 26.5 ± 1.7 μM for N1 H274Y mutant,
m
and 70 ± 6.8 μM for N2 (H9N2). The enzymatic activity of the N1 H274Y mutant appears
to be comparable to that of wild type viruses with compromised ability to bind oseltami-
vir.[16]–[30] Indeed, before starting the evaluation of new compounds, we performed
control experiments with oseltamivir free acid, MS-257 (Fig 11, a known nanomolar
inhibitor of influenza A neuraminidase)[13] and compound 4a (compound belonging to
the first series of compounds and previously tested).[11] The results are listed in Table 1.
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Molecules as candidate inhibitors of influenza A neuraminidase
Fig 10. Synthesis of the dihydroxypropoxy side chain derivatives 16e, 19e and 16f.
https://doi.org/10.1371/journal.pone.0193623.g010
The inhibition constant (K ) of oseltamivir free acid for the mutant N1 H274Y showed an
i
increase of 580-fold, compared to the wild-type. The K of MS-257 showed an increase of
i
9
5-fold. This is consistent with previously reported data, which showed MS-257 to be
more active against an Oseltamivir-resistant virus H1N1 with the H274Y mutation com-
pared to oseltamivir free acid.[31] MS-257 was also recently shown to be less susceptible
to drug resistance toward a mutant group 2 influenza Virus NA.[14] Compound 4a which
Fig 11. Compounds used for control experiments for enzyme inhibition.
https://doi.org/10.1371/journal.pone.0193623.g011
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Molecules as candidate inhibitors of influenza A neuraminidase
Table 1. Control experiments with known NAIs. K
gKong/G9/1997 H9N2).
i
was determined for N1 wild-type andH274Y (both from virus A/Anhui/1/2005 H5N1) and N2 (A/Chicken/Hon-
Compound
Ki
N1
N1-H274Y
86.6 ± 7.5 nM
16.2 ± 1.9 nM
n.d.
N2
oseltamivir free acid
MS-257[13]
0.15 ± 0.08 nM
0.17 ± 0.02 nM
9.1 ± 1.3 μM
0.67 ± 0.10 nM
0.21 ± 0.06 nM
13.2 ± 4.0 μM
4a
https://doi.org/10.1371/journal.pone.0193623.t001
was already 4 orders of magnitude less active than oseltamivir free acid against both N1
and N2, was not active toward the H274Y mutant in the range of concentrations tested
(
up to 1 mM). Compound 4a and all new compounds reported in this manuscript are
racemates; as a result, we expect that the IC50 value could be lower than the one measu-
red, if only one of the two enantiomers is active. All new compounds (16 a-f and 19 a-e),
were tested with N2, N1 and N1-H274Y and found to be inactive at 2 mM concentration.
Inhibition experiments were not carried out at compound concentrations above 2 mM.
In order to investigate the efficacy of the compounds against influenza A virus replication,
the compounds were also tested in an in vitro replication inhibition assay using A/Puerto
Rico/8/34 (PR8, H1N1) and A/Hong Kong/1/68 (HK1, H3N2) strains. Compound MS-257
(Fig 11), previously tested in cell culture assays,[31] was used as a positive control. None of the
new compounds showed replication inhibition efficacy.
Conclusion
In conclusion, we presented herein the successful synthesis of new derivatives of a bicyclo
[3.1.0]hexane scaffold containing six stereocenters and a variety of functional groups, designed
with the aid of molecular modelling to engage in productive interactions within the NA bind-
ing site. A simplified synthetic route was developed, starting from the cyclopropanation of
cyclopentenone and followed by an aziridination to achieve the common precursor that was
then functionalized with various ether side chains other than the 3-pentyl side chain, the char-
acteristic side chain in Oseltamivir. Some of these chains were installed as negative controls,
like the methylether and the ethylether, while the allyloxy, the 2-methylallyloxy and the dihy-
droxypropoxy side chains were expected by induced fit docking calculations to positively
engage in interactions in the NA binding site. However, none of the new structural variants
that we could synthesize, including those containing guanidinium groups rather than free
ammonium ions displayed activity against influenza A neuraminidases at concentrations less
than 2 mM. Unfortunately, we could not install the 3-pentyl side chain in this new set of com-
pounds. Overall, the information collected, together with the previously and here observed
confirmed activity of compound 4a, that features a 3-pentyl side chain moiety allowed us to
speculate that this side chain could be of crucial importance for productive interactions of the
bicyclo[3.1.0]hexane scaffold with influenza A neuraminidases. However, it appears that over-
all, docking calculations are overestimating the quality of the interactions generated by the
bicyclo[3.1.0]hexyl systems within the NA binding site. This may also depend on the rather
extreme flattening of the bicyclic ring system, best appreciated in the X-ray structure of 15c,
which distorts it away from the TS-like structure needed for optimal binding. We conclude
that the choice and positioning of functional groups on the bicyclo[3.1.0]hexyl system still
need to be properly tuned in order to elicit complementary interactions within the catalytic
site.
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Molecules as candidate inhibitors of influenza A neuraminidase
Materials and methods
General information: all chemicals were of analytical grade or better and were purchased from
1
13
Sigma-Aldrich unless noted otherwise. H- and C-NMR spectra were acquired on a Bruker
instrument and recorded at 600 or 400 MHz and 150 or 100 MHz, respectively. Spectra are
reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet,
q = quartet, quint = quintet, m = multiplet, bs = broad singlet), coupling constants (Hz). All
1
1
1
13
assignments were confirmed with the aid of two-dimensional H- H (COSY), H- C (HSQC)
1
13
and/or H- C (HMBC) experiments using standard pulse programs. Processing of the spectra
was performed using MestReNova software. Products numbering for spectral assignment is
clarified in the Appendix A in S3 File. Analytical thin-layer chromatography (TLC) was per-
formed on aluminum plates pre-coated with silica gel 60F-254. The developed plates were air
dried, exposed to UV light, and/or sprayed with a solution containing molybdic reagents or
permanganate reagents, and heated. Column chromatography was performed with an auto-
mated flash chromatography system. High resolution mass spectra were obtained by the elec-
trospray ionization method, using a TOF LC/MS high-resolution magnetic sector mass
spectrometer.
Synthesis
Compounds 8 and 9 were prepared accordingly to Reference 21 and 22, respectively. Com-
pound 10 was prepared accordingly to Reference 25. The analytical data were consistent with
those reported in the literature.
Compound 12. To a stirred solution of benzoic acid (730 mg, 6 mmol), sodium bicarbon-
ate (2.545 g, 30 mmol), N-Boc-sulfonylamide 10 (3.272 g, 12 mmol) in CHCl (60 mL),
3
N-isopropylethylendiamine was added (375 μL, 3 mmol) at room temperature, under nitro-
gen atmosphere. Compound 9 (1.0 g, 6 mmol) was added in one portion and the resulting
mixture was stirred at room temperature overnight. Water was added (60 mL) and the aque-
ous solution was extracted with CHCl (3 x 30 mL) and the combined organic layers were
3
washed once with saturated NaCl, dried over NaSO and evaporated. The resulting oily resi-
4
due was purified by column chromatography (Hex:EtOAc; gradient: 5–10% EtOAc in 2 CV,
1
1
0–35% EtOAc in 8 CV) to afford compound 12 as a white solid (1.234 g, 73%). H NMR
(
400 MHz, CDCl ) δ 4.15 (q, J = 7.1 Hz, 2H, CH ), 3.45 (t, J = 2.5 Hz, 1H, H ), 3.00 (ddd,
3
2Et
4
J = 4.8, 3.4, 1.2 Hz, 1H, H ), 2.91–2.85 (m, 1H, H ), 2.11–2.01 (m, 2H, H , H ), 1.45 (s, 9H,
1
3
5
6
1
3
CH3Boc), 1.25 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, CDCl ) δ 199.4 (C ), 168.4
3
2
(
COOEt), 157.6 (COBoc), 83.1 (CBoc), 61.8 (CH ), 42.9 (C ), 41.2 (C ), 30.7 (C ), 29.4 (C ),
2Et 4 3 6 1
1
2
9.4 (C ), 27.9 (CH3Boc), 14.2 (CH3Et). H NMR (400 MHz, Acetone-d ) δ 4.14 (q, J = 7.1
5
6
Hz, 2H, CH2Et), 3.58 (t, J = 2.6 Hz, 1H, H ), 2.92 (ddd, J = 4.9, 3.3, 1.1 Hz, 1H, H ), 2.89 (dt,
4
1
J = 2.6, 0.9 Hz, 1H, H ), 2.38–2.31 (t, J = 3.3, 1H, H ), 1.97–1.92 (m, 1H, H ), 1.45 (s, 9H,
CH3Boc), 1.23 (t, J = 7.1 Hz, 3H, CH3Et). HRMS (ESI) m/z calculated for [C14H19NNaO5]
3
6
5
+
3
04.11554; found 304.11603.
General procedure for aziridine opening with alcohols. To a stirred solution of com-
pound 12 (250 mg, 0.89 mmol) dissolved in alcohol (6 mL) under nitrogen atmosphere, copper
triflate (160 mg, 0.44 mmol) was added and the reaction mixture was stirred at room tempera-
ture for 8 h (TLC Hex:EtOAc 7:3). The mixture was evaporated and residue purified by col-
umn chromatography (Hex:EtOAc; gradient: 5–35% EtOAc in 10 CV).
1
Compound 13a. y = 77%. H NMR (400 MHz, CDCl ) δ 5.29 (d, J = 8.2 Hz, 1H, NHBoc),
3
4
.28–4.16 (m, 1H, H ), 4.06 (m, 2H, CH ), 3.67–3.44 (m, 3H, H , H ), 2.67–2.63 (m, 1H, H ),
4 2Et 3 7 5
13
2.45–2.29 (m, 2H, H , H ), 1.33 (s 9H, CH ), 1.26–1.06 (m, 6H, CH (8), CH ). C NMR
6
1
3Boc
3
3Et
(
101 MHz, CDCl ) δ 203.1 (C )169.8 (COOEt), 155.4 (COBoc), 80.1 (CBoc), 77.9 (C ), 64.8
3
2
4
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Molecules as candidate inhibitors of influenza A neuraminidase
(
CH2Et), 61.5 (C ), 58.6 (C ), 35.4 (C ), 30.2 (C ), 28.2 (CH ), 26.4 (C ), 15.3 (CH3Et), 14.0
7
3
1
5
3Boc
6
+
(C ). MS (ESI) m/z calculated for [C16H25NNaO6] 350.16; found 350.35.
8
1
Compound 13b. y = 82%. H NMR (400 MHz, CDCl ) δ 4.99 (bs, 1H, NHBoc), 4.23–4.03
3
(
m, 3H, CH2Et, H ), 3.74–3.52 (m, 1H, H ), 3.45 (s, 3H, CH ), 2.75 (q, J = 5.3 Hz, 1H, H ),
4
3
3-7
5
13
2
.53–2.34 (m, 2H, H , H ), 1.41 (s, 9H, CH3Boc), 1.26 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101
1
6
MHz, CDCl ) δ 203.1 (C ), 169.8 (COOEt), 155.5 (COBoc), 80.7 (CBoc), 80.0 (C ), 61.8 (CH2Et),
3
2
4
5
8.7 (C ), 57.2 (C ), 35.6 (C ), 30.0 (C ), 28.3 (CH ), 24.6 (C ), 14.2 (CH3Et). MS (ESI) m/z
3 7 1 5 3Boc 6
+
calculated for [C15H23NNaO6] 336.14; found 336.18.
1
Compound 13c. y = 65% H NMR (400 MHz, CDCl ) δ 5.99–5.86 (m, 1H, H ), 5.31 (dd,
3
8
J = 17.2, 1.5 Hz, 1H, H ), 5.22 (d, J = 10.4 Hz, 1H, H ), 4.83 (s, 1H, NHBoc), 4.34 (dd, J = 6.9,
9a
9b
5
.2 Hz, 1H, H ), 4.24–4.10 (m, 4H, H , CH ), 3.65 (bs, 1H, H ), 2.78–2.71 (m, 1H, H ), 2.51–
4 7 2Et 3 5
13
2.43 (m, 2H, H , H ), 1.44 (s, 9H, CH3Boc), 1.28 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101
6
1
MHz, CDCl ) δ 202.9 (C ), 169.8 (COOEt, COBoc), 134.4 (C ), 117.9 (C ), 77.8 (C ), 70.6 (C ),
3
2
8
9
4
7
6
1.8 (CH2Et), 58.9 (C ), 35.7 (C ), 30.5 (C ), 28.4 (C ), 24.8 (CH3Boc), 14.3 (CH3Et). MS (ESI)
3 6 5 1
+
m/z calculated for [C17H25NNaO6] 362.16; found 362.35.
1
Compound 13d. y = 55% H NMR (400 MHz, CDCl ) δ 4.99 (s, 1H, H ), 4.92 (bs, 2H, H ,
3
9a
9b
NHBoc) 4.30 (dd, J = 7.1, 5.1 Hz, 1H, H ), 4.16 (m, 2H, CH2Et), 4.04 (d, J = 2.6 Hz, 2H, H7),
4
3
1
1
.68 (bs, 1H, H ), 2.73 (q, J = 5.1 Hz, 1H, H ), 2.57–2.37 (m, 2H, H , H ), 1.74 (s, 3H, H ),
3
5
1
6
10
13
.42 (s, 9H, Boc), 1.27 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, CDCl ) δ 203.0 (C ),
3
2
69.9 (COOEt), 155.5 (COBoc), 141.8 (C ), 113.3 (C ), 77.5 (C ), 73.4 (C ), 61.8 (CH2Et), 58.7
8
9
4
7
(
C ), 35.7 (C ), 30.4 (C ), 28.3 (CH ), 24.8 (C ), 19.5 (C ), 14.2 (CH3Et). MS (ESI) m/z cal-
3 1 5 3Boc 6 10
+
culated for [C18H27NNaO6] 376.17; found 376.31.
1
Compound 13e. H NMR (400 MHz, CDCl ) δ 4.76 (s, 1H, NHBoc), 4.19 (q, J = 7.1 Hz, 2H,
3
CH2Et), 3.99 (s, 1H, H ), 3.53–3.47 (m, 1H, H ), 3.39 (s, 1H, H ), 2.72 (bs, 1H, H ), 2.49–
4
pent
3
5
2
.31 (m, 2H, H , H ), 1.56 (s, 9H, CH3Boc), 1.47 (m, 4H, CH2pent), 1.30 (t, J = 7.1 Hz, 3H,
1
6
CH3Et), 0.93 (t, J = 7.4 Hz, 6H, CH2pent), 0.87 (t, J = 7.5 Hz, 3H). MS (ESI) m/z calculated for
+
[
C19H31NNaO6] 392.20; found 392.50.
General procedure for N-Boc removal and N-acetylation starting from compounds of
general formula 13. Compound 13 was dissolved in dry CH Cl (conc. 0.5 M) under nitrogen
2
2
atmosphere, then trifluoroacetic acid (5 equiv.) was added and the reaction mixture stirred for 5
h, until starting material consumption was observed by TLC. The reaction was cooled at 0˚C
and pyridine (7.5 equiv.) was added slowly, then acetic anhydride (2 equiv.) was added and the
reaction mixture was stirred overnight at room temperature. The mixture was evaporated and
residue purified by column chromatography to afford compound of general formula 14.
1
Compound 14a. y = 69%. H NMR (400 MHz, CDCl ) δ 6.72 (d, J = 8.0 Hz, 1H, NH ), 4.27
3
Ac
(
dd, J = 7.0, 5.1 Hz, 1H, H ), 4.22–3.99 (m, 2H, CH ), 3.79 (t, J = 7.0 Hz, 1H, H ), 3.67–3.43
4
2Et
3
(
m, 2H, H ), 2.72 (ddd, J = 6.1, 5.1, 3.7 Hz, 1H, H ), 2.51–2.36 (m, 2H, H , H ), 1.96 (s, 3H,
7
5
1
6
13
CH3Ac), 1.18 (t, J = 7.1 Hz, 3H, CH3Et), 1.15 (t, J = 7.1 Hz, 3H, CH3-8). C NMR (101 MHz,
CDCl ) δ 202.8 (C ), 170.8 (CO ), 169.8 (CO ), 77.9 (C ), 64.8 (CH ), 61.6 (C ), 57.9 (C ),
3
2
Ac
OEt
4
2Et
7
3
3
5.7 (C ), 30.4 (C ), 24.5 (C ), 22.8 (CH3Ac), 15.3 (CH3Et), 14.1 (C ). MS (ESI) m/z calculated
1 5 6 8
+
for [C13H19NNaO5] 292.12; found 292.59.
1
Compound 14b. y = 63%. H NMR (400 MHz, CDCl ) δ 7.02 (d, J = 8.0 Hz, 1H, NH ),
3
Ac
4
.19–4.00 (m, 3H, H , CH ), 3.82 (t, J = 7.5 Hz, 1H, H ), 3.35 (s, 3H, CH ), 2.77–2.65 (m,
4
2Et
3
3-7
13
1H, H ), 2.44–2.41 (m, 2H, H , H ), 1.94 (s, 3H, CH3Ac), 1.19 (t, J = 7.1 Hz, 3H, CH3Et). C
5
1
6
NMR (101 MHz, CDCl ) δ 202.8 (C ), 171.1 (CO ), 169.7 (CO ), 79.5 (C ), 61.6 (CH3Et),
3
2
Ac
OEt
4
5
7.5 (C ), 56.8 (C ), 35.6 (C ), 29.8 (C ), 24.4 (C ), 22.6 (CH3Ac), 14.0 (CH3Et). MS (ESI) m/z
3 7 1 5 6
+
calculated for [C12H17NNaO5] 278.10; found 278.19.
Compound 14c. Purification by column chromatography (Hex:EtOAc; gradient: 10–70%
1
EtOAc in 7 CV, 7–100% EtOAc in 8 CV). y = 80%. H NMR (400 MHz, CDCl ) δ 6.03 (s, 1H,
3
PLOS ONE | https://doi.org/10.1371/journal.pone.0193623 February 28, 2018
12 / 22

Molecules as candidate inhibitors of influenza A neuraminidase
NH ), 5.91 (m, 1H, H ), 5.29 (dd, J = 17.2, 1.5 Hz, 1H, H ), 5.25–5.17 (m, 1H, H ), 4.35 (dd,
Ac
8
9a
9b
J = 7.2, 5.1 Hz, 1H, H ), 4.24–4.04 (m, 4H, H , CH ), 3.87 (t, J = 7.2 Hz, 1H, H ), 2.80–2.69
4
7
2Et
3
(
m, 1H, H ), 2.53–2.45 (m, 2H, H , H ), 2.03 (s, 3H, CH3Ac), 1.27 (t, J = 7.1 Hz, 3H, CH3Et).
5
6
1
1
3
C NMR (101 MHz, CDCl ) δ 202.5 (C ), 170.5 (CO ), 169.8 (CO ), 134.4 (C ), 117.9 (C ),
3
2
Ac
OEt
8
9
7
7.9 (C ), 70.5 (C ), 61.8 (CH ), 58.1 (C ), 36.0 (C ), 30.5 (C ), 24.8 (C ), 23.0 (CH3Ac), 14.2
4 7 2Et 3 1 5 6
+
(CH3Et). MS (ESI) m/z calculated for [C14H19NNaO5] 304.12; found 304.21.
1
Compound 14d. y = 52%. H NMR (400 MHz, CDCl ) δ 6.51 (d, J = 7.9 Hz, 1H, NH ),
3
Ac
4
.94 (s, 1H, H ), 4.88 (s, 1H, H ), 4.30 (dd, J = 7.0, 5.1 Hz, 1H, H ), 4.20–4.05 (m, 2H, CH2Et),
9a
9b
4
4
.02–3.93 (m, 2H, H ), 3.87 (t, J = 7.0 Hz, 1H, H ), 2.73 (ddd, J = 6.1, 5.1, 3.0 Hz, 1H, H ), 2.49
7
3
5
(
t, J = 3.0 Hz, 1H, H ), 2.45 (dd, J = 6.1, 3.0 Hz, 1H, H ), 1.99 (s, 3H, CH3Ac), 1.70 (s, 3H, H10),
6
1
13
1
.24 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, CDCl ) δ 202.6 (C ), 170.8 (CO ), 169.8
3
2
Ac
(
(
[
COOEt), 141.8 (C ), 113.2 (C ), 77.2 (C ), 73.3 (C ), 61.7 (CH ), 57.8 (C ), 35.9 (C ), 30.4
8 9 4 7 2Et 3 1
C ), 24.7(C ), 22.8 (CH ), 19.4 (C ), 14.1 (CH3Et). MS (ESI) m/z calculated for
5
6
3Ac
10
+
C15H21NNaO5] 318.13; found 318.37.
General procedure for reductive amination of compounds of general formula 14 and N-
Boc protection. Compound 14 was dissolved in a solution of 2M NH in EtOH (5 equiv.),
3
then Ti(OEt) (1.5 equiv.) was added and the reaction mixture stirred for 45 min at room tem-
4
perature. NaBH (3 equiv.) was added in one portion and the reaction mixture was diluted to a
4
concentration of 0.2 M with EtOH and stirred for 20 min. Boc O (5 equiv.) was added and the
2
reaction mixture was stirred for 2 h at room temperature. The mixture was quenched with
water, diluted with CH Cl and transferred in a separatory funnel. Saturated NaCl was added
2
2
and the aqueous solution was extracted with CH Cl . The organic phase was dried over NaSO
4
2
2
and evaporated. The residue purified by column chromatography to afford compound of gen-
eral formula 15.
1
Compound 15a. y = 65%. H NMR (400 MHz, CDCl ) δ 6.43 (d, J = 8.6 Hz, 1H, NH ),
3
Ac
5
3
1
.43 (d, J = 8.2 Hz, 1H, NHBoc), 4.13–4.06 (m, 3H, H , CH ), 3.97–3.95 (m, 1H, H ), 3.72–
2
2Et
4
.68 (m, 1H, H ), 3.62–3.40 (m, 2H, H ), 2.13–2.10 (m, 3H, H , H , H ), 1.94 (s, 3H, CH3Ac),
3
7
1
5
6
13
.40 (s, 9H, CH3Boc), 1.17 (t, J = 7.1 Hz, 3H, CH3Et), 1.14 (t, J = 7.1 Hz, 3H, CH3-8). C NMR
(
(
(
3
101 MHz, CDCl ) δ 172.5 (CO ), 170.9 (COOEt), 156.3 (COBoc), 80.7 (C ), 79.8 (CBoc), 64.9
3
Ac
4
C ), 60.9 (CH ), 55.6 (C ), 55.0 (C ), 28.5 (CH ), 28.2 (C ), 27.2 (C ), 23.4 (CH3Ac), 18.4
7
2Et
3
2
3Boc
1
5
+
C ), 15.5 (CH ), 14.2 (C ). MS (ESI) m/z calculated for [C16H26N2NaO6] 365.17; found
6
3Et
8
65.44.
1
Compound 15b. y = 76%. H NMR (400 MHz, CDCl ) δ 6.41 (s, 1H, NH ), 1.40 (s, 9H,
3
Ac
CH3Boc), 1.17 (t, J = 7.1 Hz, 3H, CH3Et), 1.14 (t, J = 7.1 Hz, 3H, CH3-8), 5.41 (d, J = 7.8 Hz, 1H,
NHBoc), 4.23–3.99 (m, 3H, H , CH ), 3.94–3.81 (m, 1H, H ), 3.81–3.66 (m, 1H, H ), 3.36 (s,
2
2Et
4
3
3
H, H ), 2.23–2.06 (m, 3H, H , H , H ), 1.95 (s, 3H, CH3Ac), 1.41 (s, 9H, CH3Boc), 1.24 (t,
7 1 5 6
13
J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, CDCl ) δ 172.3 (CO ), 171.0 (COOEt), 156.2
3
Ac
(
COBoc), 82.5 (C ), 80.0 (CBoc), 61.0 (CH2Et), 57.3 (C ), 55.6 (C ), 54.9 (C ), 29.8 (CH3Boc), 28.5
4 7 3 2
(
C ), 26.5 (C ), 23.5 (CH ), 18.3 (C ), 14.3 (CH3Et).
1
5
3Ac
6
1
Compound 15c. y = 72%. H NMR (400 MHz, CDCl ) δ 6.71 (d, J = 8.8 Hz, 1H, NH ),
3
Ac
5
.90–5.84 (m, 1H, H ), 5.55 (d, J = 8.5 Hz, 1H, NHBoc), 5.22 (dd, J = 17.2, 1.6 Hz, 1H, H9a),
8
5
.16–5.08 (m, 1H, H ), 4.22–4.11 (m, 1H, H ), 4.11–3.93 (m, 5H, CH , H , H ), 3.78–3.74
9b
2
2Et
4
7
(
m, 1H, H ), 2.23–2.02 (m, 3H, H , H , H ), 1.92 (s, 3H, CH3Ac), 1.39 (s, 9H, CH3Boc), 1.21 (t,
3 1 5 6
13
J = 7.1 Hz, 3H, (CH3Et). C NMR (101 MHz, CDCl ) δ 172.4 (CO ), 170.7 (COOEt), 156.2
3
Ac
(
COBoc), 134.8 (C ), 117.1 (C ), 80.3 (C ), 79.6 (C ), 70.3 (C ), 60.7 (CH ), 55.4 (C ), 54.7
8 9 4 Boc 7 2Et 3
(
C ), 29.6 (CH ), 28.4 (C ), 27.2 (C ), 23.2 (CH ), 18.3 (C ), 14.1 (CH3Et). MS (ESI) m/z
2
3Boc
1
5
3Ac
6
+
calculated for [C19H31N2O6] 383.22; found 383.04; m/z calculated for [C19H30N2NaO6]
405.20; found 405.20.
+
PLOS ONE | https://doi.org/10.1371/journal.pone.0193623 February 28, 2018
13 / 22

Molecules as candidate inhibitors of influenza A neuraminidase
1
Compound 15d. y = 62%. H NMR (400 MHz, CDCl ) δ 6.76 (d, J = 8.6 Hz, 1H, NH ),
3
Ac
5
.54 (d, J = 8.4 Hz, 1H), 4.91 (s, 1H, H ), 4.83 (s, 1H, H ), 4.20–4.11 (m, 1H, H ), 4.11–4.02
9a
9b
2
(
m, 2H, CH2Et), 4.01–3.83 (m, 3H, H , H ), 3.77 (m, 1H, H ), 2.17–2.02 (m, 3H, H , H , H ),
4 7 3 1 5 6
1
3
1
.91 (s, 3H, CH3Ac), 1.67 (s, 3H, H ), 1.38 (s, 9H, CH3Boc), 1.21 (t, J = 7.1 Hz, 3H, CH3Et). C
10
NMR (101 MHz, CDCl ) δ 172.5 (CO ), 170.8 (COOEt),156.3 (COBoc), 142.3 (C ), 112.6 (C ),
3
Ac
8
9
80.3 (C ), 79.6 (C ), 73.4 (C ), 60.8 (CH ), 55.4 (C ), 54.6 (C ), 28.4 (CH ), 27.2 (C , C ),
4 Boc 7 2Et 3 2 3Boc 1 5
2
3.3 (C ), 19.4 (CH ), 18.4 (C ), 14.2 (CH3Et). MS (ESI); m/z calculated for
6
3Ac
+
10
[C20H32N2NaO6] 419.22; found 419.34.
Compound 15f. Compound 14 was dissolved in a solution of 2 M NH in EtOH (5 equiv.),
3
then Ti(OEt) (4 equiv.) was added and the reaction mixture stirred for 45 min at room temper-
4
ature. NaBH (3 equiv.) was added in one portion and the reaction mixture was diluted to a
4
concentration of 0.2 M with EtOH and stirred for 1h at room temperature. The mixture was
quenched with water, diluted with CH Cl and transferred in a separatory funnel. Saturated
2
2
NaCl was added and the aqueous solution was extracted with CH Cl . The organic phase was
2
2
dried over NaSO and evaporated. The residue purified by column chromatography to afford
4
1
compound 15f. H NMR (400 MHz, CDCl ) δ 6.36 (d, J = 3.0 Hz, 1H, NH ), 5.88-5-82 (m 1H,
3
Ac
H ), 5.32–5.22 (m, 1H, H ), 5.22–5.14 (m, 2H, H , OH), 4.24–4.17 (m, 1H, H ), 4.16–4.05 (m,
8
9a
9b
2
3
H, CH2Et, H ), 4.05–3.95 (m, 2H, H ; H ), 3.30 (td, J = 7.3, 4.1 Hz, 1H, H ), 2.16–2.12 (m,
7a
4
7b
3
13
3H; H , H , H ), 1.98 (s, 3H, CH3Ac), 1.23 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz,
1
5
6
CDCl ) δ 173.2 (CO ), 172.6 (CO ), 134.5 (C ), 117.8 (C ), 80.3 (C ), 75.0 (C ), 70.4 (C ),
3
Ac
OEt
8
9
4
2
7
6
1.2 (C ), 61.0 (CH ), 30.1 (C ), 27.6 (C ), 23.0 (CH ), 18.7 (C ), 14.2 (CH ). MS (ESI) m/z
3 2Et 5 1 3Ac 6 3Et
+
+
calculated for [C14H22NO5] 284.15; found 284.15; m/z calculated for [C14H21NNaO5]
06.13; found 306.23.
General procedure for osmium-catalyzed dihydroxylation. Compound 15c (or 18c)
1 equiv.) was dissolved in dry acetone (c = 0.08 M) under nitrogen. Trimethylamine n-
3
(
oxide dihydrate (1.8 equiv.) and osmium(III) chloride (0.05 equiv.) were added and the
reaction mixture stirred for 2 h at room temperature. Completion of reaction was moni-
1
tored by H-NMR. Na S O was added and the reaction mixture was stirred for 5 minutes,
2
2
3
then filtered on a celite pad. The solvent was evaporated and the residue was purified by
C18 column chromatography (water:acetonitrile; gradient: 10–70% acetonitrile in 15 CV).
1
Compound 15e. y = 95%. H NMR (400 MHz, Methanol-d ) δ 4.20–4.09 (m, 3H, CH2Et,
4
H ), 4.07 (dd, J = 7.5, 3.8 Hz, 1H, H ), 3.78–3.70 (m, 1H, H ), 3.70–3.38 (m, 5H, H , H , H ),
2
4
8
3
7
9
2
.24–2.15 (m, 2H, H , H ), 2.14–2.07 (m, 1H, H ), 1.95 (s, 3H, CH3Ac), 1.45 (s, 9H, CH3Boc),
1 6 5
13
1.28 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, Methanol-d ) δ 174.2 (CO ),173.5
4
Ac
(
COOEt), 158.1 (COBoc), 83.0 (C ), 82.9 (C ), 80.3 (C ), 72.3 (C ), 72.2 (C ), 72.1 (C ), 71.9
4 4 Boc 8 8 9
(
C ), 64.4 (C ), 64.3 (CH ), 62.0 (C ), 56.3 (C ), 55.5 (C ), 29.6 (C ), 28.7 (CH3Boc), 28.3 (C1),
9 7 2Et 3 2 2 5
2
2.8 (CH3Ac), 19.3 (C ), 14.5 (CH3Et).
6
1
Compound 15g. y = 95%. H NMR (400 MHz, Methanol-d ) δ 4.18 (dd, J = 7.8, 4.7 Hz, 1H,
4
H ), 4.15–4.07 (m, 2H, CH ), 4.00 (dd, J = 7.7, 3.6 Hz, 1H, H ), 3.75–3.68 (m, 1H, H ),
2
2Et
4
8
3
.64–3.47 (m, 5H, H , H , H ), 2.20–2.15 (m, 2H, H , H ), 2.10–2.04 (m, 1H, H ), 1.96 (s, 3H,
7 9 3 1 6 5
13
CH3Ac), 1.25 (t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, Methanol-d ) δ 174.4 (CO ),
4
Ac
1
73.8 (COOEt), 82.6 (C ), 74.8 (C ), 72.3 (C ), 71.9 (C ), 64.4 (C ), 64.3 (CH ), 59.3 (C ), 31.1
4 2 8 9 7 2Et 3
(
C ), 28.5 (C ), 22.8 (CH ), 19.5 (C ), 14.5 (CH3Et).
5 1 3Ac 6
General procedure for the synthesis of amino acids of general formula 16. Compound
5 (1 equiv.) was dissolved in a 1:5 mixture of water and methanol (at a compound concentra-
1
tion of 0.05 M) at 0˚C. Next, 5 equivalents of a cold solution of NaOH 6 M was added and the
mixture was kept at 4˚C overnight. The solution was diluted with water and extracted once with
EtOAc. The water layer was acidified with 0.5 M HCl and extracted with EtOAc (3 times).
Organic extracts were dried over NaSO , evaporated at reduced pressure. The obtained solid
4
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Molecules as candidate inhibitors of influenza A neuraminidase
was dissolved in 1:3 mixture of TFA and dichloromethane (at a compound concentration of
0.05 M) and stirred at room temperature for 2 h. Cold diethylether was added to allow the pre-
cipitation of the product, which was triturated, precipitated by centrifugation and the solution
removed (the procedure was repeated 3 times). The white solid was then dissolved in water and
freeze-dried to give a white foam.
1
Compound 16a. y = quant. H NMR (400 MHz, D O) δ 4.32 (dd, J = 7.6, 4.6 Hz, 1H, H ),
2
4
3
.89–3.75 (m, 2H, H , H ), 3.75–3.67 (m, 1H, H ), 3.65–3.52 (m, 1H, H ), 2.44 (ddd, J = 7.5,
2
3
7a
7b
4
.6, 3.3 Hz, 1H, H ), 2.30–2.21 (m, 2H, H , H ), 1.99 (s, 3H, CH3Ac), 1.17 (t, J = 7.1 Hz, 3H,
5
1
6
13
CH3-8). C NMR (101 MHz, D O) δ 175.6 (COOH), 175.0 (CO ), 80.2 (C ), 65.8 (C ), 54.9
2
Ac
4
7
(
C ), 54.0 (C ), 27.7 (C ), 25.5 (C ), 21.7 (CH ), 18.7 (C ), 14.3 (C ). HRMS (ESI) m/z calcu-
2 3 5 1 3Ac 6 8
+
lated for [C11H18N2NaO4] 265.11588; found 265.11633.
1
Compound 16b. y = quant. H NMR (400 MHz, D O) δ 4.14 (dd, J = 7.6, 4.6 Hz, 1H, H ),
2
4
3
2
1
.82–3.67 (m, 2H, H , H ), 3.34 (s, 3H, CH ), 2.42–2.33 (m, 1H, H ), 2.20–2.16 (m, 1H, H ),
2 3 3-7 5 1
13
.14 (t, J = 3.1 Hz, 1H, H ), 1.91 (s, 3H, CH ). C NMR (101 MHz, D O) δ 175.5 (COOH),
6
3Ac
2
75.0 (CO ), 81.9 (C ), 57.0 (C ), 54.9 (C ), 53.9 (C ), 27.1 (C ), 25.5 (C ), 21.7 (CH3Ac), 18.6
Ac
4
7
2
3
5
1
-
(C ). HRMS (ESI) m/z calculated for [C10H15N2O4] 227.10373; found 227.10360.
6
1
Compound 16c. y = quant. H NMR (400 MHz, D O) δ 5.99–5.87 (m, 1H, H ), 5.34 (dq,
2
8
J = 17.3, 1.5 Hz, 1H, H ), 5.27 (dd, J = 10.4, 1.4 Hz, 1H, H ), 4.37 (dd, J = 6.8, 5.2 Hz, 1H, H ),
9a
9b
4
4
.22–4.07 (m, 2H, H ), 3.89–3.77 (m, 2H, H , H ), 2.47–2.40 (m, 1H, H ), 2.30–2.22 (m, 2H,
7 3 2 5
13
H , H ), 2.01 (s, 3H, CH ). C NMR (101 MHz, D O) δ 176.2 (COOH), 175.6 (CO ), 134.3
1
6
3Ac
2
Ac
(
C ), 119.3 (C ), 80.8 (C ), 71.6 (C ), 55.5 (C ), 54.7 (C ), 28.5 (C ), 26.4 (C ), 22.5 (CH3-Ac),
8 9 4 7 2 3 5 1
-
1
9.4 (C ). HRMS (ESI) m/z calculated for [C12H17N2O4] 253.11938; found 253.11922.
6
1
Compound 16d. y = quant. H NMR (400 MHz, D O) δ 4.94–4.88 (m, 2H, H ), 4.28–4.19
2
9
(
m, 1H, H ), 3.99–3.95 (m, 2H, H ), 3.79–3.72 (m, 2H, H , H ), 2.43–2.32 (m, 1H, H ),
4 7 2 3 1
13
2
.23–2.15 (m, 2H, H , H ), 1.91 (s, 3H, CH3Ac), 1.62 (s, 3H, CH3-10). C NMR (101 MHz,
5 6
D O) δ 175.2 (COOH), 174.8 (CO ), 141.8 (C ), 113.7 (C ), 81.3 (C ), 74.0 (C ), 54.6 (C ),
2
Ac
8
9
4
7
3
5
3.9 (C ), 27.9 (C , C ), 25.7 (CH ), 21.7 (C ), 18.4 (C ). HRMS (ESI) m/z calculated for
2 1 5 3Ac 6 10
+
[C13H20N2NaO4] 291.13153; found 291.13212.
1
Compound 16e. y = quant. H NMR (400 MHz, D O) δ 4.19 (ddd, J = 7.3, 4.6, 2.6 Hz, 1H,
2
H )), 3.79–3.64 (m, 3H, H , H ; H ), 3.59 (dd, J = 10.4, 4.0 Hz, 1H, H ), 3.54–3.34 (m, 3H, H ,
4
2
3
8
7
9
13
H ), 2.34 (dt, J = 7.3, 4.0 Hz, 1H, H ), 2.22–2.10 (m, 2H, H , H ), 1.86 (s, 3H, CH ). C
7
5
1
6
3Ac
NMR (101 MHz, D O) δ 175.3 (COOH), 175.0 (CO ), 81.2 (C ), 81.1 (C ), 70.6 (C ), 70.5
2
Ac
4
4
8
(
C ), 70.4 (C ), 70.4 (C ), 62.4 (C ), 62.4 (C ), 54.8 (C ), 54.7 (C ), 54.1 (C ), 27.7 (C ), 27.6
8 9 9 7 7 2 2 3 5
-
(C ), 25.7 (C ), 21.7 (CH ), 18.5 (C ). HRMS (ESI) m/z calculated for [C12H19N2O6]
5 1 3Ac 6
2
87.12486; found 287.12487.
1
Compound 16f. y = quant. H NMR (400 MHz, D O) δ 4.27 (dd, J = 8.0, 4.4 Hz, 1H, H ),
2
4
4
2
1
2
2
.11 (dd, J = 7.8, 4.4 Hz, 1H, H ), 3.89–3.78 (m, 1H, H ), 3.73–3.45 (m, 5H, H , H , H ), 2.37–
2
3
7
9
8
13
.27 (m, 1H, H ), 2.27–2.17 (m, 2H, H , H ), 2.00 (s, 3H, CH ). C NMR (101 MHz, D O) δ
5
1
6
3Ac
2
76.5 (COOH), 174.3 (CO ), 80.9 (C ), 73.2 (C ), 73.1 (C ), 70.2 (C ), 62.5 (C ),56.7 (C ),
Ac
4
2
8
9
7
3
+
9.5 (C )27.4 (C ), 22.1 (CH ), 18.1 (C ). HRMS (ESI) m/z calculated for [C12H18N1O7]
5
1
3Ac
6
88.10888; found 288.10854.
General procedure for guanylation starting from compounds 16. To a solution of com-
pound 16 in CH Cl (0.2M), trifluoroacetic acid (4.4 equiv) was added. After 6 h, the reaction
2
2
mixture was cooled at 0˚C and triethylamine (6.8 equiv) was added dropwise, followed by the
addition of N,N'-bis(tert-butoxycarbonyl)thiourea (1.2 equiv) and Mukaiyama’s reagent (1.2
equiv). The reaction was allowed to stir at 25˚C until completion, then the solvent was evapo-
rated. The crude material was dissolved in diethyl ether and washed with water. The organic
layer was dried over NaSO and evaporated. The crude was purified by flash chromatography.
4
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Molecules as candidate inhibitors of influenza A neuraminidase
1
Compound 18a. y = 60%. H NMR (400 MHz, CDCl ) δ 11.31 (s, 1H, NHBoc), 8.58 (d,
3
J = 7.8 Hz, 1H, NH(H2)), 6.37 (d, J = 8.5 Hz, 1H, NH ), 4.68 (ddd, J = 8.5, 7.8, 4.2 Hz, 1H, H ),
Ac
2
4
.13–4.00 (m, 2H, CH2Et), 3.97–3.92 (m, 1H, H ), 3.65 (q, J = 8.5 Hz, 1H, H ), 3.59–3.40 (m,
4
3
2
H, H ), 2.20–2.06 (m, 3H, H , H , H ), 1.90 (s, 3H, CH ), 1.45 (s, 9H, CH3Boc), 1.41 (s, 9H,
7
1
5
6
3Ac
13
CH3Boc), 1.25–1.18 (m, 3H, CH3Et), 1.18–1.07 (m, 3H, CH3-8). C NMR (101 MHz, CDCl ) δ
3
172.2 (CO ), 170.8 (CO ), 163.1(C ), 156.7 (COBoc), 152.7 (COBoc), 83.5 (CBoc), 81.3 (C4),
Ac OEt N
7
9.3 (CBoc), 64.9 (C ), 60.9 (CH ), 56.5 (C ), 53.9 (C ), 28.3 (CH3Boc), 28.1 (CH3Boc), 27.7
7
2Et
3
2
(
C ), 27.4 (C ), 23.4 (CH ), 18.4 (C ), 15.4 (CH3Et), 14.1(CH3-8). MS (ESI) m/z calculated for
1 5 3Ac 6
+
[C24H41N4O8] 513.29; found 513.46.
1
Compound 18b. y = 65%. H NMR (400 MHz, CDCl ) δ 11.33 (s, 1H, NHBoc), 8.61 (d,
3
J = 7.5 Hz, 1H, NH(H2)), 6.37 (d, J = 7.8 Hz, 1H, NH ), 4.75–4.71 (m, 1H, H ), 4.11 (q, J = 7.2
Ac
2
Hz, 2H, CH2Et), 3.85 (dd, J = 7.8, 3.3 Hz, 1H, H ), 3.67 (q, J = 7.8 Hz, 1H, H ), 3.36 (s, 3H,
4
3
CH3-7), 2.26–2.08 (m, 3H, H , H ; H ), 1.92 (s, 3H, CH3Ac), 1.47 (s, 9H, CH3Boc), 1.44 (s, 9H,
1
5
6
13
CH3Boc), 1.25 (t, J = 7.2 Hz, 3H, CH3Et). C NMR (101 MHz, CDCl ) δ 172.2 (CO ), 170.9
3
Ac
(
(
(
COOEt), 163.0 (C ), 156.7 (COBoc), 152.7 (COBoc), 83.6 (CBoc), 83.2 (C ), 79.5 (CBoc), 61.1
N
4
CH2Et), 57.3 (C ), 56.4 (C ), 53.8 (C ), 28.3 (CH3Boc), 28.1 (CH3Boc), 27.3 (C ), 27.0 (C ), 23.4
7
3
2
1
5
CH3Ac), 18.4 (C ), 14.2 (CH3Et). MS (ESI) m/z calculated for [C23H39N4O8]+ 499.27; found
6
4
99.58.
1
Compound 18c. y = 66%. H NMR (400 MHz, CDCl ) δ 11.33 (s, 1H, NHBoc), 8.60 (d,
3
J = 7.5 Hz, 1H, NH(H2)), 6.26 (d, J = 8.0 Hz, 1H, NH ), 5.89–5.84 (m, 1H, H ), 5.24 (dd,
Ac
8
J = 17.3, 1.6 Hz, 1H, H ), 5.17–5.11 (m, 1H, H ), 4.74–4.72 (m, 1H, H ), 4.14–3.96 (m, 5H,
9a
9b
2
H , CH , H ), 3.69 (q, J = 8.0 Hz, 1H, H ), 2.23–2.08 (m, 3H, H , H , H ), 1.93 (s, 3H,
7
2Et
4
3
1
5
6
13
CH3Ac), 1.48 (s, 9H, CH3Boc), 1.45 (s, 9H, CH3Boc), 1.25 (t, J = 7.1 Hz, 3H, CH3Et). C NMR
(
101 MHz, CDCl ) δ 172.2 (CO ), 170.8 (CO ), 163.1 (C ), 156.8 (COBoc), 152.8 (COBoc),
3 Ac OEt N
134.9 (C ), 117.2 (C ), 83.6 (C ), 81.1 (C ), 79.4 (C ), 70.5 (C ), 61.0 (CH2Et), 56.8 (C3),
8 9 Boc 4 Boc 7
5
3.9 (C ), 28.4 (CH3Boc), 28.2 (CH3Boc), 27.7 (C ), 27.5 (C ), 23.5 (CH ), 18.5 (C ), 14.2
2
1
5
3Ac
6
(
CH3Et).
1
Compound 18d. y = 53%. H NMR (400 MHz, CDCl ) δ 11.35 (s, 1H, NHBoc), 8.65 (d,
3
J = 7.3 Hz, 1H, NH(H2)), 6.22 (d, J = 6.9 Hz, 1H, NH ), 4.94 (s, 1H, H ), 4.87 (s, 1H, H ),
Ac
9a
9b
4
.84–4.73 (m, 1H, H ), 4.19–4.07 (m, 2H, CH ), 4.01–3.93 (m, 3H, H , H ), 3.74–3.70 (m,
2 2Et 7 2
1
H, H ), 2.24–2.12 (m, 3H, H , H , H ), 1.94 (s, 3H, CH ), 1.71 (s, 3H, CH3Ac), 1.48 (s, 9H,
3
1
5
6
3-10
13
CH3Boc), 1.46 (s, 9H, CH3Boc), 1.27 (t, J = 7.2 Hz, 3H, CH3Et). C NMR (101 MHz, CDCl ) δ
3
1
8
2
72.2 (CO ), 172.0 (CO ), 170.7 (C ), 156.6 (COBoc), 152.7 (COBoc), 142.3 (C ), 113.3 (C ),
Ac OEt N 8 9
0.8 (C ), 73.5 (C ), 61.0 (CH ), 56.6 (C ), 53.6 (C ), 28.3 (CH3Boc), 28.1 (CH3Boc), 27.5 (C1),
4
7
2Et
3
2
7.3 (C ), 23.4 (CH ), 19.4 (C ), 18.4 (C ), 14.2 (CH3Et). MS (ESI) m/z calculated for
5
3Ac
6
10
+
[C26H42N4NaO8] 561.29; found 561.76.
1
Compound 18e. y = 44%. H NMR (400 MHz, CDCl ) δ 11.33 (s, 1H, NHBoc), 8.66 (d,
3
J = 6.4 Hz, 1H, NH(H2)), 7.62 (d, J = 5.4 Hz, 1H, NH ), 7.51 (d, J = 6.0 Hz, 1H, NH ),
Ac
Ac
4
.82–4.71 (m, 1H, H ), 4.14 (q, J = 7.1 Hz, 2H, CH ), 4.02–3.98 (m, 1H, H ), 3.87–3.74 (m,
2
2Et
4
2
H, H ; H ), 3.74–3.36 (m, 4H, H , H , H ), 2.24–2.10 (m, 3H, H ; H , H ), 1.95 (s, 3H,
8
7a
7b
9
3
1
5
6
13
CH3Ac), 1.49 (s, 9H, CH3Boc), 1.46 (s, 9H, CH3Boc), 1.31–1.20 (m, 3H, CH3Et). C NMR (101
MHz, CDCl ) δ 172.0 (CO ), 171.9 (CO ), 171.8 (CO ), 162.7 (C ), 162.6 (C ), 157.0
3
Ac
Ac
OEt
N
N
(
COBoc), 157.0 (COBoc), 152.8 (COBoc), 84.1 (C ), 84.0 (CBoc), 84.0 (CBoc), 83.3 (C ), 79.9
4
4
(
CBoc), 70.6 (C ), 70.2 (C ), 63.6 (C ), 63.5 (C ), 61.3 (CH ), 58.4 (C ), 57.9 (C ), 53.6 (C ),
9 8 7 7 2Et 3 3 2
28.3 (CH3Boc), 28.2 (CH3Boc), 27.8 (C ), 27.7 (C ), 27.4 (C ), 27.2 (C ), 23.5 (CH3Ac), 18.7 (C6)
1 1 5 5
1
4.2 (CH3Et).
General procedure for the synthesis of compounds 19. Compound 18 (1 equiv.) was dis-
solved in a 1:5 mixture of water and methanol (at a compound concentration of 0.05 M) at
˚C. Next, 5 equivalents of a cold solution of NaOH 6 M was added and the mixture was kept
0
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Molecules as candidate inhibitors of influenza A neuraminidase
at 4˚C overnight. The water layer was diluted with water and acidified with a solution of HCl 6
M (5 equiv.), while stirring at 0˚C. The solution was freeze-dried and the obtained solid was
dissolved in 1:3 mixture of TFA and dichloromethane (at a compound concentration of 0.05
M) and stirred at room temperature for 2 h. Cold diethylether was added to allow the precipi-
tation of the product, which was triturated, precipitated by centrifugation and the solution
removed (the procedure was repeated 3 times). The white solid was then dissolved in water
and freeze-dried to give a white foam.
1
Compound 19a. y = 90%. H NMR (400 MHz, D O) δ 4.24 (dd, J = 8.1, 4.5 Hz, 1H, H ),
2
4
4
.12 (dd, J = 8.7, 4.4 Hz, 1H, H ), 3.78–3.65 (m, 2H, H ; H ), 3.64–3.49 (m, 1H, H ), 2.40–
2
3
7a
7b
2
.32 (m, 1H, H ), 2.32–2.25 (m, 1H, H ), 2.17 (t, J = 3.0 Hz, 1H, H ), 1.97 (s, 3H, CH3Ac), 1.16
5
1
6
13
(t, J = 7.1 Hz, 3H, CH3Et). C NMR (101 MHz, D O) δ 176.1 (COOH), 174.4 (CO ),156.7
2
Ac
(
C ), 79.7 (C ), 65.7 (C ), 55.3 (C ), 55.2 (C ), 27.3 (C ), 27.1 (C ), 21.9 (CH3Ac), 18.3 (C6),
N 4 7 2 3 5 1
-
1
4.3 (C ). HRMS (ESI) m/z calculated for [C12H19N4O4] 283.14118; found 283.14102.
8
1
Compound 19b. y = 94%. H NMR (400 MHz, D O) δ 4.07–4.03 (m, 2H, H ; H ), 3.65 (t,
2
4
2
J = 8.3 Hz, 1H, H ), 3.32 (s, 3H, CH ), 2.30 (ddd, J = 7.3, 4.4, 3.2 Hz, 1H; H ), 2.21 (ddd,
3
3-7
5
13
J = 7.3, 4.4, 3.2 Hz, 1H, H ), 2.06 (t, J = 3.2 Hz, 1H, H ), 1.88 (s, 3H, CH ). C NMR (101
1
6
3Ac
MHz, D O) δ 176.1 (COOH), 174.4 (CO ), 156.7 (C ), 81.4 (C ), 56.8 (CH ), 55.3 (C ), 55.1
2
Ac
N
4
3-7
2
(
C ), 27.3 (C ), 26.5 (C ), 21.9 (CH ), 18.2 (C ). HRMS (ESI) m/z calculated for
3 1 5 3Ac 6
-
[C11H17N4O4] 269.12553; found 269.12539.
1
Compound 19c. y = 88%. H NMR (400 MHz, D O) δ 6.04–5.83 (m, 1H, H ), 5.45–5.19
2
8
(
m, 2H, H ), 4.33 (dd, J = 8.1, 4.5 Hz, 1H, H ), 4.25–4.04 (m, 3H, H , H ), 3.78 (t, J = 8.1 Hz,
9
4
7
2
1
H, H ), 2.41–2.37 (m, 1H, H ), 2-36-2.30 (m 1H, H ), 2.21 (t, J = 3.0 Hz, 1H, H ), 2.00 (s, 3H,
3
5
1
6
13
CH3Ac). C NMR (101 MHz, D O) δ 176.7 (COOH), 175.1 (CO ),157.4 (C ), 134. 4 (C ),
2
Ac
N
8
1
19.2 (C ), 80.3 (C ), 71.4 (C ), 56.0 (C , C ), 28.2 (C ), 27.9 (C ), 22.6 (CH3Ac), 19.0 (C6).
9 4 7 2 3 5 1
+
HRMS (ESI) m/z calculated for [C13H21N4O4] 297.15573; found 297.15603.
1
Compound 19d. y = 82%. H NMR (400 MHz, CDCl ) δ 11.35 (s, 1H, NH), 8.64 (s, 1H,
3
NH(H2)), 6.21 (s, 1H, NH ), 4.95 (s, 1H, H ), 4.87 (s, 1H, H ), 4.82–4.72 (m, 1H, H ),
Ac
9a
9b
4
4
.20–4.06 (m, 1H, H ), 4.02–3.88 (m, 2H, H ), 3.79–3.69 (m, 1H, H ), 2.26–2.11 (m, 3H, H ;
2 7 3 1
13
H , H ), 1.94 (s, 3H, CH3Ac), 1.71 (s, 3H, CH3-10). C NMR (101 MHz, CDCl ) δ 172.3
5
6
3
(COOH), 170.8 (CO ), 156.7 (C ), 142.4 (C ), 112.7 (C ), 80.9 (C ), 73.6 (C ), 56.7 (C ), 53.7
Ac N 8 9 4 7 2
(
C ), 27.6 (C ), 27.4 (C ), 23.5 (CH ), 19.5 (C ), 18.6 (CH3-10).
3
5
1
3Ac
6
1
Compound 19e. y = 77% H NMR (400 MHz, D O) δ 4.15 (ddd, J = 7.9, 4.5, 1.5 Hz, 1H,
2
H ), 4.05 (dd, J = 8.7, 4.4 Hz, 1H, H ), 3.81–3.72 (m, 1H, H ), 3.72–3.58 (m, 2H; H ; H ),
4
2
8
3
7a
3
.58–3.48 (m, 2H, H , H ), 3.48–3.37 (m, 2H; H ), 2.37–2.25 (m, 1H; H ), 2.25–2.16 (m, 1H,
7b 9 9 5
13
H ), 2.12 (t, J = 3.1 Hz, 1H, H ), 1.89 (s, 3H, CH ). C NMR (101 MHz, D O) δ 175.9
1
6
3Ac
2
(
COOH), 174.4 (CO ), 156.7 (C ), 80.7 (C ), 80.7 (C ), 70.4 (C ), 70.4 (C ), 62.4 (C ), 62.4
Ac N 4 4 8 9 7
(
C ), 55.2 (C ), 55.2 (C ), 27.4 (C ), 27.1 (C ), 21.9 (CH ), 18.1 (C ). HRMS (ESI) m/z calcu-
7
2
3
5
1
3Ac
6
+
lated for [C13H23N4O6] 331.16121.; found 331.16121.
Computational studies
For modeling complexes between ligands and neuraminidase N1 we used two models, one
based on PDB entry 2HU4 with absence of the 150 cavity and one based on PDB entry 2HU0
16
with open access to the 150 cavity.[32] The Protein Preparation Wizard (PPW) was used for
curating original PDB entries.[33] The first pre-processing step PPW automatically assigned
bond orders of the ligand and add hydrogens to the protein. Second, the Epik tool was used for
estimation of the optimal protonation of the bounded ligand. In the final refinement step of
PPW the protein’s sidechains in ligand’s neighborhood are flipped so to optimize the number
of hydrogen bonding contacts. The complex was finally subjected to restrained minimization
PLOS ONE | https://doi.org/10.1371/journal.pone.0193623 February 28, 2018
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Molecules as candidate inhibitors of influenza A neuraminidase
using force field OPLS_2005.[34] In the case of PDB entry 2HU4, the Glide XP docking proto-
col with standard settings was used. The Schr o¨ dinger’s Induced Fit docking protocol has been
used in the case of initial docking of compound 4, (R = 4-phenylbenzyl). The model of the
receptor was based on PDB entry 2HU0,[32] which represents a complex between neuramini-
dase N1 and oseltamivir. The Induced Fit docking provided by Schr o¨ dinger Inc. was started by
docking of the ligand with Glide XP.[35]–[36] In order to provide the receptor site plasticity,
the procedure used reduced van der Waals radii, which were scaled by factor 0.5 for both ligand
and receptor. Up to 20 “soft docked” poses were then forwarded to the second step, which used
a Prime structure prediction for fine tuning ligand pose within receptor by reorienting nearby
side chains and subsequent minimization. In third step, each ligand is re-docked into its corre-
sponding low energy protein structures and the resulting complexes were ranked according to
Glide Score. Maestro GUI of the Schr o¨ dinger’s Suite has also been used for depiction of repre-
sentation poses of ligands within binding pocket of N1. Python script complex2img.py based on
OpenEye’s OEChem Toolkit has been used for creating interaction diagrams between N1 and
ligands.[37]
Crystal structure of 15c
Large crystals of compound 15c were obtained in a sample tube by slow evaporation from ace-
tone at T = 4˚C. Crystallization took approximately 24 h, after which a colorless transparent
prism with dimensions ꢀ 0.425 x 0.125 x 0.075 mm was selected for the single crystal X-ray
diffraction analysis.
The sample showed pleochroism (from colorless to purple) under polarized light. It was cut
from a greater crystal using a blade and polished by mechanical ablation in a drop of perfluori-
nated oil. Then, it was mounted on a glass capillary fiber with perfluorinated oil as glue. The
X-ray data collection was performed with a three-circle Bruker Smart APEX diffractometer
equipped with an APEXII CCD detector. Graphite-monochromated Mo Kα radiation was
employed throughout in conjunction with a nominal X-ray power of 50 kV x 30 mA. Raw data
were processed by means of the SAINT+ software[38] and corrected for beam anisotropy by
SADABS.[39] Finally, data were scaled and analyzed by XPREP[40] to determine the correct
space group symmetry. Eventually, a 99.8% complete set of 26608 diffraction amplitudes (4883
unique) was measured at room temperature up to a maximum Bragg angle of 54.9 deg. The
spherical atom formalism as implemented in SHELXL[41] was exploited to extract from the
diffraction data a sensible molecular model through nonlinear least-square refinements. The
agreement factors for the final least-squares model were R1(F) = 0.0660 for 2647 intense reflec-
tions (F > 4σ(F )), and 0.1261 for all the measured data, whereas the maximum and minimum
o
o
–3
Fourier residuals in the unit cell were as low as +0.35 /–0.20 eÁÅ . Crystal data of 15c:
C H N O , M = 382.45 amu, monoclinic, space group P2 /c, n˚ 14, centric, a = 21.2478(8)
19
30
2
6
1
3
Å, b = 5.1264(2) Å, c = 21.9154(8) Å, β = 116.550(2) deg V = 2135.4(2) Å , Z = 4, Z’ = 1; ρ
=
calcd
–3
–1
1.190 gÁcm , μ = 0.09 mm . CCDC 1562937 contains the supplementary crystallographic
data for this paper.[42] Full details of the X-ray structure determination are provided in the
Supporting Information.
Determination of NA Km and compounds Ki values in enzyme inhibition
experiments
A/Chicken/HongKong/G9/1997 H9N2 and A/Anhui/1/2005 H5N1 (wild-type and H274Y
mutation) were purchased from SinoBiochemical. Oseltamivir free acid and the substrate
2
-(4-methylumbelliferyl) α-D-acetylneuraminic acid sodium salt hydrate (MUNANA) were
purchased from Toronto Research Chemicals. The Michaelis constant, K , for MUNANA
m
PLOS ONE | https://doi.org/10.1371/journal.pone.0193623 February 28, 2018
18 / 22

Molecules as candidate inhibitors of influenza A neuraminidase
was obtained by measuring the initial slopes (Fluorescent Unit/min; FU/min) plotted as a
function of substrate concentration ranging from 2 μM to 1 mM in MES buffer (32.5 mM
2
-(N-morpholino)ethanesulfonic acid, 4 mM CaCl , pH 6.5) and fit using a nonlinear re-
2
gression function in GraphPad Prizm. The NA inhibition assay was performed according
to a standard method,[43] with minor modifications. Accordingly, the substrate MUNANA
is cleaved by the enzyme to yield a quantifiable fluorescent product, measured during the
assay. The fluorescence was assessed using an excitation wavelength of 365 nm and an emis-
sion wavelength of 450 nm and substrate blanks were subtracted from the sample readings.
Stock solutions of influenza viruses were freshly prepared by dissolution in MES buffer
(
32.5 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.5) at a concentration of 10 U/mL.
Oseltamivir free acid, MS-257 and all of the new compounds described herein were dis-
solved in water at 20 mM initial concentration. Dilutions of compounds were performed
in MES buffer (32.5 mM 2-(N-morpholino)ethanesulfonic acid, 4 mM CaCl , pH 6.5), rang-
2
ing from 2 mM to 5 pM and 6–10 serial dilutions were used; the concentrations depended
on the appropriate range for IC50 determination, assessed for each compound (inhibitor).
Compound 4a[11] was dissolved in DMSO at 20 mM initial concentration. Dilution of
inhibitor 4a was performed in MES buffer, ranging from 1 μM to 1 mM, not to exceed 5%
DMSO in the final experiment solution. For the inhibition assay, 100 μL of inhibitor solu-
tion (in MES buffer, 4 mM CaCl , pH 6.5),) and 5 μL of neuraminidase stock solution
2
(
10 U/mL) were preincubated for 45 min at 37˚C. The reaction was initiated by the addition
of the substrate MUNANA (8 μL of 2.5 mM solution in MES buffer, 4 mM CaCl , pH 6.5)
2
and fluorescence was measured for 5–15 min after addition of the substrate. Graphs of log10
concentration of inhibitor versus percent enzyme inhibition compared to the control were
plotted to calculate IC . The IC is the ligand concentration leading to 50% inhibition of
5
0
50
the rate of the reaction, compared to the control rate with no inhibitor, calculated using a
nonlinear regression function in GraphPad Prism. Inhibition constant K values were calcu-
i
lated using the formula K = IC / (1 + ([S]/Km)). The mean ± SD of K and K values were
i
50
m
i
obtained from two independent experiments in which the values were calculated by fitting
to dose-response curves.
Virus inhibition assay
Influenza A virus strains A/Puerto Rico/8/32 (PR8, H1N1) and A/Hong Kong/1/68 (HK1,
H3N2), both of which are mouse-adapted, were obtained from Dr. E. Brown (University of
Ottawa). The replication inhibition assay was performed according to Reference.[31] Briefly,
MDCK cells were infected with either PR8 or HK1 strain at 50 tissue culture infectious dose 50
(TCID50) in 96 wells with DMEM containing 0.0075% of trypsin (DMEM−trypsin) in the
presence or absence of test compounds. After 2 to 3 days of incubation virus replication was
visually determined either by cytopathic effects under light microscope or by immunofluores-
cent assay with chicken antiserum specific to PR8 strain (Charles River). The compounds were
initially dissolved in DMSO and then diluted at least 100 times in DMEM−trypsin. The test
was performed in quadruplicate. The inhibition effect was regarded positive when the virus
spread was not observed.
Supporting information
S1 File. Crystal structure of compound 15c.
(PDF)
PLOS ONE | https://doi.org/10.1371/journal.pone.0193623 February 28, 2018
19 / 22

Molecules as candidate inhibitors of influenza A neuraminidase
S2 File. Crystallographic information of compound 15c.
CIF)
(
1
13
S3 File. H- and C- NMR spectra of compounds.
PDF)
(
Author Contributions
Conceptualization: Cinzia Colombo.
Investigation: Cinzia Colombo, Črtomir Podlipnik, Leonardo Lo Presti, Masahiro Niikura.
Supervision: Andrew J. Bennet, Anna Bernardi.
Validation: Cinzia Colombo.
Writing – original draft: Cinzia Colombo.
Writing – review & editing: Cinzia Colombo, Andrew J. Bennet, Anna Bernardi.
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