Stereoisomers of oseltamivir-synthesis, in silico prediction and biological evaluation

Article abstract of DOI:10.1039/c6ob02673g

Oseltamivir is an important antiviral drug, which possess three chirality centers in its structure. From eight possible stereoisomers, only two have been synthesized and evaluated so far. We describe herein the stereoselective synthesis, computational activity prediction and biological testing of another three diastereoisomers of oseltamivir. These isomers have been synthesized using stereoselective organocatalytic Michael addition, cyclization and reduction. Their binding to viral neuraminidase N1 of influenza A virus was evaluated by quantum-chemical calculations and their anti-influenza activities were tested by an in vitro virus-inhibition assay. All three isomers displayed antiviral activity lower than that of oseltamivir, however, one of the stereoisomers, (3S,4R,5S)-isomer, of oseltamivir showed in vitro potency towards the Tamiflu-sensitive influenza viral strain A/Perth/265/2009(H5N1) comparable to Tamiflu.

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Stereoisomers of oseltamivir – synthesis, in silico prediction and
biological evaluation
Viktória Hajzer,a Roman Fišera,*a Attila Latika,a Július Durmis,a Jakub Kollár,b,c Vladimír Frecer,d,e
Zuzana Tučeková,f Stanislav Miertuš,e,f František Kostolanský,g Eva Varečková,g and Radovan
Šebesta*h
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Oseltamivir is an important antiviral drug, which possess three chirality centers in its structure. From eight possible
stereoisomers, only two have been synthesized and evaluated so far. We describe here stereoselective synthesis,
computational activity prediction and biological testing of another three diastereoisomers of oseltamivir. These isomers
have been synthesized using stereoselective organocatalytic Michael addition, cyclization and reduction. Their binding to
viral neuraminidase N1 of influenza A virus was evaluated by quantum-chemical calculations and their anti-influenza
activities were tested in in vitro virus-inhibition assay. All three isomers displayed antiviral activity lower than that of
oseltamivir, however, one of the stereoisomers (3S,4R,5S)-isomer of oseltamivir showed in vitro potency towards the
Tamiflu-sensitive
influenza
viral
strain
A/Perth/2009/H1N1
comparable
to
Tamiflu.
are essential for the release and spread of newly formed viral
particles from the infected host cells.3 Due to the fear of
potential pandemic flu outbreak, there has been a large
interest of both academic and industrial laboratories in the
syntheses of oseltamivir. Currently, oseltamivir is
manufactured by Roche starting from shikimic acid.4 Apart
from this procedure, however, there was a large array of
diverse syntheses developed.5 Due to their efficiency and user
friendliness, organocatalytic procedures are particularly
appealing. Enders´ discovery that prolinol silyl ether can
assemble cyclohexene core in one reaction step from an enal,
nitroalkene and aldehyde, served as inspiration for
development of organocatalytic syntheses of oseltamivir.6
Based on this precedent, Hayashi has developed a short
synthesis of oseltamivir using prolinol silyl ether-catalyzed
Introduction
Influenza is one of the most common and dangerous viral
respiratory diseases. According to the World Health
Organization (WHO), seasonal influenza epidemics are
responsible for up to 500.000 deaths each year.1
Oseltamivir phosphate (Tamiflu) is one of the most effective
antiviral drugs currently available on the market. It is included
on the WHO´s list of essential medicines,2 and was FDA
approved for the treatment and prophylaxis of severe or
complicated infections due to influenza A and B viruses.
Oseltamivir phosphate is a pro-drug of the active metabolite,
oseltamivir carboxylate, which is a selective inhibitor of viral
neuraminidase enzymes of the influenza virus. Neuraminidases
that exist in
a variety of subtypes named N1-N9 are
Michael addition to nitroacrylate as
a
key step.7 This
glycoproteins located on the surface of influenza virions and
methodology necessitated transformation of the ester group
to amino group via potentially hazardous Curtius
rearrangement. To circumvent this issue, use of Michael
acceptor, which already contains acetamido group has been
proposed.8 Further improvements has recently been suggested
by Hayashi.9 Our laboratory also investigated the possibilities
of using acetals as a starting material in the Michael addition.10
Organocatalytic Diels-Alder reaction can also serve as a basis
for oseltamivir synthesis.11
Drug resistance of influenza viruses is becoming a serious
problem, therefore, syntheses of new antiviral medicines
remain an important goal of the medicinal chemistry research.
Oseltamivir contains three stereogenic centers and thus it can
exist in the form of eight stereoisomers. Oseltamivir
phosphate itself has (3R,4R,5S) configuration. Another
stereoisomer of oseltamivir has been synthesized and its
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properties were investigated by Sartori, Zanardi and
coworkers.12 Other stereoisomers of oseltamivir have not been
described so far. There is
a possibility that different
stereoisomers of oseltamivir will give rise to altered
interactions with the chiral environment of its biological
target, the viral neuraminidase, and thus display different,
hopefully improved, antiviral activity. In this context, we
decided to synthesize three so far untested stereoisomers of
oseltamivir, predict their inhibitory activities through
molecular modeling and evaluate their antiviral efficiency
using in vitro biological testing (Figure 1).
Scheme 1.
The first step in the organocatalytic synthesis of oseltamivir
and its stereoisomers is a Michael addition. The reaction was
performed with aldehyde
of mandelic acid as acidic additive and Jørgensen-Hayashi
catalyst ((S)-C1). The corresponding Michael adducts were
2 and nitroalkene 3 in the presence
5
isolated as a mixture of syn and anti-diastereomers (Scheme
2). The diastereomers were then separated by flash
chromatography. Both diastereomers were obtained in high
enantiomeric purity, e.r. 97:3 and 98:2 respectively.
Figure 1. Chemical structures of synthesized stereoisomers of oseltamivir.
Results and discussion
Synthesis
Starting materials for the syntheses of oseltamivir isomers are
not commercially available and were prepared according to
previously described procedures. Aldehyde
2 was synthesized
Scheme 2.
by nucleophilic epoxide opening of oxiran-2-ylmethanol with
pentan-3-ol followed by periodate cleavage of the
intermediary diol (Scheme 1a).8b Nitroalkene
3
was
synthesized from nitromethane, N-methylaniline and
triethylorthoformiate (Scheme 1b).13 Phosphonate
was
Michael adduct (2R,3S)-
into a cyclohexene derivative
Michael addition and Horner-Wadsworth-Emmons reaction
(Scheme 3). The cyclohexene derivative was obtained as a
mixture of two diastereomers with d.r. 2:1 (5R/5S). These
5
and phosphonate
4, were assembled
6
via a combination of another
4
6
synthesized by aldol condensation of ethyl 2-
(diethoxyphosphoryl)acetate with formaldehyde (Scheme
1c).14
diastereoisomers were
chromatography.
again separated by
flash
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O
CO2Et
AcHN
K2CO3
18-crown-6
CH2Cl2
(3S,4R,5S)-6
O
CHO
+
NO2
+
4
d.r. 4:1, 55%
CO2Et
AcHN
3h, 70°C
NO2
O
(2R,3R)-5
AcHN
(3S,4R,5R)-6
NO2
NHAc
Scheme 3.
CO2Et
NO2
PentO
Reduction of the nitro group by zinc in aqueous acetic acid
yielded oseltamivir (1). Starting from isomer (3R,4R,5S)-6,
NOE
oseltamivir itself (3R,4R,5S)-1 was synthesized.
(3 ,4 ,5 )-
Scheme 5.
Similarly to previous isomers, reduction of the nitro group was
performed by zinc in acetic acid. In this way, reduction of
cyclohexene derivatives (3S,4R,5S) and (3S,4R,5R)-
corresponding oseltamivir diastereomers (3S,4R,5S) and
(3S,4R,5R)- (Scheme 6). Relative configurations of amines
were determined by NOESY and qDQCOSY spectra. A NOE
crosspeak between H-3 and H-5 was observed in (3S,4R,5R-
6 afforded
1
1
1,
similar with derivatives (3S,4R,5R)-6.
Scheme 4.
Starting from (R,R)-diastereomer of the Michael adduct
cyclization with phosphonate provided another two
diastereoisomers of cyclohexene derivative
Cyclization of (R,R)- provided derivative
5,
4
6
6
(Scheme 5).
with higher
5
diastereomeric ratio d.r. of 4:1 (5S/5R) than the corresponding
reaction of (R,S)- . The diastereoisomers were separated by
flash chromatography. Enantiomeric purities of these isomers
were slightly lower; e.r. 74:26 (3S,4R,5S)- and e.r. 90:10 for
(3S,4R,5R)- isomer. Lowering of the enantiomeric purity of
compounds could have been caused by non-catalysed partial
5
6
Scheme 6.
6
6
Oseltamivir is actually a pro-drug, which is hydrolyzed in acidic
media in stomach. Therefore, for biological testing, all
retro-Michael reaction during cyclization process. Relative
configuration at the C-5 carbon has been determined by
qDQCOSY and NOESY spectroscopy. An important NOE
crosspeak between H-3 and H-5 hydrogens, which has been
oseltamivir isomers
1 were hydrolyzed under basic conditions
to corresponding carboxylic acids
7
(Scheme 7).
observed, can be possible only in (3S,4R,5R)-6 derivative
(Scheme 5).
Scheme 7.
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Synthesis of remaining undescribed oseltamivir stereoisomers
would be possible by application of the opposite enantiomer
of Jørgensen-Hayashi catalyst (R)-C1 in the Michael addition
(Scheme 2). From the corresponding Michael adducts and by
following cyclization and reduction steps (Scheme 3 and 4), it
should be possible to prepare remaining undescribed
oseltamivir stereoisomers.
Molecular modelling
Molecular modelling and computational prediction of stability
of stereoisomers of the active metabolite of oseltamivir in
aqueous solution (zwitterionic oseltamivir carboxylate (
7
), Figure 3. 3D-structures of two enantiomers of oseltamivir carboxylate form mirror
images with identical total energies.
Figure 2) was carried out by means of density functional
theory (DFT). Calculations suggested relative stability of the
eight possible stereoisomers as shown in Table 1.15
In the most stable enantiomers V. (3S,4R,5R)-
7 and VI.
(3R,4S,5S)-7 of the oseltamivir carboxylate the cyclohexene
5
4
ring forms two half-chair conformations H4 and H5 with both
the 3-oxypentyl group and 5-amino groups oriented to
pseudo-axial positions, while the 4-N-acetyl group is in
pseudo-equatorial position (Figure 4). This configuration
allows formation of two intramolecular hydrogen bonds
between the carbonyl oxygen of the 4-N-acetyl and hydrogen
of 5-amino group (interatomic distance of 1.67 Å) and
between oxygen of the 3-oxypentyl group and hydrogen of 5-
Figure 2.
amino group (2.11 Å). On the other hand, the 1.8 kcal
⋅
mol-1
of
Table 1. Predicted relative stabilities ΔΔEtot,sol of stereoisomers of oseltamivir
less stable stereoisomers I. (3R,4R,5S)- and II. (3S,4S,5R)-
7
7
carboxylate in water.
the oseltamivir carboxylate also form 5H4 and 4H5 half-chair
ring conformations, however, with all three hetero-
substituents placed in pseudo-equatorial positions (Table 1). In
this configuration, only one hydrogen bond between the
carbonyl oxygen of the 4-N-acetyl and hydrogen of the 5-
amino group (1.82 Å) can be formed, which results in a lower
predicted stability of these stereoisomers. Similar observation
Cyclohexene ring
Stereoisomer a
ΔΔEtot,sol [kcal⋅mol-1] b
puckering c
5H4
I. (3R,4R,5S)-7 d
II. (3S,4S,5R)-7
III. (3S,4R,5S)-7
IV. (3R,4S,5R)-7
V. (3S,4R,5R)-7
VI. (3R,4S,5S)-7
VII. (3S,4S,5S)-7
VIII. (3R,4R,5R)-7
0.0
0.0
4H5
5H4
4H5
5H4
4H5
4H5
5H4
-1.5
-1.5
-1.8
-1.8
-0.8
-0.8
was made by Sartori et al.12 who determined from
metadynamics simulation that free energy difference between
a
stereoisomers I. (3R,4R,5S)-
7
and VI. (3R,4S,5S)-
mol-1.
7
of
oseltamivir is of the order of 1 kcal
⋅
a 3 chiral centers = 4 diastereoisomers of 2 enantiomers each, Figure 3; b ΔΔEtot,sol
– relative difference in total energies of solvated stereoisomers computed for
optimized molecular geometries (zwitterions of a total charge q = 0, Figure 2) by
DFT method (DFT-B3LYP-SCRF/6-31+G*) using Gaussian 09 18. Stereoisomer I.
(3R,4R,5S)-oseltamivir carboxylate was taken as the reference structure. Higher
c
value of ΔΔEtot,sol means lower stability of the pertinent stereoisomer;
cyclohexene ring puckering in the notation of Sartori et al.12 The conformer 4H5
has its carbon C4 placed above the plane defined by the ring double bond and
carboxylic group C2=(C1-COO-)-C6 (Fig. 3 and 4) while carbon C5 is positioned
d
below this plane; configuration of stereoisomer I. (3R,4R,5S)-7 (oseltamivir)
carboxylate corresponds to that of Tamiflu.
Figure 4. 3D-structure, cyclohexene ring puckering and intramolecular hydrogen
bonding pattern in oseltamivir stereoisomers (hydrogen bonds are shown as yellow
lines, some nonpolar hydrogens are not shown for better clarity,
equatorial).
a – axial, e –
To determine the absolute configuration of chiral compounds,
theoretical prediction of electronic CD spectra (ECD) are
frequently used.16 We have used time-dependent DFT method
to derive ECD spectra of eight stereoisomers of oseltamivir.
These theoretical ECD spectra were compared with the
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experimental spectrum of oseltamivir phosphate in water employed to derive the binding energy of inhibitors to the
published by Górecki, Table 2, Figure 5.17
relaxed enzyme using supermolecular approach described by
Frecer et al.19 Eleven amino acid residues surrounding the
bound inhibitor, oseltamivir itself and two structural water
molecules were included into the quantum motif, while the
remaining 376 residues present in the crystal structure of the
viral neuraminidase were described by molecular mechanics
and OPLS-2005 force filed.18b, 20
Table 2. Characteristic peaks in experimental and computed ECD spectra of
stereoisomers of oseltamivir in water.
Δε
λmax (nm) b
Stereoisomer
(dm3⋅mol-1·cm-
1
a
)
5.8
-9.8
2.6
12
-7
191
224
253
190
230
290
190
220
250
190
220
250
c
Table 3. Predicted relative binding affinities (ΔΔEbin,sol) of stereoisomers of oseltamivir
I. (3R,4R,5S)-7.H3PO4
carboxylate to neuraminidase N1 a of influenza A virus b
.
ΔΔEbin,sol
[kcal⋅mol-1] c
I. zwitterionic (3R,4R,5S)-7 d
V. zwitterionic (3S,4R,5R)-7 d
Stereoisomer
3
I. (3R,4R,5S)-7 d
II. (3S,4S,5R)-7
III. (3S,4R,5S)-7
IV. (3R,4S,5R)-7
V. (3S,4R,5R)-7
VI. (3R,4S,5S)-7
VII. (3S,4S,5S)-7
VIII. (3R,4R,5R)-7
0
27
-32
54
12
4
-25
37
-3
25
-37
3
VI. zwitterionic (3R,4S,5S)-7 d
-87
1
a
b
c
Δε – molar circular dichroism; λmax – wavelength of peak; ECD spectrum in
water recorded by Górecki;17 ECD spectrum computed by time-dependent DFT
d
a crystal structure of neuraminidase of type N1 was taken from Protein Data Bank
method (TDDFT-B3LYP-SCRF/6-31+G*) in water.15
(PDB entry 2HU0 23); b influenza virus – A/Viet Nam/1203/2004(H5N1); c ΔΔEbin,sol
relative differences in binding energies were computed as19
ΔΔEbin,sol
Etot,sol[N1:Oi] - Etot,sol[N1] - Etot,sol[Oi]) -
-
:
=
(
Δ
Δ
Δ
Δ
Ebin,sol,Ref for optimized solvated
molecular geometries of oseltamivir carboxylate stereoisomers [Oi],
enzyme:oseltamivir complexes [N1:Oi] and enzyme [N1] using QSite18b at the DFT-
d
B3LYP-PBSA/6-31+G*:OPLS-2005 level of theory; configuration of stereoisomer
I. (3R,4R,5S) of oseltamivir carboxylate, which corresponds to the absolute
configuration of Tamiflu, was used as the reference.
Predicted relative binding affinities in solution (ΔΔEbin,sol) of
stereoisomers of oseltamivir carboxylate to the neuraminidase
N1 (Table 3) indicate, as expected, that the enzyme inhibition
is stereospecific. Moreover, they suggest that binding of two
stereoisomers of oseltamivir to N1 will be stronger than that of
the active metabolite of Tamiflu I. (3R,4R,5S)-7-oseltamivir.
The calculations predict that the stereoisomers of oseltamivir
will inhibit the neuraminidase N1 of the influenza A virus in the
following descending order:
VII. (3S,4S,5S)-
> VIII. (3R,4R,5R)-
> II. (3S,4S,5R)- > IV. (3R,4S,5R)-
According to the calculations (Table 3), two stereoisomers of
oseltamivir VII. (3S,4S,5S)- and III. (3S,4R,5S)- can be
7
> III. (3S,4R,5S)-
7
> I. (3R,4R,5S)-
7 >
7
> VI. (3R,4S,5S)-
7
> V. (3S,4R,5R)-
7 >
7
7
Figure 5. Computed ECD spectra for three stereoisomers of oseltamivir carboxylate.
7
7
Comparison of shape of the constituent circular dichroism
Cotton effects suggested that the experimental ECD spectrum
of oseltamivir phosphate recorded in water17 matches the
expected to inhibit neuraminidase N1 stronger than Tamiflu
itself. Relatively high differences in the calculated ΔΔEbin,sol are
related to binding of polar oseltamivir stereoisomers
(zwitterions) to highly polar binding site of the enzyme (6
cationic 5 anionic residues and 2 water molecules within 4 Å
distance from the bound ligand). Therefore, even small
changes in molecular structure and geometry may result in
relatively large changes of the electrostatic component of the
interaction energy and solvation energies. In addition, chirality
of the three stereocenters in combination with the
cyclohexene ring puckering and oseltamivir recognition orients
the heterosubstituents into different directions, which results
calculated spectrum of VI. (3R,4S,5S)-
also the spectrum of stereoisomer I. with the configuration
(3R,4R,5S)- identical to that of Tamiflu.
7 and, to some extent,
7
Computational prediction of binding affinity of individual
stereoisomers of oseltamivir carboxylate to viral
neuraminidase N1 subtype of influenza
A virus (Viet
Nam/1203/2004/H5N1, PDB entry 2HU0 23) was carried out
by docking of the inhibitors to the active site in the relaxed
crystal structure of N1. Then, the hybrid quantum
mechanical/molecular mechanical (QM/MM) method18 was
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in close contacts with different residues of the N1 binding site The antiviral efficacy of three stereoisomers ((3R,4R,5R)-7,
(Fig. 6 and 7).
(3S,4R,5S)-
7,
and (3S,4R,5R)-7) of oseltamivir carboxylate
((3R,4R,5S)-7), prepared by above described procedures, has
been examined in vitro by rapid culture assay (RCA) on MDCK
cells using influenza A virus of H3N2 subtype and Tamiflu
resistant/sensitive variants of pandemic 2009 virus of H1N1
subtype. The most significant inhibition of virus replication
A/Miss (H3N2) (>90%) has been measured using the virus
dose 34 pfu/100 µl/well at the 39.06 nM concentration of
compound (3R,4R,5S)-7-oseltamivir. All other compounds
revealed lower inhibition efficacy (Table 4, Fig. 8). The
inhibition of virus replication by 50% allowed the
differentiation of antiviral potential of these compounds in the
following sequence: (3R,4R,5S)-
7 > (3R,4R,5R)-7 > (3S,4R,5S)-7
Figure 6. Superimposed structures of
8 stereoisomers of oseltamivir carboxylate
> (3S,4R,5R)-
7.
docked to the binding site of N1. Hydrogen atoms are not shown. The carboxylic group
of oseltamivir (on the right) is recognized by and strongly interacts with 3 arginines
(Arg118, Arg292 and Arg371, Fig. 7).21 This interaction defines its position and
orientation at the N1 binding site, it also co-determines relative positions of the
heterosubstituents. Side view of the superimposed molecules with the cyclohexene
ring double bond and carboxylic group C2=(C1-COO–)-C6 positioned in the horizontal
plane. Stereoisomer I. (3R,4R,5S)-7-osltamivir is shown in yellow color.
Table 4. Efficacy of inhibition of A/Mississippi/1/85(H3N2) virus replication by tested
compounds.
Tested
Concentration of tested substance (nM)
substance
90% virus inhibition
50% virus inhibition
(3R,4R,5S)-7
(3R,4R,5R)-7
(3S,4R,5S)-7
(3S,4R,5R)-7
39.1
1250
5000
5000
1.22
9.76
39.1
78.1
Inspection of enzyme-inhibitor interactions at the active site of
N1 show that major differences in the inhibitor binding
between stereoisomers VII. (3S,4S,5S)-7 and I. (3R,4R,5S)-7
occur in the hydrogen bonding pattern at the 5-amino group
(Fig. 7).
To learn whether some of above mentioned stereoisomers
inhibit replication of Tamiflu-resistant influenza A viruses, we
used Tamiflu-sensitive and Tamiflu-resistant variants of the
human pandemic 2009 virus A/Perth of H1N1 subtype (TSV-
A/Perth and TRV-A/Perth). The replication of TRV-A/Perth
virus was inhibited only up to 60% at the highest tested
concentration (i.e. at 5000 nM) of (3R,4R,5S)-7-oseltamivir-
carboxylate and none of the examined stereoisomers
exceeded its inhibition efficacy at 5000 nM (Table 5). No
inhibition of TRV virus was obtained at 625 nM concentration
of Oseltamivir stereoisomers. In contrast, all tested
compounds in the used concentration range, inhibited the
replication of TSV-A/Perth virus. The order of substances
according to their antiviral efficacy against TSV-A/Perth was
the same as was with A/Miss virus. Their antiviral efficacy on
A/Perth virus replication descended equally as in the case of
A/Miss, i.e. from (3R,4R,5S)-7, which was the most efficient, to
(3S,4R,5R)-
7, the antiviral efficacy of which was the lowest.
Figure 7. Superimposed structures of two stereoisomers of oseltamivir carboxylate I.
(3R,4R,5S)-7 (yellow color) and VII. (3S,4S,5S)-7 docked to N1 binding site and
optimized by QM/MM method.18 Yellow dashed lines represent intermolecular
hydrogen bonds. Nonpolar hydrogens were omitted for better clarity.
The calculations suggest that the synthesized stereoisomer
(3S,4R,5S)-
1 of oseltamivir (Figure 1) or III. (3S,4R,5S)-7
oseltamivir carboxylate (Table 3) should represent a more
potent inhibitor of neuraminidase N1 than the active
metabolite of Tamiflu. Whether this prediction will be carried
over also to in vitro antiviral effect on the influenza virus
infected cells was determined in biological tests.
Biological evaluation
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(A/VietNam/1203/2004(H5N1))
were
studied
by
computational methods using a combination of DFT and
molecular mechanics calculations. According to these
calculations two stereoisomers of oseltamivir showed even
greater binding affinity to the N1 neuraminidase than
oseltamivir itself. In vitro antiviral assays revealed that all three
synthesized isomers have antiviral activity, but it was lower
than that of oseltamivir. However, the stereoisomer III.
(3S,4R,5S)-7 with predicted elevated inhibitory activity against
neuraminidase N1 showed in vitro inhibitory potency towards
the Tamiflu-sensitive viral strain A/Perth/2009/H1N1
comparable to Tamiflu. Further research of remaining
stereoisomers of oseltamivir is underway in our laboratories.
Experimental
Synthesis
The solvents were purified by standard methods.22
Commercially available chemicals were used without further
purification. NMR spectra were recorded on Varian NMR
1
System 300 or 600 instrument (300 MHz for H, 151 MHz for
13C). Chemical shifts (δ) are given in ppm relative to
tetramethylsilane. Specific optical rotations were measured on
Jasco P-2000 instrument and are given in deg cm3 g–1 dm–1.
Column chromatography was performed on silica gel 60A
(0.04-0.6 mm). Thin-layer chromatography was performed on
Merck TLC-plates silica gel 60, F-254. Enantiomeric excesses
were determined by HPLC using Chiralpak and Chiralcel
columns (Daicel Chemical Industries) using n-hexane/propan-
2-ol as a mobile phase and UV detection.
Figure 8. Comparison of in vitro antiviral efficacy of Oseltamivir and its stereoisomers.
Examination of antiviral effect of compounds (3R,4R,5S)-7, (3R,4R,5R)-7, (3S,4R,5S)-7,
and (3S,4R,5R)-7 compared to the virus control (no inhibitor) by RCA on the replication
of influenza virus A/Mississippi /1/85 (H3N2). MDCK cells were infected by virus dose
34 pfu/sample. Left column: control- 0% inhibition of virus replication, medium
column-≥90% inhibition, right column-50% inhibition.
Synthetic procedures and characterization data
2-(Pentan-3-yloxy)acetaldehyde (2)
Table 5. Inhibition of TSV-A/Perth virus and TRV-A/Perth virus replication by tested
compounds.
DIBAL (800 mL, 962 mmol, 20 wt.% in hexane) was added
dropwise into cooled solution of pentan-3-ol (97.8 g, 1.11 mol,
115 mL) in CH2Cl2 (540 mL) at 0°C for 40 min. The solution was
stirred 30 min at 5°C and next 30 min at room temperature.
Subsequently, oxiran-2-ylmethanol (54.8 g, 740 mmol, 48.9
mL) was added into reaction mixture which was stirred at
room temperature for 96 h. The reaction was stopped by
pouring reaction mixture into ice with concentrated HCl. The
solution was washed with saturated solution of NaCl after
warmed at room temperature. After extraction by EtOAc, the
organic layers were dried over Na2SO4. The solid phase was
filtered off and the filtrate was concentrated under vacuum.
The crude reaction mixture was purified by reduced pressure
distillation (T = 85°C, 1.2 mbar) to provide 3-(pentan-3-
yloxy)propane-1,2-diol as a colorless oil; yield 23.4 g (19.5 %).
Compound
Inhibition of replication (%)
TSV-A/Perth virusa TRV-A/Perth virusb
5000 nMc 625 nMc
60 50
5000 nMc
100
625 nMc
90
(3R,4R,5S)-7
(3R,4R,5R)-7
(3S,4R,5S)-7
(3S,4R,5R)-7
100
80
50
10
0
0
0
0
100
85
100
60
a
b
c
TSV- Tamiflu-sensitive virus; TRV- Tamiflu-resistant virus; concentration of
tested substance.
Conclusions
We have described stereoselective synthesis, computational
evaluation and biological testing of three, so far unknown and
untested diastereoisomers of oseltamivir. These isomers have
been synthesized using stereoselective organocatalytic
Michael addition, followed by cyclization via another Michael
addition and Horner-Wadsworth-Emmons reaction. The last
step in the synthesis was nitro group reduction, which
afforded stereoisomers of oseltamivir, or after basic hydrolysis
free carboxylates for biological testing. Binding affinities of
1H NMR (300 MHz, CDCl3):
δ 3.89-3.79 (m, 1H), 3.77 – 3.44 (m,
4H), 3.17 (quint., J = 5.8 Hz, 1H), 2.98 (br s, 1H), 2.75 (br s, 1H),
1
1.60 – 1.43 (m, 4H), 0.96 – 0.83 (m, 6H). H NMR data agree
with those in the literature.8b 13C NMR (75 MHz, CDCl3):
70.6, 70.2, 64.2, 25.7, 9.5.
δ 82.7,
The solution of NaIO4 in water (5.56 g, 26.0 mmol dissolved in
28 mL water) was added dropwise into the mixture of silicagel
(50.0 g) in CH2Cl2 (260 mL). The resulting mixture was stirred
for 15 min at RT. 3-(pentan-3-yloxy)propane-1,2-diol (3.24 g,
these
derivatives
to
neuraminidase
N1
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20.0 mmol) in CH2Cl2 (20 mL) was added into the reaction column chromatography (SiO2, hex/EtOAc 1:1), to provide
mixture. The mixture was stirred at room temperature for 3h. beige crystalline substance; yield 2.3 g (67 %).
Subsequently, the silicagel from reaction mixture was filtered mp = 109–113°C (106 - 107°C).8a 1H NMR (300 MHz, CDCl3): (Z)-
off over frit. The filtrate was washed with saturated solution isomer
Na2S2O3. The solvent was evaporated under reduced pressure (d, J = 6.9 Hz, 1H), 2.28 (s, 3H). 1H NMR data for cis-isomer
δ 10.40 (s, 1H), 7.59 (dd, J = 12.0 Hz, 6.9 Hz, 1H), 6.61
and crude reaction mixture was purified by distillation under agree with those in the literature.14 13C NMR (75MHz, DMSO):
reducer pressure (T = 55°C, 12 mbar) to provide colorless oil; (Z)-isomer
δ
168.3, 131.4, 118.5, 23.7.
1H NMR (300 MHz, DMSO): mixture (Z)/(E
δ 9.76 (t, J = 1.1 Hz, 1H), 4.06 (d, J = 2Hcis/trans), 8.27 (d, J = 12 Hz, 1Htrans), 7.59 (d, J = 7 Hz, 1Hcis),
yield 1.8 g (69 %).
1H NMR (300 MHz, CDCl3):
) δ 11.09 (br s,
1.1 Hz, 2H), 3.23 (quint, J = 5.8 Hz, 1H), 1.56 (dq, J = 7.6 Hz, 3.2 7.32 (d, J = 12 Hz, 1Htrans), 6.75 (d, J = 7 Hz, 1Hcis), 2.26 (s, 1Hcis),
Hz, 4 H), 0.93 (t, J = 7.4 Hz, 6H). 1H NMR data agree with those 2.12 (s, 3Htrans).
in the literature.7a 13C NMR (75MHz, CDCl3):
25.6, 9.4.
δ201.7, 83.5, 74.5,
Ethyl-2-(diethoxyphosphoryl)prop-2-enoate (4)
The solution of formaldehyde (46.7 g, 560 mmol, 42.5 ml, 36%
in H2O) with piperidine (2.55 g, 30.3 mmol, 2.97 mL) was
(Z)-2-Nitroethen-1-amine
The mixture of nitromethane (122 g, 2.0 mol, 108 mL), N- refluxed in MeOH (600 mL) for 30 min. Ethyl-2-
methylaniline (42.9 g, 400 mmol, 43.3 mL), PTSA (2 g, 10.5 (diethoxyphosphoryl) acetate (44.8 g, 200 mmol, 40 mL) was
mmol) and (diethoxymethoxy)ethane (119 g, 800 mmol, added into the reaction mixture at room temperature. The
133 mL) was refluxed for 8 h under inert atmosphere (Ar). The mixture was refluxed for next 65 h. Piperidine (0.5 mL) was
reaction was cooled at room temperature and stirred added into RM after 24h and 48h. The solvent was evaporated
overnight. At the end reaction was finished by solvent from RM under reduced pressure. Subsequently, crude
evaporating from reaction mixture under reduced pressure. product was diluted with CHCl3 (100 mL), extracted with CHCl3.
Hexane (200 mL) was slowly added into concentrated mixture The organic layers were dried over Na2SO4. The solvent was
and
N-methyl-N-[(E)-2-nitroethenyl]aniline
began
to evaporated under reduced pressure. H3PO4 (4 mL, conc.) was
crystallize. The precipitate was filtered off, washed with added into the crude product and it was purified by vacuum
hexane (100 mL) and dried under reduced pressure for 60 min distillation (T = 90°C, 0.3 mbar), to provide compound
4 as s
at 40°C. N-methyl-N-[(E)-2-nitroethenyl] aniline was obtained colorless liquid; yield 20 g (42 %).
as pale yellow crystalline substance; yield 53 g (74 %).
1H NMR (300 MHz, CDCl3):
δ
7.00 (dd, J = 42.0, 1.8 Hz, 1H), 6.75
8.49 (dd, J = 20.4, 1.8 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.25 – 4.08
163.6
δ 147.2, 129.9, 126.4, (d, JP,C = 15.8 Hz), 143.1 (d, JP,C = 4.7 Hz), 133.2 (d, JP,C = 185.8
mp = 89.4 – 91.1°C (94°C).23 1H NMR (300 MHz, CDCl3):
δ
(d, J = 11 Hz, 1H), 7.62 – 7.14 (m, 5H), 6.85 (d, J = 10.9 Hz, 1H), (m, 4H), 1.41 – 1.29 (m, 9H). 13C NMR (75 MHz, CDCl3):
3.34 (s, 3H). 13C NMR (75 MHz, CDCl3):
120.8, 116.5, 77.1. Spectral data corresponds with literature.24 Hz), 63.5(d, JP,C = 5.9 Hz), 62.6 (d, JP,C = 5.9 Hz), 61.4, 16.2 (d,
N-methyl-N-[(E)-2-nitroethenyl]aniline (21.0 g, 118 mmol) was JP,C = 6.2 Hz), 13.9. Spectral data agree with those in the
dissolved in CHCl3 (430 mL). The mixture was cooled to 0°C and literature.14
δ
saturated by gaseous NH3 (14.7 g, 861 mmol) for 1 h at range
from 0°C to 5°C. The final saturated mixture was stored in N-[(2R,3R)-1-Nitro-4-oxo-3-(pentan-3-yloxy)butan-2-
refrigerator overnight. The crystals were filtered off next day yl]acetamide (
and washed by CHCl3. The crystalline substance was dried 2-(pentan-3-yloxy)acetaldehyde (
under reduced pressure at room temperature to provide a mL), cooled water (47 mL), mandelic acid (441 mg, 2.90 mmol)
5)
2) (2.83 g, 21.8 mmol, 2.83
beige crystalline substance; yield 9.5g (91 %).
mp= 100.1–101.2°C (101°C).13 1H NMR (300 MHz, CDCl3):
and cooled solution of (2S)-2-(diphenyl[(trimethylsilyl)oxy]
methyl)pyrrolidine (C1) (472 mg, 1.45 mmol) in 5 mL CHCl3
δ
8.44 (br s, 1H), 6.87 – 6.70 (m, 1H), 6.48 (d, J = 6.0 Hz, 1H), 5.65 were
(br s, 1H). 1H NMR data agree with those in the literature.8b nitroethenyl]acetamide (
13C NMR (75 MHz, DMSO):
151.2, 147.1 (CH(Z)), 114.0, 109.7 mL) at 0°C. Reaction mixture was stirred 2h at 0°C. The
(CH(E))(Note: 2-nitroethen-1-amine isomerizes in polar reaction was stopped by adding of saturated solution of NH4Cl
added
into
suspension
of
N-[(Z)-2-
3
) (1.89 g, 14.5 mmol) in CHCl3 (42
δ
solvents to a mixture of E and Z-isomer).
into reaction mixture. Mixture was stirred for 15 min,
extracted with CHCl3. Organic layers were dried over Na2SO4.
The solvent was evaporated under reduced pressure. The
N-[(Z)-2-Nitroethenyl]acetamide (
3)
Pyridine (8.26 g, 104 mmol, 8.4 mL) and acetic anhydride (5.33 crude product was purified by column chromatography (SiO2,
g, 52.2 mmol, 4.94.mL) were added into the cooled mixture of hex/EtOAc 1:1), to provide compound as a pale yellow oil -
(Z)-2-nitroethen-1-amine (2.30 g, 26.1 mmol), DMAP (0.64 g, mixture of isomers 1:1 (syn/anti); yield 2.5 (65 %).
5
g
5.22 mmol) at 0°C. The reaction mixture was warmed to room Diastereoisomers were isolated: (2R,3R) isomer 96% purity (dr
temperature and stirred overnight. The reaction was finished 1:28 syn/anti, er 98:2); (2R,3S) isomer 89% purity (dr 8:1
by dilution with CH2Cl2 (100 mL), washing with distillated water syn/anti, er 97:3).
(2 x 100 mL), then by NaCl saturated solution (150 mL). The (2R,3S)-5 isomer: [α]D23= + 1.1 (c = 1.0, CHCl3, 17:1 syn/anti).
organic layers were dried over Na2SO4. The solvent was 1H NMR (300 MHz, CDCl3):
δ 9.64 (s, 1H), 6.09 (d, J = 8.7 Hz,
evaporated and the crude reaction mixture was purified by 1H), 5.13 – 4.97 (m, 1H), 4.58 (d, J = 6.6 Hz, 2H), 4.09 (d, J = 3.3
8 | J. Name., 2012, 00, 1-3
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Hz, 1H), 3.40 (quint., J = 5.8 Hz, 1H), 1.99 (s, 3H), 1.64 – 1.42
(m, 4H), 0.95 (d, J = 5.6 Hz, 3H), 0.90 (d, J = 5.6 Hz, 3H). 13C Ethyl-(3R,4R,5S)-4-acetamido-5-amino-3-(pentan-3-
NMR (75 MHz, CDCl3): 201.1, 170.2, 83.6, 79.7, 74.2, 48.2, yloxy)cyclohex-1-ene-1-carboxylate ( , oseltamivir)
26.0, 25.1, 23.1, 9.5, 9.4. Spectral data agree with those in the Chlorotrimethylsilane (167 mg, 1.54 mmol, 0.19 mL) was
literature.25
added into the flask with zinc powder (1.30 g, 20 mmol) and
(2R,3R)-5 isomer: [α]D23= +50.3 (c = 1.0, CHCl3, 1:23 syn/anti). dry ether (35 mL). The mixture was stirred 15 min at room
1H NMR (300 MHz, CDCl3):
9.60 (d, J = 3.1 Hz, 1H), 6.18 (d, J = temperature under inert atmosphere. Activated zinc was
δ
1
δ
8.3 Hz, 1H), 4.91 – 4.67 (m, 2H), 4.63 – 4.46 (m, 1H), 3.93 (dd, J filtered under inert atmosphere (Ar), washed with dry ether
= 8.0, 3.1 Hz, 1H), 3.35 – 3.19 (m ,1H), 2.00 (s, 3H), 1.63 – 1.36 (2x10 mL). Ethyl-(3R,4R,5S)-4-acetamido-5-nitro-3-(pentan-3-
(m, 4H), 0.97 – 0.81 (m, 6H). 13C NMR (75 MHz, CDCl3): yloxy)cyclohex-1-ene-1-carboxylate (
6) (263 mg, 0.77 mmol)
δ
201.2, 170.6, 83.2, 80.5, 74.2, 47.3, 26.1, 24.9, 23.1, 9.5, 9.2. and acetic acid (15.6 mg, 260 mmol, 14.9 mL) were added into
Spectral data agree with those in the literature.25 HPLC: flask with activated zinc. The mixture was stirred under reflux
(Chiralpak OD-H column, 259 nm, hexane/iPro 85:15, 0.75 for 8 h. Zinc was filtered off, washed with EtOAc. The filtrate
mL.min-1) tR(syn-major) 28.6 min, tR(syn-minor) 16.6 min; tR(anti–major) was concentrated under reduced pressure. Worked reaction
12.7 min, tR(anti-minor) 10.5min.
mixture was purified by column chromatography (SiO2,
EtOAc/MeOH 2:1), to provide compound
substance; yield 134 mg (56%).
7 as a colorless solid
Ethyl-(3R,4R,5S)-4-acetamido-5-nitro-3-(pentan-3-
yloxy)cyclohex-1-ene-1-carboxylate
acetamido-5-nitro-3-(pentan-3-yloxy)cyclohex-1-ene-1-
carboxylate (
and ethyl-(3R,4R,5R)-4- HRMS m/z [M+H]+ calculated: 313.213, measured: 312.212, mp
= 90.2 – 92.1°C (107 – 108°C;26 102.5 – 103.9°C27), [α]D20= -
52.7, (c = 1.03, CHCl3), ([α]D20= - 57.2, c = 1, CHCl3),25 IR (neat):
6
)
K2CO3 (514 mg, 3.65 mmol) was added into mixture of N- 3281s (N-H), 2922m (C-H), 1710s (C=O), 1630s (C=C), 1544 (N-
[(2R,3S)-1-nitro-4-oxo-3-(pentan-3-yloxy)butan-2-yl]acetamide H). 1H NMR (300 MHz, CDCl3):
6.79 (t, J = 2.1 Hz, 1H), 5.53 (br
) (395 mg, 1.52 mmol), ethyl-2-(diethoxyphosphoryl)prop-2- d, J = 7.6 Hz, 1H), 4.29 – 4.12 (m, 3H), 3.52 (dt, J = 10.3, 8.3 Hz,
enoate ( ) (412 mg, 1.75 mmol), 18-crown-6 ether (44.6 mg, 1H), 3.35 (dd, J = 11.4, 5.7 Hz, 1H), 3.25 (ddd. J = 15.4, 10.1, 5.0
δ
(
5
4
0.167 mmol) in CH2Cl2 (20 mL), which was placed into Hz, 1H), 2.75 (dd, J = 17.7, 5.3 Hz, 1H), 2.25 – 2.08 (m, 1H), 2.04
microwave reactor. The reaction was stirred under microwave (s, 3H), 1.76 (br s, 2H), 1.62 – 1.41 (m, 4H), 1.34 -1.23 (m, 3H),
irradiation at 70°C for 2h. The reaction was stopped by adding 0.90 (td, J = 7.4, 2.4 Hz, 6H). 13C NMR (75 MHz, CDCl3):
δ 171.1,
NH4Cl into reaction mixture, extracted with CH2Cl2. Organic 166.5, 137.7, 129.6, 81.8, 74.9, 61.0, 59.1, 26.4, 25.8, 23.8,
layers were dried over Na2SO4. Solvent was evaporated and 14.3, 9.7, 9.5. Spectral data corresponds with literature.8a, 8b
crude product was purified by column chromatography (SiO2,
hex/EtOAc 2:1); to provide compound
mixture of isomers 2:1 (5R/5S); yield 337 mg (65 %). The 1-ene-1-carboxylic acid ((3R,4R,5S)-
mixture of isomers was separated. Separated isomers were 1M water solution of NaOH (27.5 mg, 0.69 mmol) was added
6 as a pale yellow oil as (3R,4R,5S)-4-Acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-
7)
isolated a colorless solid substances.
into the flask with aminoester (3R,4R,5S)-
α]D20= - 33.3 (c = mmol) dissolved in THF (14.2 mL) at 0°C. The reaction mixture
δ 6.87 – 6.81 (m, 1H), was stirred at 65°C for 1h. The reaction was finished after TLC
1 (135 mg, 0.43
(3R,4R,5S)-6 isomer: mp = 149.8 – 154.0°C;
1
1.0, CHCl3). H NMR (300 MHz, CDCl3):
[
5.78 (br. d, J = 6.7 Hz, 1H), 5.62 (ddd, J = 11.2, 10.5, 6.0 Hz, 1H), detection by adding acidic amberlite (2g). Mixture was stirred
4.95 – 4.70 (m, 1H), 4.23 (q, J = 7.1 Hz, 2H), 3.78 – 3.67 (m, 1H), for next 15 min and subsequently amberlite was filtered off
3.34 (quint., J = 5.7 Hz, 1H), 3.18 – 3.06 (m, 1H), 2.97 – 2.78 and washed by EtOH. The solvent was evaporated under
(m, 1H), 1.97 (s, 3H), 1.56 – 1.41 (m, 4H), 1.30 (t, J = 7.1 Hz, reduced pressure and worked reaction mixture was purified by
3H), 0.98 – 0.82 (m, 6H). 13C NMR (75 MHz, CDCl3):
δ 171.3, flash chromatography (reverse column, acetonitrile/H2O 19:1),
165.3, 138.1, 126.9, 82.3, 82.1, 71.9, 61.5, 56.1, 29.6, 26.3, to provide colorless powder; yield 20 mg (16%).
25.6, 23.6, 14.3, 9.7, 9.4. Spectral data corresponds with HRMS: m/z [M+H]+ calculated: 285.182, measured: 285.181.
literature.9b, 26
mp = 187.2 – 191.9°C (185 – 187°C),28 [α]D20= - 21.4 (c = 0.4 ,
H2O), ([α]D20= - 143.2, c = 0.4, H2O),28 IR (neat): 3362w (N-H),
(3R,4R,5R)-6 isomer: mp = 135.3 – 139.8°C; [α]D20= - 58.8, (c = 2956m (C-H), 1630m (C=C), 1544s (N-H)
1
0.4, CHCl3). H NMR (300 MHz, CDCl3):
δ δ 6.53 – 6.50 (m, 1H), 4.30 (d, J = 8.9
6.91 – 6.84 (m, 1H), 1H NMR (300 MHz, D2O):
5.71 (br d, J = 8.3 Hz, 1H), 4.99 (td, J = 6.9, 3.3 Hz, 1H), 4.78 Hz, 1H), 4.06 (dd, J = 11.6, 8.9 Hz, 1H), 3.60 – 3.51 (m, 2H), 2.90
(ddd, J= 8.3, 5.0 Hz, 3.4 Hz, 1H), 4.25 (q, J = 7.1 Hz, 1H), 4.17 – (dd, J = 17.1, 5.5 Hz, 1H), 2.56 – 2.43 (m, 1H), 2.10 (s, 3H), 1.68
4.04 (m, 1H), 3.61 – 3.39 (m, 1H), 3.09 – 2.95 (m, 2H), 1.99 (s, – 1.36 (m, 4H), 0.89 (dt, J = 14.4, 7.4 Hz, 6H). 13C NMR (75 MHz,
3H), 1.66 – 1.42 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H), 0.96 (t, J = 7.4 D2O):
Hz, 3H), 0.90 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3):
δ
25.4, 25.1, 22.3, 8.5, 8.4. Spectral data corresponds with
175.1, 173.7, 132.9, 132.5, 84.2, 75.6, 52.9, 49.7, 29.4,
δ
169.4, 164.4, 134.5, 127.7, 81.8, 79.3, 71.4, 60.6, 49.2, 25.6, literature.28
25.4, 24.7, 22.4, 13.3, 8.9, 8.4. Spectral data corresponds with
literature.9b,
26
HPLC (Chiralcel AD-H column, 230 nm, Ethyl-(3R,4R,5R)-4-acetamido-5-amino-3-(pentan-3-
hexane/iPro 93:7, 0.5 mL.min-1) tR(5S –major) 41.5min, tR(5S-minor) yloxy)cyclohex-1-ene-1-carboxylate ((3R,4R,5R)-
1)
26.1min, tR(5R-major) 18.2 min, tR(5R-minor) 17.5 min.
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Chlorotrimethylsilane (204 mg, 1.88 mmol, 0.24 mL) was The reaction was stirred under microwave irradiation at 70°C
added into the flask with zinc powder (1.60 g, 24.4 mmol) and for 2h. The reaction was stopped by adding NH4Cl into reaction
dry Et2O (43 mL). The mixture was stirred 15 min at room mixture, extracted with CH2Cl2. Organic layers were dried over
temperature under inert atmosphere. Activated zinc was Na2SO4. Solvent was evaporated and crude product was
filtered under inert atmosphere (Ar), washed with dry Et2O purified by column chromatography (SiO2, hex/EtOAc 2:1); to
(2x10 mL). Ethyl-(3R,4R,5R)-4-acetamido-5nito-3-(pentan-3- provide yellow oil as mixture of isomers 4:1 (5S/5R); yield 950
yloxy)cyclohex-1-ene-1-carboxylate (322 mg, 0.94 mmol) and mg (55 %). The mixture of isomers was separated once more
acetic acid (19.1 mg, 318 mmol, 18.2 mL) were added into flask by column chromatography (SiO2, CH2Cl2/EtOAc/CH3CO2H
with activated zinc. The mixture was stirred at room 9:1:0.05). Separated isomers were isolated in enantiomeric
temperature overnight. Zinc was filtered off, washed with ratio 26:74 (3S,4R,5R) and 90:10 for (3S,4R,5S) isomer.
EtOAc. The filtrate was concentrated under reduced pressure. (3S,4R,5R)-6 isomer: pale yellow oil, HRMS: m/z [M+H]+
Worked reaction mixture was purified by column calculated: 343.187, measured: 343.186, [α]D20= +58.7, (c =
1
chromatography (SiO2, EtOAc/MeOH 2:1), to provide colorless 0.73, CHCl3). H NMR (300 MHz, CDCl3)
δ 6.97 – 6.91 (m, 1H),
oil; yield 188 mg (64%).
HRMS: m/z [M+H]+ calculated: 313.213, measured: 313.212, 1H), 4.26 (q, J = 7.1 Hz, 2 H), 4.17 – 4.10 (m, 1H), 3.40 – 3.25
[α]D23= -53.1 (c = 1, CHCl3). 1H NMR (300 MHz, CDCl3):
6.86 – (m, 2H), 2.66 (dd, J = 19.1, 3.4 Hz, 1H), 2.08 (s, 3H), 1.59 – 1.38
6.76 (m, 1H), 6.21 (br d, J = 7.9 Hz, 1H), 4.22 – 4.15 (m, 3H), (m, 4H), 1.33 (t, J = 7.1 Hz, 3H), 0.91 – 0.76 (m, 6H). 13C NMR
4.00 (t, J = 4.0Hz, 1H), 3.57 (br s, 2H), 3.53 – 3.44 (m, 2H), 2.74 (75 MHz, CDCl3): 170.3, 165.7, 134.8, 129.1, 81.4, 79.2, 68.8,
6.44 (br d, J = 8.5 Hz, 1H), 4.78 – 4.71 (m, 1H), 4.71 – 4.61 (m,
δ
δ
(dd, J = 18.5, 5.3 Hz, 1H), 2.32 – 2.13 (m, 1H), 1.99 (s, 3H), 1.59 61.5, 49.3, 27.1, 26.2, 25.7, 23.4, 14.3, 9.5, 9.3.
– 1.40 (m, 4H), 1.27 (t, J = 7.1 Hz, 3H), 0.89 (dt, J = 10.8, 7.4 Hz, (3S,4R,5S)-6 isomer: colorless solid substance, HRMS: m/z
6H). 13C NMR (75 MHz, CDCl3):
δ
171.4, 166.4, 135.1, 130.7, [M+H]+ calculated: 343.187, measured: 343.186, [α]D20= + 93.5,
82.0, 72.6, 61.1, 51.8, 45.6, 29.2, 26.50, 26.49, 23.53, 14.3, (c = 1.002, CHCl3),mp
=
88.5°C – 95.8°C. 1H NMR (300 MHz,
CDCl3): δ 6.96 – 6.89 (m, 1H), 5.94 (br d, J = 8.6 Hz, 1H), 4.89
10.1, 9.4.
(dd, J = 16.8, 9.0 Hz, 1H), 4.80 – 4.59 (m, 1H), 4.23 (q, J = 7.1
Hz, 2H), 4.11 (t, J = 4.4 Hz, 1H), 3.43 – 3.27 (m, 1H), 3.17 – 2.95
(m, 2H), 1.98 (s, 3H), 1.60 – 1.40 (m, 4H), 1.30 (t, J = 7.1 Hz,
(3R,4R,5R)-4-Acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-
1-ene-1-carboxylic acid ((3R,4R,5R)-7)
1M water solution of KOH (20.2 mg, 0.36 mmol) was added 3H), 0.91 (t, J = 7.4 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H). 13C NMR (75
into the flask with aminoester (3R,4R,5R)- (56.2 mg, 0.18 MHz, CDCl3): 170.1, 165.3, 134.9, 129.4, 81.9, 81.8, 69.5,
1
δ
mmol) dissolved in THF (5.7 mL) at 0°C. The reaction mixture 61.6, 50.3, 29.0, 26.6, 25.9, 23.3, 14.3, 9.7, 9.6. HPLC: (Chiralcel
was stirred at room temperature for 3h. The reaction was IA column, 230 nm, hexane/iPro 93:7, 0.75mL.min-1): tR(5S-major)
finished after TLC detection by adding acidic amberlite (1g). 27.5 min, tR(5S-minor) 21.1 min, tR(5R-minor) 17.0 min, tR(5R-major) 16.0
Mixture was stirred for next 15 min and subsequently min.
amberlite was filtered off and washed by EtOH. The solvent
was evaporated under reduced pressure and worked reaction Ethyl-(3S,4R,5S)-4-acetamido-5-amino-3-(pentan-3-
mixture was purified by flash chromatography (reverse yloxy)cyclohex-1-ene carboxylate ((3S,4R,5S)-
column, acetonitrile/H2O 19:1), to provide colorless powder; Chlorotrimethylsilane (97.8 mg, 0.90 mmol, 0.11 mL) was
yield 21.3 mg (42%). added into the flask with zinc powder (765 mg, 11.7 mmol)
HRMS: m/z [M+H]+ calculated: 285.181, measured: 285.180, and dry Et2O (21 mL). The mixture was stirred 15 min at room
mp
151.1 – 156.5°C, [α]D20= - 54.3 (c = 0.4 , D2O), IR (neat): temperature under inert atmosphere. Activated zinc was
3292w (N-H), 2963m (C-H), 1647m (C=C), 1539s (N-H). 1H NMR filtered under inert atmosphere (Ar), washed with dry ether (2
(600 MHz, D2O): 6.63 – 6.52 (m, 1H), 4.51 – 4.49 (m, 1H), 10 mL). Ethyl-(3S,4R,5R)-4-acetamido-5nito-3-(pentan-3-
1)
=
δ
x
4.19 – 4.03 (m, 1H), 3.74 (ddd, J = 8.3, 5.5, 3.3 Hz, 1H), 3.62 – yloxy)cyclohex-1-ene-1-carboxylate (154 mg, 0.45 mmol) and
3.54 (m, 1H), 2.84 (dd, J = 17.9, 5.6 Hz, 1H), 2.47 – 2.28 (m, acetic acid (9.10 g, 152 mmol, 8.7 mL) were added into flask
1H), 2.05 (s, 3H), 1.72 – 1.35 (m, 4H), 0.95 (t, J = 7.4 Hz, 3H), with activated zinc. The mixture was stirred at room
0.90 (t, J = 7.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): 175.4, 173.6, temperature overnight. Zinc was filtered off, washed with
136.4, 128.0, 83.0, 72.7, 47.9, 46.9, 25.9, 25.6, 25.4, 21.8, 9.5, EtOAc. The filtrate was concentrated under reduced pressure.
8.2.
Worked reaction mixture was purified by column
chromatography (SiO2, EtOAc/MeOH 2:1), to provide colorless
powder; yield 109 mg (77%).
Ethyl-(3S,4R,5S)-4-acetamido-5-nitro-3-(pentan-3-
yloxy)cyclohex-1-ene carboxylate ((3S,4R,5S)-6) and ethyl- HRMS: m/z [M+H]+ calculated: 313.213:, measured: 313.211,
(3S,4R,5R)-4-acetamido-5-nitro-3-(pentan-3-yloxy)cyclohex-1-
ene carboxylate ((3S,4R,5R)-
K2CO3 (1.69 mg, 12.0 mmol) was added into mixture of N- (N-H). 1H NMR (300 MHz, CDCl3):
δ 7.01 – 6.80 (m, 1H), 6.04 (br
mp= 83.0 – 85.2°C, [α]D20= + 104.7, (c = 0.5, CHCl3), IR (neat):
3311s (N-H), 2970w (C-H), 1713s (C=O), 1636m (C=C), 1539m
6)
[(2R,3R)-1-nitro-4-oxo-3-(pentan-3-yloxy)butan-2-yl]acetamide d, J = 8.5 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H), 4.10 (t, J = 4.4 Hz,
(1.30 g, 5.0 mmol), ethyl-2-(diethoxyphosphoryl)prop-2-enoate 1H), 3.98 – 3.83 (m, 1H), 3.35 (quint, J = 5.7Hz, 1H), 3.17 – 3.00
(
4) (1.36 g, 5.75 mmol), 18-crown-6 ether (147 mg, 0.55 mmol) (m, 1H), 2.86 (dd, J = 18.2, 5.1 Hz, 1H), 2.13 – 1.95 (m, 1H),
in CH2Cl2 (66 mL), which was placed into microwave reactor. 2.06 (s, 3H), 1.64 – 1.46 (m, 4H), 1.42 (s, 2H), 1.30 (t, J = 7.1 Hz,
10 | J. Name., 2012, 00, 1-3
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3H), 0.93 (t, J = 7.4 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR (75 (3S,4R,5R)-4-Acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-
MHz, CDCl3): 169.7, 165.5, 134.0, 131.3, 80.3, 69.7, 60.0, 1-ene carboxylic acid ((3S,4R,5R)-
δ
53.6, 46.3, 33.3, 25.8, 25.1, 22.7, 13.3, 8.8, 8.7.
7)
1M water solution of NaOH (12.3 mg, 0.22 mmol) was added
into the flask with the corresponding aminoester (3S,4R,5R)-
1
Ethyl-(3S,4R,5R)-4-acetamido-5-amino-3-(pentan-3-
(37.5 mg, 0.12 mmol) dissolved in THF (3.7 mL) at 0°C. The
reaction mixture was stirred at room temperature overnight.
yloxy)cyclohex-1-ene carboxylate ((3S,4R,5R)-1)
Chlorotrimethylsilane (104.3 mg, 0.96 mmol, 0.12 mL) was The reaction was finished after TLC detection by adding acid
added into the flask with zinc powder (815 mg, 12.5 mmol) amberlite (1g). Mixture was stirred for next 15 min and
and dry Et2O (23 mL). The mixture was stirred 15 min at room subsequently amberlite was filtered off and washed by EtOH.
temperature under inert atmosphere. Activated zinc was The solvent was evaporated under reduced pressure and
filtered under inert atmosphere (Ar), washed with dry ether (2 worked reaction mixture was purified by flash chromatography
x 10 mL). Ethyl-(3S,4R,5S)-4-acetamido-5-nitro-3-(pentan-3- (reverse column, acetonitrile/H2O 19:1), to provide colorless
yloxy)cyclohex-1-ene-1-carboxylate (164 mg, 0.48 mmol) and powder; yield 8 mg (23%).
acetic acid (9.70 g, 162 mmol, 9.3 mL) were added into flask HRMS: m/z [M+H]+ calculated: 285.181, measured: 285.180,
with activated zinc. The mixture was stirred at room mp
temperature overnight. Zinc was filtered off, washed with 3331w (N-H), 2964m (C-H), 1646m (C=C), 1549s (N-H). 1H NMR
EtOAc. The filtrate was concentrated under reduced pressure. (600 MHz, D2O): 6.43 – 6.40 (m, 1H), 4.82 – 4.76 (1H, partial
=
178.0–181.8°C, [α]D20= +58.9 (c = 0.4, H2O), IR (neat):
δ
Worked reaction mixture was purified by column covered with HOD signal), 4.66 – 4.61 (m, 1H), 3.71 (ddd, J =
chromatography (SiO2, EtOAc/MeOH 2:1), to provide colorless 10.8, 5.8, 2.3 Hz, 1H), 3.53 – 3.47 (m, 1H), 2.75 (dd, J = 17.5,
powder; yield 93 mg (62%).
HRMS: m/z [M+H]+ calculated: 313.213, measured: 313.212, 4H), 0.90 (t, J = 7.5 Hz, 3H), 0.86 (t, J = 7.4 Hz, 3H). 13C NMR
mp= 102.8 – 105.5°C, [α]D20= + 46.7 (c = 0.5, CHCl3), IR(neat): (150 MHz, D2O):
176.0, 173.6, 134.1, 132.0, 81.3, 71.4, 49.1,
3356w (N-H), 2964s (C-H) 1705s (C=O), 1661s (C=C), 1552s (N- 46.8, 25.8, 25.7, 24.7, 22.0, 9.2, 8.0.
H). 1H NMR (600 MHz, CDCl3):
6.86 – 6.83 (m, 1H), 6.06 (br d,
5.8 Hz, 1H), 2.39 – 2.30 (m, 1H), 2.09 (s, 3H), 1.68 – 1.40 (m,
δ
δ
Common procedure for sample preparation for enantioselective
J = 8.2 Hz, 1H), 4.32 (ddd, J = 8.3, 5.1, 2.8Hz, 1H), 4.21 (q, J =
7.2 Hz, 2H), 4.19 – 4.12 (m, 1H), 3.33 (quint, J = 5.7 Hz, 1H),
3.17 (td, J = 5.6, 2.9 Hz, 1H), 2.62 – 2.54 (m, 1H), 2.32 (ddt,
J = 18.3, 5.9, 1.7Hz, 1H), 2.03 (s, 3H), 1.80 (br s, 2H), 1.56 –
1.45 (m, 4H), 1.30 (t, J = 7.1 Hz, 3H), 0.88 (dt, J = 10.5, 7.4 Hz,
HPLC analysis
9H-fluoren-9-ylidentriphenyl-λ⁵-phosphane was added into
flask with dissolved Michael product in toluene. The reaction
mixture was stirred at 100°C for 1h. Subsequently, reaction
mixture was cooled to room temperature and purified by
column chromatography (SiO2, hex/EtOAc) or by using
preparative TLC plate in same mixture of solvent hex:EtOAc.
6H). 13C NMR (150 MHz, CDCl3):
δ 170.8, 166.7, 136.4, 130.0,
81.4, 70.8, 61.1, 50.6, 48.6, 33.1, 26.2, 25.9, 23.7, 14.4, 9.8,
9.4.
(3S,4R,5S)-4-Acetamido-5-amino-3-(pentan-3-yloxy)cyclohex-1-
N-[(2R,3R)-4-[(9Z)-9H-Fluoren-9-ylidene]-1-nitro-3-(pentan-3-
yloxy)butan-2-yl]acetamide and N-[(2R,3S)-4-[(9Z)-9H-Fluoren-
9-ylidene]-1-nitro-3-(pentan-3-yloxy)butan-2-yl]acetamide
According to common preparation: N-[(2R,3S)-1-nitro-4-oxo-
3-(pentan-3-yloxy)butan-2-yl]acetamide (104 mg, 0.36 mmol),
ene carboxylic acid ((3S,4R,5S)-7)
1M water solution of NaOH (14.7 mg, 0.37 mmol) was added
into the flask with aminoester (71.8 mg, 0.23 mmol) dissolved
in THF (7.9 mL) at 0°C. The reaction mixture was stirred at
room temperature for 2 days. The reaction was finished after
TLC detection by adding acidic amberlite (1g). Mixture was
stirred for next 15 min and subsequently amberlite was
filtered off and washed by EtOH. The solvent was evaporated
under reduced pressure and worked reaction mixture was
purified by flash chromatography (reverse column,
acetonitrile/H2O 19:1), to provide colorless powder; yield 22.0
mg (34%).
9H-fluoren-9-ylidentriphenyl-λ⁵-phosphane (200 mg, 0.48
mmol), toluene (10 mL), CHCl3 (1.4 mL). Reaction mixture was
stirred at 100°C for 1 h. Crude mixture was purified by column
chromatography (SiO2, hex/EtOAc 2:1); to provide beige solid
substance; yield 111 mg (68%).
(2R,3R) isomer: [α]D23= +1.9 (c = 1.0, CHCl3, dr 1:1syn/anti), mp
=69.9–70.6°C. 1H NMR (300 MHz, CDCl3):
δ 7.79 – 7.60 (m, 4H),
7.44 – 7.27 (m, 4H), 6.42 (d, J = 9.4 Hz, 1H), 5.98 (d, J = 8.4 Hz,
1H), 5.46 (dd, J = 9.4, 4.1Hz, 1H), 4.96 – 4.84 (m, 1H), 4.83 –
4.67 (m, 2H), 3.37 (m, 1H), 1.94 (s, 3H), 1.56 – 1.43 (m, 4H),
HRMS: m/z [M+H]+ calculated: 285.181, measured: 285.181,
20
= 174.9 – 179.8°C, [α]D = - 24.8 (c = 0.15, H2O), IR (neat):
mp
3357w (N-H), 2971m (C-H), 1614m (C=C), 1573s (N-H). 1H NMR
(600 MHz, D2O): 6.67 – 6.43 (m, 1H), 4.29 – 4.27 (m, 1H),
0.89 (td, J = 7.4, 1.3Hz, 6H). 13C NMR (75 MHz, CDCl3):
δ 169.4,
δ
140.9, 138.6, 138.5, 137.5, 135.1, 128.3, 128.1, 126.8,126.4,
124.3, 124.2, 119.4, 119.3, 118.8, 79.7, 73.5, 69.9, 51.2, 25.5,
4.24 (dd, J = 10.9, 4.6 Hz, 1H), 3.60 – 3.54 (m,1H), 3.52 – 3.46
(m, 1H), 2.96 (dd, J = 17.6, 5.7 Hz, 1H), 2.42 – 2.35 (m, 1H),
2.11 (s, 3H), 1.65 – 1.51 (m, 4H), 0.93 (t, J = 7.5 Hz, 3H), 0.87 (t,
J = 7.4 Hz, 3H). 13C NMR (150 MHz, D2O):
δ 174.5, 174.1, 134.9,
24.5, 22.4, 9.1, 8.3. NOESY: without interaction between HA (
5.46, dd) a HB
4.96 – 4.84, m). Spectral (1H NMR) data
corresponds with literature.29
δ
(
δ
130.1, 83.4, 69.9, 50.6, 46.7, 29.7, 25.4, 25.3, 22.1, 9.1, 8.3.
According common preparation: N-[(2R,3R)-1-nitro-4-oxo-3-
(pentan-3-yloxy)butan-2-yl]acetamide (52.1 mg, 0.20 mmol),
9H-fluoren-9-ylidentriphenyl-λ
⁵
-phosphane (102 mg, 0.24
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Journal Name
mmol), toluene (5.0 mL), CHCl3 (0.8 mL). Reaction mixture was mechanics and OPLS-2005 force field.20 Only single crystal
stirred at 100°C for 1h. Crude mixture was purified by column structure configuration of the neuraminidase was considered.
chromatography (SiO2, hex/EtOAc 2:1); to provide beige solid Poisson-Boltzmann surface area (PBSA)36 implicit solvation
substance; yield 60 mg (73%).
model was used to include the effect of hydration on the
(2R,3S) isomer: [α]D23= +38.9 (c = 0.7, CHCl3, dr 1:12 syn/anti), optimized QM/MM systems.
1
mp =156.5–158.5°C. H NMR (600 MHz, CDCl3):
(m,1H), 7.77 – 7.66(m, 3H), 7.46 – 7.27 (m, 4H), 6.45 (d, J = 8.7 computed as:19
δ 8.00 – 7.93 Relative differences in enzyme-inhibitor binding energies were
Hz, 1H), 6.26 (d, J = 8.9 Hz, 1H), 5.38 (dd, J = 8.7, 6.0 Hz, 1H), ΔΔEbin,sol
4.92 (dd, J = 12.8, 6.5 Hz, 1H), 4.85 – 4.74 (m, 1H), 4.66 (dd, J = ΔEbin,sol,Ref
= (ΔEtot,sol[N1:Oi] - ΔEtot,sol[N1] - ΔEtot,sol[Oi]) -
12.8, 3.5 Hz, 1H), 3.36 – 3.25 (m, 1H), 1.91 (s, 3H), 1.58 – 1.40 for solvated optimized molecular geometries of oseltamivir
(m, 4H), 0.95 – 0.80 (m, 6H). 13C NMR (150 MHz, CDCl3):
δ
carboxylate stereoisomers [Oi], enzyme:oseltamivir complexes
170.2, 141.7, 139.8, 139.4, 138.5, 136.2, 129.2, 129.0, 128.0, [N1:Oi] and free enzyme [N1] using QSite 18b at the DFT-B3LYP-
127.5, 126.4, 125.6, 120.5, 120.3, 119.8, 80.5, 74.9, 73.0, 51.7, PBSA/6-31+G*:OPLS-2005 level of theory. Stereoisomer I.
26.5, 25.2, 23.3, 10.1, 9.2. NOESY: Interaction between HA
(δ
(3R,4R,5S) of oseltamivir carboxylate, the active form of
5.38, dd) a HB (
δ
4.85 – 4.74, m).
Tamiflu, was used as the reference structure.
Computation
Biological testing
Molecular structures of oseltamivir carboxylate stereoisomers Viruses:
Human
influenza
A
virus
(IAV)
were prepared from the crystal structure of oseltamivir bound A/Mississippi/1/85(H3N2) originated from the collection of
to viral neuraminidase of N1 subtype (PDB entry 2HU0 30) by viruses of Institute of Virology, SAS, Bratislava, Slovak
inverting the hetero-substituents attached to chiral carbon Republic). This virus was used as a model IAV of medium
atoms 3, 4 and 5. Molecular geometries of the stereoisomers virulence.37 To know whether tested compounds are effective
were optimized by quantum chemical density functional also against the Tamiflu resistant viruses, two variants of
theory (DFT)15 with polarizable continuum reaction field pandemic virus 2009 were included: A/Perth/265/2009 wild-
solvent model (PCM)31 and hybrid gradient-corrected type virus (H1N1 pdm09, A/California/7/2009-like) – carrying
32
exchange-correlation functional B3LYP
in double-zeta histidine at position 275 (275H) of the neuraminidase
gaussian basis 6-31+G* augmented with polarization and glycoprotein, Tamiflu sensitive, abbreviated as „TSV A/Perth“
33
diffuse functions
quantum chemistry software package.34
on all heavy atoms using Gaussian 09 and A/Perth/261/2009 variant virus (H1N1 pdm09,
A/California/7/2009-like) –carrying tyrosine at position 275
Electronic CD spectra of the stereoisomers were calculated by (275Y) of the neuraminidase glycoprotein – i.e. a H275Y
time-dependent density functional theory (TDDFT)15 with PCM substitution, Tamiflu resistant, abbreviated as „TRV A/Perth“.
solvent model of hydration, B3LYP functional and 6-31+G* These viruses were kindly provided by Aeron Hurt, WHO
basis set for molecular geometries of stereoisomers optimized Collaborating Centre for Reference and Research on Influenza,
in water. The Cotton effects in circular dichroism spectra was ISIRV-AVG, Melbourne, Australia. Viruses were propagated in
generated by positioning Gaussian band-shapes over first 80 fertilized chicken eggs and A/Perth variants were sequenced to
electronic transitions (rotational strengths) with a bandwidth confirm the maintenance of Tamiflu susceptibility/resistance
(FWHM) of 0.4 eV and by summing over the whole spectral markers. Virus stocks were aliquoted and stored at -70°C.
range.35
Rapid Culture Assay (RCA): The Madin-Darby canine kidney
3D-structure of the neuraminidase N1:oseltamivir carboxylate (MDCK) cells, provided by ATCC, were cultured in Dulbecco′s
complexes (influenza A virus - Viet Nam/1203/2004/H5N1) Modified Eagle′s Medium (DMEM) supplemented with 5% fetal
were prepared from the crystal structure obtained from bovine serum on 96-microwell plates (5x104 cells/100µl/well)
Protein Data Bank (PDB entry 2HU0 30) by refinement and for 24 hours. Then, medium was removed and cell monolayers
minimization 25 using OPLS-2005 force field.20 Enzyme- were infected with different dilutions of IAV stocks (a
inhibitor interactions were described by hybrid quantum 100µl/well) in Ultra MDCK serum-free medium (Lonza)
mechanical/molecular mechanical approach (QM/MM)18a containing TPCK trypsin (2 µg/mL) and 1% DMSO. The
using QSite package of Schrödinger,18b where the central infectious virus was detected after 18 hour incubation at 37°C
quantum motif (oseltamivir, side chains of 11 closest enzyme in a humid atmosphere of 5% CO2, as described.38 The optimal
residues: Glu119, Asp151, Arg152, Ile222, Arg224, Ser246, concentration of each virus strain was selected for antiviral
Glu276, Glu277, Asn294, Arg371, Tyr406 and 2 structural experiments: 34 plaque forming units (pfu)/100 µl of A/Miss
water molecules) was described at the DFT-B3LYP/6-31+G* virus, 8.9 pfu/100µl of A/Perth TRV and 11.5 pfu/100µl of
level. Frozen-orbital approximation was used to link A/Perth TSVviruses.
covalently-bonded atoms in residues at the frontiers between The antiviral efficacy of tested compounds was examined by
the QM and MM regions.18a The quantum motif was RCA modified as follows: Two-fold dilutions ranging in
embedded in classical system comprising the remaining 376 concentrations from 5000 nM to 0.078 nM of examined
amino acid residues of the viral neuraminidase present in the substances were added to MDCK cell monolayers in a volume
crystal structure, which were described by molecular of 50µl per well and subsequently the virus in a predetermined
12 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C6OB02673G
ARTICLE
8. a) S. Zhu, S. Yu, Y. Wang and D. Ma, Angew. Chem. Int. Ed.,
2010, 49, 4656-4660; b) J. Rehák, M. Huťka, A. Latika, H. Brath, A.
Almássy, V. Hajzer, J. Durmis, S. Toma and R. Šebesta, Synthesis,
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optimal concentration (50µl/well) was added. The examined
compounds as well as viruses, were diluted in serum-free Ultra
MDCK medium containing 1% DMSO and TPCK trypsin (2
µg/mL) and were incubated at 37°C in a humid atmosphere of
5% CO2. After 18 hours of infection, multiple washings of cell
monolayers with PBS and cell fixation with cold methanol at
+4°C for 15 minutes were done. Infectious virus was detected
in cell monolayers using monoclonal antibody (MAb) 107L
ChemCatChem, 2012, 4, 1007-1012.
9. a) T. Mukaiyamaꢀ, H. Ishikawaꢀ, H. Koshino and Y. HayashiꢀChem.
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10. P. Tisovský, T. Peňaška, M. Mečiarová and R. Šebesta, ACS Sust.
Chem. Eng., 2015, 3, 3429-3434.
(1.5µg/mL), specific to influenza
subsequently second antibody,
A
nucleoprotein,39 and
goat anti-mouse IgG
a
11. a) C. Suttibut, Y. Kohari, K. Igarashi, H. Nakano, M. Hirama, C.
Seki, H. Matsuyama, K. Uwai, N. Takano, Y. Okuyama, K. Osone, M.
Takeshita and E. Kwon, Tetrahedron Lett., 2011, 52, 4745-4748; b)
H. Nakano, K. Osone, M. Takeshita, E. Kwon, C. Seki, H. Matsuyama,
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This work was supported by the Slovak Research and
Development Agency under the contract no. APVV-0067-11,
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