Novel 3,4-disubstituted-Neu5Ac2en derivatives as probes to investigate flexibility of the influenza virus sialidase 150-loop

Article abstract of DOI:10.1016/j.bmc.2013.05.054

Novel 3,4-disubstituted-Neu5Ac2en derivatives have been synthesised to probe the open 150-loop conformation of influenza virus sialidases. Both equatorially and axially (epi) substituted C4 amino and guanidino 3-(p-tolyl)allyl-Neu5Ac2en derivatives were prepared, via the 4-epi-hydroxy derivative. The equatorially-substituted 4-amino derivative showed low micromolar inhibition of both group-1 (pdm09 H1N1) and group-2 (pdm57 H2N2) sialidases, and provides the first in vitro evidence that a group-2 sialidase may exhibit 150-loop flexibility.

Full text of DOI:10.1016/j.bmc.2013.05.054

Bioorganic & Medicinal Chemistry 21 (2013) 4820–4830
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Novel 3,4-disubstituted-Neu5Ac2en derivatives as probes to
investigate flexibility of the influenza virus sialidase 150-loop
⇑
Santosh Rudrawar, Jeffrey C. Dyason, Andrea Maggioni, Robin J. Thomson, Mark von Itzstein
Institute for Glycomics, Griffith University, Gold Coast Campus, Queensland 4222, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 6 June 2013
Novel 3,4-disubstituted-Neu5Ac2en derivatives have been synthesised to probe the open 150-loop con-
formation of influenza virus sialidases. Both equatorially and axially (epi) substituted C4 amino and gua-
nidino 3-(p-tolyl)allyl-Neu5Ac2en derivatives were prepared, via the 4-epi-hydroxy derivative. The
equatorially-substituted 4-amino derivative showed low micromolar inhibition of both group-1
(pdm09 H1N1) and group-2 (pdm57 H2N2) sialidases, and provides the first in vitro evidence that a
group-2 sialidase may exhibit 150-loop flexibility.
Keywords:
Influenza
Sialidase
150-loop
Neu5Ac2en
Sialic acid
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
A major role of the sialidase in the virus’ infective cycle is to
facilitate release of virus progeny from an infected cell by cleaving
Influenza remains
a
significant and continuous threat to
terminal sialic acid residues from cell surface and virus particle
associated sialoglycoconjugates that would otherwise be bound
by HA of the new virus particles and keep them clumped at the cell
surface.5 This important functional role, and the large number of
highly conserved active site amino acid residues in influenza virus
sialidases, led to the targeting of this enzyme for anti-influenza
drug development.6 Inhibitor design—based on available N2 and
N9 (group-2) NA X-ray crystal structures—targeting highly-con-
served residues of the influenza virus sialidase active site, led to
the development in the 1990s of two drugs that mimic the putative
enzyme transition state: zanamivir (RelenzaÒ) 1,7 and oseltamivir
(TamifluÒ, an orally-bioavailable pro-drug form of the sialidase
inhibitor oseltamivir carboxylate, OC 2) (Fig. 1).8 Both drugs are
effective in the prophylaxis and treatment of influenza virus infec-
tion. The worldwide stockpiling of these two anti-viral drugs as a
major component of pandemic preparedness planning highlights
the current importance of the sialidase inhibitor drug class.
Recently, the first X-ray crystal structures of influenza A virus
group-1 sialidases [(H5)N1, N4 and N8 NAs,4 and subsequently
N5 NA9] were solved. These structures showed never-before-seen
flexibility of an amino acid loop—the so-called ‘150-loop’ (residues
147–152)—that lies along one edge of the active site cavity. The
flexibility of the 150-loop leads to generation of a large cavity
(the ‘150-cavity’) adjacent to the enzyme active site when the loop
is in an ‘open’ conformation. Computational studies using molecu-
lar dynamics (MD) simulations of the avian (H5)N1 subtype sug-
gest that the 150-loop is even more flexible and able to open
into a significantly wider conformation than observed in the crys-
tal structures.10
humanity, as evidenced by seasonal epidemics that exact a high
toll in morbidity, estimated to be in the range of three to five mil-
lion cases of severe illness, and mortality, with up to half a million
deaths worldwide.1 The threat, and potentially devastating conse-
quences (an estimated global mortality of greater than 60 million2)
of adaptation of, for example, a highly pathogenic avian influenza A
(H5N1) virus to a human-to-human transmissible virus has fuelled
a drive to develop strategies to minimise, and new therapies to
prevent or treat, influenza virus infection.
Influenza A viruses, which cause the most significant disease in
humans, including seasonal epidemics and global pandemics, are
classified into subtypes based on the antigenic properties of the
virus’ surface glycoproteins, the lectin ‘haemagglutinin’ (HA: H1-
17) and the glycohydrolase ‘sialidase’ (neuraminidase, NA: N1-9).3
All of the influenza A subtypes are found in wild birds, but only three
HA subtypes (H1, H2, and H3) and two of the NA subtypes (N1 and
N2) have circulated widely in the human population.3 Influenza A
virus sialidases have been further sub-classified based upon primary
sequence, into two phylogenetically distinct groups: group-1 (N1,
N4, N5 and N8) and group-2 (N2, N3, N6, N7 and N9).4 During the last
century, influenza viruses carrying N1 (H1N1) or N2 (H2N2, H3N2)
subtypes have circulated in humans, first as pandemic strains and
then, after subsequent adaptation to humans, as seasonal epidemic
strains.3
⇑
Corresponding author. Tel.: +61 7 5552 7025; fax: +61 7 5552 9040.
E-mail address: m.vonitzstein@griffith.edu.au (M.von Itzstein).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.05.054

S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
4821
acidic residues (including Glu119, Glu227, and Asp151), and has
been targeted with the introduction of positively charged guani-
dino or amino groups as in inhibitors 1, 2 and 7.6,21 The nature
(spatial and electrostatic characteristics) of the C4 pocket when
the 150-loop is in a more open conformation, is somewhat differ-
ent to the closed 150-loop conformation.4 When the 150-loop is
open,4 Glu119 and Asp151 are seen to move away from the posi-
tions that they occupy in the closed 150-loop conformation
(Fig. 2), giving the C4 pocket a less negative electrostatic potential
when the 150-loop is open.
We were interested to examine the effect of introducing func-
tionality at the C4 position on the C3-substituted Neu5Ac2en
inhibitor scaffold. While this scaffold holds the 150-loop in the
open conformation, the flexibility of the 150-loop, and the ‘plastic-
ity/mobility’ of adjacent active site residues, as observed crystallo-
graphically in complexes of N8 NA with 24 and with different
C3-substituted Neu5Ac2en-based inhibitors,16,17 may allow the
protein to ‘mold’ to the new inhibitor structure. We therefore be-
gan a program to functionalise the C4 position of C3-substituted
Neu5Ac2en, using 3-(p-tolyl)allyl-Neu5Ac2en 6 as the template.
The initial modifications chosen were the introduction of amino
and guanidino groups at C4 in both the ‘natural’ equatorial (8
and 9) and in the axial (or epi) (10 and 11) configurations.
Fig. 1. Anti-influenza drugs (1 and 2), and influenza virus sialidase inhibitors.
To date, the 150-loop has always been seen in the ‘closed’ form
in both apo and inhibitor-complexed group-2 (N2 and N9) NA crys-
tal structures.11,12 Interestingly, the X-ray crystal structure of apo
human pdm09 H1N1 NA, despite being a group-1 sialidase, also
showed the 150-loop only in a closed conformation.13 The factors
(e.g., aa sequences) that influence the propensity for, and extent
of, 150-loop flexibility in influenza virus sialidases are currently a
matter of speculation.13,14 MD simulation studies have suggested,
however, that both human pdm09 N1, and N2 sialidases should
also exhibit 150-loop flexibility but with potentially lower popula-
tion of the open 150-loop conformations.14
The extended active site cavity of an open 150-loop conformation
sialidase, presents a potential new target for inhibitor design.4,15
We16,17 and others18–20 have begun to explore the 150-loop region
of influenza virus sialidases, introducing onto established siali-
dase-inhibitor scaffolds substituents anticipated to reach towards
the 150-cavity; for example from the C4 position of zanamivir18,20
(e.g., 318) or the ‘equivalent’ C3 position of an oseltamivir isostere
(e.g., 4),19 and from the C3 position of the natural sialidase inhibitor
2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 5) (e.g.,
6).16,17 Molecular modelling18 and STD NMR spectroscopy19 studies,
respectively, suggest that an extended alkyl substituent on the inter-
nal guanidinyl nitrogen of a zanamivir derivative (3),18 and the tria-
zole 40-substituent of oseltamivir isostere 4,19 could make additional
interactions within the 150-cavity in the open form of N1 sialidases.
We have recently published the first X-ray crystallographic evidence
of sialidase inhibitors binding the 150-cavity.16,17 C3-functionalised
Neu5Ac2en derivative 6, which inhibits both human (including
pdm09) and avian N1 NAs at the micromolar level, was shown to
bind the representative group-1 sialidase N8 with the 150-loop in
the open conformation and the C3 substituent occupying an area
of the 150-cavity.16 That study provided proof-of-concept that de-
signed compounds could lock-open the 150-loop of an influenza A
virus sialidase. In the present study, we report our further work in
this area with the synthesis and evaluation of 3,4-difunctionalised
Neu5Ac2en-based inhibitor probes of influenza A virus sialidases.
2.2. Chemistry
Two main approaches for the synthesis of the C4-N-functional-
ised C3-substituted Neu5Ac2en-based inhibitor probes 8–11 were
apparent; functionalisation at C-3 of protected 4-azido-4-deoxy-
Neu5Ac2en, using methodology developed for the synthesis of 6
from a 4-acetoxy-Neu5Ac2en derivative;17,22 or functionalisation
at C4 of a C3-substituted Neu5Ac2en derivative. In the first in-
stance, C3 allylation of a protected 4-azido-4-deoxy-Neu5Ac 2,3-
bromohydrin was attempted using the method17,22 employed to
install the allyl group at C3 on the corresponding 4-acetoxy deriv-
ative (allyltributyltin in the presence of AIBN as free radical initia-
tor). This chemistry was, however, unsuccessful on the 4-azido
substrate, resulting in polymerization and decomposition. The
alternative route to the target C4-N-functionalised 3-(p-tolyl)al-
2. Results and discussion
2.1. Structure analysis
Fig. 2. Positions of important active site residues of group-1 influenza A virus
sialidase N8 in the 150-loop open (apo form, magenta: PDB 2ht5) and closed
(complexed with Neu5Ac2en 5, green: PDB 2htr) forms. The three arginine residues
(Arg118, Arg 371, Arg292) that bind the carboxylate group of 5 hold essentially the
same positions in both the open and closed 150-loop forms. In contrast, in the 150-
loop open form (magenta) the negatively charged Asp151 and Glu119 move away
from the positions adopted in the closed 150-loop form (green), leading to a
somewhat less negatively-charged C4 pocket.
A point of focus in the development of potent influenza virus
sialidase inhibitors, has been the incorporation of functionality to
interact with conserved amino acids lining the pocket of the active
site that accommodates the C4 hydroxyl group of Neu5Ac2en 5
(designated for this work as the ‘C4 pocket’). This pocket is domi-
nated in the closed 150-loop conformation by negatively-charged

4822
S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
lyl-Neu5Ac2en derivatives requires ‘activation’ of the allylic C4 po-
sition of the C3-substituted Neu5Ac2en derivative, for installation
of an azido group. In the synthesis of 4-N-substituted Neu5Ac2en
derivatives (e.g., zanamivir), introduction of the C4 azide begins
with formation of a 4,5-oxazoline, which is either opened directly
by nucleophilic azide, or hydrolysed to the 4-epi-hydroxy deriva-
tive for further manipulation. The efficient synthesis of the 3-(p-to-
lyl)allyl-Neu5Ac2en 4,5-oxazoline 17 and the key 4-epi-hydroxy
intermediate 19 from peracetylated Neu5Ac2en derivative 12 is
outlined in Scheme 1.
Bromohydroxylation of protected Neu5Ac2en 12 using N-bro-
mosuccinimide (NBS) in aqueous acetonitrile gives a mixture of
bromohydrins 13 (trans-2,3-diaxial bromohydrin and trans-2,3-
diequatorial bromohydrin ratio = 3:1) in excellent overall yield.23
The reaction of bromohydrin mixture22 13 with allyltributyltin in
the presence of AIBN affords solely the required C3 equatorially-
allylated product 14.22,24 Ruthenium (second generation Grubbs’
catalyst) catalysed olefin-cross metathesis25 of 3-allyl derivate 14
with 4-methylstyrene in anhydrous dichloromethane at 40 °C,
affording cross-metathesis coupled product 15 in 85% yield, was
followed by acetylation of the C2 hydroxyl group of 15 to give
2-O-acetate derivative 16. The preparation of the 4,5-oxazoline
was then achieved by using a modification of the procedure de-
scribed by Zbiral and co-workers.26 Reaction of peracetylated
derivative 16 with trimethylsilyl trifluoromethanesulfonate
(TMSOTf) in ethyl acetate at 52 °C gave the 2,3-eliminated 4,5-
oxazoline derivative 17 in high yield (95%).
The next synthetic step involved introduction of the azide func-
tionality at the C4 position. The C4 azide could lead into a range of
nitrogen-based substituents—including amine, guanidino, amide,
and triazole substituents—to probe interactions in the sialidase
C4 pocket. In the case of C3-unsubstitued Neu5Ac2en derivatives,
an azide functionality can be introduced at C4 through reaction
of the 4,5-oxazoline with trimethylsilyl azide.27 However, follow-
ing literature conditions,27 the reaction of C3-functionalised oxaz-
oline intermediate 17 with trimethylsilyl azide in tert-butyl alcohol
at 80 °C resulted in an unexpected product, aziridine derivative 18.
Varying the reaction conditions with respect to solvent, tempera-
ture and equivalents of trimethylsilyl azide did not avoid aziridin-
ation. Owing to the high reactivity of the allylic double bond
towards nucleophilic azide addition, we decided to explore an
alternative method28 for introduction of azide at the C4 position.
Accordingly, 4,5-oxazoline 17 was hydrolysed with trifluoroacetic
acid in aqueous ethyl acetate to yield 4-epi-hydroxy derivative
19. Intermediate 19 was saponified to provide 4-epi-3-(p-tolyl)al-
lyl-Neu5Ac2en 20.
Reaction of 4-epi-hydroxy derivative 19 with diphenylphospho-
ryl azide28 (DPPA) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)
in benzene gave the desired 4-azido derivative 21 in good yield
(Scheme 2). This reaction proceeds with inversion of configuration
at C4. Mechanistically, the reaction takes place in two discrete
steps—formation of the reactive 4-O-phosphate followed by dis-
placement with in situ generated azide.29 It is important to men-
tion here that our effort to replace benzene as solvent with
toluene, THF or SN2 displacement-favorable DMF resulted mainly
in decomposition.
We next turned to the reduction of the C4 azide functionality.
Our initial attempts towards selective hydrogenation of the C4
azide of 21 using palladium on carbon (Pd/C 10%)30 or Lindlar cat-
alyst,27 conditions used successfully for selective azide reduction
in 4-azido-4-deoxy-Neu5Ac2en,27,30 led to unwanted side reac-
tions, for example acetate migration or even over-reduction of
the allylic and/or ring double bonds. Finally, the previously re-
ported26 method for triphenylphosphine-mediated reduction of
4-azido-4-deoxy-Neu5Ac2en derivatives, successfully gave the
desired 4-amino compound 8. The reaction of the C4 azide group
of 21 with triphenylphosphine in THF at 50 °C resulted in the
unstable P–N ylide 22 which upon in situ hydrolysis with aque-
ous sodium hydroxide31 afforded target 4-amino derivative 8 in
good yield.
To install the more basic guanidino group at C4, the amino
derivative can be reacted with N,N0-bis-t-butoxycarbonyl-1H-pyra-
zole-1-carboxamidine (bisBocPCH).31 Attempted guanidinylation
of the 4-amino group of fully deprotected acid 8, however, was
unsuccessful. We therefore explored an alternative strategy as
shown in Scheme 3. The C4 azide derivative 21 was de-O-acety-
Scheme 1. Reagents and conditions: (a) NBS, MeCN/H2O (2.5:1), 80 °C, 2 h (70% di-axial bromohydrin, 24% di-equatorial bromohydrin, combined yield 94%); (b) Bu3SnAll,
AIBN, toluene, 100 °C, 8 h (68%); (c) Grubbs’ catalyst 2nd generation (15 mol %), 4-methylstyrene, DCM, 40 °C, 48 h (85%); (d) Ac2O, pyridine, DMAP, rt, 16 h (95%); (e) TMSOTf,
ethyl acetate, 60 °C, 2 h (95%); (f) azidotrimethylsilane, tert-butyl alcohol, 80 °C, 4 h (68%); (g) TFA, H2O, ethyl acetate, rt, 16 h (96%) and (h) aq NaOH, MeOH/H2O (1:1), 5 °C-rt,
16 h (90%).

S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
4823
the product with overall retention of configuration at C4.33,34 In the
next sequence, base-catalysed de-O-acetylation of 27 gave triol
methyl ester 28. The azide of 28 was then reduced with H2S in pyr-
idine to give the 4-epi-amino derivative 29 in 84% yield. Finally, de-
esterification of amine 29 using NaOH in aqueous MeOH gave de-
sired 4-epi-amino derivative 10 in 68% yield, along with a side
product resulting from acetate migration to give 4-acetamido-4-
deoxy-4-epi-Neu2en derivative 30. 4-epi-Amino derivative 29
was efficiently converted into the 4-epi-guanidino compound 31
by reaction with bisBocPCH. Subsequent processing of 31 as for
the C4 equatorially-substituted analogue 25—deprotection of Boc
groups and de-esterification—provided the 4-epi-guanidino deriva-
tive 11 in good yield. This is, to the best of our knowledge, the first
reported example of a 4-epi-guanidino substituent on a Neu5Ac2en
scaffold.
Scheme 2. Reagents and conditions: (a) DPPA, DBU, benzene, 5–10 °C, 15 min, rt,
5 h (85%); (b) PPh3, THF, 50 °C, 10 min. and (c) aq NaOH, 50 °C, 2 h (72% over 2
steps).
2.3. Biological evaluation
The synthesised 3,4-disubstituted Neu5Ac2en derivatives 8–11,
were evaluated19 for their ability to inhibit the hydrolysis of the
lated using sodium methoxide to give triol methyl ester 23. Reduc-
tion of the azide of 23 with H2S in pyridine32 gave the amino deriv-
ative 24 in 80% yield. Guanidinylation of the 4-amino group in 24
with bisBocPCH successfully produced the protected guanidine
derivative 25. It is noteworthy that the reaction required several
days for complete consumption of starting material; reaction time
was not improved upon addition of excess guanidinylating reagent
or raising the reaction temperature to 40 °C. The longer reaction
time (as compared to 18–20 h reaction time for C3-unsubstituted
Neu5Ac2en derivatives31) may be due to the steric effect of the
C3 p-tolylallyl substituent, which could make access to the bulky
guanidinylating reagent difficult. Removal of the Boc protecting
groups of 25 by treatment with TFA, followed by de-esterification
with aqueous sodium hydroxide afforded 4-guanidino derivative
9 in good yield.
The syntheses of the corresponding 4-epi-(axially) configured
amino and guanidino derivatives also began with 4-epi-hydroxy
derivative 19, following the route shown in Scheme 4. Treatment
of the peracetylated methyl ester of 4-epi-3-(p-tolyl)allyl-Neu5A-
c2en (26), obtained by acetylation of 19, with sodium azide in
aqueous THF in the presence of a catalytic amount of tetrakis(tri-
phenylphosphine)palladium(0) resulted in formation of 4-epi-azi-
do derivative 27 in 82% yield. This reaction was highly
stereoselective and no detectable amount of the other C4 isomer
21 was observed by 1H NMR spectroscopy. It is reported33,34 that
palladium(0)-catalysed allylic azidation proceeds with oxidative
fluorogenic substrate 2-a
-(40-methylumbelliferyl)-N-acetylneu-
raminic acid (MUN) by group-1 (pdm09 N1) and group-2 (pdm57
N2) influenza A virus sialidases. The sialidase inhibitory activities
of 8–11, the parent template 3-(p-tolyl)allyl-Neu5Ac2en 6, and
the natural sialidase inhibitor Neu5Ac2en 5, are presented in
Table 1.
Against the N1 sialidase, the C4-amino derivative 8 shows com-
parable low micromolar inhibition to the parent (C4-hydroxy)
template 6. Interestingly, introduction of the larger guanidino
group at C4 (compound 9) results in a marked 100-fold weaker
inhibition. This is in stark contrast to the C3-unsubstituted ana-
logue, 4-deoxy-4-guanidino-Neu5Ac2en 1 (zanamivir; IC50 7 nM)
which binds the closed 150-loop conformation of the enzyme,
and which shows significantly stronger inhibitory activity than
its corresponding 4-amino analogue 7.7,21 The X-ray crystal struc-
ture of zanamivir 1 in complex with ‘closed’ group-2 sialidase N9,
shows interaction of each of the three nitrogen atoms of the C4
guanidino group with one or more of the residues Glu119,
Glu227 and Asp151.7,35 The altered orientations of Glu119 and
Asp151 in an open 150-loop conformation sialidase, would reduce
the potential for this strong interaction. In addition, the two acidic
residues Glu119 and Asp151 generally mask the positive charge re-
lated to residue Arg156 which also borders the C4 pocket. As
Glu119 and Asp151 move further away, Arg156 sits in a more ex-
posed position on the lip of the 150-cavity. Adverse interaction of
the larger guanidino group at the C4 position with Arg156 could
contribute to the observed weaker inhibition of guanidino deriva-
tive 9.
addition of the palladium catalyst, yielding a (p-allyl)palladium
complex with inversion of configuration. Subsequent nucleophilic
attack by azide also occurs with inversion of configuration to yield
Scheme 3. Reagents and conditions: (a) NaOMe, MeOH, 5 °C to rt, 1 h (90%); (b) H2S, pyridine, rt, 16 h (80%); (c) bisBocPCH, Et3N, MeOH/THF, rt, 7 d (72%); (d) TFA, DCM, 5 °C
to rt, 5 h, (e) aq NaOH, MeOH/H2O (1:1), 5 °C to rt, 16 h (83% over 2 steps).

4824
S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
Scheme 4. Reagents and conditions: (a) Ac2O, DMAP, pyridine, rt, 16 h (96%); (b) NaN3, Pd(Ph3P)4, THF/H2O, 50 °C, 16 h (82%); (c) NaOMe, MeOH, 5 °C to rt, 2 h (90%); (d) H2S,
pyridine, rt, 16 h (84%); (e) aq NaOH, MeOH/H2O (1:1), 5 °C to rt, 16 h (68%); (f) bisBocPCH, Et3N, MeOH/THF, rt, 3 d (85%) and (g) TFA, DCM, 5 °C to rt, 5 h; (80% over 2 steps).
Table 1
reducing their interaction with a basic 4-epi-substituent, providing
a rationale for the observed weaker affinity of 4-epi-amino deriva-
tive 10. For the 4-epi-guanidino derivative 11, as observed in pre-
vious modelling studies on the closed 150-loop conformation,21
the confined space below the inhibitor in this area of the active site
appears to preclude favourable binding of the larger guanidino
substituent in the axial orientation; based on the inhibition values
for 11 this aspect appears to be more pronounced in the N2 than in
the more flexible N1 sialidase.
In vitro inhibition of influenza A virus N1 and N2 sialidases by 3,4-disubstituted-
Neu5Ac2en derivatives 8–11
Interestingly, in terms of inhibition of group-1 (N1) versus
group-2 (N2) sialidases, parent inhibitor 6 shows comparable inhi-
bition of both enzymes, and equatorially C4-substituted deriva-
tives 8 and 9 show 10-fold greater inhibitory activity against the
N2 subtype. Both the pdm09 N113 and pdm57 N212 sialidases have
been crystallised to date only in closed 150-loop conformations.
The level of inhibition of in particular 6 and 8 against these en-
zymes, however, is comparable to that seen for 6 against avian
(H5)N1 NA which has been shown crystallographically4 to have a
‘flexible’ 150-loop. Assuming—based on this similar low micromo-
lar level of inhibition, and the demonstrated16 binding of 6 to the
open 150-loop conformation of group-1 sialidase N8—that the
compounds are binding the sialidases in similar conformations,
this represents the first in vitro evidence, supporting in silico obser-
vations,10,14 that group-2 sialidases may also possess some degree
of flexibility in the 150-loop. It is tempting to speculate that the
greater tendency for the 150-loop to remain closed in group-2 sia-
lidases,14 may lead to a more favourable binding environment for
the amine-based C4 substituents of 8 and 9, than does the more
freely opening group-1 NA.
a
Compd
R1
R2
IC50
(lM)
b
c
N1
N2
6
8
9
10
11
5
OH
NH2
NHC(NH)NH2
H
H
H
H
NH2
6.5
3.5
3.2
0.1
42
336
465
730
405
d
H
NHC(NH)NH2
NI
1.8
e
Neu5Ac2en
0.7
a
Results are given as means for 6 experimentally determined values. Benchmark
inhibitor 4-deoxy-4-guanidino-Neu5Ac2en 1: N1, IC50 0.007
l
M; N2, IC50 0.005
l
M.
b
N1 [A/California/04/2009 (pdm09 H1N1)].
N2 [A/R1/5+/1957 (H2N2)].
No inhibition at 1 mM inhibitor concentration.
c
d
e
N1 [A/Paris/2590/2009 (pdm09 H1N1)]; Inhibition data (Ki) from Ref. 16.
In comparison to the equatorially-substituted derivatives 8 and
9, the corresponding axially-substituted (4-epi) isomers 10 and 11
show overall weak inhibitory activity. A C4 substituent in the epi-
configuration is oriented towards the floor of the active site cavity.
Previously observed sub-micromolar influenza virus sialidase inhi-
bition by 4-epi-amino-4-deoxy-Neu5Ac2en,21 has been proposed
to be due to interaction of the epi-amino group with Glu227, which
sits under the inhibitor on the floor of the active site.21 When the
150-loop is open, Glu119 flips over and forms a very close
interaction with Glu227 (less than 2 Å distant). pKa calculations
suggest that either one or both of these normally acidic residues
would be protonated, making them electrostatically neutral and
2.4. Conclusion
In summary, the synthesis of novel 3,4-disubstituted-Neu5A-
c2en derivatives 4-amino- (8) and 4-guanidino- (9) 4-deoxy-3-
(p-tolyl)allyl-Neu5Ac2en and their corresponding 4-epi analogues
(10 and 11), as inhibitor-based probes of influenza virus sialidases,
has been efficiently achieved via the key 4-epi-hydroxy-3-(p-toly-
l)allyl-Neu5Ac2en derivative 19. The equatorially-substituted
derivatives 8 and 9 showed the more potent sialidase inhibition,

S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
4825
with the 4-amino derivative 8 in particular giving low micromolar
inhibition of both group-1 (pdm09 N1) and group-2 (pdm57 N2)
sialidases, and providing the first in vitro evidence that a group-2
sialidase may exhibit 150-loop flexibility. The intermediate 4-azi-
do-4-deoxy-3-(p-tolyl)allyl-Neu5Ac2en derivative 21 is an impor-
tant precursor that could lead into a range of nitrogen-based
substituents—including amides, and triazoles—to further probe
interactions in the sialidase C4 pocket.
trated to dryness under vacuum. The crude product is dissolved
in water, the pH of the solution is adjusted to pH 7 using aq NaOH
(1 M), and the solution is lyophilised. Target compounds were sub-
sequently purified by reversed phase HPLC.
3.1.3. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-3-
C-[30-(p-tolyl)prop-20-enyl]-b-
D-erythro-L-gluco-non-2-
ulopyranosonate (15)
To a solution of allyl compound 14 (3.2 g, 6.02 mmol) in anhy-
drous DCM (200 mL) under N2 was added 4-methylstyrene
(7.94 mL, 60.20 mmol) at room temperature. Subsequently, ruthe-
nium catalyst, Grubbs catalyst (2nd generation) (254 mg,
0.30 mmol, 10 mol %) was added to the reaction mixture, which
was then stirred at 40 °C for 24 h. TLC analysis showed presence
of starting material (ꢁ40%), then a further amount of Grubbs cata-
lyst (2nd generation) (254 mg, 0.30 mmol, 5 mol %) was added and
the reaction mixture was stirred at 40 °C for 24 h (complete disap-
pearance of the starting material by TLC analysis). The solution was
concentrated under reduced pressure and the residue was purified
by flash chromatography on silica (acetone/hexane, 3:7) to afford
the cross-coupled product 15 as a white solid (3.17 g, yield 85%).
Rf 0.60 (EtOAc); 1H NMR (300 MHz, CDCl3): d 1.84, 1.94, 1.99,
2.07, 2.09 (5ꢀ s, 15H, NHCOCH3, OCOCH3 ꢀ4), 2.15 (m, 1H, –
CH2–), 2.20 (m, 1H, H-3), 2.28 (s, 3H, p-tolyl CH3), 2.60 (m, 1H, –
CH2–), 3.62 (s, 3H, COOCH3), 4.46 (dd, J = 12.3, 6.9 Hz, 1H, H-9),
4.12 (dd, J = 10.8, 2.4 Hz, 1H, H-6), 4.21 (ddd, J = 10.5, 10.2,
9.9 Hz, 1H, H-5), 4.32 (dd, J = 12.0, 2.4 Hz, 1H, H-90), 4.33 (d,
J = 1.5 Hz, 1H, 2-OH, D2O exchangeable), 5.03 (dd, J = 10.8, 9.9 Hz,
1H, H-4), 5.16 (m, 1H, H-8), 5.28 (dd, J = 6.9, 2.1 Hz, 1H, H-7),
5.41 (d, J = 10.2 Hz, 1H, NH), 5.91 (m, 1H, –CH@), 6.20 (d,
J = 15.9 Hz, 1H, @CH-Ph), 7.04 (d, J = 8.1 Hz, 2H, Ph), 7.14 (d,
J = 8.1 Hz, 2H, Ph); 13C NMR (75.5 MHz, CDCl3): d 20.8, 20.9, 21.0,
21.2 (OCOCH3 ꢀ4), 23.1 (NHCOCH3), 29.3 (p-tolyl CH3), 31.5
(–CH2–), 44.6 (C-3), 49.6 (C-5), 53.9 (COOCH3), 62.7 (C-9), 67.8
(C-7), 70.4 (C-8), 70.6 (C-6), 73.6 (C-4), 96.4 (C-2), 125.4 (–CH@),
125.8 (Ph), 129.3 (Ph), 131.8 (@CH-Ph), 134.2 (Ph q carbon),
137.1 (Ph q carbon), 169.4 (C-1), 170.3, 170.4, 170.5, 170.7, 171.6
(NHCOCH3, OCOCH3 ꢀ4); LRMS [C30H39NO13] (+ve ion mode) (m/
z): 644 [M+Na]+.
3. Experimental
3.1. Chemistry
3.1.1. General information
Reagents and dry solvents purchased from commercial sources
were used without further purification. Solvents used for chroma-
tography were distilled prior to use. All anhydrous reactions were
carried out under an atmosphere of nitrogen or argon, using oven-
dried glassware. Reactions were monitored using TLC on alumin-
ium plates coated with Silica Gel 60 F254 (E. Merck) and visualised
by UV light (254 nm) where applicable, and by application of eth-
anolic H2SO4 (5% v/v) and heating. Compounds were purified by
flash chromatography on Silica Gel 60 (0.040–0.063 mm). NMR
spectra were recorded on a Bruker Avance 300 or 600 MHz spec-
trometer. 1H NMR and 13C NMR chemical shifts (d) are reported
in parts per million, relative to the residual solvent peak as internal
reference [CDCl3: 7.26 (s) for 1H; 77.0 (t) for 13C; D2O: 4.67 (s) for
1H; CD3CN: 1.32 for 13C; CD3OD: 3.31 for 1H; 49.0 for 13C]. 2D COSY
and HSQC experiments were run to support assignments. Low res-
olution mass spectra (LRMS) were acquired in positive or negative
ion mode as indicated, on a Bruker Daltonics Esquire 3000 ESI MS;
high-resolution mass spectrometry (HRMS) data was acquired at
the Griffith University FTMS Facility on a Bruker Daltonics Apex
III 4.7e Fourier Transform MS, fitted with an Apollo ESI source. Re-
versed phase (RP) HPLC purification was performed on an Agilent
HP1100 series system equipped with a C18 column [Synergi
4
5
l
l
m, Hydro-RP 80 Å (250 ꢀ 10 mm), Phenomenex; or, AQUA
m, C18 (250 ꢀ 10 mm), Phenomenex],
N-Acetylneuraminic acid (5-acetamido-3,5-dideoxy-D-glycero-
D
-galacto-non-2-ulosonic acid) was purchased from Jülich Chiral
Solutions GmbH (Jülich, Germany) and Carbosynth UK.
Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-3,5-
-glycero-
-galacto-non-2-enonate (12)36 and bromohy-
methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3-bromo-3,
-erythro- -gluco-non-2-ulopyranosonate, and methyl
5-acetamido-4,7,8,9-tetra-O-acetyl-3-bromo-3,5-dideoxy- -ery-
thro-
-mano-non-2-ulopyranosonate (13)23 were prepared accord-
ing to literature procedures. Syntheses of methyl 5-acetamido-4,
7,8,9-tetra-O-acetyl-3,5-dideoxy-3-C-(prop-20-enyl)-
/b- -erythro-
3.1.4. Methyl 5-acetamido-2,4,7,8,9-penta-O-acetyl-3,5-
dideoxy-3-C-[30-(p-tolyl)prop-20-enyl]-b-
2-ulopyranosonate (16)
D-erythro-L-gluco-non-
dideoxy-
drins
5-dideoxy-
D
D
A solution of compound 15 (3.1 g, 4.98 mmol) in anhydrous
pyridine (22 mL) under N2 was treated with acetic anhydride
(18 mL) and DMAP (59.8 mg, 0.49 mmol). The reaction mixture
was stirred at room temperature for 16 h. The progress of the reac-
tion was monitored by TLC analysis. The reaction mixture was
evaporated to dryness, taken up in EtOAc (60 mL) and the EtOAc
solution was washed successively with 0.1 N HCl (20 mL), water
(20 mL), and satd aq NaCl (25 mL). The organic phase was dried
(Na2SO4), filtered and the filtrate evaporated under reduced pres-
sure. The residue was purified by flash chromatography on silica
(EtOAc/hexane, 4:1) to afford the title compound 16 as a white so-
lid (3.13 g, yield 95%). Rf 0.50 (EtOAc); 1H NMR (300 MHz, CDCl3): d
1.83, 1.86, 2.00, 2.01, 2.12, 2.15 (6ꢀ s, 18H, NHCOCH3, OCOCH3 ꢀ5),
2.20 (m, 1H, –CH2–), 2.26 (m, 1H, H-3), 2.28 (s, 3H, p-tolyl CH3),
2.56 (m, 1H, –CH2–), 3.75 (s, 3H, COOCH3), 3.91 (dd, J = 10.5,
2.4 Hz, 1H, H-6), 4.11 (dd, J = 12.6, 6.9 Hz, 1H, H-9), 4.20 (ddd,
J = 10.5, 10.2, 10.2 Hz, 1H, H-5), 4.54 (dd, J = 12.3, 2.4 Hz, 1H, H-
90), 4.94 (m, 1H, H-8), 5.17 (dd, J = 10.5, 10.2 Hz, 1H, H-4), 5.26
(d, J = 9.6 Hz, 1H, NH D2O exchangeable), 5.33 (m, 1H, H-7), 5.96
(m, 1H, –CH@), 6.21 (d, J = 15.6 Hz, 1H, @CH-Ph), 7.05 (d,
J = 7.8 Hz, 2H, Ph), 7.14 (d, J = 8.1 Hz, 2H, Ph); 13C NMR
(75.5 MHz, CDCl3): d 20.8, 21.0, 21.1, 21.4 (OCOCH3 ꢀ5), 23.0
(NHCOCH3), 29.3 (p-tolyl CH3), 30.2 (–CH2–), 45.9 (C-3), 49.3 (C-
a-
D
L
a-D
L
a
D
L-
gluco-non-2-ulopyranosonate (14) was carried out as previously
described.17,22 The experimental details and physical data for new
compounds are provided below. The purities of all intermediate
compounds were determined to be >90% by 1H and 13C NMR (see
Supplementary data). The purity of target compounds was P95%
by HPLC analysis.
3.1.2. General method for base-catalysed compound
deprotection
A solution of compound (0.05 mmol) in MeOH/H2O 1:1 (4 mL)
at 5 °C is adjusted to pH 13 using aq NaOH (1 M). The solution is
then stirred at room temperature and the progress of reaction is
monitored by TLC analysis (EtOAc/MeOH/H2O, 7:2:1). At
completion of the reaction, AmberliteÒ IR-120 (H+) resin is added
to adjust to pH 3, the reaction mixture is filtered, the resin is
washed with MeOH/H2O 1:1 (25 mL), and the filtrate is concen-

4826
S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
5), 53.3 (COOCH3), 62.1 (C-9), 68.1 (C-7), 72.3 (C-8), 72.5 (C-6, C-4),
99.5 (C-2), 125.8 (–CH2–CH@, Ph), 129.3 (Ph), 130.9 (@CH-Ph),
134.3 (Ph q carbon), 137.0 (Ph q carbon), 165.8 (C-1), 167.9,
170.3, 170.6, 170.9, 171.3 (NHCOCH3, OCOCH3 ꢀ5) LRMS
[C32H41NO14] (+ve ion mode) (m/z): 686 [M+Na]+.
were washed with successively with aq sodium hydrogen carbon-
ate (5 mL) followed by brine (4 mL). The combined organic extracts
were then dried (Na2SO4), and evaporated to dryness. The crude
product was purified by column chromatography on silica
(EtOAc/acetone) to yield a white solid assigned as the title aziridine
derivative 18 (184 mg, yield 68%). Rf = 0.5 (EtOAc/acetone, 3:2); IR
vmax/cmꢂ1: 2101, 1738, 1660, 1369, 1212, 1042, 734; 1H NMR
(300 MHz, CDCl3): d 1.99, 2.12, 2.13, 2.32 (4ꢀ s, 12H, NHCOCH3,
OCOCH3 ꢀ3), 2.40 (s, 3H, p-tolyl CH3), 2.83 (dd, J = 17.7, 3.9 Hz,
1H, –CH2–), 3.17 dd, J = 17.7, 7.5 Hz, 1H, –CH2–), 3.63 (m, 1H, –
CH2–CH@), 3.75 (m, 1H, H-5), 3.82 (s, 3H, COOCH3), 4.11 (d.
J = 9.9 Hz, 1H, H-4), 4.22 (dd, J = 12.3, 7.2 Hz, 1H, H-9), 4.33 (dd,
J = 10.2, 1.5 Hz, 1H, H-6), 4.61 (d, J = 6.3 Hz, 1H, @CH-Ph), 4.81
(dd, J = 12.6, 2.4 Hz, 1H, H-90), 5.36 (m, 1H, H-8), 5.46 (dd, J = 4.5,
1.8 Hz, 1H, H-7), 5.73 (d, J = 9.0 Hz, 1H, NH), 7.24 (br s, 4H, Ph);
LRMS [C28H35N5O10] (+ve ion mode) (m/z): 602 [M+H]+.
3.1.5. Methyl 5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-3,5-
dideoxy-3-C-[30-(p-tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-
enonate (19)
To a solution of glycosyl acetate 16 (1.8 g, 2.71 mmol) in anhy-
drous ethyl acetate (50 mL) under N2 was added dropwise trimeth-
ylsilyl trifluoromethanesulfonate (1.42 mL, 7.88 mmol). After the
addition was complete the temperature was raised to 60 °C and
the reaction was stirred for 2 h. The progress of the reaction was
monitored by TLC analysis. After complete consumption of starting
material the reaction mixture was allowed to cool and was poured
into a vigorously stirred mixture of ice-cold saturated aq sodium
hydrogen carbonate (20 mL) and solid sodium hydrogen carbonate
(1 g). The layers were separated and the aqueous layer re-extracted
with ethyl acetate (2 ꢀ 60 mL). The combined organic layers were
evaporated and the intermediate 4,5-oxazoline compound 17 was
isolated as a white foam (1.39 g, 2.56 mmol, yield 95%). The oxaz-
oline was observed on TLC at Rf = 0.7 using ethyl acetate and prod-
uct formation was confirmed by MS (LRMS [C28H33NO10] (m/z):
(+ve ion mode) 566 [M+Na]+. It was hydrolysed without purifica-
tion. The crude oxazoline 17 was dissolved in ethyl acetate
3.1.7. 5-Acetamido-2,6-anhydro-3,5-dideoxy-3-C-[30-(p-
tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-enonic acid (20)
Compound 19 (78 mg, 0.14 mmol) was deprotected according
to the general procedure for 16 h. The crude product was purified
by RP-HPLC [column, Synergi; isocratic elution with 25% MeCN in
water (0.05% TFA); flow rate 3 mL minꢂ1; column temperature
40 °C: retention time 16.58–19.30 min] and then lyophilised to
give the title compound 20 as a white solid (52 mg, 90%). Rf = 0.4
(EtOAc/MeOH/H2O, 7:2:1); 1H NMR (300 MHz, D2O): d 2.07 (s,
3H, NHCOCH3), 2.33 (s, 3H, p-tolyl CH3), 3.12 (dd, J = 14.4, 7.2 Hz,
1H, –CH2–), 3.48 (m, 1H, –CH2–), 3.46–3.51 (m, 2H, H-7, H-9),
3.62–3.71 (m, 2H, H-8, H-90), 4.18–4.21 (m, 3H, H-4, H-5, H-6),
6.29 (m, 1H, –CH@), 6.50 (d, J = 15.9 Hz, 1H, @CH-Ph), 7.23 (d,
J = 7.5 Hz, 2H, Ph), 7.38 (d, J = 7.5 Hz, 2H, Ph); 13C NMR (125 MHz,
D2O): d 20.1 (p-tolyl CH3), 21.7 (NHCOCH3), 34.0 (–CH2–), 47.5
(C-5), 62.1 (C-9), 71.6 (C-6, C-7), 77.4 (C-4, C-8), 119.0 (C-3),
125.3 (–CH@), 126.1 (Ph), 129.3 (Ph), 132.0 (@CH-Ph), 134.3 (Ph
q carbon), 137.8 (Ph q carbon), 141.7 (C-2), 171.0 (C-1), 173.3
(NHCOCH3); LRMS [C21H27NO8] (m/z): (+ve íon mode) 444
[M+Na]+, (ꢂve íon mode) 420 [MꢂH]ꢂ; HRMS (ESI) (m/z):
[M+Na]+ calcd for C21H27NO8Na1 444.1629; found: 444.1642.
(40 mL). After the addition of trifluoroacetic acid (350 lL) and
water (1 mL) the reaction mixture was stirred at room temperature
for 16 h. The reaction mixture was diluted with more ethyl acetate
(40 mL) and neutralised with NaHCO3 solution. The organic layer
was separated, dried (Na2SO4), filtered and evaporated. The crude
product was purified by column chromatography on silica
(EtOAc/hexane) to yield the title compound 19 as a white foam
(1.38 g, yield 96%). Rf = 0.45 (EtOAc); 1H NMR (300 MHz, CDCl3):
d 1.91 (s, 3H, NHCOCH3), 2.04, 2.05, 2.08 (3ꢀ s, 9H, OCOCH3 ꢀ3),
2.30 (s, 3H, p-tolyl CH3), 3.35 (dd, J = 14.4, 7.5 Hz, 1H, –CH2–),
3.45 (dd, J = 14.4, 6.9 Hz, 1H, –CH2–), 3.79 (s, 3H, COOCH3), 4.09–
4.17 (m, 3H, H-4, H-6, H-9), 4.32 (ddd, J = 11.1, 10.2, 3.9 Hz, 1H,
H-5), 4.75 (dd, J = 12.3, 2.4 Hz, 1H, H-90), 5.29 (m, 1H, H-8), 5.42
(m, 1H, H-7), 5.83 (d, J = 9.9 Hz, 1H, NH; D2O exchanged), 6.14
(m, 1H, @CH–), 6.43 (d, J = 15.9 Hz, 1H, @CH-Ph), 7.08 (d,
J = 8.1 Hz, 2H, Ph), 7.21 (d, J = 8.1 Hz, 2H, Ph); 13C NMR
(75.5 MHz, CDCl3): d 20.8, 20.9, 21.1 (NHCOCH3, OCOCH3 ꢀ3),
23.2 (p-tolyl CH3), 33.1 (–CH2–), 46.3 (C-5), 52.2 (COOCH3), 62.4
(C-9), 65.5 (C-6), 67.8 (C-7), 71.9 (C-8), 72.4 (C-4), 121.4 (C-3),
125.9 (–CH@), 126.1 (Ph), 129.2 (Ph), 132.1 (@CH-Ph), 134.0 (Ph
q carbon), 137.2 (Ph q carbon), 140.4 (C-2), 162.8 (C-1), 170.0,
3.1.8. Methyl 5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-
azido-3,4,5-trideoxy-3-C-[3’-(p-tolyl)prop-20enyl]-
galacto-non-2-enonate (21)
D-glycero-D-
A mixture of 4-hydroxy derivative 19 (1.23 g, 2.2 mmol) and
diphenylphosphoryl azide (0.58 mL, 2.70 mmol) were dissolved
in anhydrous benzene (26 mL) under N2. The mixture was cooled
to 5–10 °C and DBU (0.4 mL, 2.70 mmol) was added under nitro-
gen. The reaction mixture was stirred at this temperature for
15 min and then ice bath was removed, reaction was continued
at room temperature. After stirring for 5 h at room temperature,
170.3, 170.5, 170.7 (NHCOCH3, OCOCH3 ꢀ3); LRMS [C28H35NO11
]
(+ve ion mode) (m/z): 584 [M+Na]+.
additional amounts of diphenylphosphoryl azide (120
lL,
3.1.6. Methyl 5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-
0.54 mmol) and DBU (80 L, 0.54 mmol) were added and stirring
l
azido-3,4,5-trideoxy-3-C-[30-(p-tolyl)aziridin-20-methyl]-
D
-
was continued for 2 h. To the mixture was added ethyl acetate
(20 mL) and 1 N HCl (5 mL). After stirring for a few minutes, the or-
ganic layer was separated, rewashed with 1 N HCl (4 mL), dried
(Na2SO4), filtered, evaporated to dryness. The crude product was
purified by column chromatography on silica (EtOAc/hexane) to
yield the title compound 21 as a white foam (1.09 g, yield 85%).
Rf = 0.40 (EtOAc); 1H NMR (300 MHz, CDCl3): d 1.94, 2.02, 2.04,
2.11 (4ꢀ s, 12H, NHCOCH3, OCOCH3 ꢀ3), 2.30 (s, 3H, p-tolyl CH3),
3.15 (dd, J = 14.7, 8.1 Hz, 1H, –CH2–), 3.76 (m, 1H, –CH2–), 3.78
(s, 3H, COOCH3), 4.11 (m, 2H, H-5, H-9), 4.24 (d, J = 8.4 Hz, 1H, H-
4), 4.30 (dd, J = 9.6, 2.7 Hz, 1H, H-6), 4.60 (dd, J = 12.6, 3.0 Hz, 1H,
H-90), 5.26 (m, 1H, H-8), 5.42 (dd, J = 5.1, 2.7 Hz, 1H, H-7), 5.62
(d, J = 9.0 Hz, 1H, NH), 6.06 (m, 1H, –CH@), 6.46 (d, J = 15.6 Hz,
1H, @CH-Ph), 7.08 (d, J = 7.8 Hz, 2H, Ph), 7.23 (d, J = 8.1 Hz, 2H,
Ph); 13C NMR (75.5 MHz, CDCl3): d 20.7, 20.8, 20.9, 21.1 (NHCOCH3,
glycero-D-galacto-non-2-enonate (18)
To a solution of oxazoline 17 (244 mg, 0.45 mmol) in anhydrous
tert-butyl alcohol (10 mL) under N2 was added dropwise azidotri-
methylsilane (209 mg, 1.82 mmol). After the addition was com-
plete the temperature was raised to 80 °C and the reaction was
stirred for 4 h. The progress of the reaction was monitored by
TLC analysis. After complete consumption of starting material the
reaction mixture was allowed to cool and was poured into a vigor-
ously stirred mixture of ice-cold saturated aq sodium nitrite
(10 mL) and then hydrochloric acid (0.1 N, 5 mL) was added over
a period of 10 min. Ethyl acetate (30 mL) and water (10 mL) were
then added and the organic layer was separated off and washed
with water (2 ꢀ 5 mL). The combined aqueous layers were ex-
tracted with ethyl acetate (10 mL) and the combined organic layers

S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
4827
OCOCH3 ꢀ3), 23.2 (p-tolyl CH3), 31.3 –CH2–), 49.1 (C-5), 52.3
3.1.11. Methyl 5-acetamido-2,6-anhydro-4-amino-3,4,5-
(COOCH3), 62.0 (C-4), 62.1 (C-9), 67.6 (C-7), 71.0 (C-8), 75.5 (C-6),
120.7 (C-3), 125.1 (–CH@), 126.1 (Ph), 129.2 (Ph), 132.4 (@CH-
Ph), 134.3 (Ph q carbon), 137.2 (Ph q carbon), 141.1 (C-2), 162.3
(C-1), 170.2, 170.3, 170.4, 170.5 (NHCOCH3, OCOCH3 ꢀ3); LRMS
[C28 H34N4O10] (+ve ion mode) (m/z): 609 [M+Na]+; HRMS (ESI)
(m/z): [M+Na]+ calcd for C28H34N4NaO10 609.2167; found:
609.2163; vmax/cmꢂ1 2100, 1743, 1660, 1370, 1215, 1040.
trideoxy-3-C-[30-(p-tolyl)prop-2’enyl]-
D-glycero-D-galacto-non-
2-enonate (24)
H2S gas was gently bubbled into a stirred mixture of the azide
compound 23 (300 mg, 0.65 mmol) in anhydrous pyridine
(10 mL) at room temperature for 16 h. The mixture was flushed
with nitrogen for 10 min and then evaporated to dryness keeping
the bath temperature below 40 °C. The residue was purified by col-
umn chromatography on silica (EtOAc/MeOH/Et3N, 25:10:1) to af-
ford title compound 24 as an off-white solid (226 mg, 80%). Rf = 0.4
3.1.9. 5-Acetamido-4-amino-2,6-anhydro-3,4,5-trideoxy-3-C-
[30-(p-tolyl)prop-20enyl]-
D
-glycero-
D
-galacto-non-2-enonic acid
(MeOH/EtOAc, 3:7); 1H NMR (300 MHz, CD3OD):
d
2.00
(8)
(NHCOCH3), 2.30 (s, 3H, p-tolyl CH3), 3.21 (dd, J = 15.3, 8.1 Hz,
1H, –CH2–), 3.54–4.04 (m, 11H, COOCH3, H-4, H-5, H-6, H-7, H-8,
H-9, H-90, 1H –CH2–), 6.17 (m, 1H, –CH@), 6.51 (d, J = 15.6 Hz,
1H, @CH-Ph), 7.08 (d, J = 8.1 Hz, 2H, Ph), 7.24 (d, J = 8.1 Hz, 2H,
Ph); 13C NMR (75.5 MHz, CD3OD): d 21.2 (p-tolyl CH3), 22.7
(NHCOCH3), 31.6 (–CH2–), 52.5 (COOCH3), 52.7 (C-5), 53.3 (C-4),
64.9 (C-9), 70.5 (C-8), 71.3 (C-7), 77.7 (C-6), 123.6 (C-3), 127.0
(Ph), 127.5 (–CH@), 129.9 (Ph), 132.7 (@CH-Ph), 136.0 (Ph q car-
To a solution of azide 21 (100 mg, 0.17 mmol) in dry tetrahy-
drofuran (5 mL) was added triphenylphosphine (89 mg,
0.34 mmol) at room temperature under N2. The reaction mixture
was stirred for 10 min at 50 °C. To the reaction solution were added
water (2 mL) and 25% aqueous sodium hydroxide (13.6 mg,
0.34 mmol) at 50 °C, followed by stirring for 2 h at the same tem-
perature. The reaction solution was cooled to 0 °C, concentrated
hydrochloric acid (100
l
L) was added and the mixture was allowed
bon), 138.1 (Ph q carbon), 142.5 (C-2), 165.2 (C-1), 175.1
to stand for 10 min. Subsequently, the aqueous layer was separated
and solvent was evaporated to dryness. To remove the formed tri-
phenylphosphane oxide and less polar impurities, the crude prod-
uct was chromatographed over 10 g of Florisil with
dichloromethane/methanol/0.2 M ammonium hydroxide in water
(4:4:1). The crude product was purified by RP-HPLC [column, Syn-
ergi; isocratic elution with 20% MeCN in water; flow rate
3 mL minꢂ1; column temperature 36 °C: retention time 12.50–
16.48 min] and then lyophilised to give the title compound 8 as
white solid (52 mg, 72% over 2 steps). Rf = 0.35 (1-propanol/H2O/
acetic acid, 15:4:0.5); 1H NMR (300 MHz, D2O): d 2.04 (s, 3H,
NHCOCH3), 2.33 (s, 3H, p-tolyl CH3) 3.04 (dd, J = 16.2, 7.2 Hz, 1H,
–CH2–), 3.40 (dd, J = 16.5, 5.7 Hz, 1H, –CH2–), 3.64–3.70 (m, 2H,
H-7, H-9), 3.87–3.95 (m, 2H, H-8, H-90), 4.05 (d, J = 8.4 Hz, 1H, H-
4), 4.26 (m, 1H, H-6), 4.40 (dd, J = 9.6, 8.7 Hz, 1H, H-5), 6.25 (m,
1H, –CH@), 6.57 (d, J = 15.9 Hz, 1H, @CH-Ph), 7.24 (d, J = 8.1 Hz,
2H, Ph), 7.39 (d, J = 8.1 Hz, 2H, Ph); 13C NMR (75.5 MHz, D2O): d
20.0 (p-tolyl CH3), 21.9 (NHCOCH3), 29.6 (–CH2–), 48.3 (C-5), 51.6
(C-4), 62.9 (C-9), 68.2 (C-7), 69.5 (C-8), 74.9 (C-6), 105.3 (C-3),
126.0 (Ph), 126.3 (–CH2–CH@), 129.4 (Ph), 131.0 (@CHPh), 134.2
(Ph q carbon), 137.9 (Ph q carbon), 149.5 (C-2), 170.6 (C-1), 174.7
(NHCOCH3); LRMS [C21H28N2O7] (ꢂve íon mode) (m/z): 419
[MꢂH]ꢂ; HRMS (ESI) (m/z): [M+Na]+ calcd for C21H28N2O7Na1
443.1789; found: 443.1787.
(NHCOCH3); LRMS [C22H30N2O7] (ꢂve ion mode) (m/z): 433
[MꢂH]ꢂ; HRMS (ESI) (m/z): [M+Na]+ calcd for C22H29N2O7
433.1980; found: 433.1978.
3.1.12. Methyl 5-acetamido-2,6-anhydro-4-[2,3-bis(tert-
butoxycarbonyl)guanidino]-3,4,5-trideoxy-3-C-[30-(p-
tolyl)prop-20enyl]-
D
-glycero-
D-galacto-non-2-enonate (25)
A solution of 24 (120 mg, 0.28 mmol), triethylamine (108
lL,
0.86 mmol) and [(tert-butoxycarbonyl)imino]pyrazol-1-ylmethylc-
arbamic acid tert-butyl ester (bisBocPCH; 128 mg, 0.41 mmol) in a
mixture of dry tetrahydrofuran (10 mL) and dry methanol (3 mL)
was stirred under nitrogen at room temperature for 7 days. The
progress of the reaction was monitored by TLC analysis. Then the
reaction mixture was evaporated to dryness and the crude product
was purified by column chromatography on silica (EtOAc/MeOH)
to yield the title compound 25 as a white solid (134 mg, 72%).
Rf = 0.8 (MeOH/EtOAc, 1:9); 1H NMR (300 MHz, CDCl3): d 1.40,
1.48 (2ꢀ s, 18H, NBoc), 2.01 (s, 3H, NHCOCH3), 2.30 (s, 3H, p-tolyl
CH3), 3.19 (dd, J = 14.7, 6.6 Hz, 1H, –CH2–), 3.48 (dd, J = 14.7, 6.3 Hz,
1H, –CH2–), 3.62 (d, J = 8.1 Hz, 1H, H-7), 3.79–3.87 (m, 4H, COOCH3,
H-9), 3.91-3.95 (m, 2H, H-8, H-90), 4.07–4.17 (m, 2H, H-5, H-6),
5.24 (br s, 1H, NH), 5.41 (m, 1H, H-4), 6.09 (m, 1H, @CH–), 6.30
(d, J = 15.9 Hz, 1H, @CH-Ph), 7.07 (d, J = 8.1 Hz, 2H, Ph), 7.21 (d,
J = 8.1 Hz, 2H, Ph), 8.33 (br s, 1H, NH), 8.90 (br s, 1H, NH); LRMS
[C33H48N4O11] (+ve íon mode) (m/z): 699 [M+Na]+.
3.1.10. Methyl 5-acetamido-2,6-anhydro-4-azido-3,4,5-
trideoxy-3-C-[30-(p-tolyl)prop-2’enyl]-
2-enonate (23)
D
-glycero-
D-galacto-non-
3.1.13. 5-Acetamido-2,6-anhydro-3,4,5-trideoxy-4-guanidino-3-
C-[30-(p-tolyl)prop-20enyl]-
D-glycero-D-galacto-non-2-enonic
To a solution of compound 21 (440 mg, 0.72 mmol) in anhy-
drous methanol (15 mL) at 5 °C was added 1 M sodium methoxide
solution (0.74 mL, 0.72 mmol) under N2. The reaction mixture was
stirred at room temperature for 1 h and the progress of the reaction
was monitored by TLC analysis. After complete consumption of
starting material AmberliteÒ IR-120 (H+) resin was added to adjust
pH 6.5, the reaction mixture was filtered, the resin was washed
with methanol (30 mL) and the filtrate was concentrated to dry-
ness under vacuum. The crude product was purified by column
chromatography on silica to yield the title compound 23 as a white
foam (310 mg, yield 90%). Rf = 0.45 (MeOH/EtOAc, 0.5:10); 1H NMR
(300 MHz, CD3OD): d 2.00 (s, 3H, NHCOCH3), 2.30 (s, 3H, p-tolyl
CH3), 3.16 (dd, J = 15.0, 8.4 Hz, 1H, –CH2–), 3.56 (d, J = 9.3 Hz, 1H,
H-4), 3.63–3.87 (m, 8H, COOCH3, H-5, H-6, H-9, H-90, 1H –CH2–),
4.16 (m, 1H, H-8), 4.27 (m, 1H, H-7), 6.14 (m, 1H, –CH@), 6.50 (d,
J = 15.9 Hz, 1H, @CH-Ph), 7.09 (d, J = 8.1 Hz, 2H, Ph), 7.25 (d,
J = 8.1 Hz, 2H, Ph); LRMS [C22H28N4O7] (+ve íon mode) (m/z): 483
[M+Na]+.
acid (9)
To a solution of compound 25 (60 mg, 0.09 mmol) in anhydrous
dichloromethane (10 mL) at 5 °C was added trifluoroacetic acid
(2 mL) under N2. The reaction mixture was stirred at room temper-
ature for 5 h. The progress of the reaction was monitored by TLC
analysis (Rf = 0.1; MeOH/EtOAc, 3:7). The solvent was removed un-
der vacuum. The crude product was deprotected according to the
general procedure for 16 h. The crude product was purified by
RP-HPLC [column, Synergi; isocratic elution with 15% MeCN in
water; flow rate 3 mL minꢂ1; column temperature 36 °C: retention
time 6.50–8.32 min] and then lyophilised to give the title com-
pound (9) as a white solid (34 mg, 83% over 2 steps). Rf = 0.35 (1-
propanol/H2O/acetic acid, 15:4:0.5); 1H NMR (300 MHz, D2O): d
2.00 (s, 3H, NHCOCH3), 2.33 (s, 3H, p-tolyl CH3), 3.00 (dd, J = 15.3,
8.7 Hz, 1H, –CH2–), 3.31 (dd, J = 15.0, 4.2 Hz, 1H, –CH2–), 3.62–
3.68 (m, 2H, H-7, H-9), 3.86–3.93 (m, 2H, H-8, H-90), 4.26–4.39
(m, 3H, H-4, H-5, H-6), 6.18 (m, 1H, –CH@), 6.46 (d, J = 15.9 Hz,
1H, @CH-Ph), 7.24 (d, J = 8.1 Hz, 2H, Ph), 7.36 (d, J = 8.1 Hz, 2H,

4828
S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
Ph); 13C NMR (75.5 MHz, D2O):
d
20.1 (p-tolyl CH3), 21.8
20.7, 20.8, 20.9, 21.1 (NHCOCH3, OCOCH3 ꢀ3), 23.2 (p-tolyl CH3),
(NHCOCH3), 31.2 (–CH2–), 53.1 (C-5), 62.9 (C-9), 68.1 (C-7), 69.6
(C-8), 75.1 (C-6), 106.6 (C-3), 126.0 (Ph), 126.7 (–CH2–CH@),
129.3 (Ph), 131.4 (@CH-Ph), 134.4 (Ph q carbon), 137.7 (Ph q car-
bon), 148.1 (C-2), 157.0 (C-1), 170.8 (NHCOCH3), (C-4 not ob-
served). LRMS [C22H30N4O7] (m/z): (+ve ion mode) 463 [M+H]+,
(ꢂve ion mode) 461 [MꢂH]ꢂ; HRMS (ESI) (m/z): [M+Na]+ calcd
for C22H30N4O7Na1 485.2007; found: 485.2014.
33.5 (–CH2–), 45.4 (C-5), 52.4 (COOCH3), 59.7 (C-4), 62.1 (C-9),
67.4 (C-7), 71.3 (C-8), 72.7 (C-6), 118.3 (C-3), 124.5 (–CH@),
126.2 (Ph), 129.2 (Ph), 132.8 (@CH-Ph), 134.0 (Ph q carbon),
137.3 (Ph q carbon), 141.1 (C-2), 162.4 (C-1), 169.8, 170.0, 170.2,
170.6 (NHCOCH3, OCOCH3 ꢀ3); LRMS [C28H34N4O10] (+ve ion
mode) (m/z): 609 [M+Na]+; HRMS (ESI) (m/z): [M+Na]+ calcd for
C28H34N4NaO10 609.2167; found: 609.2159.
3.1.14. Methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-2,6-anhydro-
3.1.16. Methyl 5-acetamido-2,6-anhydro-4-azido-3,4,5-
3,5-dideoxy-3-C-[30-(p-tolyl)prop-20enyl]-
D
-glycero-
D-talo-non-
trideoxy-3-C-[30-(p-tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-
2-enonate (26)
enonate (28)
To a solution of compound 19 (2 g, 3.55 mmol) in anhydrous
pyridine (10 mL) was added acetic anhydride (5 mL) and 4-
(dimethylamino)pyridine (43.5 mg, 1 mol %) at room temperature
under N2. The reaction mixture was stirred at room temperature
for 16 h (complete disappearance of starting material by TLC anal-
ysis). The reaction mixture was evaporated to dryness, taken up in
ethyl acetate (50 mL) and washed successively with 0.1 N HCl, H2O,
and aq NaCl. The organic phase was dried (anhydrous Na2SO4), fil-
tered and evaporated under reduced pressure, and the residue
purified by flash column chromatography on silica gel (EtOAc/hex-
ane, 4:1) to afford the title compound 26 as a white solid (2.06 g,
96%). Rf = 0.5 (EtOAc); 1H NMR (300 MHz, CDCl3): d 1.86, 2.03,
2.04, 2.06, 2.07 (5ꢀ s, 15H, NHCOCH3, OCOCH3 ꢀ4), 2.29 (s, 3H,
p-tolyl CH3), 3.09 (dd, J = 15.3, 7.2 Hz, 1H, –CH2–), 3.34 (dd,
J = 15.3, 6.6 Hz, 1H, –CH2–), 3.80 (s, 3H, COOCH3), 4.10–4.18 (m,
2H, H-6, H-9), 4.51 (ddd, J = 11.1, 4.2, 3.9 Hz, 1H, H-5), 4.78 (dd,
J = 12.3, 2.4 Hz, 1H, H-90), 5.26-5.30 [m, 2H, H-8, NH (D2O ex-
changed)], 5.46 (dd, J = 4.5, 2.4 Hz, 1H, H-7), 5.56 (d, J = 3.9 Hz,
1H, H-4), 6.05 (m, 1H, –CH@), 6.36 (d, J = 15.6 Hz, 1H, @CH-Ph),
7.06 (d, J = 7.8 Hz, 2H, Ph), 7.21 (d, J = 8.1 Hz, 2H, Ph); 13C NMR
(75.5 MHz, CDCl3): d 20.7, 20.8, 20.9, 21.0, 21.1 (NHCOCH3, OCOCH3
ꢀ4), 23.1 (p-tolyl CH3), 32.8 (–CH2–), 44.9 (C-5), 52.3 (COOCH3),
62.3 (C-9), 67.1 (C-4), 67.6 (C-7), 71.9 (C-8), 73.0 (C-6), 118.7 (C-
3), 125.2 (–CH@), 126.1 (Ph), 129.1 (Ph), 131.7 (@CH-Ph), 134.4
(Ph q carbon), 137.0 (Ph q carbon), 141.8 (C-2), 162.4 (C-1),
169.6, 170.0, 170.2, 170.5, 170.6 (NHCOCH3, OCOCH3 ꢀ4); LRMS
[C30H37NO12] (+ve mode) (m/z): 626 [M+Na]+.
To a solution of compound 27 (350 mg, 0.60 mmol) in anhy-
drous methanol (15 mL) at 5 °C was added 1 M sodium methoxide
solution (0.59 mL, 0.60 mmol) under N2. The reaction mixture was
stirred at room temperature for 2 h and the progress of the reaction
was monitored by TLC analysis. After complete consumption of
starting material AmberliteÒ IR-120 (H+) resin was added to adjust
pH 6.5, the reaction mixture was filtered, the resin was washed
with methanol (30 mL) and the filtrate was concentrated to dry-
ness under vacuum. The crude product was purified by column
chromatography on silica to yield the title compound 28 as a white
foam (247 mg, yield 90%). Rf = 0.45 (MeOH/EtOAc, 0.5:10); 1H NMR
(300 MHz, CDCl3): d 2.07 (s, 3H, NHCOCH3), 2.31 (s, 3H, p-tolyl
CH3), 2.98 (dd, J = 15.0, 8.7 Hz, 1H, –CH2–), 3.57 (dd, J = 9.0,
1.2 Hz, 1H, H-7), 3.77–3.90 (m, 6H, H-9, H-90, 1H –CH2–, COOCH3),
3.97–4.01 (m, 2H, H-6, H-8), 4.09 (d, J = 4.2 Hz, 1H, H-4), 4.27 (m,
1H, H-5), 6.07 (m, 1H, –CH@), 6.19 (d, J = 8.4 Hz, 1H, NH), 6.42 (d,
J = 15.9 Hz, 1H, @CH-Ph), 7.09 (d, J = 7.8 Hz, 2H, Ph), 7.23 (d,
J = 8.1 Hz, 2H, Ph); LRMS [C22H28N4O7] (+ve ion mode) (m/z): 483
[M+Na]+.
3.1.17. Methyl 5-acetamido-2,6-anhydro-4-amino-3,4,5-
trideoxy-3-C-[30-(p-tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-
enonate (29)
H2S gas was gently bubbled into a stirred mixture of the azide
compound 28 (240 mg, 0.52 mmol) in anhydrous pyridine
(10 mL) at room temperature for 16 h. The mixture was flushed
with nitrogen for 10 min and then evaporated to dryness keeping
the bath temperature below 40 °C. The residue was purified by col-
umn chromatography on silica (EtOAc/MeOH/Et3N, 25:10:1) to af-
ford the title compound 29 as an off-white solid (190 mg, 84%).
Rf = 0.4 (MeOH/EtOAc, 3:7); LRMS [C22H30N2O7] (m/z): (+ve ion
mode) 457 [M+Na]+, (ꢂve ion mode) 433 [MꢂH]ꢂ; HRMS (ESI)
(m/z): [M+Na]+ calcd for C22H30N2NaO7 457.1945; found:
457.1936.
3.1.15. Methyl 5-acetamido-7,8,9-tri-O-acetyl-2,6-anhydro-4-
azido-3,4,5-trideoxy-3-C-[30-(p-tolyl)prop-20enyl]-
talo-non-2-enonate (27)
D-glycero-D-
To a mixture of compound 26 (1.2 g, 2 mmol) in tetrahydrofu-
ran (28.8 mL) and H2O (12 mL) was added NaN3 (258 mg, 4 mmol)
and tetrakis(triphenylphosphine)palladium(0) (114 mg, 5 mol %)
under an atmosphere of nitrogen. The resulting mixture was stir-
red at 50 °C for 16 h (complete disappearance of starting material
by TLC analysis). The reaction mixture was concentrated to dry-
ness, and the residue was partitioned between diethyl ether
(25 mL) and H2O (10 mL). The organic layer was washed succes-
sively with 2 N HCl (2 ꢀ 10 mL), saturated NaHCO3 solution
(2 ꢀ 10 mL) and H2O (2 ꢀ 10 mL), and dried (Na2SO4). After solvent
removal the residue was purified by column chromatography on
silica (EtOAc/hexane, 3:2) to afford title compound 27 as a white
3.1.18. 5-Acetamido-2,6-anhydro-4-amino-3,4,5-trideoxy-3-C-
[30-(p-tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-enonic acid
(10)
Compound 29 (100 mg, 0.23 mmol) was deprotected according
to the general procedure for 16 h. The crude product was purified
by RP-HPLC [column, Synergi; isocratic elution with 25% MeCN in
water (0.05% TFA); flow rate 3 mL minꢂ1; column temperature
40 °C: retention time 10.80–12.39 and 14.22–18.67 min] and then
lyophilised to give the title compound 10 as a white solid (65 mg,
68%) and impurity 30 (23 mg, 24%). Rf = 0.4 (1-propanol/H2O/acetic
acid, 15:4:0.5); 10: 1H NMR (300 MHz, D2O): d 2.08 (s, 3H,
NHCOCH3), 2.33 (s, 3H, p-tolyl CH3), 3.03 (dd, J = 16.2, 7.5 Hz, 1H,
–CH2–), 3.38 (dd, J = 16.2, 4.8 Hz, 1H –CH2–), 3.66–3.72 (m, 2H,
H-7, H-9), 3.88–4.05 (m, 3H, H-4, H-8, H-90), 4.48 (apparent s,
2H, H-5, H-6), 6.24 (m, 1H, –CH@), 6.52 (m, 1H, @CH-Ph), 7.24 (d,
J = 8.1 Hz, 2H, Ph), 7.39 (d, J = 8.1 Hz, 2H, Ph); 13C NMR
(75.5 MHz, D2O + CD3CN): d 21.7 (p-tolyl CH3), 21.8 (NHCOCH3),
31.2 (–CH2–), 45.2 (C-5), 46.7 (C-4), 62.9 (C-9), 68.0 (C-7), 69.4
solid (960 mg, 82%). Rf = 0.6 (EtOAc/hexane, 4:1); IR vmax/cmꢂ1
:
2100, 1732, 1370, 1213, 1043, 909, 730; 1H NMR (300 MHz, CDCl3):
d 1.94, 2.04, 2.05, 2.06 (4ꢀ s, 12H, NHCOCH3, OCOCH3 ꢀ3), 2.30 (s,
3H, p-tolyl CH3), 3.05 (dd, J = 15.0, 8.4 Hz, 1H, –CH2–), 3.75 (dd,
J = 15.0, 5.7 Hz, 1H, –CH2–), 3.80 (s, 3H, COOCH3), 4.02–4.13 (m,
3H, H-4, H-6, H-9), 4.47 (ddd, J = 10.8, 4.2, 4.2 Hz, 1H, H-5), 4.68
(dd, J = 12.3, 2.4 Hz, 1H, H-90), 5.26 (m, 1H, H-8), 5.43 (dd, J = 5.1,
2.1 Hz, 1H, H-7), 5.64 (d, J = 10.2 Hz, 1H, NH), 6.07 (m, 1H, –
CH@), 6.43 (d, J = 15.6 Hz, 1H, @CH-Ph), 7.08 (d, J = 7.8 Hz, 2H,
Ph), 7.23 (d, J = 8.1 Hz, 2H, Ph); 13C NMR (75.5 MHz, CDCl3): d

S. Rudrawar et al. / Bioorg. Med. Chem. 21 (2013) 4820–4830
4829
(C-8), 69.6 (C-6), 125.8 (Ph), 125.9 (Ph), 126.4 (–CH2–CH@), 128.9
(Ph), 129.0 (Ph), 131.1 (@CH-Ph), 134.2 (Ph q carbon), 136.9 (Ph
q carbon), 149.5 (C-2), 169.7 (C-1), 173.3 (NHCOCH3), (C-3 not ob-
served). LRMS [C21H28N2O7] (m/z): (+ve íon mode) 443 [M+Na]+,
(ꢂve íon mode) 419 [MꢂH]ꢂ; HRMS (ESI) (m/z): [M+Na]+ calcd
for C21H28N2O7Na1 443.1789; found: 443.1802.
4.18 (m, 2H, H-4, H-6), 4.41 (dd, J = 11.4, 4.5 Hz, 1H, H-5), 6.21
(m, 1H, –CH@), 6.48 (m, 1H, @CH-Ph), 7.22 (d, J = 8.1 Hz, 2H, Ph),
7.37 (d, J = 8.1 Hz, 2H, Ph); 13C NMR (75.5 MHz, D2O): d 19.9 (p-tolyl
CH3), 21.3 (NHCOCH3), 33.1 (–CH2–), 46.2 (C-5), 48.5 (C-4), 62.7 (C-
9), 67.6 (C-7), 69.5 (C-8), 70.1 (C-6), 104.9 (C-3), 125.8 (Ph), 126.3 (–
CH2–CH@), 129.0 (Ph), 130.8 (@CH-Ph), 134.2 (Ph q carbon), 137.4
(Ph q carbon), 147.8 (C-2), 156.4 (C-10), 170.7 (C-1), 173.6
(NHCOCH3); LRMS [C22H30N4O7] (m/z): (+ve ion mode) 463
[M+H]+, (ꢂve ion mode) 461 [MꢂH]ꢂ; HRMS (ESI) (m/z): [M+Na]+
calcd for C22H30N4O7Na1 485.2007; found: 485.2004.
3.1.19. 4-Acetamido-2,6-anhydro-5-amino-3,4,5-trideoxy-3-C-
[30-(p-tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-enonic acid
(30)
1H NMR (600 MHz, D2O): d 1.84 (s, 3H, NHCOCH3), 2.29 (s, 3H,
p-tolyl CH3), 3.05 (dd, J = 15.0, 7.8 Hz, 1H, –CH2–), 3.15 (dd,
J = 15.0, 6.6 Hz, 1H, –CH2–), 3.65–3.71 (m, 3H, H-5, H-7, H-9),
3.87 (dd, J = 12.0, 2.4 Hz, 1H, H-90), 3.95 (m, 1H, H-8), 4.15 (d,
J = 10.8 Hz, 1H, H-6), 4.68 (d, J = 4.2 Hz, 1H, H-4), 6.18 (m, 1H, –
CH@), 6.40 (d, J = 15.6 Hz, 1H, @CH-Ph), 7.19 (d, J = 7.8 Hz, 2H,
Ph), 7.33 (d, J = 7.8 Hz, 2H, Ph); 13C NMR (125 MHz, D2O + CD3CN):
d 20.6 (p-tolyl CH3), 22.1 (NHCOCH3), 33.0 (–CH2–), 45.9 (C-5), 49.1
(C-4), 63.1 (C-9), 67.9 (C-7), 69.9 (C-8), 70.2 (C-6), 106.1 (C-3),
126.4 (Ph), 127.3 (–CH2–CH@), 129.6 (Ph), 131.1 (@CH-Ph), 134.9
(Ph q carbon), 137.5 (Ph q carbon), 147.9 (C-2), 170.0 (C-1), 175.0
(NHCOCH3). LRMS [C21H28N2O7] (m/z): (+ve ion mode) 443
[M+Na]+, (ꢂve ion mode) 419 [MꢂNa]ꢂ; HRMS (ESI) (m/z):
[M+Na]+ calcd for C21H28N2O7Na1 443.1789; found: 443.1808.
3.2. Biological evaluation
Sialidases: Recombinant influenza A virus N1 [A/California/04/
2009 (pdm09 H1N1)], and N2 [A/R1/5+/1957 (H2N2)] sialidases
were expressed in a baculovirus expression system as previously
described,13 from plasmids kindly provided by George F. Gao (Insti-
tute of Microbiology, Chinese Academy of Sciences, Beijing).
Sialidase inhibition assay: Compounds were assayed for their
capacity to inhibit influenza virus sialidase catalysed hydrolysis
of the fluorogenic substrate 4-methylumbelliferyl N-acetyl-a-D-
neuraminide (MUN) (Sigma–Aldrich), using a modification37 of
the method of Potier et al.38 Assays were carried-out in a 96-well
plate format using the reported19 procedure, modified by the addi-
tion of pre-incubation of the inhibitor with enzyme, before addi-
tion of the substrate. Stock solutions of the substrate MUN,
inhibitor and sialidase were prepared in the reaction buffer
(50 mM sodium acetate, 6 mM CaCl2, pH 5.5). The reaction mixture
3.1.20. Methyl 5-acetamido-2,6-anhydro-4-[2,3-bis(tert-
butoxycarbonyl)guanidino]-3,4,5-trideoxy-3-C-[30-(p-
tolyl)prop-20enyl]-
D-glycero-
D-talo-non-2-enonate (31)
A solution of 29 (241 mg, 0.55 mmol), triethylamine (217
lL,
with a final volume of 10 lL was prepared in a 96-well solid black
0.86 mmol) and [(tert-butoxycarbonyl)imino]pyrazol-1-ylmethylc-
arbamic acid tert-butyl ester (bisBocPCH; 344 mg, 1.11 mmol) in a
mixture of dry tetrahydrofuran (10 mL) and dry methanol (3 mL)
was stirred under nitrogen at room temperature for 3 days. The
progress of the reaction was monitored by TLC analysis. The reac-
tion mixture was evaporated to dryness and the crude product was
purified by column chromatography on silica (EtOAc/MeOH) to
yield the title compound 31 as a white solid (318 mg, 85%).
Rf = 0.8 (MeOH/EtOAc, 1:9); 1H NMR (300 MHz, CDCl3): d 1.42 (s,
9H, Boc), 1.45 (s, 9H, Boc), 2.04 (s, 3H, NHCOCH3), 2.29 (s, 3H, p-to-
lyl CH3), 3.12 (dd, J = 15.6, 7.8 Hz, 1H, –CH2–), 3.46–3.54 (m, 2H, H-
5, 1H –CH2–), 3.79–3.91 (m, 5H, H-9, H-90, COOCH3), 3.98–4.13 (m,
3H, H-4, H-6, H-8), 4.65 (m, 1H, H-7), 5.67 (s, 1H, AcNH), 6.06 (m,
1H, –CH@), 6.35 (d, J = 15.9 Hz, 1H, @CH-Ph), 7.05 (d, J = 7.8 Hz,
2H, Ph), 7.18 (d, J = 8.1 Hz, 2H, Ph), 8.40 (d, J = 6.0 Hz, 1H, C4–
NH), 8.78 (d, J = 4.5 Hz, 1H, guanidine NHBoc); LRMS
[C33H48N4O11] (+ve ion mode) (m/z): 699 [M+Na]+.
plate on ice. All inhibition assays were done in sixplicate over an
inhibitor concentration range of 1 mM to 1 nM (10-fold dilutions),
with a MUN concentration of 0.1 mM. The sialidase was pre-incu-
bated with the inhibitor for 15 min at room temperature, and then
the reaction was started by the addition of the substrate MUN. The
components were combined by brief centrifugation at up to
1000 rpm for approximately 10 s. Immediately after centrifugation
the reaction was incubated at 37 °C with 900 rpm shaking for
30 min. After incubation at 37 °C for 30 min, the reactions were
stopped by the addition of 0.25 mL of 0.2 M glycine buffered with
NaOH to pH 10. Neuraminidase activity was determined by spec-
trofluorometric measurement of the 4-methylumbelliferone re-
leased (excitation 365 nm, emission 445 nm) in
a Viktor 3
multiplate reader (Perkin Elmer); reading at 1 s per well. Sample
measurements were corrected for background fluorescence that
was not produced by the enzyme-catalyzed hydrolysis of the sub-
strate, by subtracting a blank sample that contained MUN in the
reaction buffer. Data analysis was carried out using SigmaPlot En-
zyme Kinetics Module (Systat Software).
3.1.21. 5-Acetamido-2,6-anhydro-3,4,5-trideoxy-4-guanidino-3-
C-[30-(p-tolyl)prop-20enyl]-
D-glycero-D-talo-non-2-enonic acid
(11)
Acknowledgements
To a solution of compound 31 (100 mg, 0.14 mmol) in anhy-
drous dichloromethane (10 mL) at 5 °C was added trifluoroacetic
acid (2 mL) under N2. The reaction mixture was stirred at room
temperature for 5 h. The progress of the reaction was monitored
by TLC analysis (Rf = 0.15, MeOH/EtOAc, 3:7). The solvent was re-
moved under vacuum. The resulting crude product was deprotected
according to the general procedure for 16 h. The crude product was
purified by RP-HPLC [column, Synergi; isocratic elution with 15%
MeCN in water; flow rate 3 mL minꢂ1; column temperature 36 °C:
retention time 5.80–7.92 min] and then lyophilised to give the title
compound 11 as a white solid (54 mg, 80% over 2 steps). Rf = 0.25
(1-propanol/H2O/acetic acid, 15:4:0.5); 1H NMR (300 MHz, D2O):
d 2.00 (s, 3H, NHCOCH3), 2.32 (s, 3H, p-tolyl CH3), 3.10 (dd,
J = 15.6, 7.8 Hz, 1H, –CH2–), 3.23 (dd, J = 15.6, 6.6 Hz, 1H, –CH2–),
3.60–3.70 (m, 2H, H-7, H-9), 3.87–3.96 (m, 2H, H-8, H-90), 4.11–
Yan Wu and Raphael Böhm are thanked for their excellent assis-
tance. M.v.I. gratefully acknowledges the financial support of the
Honda Foundation (Australia) in a grant to the Institute for Glyco-
mics, the National Health and Medical Research Council (NHMRC),
and the Australian Research Council (ARC).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.05.054.
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