Synthesis and inhibitory activity of sialic acid derivatives targeted at viral sialate-O-acetylesterases

Article abstract of DOI:10.1016/j.ejmech.2011.04.008

A series of sialosides modified at the 4- and 9-hydroxy group were synthesised and tested for inhibition of the viral haemagglutinin-esterase activity from various Orthomyxoviruses and Coronaviruses. While no inhibition of the sialate-4-O-acetylesterases

Full text of DOI:10.1016/j.ejmech.2011.04.008

European Journal of Medicinal Chemistry 46 (2011) 2852e2860
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and inhibitory activity of sialic acid derivatives targeted at viral
sialate-O-acetylesterases
,
Mathew Stanley a, Juliane Mayr b, Wolfgang Huber b, Reinhard Vlasak b, Hansjörg Streicher a
*
a Department of Chemistry and Biochemistry, University of Sussex, Brighton, BN1 9QG, UK
b Department of Molecular Biology, University of Salzburg, Salzburg, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of sialosides modified at the 4- and 9-hydroxy group were synthesised and tested for inhibition
of the viral haemagglutinin-esterase activity from various Orthomyxoviruses and Coronaviruses. While
no inhibition of the sialate-4-O-acetylesterases from mouse hepatitis virus strain S or sialodacryoadenitis
virus was found, a 9-O-methyl derivative displayed inhibitory activity against recombinant sialate-9-O-
acetylesterase from influenza C virus.
Received 25 November 2010
Received in revised form
25 March 2011
Accepted 2 April 2011
Available online 8 April 2011
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Hemagglutinin-esterase
Sialate-O-acetylesterase
Influenza C virus
Sialic acid
Sialoside
Inhibitor
Bovine coronavirus
Mouse hepatitis virus
Sialodacryoadenitis virus
1. Introduction
biological events include cell differentiation, tumor growth,
immunity, apoptosis, microbial infections and in particular cancer
Sialic acids are a group of biologically important 9-carbon sugars
which decorate, ketosidically linked to cell surface glycoconjugates,
the glycocalix and thus the cells of higher organisms. At this
exposed position, they serve as receptors for sialic acid-recognising
proteins and are consequently involved in wide variety of biological
events, both pathogenic and non-pathogenic [1e3]. One of the
most common modifications of sialic acids in mammals is O-acet-
ylation. It occurs either at C-4 or at any position within the glycerol
side chain of sialic acid and multiple acetylations are possible
(Fig. 1) [4,5].
Sialate-O-acetylation has attracted increased interest in recent
years due its abundance and involvement in many, including
pathological, biological processes. For instance, acetylation may
promote or hinder recognition of sialic acid by proteins, cells or
pathogens. O-acetylation may also slow down the activity of
degradative enzymes such as sialate lyases or sialidases [3]. Related
where they are considered markers for certain skin tumors and
a form of leukaemia [2e7]. Another important function of O-acet-
ylation is masking of siglec binding sites. CD22, a siglec regulating
the activity of the B-cell receptor, binds to a2-6 linked sialic acids,
and binding can be masked by 9-O-acetylation [8]. This masking is
regulated by the cellular sialate-O-acetylesterase, which thereby
also regulates B-cell receptor signal strength [9]. Loss of function of
the esterase activity results in autoimmune disease [10,11].
Besides these functions, sialic acids are also used as docking
platforms for viral pathogens. Several RNA viruses which infect the
respiratory and gastrointestinal tract utilize sialic acids as
a receptor determinant. To facilitate release of progeny virus from
infected cells, a number of viruses express “receptor-destroying
enzymes” (RDE), which are targets for antiviral drugs. The best
known are the sialidase-inhibitors Zanamivir (Relenza) and Osel-
tamivir (Tamiflu). Besides sialidases, the haemagglutinin-esterases
(HE) of influenza C virus, isavirus, betacoronaviruses and tor-
oviruses represent another class of RDEs. They are sialate-O-ace-
tylesterases (SOAE) hydrolysing O-acetyl esters of O-acetylated
sialic acid derivatives (Fig.1). Two main subtypes of HEs are known:
* Corresponding author.
E-mail address: h.streicher@sussex.ac.uk (H. Streicher).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.04.008

M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
2853
Table 1
Selection of HE expressing viruses of three different taxa and their substrate
specificity.
Virus
Substrate specificity of HE
Orthomyxovirus
Influenza C virus (INF-C)
Infectious salmon anaemia virus (ISAV)
Betacoronavirus
Neu5,9Ac2
Neu4,5Ac2
Human coronavirus strain OC43 (HCoV-OC43) Neu5,9Ac2
Bovine coronavirus (BCoV)
Rat sialoadacryoadenitis coronavirus (SDAV)
Mouse hepatitis virus strain S (MHV-S)
Torovirus
Neu5,9Ac2
Neu4,5Ac2
Neu4,5Ac2
Bovine torovirus (BToV)
Porcine torovirus (PToV)
Neu5,7(8),9Ac3 and Neu5,9Ac2
Neu5,9Ac2
Fig. 1. Structures of acetylated sialic acids and action of viral sialate-O-acetylesterases
(SOAEs).
Enzymes from four different viruses, influenza C virus (INF-C)
[36,40e42], bovine coronavirus (BCoV) [43,44], mouse hepatitis
virus strain S (MHV-S) [45,46] and sialodacryoadenitis virus (SDAV)
[47] were investigated.
sialate-4-O-acetylesterases (4-SOAE) and sialate-9-O-acetyles-
terases (9-SOAE).
HE expressing viruses include human pathogens like influenza C
viruses, the respiratory human coronavirus OC43 (HCoV OC43) and
HKU1 (HCoV HKU1), several important animal betacorona- and
toroviruses and infectious salmon anaemia virus (ISAV), a piscine
orthomyxovirus. HCoVs account for 10e30% of respiratory infec-
tions, in particular, the common cold, but they can also cause
gastroenteritis and neurological disorders [12,13]. The infections
are usually mild and subclinical, but strains related to OC43 and
HKU1 were associated with severe human disease [14,15]. Beta-
coronaviruses have attracted attention after the outbreak of SARS
CoV in 2002/2003. This genus consists of human and animal
viruses. Coronaviruses are able to cross the animal-to-human
species barrier: bat-to-human in case of SARS CoV [16,17] and
bovine-to-human in case of BCoV leading to HCoV OC43 [18]. Tor-
oviruses (ToV) are evolutionary related to coronaviruses [19]. ToV
are associated with asymptomatic enteric infections in pigs [20].
Studies suggest that they are highly prevalent in swine populations
[21,22]. The closely related bovine toroviruses (BToV) are impli-
cated with serious or even fatal infections [23e25]; they are found
worldwide [26e30]. ToV are also associated with human gastro-
enteritis. In fecal samples from children and adults with diarrhoea,
torovirus antigens were detected by ELISA [31,32], immunoelectron
microscopy [33e35] and by reverse transcription polymerase chain
reaction with primers covering a highly conserved region of the ToV
genomes [35].
2. Inhibitor design
The allyl group was chosen as an aglycon mimetic in target allyl
sialosides 1e4 and control sialoside 5 because it offers a range of
selective chemical methods, such as e. g. olefin metathesis, for
further functionalisation or immobilisation of the inhibitors. To
probe the active sites of 4-SOAE and 9-SOAE, two types of modifi-
cations of the positions 4 and 9 of the sialosides were introduced
(Fig. 2).
Firstly, methylation resulting in target structures 1 and 3 should
yield information whether additional hydrophobic interactions
could contribute to more efficient binding and about the role of the
respective hydroxyl group as hydrogen bond donor. Secondly,
methylphosphonate groups were introduced as mimetics of the
suspected tetrahedral intermediate of acetate hydrolysis. In theory,
compounds 2 and 4 could interact with active site amino acids
stabilising this polar transition state, including the ‘oxyanion hole’
common in serine esterases [48].
3. Syntheses
a-Allyl sialoside 6, which serves as intermediate for all
syntheses, was conveniently synthesised in high yield in 4 steps
from commercially available N-acetylneuraminic acid using a well-
established Koenigs-Knorr methodology (Scheme 1) [49,50].
Control compound 5 was obtained from 6 through saponification.
Selective methylation of the 4-OH was then made possible by
blocking positions 8 and 9 as the isopropylidene ketal through acid
catalysed reaction of 6 with 2,2-dimethoxypropane to give 7 in 85%
yield. Alkylation of 7 under Williamson-conditions followed by
acid-mediated ketal hydrolysis and basic saponification of the
methyl ester gave crude inhibitor 1 which was purified by gel
permeation chromatography. For the introduction of a phospho-
nate group at position 4, hydroxyl groups 8 and 9 were protected as
Regarding the potential for transmission of betacoronaviruses
from animal to human and the danger of the emergence of further
epidemics, efficient treatment(s) would be of great interest
(Table 1).
Interestingly, the comparison of the crystal structures of influ-
enza C virus HEF [36], BCoV HE [37] and two ToV HEs [38] shows
highly conserved sites of the SOAE domains. Therefore, the SOAE
active site is probably an excellent target for broad-spectrum
antivirals against sialate-O-acetylesterases of both orthomyxo-
and coronaviruses.
The successful development and introduction of the anti-
influenza drugs Tamiflu and Relenza which are inhibitors of the
‘receptor-destroying’ sialidase from influenza virus has demon-
strated that such an approach is promising.
Earlier we postulated two essential pharmacophoric groups of
Neu5,9Ac2 in correct spatial arrangement required for strong sub-
strateeenzyme interaction with sialate-9-O-acetylesterases: the 9-
O-acetyl and the alpha-C2 carboxylate group [39]. Although no
investigations about the substrateeenzyme interactions of sialate-
4-O-acetylesterases are available, a similar mechanism as for 9-O-
acetylesterases is suggested.
Fig. 2. Structures of target sialosides 1e5. 1: R1 ¼ H, R2 ¼ CH3. 2: R1 ¼ H, R2 ¼ P(O)
(CH3)OꢀNH4þ. 3: R1 ¼ CH3, R2 ¼ H. 4: R1 ¼ P(O) (CH3)OꢀNH4þ, R2 ¼ H. 5: R1 ¼ R2 ¼ H.
In light of these, we embarked on a study which aims to find
competitive inhibitors of 9- and 4-SOAE, which are serine esterases.

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M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
used as a negative control. No sialidase activity against any of the
inhibitors was detected in the esterase preparations. The produc-
tion, isolation and purification of the viruses and enzymes as well
as the assays are described in the experimental section.
4.2. Inhibition results
Sialate-O-acetylesterases were incubated in the presence of
different sialoside concentrations. Table 2 summarises the inhibi-
tory effect of 9-modified sialosides 3 and 4 toward the 9-SOAE
enzymes determined by pNPA assay. We identified the 9-O-methyl
sialoside 3 as a potential inhibitor of HE12-GFP. At a concentration
of 5 mM compound 3 the inhibition is approximately 86%. Analysis
by fluorimetric HPLC revealed a 10e15% inhibition by 3 of
Neu5,9Ac2-hydrolysis by HE12-GFP at a concentration of 1 mM,
thus confirming the results of the pNPA assay (data not shown).
Less inhibitory effect of 3 was detected with the whole influenza
virus particles. We suggest that the different conformation of the
haemagglutinin protein (recombinant HE12-GFP ¼ monomeric
protein; whole virus ¼ trimeric protein) may contribute to the
differences in the inhibitory effect of sialoside 3. The 9-SOAE of
BCoV was also less effected by 3. According to [39], it seems that the
BCoV is more dependent on the aglycon moiety than the influenza
C virus esterase. It may be that the allyl group of the sialosides has
an effect on the enzymatic reaction and consequently less inhibi-
tion was detected.
Comparison of the inhibitory activities of 9-modified sialosides
3 and 4 suggests that the negatively charged methylphosphonate
does not induce detectable inhibition when compared to the
unmodified sialoside 5. There is however, a significant effect of the
small, hydrophobic, methyl group at the same position indicating
that inhibition can be improved by further modifications at this
position. This effect has so far been seen only with the recombinant
esterase from the influenza C virus, not with whole virus. It can be
speculated that this is a result of it being a monomer rather than the
native trimer, but that will require further studies with optimised
inhibitors of higher affinity.
Scheme 1. Synthesis of reference compound 5 and intermediate 6. (a) 1. MeOH, IR-
120(Hþ). 2. AcCl, AcOH. 3. CH2]CH2CH2OH, Ag2CO3, AgClO4. 4. NaOMe, MeOH. (b) 1.
NaOH. 2. Gpc (0.1 M NH4HCO3).
the cyclic carbonate using diphosgene and dimethylaminopyridine
to give 8. Introduction of the phosphonate was achieved with
methyl methylphosphonyl chloride and Hünig’s base to give
phosphonate diester 9. The position of the phosphonylation was
confirmed by acetylation of the remaining hydroxyl group at
position 7 and analysis of the product (not shown). Selective
cleavage of the phosphonic acid methyl ester with thiophenol and
triethylamine followed by basic hydrolysis of all other ester groups
and purification furnished compound 2 in good yield. (Scheme 2).
For methylation at position 9, compound 6 was converted into
the 8,9-epoxide by subsequent treatment with toluenesulfonyl
chloride in pyridine and sodium methoxide in methanol to give 10.
Acid-mediated opening of the epoxide in methanol gave the 9-
methyl ether and, after saponification of the methyl ester and
purification, target inhibitor 3. For the 9-phosphonate, direct reac-
tion of 5 with methyl methylphosphonylchloride and Hünig’s base
followed by per-O-acetylation to yield 11 proved to be the best
route. Deprotection of 10 was carried out as described for 9 and thus
inhibitor 4 was obtained in 20% overall yield (4 steps) (Scheme 3).
4. Inhibition of viral sialate-O-acetylesterases
4.1. General
Inhibition of the SOAE activity of three viruses, influenza C virus
(INF-C), bovine coronavirus (BCoV) and mouse hepatitis virus strain
S (MHV-S) and of two chimeric recombinant viral haemagglutinin
esterases, from influenza C/Cal/78 virus (HE12-GFP) and sialoda-
cryoadenitis virus (SDAV-HE) was investigated.
For influenza C virus esterase, these results are in line with those
from an earlier study where a Ki of 4.2 mM was determined for
a 9-acetamido-9-deoxy-sialic acid derivative [51].
The inhibitory effect of compounds 3 and 4 toward the 9-SOAE
activities of HE12-GFP, INF-C virus and BCoV and of compounds 1
and 2 toward the 4-SOAE activity of SDAV-HE and MHV-S were
determined by pNPA assay and fluorimetric HPLC. Compound 5 was
Table 3 summarises the inhibitory behaviour of compounds 1
and 2 toward MHV-S and SDAV-HE determined by the pNPA assay.
No significant inhibition was observed even at concentrations of
5 mM, indicating that there is less scope for modification of the 4-
Scheme 2. Syntheses of sialosides modified at the hydroxy group in position 4. (a) DMP, TsOH, acetone. (b) 1. DMS, NaH, acetonitrile. 2. 80% AcOH, then NaOH. 3. Gpc (0.1 M
NH4HCO3). (c) Diphosgene, DMAP, CH2Cl2. (d) ClP(O) (OCH3)CH3, EtNiPr2, CH2Cl2. (e) 1. PhSH, NEt3, THF. 2. Pyridine, NEt3, H2O. 3. Gpc (0.1 M NH4HCO3).

M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
2855
Scheme 3. Syntheses of sialosides modified at the hydroxy group in position 9. (a) 1. TsCl, pyridine. 2. NaOMe, MeOH. (b) 1. MeOH, IR-120(Hþ). 2. 0.05 M NaOH, dioxane. 3. Gpc
(0.1 M NH4HCO3). (c) 1. ClP(O) (OCH3)CH3, EtNiPr2, CH2Cl2. 2. Ac2O, pyridine. (d) 1. PhSH, NEt3, THF. 2. 0.05 M NaOH, dioxane. 3. Gpc (0.1 M NH4HCO3).
hydroxy group. It should be kept in mind as well that the mode of
action of 4-SOAE is may be different to that of 9-SOAE.
Unmodified control sialoside 5 (negative control) did not show
detectable inhibition of any of the enzymes. 3,4-Dichlor-
oisocoumarin (positive control) was highly reactive towards all
esterase activities at 0.1 mM concentration.
system equipped with glass columns packed with LiChroprep Si 60
(15e25 m) from Merck, Darmstadt, Germany. Solvents for chroma-
tography were used as received except for toluene and ethyl acetate,
which were distilled beforeuse. Gelpermeationchromatography was
carried out in the 1e10 mg scale on an XK 16/70 column (bed volume
130 mL), from Amersham packed with Sephadex G-10 (particle size
m
40e120 mm) and 0.1M NH4HCO3 as buffer. Detection was achieved
with a differential refractometer from Knauer, Berlin, Germany.
1H NMR, 13C NMR, 31P NMR and all multidimensional spectra
were recorded on Varian VNMRS spectrometers (600 MHz,
500 MHz or 400 MHz). Chemical shifts in 1H and 13C NMR spectra
were referenced to the residual proton resonance of the respective
deuterated solvents, CDCl3 (7.26 ppm), D2O (4.80 ppm) and CD3OD
(3.31 ppm) respectively. For 31P NMR spectra H3PO4 was used as
external standard (0 ppm).
HR-ESI-MS spectra were recorded on a Bruker Daltonics Apex III
in positive mode with MeOH and/or H2O as solvent. Where
possible, HR-ESI-MS has been used to characterise compounds
which have been synthesised.
5. Conclusions
We have synthesised a set of modified sialosides useful for
probing the active sites of 4- and 9-sialate-O-acetylesterase
enzymes. We have screened the compounds for inhibition of a set
of viral SOAE’s and while no inhibition of 4-SOAE could be detected,
a 9-O-methyl derivative showed inhibition of the recombinant
SOAE from influenza C virus. Further studies on how his can be
exploited to develop high-affinity inhibitors of the enzyme as
potential lead compounds for drug development are under way.
6. Experimental section
Abbreviations: EA: ethyl acetate; DCM: dichloromethane; Tol:
toluene. THF: tetrahydrofuran.
6.1. General
Whereanhydroussolventswererequiredforreactions, thesewere
purchased (anhydrous) and used as received. DCM was doubly
distilled (over CaH2) before use. Fine chemicals were purchased from
Aldrich-, Sigma- or Acros-Chemicals and were of the highest purity
available. Reactions were monitored via thin layer chromatography
6.2. Chemistry
6.2.1. Ammonium (allyl 5-acetamido-3,5-dideoxy-4-O-methyl-
D-
glycero- -galacto-2-nonulopyranosidonate) (1)
a-D
Compound 7 (55.6 mg, 0.138 mmol) was dissolved in dry CH3CN
(TLC) using pre-coated silica sheets with fluorescent indicator UV254
.
(0.7 mL), under N2 (g) and was cooled to 0 ꢁC (ice bath). DMSO4
Compound detection was achieved by UV absorption and by devel-
oping plates by staining with a molybdenum phosphate reagent (20 g
ammonium molybdate and 0.4 g cerium(IV) sulphate in 400 mL of 10%
aqueous sulphuric acid) with subsequent heating.
Chromatographic purification was performed using silica gel 60A
‘Davisil’ (particle size 35e70 mm) from Fisher Scientific, UK. Silica-
(24.5
(6.6 mg, 0.276 mmol). The resulting suspension was allowed to stir
for 4 h at 0 ꢁC, more DMSO4 (24.5
L, 0.259 mmol) was added and the
suspension was left to stir overnight. When TLC indicated comple-
tion of the reaction, the reaction was quenched with excess NH4Cl(s)
and the solution was diluted with DCM (3 mL), filtered over Celite
and evaporated to dryness. After flash chromatography (Tol:Acetone
mL, 0.259 mmol) was added to the solution, followed by NaH(s)
m
based MPLC chromatography was carried out on the Büchi Sepacore
Table 2
Table 3
Inhibitory effect of 9-modified sialosides 3 and 4 on the 9-SOAE enzymes from INF-C
Inhibitory effect of 4-modified sialosides 1 and 2 on the 4-SOAE enzymes from
virus and BCoV.
MHV-S and SDAV.
Sialoside concentration
HE12-GFP
INF-C virus
BCoV
Sialoside concentration
MHV-S
SDAV-HE
3
4
3
4
3
4
1
2
1
2
0.5 mM
1 mM
2 mM
5 mM
22.95%a
22.42%
33.03%
85.79%
1.06%
6.08%
7.91%
e
7.01%
8.63%
7.05%
e
<0%
3.85%
7.39%
e
2.86%
9.00%
<0%
e
9.78%
2.70%
<0%
e
0.1 mM
0.5 mM
1 mM
4.00%a
0%
8.31%
7.73%
5.00%
2.36%
11.89%
7.81%
2.13%
2.56%
0.51%
8.73%
8.30%
2.89%
0.65%
10.96%
5 mM
a
a
Inhibition (in %) of 9-SOAE-catalysed hydrolysis of p-nitrophenyl acetate.
Inhibition (in %) of 4-SOAE-catalysed hydrolysis of p-nitrophenyl acetate.

2856
M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
1:1) the corresponding 4-O-methyl ether was obtained as a clear oil
(29.3 mg, 51%). Rf : 0.24 (Tol:Acetone 2:1). HR-ESI-MS (m/z)
[M þ Na]þ calculated for C19H31NO9Na: 440.1897. Found : 440.1891.
1H NMR (600 MHz, CDCl3) dH: 1.36, 1.39 (2s, 6H, eC(CH3)2), 1.73 (dd,
1H, J ¼ 12.1, 12.2 Hz, H3ax), 2.05 (s, 3H, eNHCOCH3), 2.87 (dd, 1H,
J ¼ 4.5,12.8 Hz, H3eq), 3.34 (s, 3H, eOCH3), 3.37 (d,1H, J ¼ 10.5 Hz, H6),
3.42 (ddd, 1H, J ¼ 4.5, 10.7, 10.9, H4), 3.56 (dd, 1H, J ¼ 5.6, 5.7 Hz, H7),
3.78 (s, 3H, eCO2CH3), 3.87 (m, 1H, H5), 4.04e4.13 (m, 3H, H9, H90,
- CHH’eCH ¼ CH2), 4.28e4.33 (m, 2H, H8, CHH’eCH ¼ CH2), 4.42 (d,
1H, J ¼ 4.9 Hz, eOH), 5.15 (d, 1H, J ¼ 10.5 Hz, eCH2eCH ¼ CHcisH),
5.25 (d,1H, J ¼ 17.2 Hz, eCH ¼ CHtransH), 5.39 (d,1H, J ¼ 7.4 Hz, eNH),
5.86 (ddd, 1H, J ¼ 55.6, 10.7, 16.4 Hz, eCH2eCH ¼ CH2). 13C NMR
(151 MHz, CDCl3) dC: 23.47 (eNHCOCH3), 25.75, 26.99 (eC(CH3)2),
36.42 (C3), 51.27 (C5), 52.54 (eCO2CH3), 55.57 (eOCH3), 65.47
(eCH2CH]CH2), 66.93 (C9), 69.86 (C7), 74.76 (C6), 75.44 (C8), 75.94
(C4), 99.21 (C2), 108.71 (eC(CH3)2), 117.31 (eCH2CH]CH2), 134.03,
(eCH2CH]CH2), 169.20 (C1), 173.85 (eNHCOCH3).
The methyl ether (19.9 mg, 0.0477 mmol) was dissolved in 80%
aqueous AcOH (1.5 mL), the mixture was stirred for 2 h, the solvent
was evaporated and the oily product was then co-evaporated with
toluene and dried in vacuo. Aqueous NaOH (1.5 mL, 0.1 M) was
added to the oil and the resulting solution was left to stir for 2 h.
After completion, the solution was neutralised using Amberlite IR-
120 (Hþ) ion-exchange resin, the resin was removed and the
solvent removed in vacuo yielding 1 (17.9 mg, wqu). A sample of 1
(9.9 mg) was purified by gel permeation chromatography which
was then lyophilised to give a white powder. HR-ESI-MS (m/z)
1H, J ¼ 1.5, 10.4 Hz, H6), 3.62 (dd,1H, J ¼ 5.8,11.4 Hz, H9), 3.74 (dd,1H,
J ¼ 10.1,10.2 Hz, H5), 3.83 (dd,1H, J ¼ 2.5,11.4 Hz, H9’), 3.86 (m,1H, H8),
4.01 (ddd, 1H, J ¼ 1.2, 5.6, 12.6 Hz, eCHH’eCH ¼ CH2), 4.26e4.32 (m,
2H, H4, eCHH’eCH ¼ CH2), 5.10 (dd, 1H, J ¼ 1.4, 10.5 Hz,
eCH2eCH
¼
CHHcis), 5.25 (dd, 1H,
J
¼
1.7, 17.3 Hz,
eCH2eCH ¼ CHHtrans), 5.90 (m, 1H, eCH2eCH ¼ CH2). 13C NMR
(126 MHz, MeOH-D4) dC: w13.68 (d, J ¼ 139.2 Hz, PeCH3), 22.94
(eNHCOCH3), 41.49 (C3), 53.69 (C5), 64.76 (C9), 66.51 (eCH2CH]
CH2), 70.52 (C7), 71.51 (C4), 72.92 (C8), 75.28 (C6),100.57 (C2),116.92
(eCH2CH]CH2), 135.92 (eCH2CH]CH2), 172.77 (C1), 176.05
(eNHCOCH3). 31P NMR (400 MHz, MeOH-D4) dP: 24.73 (s).
6.2.3. Ammonium (allyl 5-acetamido-9-O-methyl-3,5-dideoxy-d-
glycero-a-d-galacto-2-nonulopyranosidonate) (3)
A stirred solution of epoxide 10 (50 mg, 0.145 mmol) in meth-
anol (1.2 mL) was acidified (to pH 2) with Amberlite IR-120 (Hþ)
ion-exchange resin, the resin was filtered off and the solvent
evaporated from the solution to leave an oily residue. The crude
product was purified by flash chromatography (EA / EA:MeOH;
5:1) to yield the 9-O-methyl ether (16.4 mg, 45%). Rf : 0.38
(EA:MeOH; 5:1). HR-ESI-MS (m/z) [M þ Na]þ calculated for :
C16H21NO9Na: 400.1584. Found: 400.1578. 1H NMR (500 MHz,
CDCl3) dH: 2.04 (s, 3H, -NHCOCH3), 1.96 (dd, 1H, J ¼ 11.7, 12.2 Hz,
H3ax), 2.74 (bs, 1H, eOH), 2.81 (dd, 1H, J ¼ 4.3, 13.3 Hz, H3eq),
3.39e3.43 (m, 1H, H6), 3.43 (s, 3H, eOCH3), 3.53 (dd, 1H, J ¼ 3.3,
9.0 Hz, H7), 3.65e3.80 (m, 5H, H4, H5, H9, H9’, OH), 3.85 (s, 3H,
eCO2CH3), 3.92 (dd, 1H, J ¼ 5.9, 12.5 Hz, eCHH’eCH ¼ CH2), 4.01
(dd, 1H, J ¼ 3.5, 8.7 Hz, H8), 4.27 (dd, 1H, J ¼ 5.4, 12.6 Hz,
eCHH’eCH ¼ CH2), 4.67 (bs, 1H, OH), 5.15 (d, 1H, J ¼ 10.5 Hz,
CH2eCH ¼ CHcisH), 5.23 (dd, 1H, J ¼ 1.0, 17.2 Hz, eCH ¼ CHtransH),
5.83 (ddd, 1H, J ¼ 5.7, 10.8, 16.3 Hz, eCH2eCH ¼ CH2), 6.73 (d, 1H,
J ¼ 6.8 Hz, eNH). 13C NMR (126 MHz, CDCl3) dC: 23.07 (eNHCOCH3),
40.79 (C3), 53.33, 53.42 (C5, eCO2CH3), 59.21 (eOCH3), 65.42
(eCH2eCH ¼ CH2), 68.15 (C4), 68.79 (C7), 69.98 (C8), 73.86, 73.87
[M
þ
Na]þ calculated for
: C15H25NO9Na: 386.1427. Found:
386.1422. 1H NMR (600 MHz, D2O) dH: 1.65 (dd, 1H, J ¼ 12.0,12.1 Hz,
H3ax), 2.10 (s, 3H, eNHCOCH3), 2.99 (dd, 1H, J ¼ 4.5, 12.6 Hz, H3eq),
3.48 (s, 3H, eOCH3), 3.52 (m, 1H, H4), 3.65 (d, 1H, J ¼ 8.9 Hz, H7),
3.72 (m,1H, H9), 3.81 (dd,1H, J ¼ 1.5,10.5 Hz, H6), 3.91e4.00 (m, 3H,
H5, H8, H9’), 4.12 (ddd,1H, J ¼ 1.2, 5.7,12.2 Hz, CHH’eCH ¼ CH2), 4.33
(ddd, 1H, J ¼ 1.1, 6.0, 12.1 Hz, CHH’eCH ¼ CH2), 5.30 (d, 1H,
J ¼ 10.3 Hz, eCH2eCH ¼ CHcisH), 5.41 (dd, 1H, J ¼ 1.4, 17.3 Hz,
eCH ¼ CHtransH), 6.02 (m,1H, eCH2eCH ¼ CH2). 13C NMR (151 MHz,
D2O) dC: 21.97 (eNHCOCH3), 36.90 (C3), 50.22 (C5), 56.34 (eOCH3),
62.68 (C9), 65.92 (eCH2CH]CH2), 68.19 (C7), 71.44 (C8), 72.77 (C6),
77.20 (C4), 100.38 (C2), 118.22 (eCH2CH]CH2), 133.67, (eCH2CH]
CH2), 172.94 (C1), 174.93 (eNHCOCH3).
(C6, C9), 98.58 (C2), 117.46 (eCH2eCH
(eCH2eCH ¼ CH2), 169.82 (C1), 173.72 (eNHCOCH3).
¼
CH2), 133.73
The ether (5 mg, 0.0132 mmol) was dissolved in dioxane (0.5 mL)
and NaOH (0.05M, 0.5 mL) was added with stirring. The mixture was
stirred for 2 h at room temperature, neutralised with Amberlite
IR-120 (Hþ) ion-exchange resin, filtered and the solvent was
evaporated in vacuo. The residue was purified by gel permeation
chromatography to afford compound 3 (5 mg, qu.) as a powder. HR-
ESI-MS (m/z) [M þ Na]þ calculated for: C15H25NO9Na: 386.1427.
Found 386.1422. 1H NMR (500 MHz, D2O) dH: 1.76 (dd, 1H, J ¼ 12.1,
12.3 Hz, H3ax), 2.11 (s, 3H, eNHCOCH3), 2.82 (dd, 1H, J ¼ 4.6, 12.5 Hz,
H3eq), 3.48 (s, 3H, eOCH3), 3.61e3.68 (m, 2H, H7, H9), 3.72e3.83 (m,
3H, H4, H6, H9’), 3.90 (dd,1H, J ¼ 10.1,10.2 Hz, H5), 4.05 (dd,1H, J ¼ 7.7,
7.7 Hz, H8), 4.10 (dd, 1H, J ¼ 5.8, 12.2 Hz, eCHH’eCH ¼ CH2), 4.32
(dd, 1H, J ¼ 6.1, 12.1 Hz, eCHH’eCH ¼ CH2), 5.30 (d, 1H, J ¼ 10.4 Hz,
eCH2eCH ¼ CHcisH), 5.40 (d, 1H, J ¼ 17.4 Hz, eCH ¼ CHtransH), 6.02
(ddd,1H, J ¼ 6.0,11.2,16.6 Hz, eCH2eCH ¼ CH2). 13C NMR (126 MHz,
D2O) dC: 21.94 (eNHCOCH3), 40.40 (C3), 51.76 (C5), 58.36 (eOCH3),
65.96 (eCH2eCH ¼ CH2), 68.17, 68.19 (C4, C7), 70.03 (C8), 72.46 (C6),
6.2.2. Diammonium (allyl 5-acetamido-3,5-dideoxy-4-O-(P-
methylphosphonyl)- D-glycero-a-d-galacto-2-
nonulopyranosidonate) (2)
Under an N2 (g) atmosphere, compound 9 (18 mg, 0.0305 mmol)
was dissolved in anhydrous THF (0.7 mL). Triethylamine (60 L,
0.427 mmol) was added followed by a few minutes of stirring. Thi-
ophenol (22 L, 0.213 mmol) was added and the solution was stirred
overnight. Over the course of 4 days, gentle heating (35e40 ꢁC) and
a further 4 portions of triethylamine (60 L, 0.427 mmol) and thio-
phenol (22 L, 0.213 mmol) were added whilst maintaining the
m
m
m
m
solvent volume (w0.5e0.7 mL). Once TLC analysis had indicated that
the reaction had progressed no further, the solvent was evaporated in
vacuo and purified on
a
short silica plug (EA:MeOH;
73.14 (C9), 100.53 (C2), 118.14 (eCH2eCH
(eCH2eCH ¼ CH2), 173.38 (C1), 174.94 (eNHCOCH3).
¼
CH2), 133.67
5:1 / DCM:MeOH; 1:1 / MeOH) to give the deprotected phos-
phonate intermediate which was lyophilised with dioxane/H2O
(9 mg, 96%). The deprotected phosphonate intermediate was then
heated to 50 ꢁC with pyridine:NEt3:H2O (1:1:1, 0.75 mL). After stir-
ring overnight, the solvent was evaporated in vacuo and the residue
was purified by gel permeation chromatography to afford compound
2 (9 mg, 76%) as a white solid after lyophilisation. HR-ESI-MS (m/z)
[M þ Na]þ calculated for: C15H26NOPNa: 450.1141. Found: 450.1136.
1H NMR (500 MHz, MeOH-D4) dH: 1.29 (d, 1H, J ¼ 16.6 Hz, P-CH3),
1.84 (dd,1H,12.1,12.3 Hz, H3ax), 2.03 (s, 3H, eNHCOCH3), 2.91 (dd,1H,
J ¼ 4.7, 12.6 Hz, H3eq), 3.53 (dd, 1H, J ¼ 1.5, 9.0 Hz, H7), 3.57 (dd,
6.2.4. Diammonium (allyl 5-acetamido-3,5-dideoxy-9-O-(P-
methylphosphonyl)- D-glycero-a-d-galacto-2-
nonulopyranosidonate) (4)
Under an N2 (g) atmosphere, compound 11 (14 mg, 0.0240 mmol)
was dissolved in anhydrous THF (0.7 mL). Triethylamine (47 L,
0.336 mmol) was added followed by a few minutes of stirring. Thi-
ophenol (17 L, 0.168 mmol) was then added and the solution was
stirred overnight. Over the course of 4 days, a further 4 portions of
triethylamine (47 L, 0.336 mmol) and thiophenol (17 L,
m
m
m
m

M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
2857
0.168 mmol) were added whilst maintaining the THF solvent volume
(w0.5e0.7 mL). Once TLC analysis had indicated that the reaction
had progressed no further, the solvent was evaporated in vacuo and
purified on a short silica plug (DCM:MeOH; 5:1 / 2:1 / MeOH) to
give the deprotected phosphonate intermediate which was lyophi-
lisedwithdioxane/H2O(10.2mg, 74%).Thedeprotectedphosphonate
intermediate (10 mg) was then treated with a 1:1 mixture of dioxane
and 0.05 M NaOH (aq.) at approximately 4e5 ꢁC for 2 h after which
the solution was neutralised with ion-exchange resin Amberlite IR-
120. After filtration, the solvent was removed and the residue was
purified by gel permeation chromatography to afford compound 4
(9.6 mg, wqu.) after lyophilisation. HR-ESI-MS (m/z) [M þ Na]þ
calculated for: C15H26NOPNa: 450.1141. Found: 450.1136. 1H NMR
(500 MHz, D2O) dH: 1.41 (d, 3H, J ¼ 16.4 Hz, PeCH3), 1.79 (dd, 1H,
J ¼ 12.1, 12.2 Hz, H3ax), 2.11 (s, 3H, eNHCOCH3), 2.80 (dd, 1H, J ¼ 4.3,
12.5 Hz, H3eq), 3.74 (d,1H, J ¼ 8.4 Hz, H7), 3.76e3.82 (m,1H, H4), 3.87
(d,1H, J ¼ 10.3 Hz, H6), 3.93 (dd,1H, J ¼ 9.9,10.1 Hz, H5), 4.02e4.09 (m,
2H,H8, H9), 4.12(dd,1H,J¼ 5.7,12.0Hz,eCHH’eCH¼ CH2), 4.15e4.22
(m, 1H, H9’), 4.34 (dd, 1H, J ¼ 6.1, 12.1 Hz, eCHH’eCH ¼ CH2), 5.31
(d, 1H, J ¼ 10.2 Hz, eCH2eCH ¼ CHcisH), 5.41 (d, 1H, J ¼ 17.3 Hz, eCH
(C4), 70.38 (C7), 72.60 (C8), 75.11 (C6),100.16 (C2),117.09 (eCH2CH]
CH2), 135.58 (eCH2CH]CH2), 171.15 (C1), 175.35 (eNHCOCH3).
6.2.7. Methyl (allyl 5-acetamido-3,5-dideoxy-8,9-O-
isopropylidene-D-glycero-a-D-galacto-2-nonulopyranosid)onate (7)
Compound 6 (56 mg, 0.154 mmol) was dissolved in dry acetone
(1.85 mL) under a atmosphere of N2(g). 2,2-Dimethoxypropane
(200 mL) was added to the solution, followed by p-TsOH$H2O (7 mg).
The solution was left to stir at room temperature, under N2, for 2 h.
Once complete, NEt3 (few drops) was added to the solution to
neutralise the reaction and the solvent was evaporated to dryness.
After flash chromatography (Tol:Acetone 1:1), a clear oil was
obtained, which after further drying in vacuo yielded 7 as a white
foam (53 mg, 85%). Rf : 0.52 (Tol:Acetone 1:1). HR-ESI-MS (m/z)
[M þ Na]þ calculated for C18H29NO9Na : 426.1735. Found : 426.1734.
1H NMR (500 MHz, CDCl3) dH: 1.34, 1.38 (2s, 6H, eC(CH3)2), 1.79 (dd,
1H, J ¼ 12.0, 12.2 Hz, H3ax), 2.02 (s, 3H, eNHCOCH3), 2.69 (dd, 1H,
J ¼ 4.7,12.8 Hz, H3eq), 3.43 (d,1H, J ¼ 10.4 Hz, H6), 3.60 (dd,1H, J ¼ 5.6,
5.7 Hz, H7), 3.69 (m, 1H, H4), 3.74e3.81 (m, 4H, H5, eCO2CH3),
3.98e4.11 (m, 4H, H9, H90, eOH, eCHH’eCH ¼ CH2), 4.14 (d, 1H,
J ¼ 5.7 Hz, eOH), 4.21e4.27 (m, 2H, eCHH’eCH ¼ CH2, H8), 5.13 (ddd,
1H, J ¼ 1.1,1.4,10.5 Hz, eCH2eCH ¼ CHcisH), 5.23 (ddd,1H, J ¼ 1.4,1.6,
17.2 Hz, eCH ¼ CHtransH), 5.83 (ddd, 1H, J ¼ 5.6, 10.6, 16.3 Hz,
eCH2eCH ¼ CH2), 6.59 (d, 1H, J ¼ 8.0 Hz, eNH). 13C NMR (126 MHz,
CDCl3) dC: 23.30 (eNHCOCH3), 25.64, 26.93 (eC(CH3)2), 40.62 (C3),
52.67 (eCO2CH3), 53.19 (C5), 65.53 (eCH2CH]CH2), 66.55 (C9),
67.94 (C4), 69.79 (C7), 74.66 (C6), 75.88 (C8), 99.15 (C2), 108.75
(eC(CH3)2), 117.27 (eCH2CH]CH2), 134.02, (eCH2CH]CH2), 169.17
(C1), 173.50 (eNHCOCH3).
¼ CHtransH), 6.01 (ddd,1H, J ¼ 5.7,11.3,16.1 Hz, eCH2eCH ¼ CH2). 13
C
NMR (126 MHz, D2O) dC: w10.80 (d, J ¼ 137 Hz, PeCH3), 22.07
(eNHCOCH3), 40.02 (C3), 51.83 (C5), 65.04 (d, J ¼ 5 Hz, C9), 65.90
(eCH2eCH ¼ CH2), 67.64 (C7), 68.01 (C4), 70.13 (d, J ¼ 7 Hz, C8), 72.62
(C6), 99.96 (C2),118.33 (eCH2eCH ¼ CH2),133.57 (eCH2eCH ¼ CH2),
172.61 (C1), 174.95 (eNHCOCH3).
31P NMR (162 MHz, D2O) dP: 28.45 (s).
6.2.5. Ammonium (allyl 5-acetamido-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosidonate) (5)
Compound 6 (10 mg, 0.0275 mmol) was dissolved in dioxane
(0.5 mL) with stirring. Aqueous NaOH (0.1 M, 0.5 mL) was added
and the solution was stirred for 2 h. The solution was then neu-
tralised with Amberlite IR-120 (Hþ) ion-exchange resin, filtered and
the solvent was removed in vacuo. The crude residue was then
purified by gel permeation chromatography and lyophilised to give
a white powder (9 mg, qu). HR-ESI-MS (m/z) [M þ Na]þ calculated
for : C14H23NO9Na: 372.1271 Found: 372.1265. 1H NMR (500 MHz,
D2O) dH: 1.74 (dd, 1H, J ¼ 12.1, 12.1 Hz, H3ax), 2.11 (s, 3H,
eNHCOCH3), 2.83 (dd, 1H, J ¼ 4.4, 12.4 Hz, H3eq), 3.66 (d, 1H,
J ¼ 8.9 Hz, H7), 3.69e3.82 (m, 3H, H4, H6, H9), 3.85e3.99 (m, 3H, H5,
H8, H9’), 4.09 (dd, 1H, J ¼ 5.7, 12.0 Hz, eCHH’eCH ¼ CH2), 4.32 (dd,
1H, J ¼ 6.2, 12.0 Hz, eCHH’eCH ¼ CH2), 5.30 (d, 1H, J ¼ 10.4 Hz,
eCH2eCH ¼ CHcisH), 5.40 (d, 1H, J ¼ 17.3 Hz, eCH ¼ CHtransH), 6.02
(ddd, 1H, J ¼ 5.9, 11.4, 16.5 Hz, eCH2eCH ¼ CH2). 13C NMR
(500 MHz, D2O) dC: 22.01 (eNHCOCH3), 40.45 (C3), 51.88 (C5),
62.60 (C9), 65.95 (eCH2CH]CH2), 68.21 (C4, C7), 71.69 (C8), 72.65
(C6), 100.59 (C2), 118.19 (eCH2CH]CH2), 133.76, (eCH2CH]CH2),
173.42 (C1), 175.06 (eNHCOCH3).
6.2.8. Methyl (allyl 5-acetamido-3,5-dideoxy-8,9-O-carbonyl-
glycero- -galacto-2-nonulopyranosid)onate (8)
D-
a-D
Compound 6 (345 mg, 0.949 mmol) and 4-dimethylaminopyridine
(278 mg, 2.28 mmol) were dried in vacuo for approximately 1 h and
then placed under an atmosphere of N2. Dry DCM (20 mL) was added
via syringe and the resulting suspension formed was ultrasonicated for
a few minutes then cooled to approximately 0 ꢁC. Diphosgene (152
mL,
1.23 mmol) was added via microsyringe to the white suspension with
vigourous stirring. When TLC indicated completion of the reaction, the
reaction was quenched by pouring the solution into 40 mL of cold 10%
KH2PO4. The DCM layer was separated and the aqueous layer was
extracted with ethyl acetate (4 ꢂ 25 mL). The organic phases were then
dried over MgSO4 and the solvents were evaporated. After flash chro-
matography (EA:MeOH; 15:1), compound 8 was obtained as a clear oil
(220 mg, 60%). Rf : 0.63 (EA:MeOH; 5:1). HR-ESI-MS (m/z) [M þ Na]þ
calculated for C16H23NO10Na : 412.1220. Found: 412.1214. 1H NMR
(500 MHz, MeOH-D4)dH:1.74 (dd,1H, J¼ 12.3,12.3 Hz, H3ax), 2.01(s, 3H,
eNHCOCH3), 2.66 (dd,1H, J ¼ 4.6,12.8 Hz, H3eq), 3.53 (d,1H, J ¼ 10.5 Hz,
H6), 3.61 (ddd,1H, J ¼ 4.5,10.6,12.9 Hz, H4), 3.75e3.79 (m,1H, H5), 3.80
(s, 3H, eCO2CH3), 3.93 (m, 1H, H7), 3.97 (dd, 1H, J ¼ 5.3, 12.6 Hz,
eCHH’eCH ¼ CH2), 4.19 (dd, J ¼ 4.2, 12.8 Hz, eCHH’eCH ¼ CH2), 4.55
(dd,1H, J ¼ 8.4, 8.4 Hz, H9), 4.70 (dd,1H, J ¼ 7.2, 7.9 Hz, H9’), 4.95 (m,1H,
H8), 5.14 (d, 1H, J ¼ 10.5 Hz, eCHH’eCH ¼ CHHcis), 5.26 (d, 1H,
J ¼ 17.4 Hz, eCHH’eCH ¼ CHHtrans), 5.86 (dddd, 1H, J ¼ 1.08, 5.2, 10.5,
16.8, eCHH’eCH ¼ CH2). 13C NMR (126 MHz, MeOHeD4) dC: 22.91
(eNHCOCH3), 41.61 (C3), 53.25 (C5), 53.81 (eCO2CH3), 66.44
(eCH2CH]CH2), 67.39 (C9), 68.32 (C4), 69.37 (C7), 76.35 (C6), 79.78
(C8), 100.64 (C2), 117.09 (eCH2CH]CH2), 135.36 (eCH2CH]CH2),
157.53 (eOeC(O)eOe), 170.05 (C1), 175.25 (eNHCOCH3).
6.2.6. Methyl (allyl 5-acetamido-3,5-dideoxy-d-glycero-a-d-
galacto-2-nonulopyranosid)onate (6)
Compound 6 was synthesised as described in the literature [20].
Rf : 0.16 (DCM:MeOH 7:1). HR-ESI-MS (m/z) [M þ Na]þ calculated
for C15H25NO9Na : 386.1427. Found: 386.1421.
1H NMR (500 MHz, MeOH-D4) dH: 1.76 (dd, 1H, J ¼ 12.5, 12.5 Hz,
H3ax), 2.00 (s, 3H, eNHCOCH3), 2.70 (dd, 1H, J ¼ 4.6, 12.8 Hz, H3eq),
3.51 (d,1H, J ¼ 9.1 Hz, H7), 3.58 (d,1H, J ¼ 10.5 Hz, H6), 3.61e3.68 (m,
2H, H4, H9), 3.77 (dd,1H, J ¼ 10.0,10.2 Hz, H5), 3.83 (s, 3H, eCO2CH3),
3.84e3.87 (m, 2H, H8, H9’), 3.99 (dd, 1H, J ¼ 5.4, 12.6 Hz,
eCHH’eCH ¼ CH2), 4.29 (dd,1H, J ¼ 5.2,12.9 Hz, eCHH’eCH ¼ CH2),
5.12 (d,1H, J ¼ 10.3 Hz, eCHH’eCH ¼ CHHcis), 5.23 (d,1H, J ¼ 17.3 Hz,
eCHH’eCH ¼ CHHtrans), 5.87 (m, 1H, eCHH’eCH ¼ CH2). 13C NMR
6.2.9. Methyl (allyl 5-acetamido-8,9-O-carbonyl-3,5-dideoxy-4-O-
(O,P-dimethylphosphonyl)- D-glycero-a-D-galacto-2-
nonulopyranosid)onate (9)
(126 MHz, CDCl3) dC
:
22.82 (eNHCOCH3), 41.86 (C3), 53.50
Compound 8 (75 mg, 0.193 mmol) was placed in a flask under
(eCO2CH3), 53.99 (C5), 64.88 (C9), 66.37 (eCH2CH]CH2), 68.67
an N2 (g) atmosphere. Dry DCM (2 mL) was added via syringe

2858
M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
resulting in a suspension which required ultrasonication. N,N-dii-
sopropylethylamine (67 L, 0.386 mmol) was added to the suspen-
117.50 (eCH2CH]CH2), 128.13, 130.19 (H3C(Ar)C(Ar)C(H)e, H3C(Ar)
C(Ar)C(H)e), 132.61 (eS(Ar)C(Ar)C(H)e), 133.68 (eCH2CH]CH2),
145.28 (eS(Ar)C(Ar)C(H)e), 169.89 (C1), 174.22 (eNHCOCH3).
The tosylate (194 mg, 0.375 mmol) was dissolved in in anhy-
drous MeOH (2.4 mL). Freshly prepared NaOMe (in anhydrous
MeOH) (0.3 mL, 0.5 M) was added dropwise to the stirring solution.
Further 0.1 mL portions of NaOMe (0.5 M) were added as required
(a total of 0.7 mL NaOMe was used). The reaction was monitored by
TLC. On completion, the solution was neutralised with Amberlite
IR-120 (Hþ) ion-exchange resin. The resin was removed by filtration
and the solvent was removed in vacuo. Flash chromatography of the
crude material (EA:MeOH; 15:1 þ 0.5% NEt3) gave epoxide 10 as
a white foam (87 mg, 68%). Rf : 0.34 (EA:methanol; 10:1). HR-ESI-
MS (m/z) [M þ Na]þ calculated for C15H23NO8Na : 368.1321.
Found: 368.1316. 1H NMR (500 MHz, CDCl3) dH: 1.81 (dd, 1H,
J ¼ 12.1, 12.3 Hz, H3ax), 2.00 (s, 3H, eNHCOCH3), 2.71 (dd, 1H, J ¼ 4.6,
12.7 Hz, H3eq), 2.77 (dd, 1H, J ¼ 2.5, 5.1 Hz, H9), 2.83 (dd, 1H, J ¼ 4.3,
4.6 Hz, H9’), 3.24 (m, 1H, H8), 3.38 (dd, 1H, J ¼ 4.8, 4.8 Hz, H7), 3.55
(dd, 1H, J ¼ 0.9, 10.4 Hz, H6), 3.65 (m, 1H, H4), 3.76e3.82 (m, 1H, H5),
3.93 (bs, 1H, eOH), 3.96 (dd, 1H, J ¼ 5.9, 12.6 Hz, eCHH’eCH ¼ CH2),
4.25 (m, 1H, eCHH’eCH ¼ CH2), 4.27 (d, 1H, J ¼ 5.7 Hz, eOH), 5.12
(dd, 1H, J ¼ 1.3, 10.5 Hz, eCH2eCH ¼ CHHcis), 5.21 (dd, 1H, J ¼ 1.5,
17.2 Hz, eCH2eCH ¼ CHHtrans), 5.82 (ddd, 1H, J ¼ 5.6, 10.8, 16.1 Hz,
eCH2eCH ¼ CH2), 6.57 (d, 1H, J ¼ 7.7 Hz, eNH). 13C NMR (126 MHz,
CDCl3) dC: 23.31 (eNHCOCH3), 40.90 (C3), 46.15 (C9), 52.06 (C8),
52.92 (C5), 53.10 (eCO2CH3), 65.64 (eCH2CH]CH2), 67.91 (C4),
69.55 (C7), 75.44 (C6), 99.11 (C2), 117.35 (eCH2CH]CH2), 134.07
(eCH2CH]CH2), 169.29 (C1), 173.72 (eNHCOCH3).
m
sion followed by further ultrasonication. With continuous stirring,
the suspension was cooled to approximately ꢀ15 ꢁC. Methyl meth-
ylphosphonyl chloride (27 mL, 0.270 mmol) was added dropwise to
the solution via microsyringe. The reaction was allowed to warm to
room temperature and was stirred overnight. The reaction was again
then cooled to ꢀ15 ꢁC, where a further portion of N,N-diisopropyle-
thylamine (67
chloride (27
mL, 0.386 mmol) and Methyl methylphosphonyl
L, 0.270 mmol) was added. Once TLC indicated
m
completion, the reaction was quenched by the addition of MeOH
(1 mL) and a small spatula of NaHCO3 (s). After stirring for 10 min, the
solvent was removed in vacuo and the product was purified by flash
chromatography (EA:MeOH; 7:1 / 5:1) to give 9 (57 mg, 60%). Rf :
0.21 (EA:MeOH; 10:1). HR-ESI-MS (m/z) [M þ Na]þ calculated for
C18H28NO12P Na : 504.1247. Found 504.1241. 1H NMR (500 MHz,
CDCl3) dH: 1.53, 1.47 (2d, 3H, J ¼ 17.7, 17.8 Hz, PeCH3), 2.01 (m, 1H,
H3ax), 2.06 (s, 3H, eNHCOCH3), 2.73, 2.78 (2dd, 1H, J ¼ 5.0, 12.8, 4.9,
12.9 Hz, H3eq), 3.25 (d,1H, J ¼ 10.3 Hz, H6), 3.66, 3.75 (2d, 3H, J ¼ 11.5,
11.2 Hz, PeOCH3), 3.81, 3.82 (2s, 3H, eCO2CH3), 3.83e3.92 (m, 1H,
H5), 4.02 (dd, 1H, J ¼ 5.4, 12.4 Hz, eCHH’eCH2 ¼ CH2), 4.22 (dd, 1H,
J ¼ 5.1,12.6 Hz, CHH’eCH2 ¼ CH2), 4.40, 4.53 (m,1H, H4), 4.49 (dd,1H,
J ¼ 8.5 Hz, H9), 4.65 (m, 1H, H9’), 4.74 (bs, 1H, eOH), 4.87 (m, 1H, H8),
5.16 (d, 1H, J ¼ 10.5 Hz, eCH2eCH ¼ CHHcis), 5.24 (d, 1H, J ¼ 17.3 Hz,
eCH2eCH ¼ CHHtrans), 5.82 (ddd, 1H, J ¼ 5.4, 10.5, 16.1 Hz,
eCH2eCH ¼ CH2), 7.43 (m, 1H, eNH). 13C NMR (126 MHz, CDCl3) dC
:
w10.85, w11.18 (2d, J ¼ 143.41, 146.06 Hz, PeCH3), 23.14 (2s,
eNHCOCH3), 38.95 (C3), 52.00 (C5), 52.27 (d, J ¼ 7.4 Hz, PeOCH3),
53.09, 53.16 (eCO2CH3), 65.65 (eCH2eCH ¼ CH2), 66.85, 66.89 (C9),
69.03, 69.05 (C7), 71.68, 71.72 (C4), 74.99 (C6), 75.86, 75.91 (C8),
6.2.11. Methyl (allyl 5-acetamido-4,7,8-tri-O-acetyl-3,5-dideoxy-9-
99.04, 99.13 (C2), 117.55 (eCH2eCH
¼
CH2), 133.57, 133.59
O-(O,P-dimethylphosphonyl)-D-glycero-a-D-galacto-2-
nonulopyranosid)onate (11)
Under an N2 (g) atmosphere, compound 6 (77 mg, 0.212 mmol)
was suspended in dry DCM (2 mL) and the mixture was ultra-
(eCH2eCH ¼ CH2), 155.15 (eOeC(O)eOe), 168.38, 168.41 (C1),
174.23, 174.32 (eNHCOCH3).
31P NMR (126 MHz, CDCl3) dP: 34.72 (bs).
sonicated. Diisopropylethylamine (81
mL, 0.466 mmol) was added
6.2.10. Methyl (allyl 5-acetamido-8,9-anhydro-3,5-dideoxy-d-
to the suspension followed by further ultrasonication. With
glycero-
a
-d-galacto-2-nonulopyranosid)onate (10)
continuous
stirring,
the
suspension
was
cooled
to
Compound 6 (560 mg, 1.54 mmol) was co-evaporated with dry
pyridine and was then suspended in dry pyridine (12 mL) under N2
(g). The resulting suspension was cooled to to 0 ꢁC (ice bath) and
toluenesulfonyl chloride (293 mg, 1.54 mmol) was quickly added to
the stirred solution.
approximately ꢀ15 ꢁC. Once acclimatised to temperature, methyl
methylphosphonyl chloride (29 mL, 0.297 mmol) was added drop-
wise to the solution via microsyringe. After 5 h the reaction was
again cooled to ꢀ15 ꢁC, where
a further portion of N,N-
diisopropylethylamine (81
mL, 0.466 mmol) and methyl methyl-
After 20 min, the solution was allowed to warm up to room
temperature and was subsequently stirred for a further 5 h after
which another portion of toluenesulfonyl chloride (293 mg,
1.54 mmol) was added. The solution was left to stir overnight. Once
completed, solvent was removed in vacuo and the crude product
was purified by flash chromatography (EA / EA:MeOH; 5:1) to
yield the 9-O-tosylate as a white foam (470 mg, 59%). Rf : 0.70
(EA:methanol; 4:1). HR-ESI-MS (m/z) [M þ Na]þ calculated for
C22H31NO11SNa : 540.1516. Found: 540.1510. 1H NMR (500 MHz,
CDCl3) dH: 1.81 (dd, 1H, J ¼ 11.7, 11.9 Hz, H3ax), 2.01 (s, 3H,
eNHCOCH3), 2.41 (s, 3H, e(Ar)CeCH3), 2.74 (dd, 1H, J ¼ 3.8, 13.8 Hz,
H3eq), 3.40 (d, 1H, J ¼ 9.2 Hz, H6), 3.45 (d, 1H, J ¼ 8.8 Hz, H7),
3.64e3.75 (m, 2H, H4, H5), 3.65e3.77 (bs, 1H, eOH), 3.77 (s, 3H,
eCO2CH3), 3.86 (dd,1H, J ¼ 5.7,12.6 Hz, eCHH’eCH ¼ CH2), 3.91 (bs,
1H, eOH), 4.04 (m, 1H, H8), 4.12e4.20 (m, 2H, H9,
eCHH’eCH ¼ CH2), 4.29 (m, 1H, H9’), 4.68 (bs, 1H, eOH), 5.10 (dd,
1H, J ¼ 0.8,10.5 Hz, eCH2eCH ¼ CHHcis), 5.18 (dd,1H, J ¼ 1.1,17.3 Hz,
eCH2eCH ¼ CHHtrans), 5.77 (ddd, 1H, J ¼ 5.6, 10.8, 16.2 Hz,
eCH2eCH ¼ CH2), 6.61 (d, 1H, J ¼ 7.0 Hz, eNH), 7.32 (d, 2H,
J ¼ 8.2 Hz, e(Ar)CeH), 7.75 (d, 2H, J ¼ 8.3 Hz, eSeC(Ar)CeH). 13C
NMR (126 MHz, CDCl3) dC: 21.83 ((Ar)CeCH3), 23.22 (eNHCOCH3),
40.59 (C3), 52.99 (C5), 53.56 (eCO2CH3), 65.49 (eCH2CH]CH2),
67.69 (C4), 68.70 (C7), 69.29 (C8), 72.22 (C9), 73.72 (C6), 98.76 (C2),
phosphonyl chloride (29 mL, 0.297 mmol) was added. The reaction
was left to stir overnight. Once TLC indicated completion, the
reaction was quenched by the addition of MeOH (1 mL) and a small
spatula of NaHCO3 (s). After stirring for 10 min, the solvent was
removed in vacuo and the product was purified by flash chroma-
tography (EA:MeOH; 10:1 / 5:1) to give the 9-O-phosphonate
(27 mg, 30%). Rf
: 0.25 (EA:MeOH; 10:1). HR-ESI-MS (m/z)
[M þ Na]þ calculated for C17H30NO11PNa : 478.1454. Found
478.1449. 1H NMR (500 MHz, CDCl3) dH: 1.54 (dd, 3H, J ¼ 2.2,
17.6 Hz, PeCH3), 1.75 (dd, 1H, J ¼ 12.2, 12.3 Hz, H3ax), 2.00 (s, 3H,
eNHCOCH3), 2.69 (dd, 1H, J ¼ 4.6, 12.8 Hz, H3eq), 3.52 (ddd, 1H,
J ¼ 1.4, 9.9, 10.1 Hz, H7), 3.62 (dd, 1H, J ¼ 1.4, 10.5 Hz, H6), 3.66 (m,
1H, H4), 3.75 (dd, 3H, J ¼ 1.7, 11.2 Hz, PeOCH3), 3.75e3.80 (m, 1H,
H5), 3.98e4.02 (m, 1H, H8), 3.98e4.02 (m, 1H, eCHH’eCH ¼ CH2),
4.15 (m, 1H, H9), 4.26e4.33 (m, 1H, eCHH’eCH ¼ CH2), 4.26e4.33
(m, 1H, H9’), 5.12 (dd, 1H, J ¼ 1.1, 10.5 Hz, eCH2eCH ¼ CHHcis), 5.24
(dd,1H, J ¼ 1.7, 17.2 Hz, eCH2eCH ¼ CHHtrans), 5.87 (ddd, 1H, J ¼ 5.5,
10.6, 16.2 Hz, eCH2eCH ¼ CH2). 13C NMR (126 MHz, CDCl3) dC
:
w9.15, w10.29 (2d, J ¼ 144 Hz, PeCH3), 22.68 (eNHCOCH3), 41.69
(C3), w52.91, 53.83, 53.85 (s, m, C5, PeOCH3), 53.28 (eCO2CH3),
66.26 (eCH2eCH ¼ CH2), 68.46 (C4), 69.12, 69.30 (2d, J ¼ 6.3, 6.4 Hz,
C9), 69.80, 69.94 (C7), 70.80, 70.90 (2d, J ¼ 6.4, 6.8 Hz, C8), 74.70
(C6), 100.12 (C2), 116.94 (eCH2eCH
¼
CH2), 135.47

M. Stanley et al. / European Journal of Medicinal Chemistry 46 (2011) 2852e2860
2859
(eCH2eCH ¼ CH2), 170.84 (C1), 175.16 (eNHCOCH3). 31P NMR
6.3.4. pNPA assay
An esterase activity was incubated in the presence of a sialoside
at room temperature for 30 min. For control inhibition reactions,
(162 MHz, CDCl3) dP: 34.24 (s), 34.34 (s).
The phosphonate (23 mg, 0.0520 mmol) was dissolved inpyridine
(0.5 mL) and acetic anhydride (0.5 mL). The flask was stoppered and
allowed to stirovernight. Once the reactionwas complete, the solvent
was removed in vacuo. Repeated co-evaporation with toluene, fol-
lowed by flash chromatography (EA:MeOH 20:1 / 10:1) gave 11
(26 mg, 90%). Rf : 0.45 (EA:MeOH; 10:1). HR-ESI-MS (m/z) [M þ Na]þ
calculated for : C23H36NO14PNa : 604.1771. Found: 604.1766. 1H NMR
(500 MHz, CDCl3) dH: 1.45 (dd, 1H, J ¼ 3.4, 17.6 Hz, P-CH3), 1.85,
2.12e2.14 (s,m, 9H, 3 ꢂ eOCOCH3), 2.00 (s, 3H, eNHCOCH3),1.95 (dd,
1H, J ¼ 12.4, 12.6 Hz, H3ax), 2.60 (dd, 1H, J ¼ 4.6, 12.8 Hz, H3eq), 3.68,
3.72 (2d, 3H, J ¼ 11.1, 11.2 Hz, PeOCH3), 3.87 (dd, 1H, J ¼ 5.9, 12.8 Hz,
eCHH’eCH ¼ CH2), 3.95e4.13 (m, 3H, H5, H6, H9), 4.21e4.33 (m, 2H,
H9’, eCHH’eCH ¼ CH2), 4.87 (m, 1H, H4), 5.14 (dd,1H, J ¼ 1.4, 10.4 Hz,
eCH2eCH ¼ CHHcis), 5.22 (d, 1H, J ¼ 10.0 Hz, eNH), 5.26 (d, 1H,
J ¼ 17.2 Hz, eCH2eCH ¼ CHHtrans), 5.30 (m, 1H, H7), 5.37 (m, 1H, H8),
5.84 (ddd, 1H, J ¼ 5.5, 10.7, 16.1 Hz, eCH2eCH ¼ CH2). 13C NMR
(126 MHz, CDCl3) dC: w10.49, w10.57 (2d, J ¼ 145.1, 145.4 Hz, PeCH3),
21.00, 21.27, 23.35 (3 ꢂ eOCOCH3, eNHCOCH3), 38.17 (C3), 49.69,
49.73 (C5), w52.31 (m, PeOCH3), 52.83 (eCO2CH3), 63.88 (m, C9),
66.02 (eCH2eCH ¼ CH2), 67.54 (C7), 69.18, 69.23 (C4), w69.60,
w69.82 (2d, J ¼ 6.5, 6.5 Hz, C8), 72.65, 72.68 (C6), 98.70 (C2), 117.38
(eCH2eCH ¼ CH2), 133.76 (eCH2eCH ¼ CH2), 168.49, 168.52 (C1),
170.16, 170.25, 170.35, 171.06 (3 ꢂ eOCOCH3, eNHCOCH3). 31P NMR
(162 MHz, CDCl3) dP: 32.51 (s), 32.73 (s).
100
m
M 3,4-dichloroisocoumarin was incubated with the different
esterases [52]. 10
A400 was monitored.
ml pNPA and PBS pH 7.4 was added to 1 ml and the
6.3.5. Fluorimetric HPLC analysis
Reverse-phase high-pressure liquid chromatography (HPLC)
analysis of sialic acids was performed as described previously [45].
10 mU esterase and 1 mM sialosides were added to PBS pH 7.4 and
incubated at room temperature for 30 min.10 mg glycosidically bound
sialic acids (BSM) or free O-acetylated sialic acids (synthesised in our
lab) were added and incubated at 37 ꢁC for various time periods
(20e240 min). Samples containing glycosidically bound sialic acids
were first hydrolysed with 2 M propionic acid for 2 h at 80 ꢁC. The
further processing of the samples was described elsewhere [53].
Appendix. Supplementary material
Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2011.04.008.
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