Synthesis and biological evaluation of selective phosphonate-bearing 1,2,3-triazole-linked sialyltransferase inhibitors

Article abstract of DOI:10.1039/d1md00079a

The critical role of sialyltransferase (ST) enzymes in tumour cell growth and metastasis, as well as links to multi-drug and radiation resistance, has seen STs emerge as a target for potential antimetastatic cancer treatments. One promising class of ST inhibitors that improve upon the pharmacokinetic issues of previous inhibitors is the 1,2,3-triazole-linked transition-state analogues. Herein, we present the design and synthesis of a new generation of 1,2,3-triazole-linked sialyltransferase inhibitors, along with their biological evaluation demonstrating increased potency for phosphonate bearing compounds. The six most promising inhibitors presented in this work exhibited a greater number of binding modes for hST6Gal I over hST3Gal I, withK_iranging from 3-55 μM. This work highlights phosphonate bearing triazole-linked compounds as a promising class of synthetically accessible ST inhibitors that warrant further investigation.

Full text of DOI:10.1039/d1md00079a

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RESEARCH ARTICLE
Synthesis and biological evaluation of selective
phosphonate-bearing 1,2,3-triazole-linked
sialyltransferase inhibitors†
a
Cite this: DOI: 10.1039/d1md00079a
Christopher Dobie,
Andrew P. Montgomery, a Rémi Szabo,a
ab
ab
*
Haibo Yu
and Danielle Skropeta
The critical role of sialyltransferase (ST) enzymes in tumour cell growth and metastasis, as well as links to
multi-drug and radiation resistance, has seen STs emerge as a target for potential antimetastatic cancer
treatments. One promising class of ST inhibitors that improve upon the pharmacokinetic issues of previous
inhibitors is the 1,2,3-triazole-linked transition-state analogues. Herein, we present the design and synthesis
of a new generation of 1,2,3-triazole-linked sialyltransferase inhibitors, along with their biological evaluation
demonstrating increased potency for phosphonate bearing compounds. The six most promising inhibitors
presented in this work exhibited a greater number of binding modes for hST6Gal I over hST3Gal I, with Ki
ranging from 3–55 μM. This work highlights phosphonate bearing triazole-linked compounds as a
promising class of synthetically accessible ST inhibitors that warrant further investigation.
Received 9th March 2021,
Accepted 19th June 2021
DOI: 10.1039/d1md00079a
rsc.li/medchem
sialyltransferases (ST3Gal I–VI), two beta-galactoside α2-6-
Introduction
sialyltransferases
sialyltransferases
(ST6Gal
(ST6GalNAc-I–VI),
I–II),
six
GalNAc
six
α2-6-
α2-8-
N-Acetylneuraminic acid (Neu5Ac, or sialic acid) is a 9-carbon,
charged monosaccharide located at the terminus of many
human cell-surface glycans. Increased cell surface sialylation
due to overexpression of sialyltransferase enzymes (STs) in
tumoural cells is a well-established hallmark of cancer,
making the development of ST inhibitors a potential route for
new cancer treatments and biomarkers.1–3 Hypersialylation
has been implicated in increased metastatic potential through
integrin and selectin mediated processes,4–6 immune evasion
by binding to Siglecs on immune cells,7,8 and improving
tumour cell survival by inhibition of apoptotic signalling.9
Increased surface sialylation has also been linked to
resistance to chemotherapeutics such as cisplatin and
paclitaxel,10,11 and radioresistance in colorectal cancers.12,13
Sialylation also plays a key role in viral infections.14
and
sialyltransferases (ST8Sia-I–VI, among which ST8Sia-II and
ST8Sia-IV are polysialyltransferases).17–19 In humans, STs all
utilise the common donor cytidine monophosphate Neu5Ac
(CMP-Neu5Ac) making selective inhibitor design a challenge.
The various ST subtypes are highly overexpressed up to 50%
or more in several cancers including colorectal,20,21 breast,22
and pancreatic cancer.23 Selective targeting of the specific ST
subtypes over-expressed in a particular cancer is crucial in
order to avoid off-target effects such as liver and kidney
dysfunction exhibited for pan ST inhibitors.24 As part of an
ongoing study into ovarian and pancreatic cancer, this study
is aimed at selective inhibition of human ST3Gal I and
ST6Gal I as two of the most well studied STs in terms of their
potential as anti-metastatic targets.1–3
Sialyltransferase enzymes belong to the glycosyltransferase
family and facilitate the transfer of Neu5Ac to an acceptor
Previous studies into ST inhibitors have identified
transition-state analogues as the most potent inhibitors to
date (Fig. 1).2 These compounds mimic the planar
oxocarbenium-like transition-state of the ST mechanism. One
of the most potent ST inhibitors in the literature is the
phosphodiester-linked cytidine compound (R)-1 (Ki = 19 nM,
hST6GAL I) inspired by the donor molecule CMP-Neu5Ac,
with an aromatic m-phenoxy system replacing the sialic acid,
and a phosphonate group replacing the carboxylate of CMP-
Neu5Ac.25,26 More recent research into ST inhibitors has
uncovered the highly potent cyclopentyl (R-2, Ki = 28 nM,
glycan
N-acetylgalactosamine (GalNAc) or another sialic acid (Sia)
residue.15,16 Eukaryotic sialyltransferases (ST) are
chain
terminated
by
a
galactose
(Gal),
a
glycosyltransferase subset including six beta-galactoside α2-3-
a Molecular Horizons and School of Chemistry & Molecular Bioscience, Faculty of
Science, Medicine and Health, University of Wollongong, NSW 2522, Australia.
E-mail: skropeta@uow.edu.au
b Illawarra Health and Medical Research Institute, Wollongong, NSW 2522,
Australia
hST6Gal I), and amide (3 Ki
phosphodiester-linked compounds.
= 19 nM, hST6Gal I)
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1md00079a
Potential
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Fig. 1 Structures of literature sialyltransferase inhibitors 1–5, and the general structure of the target triazole-linked compounds of this study.
pharmacokinetic issues with the phosphodiester linkage (due
to metabolism by phosphatases in vivo)27 has led to the
investigation by computational methods of carbamate and
1,2,3-triazole linkers as bioisosteric replacements.28–30
utilising an indium triflate catalyst at reflux to give the
deprotected 5′-O-progargyluridine (8) in quantitative yield.
The overall yield of 8 from uridine was 69%.
The α-azidophosphonates were synthesised from a series
of α-hydroxyphosphonates (10a–g, Scheme 2), prepared by
reacting an aromatic aldehyde (9) and dibenzyl phosphite.35
The α-hydroxyphosphonate then underwent a Mitsunobu
reaction to yield the α-azidophosphonate (11a–g).36
A study into the synthesis and biological evaluation of
carbamate linked ST inhibitors produced the p-fluorinated
aryl derivative (4), with a carbamate moiety replacing the
phosphate linker (Fig. 1).31 While less active than the parent
compound, the carbamate-based derivative showed
promising low micromolar activity (4, Ki = 1.1 μM against
hST6GAL I), alongside improved drug-likeness and synthetic
accessibility as a result of replacing the cytidine moiety with
uridine and the charged phosphodiester with a carbamate
group. Following on from this, a series of neutral 1,2,3-
These two key fragments were then coupled via a copper
azide-alkyne cycloaddition (CuAAC) in the presence of Cu(I),
generated from Cu(OAc)2 reduced with sodium ascorbate. A
wide range of solvent systems have been utilised in the
literature with these CuAAC “click” reactions, although many
are quite polar (such as ACN/H2O)37,38 and would not be
suitable for the highly lipophilic α-azidophosphonates. The
solvent system used for this click reaction was 1 : 1 THF/H2O,
with the copper and sodium ascorbate dissolved in water and
then added to the nucleoside and phosphonate fragments
dissolved in neat THF, followed by vigorous stirring to aid
solubility of the reagents in the reaction. Formation of the
1,4-disubstituted product was confirmed by the presence of
the characteristic proton peak at around 8.5–8.8 ppm,
attributed to the proton at C5 of the 1,2,3-triazole ring and
showing correlations to both C5 and the chiral carbon.
Once coupled, the fully protected triazole compounds
(12a–g) were deprotected via catalytic hydrogenation with a
Pd/C catalyst and hydrogen gas in methanol over 4 hours.
The fully deprotected compounds (13a–g) were purified and
the diastereomers separated via C18 reverse phase HPLC
triazole linked-compounds such as
5 have also been
developed and evaluated against hST6Gal I (84% inhibition
at 100 μM).32 As the phosphonate moiety is often linked to
potency in ST inhibitors,2 herein
a series of novel
phosphonate-bearing, ether-linked 1,2,3-triazole based
compounds have been synthesised aimed at generating a
selective, high affinity and accessible ST6Gal I inhibitor for
further large-scale biological studies (Fig. 1).
Results and discussion
To probe the effect of increased linker length on ST
inhibition, we synthesised a novel series of 14 ether-linked,
1,2,3-triazole based derivatives, examined their comparative
binding energies and evaluated their selectivity and
inhibitory activity towards human ST6Gal I and ST3Gal I in a
luminescence-based microplate assay. Synthesis of the target
ether-linked molecules was achieved by the coupling of 5′-O-
propargyluridine with an α-azidophosphonate. The 5′-O-
propargyluridine fragments were synthesised starting from
commercially available uridine (6) via a similar method to
Sun et al. (Scheme 1).33 First, the 2′,3′-diol was protected as
an acetonide, allowing for successful propargyl addition at
the 5′-alcohol utilising sodium hydride and propargyl
bromide, to yield the protected compound 7. The acetonide
was removed via the method of Golden et al., (ref. 34)
Scheme
1 Synthesis of 5′-O-propargyluridine (8). Reagents and
conditions: (a) (i) acetone, H2SO4, 0 °C to r.t, overnight, >99%; (ii)
propargyl bromide, NaH, THF, 0 °C to r.t, overnight, 70%; (b) In(OTf)3,
ACN/H2O (9 : 1), reflux, 6 h, >99%.
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Scheme 2 Synthesis of novel dibenzyl α-azidophosphonates 11a–g,
their CuAAC coupling to alkynyluridine 8, and purification to form
ether-linked 1,2,3-triazoles 13a–g. Reagents and conditions: (a)
dibenzyl phosphite, TEA, DCM, r.t., overnight, 60–85%; (b) HN3,
toluene, DIAD, PPh3, THF, 0 °C to r.t., 80–99%; (c) 8, Cu(OAc)2, sodium
ascorbate, r.t., overnight; (d) (i) Pd/C, H2, MeOH, 4 h; (ii) RP-HPLC,
IR120 Na+. R1 groups and yields are shown in Table 1.
Fig. 2 The key compounds used in the docking study against hST3Gal
I and hST6Gal I to probe the effects of linker length on binding, along
with compound 5.
using
a mixture of methanol or acetonitrile and a
triethylammonium bicarbonate buffer, which yielded the
purified compounds as their triethylammonium salts. These
were further exchanged with sodium by shaking with
Amberlite IR120 Na+ form in MilliQ water for 1 hour
(repeated three times). The resulting aqueous solution was
lyophilised to yield the final product as an amorphous white
powder. A small amount of styrene-divinylbenzene impurity
(leached from the ion exchange resin) was observed in the H
NMR spectra of compounds 13a/b as small peaks at 7.9–8.2
ppm.
at the active site of each enzyme. Receptor structures were
prepared for docking using AutoDockTools (ADT) version
4.2.6.42 In the docking procedure, the receptor is treated as
rigid and no explicit waters have been included. The top
ranked models for each compound tested based on the
binding energies calculated by AD Vina were evaluated based
upon comparison to the position of the phosphodiester lead
compound (R-1) in the simulated snapshots VMD v1.9.4.43
The mean binding energies for all compounds across the
snapshots are reported in Table 2.
1
The docking against ST6Gal I gave the greatest number of
binding modes (n = 6) that were used to calculate the binding
energies. Overall, the three literature compounds (R)-1, (R)-2,
and (R)-3 all had the lowest calculated binding energies against
ST6Gal I (of −9.6, −9.0 and −9.4 kcal mol−1 respectively), with
the cyclopentyl compound (R)-2 having the lowest binding
energy by a statistically significant margin. This may be due to
fewer possible protein-ligand interactions identified by AD Vina
for these compounds. The docking against ST6Gal I also
suggests that increasing the length of the linker in these ST
inhibitors may not negatively affect binding, as there was no
statistical significance in the results of the triazole compounds,
regardless of linker length. As shown in Fig. 3, this may be due
to the large size of the binding pocket of ST6Gal I, which
normally needs to accommodate both the CMP-Neu5Ac donor
Computational analysis
To assess the comparative binding of the triazole compounds
of our previous study and the effects of increased linker
length on binding, a series of molecular docking simulations
were undertaken. Docking was performed with literature
phosphodiester-linked compounds (Fig. 1) developed by
Skropeta et al. (R-1),25 Li et al. (R-2),26 and Guo et al. (R-3,
S-3).39 The S diastereomer of the amide compound 3 was
included as that study did not include a determination of
stereochemistry, similar to the synthesis seen here. Five
1,2,3-triazoles were also included to test the effects on
binding of replacing cytidine (R-1) with uridine (R-14), the
presence of a phosphonate (R-5 and R-15), and increasing
linker length (R-16, R-17 vs. R-13a), as shown in Fig. 2.
substrate and an acceptor glycan chain.
A free energy
These compounds were docked into six snapshots from
100 ns of molecular dynamics simulations performed on
ST6Gal I (PDB ID: 4JS2) as described by Montgomery et al.29
In addition to the docking into hST6Gal I, molecular
dynamics simulations were also conducted with a homology
model of hST3Gal I from SWISS-MODEL (UNIPROT ID:
Q11201, model version 3.0.0),40 for 100 ns with the
phosphodiester lead compound (R)-1, and snapshots taken at
0, 20, 40, 60, 80 and 100 ns timepoints for docking.
perturbation study with hST6Gal I by Montgomery et al.30 has
indicated that the flexibility of this binding pocket also
compensates for the added rigidity of the triazole group,
allowing the triazole to effectively mimic the phosphodiester
linker of traditional ST inhibitors.
Potential selectivity for ST6Gal I over ST3Gal I is observed,
as more acceptable binding modes (n) were obtained for the
compounds assessed against ST6Gal I, particularly for the
ether-linked 1,2,3-triazole compound (R)-13a, as an example
of the compounds synthesised herein. In some cases, no
calculated binding modes correlated with the results for the
Docking was performed with AutoDock Vina (AD Vina)
version 1.1.2 (ref. 41) for a box of 30 Å × 30 Å × 30 Å centred
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Table 1 Yields for protected triazoles 12a–g from the copper azide-alkyne cyclisation coupling of 5′-O-propargyluridine (8) and α-azidophosphonates
(11a–g), followed by their deprotection to form the final deprotected 1,2,3-triazole series in their Na+ salt form (13a–g)
R1
Click yield (R2: OBn)a
Deprotection yield (R2: O−Na+)b
12a: 51%
13a-(s): 99%
13a-(l): 91%
12b: 52%
12c: 66%
12d: 57%
13b-(s): 94%
13b-(l): 99%
13c-(s): 97%
13c-(l): 91%
13d-(s): 92%
13d-(l): 62%
12e: 60%
12f: 52%
13e-(s): 92%
13e-(l): 85%
13f-(s): 95%
13f-(l): 92%
12g: 57%
13g-(s): 96%
13g-(l): 92%
a
b
Yield of corresponding 1,2,3-triazole linked product from CuAAC click coupling (12a–g, Scheme 2). Yield of final compound after catalytic
hydrogenation deprotection and RP-HPLC separation and purification of diastereomers (13a–g, Scheme 2).
Table 2 Mean binding energies of 1,2,3-triazole linked and literature ST inhibitors. See Fig. 1 and 2 for structures
Compound
Binding energya (kcal mol−1
)
Phosphodiester
Nucl.
ST3Gal I
nb
ST6Gal I
nb
Ref
(R)-1
(R)-2
(R)-3
(S)-3
(R)-14
Triazole
(R)-5
(R)-13a
(R)-15
(R)-16
(R)-17
Cyt
Cyt
Cyt
Cyt
Uri
−9.2 0.3
−9.5 0.3
−9.2 0.2
−9.8 0.3
−9.8 0.2
3
3
3
3
3
−9.6 0.2
−9.0 0.1
−9.4 0.2
−9.8 0.2
−9.8 0.2
6
6
6
6
6
25
26
40
40
—
Uri
Uri
Uri
Uri
Uri
−9.9 0.1
−10.1 −0.7
−10.1 0.3
−9.9 0.3
−9.9 0.4
5
4
6
5
5
−10.1 0.1
−10.0 0.2
−10.1 0.2
−10.2 0.3
−10.4 0.3
6
5
6
6
6
32
—
—
—
—
a
Arithmetic mean of the binding energy SEM obtained from docking into six snapshots of the ST3Gal I (UNIPROT ID: Q11201) and ST6Gal I
b
(PDB ID: 4JS2) simulations with the phosphodiester lead compound (R)-1. Number of snapshots (max. possible is 6) with acceptable binding
modes that were used to calculate the binding energy in the column immediately to the left.
molecular dynamics simulation. In part due to the high
variance of binding conformations and averaging of only
snapshots where there was deemed an acceptable binding
mode, there was no statistical significance between the
obtained binding energies against ST3Gal I. Due to these
higher errors, there was no significant difference for any of
the compounds between their average binding energies
against ST3Gal I and ST6Gal I.
When compared to results from our previous investigation
with the crystal structure of hST6Gal I, the observed
interactions are largely similar.31 Here, we also see
importance of hydrophobic contacts for binding of the ether-
linked triazole compounds, as the aromatic structures of the
phenyl rings and triazole itself participate in interactions
with hydrophobic residues. These results highlight the
importance of the acceptor binding region of the ST6Gal I
active site for inhibitor design.
Fig.
4
shows the ligand-receptor interactions for
phosphodiester (R)-1 and triazole-linked (R)-13a for their
binding modes in the crystal structure of hST6Gal I. In
particular, the hydrogen bonding interaction between the
Tyr354 OH and the ligand phosphonate group is substituted
for a hydrophobic interaction between the Tyr354 ring and the
triazole CH, which could potentially contribute to the higher
exhibited binding energy from the docking calculations.
Biological evaluation
Biological assessment of the activity of these compounds
against recombinant human ST3Gal I and ST6Gal I was
carried out based on the CMP-Glo™ assay developed by
Promega and detailed by Das et al.45 CMP-Neu5Ac was used
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Fig.
4
Predominant interactions from binding modes of the
phosphodiester-linked lead compound (R)-1 and ether-linked triazole
compound (R)-13a with the hST6Gal I crystal structure (PDB ID: 4JS2),
analysed with Discovery Studio Visualizer.44 Hydrogen bonds are
shown in red, and hydrophobic contacts in blue.
hST3Gal I in the range of 28–49% inhibition at 100 uM were
also the most active against hST6Gal I in the range of 57–
78% at 10 uM (e.g. see compounds 4, 13a, 13c, 13f). Due to
this increased activity against hST6Gal I over hST3Gal I, it
appears that these compounds show a degree of selectivity
towards hST6Gal I.
Consistent with previous literature results on ST
inhibitors, there are relatively small differences in activity
between diastereomers of the most active inhibitors against
hST6Gal I, for example 13f-(s) and 13f-(l) (Table 3). These
results also confirm the positive impact of the phosphonate
on potency, as shown by comparison of the ‘sans-
phosphonate’ compound 5, which showed 84% inhibition at
100 μM, compared with the phosphonate bearing 13a-(s) and
13a-(l) (Ki = 40 and 50 μM, resp.). The higher activities of the
m-phenoxy compounds (13a and 13c) are consistent with
previous studies, with compounds such as (R)-1, (R)-2, and 3
all possessing a phenyl group in this position.
Fig. 3 Comparison of binding modes in ST6Gal I (PDB ID: 4JS2)
between the (A) co-crystallised substrate CMP; (B) phosphodiester lead
compound (R)-1; and (C) ether-linked 1,2,3-triazole based compound
(R)-13a. The surface representation highlights the presence of H-bond
donors in pink and H-bond acceptors in green. Figure made with
Discovery Studio Visualiser.44
as the donor and Gal-β1,3-GalNAc and LacNAc as the
acceptors for hST3Gal I and hST6Gal I, respectively. CMP
produced as a by-product of the ST reaction was detected
using a luciferase reaction producing luminescence with a
linear response to the concentration of CMP. With acceptor
concentration set at 1 mM, the Km for CMP-Neu5Ac was
calculated as 37.16 5.42 μM for hST6Gal I, comparable to
previously reported values.26 The percentage inhibition of all
compounds at 10 and 100 μM (with 1 mM acceptor and 100
μM CMP-Neu5Ac) was initially calculated, and for those
compounds with over 50% inhibition at 10 μM, Ki's were
determined. Inhibition data for compounds against hST3Gal
I is only shown for an inhibitor concentration of 100 μM as
they were either only mildly or completely inactive against
the enzyme. A summary of the inhibitor screening is shown
in Table 3, with the previously described carbamate (4) and
triazole (5) compounds included as references.31,32
Broadly, this series of compounds exhibited promising
activity against hST6Gal I, with the m-phenoxy, m-phenoxy-p-
fluoro, and m-1,1,2,2-tetrafluroethoxy (13a, c, and f, resp.)
having Ki's in the mid-to-low micromolar range. The reduced
activity of the compounds from this study against hST3Gal I
correlates with the reduced binding affinity with the
homology model observed in the docking study. Interestingly,
it was noted that the compounds that showed activity against
The most potent inhibitor in the series investigated here
is the tetrafluoro compound 13f-(l) (Ki = 3.4
0.6 μM;
hST6Gal I), which could be due to the fluorinated substituent
participating in similar binding interactions with the enzyme
as the glycerol chain on the natural ST donor molecule CMP-
Neu5Ac, as fluorine is known to participate in hydrogen
binding-like interactions.46 The fluorine moiety could also
participate in hydrophobic interactions with the enzyme
binding site, similar to the m-phenoxy moiety on the
literature compound (R)-1 and the other active triazole
compounds 13a and 13c.
A wide array of assays has been used to evaluate the
effectiveness of ST inhibitors based on HPLC, MS,
fluorescence or luminescence activity readouts and on a
range of both bacterial and non-human mammalian ST
enzymes, with varying sequence homology. This makes it
difficult to compare inhibitor activity values across studies.
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Table 3 Inhibitory activity of compounds against hST3Gal I and hST6Gal I
hST6Gal I
hST3Gal I
Compound
Inhibition at 10 μM (%)
Km or Ki (μM)
37
1.1 0.1
—
40 10
50 10
—
Km/Ki
Inhibition at 100 μM (%)
CMP-Neu5Ac
4-(l)
5-(s/l)
—
59
84a
62
26
41
10
57
59
44
45
39
23
65
78
25
21
5
—
32.5
—
1.0
0.7
—
—
38
43
28
2
—
—
43
9
—
—
—
—
49
39
—
—
13a-(s)
13a-(l)
13b-(s)
13b-(l)
13c-(s)
13c-(l)
13d-(s)
13d-(l)
13e-(s)
13e-(l)
13f-(s)
13f-(l)
—
—
6
1
6.0
1.1
—
—
—
34
—
—
—
—
6
—
6.7 0.6
3.4 0.6
—
5.6
11.0
—
13g-(s)
13g-(l)
—
—
a
Inhibition for this compound was recorded at 100 μM inhibitor concentration.
Herein, the three most active compounds exhibited Ki values
of <10 μM in a luminescence-based assay over a single hour
incubation period. Recent studies on a murine breast cancer
model showed effectiveness in vivo, inhibiting tumour
growth, reducing angiogenesis, and delaying metastasis of
MDA-MB-231 cells. In this case, the inhibitors also showed
activity in the 1–10 μM range against human ST6Gal I and rat
ST3Gal III in an enzymatic assay.47
activity against hST6Gal I, further investigation is underway
on this series evaluating the effect of inhibiting sialylation on
cell migration, invasion and tumour spheroid formation in
pancreatic, ovarian and hepatic cancer. While panels of all 20
human ST enzymes are not yet commercially available,
greatly expanded ST inhibitor screening capabilities in recent
years19,48,49 enables the screening of our inhibitors against a
wider panel of human ST enzymes in the future, along with
computational studies on newly released ST crystal
structures.50 Overall, the series herein is a promising step
forward in the development of more metabolically stable,
synthetically accessible and selective ST inhibitors.
Conclusions
In conclusion, a series of 14 phosphonate-bearing, uridine-
based 1,2,3-triazole compounds (13a–g) were synthesised and
investigated for their activity as ST inhibitors and to probe
any potential selectivity between two of the most
therapeutically relevant STs (hST3Gal I and hST6Gal I). To
achieve this, a series of novel α-azidophosphonates (11a–g)
were synthesised and coupled to 5′-O-propargyluridine (8) via
CuAAC chemistry, followed by deprotection and purification
Materials and methods
General docking procedure
Ligands were docked into the binding site of the hST3Gal I
homology model (UNIPROT ID: Q11201, model version
3.0.0)40 and hST6Gal I crystal structure (PDB ID: 4JS2) with
AutoDock Vina (AD-Vina) version 1.1.2.41 Receptor structures
were prepared for docking using AutoDockTools (ADT)
version 4.2.6.43 The three-dimensional structure of the
inhibitors were prepared utilising ChemDraw 19.0 and
Avogadro v1.1.1.51 ADT was used to assign both rigid and
rotatable bonds and to remove non-polar hydrogens. Docking
was performed for a box of 30 Å × 30 Å × 30 Å centred at the
active site. These conditions have been used in our previous
computational work.28,29 The receptor is treated as rigid and
no explicit waters have been included. The top ranked
models for each compound tested based on the binding
energies calculated by AD-Vina were evaluated based upon
comparison to the position of the ligand in the molecular
dynamics simulations of hST3Gal I and hST6Gal I with VMD
v1.9.2.43 Statistical analysis was undertaken on the calculated
binding energies for each compound to determine if they
to yield
a series of 14 phosphonate-bearing triazole
compounds (13a–g). These transition-state analogues were
then tested for activity against both hST3Gal I and hST6Gal I
in vitro. The series appeared to exhibit selectivity for hST6Gal
I over ST3Gal I in terms of a larger number of binding
modes, with 6 compounds – 13a-(s), 13a-(l), 13c-(s), 13c-(l),
13f-(s) and 13f-(l) – showing promising activities (Ki = 3.4–
53.7 μM; hST6Gal I) compared to other reported ST
inhibitors.31,47
These
experimental
results
support
previous
computational work that suggested 1,2,3-triazoles would be a
suitable non-charged linker to replace the phosphodiester
linker of lead literature compounds such as (R)-1. The ether-
triazole series is also highly synthetically accessible, with only
six steps required to yield final compounds, with an overall
yield from uridine of up to 44%. With this encouraging
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were statistically different from one another, using a two-
tailed students t-test. Results were deemed significant for P <
0.05.
compounds. 1H, 13C, 31P, and 19F NMR spectra for these
compounds are provided in the ESI.†
Disodium 5′-O-[1-(phosphonato-3-phenoxyphenylmethyl)-
1,2,3-triazol-4-yl]methyluridine (13a). From 12a (40.4 mg, 52.6
μmol): purification by RP-HPLC, converted to sodium salts by
ion-exchange (IR 120 Na+) in water, and lyophilised to give
the purified compounds 13a-(s) (16.0 mg, 99%), 13a-(l) (15.2
mg, 91%). 13a-(s): HPLC (prep. RP-C18, 34–39% MeOH): tR:
17.1 min. [α]2D5 = +8.4 (c = 0.14, H2O). 1H NMR (400 MHz,
D2O): 8.62 (s, 1H), 7.60 (d, 1H, J = 8.0 Hz), 7.17 (t, 1H, J = 7.4
Hz), 4.85–4.72 (m, 2H), 5.38 (d, 1H, J = 8.3 Hz), 7.39–7.35 (m,
4H), 6.94–6.88 (m, 3H), 6.83 (s, 1H), 5.90 (d, 1H, J = 18.3 Hz),
5.89 (d, 1H, J = 4.5 Hz), 4.25 (m, 1H), 4.21 (t, 1H, J = 4.9 Hz),
4.17 (t, 1H, J = 4.7 Hz), 3.93–3.91 (m, 1H), 3.81–3.78 (m, 1H).
13C NMR (100 MHz, D2O): 162.2, 157.9, 157.7, 154.1, 145.0,
142.0, 141.3, 131.24, 131.15, 126.6, 124.9, 124.65, 124.61,
119.6, 119.0, 103.5, 90.0, 84.6, 75.5, 71.5, 70.1, 66.4 (d, J =
124.1 Hz), 64.7. 31P NMR (162 MHz, D2O): 8.36. ESI-HRMS
(m/z): calculated for C25H26N5O10P− [M − H]−: 586.1339, found
586.1350. 13a-(l): HPLC (prep. RP-C18, 34–39% MeOH): tR:
19.9 min. [α]2D5 = −9.9 (c = 0.17, H2O). 1H NMR (500 MHz,
D2O): 8.68 (s, 1H), 7.45–7.42 (m, 3H), 7.39–7.38 (m, 1H),
7.35–7.33 (m, 1H), 7.23 (t, 1H, J = 7.4 Hz), 7.01 (d, 2H, J = 8.0
Hz), 6.95 (d, 1H, J = 8.4 Hz), 6.92 (s, 1H), 5.91 (d, 1H, J = 18.5
Hz), 5.90 (d, 1H, J = 3.8 Hz), 5.53 (d, 1H, J = 7.9 Hz), 4.83–
4.80 (m, 2H), 4.26 (m, 1H), 4.24–4.21 (m, 2H), 4.01–3.99 (m,
1H), 3.91–3.88 (m, 1H). 13C NMR (125 MHz, D2O): 162.6,
158.0, 157.8, 154.6, 144.8, 141.8, 141.3, 131.29, 131.19, 130.5,
126.5, 125.0, 124.8, 119.6, 119.1, 119.0, 103.5, 90.1, 84.3, 75.4,
71.1, 70.2, 66.5 (d, J = 124.5 Hz), 64.6. 31P NMR (162 MHz,
D2O): 10.77. ESI-HRMS (m/z): calculated for C25H26N5O10P−
[M − H]−: 586.1339, found 586.1329.
Molecular dynamics simulations
For molecular dynamics simulations of the hST3Gal
I
homology model with compound (R)-1, the ligand was
docked into the crystal structure using AD Vina according to
the method above. The system was prepared via the same
method as in previous work, with the same forcefields and
conditions on the simulation.28 Molecular dynamic
simulations were conducted with NAMD 2.12.52
General chemistry
All chemicals were purchased from Sigma Aldrich/Merck
(USA), Carbosynth (UK) or ChemSupply (SA) and used as
supplied or purified by standard methods. All reactions were
performed under anhydrous conditions unless stated
otherwise and monitored by TLC with Merck silica gel 60 F254
aluminium backed plates and visualised by UV or staining.
Column chromatography was performed under ‘flash’
1
conditions on silica gel 60 (40–63 μm mesh). H, 13C, 19F, 31P,
and 2D NMR spectra were acquired on Bruker Avance Neo
500 and 400 MHz NMR instruments. Chemical shifts for 1H
and 13C NMR are given in ppm relative to residual solvent
peaks or TMS. 19F NMR shifts are reported against an
external reference of 0.05% α,α,α-trifluorotoluene in CDCl3,
and 31P shifts against an external reference of 85% H3PO4 in
D2O. Optical rotation was measured using a Jasco P-2000
polarimeter with specific rotation reported in degrees, and
concentration (c) reported in g/100 mL. High resolution
electrospray ionisation mass spectrometry (HRMS) was
performed on a Waters XEVO G2 Q-TOF spectrometer with
leucine enkephalin (LeuEnk) as an internal standard.
Disodium
5′-O-[1-(phosphonato-3-cyclopentoxyphenyl
methyl)-1,2,3-triazol-4-yl]methyluridine (13b). From 12b (172
mg, 0.163 mmol): purification by RP-HPLC, converted to
sodium salts by ion-exchange (IR 120 Na+) in water, and
lyophilised to give the purified compounds 13b-(s) (20.2 mg,
94%), 13b-(l) (21.3 mg, 99%). 13b-(s): HPLC (prep. RP-C18,
Analytical RP-HPLC was performed with
a Shimadzu
Prominence-I LC2030C system with a PDA detector (190–800
nm), with a Luna C18 (2) 100 Å (Phenomenex, 3 μm, 4.6 ×
150 mm) column. Preperative RP-HPLC was performed with a
Shimadzu Prominence LC-20AP system with a PDS detector
(190–800 nm) using a Prep C18 (Shimadzu, 5 μm, 20 × 150
mm) column. The purity of all final compounds was
determined to be ≥95% based on 1H NMR data (see ESI†).
Synthesis and characterisation of novel compounds not
shown here (10e–f, 11a–g, and 12a–g) are detailed in the ESI.†
1
34–39% MeOH): tR: 17.6 min. [α]2D5 = −19.0 (c = 0.1, H2O). H
NMR (500 MHz, D2O): 8.67 (s, 1H), 7.64 (d, 1H, J = 8.2 Hz),
7.30 (t, 1H, J = 7.9 Hz), 7.13 (d, 1H, J = 7.9 Hz), 6.89–6.86 (m,
2
2H), 5.96 (d, 1H, J = 4.3 Hz), 5.84 (d, 1H, J(H,P) = 18.3 Hz),
5.33 (d, 1H, J = 8.5 Hz), 4.86–4.75 (m, 2H), 4.81 (m, 1H), 4.28–
4.20 (m, 3H), 3.96 (m, 1H), 3.84 (m, 1H), 1.95–1.86 (m, 2H),
1.71–1.61 (m, 6H). 13C NMR (125 MHz, D2O): 170.4, 162.5,
158.4, 155.4, 144.9, 142.0, 140.9, 130.9, 130.5, 127.2, 126.5,
122.2, 116.4, 116.1, 103.7, 90.1, 84.6, 81.8, 75.6, 71.6, 70.4,
Deprotection of triazoles
1
64.9, 62.0 (d, J(C,P) = 125.0 Hz), 33.6, 24.9. 31P NMR (162
Protected triazole compounds (1 equiv.) was dissolved in 10
mL MeOH and stirred with 10% (w/w) Pd/C catalyst under an
atmosphere of hydrogen gas. After completion (judged by
ESI-MS), the reaction mixture was filtered over celite and
dried on a rotary evaporator to give a crude mixture of
deprotected diastereomers. The diastereomers were separated
via RP-HPLC, converted to their sodium salts by ion-exchange
(IR 120 Na+) in water, and lyophilised to give the purified
MHz, D2O): 7.90. ESI-HRMS (m/z): calculated for C24H30N5-
O
10P [M − H]−: 578.1646, found 578.1652. 13b-(s): HPLC
(prep. RP-C18, 34–39% MeOH): tR: 20.2 min. [α]2D5 = −14.8 (c =
0.1, H2O). 1H NMR (500 MHz, D2O): 8.72 (s, 1H), 7.36–7.22
(m, 2H), 7.11–7.07 (m, 1H), 6.91–6.85 (m, 2H), 5.89 (m, 1H),
2
5.84 (d, 1H, J(H,P) = 18.4 Hz), 5.34 (d, 1H, J = 8.4 Hz), 4.86–
4.75 (m, 3H), 4.27–4.21 (m, 3H), 4.04–3.85 (m, 2H), 2.00–1.89
(m, 2H), 1.76–1.61 (m, 6H). 13C NMR (125 MHz, D2O): 168.7,
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161.3, 157.0, 143.5, 140.3, 139.4, 129.5, 125.9, 125.1, 120.9,
114.9, 114.8, 102.2, 88.8, 82.9, 80.5, 74.2, 69.8, 69.0, 65.5 (d,
1J(C,P) = 121.9 Hz), 63.2, 32.8, 32.2, 23.5. 31P NMR (162 MHz,
D2O): 7.98. ESI-HRMS (m/z): calculated for C24H30N5O10P [M
− H]−: 578.1649, found 578.1652.
(t, 1H, J = 4.6 Hz), 4.14 (t, 1H, J = 5.1 Hz), 3.99 (m, 1H), 3.87
(m, 1H). 13C NMR (125 MHz, D2O): 177.4, 163.2 (d, J = 242.4
Hz), 160.3, 144.8, 140.7, 135.0, 131.0 (d, J = 5.3 Hz), 130.9 (d,
J = 4.4 Hz), 126.5, 116.4 (d, J = 22.3 Hz), 104.0, 90.5, 83.7,
75.4, 71.1, 70.5, 66.2 (d, J = 128.4 Hz), 64.6. 31P NMR (162
MHz, D2O): 8.33. 19F NMR (376 MHz, D2O): −115.4. ESI-
Disodium 5′-O-[1-(phosphonato-3-phenoxy-4-fluorophenyl
methyl)-1,2,3-triazol-4-yl]methyluridine (13c). From 12c (100
mg, 0.120 mmol): purification by RP-HPLC, converted to
sodium salts by ion-exchange (IR 120 Na+) in water, and
lyophilised to give the purified compounds 13c-(s) (38.1 mg,
97%), 13c-(l) (35.8 mg, 91%). 13c-(s): HPLC (prep. RP-C18,
HRMS (m/z): calculated for
C
19H21FN5O9P− [M
−
H]−:
512.0983, found 512.0982.
Disodium
5′-O-[1-(phosphonato-3-trifluoromethylphenyl
methyl)-1,2,3-triazol-4-yl]methyluridine (13e). From 12e (189
mg, 0.186 mmol): purification by RP-HPLC, converted to
sodium salts by ion-exchange (IR 120 Na+) in water, and
lyophilised to give the purified compounds 13e-(s) (52.1 mg,
92%), 13e-(l) (48.1 mg, 85%). 13e-(s): HPLC (prep. RP-C18,
26–31% MeOH): tR: 16.2 min. [α]2D5 = −2.6 (c = 0.3, H2O). 1H
NMR (500 MHz, D2O): 8.80 (s, 1H), 7.79 (d, 1H, J = 8.0 Hz),
7.71 (d, 1H, J = 7.5 Hz), 7.66–7.62 (m, 2H), 7.64 (d, 1H, J = 8.0
Hz), 6.05 (d, 1H, J = 21.8 Hz), 6.03 (d, 1H, J = 4.3 Hz), 5.19 (d,
1H, J = 8.2 Hz), 4.97–4.87 (m, 2H), 4.42 (m, 1H), 4.39 (t, 1H, J
= 4.5 Hz), 4.35 (t, 1H, J = 4.8 Hz), 4.10 (m, 1H), 3.97 (m, 1H).
13C NMR (125 MHz, D2O): 166.8, 152.8, 148.2, 145.2, 142.3,
139.8, 133.0, 130.5, 126.6, 125.8, 125.0, 102.9, 90.0, 84.9, 75.7,
71.6, 70.2, 66.1 (d, J = 125.7 Hz), 64.7. 31P NMR (162 MHz,
D2O): 8.01. 19F NMR (376 MHz, D2O): −62.2. ESI-HRMS (m/z):
calculated for C20H21F3N5O9P− [M − H]−: 562.0951, found
562.0961. 13e-(l): HPLC (prep. RP-C18, 26–31% MeOH): tR:
20.7 min. [α]2D5 = +4.9 (c = 0.25, H2O). 1H NMR (400 MHz,
D2O): 8.81 (s, 1H), 7.78 (d, 1H, J = 7.4 Hz), 7.73 (d, 1H, J = 7.0
Hz), 7.72 (s, 1H), 7.63 (t, 1H, J = 7.6 Hz), 7.36 (d, 1H, J = 8.1
Hz), 6.08 (d, 1H, J = 18.2 Hz), 5.96 (d, 1H, J = 3.9 Hz), 5.44 (d,
1H, J = 8.0 Hz), 4.97–4.88 (m, 2H), 4.36–4.31 (m, 3H), 4.12 (m,
1H), 4.02 (m, 1H). 13C NMR (100 MHz, D2O): 166.7, 152.6,
145.0, 142.0, 139.6, 132.8, 130.4, 126.5, 125.7, 125.3, 103.2,
89.8, 84.4, 75.2, 71.1, 70.3, 66.1 (d, J = 123.3 Hz), 64.4. 31P
NMR (162 MHz, D2O): 8.15. 19F NMR (376 MHz, D2O): −62.1.
ESI-HRMS (m/z): calculated for C20H21F3N5O9P− [M − H]−:
562.0951, found 562.0932.
1
34–39% MeOH): tR: 17.2 min. [α]2D5 = +1.5 (c = 0.1, H2O). H
NMR (500 MHz, D2O): 8.58 (s, 1H), 7.54 (d, 1H, J = 7.9 Hz),
7.38–7.35 (m, 1H), 7.30 (t, 2H, J = 8.1 Hz), 7.20 (dd, 1H, J =
11.1, 8.7 Hz), 7.09 (t, 1H, J = 7.4 Hz), 6.89 (d, 2H, J = 8.0 Hz),
6.86 (d, 1H, J = 8.0 Hz), 5.84 (d, 1H, J = 4.2 Hz), 5.79 (d, 1H,
2J(H,P) = 18.5 Hz), 5.29 (d, 1H, J = 7.8 Hz), 4.71 (m, 2H), 4.16
(m, 1H), 4.09 (m, 2H), 3.80 (m, 2H). 13C NMR (125 MHz,
1
D2O): 162.9, 158.4, 155.8, 145.1, 141.9, 138.0 (d, J(C,F) = 283.8
Hz), 131.3, 127.3, 126.8, 124.8, 118.5, 117.9, 103.7, 90.4, 84.5,
1
75.8, 71.5, 70.2, 66.0 (d, J(C,P) = 125.2 Hz), 64.9. 31P NMR
(162 MHz, D2O): 7.68. 19F NMR (376 MHz, D2O): −132.8. ESI-
HRMS (m/z): calculated for C25H24F4N5O10P− [M
−
H]−:
604.1245, found 604.1232. 13c-(l): HPLC (prep. RP-C18, 34–
39% MeOH): tR: 19.8 min. [α]2D5 = −17.2 (c = 0.11, H2O). 1H
NMR (500 MHz, D2O): 8.16 (s, 1H), 7.86 (d, 1H, J = 8.2 Hz),
2
6.90–6.88 (m, 2H), 6.54 (d, 1H, J(H,P) = 22.2 Hz), 5.88 (d, 1H, J
= 4.5 Hz), 5.53 (d, 1H, J = 8.3 Hz), 5.03–4.94 (m, 4H), 4.68 (m,
2H), 4.12 (m, 3H), 3.79 (m, 2H). 13C NMR (125 MHz, D2O):
1
162.8, 158.3, 154.7, 143.6, 141.8, 137.9 (d, J(C,F) = 275.5 Hz),
131.3, 130.5, 127.3, 126.5, 124.8, 122.5, 118.0, 116.8, 102.3,
1
90.2, 84.3, 75.6, 71.1, 70.2, 66.0 (d, J(C,P) = 124.1 Hz), 64.7 31P
NMR (162 MHz, D2O): 7.89. 19F NMR (376 MHz, D2O): −133.5.
ESI-HRMS (m/z): calculated for C25H24F4N5O10P− [M − H]−:
604.1245, found 604.1238.
Disodium
5′-O-[1-(phosphonato-4-fluorophenylmethyl)-
1,2,3-triazol-4-yl]methyluridine (13d). From 12d (69.0 mg,
0.0995 mmol): purification by RP-HPLC, converted to sodium
salts by ion-exchange (IR 120 Na+) in water, and lyophilised
to give the purified compounds 13d-(s) (25.5 mg, 92%), 13d-
(l) (17.0 mg, 62%). 13d-(s): HPLC (prep. RP-C18, 15–20%
MeOH): tR: 27.2 min. [α]2D5 = −21.8 (c = 0.1, H2O). 1H NMR
(500 MHz, D2O): 8.68 (s, 1H), 7.51–7.46 (m, 3H), 7.13–7.08
(m, 2H), 6.01 (m, 1H), 5.89 (d, 1H, J = 17.8 Hz), 5.36 (d, 1H, J
= 7.9 Hz), 4.89–4.87 (m, 2H), 4.31–4.27 (m, 3H), 3.97 (m, 1H),
3.88 (m, 1H). 13C NMR (125 MHz, D2O): 172.5, 163.2 (d, J =
242.9 Hz), 156.9, 145.1, 141.6, 135.1, 131.04 (d, J = 4.6 Hz),
130.97 (d, J = 4.6 Hz), 126.4, 116.5 (d, J = 21.7 Hz), 103.7,
90.0, 84.7, 75.6, 72.0, 70.5, 66.1 (d, J = 126.6 Hz), 64.9. 31P
NMR (162 MHz, D2O): 8.44. 19F NMR (376 MHz, D2O): −112.9.
ESI-HRMS (m/z): calculated for C19H21FN5O9P− [M − H]−:
512.0983, found 512.1000. 13d-(l): HPLC (prep. RP-C18, 15–
20% MeOH): tR: 30.4 min. [α]2D5 = −6.4 (c = 0.16, H2O). 1H
NMR (500 MHz, D2O): 8.68 (s, 1H), 7.39–7.37 (m, 2H), 7.01
(m, 3H), 5.88 (d, 1H, J = 4.0 Hz), 5.83 (d, 1H, J = 18.2 Hz),
5.44 (d, 1H, J = 7.4 Hz), 4.80–4.77 (m, 2H), 4.20 (m, 1H), 4.17
Disodium 5′-O-[1-(phosphonato-3-(1,1,2,2-tetrafluoroethoxy)
phenylmethyl)-1,2,3-triazol-4-yl]methyluridine (13f). From 12f
(86.4 mg, 0.110 mmol): purification by RP-HPLC, converted to
sodium salts by ion-exchange (IR 120 Na+) in water, and
lyophilised to give the purified compounds 13f-(s) (34.1 mg,
95%), 13f-(l) (33.0 mg, 92%). 13f-(s): HPLC (prep. RP-C18, 12–
1
16% ACN): tR: 16.3 min. [α]2D5 = −7.0 (c = 0.16, H2O). H NMR
(400 MHz, D2O): 8.67 (s, 1H), 7.57 (d, 1H, J = 8.4 Hz), 7.43 (d,
1H, J = 7.6 Hz), 7.38 (t, 1H, J = 7.8 Hz), 7.15 (d, 1H, J = 7.7 Hz),
2
3
7.08 (s, 1H), 6.21 (tt, 1H, J(H,F) = 52.4 Hz, J(H,F) = 2.7 Hz), 5.87
(d, 1H, J = 4.4 Hz), 5.86 (d, 1H, 2J(H,P) = 18.0 Hz), 5.07 (d, 1H, J =
8.0 Hz),4.81–4.72 (m, 2H), 4.24–4.15 (m, 3H), 3.88 (m, 2H). 13C
NMR (100 MHz, D2O): 162.5, 155.5, 149.1, 144.3, 141.0, 140.7,
1
130.5, 127.2, 126.0, 121.4, 120.9, 116.6 (t, J(C,F) = 271.0 Hz),
1
108.0 (t, J(C,F) = 245.8 Hz), 102.6, 89.7, 83.6, 75.1, 70.5, 69.3,
65.6 (d, 1J(C,P) = 123.6 Hz), 64.1. 31P NMR (162 MHz, D2O): 7.59.
19F NMR (376 MHz, D2O): −88.4, −137.7. ESI-HRMS (m/z):
calculated for C21H21F4N5O10P [M − H]−: 610.0962, found
610.0961. 13f-(l): HPLC (prep. RP-C18, 12–16% ACN): tR: 19.4
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min. [α]2D5 = −3.8 (c = 0.11, H2O). 1H NMR (400 MHz, D2O): 8.54
(s, 1H), 7.23–7.18 (m, 2H), 7.04 (d, 1H, J = 7.9 Hz), 7.00 (d, 1H, J
= 7.1 Hz), 6.93 (s, 1H), 6.08 (tt, 1H, 2J(H,F) = 52.6 Hz, 3J(H,F) = 3.0
Hz), 5.70 (d, 1H, 2J(H,P) = 15.9 Hz), 5.67 (d, 1H, J = 5.6 Hz), 5.15
(d, 1H, J = 7.7 Hz), 4.62 (m, 2H), 4.03–3.99 (m, 3H), 3.82–3.68
Wollongong. H. Y. is the recipient of an Australian Research
Council (ARC) Future Fellowship (Project number
FT110100034) and A. M. received an Australian Government
Research Training Program Award. This research was in part
supported by an ARC Discovery Project (DP170101773), and
with resources at the NCI National Facility at the Australian
National University through the National Computational
Merit Allocation Scheme. We also thank Dr Andrew Tague for
his technical support.
(m, 2H). 13C NMR (100 MHz, D2O): 161.7, 156.8, 148.4, 143.5,
1
140.9, 140.1, 129.7, 126.3, 125.3, 120.5, 120.4, 116.6 (t, J(C,F)
=
1
271.0 Hz), 107.8 (t, J(C,F) = 245.0 Hz), 102.2, 88.9, 82.7, 74.1,
69.7, 68.9, 65.0 (d, J(C,P) = 126.0 Hz), 63.1. 31P NMR (162 MHz,
1
D2O): 7.60. 19F NMR (376 MHz, D2O): −88.3, −137.7. ESI-HRMS
(m/z): calculated for C21H21F4N5O10P [M − H]−: 610.0962, found
610.0958.
Notes and references
Disodium 5′-O-[1-(phosphonatobenzothiophen-3-ylmethyl)-
1,2,3-triazol-4-yl]methyluridine (13g). From 12g (42.9 mg, 58.6
μmol): purification by RP-HPLC, converted to sodium salts by
ion-exchange (IR 120 Na+) in water, and lyophilised to give
the purified compounds 13g-(s) (16.8 mg, 96%), 13g-(l) (16.2
mg, 92%). 13g-(s): HPLC (prep. RP-C18, 26–31% MeOH): tR:
22.1 min. [α]2D5 = −13.9 (c = 0.1, H2O). 1H NMR (500 MHz,
CDCl3): 8.31 (s, 1H), 8.08 (s, 1H), 8.01 (s, 1H), 7.67 (d, 1H, J =
7.9 Hz), 7.35 (m, 1H), 7.10 (m, 2H), 6.96 (d, 1H, J = 7.9 Hz),
6.04 (d, 1H, J = 17.0 Hz), 5.47 (d, 1H, J = 3.7 Hz), 4.94 (d, 1H,
J = 8.1 Hz), 4.61–4.43 (m, 2H), 3.93 (m, 1H), 3.86 (t, 1H, J =
5.1 Hz), 3.81 (t, 1H, J = 4.2 Hz), 3.74–3.72 (m, 1H), 3.58–3.56
(m, 1H). 13C NMR (125 MHz, CDCl3): 162.9, 161.3, 144.5,
140.6, 139.0, 131.2, 130.2, 126.8, 125.7, 125.2, 124.0, 122.1,
102.5, 89.7, 83.5, 75.3, 70.3, 68.4, 63.8, 59.3 (d, J = 122.7 Hz).
31P NMR (162 MHz, CDCl3): 8.19 ESI-HRMS (m/z): calculated
for C21H22N5O9PS [M − H]−: 550.0798, found 550.0789. 13g-(l):
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Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank Phil Clingan, Maxine Stewart and the Illawarra
Cancer Carers for financial support including
scholarship for C. D. and R. S. matched by the University of
a
PhD
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