Design, synthesis and binding properties of a fluorescent α9β1/α4β1 integrin antagonist and its application as an in vivo probe for bone marrow haemopoietic stem cells

Article abstract of DOI:10.1039/c3ob42332h

The α₉β₁ and α₄β ₁ integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α₄β₁ integrin have been thoroughly investigated with respect to HSC function, the role of α₉β₁ integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α₉β ₁ integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(i)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α₉β₁ integrin with potent cross-reactivity against α₄β₁ integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α₉β ₁ and α₄β₁ integrins, which showed faster binding to α₄β₁ integrin relative to α₉β₁, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α₉β ₁ and α₄β₁ integrins; (2) investigating the biochemical properties of α₉β ₁ and α₄β₁ integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.

Full text of DOI:10.1039/c3ob42332h

Organic &
Biomolecular Chemistry
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PAPER
Design, synthesis and binding properties of a
fluorescent α9β1/α4β1 integrin antagonist and its
application as an in vivo probe for bone marrow
haemopoietic stem cells†
Cite this: Org. Biomol. Chem., 2014,
12, 965
Benjamin Cao,a Oliver E. Hutt,a Zhen Zhang,a Songhui Li,a Shen Y. Heazlewood,a
Brenda Williams,a Jessica A. Smith,a David N. Haylock,a,b G. Paul Savage*a and
Susan K. Nilssona,b
The α9β1 and α4β1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have
important roles in stem cell regulation and trafficking. Although the roles of α4β1 integrin have been
thoroughly investigated with respect to HSC function, the role of α9β1 integrin remains poorly character-
ised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to
determine cell-associated protein localisation and activation, and to elucidate the mechanism of small
molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent
cell binding properties of a new fluorescent α9β1 integrin antagonist (R-BC154), which was based on a
series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cyclo-
addition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154
exhibits high nanomolar binding affinities to α9β1 integrin with potent cross-reactivity against α4β1 integrin
under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differ-
ences in the binding kinetics between α9β1 and α4β1 integrins, which showed faster binding to α4β1 integrin
relative to α9β1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of
binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to
mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1)
screening small molecule inhibitors of α9β1 and α4β1 integrins; (2) investigating the biochemical properties
of α9β1 and α4β1 integrin binding and (3) investigating integrin expression and activation on defined cell
phenotypes in vivo.
Received 22nd November 2013,
Accepted 11th December 2013
DOI: 10.1039/c3ob42332h
www.rsc.org/obc
In mammals, 18 α-chains and 8 β-chains have been identi-
fied, with 24 different and unique αβ combinations described
Introduction
Integrins are non-covalently linked αβ heterodimeric trans- to date.4 The α4β1 integrin (very late antigen-4; VLA-4) is
membrane proteins that function primarily as mediators of expressed primarily on leukocytes and are known to be recep-
cell adhesion and cell signalling processes.1 Owing to their tors for vascular cell adhesion molecule-1 (VCAM-1), fibro-
roles in tumour development, metastasis and haemostasis, nectin and osteopontin (Opn).5,6 The α4β1 integrin is a key
integrins have been inextricably linked to several pathological regulator of leukocyte recruitment, migration and activation
conditions including cancer, infection, thrombosis and auto- and has important roles in inflammation and autoimmune
immune disease and are acknowledged therapeutic targets for disease.7 Accordingly, significant effort has been focused on
small molecule intervention.2,3
the development of small molecule inhibitors of α4β1 integrin
function for the treatment of asthma, multiple sclerosis and
Crohn’s disease, with several candidates progressing to phase I
and II clinical trials.8 The related β1 integrin, α9β1, shares
many of the structural and functional properties as α4β1 and
also binds to several of the same ligands including VCAM-1
and Opn.9–12 Additionally, α9β1 integrin are receptors for vas-
cular endothelial growth factors (VEGF), nerve growth factor
aCSIRO Materials Science and Engineering, Bag 10, Clayton Sth MDC, VIC 3169,
Australia. E-mail: Paul.Savage@csiro.au; Fax: +61 3 9543 2376;
Tel: +61 3 9545 2523
bAustralian Regenerative Medicine Institute, Monash University, Wellington Rd,
Clayton, VIC 3800, Australia
†Electronic supplementary information (ESI) available: Supplementary figures
S1–3 and copies of 1H, 13C, COSY and HSQC NMR spectra. See DOI: 10.1039/
c3ob42332h
(NGF) and tenascin-C.11,13,14 Unlike α4β1, which has
a
restricted expression that is largely on leukocytes, the cellular
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expression of α9β1 is widespread and there is evidence to molecule α9β1 integrin antagonist, which we could use for
support its involvement in wound healing, angiogenesis and in vitro screening applications to identify novel integrin inhibi-
tumour development.12,14–16
tors as potential HSC mobilisation agents and for in vivo
Previously, both α4β1 and α9β1 integrins have been shown to binding experiments to identify cell phenotypes that are tar-
be expressed by haemopoietic stem cells (HSC), which are the geted by this class of inhibitors. In the search for a suitable
bone marrow stem cells responsible for the production of all small-molecule that could be adapted for fluorescent labelling
blood cells throughout life.17–19 In this context, the integrins we identified the N-phenylsulfonyl proline peptidomimetic 2,
α4β1 and α9β1 are primarily involved in the sequestration and which bears structural resemblance to BIO5192 (1) (Fig. 1).12
recruitment of HSC to the bone marrow as well as the main- In contrast to BIO5192 (1), which is a potent and selective α4β1
tenance of HSC quiescence, a key characteristic for long-term antagonist, compound 2 is a sub-nanomolar inhibitor of α9β1
repopulating stem cells.18 HSC regulation by α4β1 and α9β1 but also shows potent cross reactivity against α4β1 integrin.12
integrins is mediated through interactions with VCAM-1 and In this seminal work, [35S]-labelled 2 was developed as a probe
Opn, which are expressed and/or secreted by bone-lining for investigating the functional differences between α4β1 and
osteoblasts, endothelial cells and other cells of the bone α9β1 integrins. While radiolabelling techniques are highly sen-
marrow environment.18 Recently, DiPersio and co-workers sitive, issues associated with radioisotope labelling (costly,
identified a selective small molecule α4β1 integrin antagonist, hazardous, difficult to handle and dispose, and isotope stabi-
BIO5192 (1), which is capable of mobilising bone marrow HSC lity) make their applications restricted. Moreover, radiolabelled
and progenitor cells into the peripheral circulation in mice. probes cannot be effectively used in a number of biological
(Fig. 1).20 This study validated small molecule inhibitors of applications as they are not compatible with flow cytometry or
α4β1 integrin as effective HSC mobilisation agents, which may standard microscopy techniques.
have potential applications in peripheral blood stem cell trans-
Herein we report the design, synthesis and binding pro-
plants for the treatment of blood disorders and cancers. perties of a fluorescent high-affinity small-molecule α9β1/α4β1
Despite the structural and functional similarities between α4β1 integrin probe that can be used for (1) in vitro inhibitor screen-
and α9β1, the role of α9β1 integrin in this regard remains ing; (2) in vitro investigations of α9β1 and α4β1 integrin binding
unexplored.
activity and (3) in vivo labelling experiments using multi-par-
While α4β1 integrin has been rigorously studied within the ameter flow cytometry. Interestingly, several potent α2β1 integ-
medicinal chemistry community, α9β1 integrin has received rin inhibitors such as 3, which also possess the
considerably less attention as a target for small molecule inter- N-phenylsulfonyl proline core scaffold have also been reported
vention. Our ongoing interests in α9β1 integrin in HSC biology (Fig. 1).21,22 Thus, any synthetic methodologies developed for
led us to pursue the development of a fluorescent small fluorescent labelling of α9β1 antagonists could also encompass
α4β1, α4β7 and α2β1 integrin antagonists.
Results and discussion
Design and synthesis of a fluorescent α4β1/α9β1 integrin probe
Our general strategy for the fluorescent labelling of compound
2 was based on established structure activity relationships
(Fig. 2).12,23 The pyrrolidine carbamate is important for ensur-
ing high binding affinities to α9β1 integrin and substitution of
the tyrosine phenyl group is not well tolerated.12 The acid func-
tionality has been shown to be essential for activity.24 While
lipophilic substitution on the sulfonyl phenyl group is tole-
rated, the installation of a suitable linker at this position would
Fig. 1 Structures of α4β1 selective integrin antagonist and HSC mobilis-
ation agent 1 (BIO5192), α9β1/α4β1 integrin antagonist 2 and α2β1 integrin
antagonist 3. The common N-phenylsulfonyl proline core is highlighted
in bold.
Fig. 2 Structure activity relationship for N-phenylsulfonyl proline-
based α9β1 integrin antagonists.
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not necessarily fulfil these requirements.23 However, functio-
nalisation with a wide range of substituents at the 4-position
of the proline ring in the trans-configuration can be tole-
rated.25,26 Thus, it was reasonable to suggest that incorporation
of a suitable linker, such as a polyethylene glycol (PEG) linker
at the C4-position of the proline residue would preserve the
bioactive conformation of the dipeptide and provide distan-
cing from the steric bulk of the fluorescent component. There-
fore, our initial efforts focused on identifying an efficient
strategy for installing a trans-configured bifunctional PEG
linker at the C4-position of compound 2 for subsequent conju-
gation to a fluorescent tag.
We have previously reported on lactone 4 as a versatile
synthon for accessing 4-cis-hydroxy proline based dipeptides
through direct acylation with protected amino acids.27 Sub-
sequent activation of the 4-cis-hydroxy group followed by SN2
displacement with nucleophiles would then provide the
desired 4-trans-configured proline derivatives. Thus, we envi-
saged a variety of C4-functionalised derivatives of compound 2
could be acquired starting from lactone 4 and tyrosine deriva-
tive 7 (Scheme 1). Lactone 4 was readily prepared by treatment
of N-phenylsulfonyl-trans-4-hydroxy-L-proline under Mitsunobu
conditions employing DIAD and PPh3.27 The tyrosine deriva-
tive 7 was synthesised from protected 5 by treatment with
pyrrolidine carbonyl chloride in the presence of K2CO3 to give
intermediate 6, followed by removal of the Cbz protecting
group. Exposure of lactone 4 to tyrosine derivative 7 under
biphasic conditions afforded the dipeptide 8 in 89% yield as a
single diastereoisomer. This method takes advantage of the
activated nature of the bicyclic lactone and allows clean con-
version to the 4-cis-hydroxy proline dipeptides without resort-
ing to dehydrative peptide coupling. Initial attempts in
installing the PEG linker focused on the direct displacement
of cis-configured triflate 9 with the amino PEG derivative 10,
which could be readily obtained from commercially accessible
4,7,10-trioxa-1,13-tridecanediamine.28 SN2 displacement of
4-cis-triflates with amines has been reported to give the corres-
ponding trans-amino proline derivatives, which would be
attractive in the current context given the low steric bulk of the
resultant linkage.26 Accordingly, the hydroxyl group of com-
pound 8 was converted to the corresponding triflate 9 prior to
treatment with the PEG derivative 10, which gave the PEGy-
lated product 11, albeit in disappointing yields (27% over 2
steps) (Scheme 1).
Scheme 1 Synthesis of PEGylated derivative 11 via triflate displacement
approach.
Although no major side products were isolated during the
formation of either the triflate 9, or the PEG derivative 10, a
possible rationalisation for the poor yields of the N-alkylation
reaction was intermediate formation of the trifluoromethane-
sulfonyl imidate. Therefore, the simplified cis-hydroxy com-
pound 12, which lacks
a secondary amide was also
investigated. Conversion of 12 to the corresponding triflate
and then treatment with PEG amine 10 gave the trans-amino-
proline derivative 13, but again in a moderate 30% yield over
two steps (Scheme 2a). This suggests concomitant formation
of a trifluoromethanesulfonyl imidate intermediate is unlikely
Scheme 2 Attempted N-alkylation of the C4 position of proline deriva-
tives 12 and 14 with PEG amine 10 via (a) triflate displacement and (b)
to be the principal cause for the low yields obtained for the reductive amination, respectively.
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PEGylation of dipeptide 9. The poor reaction outcomes mesylate, which was subsequently displaced with sodium
achieved from SN2 displacement of triflate intermediates azide to give the trans-azido proline ester 16 in 88% yield over
prompted us to pursue the derivative 13 via reductive amin- 2 steps. Hydrolysis of the methyl ester of 16 gave the proline
ation of the ketone 14. Thus, the ketone 14, which was acid 17, which was then reacted with the tyrosine derivative 7
obtained from pyridinium chlorochromate (PCC) oxidation of under standard HBTU coupling conditions to furnish the
alcohol 12, was treated with NaBH(OAc)3 and the PEG amine dipeptide 18 in 93% yield. Alternatively, dipeptide 18 is also
10, to give the desired 13 as the minor component of a accessible from the cis-alcohol 8 (from Scheme 1). Con-
2 : 1 mixture of cis : trans isomers in a 30% yield (Scheme 2b). veniently, mesylation and subsequent azide displacement of
Thus, the poor yields observed for both direct displacement of alcohol 8 proceeded smoothly to furnish product 18, which
cis-triflates and reductive amination using PEG amine sub- was obtained without the necessity for chromatographic
strates was attributed to a combination of low reaction purification.
efficiency and the recalcitrant nature of these compounds
With the PEG alkyne 15 and azide 18 in hand, attention
toward purification. Although we could repeatedly access the turned to their coupling using the CuAAC reaction (Scheme 4).
PEG-coupled compound 11, the low yields made this sequence Satisfyingly, treatment of 15 and 18 with CuSO4, sodium ascor-
untenable and we sought more efficient strategies for incorpor- bate and the Cu(I)-stabilising ligand tris[(1-benzyl-1H-1,2,3-
ation of a PEG linker whilst preserving the bioactive nature of triazol-4-yl)methyl]amine (TBTA) gave the 1,4-disubstituted
the dipeptide.
triazole 19 in virtually quantitative yield. Hydrolysis of methyl
Reger and co-workers reported that introducing N-linked ester 19 gave the acid 20 and following removal of the Cbz
aromatic heterocycles (e.g. imidazoles, triazoles, tetrazoles and group by hydrogenolysis, the fully deprotected PEG-functiona-
benzimidazoles) at the 4-position of the proline residue is well lised integrin antagonist 21 was obtained in good yields (87%
tolerated such that α4β1 integrin binding activity was not detri- over 2 steps). Finally, treatment of amine 21 with NHS-rhod-
mentally affected.29 Based on this observation, we anticipated amine under aqueous conditions gave the fluorescent labelled
that attachment of a PEG linker via a triazole might also be integrin antagonist 22 in 38% yield as a 5 : 1 mixture of 5- and
tolerated for α9β1 integrin binding. Consequently, this would 6-carboxytetramethylrhodamine regioisomers after purifi-
allow installation of the PEG linker using the Cu(I)-catalyzed cation by C18 reversed phase chromatography.
azide alkyne cycloaddition (CuAAC) reaction between an
alkyne functionalised PEG derivative 15 and a trans-azido between the fluorophore and the bioactive molecule is
integrin antagonist 18 (Scheme 3).30,31
required to prevent the fluorescent tag from interfering with
Generally, it is appreciated that a sufficiently long spacer
The alkyne functionalised PEG derivative 15 was obtained small molecule–receptor interactions.32 To assess the impor-
in one step from 10 by condensation with propionic acid tance of linker length for this class of compounds, compound
under DCC coupling conditions (Scheme 3a). The synthesis of 25 (R-BC154), which lacks the PEG-spacer was also syn-
trans-azido functionalised dipeptide 18 was readily achieved thesised. Thus, hydrolysis of the methyl ester 18 with NaOH
from lactone 4 (Scheme 3b). Treatment of 4 with Na2CO3 in gave the deprotected azide inhibitor 23, which was sub-
MeOH afforded the cis-hydroxy proline ester 12 in 80% yield. sequently reacted with N-propynyl sulforhodamine B 2433
The cis-alcohol of 12 was converted to the corresponding (ESI†) in the presence of CuSO4, sodium ascorbate and TBTA
Scheme 3 Synthesis of (a) alkyne-functionalised PEG derivative 15 and (b) trans-azido N-phenylsulfonyl proline derivative 18.
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Scheme 5 Synthesis of lissamine rhodamine B-labelled integrin antag-
onist 25 (R-BC154).
(Kd = 38.0 nM) under Ca2+/Mg2+ conditions (Fig. 3a and b).
Interestingly, when compared to compound 22, R-BC154 was
associated with only a 1.9-fold and 1.5-fold reduction in
binding affinity to α4β1 and α9β1 integrins, respectively. These
results suggest that both α4β1 and α9β1 integrins can indeed
tolerate significant steric encumbrances at the 4-position of
the proline residue for this class of N-phenylsulfonyl proline-
based integrin antagonists. This observation indicates that
there may be minimal benefits for the incorporation of a PEG
linker. Nevertheless, while linkers separating the bioactive
component from the fluorophore might not be necessary in
this example, the incorporation of an amine-functionalised
PEG linker (e.g. compound 21) might find utility in other appli-
Scheme 4 Synthesis of 5(6)-carboxytetramethylrhodamine-labelled
PEG-linked integrin antagonist 22.
to give the fluorescent labelled 25 (R-BC154) in 43% yield after
purification by HPLC (Scheme 5).
In vitro binding properties of 22 and 25 (R-BC154) to α4β1 and
α9β1 integrins
With fluorescent probes 22 and 25 (R-BC154) in hand, we cations such as the functionalisation of surfaces, resins and
assessed their integrin dependent cell binding properties microbeads for cell adherence, ex vivo culture and cell iso-
using α4β1 and α9β1 over-expressing human glioblastoma LN18 lation purposes.34,35
cell lines that we generated (ESI Fig. S1 and S2†). When each
The surprisingly high affinities of R-BC154 to α4β1/α9β1
LN18 cell line was treated with 22 and R-BC154 (25) under integrins prompted us to explore its binding properties
physiological mimicking conditions (1 mM Ca2+/Mg2+), both further. Like many integrin ligands, the affinity and binding
compounds were found to bind α4β1 and α9β1 LN18 cells in a kinetics of R-BC154 is also dependent on the activation state
dose-dependent manner (Fig. 3a and b). Virtually no binding of integrins, which can be regulated by divalent metal
was observed in the control cell line, which lacks integrin cations.36,37 As expected, no integrin binding was observed in
expression indicating that binding is integrin specific (Fig. 3a the absence of cations (ESI Fig. S3†). However, in the presence
and b). Both the PEG-linked compound 22 and R-BC154 (25) of 1 mM Mn2+, conditions known to induce integrins to adopt
bound α9β1 integrin with greater selectivity than α4β1 integrin a higher affinity binding confirmation, we observed greater
as determined by their calculated dissociation constants (Kd). overall binding to both α4β1 and α9β1 over-expressing LN18 cell
Specifically, compound 22 binds α9β1 (Kd = 8.4 nM) with 2.4- lines (Fig. 3b and c). Additionally, under Mn2+ activation a 3.1-
fold greater affinity than α4β1 (Kd = 20.1 nM) and R-BC154 has fold increase in the binding affinity of R-BC154 towards α4β1
3 times greater affinities for α9β1 (Kd = 12.7 nM) relative to α4β1 (Kd = 12.4 nM) was observed when compared to Ca2+/Mg2+
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Fig. 3 Saturation binding experiment of compound 22 and R-BC154 to control (no integrins; cross-dotted line), α4β1 (circle-dashed line) and α9β1
(square-solid line) LN18 cells with (a) compound 22 in the presence of 1 mM Ca2+/Mg2+ (open symbol) and (b) R-BC154 in the presence of either
1 mM Ca2+/Mg2+ (open symbol) or 1 mM Mn2+ (closed symbol). Data shown are expressed as mean fluorescence intensity (MFI) SEM (n = 3). (c)
Fluorescence microscopy images of over-expressing and control LN18 cells stained with 50 nM R-BC154 (red) under Ca2+/Mg2+ and Mn2+ con-
ditions. Cells were counterstained with DAPI (blue).
Fig. 4 Kinetics measurements of R-BC154 binding to LN18 cells. Association rates for binding of R-BC154 to (a) α4β1 (circle-dashed line) and (b)
α9β1 (square-solid line) integrins were determined in the presence of 1 mM Ca2+/Mg2+ (open symbol) and 1 mM Mn2+ (closed symbol) in TBS buffer
by treatment of cells with 50 nM R-BC154 for 0, 0.5, 1, 2, 3, 5, 10, 15 and 20 minutes at 37 °C. (c) Dissociation rate measurements for binding of
R-BC154 to α4β1 (circle-dashed line) and α9β1 (square-solid line) integrins were determined in the presence of 1 mM Ca2+/Mg2+ (open symbol) and
1 mM Mn2+ (closed symbol) in TBS buffer at 0, 2.5, 5, 15, 30, 45 and 60 minutes. Data shown are expressed as % mean of maximum fluorescence
SEM (n = 3) and plotted as a function of time. On-rate data were fitted to a two-phase association function for all curves (R2 > 0.997). Off-rate data
were fitted to a one-phase exponential decay function for all curves except α4β1 (Ca2+/Mg2+), which was fitted to a two-phase exponential decay
function (R2 > 0.999).
conditions (Kd = 38.0 nM) (Fig. 3b). Despite the greater overall complex (koff = 0.717 and 0.014 min−1) compared to its α9β1
level of α9β1 integrin binding that was induced by the addition (koff = 0.054 and <0.01 min−1) counterpart (Fig. 4c and
of Mn2+, a minimal change in the binding affinity was evident Table 1). In addition, the koff values for R-BC154 binding to
when compared to Ca2+/Mg2+ conditions (Kd = 14.4 nM vs. 12.7 α4β1 and α9β1 integrins in the presence of Mn2+ was signifi-
nM, respectively) (Fig. 3b).
cantly slower compared to Ca2+/Mg2+ conditions, with greater
The differences in the biochemical properties of α4β1 and than 60% of R-BC154 still bound after 60 min (Fig. 4c). The
α9β1 integrins were further investigated by measuring the kine- slower off-rates observed under these conditions suggests
tics of R-BC154 binding. Association rate measurements Mn2+ acts to stabilise the ligand bound conformation and is
showed R-BC154 binding under Ca2+/Mg2+ conditions was consistent with previous reports using radiolabelled sub-
faster relative to Mn2+ conditions for both α4β1 and α9β1 integ- strates.12 Thus, while faster on-rates and off rates are observed
rins (Fig. 4a and b, respectively). Calculation of the on-rate with Ca2+/Mg2+ conditions, Mn2+ activation is associated with
constants (kon) showed R-BC154 binding to α4β1 integrin (kon
=
slower on- and off-rates for α4β1 and α9β1 integrin binding.
0.094 nM−1 min−1) was faster than α9β1 binding (kon = 0.061 Consequently, competitive inhibition assays using R-BC154 for
nM−1 min−1) under physiological conditions (Table 1). Never- in vitro screening of small molecule integrin inhibitors under
theless, similar kon values were observed under Mn2+ activation Ca2+/Mg2+ conditions is preferred as the exceedingly slow off
for both α4β1 (kon = 0.038 nM−1 min−1) and α9β1 integrins (kon rates under Mn2+ activation would require much longer incu-
∼ 0.04 nM−1 min−1) (Table 1).
bation times (data not shown). Our results suggest that
The off-rate kinetics of R-BC154 binding was determined by although α4β1 integrin binds slightly faster to this class of
dissociation experiments. Under both Ca2+/Mg2+ and Mn2+ N-phenylsulfonyl proline-based antagonists compared to α9β1,
states, dissociation rates were faster for the R-BC154-α4β1 more prolonged binding is observed for α9β1 integrin
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Table 1 Summary of R-BC154 binding properties to α4β1 and α9β1 over-
expressing LN18 cells in the presence of Ca2+/Mg2+ or Mn2+
a
b
e
kobs
koff
(min−1
kon
Conditions
(min−1
)
)
(nM−1 min−1
)
α4β1 cells
α9β1 cells
1 mM Ca2+/Mg2+
1 mM Mn2+
5.426
1.891
3.117
2.035
0.717c
0.014
0.094
0.038
0.061
∼0.04
1 mM Ca2+/Mg2+
1 mM Mn2+
0.054
<0.01d
a The observed association rate (kobs) represents the fast phase of
binding and accounts for >60% and >80% of R-BC154 binding to α4β1
and α9β1 integrins, respectively. b Data from the dissociation
experiment represented in Fig. 4c was fitted to
a one-phase
exponential decay function (unless otherwise stated) and dissociation
rate constants (koff) extrapolated from the curve. c Dissociation data for
R-BC154 binding to α4β1 LN18 cells in the presence of Ca2+/Mg2+ was
fitted to a two-phase dissociation curve and koff was determined from
the fast-phase of the curve, which accounted for >60% of the
dissociation. d Dissociation of the R-BC154-α9β1 integrin complex
under Mn2+ activation was too slow (>55% still bound after 120 min;
data not shown) to accurately calculate off-rates; koff value was
estimated based on an approximate half-life of ∼100 min. e The
association rate constant (kon) was calculated using the formula (kobs
koff)/[R-BC154 concentration = 50 nM].
−
(Table 1). Thus, under physiologically relevant conditions, this
class of dual α4β1/α9β1 integrin inhibitors might be expected to
elicit greater affects against α9β1 integrin-dependent inter-
actions in vivo owing to their significantly slower off-rates to
α9β1 despite the higher association rates observed for α4β1
integrin.
Fig. 5 Flow cytometric histogram plots of (a) bone marrow haemo-
poietic progenitor cells (LSK) and (b) HSC (LSKSLAM) isolated from
untreated (grey lines) and R-BC154 (10 mg kg−1) injected (red lines)
C57Bl/6 mice. Data is representative of 3 biological samples. Fluorescent
microscopy images (inset) of FACS sorted progenitor cells (Lineage−Sca-
1+c-Kit+) isolated from (c) untreated and (d) R-BC154 injected mice.
Sca-1+ (blue), c-Kit+ (green), R-BC154+ (red).
In vivo binding of R-BC154 to bone marrow HSC and
progenitor cells
Our in vitro binding data demonstrated that R-BC154 is a high
affinity α4β1 and α9β1 integrin antagonist, whose binding
activity is highly dependent on integrin activation. Thus, we
speculated whether R-BC154 could be used in in vivo binding
experiments to investigate α4β1/α9β1 integrin activity on
defined populations of HSC. To date, assessing integrin
activity on HSC has relied primarily on in vitro or ex vivo stain-
ing of bone marrow cells or purified HSC using fluorescent
labelled antibodies. Whilst ex vivo staining provides confir-
mation of integrin expression by HSC, investigation of integrin
activation in their native state within bone marrow can only be
determined through in vivo binding experiments, as the
complex bone marrow microenvironment cannot be ade-
quately reconstructed in vitro.
also confirmed by fluorescence microscopy on purified popu-
lations of progenitor cells (Lineage−Sca-1+c-Kit+) (Fig. 5c and d).
R-BC154 labelled progenitor cells exhibited a fluorescence halo
indicating R-BC154 binding was primarily cell surface, which
is consistent with integrin-binding. Our in vivo binding results
indicate that this class of α4β1/α9β1 integrin antagonists are
capable of binding to extremely rare populations of haemo-
poietic progenitor cells and HSC, which represent only 0.2%
and 0.002% of mononucleated cells within murine bone
marrow, respectively.38
The α4β1 and α9β1 integrins are recognised to be important
modulators of HSC lodgement within bone marrow through
binding to VCAM-1 and Opn.18 Compound 2 has been shown
to inhibit binding of α4β1 and α9β1 integrins to both VCAM-1
and Opn in vitro with nanomolar inhibitory potencies.12 Our
in vivo binding results using R-BC154 indicate that the α4β1 and
α9β1 integrins expressed by HSC are in an active binding con-
formation in situ. This suggests that small molecule α4β1/α9β1
integrin antagonists such as 2, not only bind directly to bone
marrow HSC, but they might also be capable of inhibiting
α4β1/α9β1 dependent adhesive interactions and potentially
To assess whether R-BC154 and this class of N-phenylsulfo-
nyl proline-based peptidomimetics could bind directly to HSC,
we injected R-BC154 (10 mg kg−1) intravenously into mice and
analysed R-BC154 labelling of phenotypically defined bone
marrow progenitor cells (LSK cell; lineage−Sca-1+c-Kit+) and
HSC (LSKSLAM cell; LSKCD48−CD150+) using multi-colour
flow cytometry (Fig. 5a and b). Increased cell-associated fluo-
rescence as a result of R-BC154 binding was observed for both
progenitor cells and HSC populations that were isolated from
R-BC154 injected mice when compared to bone marrow from
un-injected mice. Furthermore, in vivo R-BC154 binding was
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serve as effective agents for inducing the mobilisation of bone ThermoNicolet 6700 spectrometer using a SmartATR (attenu-
marrow HSC into the peripheral circulation. Assessment of the ated total reflectance) attachment fitted with a diamond
HSC mobilisation properties of α4β1/α9β1 integrin antagonists window. Proton (1H) and carbon (13C) NMR spectra were
in the context of peripheral blood stem cell transplants will be recorded on a BrukerAV400 spectrometer at 400 and 100 MHz,
1
reported elsewhere.
respectively. H NMR are reported in ppm using a solvent as
an internal standard (CDCl3 at 7.26 ppm). Proton-decoupled
13C NMR (100 MHz) are reported in ppm using a solvent as an
internal standard (CDCl3 at 77.16 ppm). High resolution mass
spectrometry was acquired on either a WATERS QTOF II
Conclusion
In summary, we report the synthesis of two N-phenylsulfonyl (CMSE, Clayton, VIC 3168) or a Finnigan hybrid LTQ-FT mass
proline-based fluorescent integrin antagonists, 22 and 25 spectrometer (Thermo Electron Corp.) (Bio21 Institute, Univer-
(R-BC154), both exhibiting high nanomolar binding affinities sity of Melbourne, Parkville, VIC 3010) employing electrospray
to α9β1 and α4β1 integrins. Using the CuAAC reaction, we con- ionisation (ESI).
jugated either an alkyne-functionalised PEG-linker or fluoro-
(1S,4S)-5-(Phenylsulfonyl)-2-oxa-5-azabicyclo[2.2.1]heptan-
phore to 4-azido N-phenylsulfonyl proline based 3-one (4). Diisopropylazocarboxylate (1.87 ml, 9.48 mmol) was
peptidomimetics, which is a strategy that may also be appli- added dropwise over 20 min to a stirred suspension of N-phe-
cable to other integrin inhibitors (e.g. α2β1 antagonists) posses- nylsulfonyl-trans-4-hydroxy-L-proline (2.45 g, 9.03 mmol)
sing similar proline derived scaffolds. By comparing the and triphenylphosphine (2.49 g, 9.48 mmol) in CH2Cl2
binding properties of 22 and R-BC154, we showed that a long (150 ml) at 0 °C under N2. The reaction was warmed to rt and
linker separating the fluorophore from the bioactive com- stirred overnight, concentrated under reduced pressure and
ponent may not be significantly advantageous to integrin the residue purified by flash chromatography (50 : 50 EtOAc–
binding affinity or α9/α4 selectivity. Our in vitro binding kine- pet. spirits) to give the lactone 4 (1.77 g, 78%) as a colourless
tics experiments using R-BC154 demonstrated that this class solid. Spectroscopic data is identical to previously reported
of N-phenylsulfonyl proline-based inhibitors bind faster to values.27
α4β1 integrin but exhibits more prolonged binding to α9β1
(S)-4-(2-(((Benzyloxy)carbonyl)amino)-3-methoxy-3-oxopropyl)-
integrin. Finally, we provide evidence that R-BC154 is capable phenyl pyrrolidine-1-carboxylate (6). 1-Pyrrolidinecarbonyl
of binding haemopoietic progenitor cells and HSC within mice chloride (336 μl, 3.04 mmol) was added to a Biotage™ micro-
bone marrow in vivo, indicating this class of inhibitors might wave vial containing a mixture of the tyrosine 5 (500 mg,
be capable of inhibiting HSC dependent integrin interactions. 1.52 mmol) and K2CO3 (420 mg, 3.04 mmol) in CH3CN (9 ml).
Thus, R-BC154 is a useful high affinity, activation dependent The mixture was heated to 100 °C in a microwave reactor for
integrin probe, which can be used to investigate α9β1 and α4β1 45 min, diluted with H2O and then stirred for 30 min. The
integrin binding activity both in vitro and in vivo.
aqueous layer was extracted with EtOAc (2 × 20 ml) and the
combined organic phases washed with sat. aq. NaHCO3, dried
(MgSO4) and concentrated under reduced pressure. The
residue was recrystallised (EtOAc–pet. spirits) to give the carba-
mate 6 (484 mg, 75%) as colourless crystals, mp 116–117 °C;
[α]D +42.5 (c 0.81 in CHCl3); δH (400 MHz, CDCl3) 1.94 (4 H, m,
Methods
General methods
All starting materials, reagents, and solvents were obtained (CH2)2), 3.09 (2 H, m), 3.47 (2 H, t, J = 6.5 Hz), 3.55 (2 H, t, J =
from commercial sources and used without further purifi- 6.5 Hz), 3.71 (3 H, s), 4.64 (1 H, m), 5.10 (2 H, s), 5.22 (1 H, d,
cation unless otherwise stated. All anhydrous reactions were J = 8.0 Hz), 7.03–7.38 (9 H, m); δC (100 MHz, CDCl3) 25.0, 25.8,
performed under a dry nitrogen atmosphere. Diethyl ether, 37.5, 46.3, 46.4, 52.3, 54.8, 67.00, 121.9 (2 C), 128.1 (2 C),
dichloromethane, tetrahydrofuran and toluene were dried by 128.1, 128.5 (2 C), 130.0 (2 C), 132.4, 136.2, 150.6, 153.0, 155.6,
passage through two sequential columns of activated neutral 171.9; ν/cm−1 3331, 2954, 1719, 1698; 1511, 1402, 1345, 1216,
alumina on the Solvent Dispensing System built by J. C. Meyer 1061, 1020, 866, 754, 699; HRMS (ESI+) m/z 449.1686
and based on the original design by Grubbs and co-workers.39 (C23H26N2NaO6 [M + Na]+ requires 449.1689).
Petroleum spirits refers to the fraction boiling at 40–60 °C.
(S)-4-(2-Amino-3-methoxy-3-oxopropyl)phenyl pyrrolidine-
Thin layer chromatography (TLC) was performed on Merck 1-carboxylate (7). A mixture of protected tyrosine 6 (950 mg,
pre-coated 0.25 mm silica aluminium-backed plates and visu- 1.13 mmol) and Pd/C (10%, 50 mg) in MeOH (40 ml) was
alised with UV light and/or dipping in ninhydrin solution or purged 3 times with H2. The mixture was stirred under a H2
phosphomolybdic acid solution followed by heating. Purifi- atmosphere for 2 h at which point TLC indicated complete
cation of reaction products was carried out by flash chromato- consumption of starting material. The mixture was filtered
graphy using Merck Silica Gel 60 (230–400 mesh) or reverse through a layer of Celite and the filtrate concentrated under
phase C18 silica gel. Melting points were recorded on a Reich- reduced pressure to give the crude amine 7 (637 mg, 98%) as a
ert-Jung Thermovar hot-stage microscope melting point appar- colourless oil, which set solid upon standing. A small portion
atus. Optical rotations were recorded on a Perkin Elmer Model was further purified by flash chromatography (5 : 95 to 10 : 90
341 polarimeter. FTIR spectra were obtained using
a
MeOH–CH2Cl2) for characterisation, [α]D −11.6 (c 1.09 in
972 | Org. Biomol. Chem., 2014, 12, 965–978
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MeOH); δH (400 MHz, CDCl3) 1.97 (4 H, m), 2.91 (1 H, dd, J = 6.6 Hz), 3.46–3.60 (14H, m), 3.76 (3H, s), 4.09 (1H, dd, J = 3.0,
7.1, 13.6 Hz), 3.02 (1 H, dd, J = 6.1, 13.6 Hz), 3.42 (2 H, t, J = 8.9 Hz), 4.85 (1H, td, J = 5.7, 7.8 Hz), 5.07 (2H, s), 5.42 (1H,
6.5 Hz), 3.43 (2 H, t, J = 6.5 Hz), 3.68 (3 H, s), 4.84 (2 H, br s), brs), 7.05 (2H, d, J = 8.6 Hz), 7.14 (2H, d, J = 8.6 Hz), 7.21 (1H,
3.70 (1 H, dd, J = 6.2, 7.1 Hz), 7.05 (2 H, m), 7.21 (2 H, m); δC d, J = 7.8 Hz), 7.28–7.35 (5H, br m), 7.53 (2H, t, J = 7.5 Hz), 7.60
(100 MHz, CDCl3) 25.9, 26.7, 41.1, 47.5, 47.5, 52.4, 56.7, 123.0 (1H, br t, J = 7.04), 7.81 (1H, br d, J = 7.05 Hz); δC (100 MHz,
(2 C), 131.2 (2 C), 135.6, 151.6, 155.1, 176.1; ν/cm−1 3311, 2954, CDCl3) 25.1, 25.9, 29.6, 30.1, 36.5, 37.5, 39.4, 45.9, 46.5, 46.6,
2878, 1714, 1510, 1399, 1344, 1214, 1168, 1086, 1062, 1020, 52.6, 53.3, 54.7, 56.5, 61.6, 66.6, 69.8, 69.7, 70.3, 70.3, 70.67,
864, 755; HRMS (ESI+) m/z 293.1497 (C15H20N2O4SH [M + H]+ 70.72, 122.0 (2 C), 128.1 (2 C), 128.2 (2 C), 128.6 (3 C), 129.4
requires 293.1496).
(2 C), 130.2 (2 C), 133.0, 133.5, 136.0, 137.0, 150.7, 153.2,
4-((S)-2-((2S,4S)-4-Hydroxy-1-(phenylsulfonyl)pyrrolidine-2- 156.6, 170.9, 171.6; ν/cm−1 3334, 2877, 2341, 1706, 1521.
carboxamido)-3-methoxy-3-oxopropyl)phenyl pyrrolidine-1-car- HRMS (ESI+) m/z 904.3770 (C44H59NaN5O12S [M + Na]+ requires
boxylate (8). The lactone 4 (466 mg, 1.84 mmol) and the 904.3779).
amine 7 (510 mg, 1.76 mmol) in toluene–H2O (5 : 1, 6 ml) was
Methyl
(2S,4S)-4-hydroxy-1-(phenylsulfonyl)pyrrolidine-2-
stirred at 80 °C for 2 d and then diluted with EtOAc and carboxylate (12). A mixture of lactone 4 (1.74 g, 6.88 mmol)
washed with 1 M HCl, sat. aq. NaHCO3, brine, dried (MgSO4) and Na2CO3 (3.65 g, 34.4 mmol) was stirred in MeOH (50 ml)
and concentrated under reduced pressure. The residue was at rt overnight. The residue was concentrated, taken up in
purified by flash chromatography (100% EtOAc to 5% MeOH– EtOAc (100 ml), and H2O and the organic phase separated.
EtOAc) to give the alcohol 8 (851 mg, 89%) as a colourless The aqueous phase was extracted with EtOAc (2 × 30 ml) and
foam, [α]D −31.4 (c 1.66 in MeOH); δH (400 MHz, CDCl3) 1.57 the combined organic phases washed with brine, dried
(1 H, m), 1.89–2.00 (5 H, m), 2.94 (1 H, dd, J = 9.5, 14.0 Hz), (MgSO4) and concentrated under reduced pressure to give the
3.13 (1 H, dd, J = 4.0, 10.5 Hz), 3.23 (1 H, d, J = 10.5 Hz), 3.35 methyl ester 12 (1.57 g, 80%) as a colourless solid. Spectro-
(1 H, dd, J = 5.0, 14.0 Hz), 3.40 (2 H, t, J = 6.5 Hz), 3.51–3.56 scopic data is consistent with reported values and the material
(3 H, m), 3.78 (3 H, s), 4.03 (1 H, m), 4.12 (1 H, d, J = 9.0 Hz), was used without further purification.27
4.75 (1 H, m), 6.99 (2 H, d, J = 8.3 Hz), 7.07 (1 H, d, J = 7.5 Hz),
(2S,4R)-Methyl-4-((3-oxo-1-phenyl-2,8,11,14-tetraoxa-4-aza-
7.19 (2 H, 8.3 Hz), 7.51–7.63 (3 H, m), 7.83 (2 H, d, J = 7.5 Hz); heptadecan-17-yl)amino)-1-(phenylsulfonyl)-pyrrolidine-2-car-
δC (100 MHz, CDCl3) 25.1, 25.9, 37.0, 37.6, 46.5, 46.6, 52.6, boxylate (13). Proline ester 12 (100 mg, 0.35 mmol) was
53.3, 57.8, 61.7, 69.7, 122.5 (2 C), 127.9 (2 C), 129.4 (2 C), 130.5 dissolved in dry CH2Cl2 (1 ml) under N2 at −20 °C. DIPEA
(2 C), 133.4, 133.8, 136.3, 150.2, 154.1, 171.6, 171.7; ν/cm−1 (192 μl, 0.53 mmol) was added followed by Tf2O (89 μl,
3408, 1743, 1718, 1701, 1663; HRMS (ESI+) m/z 568.1723 0.53 mmol) dropwise over 10 min. The mixture was stirred at
(C26H31NaN3O8S [M + Na]+ requires 568.1730).
−20 °C for 2 h and then PEG amine 10 (248 mg, 0.70 mmol) in
4-((R)-3-Methoxy-3-oxo-2-((2S,4R)-4-((3-oxo-1-phenyl-2,8,11,14- CH2Cl2 (3 ml) was added dropwise at −20 °C. The reaction was
tetraoxa-4-azaheptadecan-17-yl)amino)-1-(phenylsulfonyl)pyrroli- stirred for 1 h at −20 °C and then warmed to rt for 4 h. Satu-
dine-2-carboxamido)propyl)phenyl pyrrolidine-1-carboxylate (11). rated NaHCO3 and EtOAc were added and the reaction mixture
Alcohol 8 (105 mg, 0.19 mmol) was dissolved in dry CH2Cl2 extracted, washed with water, dried over MgSO4 and the
(2 ml) under N2 at −20 °C. DIPEA (99 μl, 0.57 mmol) was solvent removed under reduced pressure. The crude material
added followed by Tf2O (50 μl, 0.57 mmol) dropwise over was purified by flash chromatography (100% CH2Cl2 then 5%
30 min. The reaction was stirred for 2 h at −20 °C and then MeOH–CH2Cl2 with 1% NEt3) to give 13 as a pale yellow oil
quenched with sat. aq. NaHCO3, diluted with EtOAc and the (319 mg, 30%), δH (200 MHz, CDCl3) 1.62 (1H, br t, J = 4.0 Hz),
organic phase separated. The organic phase was washed with 1.75–1.85 (4H, m), 1.95–2.05 (1H, m), 2.12–2.21 (1H, m), 2.59
H2O, 2% citric acid, sat. aq. NaHCO3 and brine. The aqueous (2H, br s), 3.17 (1H, br s), 3.31 (2H, q, J = 6.3 Hz), 3.42 (1H, t,
phase was extracted with EtOAc (2 times) and the combined J = 5.7 Hz), 3.45–3.69 (13H, m), 3.70 (3H, s), 4.37 (1H, dd, J =
organic phases dried (MgSO4) and the residue concentrated 5.7, 8.7 Hz), 5.08 (2H, s), 5.40 (1H, br s), 7.27–7.38 (5H, m),
under reduced pressure to give the crude triflate. To this 7.52 (2H, t, J = 7.5), 7.57–7.60 (1H, m), 7.84–7.89 (2H, m); δC
residue was added the PEG amine 10 (141 mg, 0.39 mmol) in (100 MHz, CDCl3) 29.5, 29.7, 37.0, 39.3, 45.7, 52.6, 53.4, 56.9,
dry THF (200 μl) and the reaction stirred overnight at rt. The 59.6, 66.6, 69.7, 69.7, 70.2 (2 C), 70.60, 70.62, 127.7 (2 C), 128.1
mixture was diluted with 10% butan-2-ol–EtOAc (20 ml) and (2 C), 128.2, 128.6 (2 C), 129.1 (2 C), 133.0, 136.9, 138.0, 156.6,
the organic phase washed with sat. aq. NaHCO3, brine, dried 172.5; ν/cm−1 3400, 3300, 2980, 2925, 2868, 1787, 1710, 1526;
(MgSO4) and concentrated under reduced pressure. The crude HRMS (EI+) m/z 622.2795 (C30H43N3O9SH [M + H]+ requires
residue was purified by flash chromatography (2% to 3% 622.2798).
MeOH–CH2Cl2) to give 11 (46 mg, 27% over 2 steps) as a col-
Methyl (S)-4-oxo-1-(phenylsulfonyl)pyrrolidine-2-carboxylate
ourless oil. A small portion was further purified by C18-silica (14). A mixture of proline ester 12 (300 mg, 1.05 mmol) and
gel chromatography (40% H2O–MeCN) for characterisation, δH PCC (907 mg, 4.2 mmol) in dry CH2Cl2 (20 ml) was stirred for
(400 MHz, CDCl3) 1.40 (1H, m), 1.55 (2H, m), 1.77 (2H, m), 2 d at rt. The mixture was filtered through a plug of silica and
1.93 (4H, m), 2.12 (1H, m), 2.43 (2H, m), 2.82 (1H, dd, J = 7.8, the solvent removed under reduced pressure to obtain the
9.2 Hz), 2.94 (1 H, m), 3.02 (1 H, dd, J = 7.8, 14.0 Hz), ketone 14 (260 mg, 88%) as colourless crystals, mp
3.23–3.32 (4 H, m), 3.38 (2H, t, J = 6.1 Hz), 3.44 (2H, t, J = 89.5–90.3 °C (lit40 83.5–84.5 °C); δH (200 MHz, CDCl3) 2.56
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(1H, dd, J = 3.0 18.2 Hz), 2.82 (1H, dd, J = 9.3, 18.2 Hz), 3.59 ddd, J = 0.7, 3.0, 11.0 Hz), 3.71 (1 H, dd, J = 5.0, 11.0 Hz), 3.76
(3H, s), 3.76 (1H, d, J = 17.4 Hz), 3.90 (1H, d, J = 17.4 Hz), 4.81 (3 H, s), 4.18–4.22 (1 H, m), 4.30 (1 H, t, J = 7.5 Hz), 7.53–7.58
(1H, dd, J = 3.0, 9.3 Hz), 7.50–7.70 (3H, m), 7.81–7.90 (2H, m); (2 H, m), 7.61–7.65 (1 H, m), 7.87–7.89 (2 H, m); δC (100 MHz,
δC (75 MHz, CDCl3) 41.7, 52.8, 52.9, 57.6, 127.6, 129.6, 133.7, CDCl3) 36.5, 52.9, 53.2, 59.45, 59.51, 127.6 (2 C), 129.3 (2 C),
137.7, 170.8, 206.2; ν/cm−1 3002, 2961, 1763, 1747; HRMS (EI+) 133.3, 137.6, 171.9; ν/cm−1 2101, 1747, 1445, 1345, 1207, 1158,
m/z 306.0412 (C12H13NO5SNa [M + Na]+ requires 306.0412).
1095, 1017, 758, 722, 696; HRMS (ESI+) m/z 311.0808
(C12H14N4O4SH [M + H]+ requires 311.0809); m/z 328.1073
(C12H14N4O4SNH4 [M + NH4]+ requires 328.1074).
Reductive amination method
The PEG amine 10 (189 mg, 0.53 mmol) was added to a solu-
(2S,4R)-4-Azido-1-(phenylsulfonyl)pyrrolidine-2-carboxylic
tion of the ketone 14 (150 mg, 0.53 mmol) in dry CH2Cl2 acid (17). The methyl ester 16 (586 mg, 1.89 mmol) in 3 : 1
(2 ml) under N2. Na(OAc)3BH (156 mg, 0.74 mmol) was added, EtOH–THF (40 ml) was treated with 0.2 M NaOH (12.3 ml,
followed by acetic acid (30 μl, 0.53 mmol) and the reaction 2.45 mmol). The mixture was stirred for 3 h at rt and then con-
stirred for 24 h at rt. Additional Na(OAc)3BH (0.5 eq.) was centrated under reduced pressure. The crude material was
added and the reaction stirred for a further 24 h. The reaction diluted with ether and the aqueous phase separated. The
was quenched with NaOH (1 M) and extracted with CH2Cl2, organic layer was extracted with 0.2 M NaOH (2 × 10 ml) and
washed with H2O, dried and the solvent removed under the combined aqueous extract was acidified with 10% HCl.
reduced pressure. The crude material was purified by flash The aqueous layer was extracted with CHCl3 (4 × 30 ml) and
chromatography (CH2Cl2 to 5% MeOH–CH2Cl2 with 1% NEt3) the combined organic phases washed with brine, dried
to give 13 (99 mg, 30%) as a 2 : 1 mixture of cis : trans (MgSO4) and concentrated under reduced pressure. The crude
diastereoisomers.
material was purified by flash chromatography (5% MeOH–
Benzyl (15-oxo-4,7,10-trioxa-14-azaheptadec-16-yn-1-yl)carba- CH2Cl2 with 0.5% AcOH) to give acid 17 (492 mg, 88%) as a
mate (15). Diisopropylcarbodiimide (291 μl, 1.86 mmol) was colourless oil, [α]D −34.4 (c 0.82 in MeOH); δH (400 MHz,
added to a mixture of PEG amine 10 (0.55 g, 1.55 mmol) and CDCl3) 2.18–2.32 (2 H, m), 3.40 (1 H, m), 3.73 (1 H, dd, J = 4.8,
propiolic acid (96 μl, 1.55 mmol) in dry CH2Cl2 (5 ml) at 0 °C. 11.5 Hz, H5′), 4.22 (1 H, m, H4), 4.29 (1 H, t, J = 7.7 Hz), 7.56
The reaction was warmed to rt and stirred for 2 h and then (2 H, m), 7.64 (1 H, m), 7.88 (2 H, m), 10.72 (1 H, br s); δC
concentrated. The residue was taken up in EtOAc and the urea (100 MHz, CDCl3) 36.1, 53.4, 59.5, 127.6 (2 C), 129.4 (2 C),
byproduct precipitate removed by filtration through a layer of 133.6, 136.9, 176.6; ν/cm−1 3500–2500, 2107, 1731; HRMS
celite filtered off through a layer of celite to remove the urea (ESI+) m/z 319.0472 (C11H12N4NaO4S [M + Na]+ requires
byproduct. The organic filtrate was concentrated under 319.0472).
reduced pressure and the residue purified by flash chromato-
graphy (80% EtOAc–pet. spirits to 100% EtOAc to 5% MeOH– boxamido)-3-methoxy-3-oxopropyl)phenyl pyrrolidine-1-carb-
EtOAc) to give the alkyne 12 (472 mg, 75%) as a colourless oil, oxylate (18). O-(Benzotriazol-1-yl)-N,N,N’,N’-tetramethyl
4-((S)-2-((2S,4R)-4-Azido-1-(phenylsulfonyl)pyrrolidine-2-car-
δH (400 MHz, CDCl3) major rotamer: 1.76 (4 H, m), 2.74 (1 H, uronium hexafluorophosphate (HBTU) (693 mg, 1.83 mmol)
s), 3.31 (2 H, dd, J = 6.1, 12.3 Hz), 3.39 (2 H, dd, J = 5.9, 12.3 was added to a stirred mixture of acid 17 (491 mg, 1.66 mmol)
Hz), 3.51–3.64 (12 H, m), 5.08 (2 H, br s), 5.34 (1 H, br s), 6.82 and N,N-diisopropylethylamine (DIPEA) (578 μl, 3.32 mmol) in
(1 H, br s), 7.27–7.35 (5 H, m); δC (100 MHz, CDCl3) DMF (6 ml) at 0 °C and stirred for 10 min under N2. Amine 7
major rotamer; 23.6, 28.6, 38.6, 39.5, 66.6, 69.8. 70.1, 70.3 (484 mg, 1.66 mmol) in DMF (6 ml) was then added dropwise
(2 C), 70.6, 70.7 (2 C), 72.7, 128.2, 128.3, 128.6 (3 C), 136.9, and the combined mixture warmed to rt and stirred overnight.
152.2, 156.6; ν/cm−1 3292, 2924, 2871, 2105, 1704, 1645, 1535; The mixture was diluted with EtOAc, washed sequentially with
HRMS (ESI+) m/z 407.2177 (C21H31N2O6Na [M + Na]+ requires 5% HCl, sat. aq. NaHCO3, brine, dried (MgSO4) and concen-
407.2182).
trated under reduced pressure. The residue was purified by
Methyl (2S,4R)-4-azido-1-(phenylsulfonyl)pyrrolidine-2-car- flash chromatography (3% MeOH–CH2Cl2) to give the dipep-
boxylate (16). MsCl (304 μl, 3.93 mmol) was added to a tide 7 (928 mg, 98%) as a pale yellow foam, [α]D −4.6 (c 0.85 in
mixture of the alcohol 12 (934 mg, 3.27 mmol) and triethyl- CHCl3); δH (400 MHz, CDCl3) 1.67 (1 H, m), 1.86–1.98 (4 H, m),
amine (620 μl, 4.48 mmol) in dry CH2Cl2 (20 ml) at 0 °C under 2.04 (1 H, dt, J = 5.5, 13.2 Hz), 2.99 (1 H, dd, J = 8.5, 14.0 Hz),
N2. The mixture was stirred for 2 h, diluted with CH2Cl2 and 3.19 (1 H, dd, J = 4.5, 10.9 Hz), 3.30 (1 H, dd, J = 5.5 14.0 Hz),
washed sequentially with 5% HCl, sat. aq. NaHCO3, brine, 3.44 (2 H, t, J = 6.5 Hz), 3.48 (1 H, dd, J = 5.5, 11.5 Hz), 3.53
dried (MgSO4) and concentrated under reduced pressure to (2 H, t, J = 6.5 Hz), 3.78 (3 H, s), 3.82 (1 H, m), 4.09 (1 H, dd,
give the crude mesylate as a pale yellow oil. This residue was J = 5.5, 8.4 Hz), 4.87 (1H, dt, J = 8.3, 8.3, 5.5 Hz), 7.05, 7.15
taken up in DMSO (13 ml) and treated with NaN3 (638 mg, (4 H, 2 × d, J = 8.5 Hz), 7.19 (1 H, d, J = 8.2 Hz), 7.53–7.57 (2 H,
9.81 mmol) and the mixture stirred overnight at 80 °C. The m), 7.62–7.66 (1 H, m), 7.83–7.86 (2 H, m); δC (100 MHz,
reaction was diluted with EtOAc and washed with H2O, brine, CDCl3) 25.1, 25.9, 35.6, 37.5, 46.4, 46.6, 52.7, 53.1, 53.8, 58.9,
dried (MgSO4) and concentrated. The residue was purified by 61.1, 122.1 (2 C), 128.0 (2 C), 129.5 (2 C), 130.2 (2 C), 132.9,
recrystallisation (MeOH) to give 16 (874 mg, 86% over 2 steps) 133.7, 136.0, 150.7, 153.1, 169.9, 171.5; ν/cm−1 3316,
as colourless needles, mp 98–100 °C, [α]D −33.4 (c 1.02 in 2975, 2880, 2105, 1716, 1682; HRMS (ESI+) m/z 593.1789
CHCl3); δH (400 MHz, CDCl3) 2.17–2.21 (2 H, m), 3.43 (1 H, (C26H30N6NaO7S [M + Na]+ requires 593.1789).
974 | Org. Biomol. Chem., 2014, 12, 965–978
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Synthesis of 18 via alcohol 8
53.6, 57.9, 60.7, 66.5, 69.7, 69.7, 70.3, 70.5, 70.6, 70.7, 122.2
(2 C), 126.1, 127.7 (2 C), 128.1, 128.2, 128.5 (3 C), 129.7 (2 C),
Methanesulfonyl chloride (92 μl, 1.18 mmol) was added to a 130.3 (2 C), 133.1, 133.9, 135.5, 136.9, 143.2, 150.6, 153.3,
stirred mixture of the alcohol 8 (251 mg, 0.394 mmol) and tri- 156.6, 159.8, 169.0, 171.5; ν/cm−1 3331, 2951, 2875, 1714, 1667,
ethylamine (170 ml, 1.22 mmol) in dry CH2Cl2 at 0 °C under 1575, 1512; HRMS (ESI+) m/z 999.3891 (C47H60NaN8O13S
N2. The reaction was stirred for 1 h at 0 °C and then warmed [M + Na]+ requires 999.3893).
to rt and stirred for a further 1 h. The reaction was diluted
(R)-2-((2S,4R)-4-(4-((3-(2-(2-(3-Aminopropoxy)ethoxy)ethoxy)-
with CH2Cl2 and washed sequentially with 5% HCl, sat. aq. propyl)carbamoyl)-1H-1,2,3-triazol-1-yl)-1-(phenylsulfonyl)pyrroli-
NaHCO3 and brine. The organic phase was dried (MgSO4) and dine-2-carboxamido)-3-(4-((pyrrolidine-1-carbonyl)oxy)phenyl)-
concentrated under reduced pressure to give the intermediate propanoic acid (21). NaOHaq (1.13 ml, 0.225 mmol, 0.2 M) was
mesylate (4-((S)-3-methoxy-2-((2S,4S)-4-((methylsulfonyl)oxy)-1- added to a stirred mixture of methyl ester 19 (110 mg,
(phenylsulfonyl)pyrrolidine-2-carboxamido)-3-oxopropyl)phenyl 0.113 mmol) in EtOH–THF (2 : 1, 3 ml) and stirred overnight at
pyrrolidine-1-carboxylate) (270 mg, quant) as a colourless rt. The reaction was quenched with 1 M HCl, diluted with
foam, which was used without further purification, [α]D −13.5 EtOAc and the organic phase separated. The aqueous phase
(c 1.0 in CHCl3); δH (400 MHz, CDCl3) 1.81 (1 H, m), 1.89–1.96 was extracted twice with CHCl3 and the combined organic
(4 H, m), 2.63 (1 H, br d), 2.82 (3 H, s), 3.06–3.18 (2 H, m), 3.44 phases dried (MgSO4) and concentrated under reduced
(2 H, t, J = 6.4 Hz), 3.50–3.55 (3 H, m), 3.68 (1 H, dt, J = 1.3, pressure to give the crude acid 20 (100 mg). A mixture of the
12.5 Hz), 3.71 (3 H, s), 4.63 (1 H, dd, J = 2.0, 10.1 Hz), 4.73 crude acid and 10% Pd/C (50 mg, 50% H2O) in MeOH–H2O
(1 H, dd, J = 6.7, 13.4 Hz), 5.02 (1 H, tt, J = 1.4, 4.7 Hz), 7.07 (5 : 1, 12 ml) was stirred under a H2 atmosphere for 2 h at rt.
(2 H, d, J = 8.6), 7.16 (2 H, d, J = 8.6 Hz), 7.39 (1 H, d, J = The mixture was filtered through a layer of Celite, concentrated
7.5 Hz), 7.56 (2 H, t, J = 7.6 Hz), 7.64–7.68 (1 H, m), 7.81–7.84 under reduced pressure and the residue purified using a C18
(2 H, m); δC (100 MHz, CDCl3) 25.1, 25.9, 35.5, 37.4, 38.9, 46.5, reversed phase cartridge (100% H2O to 50% MeOH–H2O). The
46.5, 52.5, 54.0, 55.4, 61.0, 78.0, 122.1 (2 C), 128.0 (2 C), 129.8 purified material was lyophilised to give the amine 21
(2 C), 130.1 (2 C), 132.8, 134.1, 135.4, 150.7, 153.1, 169.8, (81.3 mg, 87% over 2 steps) as a colourless fluffy powder, [α]D
171.3; ν/cm−1 3409, 2954, 2880, 1715, 1678, 1511; HRMS (ESI+) −16.0 (c 0.49 in MeOH); δH (400 MHz, D2O) 1.81 (4 H, m), 1.91
m/z 646.1497 (C27H33NaN3O10S2 [M + Na]+ requires 646.1505).
(4 H, m), 2.38 (1 H, m), 2.98–3.09 (4 H, m), 3.21–3.30 (3 H, m),
The above mesylate (97 mg, 0.16 mmol) was taken up in 3.30 (2 H, m), 3.45 (2 H, t, J = 6.8 Hz), 3.59–3.66 (12 H, m), 3.89
DMSO (1.5 ml) and treated with NaN3 (30.4 mg, 0.47 mmol) (1 H, d, J = 12.9 Hz), 4.04 (1 H, dd, J = 4.8, 12.9 Hz), 4.41 (1 H,
and the mixture stirred overnight at 80 °C. The reaction was t, J = 8.2 Hz), 4.53 (1 H, dd, J = 5.3, 7.4 Hz), 5.00 (1 H, br s),
diluted with EtOAc and washed with H2O, brine, dried 6.99 (2 H, d, J = 8.5 Hz), 7.306–7.356 (4 H, m), 7.43–7.50 (3 H,
(MgSO4) and concentrated to give azide 18 (82 mg, 92%). Spec- m), 7.99 (1 H, s); δC (100 MHz, D2O with acetone) 25.2, 25.8,
troscopic data were consistent with those reported above.
27.2, 29.1, 35.5, 36.9, 37.2, 38.3, 47.1, 47.1, 49.5, 55.1, 56.7,
4-((R)-3-Methoxy-3-oxo-2-((2S,4R)-4-(4-((3-oxo-1-phenyl-2,8,11,14- 60.4, 61.7, 68.9, 69.3, 70.1, 70.2, 70.3, 122.6 (2 C), 125.8, 127.5
tetraoxa-4-azaheptadecan-17-yl)carbamoyl)-1H-1,2,3-triazol-1-yl)-1- (2 C), 130.2 (2 C), 131.2 (2 C), 134.5, 135.1, 135.6, 142.9, 150.4,
(phenylsulfonyl)pyrrolidine-2-carboxamido)propyl)phenyl pyrroli- 155.6, 161.9, 173.2, 175.8; ν/cm−1 3382, 3064, 2950, 2878, 1706,
dine-1-carboxylate (19). Sodium ascorbate (4.4 mg, 22.2 μmol), 1658, 1511; HRMS (ESI+) m/z 829.3550 (C38H52N8O11S [M + H]+
CuSO4 (224 μl, 2.24 μmol, 0.01 M in H2O) and TBTA (281 μl, requires 829.3549).
2.81 μmol, 0.01 M in THF) were added sequentially to a
5(6)-Carboxytetramethyl rhodamine labelled compound
mixture of the azide 18 (32 mg, 56.1 μmol) and the alkyne 15 (22). A mixture of the amine 21 (9.5 mg, 11.3 μmol) in 0.2 M
(25 mg, 61.8 μmol) in DMF (1 ml). The reaction was stirred at NaHCO3 (1 ml) was treated with 5(6)-carboxytetramethyl rho-
60 °C for 2 h and then diluted with EtOAc (20 ml). The organic damine N-succinimidyl ester (NHS-rhodamine, Thermo Scien-
phase was washed with sat. aq. NaHCO3, brine, dried (MgSO4) tific) (8.9 mg, 16.9 μmol) and the mixture overnight at rt. The
and concentrated under reduced pressure. The residue was reaction was quenched with acetic acid, concentrated and the
purified by flash chromatography (21 : 2 : 1 : 1 EtOAc–acetone– residue purified by reversed phase chromatography (50%
MeOH–H2O) to give the triazole product 19 (54 mg, 99%) as a MeOH–H2O to 100% MeOH) to give the rhodamine labelled
colourless oil, [α]D +10.7 (c 1.0 in CHCl3); δH (400 MHz, CDCl3) compound 22 (5.3 mg, 38%) as a purple powder after lyophilisa-
1.72–1.94 (8 H, m), 2.14 (1 H, dt, J = 12.8, 13.9 Hz), 2.54 (1 H, tion. Compound 22 was isolated as a 5 : 1 mixture of regio-
ddd, J = 2.3, 6.5, 12.9 Hz), 2.92 (1 H, dd, J = 10.0, 13.9 Hz), 3.29 isomers; δH (400 MHz, d4-methanol) major isomer: 1.80–1.99
(2 H, dd, J = 6.0, 12.3 Hz), 3.38 (1 H, dd, J = 4.8 Hz, 13.9 Hz), (9 H, m), 2.37 (1 H, dt, J = 6.6, 13.5 Hz), 2.70 (1 H, m), 3.08
3.41–3.63 (19 H, m), 3.80 (3 H, s), 3.83 (1 H, m), 4.29 (1 H, dd, (1 H, dd, J = 7.5, 13.9 Hz), 3.22–3.26 (1 H, m), 3.26 (6 H, s),
J = 2.0, 8.7 Hz), 4.65 (1 H, m), 4.94 (1 H, dt, J = 4.9, 9.8 Hz), 3.27 (6 H, s), 3.35 (2H, t, J = 6.3 Hz), 3.43 (2H, t, J = 6.3 Hz),
5.06 (2 H, br s), 5.44 (1 H, br t, J = 5.7 Hz), 7.04 (2 H, d, J = 3.48–3.66 (17 H, m), 3.74 (1 H, dd, J = 3.7, 12.0 Hz), 3.91 (1 H,
8.5 Hz), 7.23 (2 H, d, J = 8.5 Hz), 7.28–7.33 (5 H, m), 7.38–7.43 dd, J = 6.0, 12.0 Hz), 4.45 (1 H, t, J = 7.1 Hz), 4.61 (1 H, t, J =
(2 H, m), 7.52 (2 H, t, J = 7.7 Hz), 7.62 (1 H, t, J = 7.5 Hz), 7.79 5.6 Hz), 4.94 (1 H, m), 6.86 (2 H, br s), 6.95–6.99 (4 H, m), 7.16
(2 H, d, J = 7.3 Hz), 8.18 (1 H, s); δC (100 MHz, CDCl3) 25.1, (2 H, d, J = 9.5 Hz), 7.28 (2 H, d, J = 8.1 Hz), 7.35–7.42 (3 H, m),
25.9, 29.4, 29.5, 33.7, 37.2, 37.9, 39.4, 46.4, 46.6, 52.7, 53.2, 7.51 (1 H, t, J = 7.3 Hz), 7.60–7.63 (2 H, m), 8.06–8.08 (2 H, m),
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8.56 (1 H, s); δC (100 MHz, d4-methanol) major isomer: 25.9, 131.6 (2 C), 132.3, 133.8, 133.9, 134.4, 135.26, 135.34, 138.3,
26.7, 30.4, 36.6, 38.0, 38.1, 38.9, 40.9 (4 C), 47.47, 47.53, 55.9, 144.1, 144.8, 146.9, 151.7, 155.2, 157.16, 157.17, 157.2, 157.8,
60.3, 62.3, 70.3, 70.5, 71.35, 71.4, 71.6 (2 C), 97.3 (2 C), 114.8 (2 159.4, 173.1, 173.8; ν/cm−1 3088–3418, 2977, 2876, 1711, 1649,
C), 115.2 (2 C), 122.7 (4 C), 126.3, 128.6 (2 C), 130.0, 130.1, 1588; HRMS (ESI+) m/z 1174.3447 (C55H61N9NaO13S3 [M + Na]+
130.5 (2 C), 131.1, 131.7 (4 C), 132.5 (2 C), 134.4 (2 C), 136.0, requires 1174.3443). For in vitro and in vivo testing, the free
137.2, 137.3, 138.1, 144.0, 151.6, 155.1, 158.7 (2 C), 159.0 (2 C), acid of 25 (11.7 mg, 9.97 μmol) was dissolved in 0.01 M NaOH
161.6, 162.0, 168.7, 172.6; HRMS (ESI+) m/z 1241.4970 (997 μl, 9.97 μmol) and the dark purple solution filtered
(C63H72N10O15SH [M + H]+ requires 1241.4972).
through a 0.45 μm syringe filter unit. The product was lyophi-
(S)-2-((2S,4R)-4-Azido-1-(phenylsulfonyl)pyrrolidine-2-carbox- lised to give the sodium salt of 25 (11.6 mg, 99%) as a fluffy
amido)-3-(4-((pyrrolidine-1-carbonyl)oxy)phenyl)propanoic acid purple powder.
(23). The methyl ester 18 (420 mg, 0.737 mmol) in EtOH
(10 ml) was treated with 0.2 M NaOH (4.05 ml, 0.811 mmol)
Flow cytometry
and stirred at rt for 1 h. The mixture was concentrated under Flow cytometric analysis was performed using an LSR II (BD
reduced pressure to remove EtOH and the aqueous phase acid- Biosciences) equipped as previously described.18 Both carboxy-
ified with 10% HCl. The aqueous phase was extracted with tetramethylrhodamine (compound 22) and Lissamine Rhoda-
CHCl3 (4 × 10 ml) and the combined organic phases were mine B (R-BC154) were excited at 561 nm (yellow-green laser)
washed with brine, dried (MgSO4) and concentrated under and detected at 585
20 nm. Fluorescence activated cell
reduced pressure. The crude material was purified by flash sorting of haemopoietic progenitor cells was performed using
chromatography (10% MeOH–CH2Cl2 with 0.5% AcOH) to give a Cytopeia Influx (BD Biosciences) cell sorter equipped as pre-
acid 23 (384 mg, 94%) as a pale yellow foam, [α]D −0.7 (c 1.00 viously described.18
in CHCl3); δH (400 MHz, CDCl3) 1.67–1.73 (1 H, m), 1.89–1.96
(5 H, m), 3.10 (1 H, dd, J = 8.0, 13.8 Hz), 3.21 (1 H, dd, J = 4.0,
Cell lines
11.5 Hz), 3.38 (1 H, dd, J = 5.3, 14.0 Hz), 3.44–3.55 (5 H, m), Stable LN18 cells (ATCC number: CRL-2610) over-expressing
3.81 (1 H, m), 4.11 (1 H, t, J = 6.5 Hz), 4.89 (1 H, m), 7.05, 7.22 integrin α4β1 (LN18 α4β1) or α9β1 (LN18 α9β1 were generated by
(4 H, 2 × d, J = 8.0 Hz), 7.41 (1 H, d, J = 6.8 Hz), 7.53–7.64 (3 H, retroviral transduction using the pMSCV-hITGA4-IRES-hITGB1
m), 7.85 (2 H, d, J = 7.5 Hz); δC (100 MHz, CDCl3) 25.0, 25.8, and pMSCV-hITGA9-IRES-hITGB1 vectors as previously
36.1, 36.8, 46.5, 46.6, 53.2, 53.9, 58.9, 61.2, 122.0 (2 C), 128.0 described.18 Transduced cells were selected by two rounds of
(2 C), 129.4 (2 C), 130.5 (2 C), 133.6, 133.7, 136.0, 150.5, 153.6, FACS using 2.5 μg ml−1 PE-Cy5-conjugated mouse-anti-human
170.9, 173.7; ν/cm−1 3329, 2977, 2881, 2105, 1706, 1672; HRMS α4 antibody (BD Bioscience) or 20 μg ml−1 of mouse-anti-
(ESI+) m/z 557.1817 (C25H29N7O6S [M + H]+ requires 557.1813).
human α9β1 antibody (Millipore) in PBS–2% FBS, followed by
R-BC154 (25). The azide 23 (12 mg, 22 μmol) and N-propy- 0.5 μg ml−1 of PE-conjugated goat-anti-mouse IgG (BD Bio-
nyl sulforhodamine B 24 (14 mg, 24 μmol) in DMF (2 ml) were science). Silencing of α4 expression in LN18 and LN18 α9β1
treated with CuSO4 (86 μl, 0.86 μmol, 0.01 M in H2O), sodium cells was performed as described above using pSM2c-shITGA4
ascorbate (430 μl, 4.3 μmol, 0.01 M in H2O) and tris[(1-benzyl- (Open Biosystems). α4-silenced LN18 cells (control cell line;
1H-1,2,3-triazol-4-yl)methyl]amine (TBTA) (108 μl, 1.08 μmol, LN18 SiA4) and LN18 α9β1 (LN18 α9β1 SiA4) were negatively
0.01 M in DMF). The mixture was stirred at 60 °C for 2 h at selected for α4 expression using FACS.
which point TLC indicated formation of a new fluorescent
product. The mixture was concentrated under reduced
Immunohistochemistry
pressure and the residue partly purified by flash chromato-
Antibody staining. LN18 SiA4 (control cell line), LN18 α4β1,
graphy (40 : 10 : 1 CHCl3–MeOH–H2O with 0.5% AcOH). This and LN18 α9β1 cells were stained with 2.5 μg ml−1 of mouse-
material was further purified by HPLC (50%–98% MeCN–H2O anti-human α4 antibody (BD Bioscience), 4 μg ml−1 of mouse-
(0.1% TFA) gradient over 15 minutes; Rt = 14.9 min) to give anti-human α9β1 antibody (Millipore) or 4 μg ml−1 of mouse
pure 25 (10.6 mg, 43%) as a purple glass, δH (400 MHz, d4- isotype control (BD Bioscience) in PBS–2% FBS for one hour,
methanol) 1.27–1.31 (12 H, dt, J = 7.0, 3.5 Hz), 1.91–1.98 (4 H, followed by 5 μg ml−1 of Alexa Fluor 594 conjugated goat-anti-
m), 2.29–2.35 (1 H, m), 2.71–2.78 (1 H, m), 3.08 (1 H, dd, J = mouse IgG1 for 1 h and then washed with PBS–2% FBS three
7.5, 13.8 Hz), 3.22 (1 H, dd, J = 5.3, 13.8 Hz), 3.41 (2 H, t, J = times.
6.5 Hz), 3.54 (2 H, t, J = 6.5 Hz), 3.63–3.70 (8 H, m), 3.85 (1 H,
R-BC154 (25) staining. Cultured LN18 SiA4 (control cell
dd, J = 3.5, 12.0 Hz), 3.97 (1 H, dd, J = 5.6, 11.6 Hz), 4.21 (2 H, line), LN18 α4β1, and LN18 α9β1 cells were treated with
d, J = 1.4 Hz), 4.41 (1 H, t, J = 7.3 Hz), 4.72 (1 H, dd, J = 5.4, R-BC154 (50 nM) in TBS–2% FBS (50 mM TrisHCl, 150 mM
7.5 Hz), 5.08 (1 H, m), 6.91 (2 H, t, J = 2.2 Hz), 6.98–7.04 (4 H, NaCl, 2 mM glucose, 10 mM Hepes, pH 7.4) containing 1 mM
m), 7.11 (2 H, t, J = 9.0 Hz), 7.30 (2 H, d, J = 8.6 Hz), 7.40 (1 H, CaCl2–MgCl2 or 1 mM MnCl2) and incubated for 20 min at
d, J = 8.0 Hz), 7.44 (2 H, t, J = 7.5 Hz), 7.58–7.68 (4 H, m), 8.00 37 °C and then washed with TBS–2% FBS three times. The
(1 H, dd, J = 1.9, 8.0 Hz), 8.37 (1 H, d, J = 1.8 Hz); δC (100 MHz, stained cells were fixed with 4% paraformaldehyde in PBS for
d4-methanol) 12.9 (4 C), 25.9, 26.7, 37.1, 37.6, 39.0, 46.8 (4 C), 5 min, washed with water three times and then stained with
47.5, 47.6, 54.8, 55.7, 60.3, 62.1, 97.0 (2 C), 115.0 (2 C), 115.26, 2.5 μg ml−1 of DAPI. The cells were mounted in Vectorshield,
115.29, 122.9 (2 C), 123.9, 127.5, 128.6 (2 C), 129.3, 130.5 (2 C), washed with water, coverslipped and stored at 4 °C overnight
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before images were taken under fluorescent microscope In vivo bone marrow binding assay
(Olympus BX51).
R-BC154 (25) in PBS (10 mg kg−1) was injected intravenously
into C57 mice. After 5 min, bone marrow cells were isolated as
previously described.41,42 Briefly, one femur, tibia and iliac
crest were excised and cleaned of muscle. After removing the
epi- and metaphyseal regions, bones were flushed with PBS–
2% FBS to obtain whole bone marrow, which were washed
with PBS–2% FBS and then immunolabelled for flow cytome-
try. For analysis of R-BC154 (25) binding, the following anti-
body combinations were chosen to minimise emission spectra
overlap. For staining progenitor cells (LSK; Lineage−Sca-1+c-
kit+) and HSC (LSKSLAM; LSKCD150+CD48−), cells were
labelled with a lineage cocktail (CD3, Ter-119, Gr-1, Mac-1,
B220; all antibodies APC-Cy7 conjugated), anti-Sca-1-PB, anti-
c-kit-AF647, anti-CD48-FITC and anti-CD150-BV650.
Saturation binding experiments
Cultured α4β1, α9β1 and control LN18 cells (0.5 × 106 cells) were
treated with 100 μl of either compound 22 or 25 (R-BC154) at
0, 1, 3, 10, 30 and 100 nM in TBS–2% FBS (containing either
no cations, 1 mM CaCl2–MgCl2 or 1 mM MnCl2). The cells
were incubated at 37 °C for 60 min, washed once with TBS–2%
FBS, dry pelleted and resuspended in the relevant binding
buffer for flow cytometric analysis. Mean channel fluorescence
was plotted against concentration and fitted to a one-site sat-
uration ligand binding curve using GraphPad Prism 6. The dis-
sociation constant, Kd was determined from the curves.
Off-rate kinetics measurements
For assessing R-BC154 binding on sorted populations of
progenitor cells (LSK cells), whole bone marrow was harvested
from untreated and treated (R-BC154; 10 mg kg−1) mice
(3 mice per group). Lineage positive cells were immuno-
labelled using a lineage cocktail (B220, Gr-1, Mac-1 and Ter-
119) and then removed by immunomagnetic selection with
sheep anti-rat conjugated Dynabeads (Invitrogen) according to
the manufacturer’s instructions. The resultant lineage depleted
cells were stained with anti-Sca-1-PB and anti-c-kit-FITC.
Immunolabelled cells were sorted on Sca-1+c-kit+ using a
Cytopeia Influx (BD Biosciences) cell sorter and imaged using
an Olympus BX51 microscope.
Eppendorf vials containing α4β1 or α9β1 LN18 cells (0.5 × 106
cells) were treated with 50 nM of R-BC154 (100 μl in TBS–2%
FBS containing either 1 mM CaCl2–MgCl2 or 1 mM MnCl2 at
37 °C until for 30 min, washed once with the relevant binding
buffer and dry pelleted. The cells were treated with 500 nM of
an unlabelled competing inhibitor (100 μl, in TBS–2% FBS
containing either 1 mM CaCl2–MgCl2 or 1 mM MnCl2) at 37 °C
for the times indicated (0, 2.5, 5, 15, 30, 45, 60 min). The cells
were diluted with cold TBS–2% FBS (containing the relevant
cations), pelleted by centrifugation, washed once and resus-
pended (∼200 μl) in binding buffer for flow cytometric analy-
sis. Mean channel fluorescence was plotted against time and
the data was fitted to either a one-phase or two-phase exponen-
tial decay function using GraphPad Prism 6. The off-rate, koff
was extrapolated from the curves.
Acknowledgements
The authors thank Dani Cardozo for assistance with animal
experimentation, Andrea Reitsma and Chad Heazlewood for
technical assistance, Stuart Littler for HPLC, Jo Cosgriff, Carl
Braybrook and Michael Leeming for mass spectrometry and
Jack Ryan for helpful discussions.
On-rate kinetics measurements
Eppendorf vials containing α4β1 or α9β1 LN18 cells (0.5 × 106
cells) in 50 μl TBS–2% FBS containing either 1 mM CaCl2–
MgCl2 or 1 mM MnCl2 were pre-activated in a heating block
for 20 min at 37 °C. 100 nM R-BC154 (50 μl – final concen-
tration = 50 nM) in the relevant TBS–2% FBS (with relevant
cations) was added to each tube and after 0, 0.5, 1, 2, 3, 5, 10,
15 and 20 min incubation at 37 °C, the tubes were quenched
by the addition of 3 ml of TBS–2% FBS (with relevant cations).
The cells were washed once TBS–2% FBS (with relevant
cations), pelleted by centrifugation and resuspended (200 μl)
in the relevant binding buffer for flow cytometric analysis.
Mean channel fluorescence was plotted against time and the
data was fitted to either a one-phase or two phase association
function using GraphPad Prism 6. The observed on-rate, kobs
was extrapolated from the curves and kon was calculated using
(kobs − koff)/[R-BC154 = 50 nM].
References
1 R. O. Hynes, Cell, 1992, 69, 11–25.
2 E. A. Clark and J. S. Brugge, Science, 1995, 268, 233–239.
3 D. Cox, M. Brennan and N. Moran, Nat. Rev. Drug. Discov-
ery, 2010, 9, 804–820.
4 J. D. Humphries, A. Byron and M. J. Humphries, J. Cell Sci.,
2006, 119, 3901–3903.
5 K. J. Bayless, G. A. Meininger, J. M. Scholtz and G. E. Davis,
J. Cell Sci., 1998, 111, 1165–1174.
6 R. R. Lobb and M. E. Hemler, J. Clin. Invest., 1994, 94,
1722–1728.
7 R. R. Lobb and S. P. Adams, Expert Opin. Invest. Drugs,
1999, 8, 935–945.
8 M. Millard, S. Odde and N. Neamati, Theranostics, 2011, 1,
154–188.
9 Y. Taooka, J. Chen, T. Yednock and D. Sheppard, J. Cell
Biol., 1999, 145, 413–420.
Mice
C57Bl/6 mice were bred at Monash Animal Services (Monash
University, Clayton, Australia). Mice were 6–8 weeks old and
sex-matched for experiments. All experiments were approved
by Monash Animal Research Platform ethics committee
(MARP/2012/128).
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Paper
Organic & Biomolecular Chemistry
10 Y. Yokosaki, N. Matsuura, T. Sasaki, I. Murakami, 25 R. B. Pepinsky, W. C. Lee, M. Cornebise, A. Gill,
H. Schneider, S. Higashiyama, Y. Saitoh, M. Yamakido,
Y. Taooka and D. Sheppard, J. Biol. Chem., 1999, 274,
36328–36334.
K. Wortham, L. L. Chen, D. R. Leone, K. Giza,
B. M. Dolinski, S. Perper, C. Nickerson-Nutter, D. Lepage,
A. Chakraborty, E. T. Whalley, R. C. Petter, S. P. Adams,
R. R. Lobb and D. M. Scott, J. Pharmacol. Exp. Ther., 2005,
312, 742–750.
11 H. Takahashi, T. Isobe, S. Horibe, J. Takagi, Y. Yokosaki,
D. Sheppard and Y. Saito, J. Biol. Chem., 2000, 275, 23589–
23595.
12 R. B. Pepinsky, R. A. Mumford, L. L. Chen, D. Leone,
S. E. Amo, G. Van Riper, A. Whitty, B. Dolinski, R. R. Lobb,
D. C. Dean, L. L. Chang, C. E. Raab, Q. Si, W. K. Hagmann
and R. B. Lingham, Biochemistry, 2002, 41, 7125–7141.
26 L. S. Lin, T. Lanza, J. P. Jewell, P. Liu, C. Jones,
G. R. Kieczykowski, K. Treonze, Q. Si, S. Manior, G. Koo,
X. C. Tong, J. Y. Wang, A. Schuelke, J. Pivnichny, R. Wang,
C. Raab, S. Vincent, P. Davies, M. MacCoss, R. A. Mumford
and W. K. Hagmann, J. Med. Chem., 2009, 52, 3449–3452.
13 N. E. Vlahakis, B. A. Young, A. Atakilit, A. E. Hawkridge, 27 J. A. Smith, G. P. Savage and O. E. Hutt, Aust. J. Chem.,
R. B. Issaka, N. Boudreau and D. Sheppard, J. Biol. Chem.,
2007, 282, 15187–15196.
14 E. M. Walsh, R. Kim, L. Del Valle, M. Weaver, J. Sheffield,
2011, 64, 1509–1514.
28 C. E. Jakobsche, P. J. McEnaney, A. X. Zhang and
D. A. Spiegel, ACS Chem. Biol., 2012, 7, 315–320.
P. Lazarovici and C. Marcinkiewicz, Neuro-Oncology, 2012, 29 T. S. Reger, J. Zunic, N. Stock, B. W. Wang, N. D. Smith,
14, 890–901.
B. Munoz, M. D. Green, M. F. Gardner, J. P. James,
W. C. Chen, K. Alves, Q. Si, K. M. Treonze, R. B. Lingham
and R. A. Mumford, Bioorg. Med. Chem. Lett., 2010, 20,
1173–1176.
15 P. Singh, C. Chen, S. Pal-Ghosh, M. A. Stepp, D. Sheppard
and L. Van De Water, J. Invest. Dermatol., 2009, 129, 217–
228.
16 I. Staniszewska, S. Zaveri, L. Del Valle, I. Oliva, 30 C. W. Tornoe, C. Christensen and M. Meldal, J. Org. Chem.,
V. L. Rothman, S. E. Croul, D. D. Roberts, D. F. Mosher, 2002, 67, 3057–3064.
G. P. Tuszynski and C. Marcinkiewicz, Circ. Res., 2007, 100, 31 V. V. Rostovtsev, L. G. Green, V. V. Fokin and
1308–1316.
K. B. Sharpless, Angew. Chem., Int. Ed., 2002, 41, 2596–
17 J. Teixido, M. E. Hemler, J. S. Greenberger and
P. Anklesaria, J. Clin. Invest., 1992, 90, 358–367.
18 J. Grassinger, D. N. Haylock, M. J. Storan, G. O. Haines,
B. Williams, G. A. Whitty, A. R. Vinson, C. L. Be, S. H. Li,
2599.
32 T. C. Liu, J. R. Nedrow-Byers, M. R. Hopkins and
C. E. Berkman, Bioorg. Med. Chem. Lett., 2011, 21, 7013–
7016.
E. S. Sorensen, P. P. L. Tam, D. T. Denhardt, D. Sheppard, 33 K. E. Beatty and D. A. Tirrel, Bioorg. Med. Chem. Lett., 2008,
P. F. Choong and S. K. Nilsson, Blood, 2009, 114, 49–59. 18, 5995–5999.
19 T. D. Schreiber, C. Steinl, M. Essl, H. Abele, K. Geiger, 34 A. H. Broderick, S. M. Azarin, M. E. Buck, S. P. Palecek and
C. A. Muller, W. K. Aicher and G. Klein, Haematol.-Hematol.
J., 2009, 94, 1493–1501.
20 P. Ramirez, M. P. Rettig, G. L. Uy, E. Deych, M. S. Holt,
D. M. Lynn, Biomacromolecules, 2011, 12, 1998–2007.
35 G. Delaittre, A. M. Greiner, T. Pauloehrl, M. Bastmeyer and
C. Barner-Kowollik, Soft Matter, 2012, 8, 7323–7347.
J. K. Ritchey and J. F. DiPersio, Blood, 2009, 114, 1340– 36 A. P. Mould, J. Cell Sci., 1996, 109, 2613–2618.
1343.
37 E. F. Plow, T. K. Haas, L. Zhang, J. Loftus and J. W. Smith,
21 S. Choi, G. Vilaire, C. Marcinkiewicz, J. D. Winkler,
J. Biol. Chem., 2000, 275, 21785–21788.
J. S. Bennett and W. F. DeGrado, J. Med. Chem., 2007, 50, 38 J. Grassinger, D. N. Haylock, B. Williams, G. H. Olsen and
5457–5462. S. K. Nilsson, Blood, 2010, 116, 3185–3196.
22 M. W. Miller, S. Basra, D. W. Kulp, P. C. Billings, S. Choi, 39 A. B. Pangborn, M. A. Giardello, R. H. Grubbs, R. K. Rosen
M. P. Beavers, O. J. T. McCartye, Z. Y. Zou, M. L. Kahn, and F. J. Timmers, Organometallics, 1996, 15, 1518–1520.
J. S. Bennett and W. F. DeGrado, Proc. Natl. Acad. Sci. U. S. 40 R. H. Andreatta, V. Nair and A. V. Roberston, Aust. J. Chem.,
A., 2009, 106, 719–724. 1967, 20, 2701–2713.
23 O. E. Hutt, S. Saubern and D. A. Winkler, Bioorg. Med. 41 D. N. Haylock, B. Williams, H. M. Johnston, M. C. P. Liu,
Chem., 2011, 19, 5903–5911.
K. E. Rutherford, G. A. Whitty, P. J. Simmons, I. Bertoncello
24 S. Venkatraman, J. Lim, M. Cramer, M. F. Gardner,
and S. K. Nilsson, Stem Cells, 2007, 25, 1062–1069.
J. James, K. Alves, R. B. Lingham, R. A. Mumford and 42 J. Grassinger, B. Williams, G. H. Olsen, D. N. Haylock and
B. Munoz, Bioorg. Med. Chem. Lett., 2005, 15, 4053–4056. S. K. Nilsson, Cytokine, 2012, 58, 218–225.
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