Synthesis and biological activity of a CXCR4-targeting bis(cyclam) lipid

Article abstract of DOI:10.1039/c8ob01439f

A bis(cyclam)-capped cholesterol lipid designed to bind C-X-C chemokine receptor type 4 (CXCR4) was synthesised in good overall yield from 4-methoxyphenol through a seven step synthetic route, which also provided a bis(cyclam) intermediate bearing an octa

Full text of DOI:10.1039/c8ob01439f

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Synthesis and biological activity of a CXCR4-targeting bis(cyclam)
lipid
Received 00th January 20xx,
Accepted 00th January 20xx
Anna D. Peters,a,b,† Catriona McCallion,a,b,c,† Andrew Booth,a,b Julie A. Adams,c Karen Rees-Unwin,d
Alain Pluen,e John Burthem,c,d,* Simon J. Webba,b,*
DOI: 10.1039/x0xx00000x
A bis(cyclam)-capped cholesterol lipid designed to bind C-X-C chemokine receptor type 4 (CXCR4) was synthesised in good
overall yield from 2-methoxyphenol through a seven step synthetic route, which also provided a bis(cyclam) intermediate
bearing an octaethyleneglycol-primary amine that can be easily derivatised. This bis(cyclam)-capped cholesterol lipid was
water soluble and self-assembled into micellar and non-micellar aggregates in water at concentrations above 8 μM. The
bioactivity of the bis(cyclam)-capped cholesterol lipid was assessed using primary chronic lymphocytic leukaemia (CLL)
cells, first with a competition binding assay then with a chemotaxis assay along a C-X-C motif chemokine ligand 12
(CXCL12) concentration gradient. At 20 μM, the bis(cyclam)-capped cholesterol lipid was as effective as the commercial
drug AMD3100 for preventing the migration of CLL cells, despite a lower affinity for CXCR4 than AMD3100.
www.rsc.org/
lymphocyte interactions with cells in the microenvironment,
Introduction
potentially
blocking
interactions
that
promote
chemoprotection and proliferation of the CLL cells.3,4,5
Furthermore, when used in conjunction with standard
therapies for other cancers it increased cancer cell death6,7,8,9
and forced breast cancer cells out of the bone marrow and
into the vasculature in vivo.10
The mammalian C-X-C chemokine receptor type 4 (CXCR4) is a
key cell-surface protein that can have a major role in cancer
and other illnesses. CXCR4 is targeted by bis(cyclam)
containing drugs, which disrupt interactions between the
receptor and its conjugate ligand, C-X-C motif chemokine
ligand 12 (CXCL12).1 The bis(cyclam) compound class came to
particular prominence in the 1990s due to the central role of
CXCR4 in HIV infection, as certain forms of the virus were
found to bind and enter T-cells through the receptors CXCR4
and CCR5.1 The current leader in the bis(cyclam) class of drugs,
AMD3100 (plerixafor, Figure 1), is used in the clinic in the
context of haematopoietic stem cell transplants. This usage
exploits the role of CXCR4 in the retention of stem cells in the
bone marrow (BM): AMD3100 disrupts the CXCR4-CXCL12 axis,
mobilising the stem cells from the BM and into the
vasculature, where the cells can be collected and used for
transplant.1,2
Research into the use of AMD3100 to treat chronic
lymphocytic leukaemia (CLL) has found that it can disrupt CLL
a.School of Chemistry, University of Manchester, Oxford Road, Manchester M13
9PL, United Kingdom.
b.Manchester Institute of Biotechnology, University of Manchester, 131 Princess St,
Manchester M1 7DN, United Kingdom. E-mail: S.Webb@manchester.ac.uk
c. Department of Haematology, Manchester Royal Infirmary, Manchester University
NHS Foundation Trust, Manchester M13 9WL, United Kingdom
d.Division of Cancer Sciences, University of Manchester, Manchester, United
Kingdom. E-mail: john.burthem@cmft.nhs.uk
Figure 1. Bis(cyclam) conjugates 1, 2 and AMD3100.
e. Division of Pharmacy and Optometry, School of Health Sciences, University of
Manchester, M13 9PL, United Kingdom.
Combination therapies that include AMD3100 have
reached clinical trial in a range of cancers, both leukaemic and
in solid tumours. It has been applied to enhance
† Contributed equally to the work.
Electronic Supplementary Information (ESI) available: Spectra for new compounds,
VT NMR spectra, HPLC traces, DLS histograms, AFM images and Western Blots].
See DOI: 10.1039/x0xx00000x
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immunotherapy and standard chemotherapeutics,11,12
Journal Name
2, a lipid analogue of AMD3100
In this work we present
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underscoring the importance of the interaction between (Figure 1). This compound features aDOshI:o10rt.10o39c/tCa8(eOtBh0y1l4e3n9eF
CXCR4 and cell chemotaxis, both with regards to metastasis glycol) tether attached to the central aromatic ring, designed
and retention within chemoprotective niches.
The high affinity, small size, and well-established synthetic end of this tether we have attached a lipid anchor, a
chemistry of the bis(cyclam) moiety make modification of this cholesteryl group, which may lead to the self-assembly of
to provide flexibility to the bis(cyclam) binding group. At the
2
motif attractive, particularly for targeting cancer cells where into nanoparticles that are taken up by cells or provide
the CXCR4 receptor is over-expressed. The high affinity of this additional interactions with the membrane of the target cell.
class of molecule for CXCR4 (Ki = 74 nM for AMD3100)13 occurs Several groups have found that adding a lipophilic region to a
because the bis(cyclam) motif binds tightly to two aspartate drug can improve targeting of the plasma membrane, leading
residues in CXCR4,14 specifically through interactions between to enhanced inhibition of HIV-1 infection, hepatitis B infection,
the cyclams and the Asp171 and Asp262 residues, interactions BACE1 activity and GPCR signalling.24 Similarly, it was hoped
that were uncovered through the introduction of point that cholesterol conjugation may reduce dimensionality and
mutations in the receptor.13,15 It has been found that these increase surface concentrations of
2, improving its affinity for
interactions are strengthened if transition metal ions are membrane receptors in comparison to non-lipidated
complexed within the cyclam groups, with Ki values decreased counterparts.25
approximately six-fold for zinc(II)-complexed AMD3100 when
The synthetic pathway towards 2 contains only four steps
incubated with CXCR4-expressing COS-7 cells.16 Several groups from commercially available advanced intermediates, or seven
have used bis(cyclam) motifs for targeted drug delivery steps from commonly available reagents. This synthetic
to,17,18,19 or imaging of,19,20 cancer cells. Oupický et al. made pathway will also provide amine-terminated bis(cyclam)
1,
bis(cyclam) polymers conjugated through the cyclams, and which could be easily conjugated to fluorophores and
found the resulting nanoparticles were specifically taken up by nanoparticles for targeting of CXCR4-overexpressing cells.
cells overexpressing CXCR4. These polymers were used to
deliver DNA as a therapeutic cargo; however, the affinity of
the bis(cyclam) polymer for CXCR4 (measured in terms of
Results and discussion
antagonism against CXCR4 redistribution, EC50 = 103 ng/mL)
was significantly lower than AMD3100 alone
Synthesis of 1 and 2
Compound was designed to maintain both high affinity for
1
(EC50 = 2 ng/mL).17,18 A modified polymer with pendant and
therefore less hindered cyclam groups was produced by the
authors and found to have increased affinity (EC50 = 21.3-56.8
ng/mL, depending upon formulation) in comparison to
previous polymeric forms.21 An analogue of AMD3100 for
targeted imaging was created by Poty et al. using a seven step
synthesis.20,22 This targeted PET radioligand had a 68Ga
complex appended to a bis(cyclam) core; the IC50 values for
the compounds after attachment to the chelating group varied
between 121-1485 nM, in comparison to 14 nM for AMD3100
(found in a competition assay against 125I-CXCL12).22
CXCR4 and allow conjugation to biomolecules, drug delivery
vehicles or imaging agents. Within the bis(cyclam) family of
drugs, it is thought that the interaction with the CXCR4
receptor occurs predominantly through the cyclams, although
changes to the spacer between the cyclam moieties can
change overall geometry and conformation, and therefore
alter affinity for the receptor.20,22,26 The structure of
1 includes
an octa(ethylene glycol) (OEG) tether terminated with a
primary amine, which is conjugated to the central aromatic
region. This allows the bis(cyclam) moieties to be unimpeded
while allowing simple conjugation to a wide range of different
cargo.
Although AMD3100 remains the only FDA-approved
CXCR4-antagonist in clinical use, a variety of other CXCR4-
antagonists have also been developed. AMD070 is another
small molecule CXCR4 antagonist, which is of particular
interest due to its oral bioavailability. It has reached Phase I/II
clinical trials for use in the treatment of HIV, where its oral
bioavailability is particularly desirable.23 Cyclic pentapeptides
have been extensively investigated since T140, a cyclic
pentapeptide derived from horseshoe crab, was shown to
have high affinity for CXCR4, albeit with low bioavailability.
From structure-activity relationship studies on T140, a library
of cyclic pentapeptide CXCR4 antagonists with high affinity to
the receptor has since been developed, but bioavailability
The initial synthetic route was designed to provide key
intermediate
hoped would provide >100 mg of
commercially available sulfonyl chloride 3a. The primary amine
of can then be easily conjugated to a lipid anchor. This
1
in only three steps (Scheme 1), which we
1
,
starting from
1
synthetic approach was inspired by established methods for
the synthesis of AMD3100, where triply protected cyclams are
conjugated to the central aromatic region through
bromomethyl “arms”.27
Commercially available advanced intermediate 3a was
reacted with Boc protected OEG amine
sulfonamide 5a in good yield (63 %). However analysis of the
product showed multiplets at 4.62 and 4.52 ppm in the 1H
NMR spectrum and several peaks in the mass spectrum, both
of which indicated partial displacement of the bromides by
chloride during the reaction (the H NMR spectrum shows ca.
50% exchange, with mass spectrometry showing peaks for the
bis(chloride) (m/z 693), mixed chloride/bromide (m/z 739) and
4 to give crude
generally remains low.23 Nevertheless, BL8040,
a T140
analogue that is orally bioavailable, is an inverse agonist of
CXCR4 that induces apoptosis of CXCR4-dependent cells in
vitro. It has reached Phase II clinical trials for use alongside
standard chemotherapeutics in acute myeloid leukaemia.23b,c
1
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1
bis(bromide) (m/z 783) products, see the ESI). Treating the temperature sharpened all the resonances, andVtiehweArtiHcleNOnMlinRe
DOI: 10.1039/C8OB01439F
6 at 345 K in CD3CN gave a sharp spectrum that
product mixture under Finkelstein conditions reinstated the spectrum of
bromides to give 5a, but the intermediate compound with a permitted assignment of the resonances (see the ESI). Despite
mixture of chloride and bromides was found to be sufficiently the successful purification of , Boc deprotection of this
6
reactive for the next synthetic step.
compound using standard conditions (e.g. TFA and TFAA in
CH2Cl2 at 0 °C), proved to be a challenging step. The removal of
all seven protecting groups was relatively slow, with NMR
spectroscopy showing that several Boc groups were still
present even after 4 h at room temperature. The difficulty in
removing all of the Boc groups was ascribed to the poor
solubility of the partially deprotected intermediates. However,
extending the reaction times and increasing the temperature
led to significantly increased degradation of the product;
analytical HPLC of the poorly soluble mixture showed several
additional peaks were formed after 1 h and 2 h in the TFA
mixture. Changing the deprotection conditions to HCl in
acetonitrile led to successful deprotection and compound
1
could be obtained after HPLC purification (as the acetate salt
1
due to acetic acid in the eluent) and characterised by H NMR
spectroscopy. However the yield for the key intermediate
was never higher than 10 % for these two steps from 5a
1
.
Scheme 2. Synthesis of 3-(2,5-bis(chloromethyl)-4-methoxyphenoxy)propane-1-
sulfonyl chloride 3b. i) 1,3-Propanesultone, NaOH, MeOH/1,4-dioxane; ii) SOCl2, DMF;
iii) Paraformaldehyde, CH3CO2H/HCl.
Scheme 1. Four step synthesis of 2. i) 1.2 mol eq. DIPEA, 30 min sonication, 30 min
stirring in dry DMF; ii) (Boc)3cyclam (2.4 eq.), excess NaHCO3, heated to reflux for 48 h
in dry acetonitrile; iii) Cyclam (10 eq.), excess NaHCO3, heated to reflux for 48 h in dry
acetonitrile; iv) TFA, TFAA, CH2Cl2, RT, 4 h; v) 8 mol. eq. HCl in dry acetonitrile, 45 min;
vi) Cholesteryl 3β-(N-hydroxysuccinimidyl)carbamate, excess NaHCO3 in dry
acetonitrile, 70 °C, overnight; vii) Cholesteryl 3β-(N-hydroxysuccinimidyl)carbamate,
stir overnight in PBS (pH 7.4).
To improve the yield of
1, two improvements in the
synthesis were developed. Firstly although the advanced
intermediate 3a can be obtained commercially, this material is
expensive and supply can be unreliable. We therefore decided
to synthesise on a larger scale the corresponding chloride
analogue 3b, which is available in three simple steps (Scheme
2), then compare the reactivity of this bis(chloride) to that of
the bis(bromide) 3a. Secondly, an alternative subsequent
route was devised, to give less protected intermediate
Literature precedent was followed to install two cyclams
onto the central aromatic linker, using commercially available
tri-Boc protected cyclam.27 This step gave a moderate yield of
the crude product, which required purification by HPLC. After
purification on a C8 reverse phase column, using 0.1% (v/v)
acetic acid in either water or acetonitrile as eluent, the
compound 7 by using less expensive unprotected cyclam.
In the first of these modifications, 3-(2’,5’-
bis(chloromethyl)-4’-methoxyphenoxy)propane-1-sulfonyl
chloride 3b was obtained in an overall yield of 59 % in three
protected bis(cyclam)
solubility profile of the product
in many common solvents. Furthermore, the resonances in the
6
was obtained in 10 % yield. The
6
was poor, with low solubility
simple and fast synthetic steps from 4-methoxyphenol
8,
following a modification of published procedures (Scheme 2).29
Several key adjustments were discovered to be indispensable
for this synthesis to be efficient. First, the activation of the
1
room temperature H NMR spectrum of
6 in CD3CN were all
broad, as often observed for Boc substituted cyclams;28 this
made it difficult to assess the purity of the product from this
step. However, VT NMR showed that increasing the
propanesulfonate
be carried out using a concentrated suspension of
9
to the propanesulfonyl chloride 10 has to
; even a
9
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small 20 % increase in the amount of solvent caused a 30 % dry acetonitrile, and a solution of cholesteryl-NHS carbamate
View Article Online
DOI: 10.1039/C8OB01439F
decrease in the yield of 10. Furthermore using diethyl ether for was added dropwise over 5 h. MALDI analysis of the fractions
the extraction of 10 turned out to be crucial to obtain a good obtained after HPLC purification of the crude reaction mixture
yield. Compound 3b initially precipitates out as a colloid, but showed that the desired adduct had formed, mixed with both
reheating the suspension at 55 °C gives a thicker precipitate unreacted
that can be easily filtered off. Reaction of 3b with Boc- conjugated. Unfortunately, this method gave a yield <20 %,
protected PEG amine gave sulfonamide 5b in good yield (75 due in part to the insolubility of the reactants in a shared
1 and the adduct with two cholesterol units
4
%, Scheme 1). Intermediate 3b was used without further solvent and also by loss on the HPLC. To increase the solubility
transformation of the reactive arms from chloride to bromide of both reagents, the reaction temperature was elevated to 70
as the chloromethyl groups were found to be sufficiently °C. However the yield once again decreases, which might be
reactive in subsequent steps.
In the second modification, the reaction of sulfonamides 5a Finally, by reducing the cholesteryl-NHS addition time to 0.5 h,
or 5b with unprotected cyclam to give was proposed. This not sonicating, reacting overnight at 75 °C, and adding
was hoped to be an economical alternative since only one Boc cholesteryl-NHS to the amine in dry acetonitrile in a two to
group must be removed from to give , allowing shorter one ratio, the final product was produced after HPLC
due to degradation of the product at high temperatures.
7
1
7
1
2
deprotection times with concomitantly less degradation and purification in a yield of 36 %, which was a significant
fewer side-products. A ten-fold molar excess of cyclam was improvement to the previous procedures.
reacted with 5a or 5b under the same conditions employed to
make 6. After 48 h heating at reflux, the excess cyclam was
Self-assembly of 2 in aqueous media
filtered off, then the residue was purified using HPLC (reverse Bis(cyclam)
2 was designed to be amphiphilic, with the
phase C8 column, mobile phase 0.1% (v/v) acetic acid in either lipophilic cholesterol region driving self-assembly into
water or acetonitrile) to afford the mono-Boc protected nanoparticles or enhancing interactions with the membrane of
product
7 in a reasonable yield (e.g. 45 % from 5b). This was a CLL cells. To assess the former, the self-assembly of 2 in water,
significant improvement in yield compared to the previous both dynamic light scattering (DLS) and NMR spectroscopy
synthetic route and much of the excess cyclam could be were employed.
recovered for future use. Unlike analogue
protecting groups, the H NMR spectrum of
6
with seven Boc
1
7 was as sharp at
room temperature as that of
6 at elevated temperature (75 °C)
and sharpens further as the temperature increase above room
temperature (see the ESI), which indicates the role of multiple
Boc groups in the slow conformational interconversion found
for 6. Compound 7 was then deprotected with hydrochloric
acid in acetonitrile (8 eq. of acid, 37 % HCl in dry acetonitrile)
at room temperature for 25 min. After purification on alumina
the deprotected amine
yield (90 %). The overall yield of
1
was achieved in almost quantitative
from 3-(2’,5’-
1
bis(chloromethyl)-4’-methoxyphenoxy)propane-1-sulfonyl
chloride 3b was 28 %, with 161 mg of key compound
obtained for further conjugation.
1
Following the successful synthesis of
1 in good yield, the
acylation of the primary amine at the end of the OEG over the
secondary amines in the cyclam groups was assessed. Initial
investigations into the synthesis of the cholesterol conjugate
showed that reaction with cholesterol chloroformate in DMF
with DIPEA led to over-acylation, with up to three cholesterol
units becoming conjugated to
the secondary amines of the cyclam ring; MALDI mass
spectrometry showed unreacted , with additional peaks
1, presumably by reaction with
1
showing increases of 415 mass units. To overcome this
problem, the reactivity of the acyl carbon was modulated by
synthesising the corresponding N-hydroxysuccinimide (NHS)
carbamate by reaction of the chloroformate with NHS in the
Figure 2. a) 1H NMR spectrum of 2 in D2O (13.9 mM), showing broad peaks due to self-
assembly. Acetone added as a non-aggregating reference compound (sharp peak at 2.2
ppm). b) Dynamic light scattering (DLS) measurements of the hydrodynamic radii (rh) of
2 dispersed in water. Each sample measured ten times and each preparation measured
in triplicate. The hydrodynamic radius distribution was bimodal, with a population with
rh = 0 to 10 nm region (blue) and rh = 10 to 40 nm (red). Each point indicates a mean
radius measured on an individual run, while the black bars indicate the median and
interquartile range across all measurements.
presence of sodium hydrogen carbonate. Although
cholesteryl-NHS carbamate displayed rather different solubility
profiles ( is most soluble in water), sonication was able to
dissolve both reagents in acetonitrile to some extent. A slight
excess of the amine was used (1.2 eq.), as a dilute solution in
1 and the
1
1
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1H NMR spectroscopy was first used to confirm the self-
ARTICLE
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DOI: 10.1039/C8OB01439F
assembly of molecule in aqueous media. Despite giving sharp
2
1
peaks in the H NMR spectrum when dissolved in CDCl3 (see
the ESI), when dispersed in D2O significant signal broadening
was observed in the spectrum (Figure 2 a). Such broadening is
consistent with the presence of self-assembled particles or
aggregates. An internal control, acetone, presented a sharp
peak in the same spectrum.
Further to this outcome, the size of the self-assembled
particles was investigated using DLS. Following the dispersal of
molecule 2 in pure water by sonication, at concentrations from
2.56 nM to 1 mM, the hydrodynamic radii (rh) of any particles
in suspension were measured. These concentrations were
selected as being within the range proposed for bioanalytical
studies for targeting of CXCR4 on cells by 2 in suspension.
The presence of aggregates was observed at
concentrations above 8 μM. This was the lowest concentration
for which particles could be reliably observed and suggests
that the critical aggregation concentration of
2 lies below this
concentration. From 8 μM to 1 mM of , the rh distribution of
2
the particles was found to be bimodal, with one population
with radii between 0.9-1.7 nm, and another population with
radii between 21.5-29.1 nm (Figure 2 b). Similar cholesteryl-
PEG conjugates have been shown to form micelles. Chen et al.
reported that micellar cholesterol-PEG2000-FITC had
a
hydrodynamic radius of 6.1 ± 1.2 nm,30 while Yu et al. found
that docetaxel-loaded cholesterol-PEG2000 micelles had an
average hydrodynamic radius of 6.9 ± 0.3 nm.31 Micelles
formed of
2 should be significantly smaller than those formed
of PEG2000 given the 6-fold smaller chain length. The
population of objects with rh of 0.9-1.7 nm would therefore be
consistent with micelles composed of 2, with the population of
larger objects perhaps due to further aggregation into
vesicular, tape-like or sheet-like structures. Imaging of
suspensions of
2 (100 μM) by atomic force microscopy (AFM)
revealed a large number of self-assembled particles with radii
typically in the range 6-40 nm, as well as some larger
aggregates (e.g. 160 nm radius) (see the ESI). Both micelles
and these larger structures would exhibit polyvalent displays
of bis(cyclam) on the surface, which may enhance binding to
cell surface CXCR4. The larger nanoscale objects may also be
subject to endocytosis by targeted cells.32
Biological activity of 2
Figure 3. a) Competition assay of either AMD3100,
labelled CXCR4 antibody (PE-α-CXCR4) for CXCR4 on primary CLL cells; AMD3100
(●, blue curve); , red curve); , pink curve). Dose response curves
assessed through flow cytometric analysis of a competition assay with PE-α-
CXCR4, where both and show equivalent efficacy, but reduced potency
1, or 2 against phycoerythrin-
Receptor affinity (competition assay): Lipid
2 should interact
with cells over-expressing CXCR4 through the bis(cyclam) motif
binding to the receptor, but may have secondary interactions
involving cholesterol and the cell membrane that may offer
advantages over AMD3100. The self-assembly of the
molecules in aqueous media could either increase or reduce
1
(
■
2 (▲
1
2
compared to AMD3100. b) Western Blot of phosphorylated ERK (p-ERK) and total
ERK. CXCL12 strongly induces phosphorylation of ERK compared with
unstimulated control. This is blocked by
2 (20 µM) or AMD3100 (20 µM); neither
the affinity of
multiple binding interactions or the bis(cyclam) may become
hidden upon self-assembly. The binding affinity of for CXCR4
expressed by primary CLL cells was therefore assessed using a
competition assay with phycoerythrin-labelled CXCR4 antibody
2, as polyvalent nanoparticles may either form
drug alone induces signalling. c) Cell migration in response to CXCL12. CLL cell
chemotaxis up a CXCL12 concentration gradient (200 ng/mL, after 1.5 h) through
a filter migration (Transwell) assay. Pre-incubation (20 μM, 3 h) with
2 or AMD
2
3100 reduced migration, comparable to nil stimulation. Statistical analysis
performed using an ordinary one-way ANOVA, with Tukey’s multiple
comparisons test; n = 3. ns = not significantly different. ** p ≤ 0.01.
compared both to unconjugated 1 and to AMD3100 alone.
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inhibits CXCL12-induced cell chemotaxis in cuVlietwurAert.icle OTnlhinee
DOI: 10.1039/C8OB01439F
A dose-dependent decrease in fluorescence with increasing chemotaxis of cells up a CXCL12 concentration gradient was
concentration of either or AMD3100 (used as a control) assessed using a filter migration (Transwell) assay; aliquots of
was observed (Figure 3a), which confirms specific binding of cell suspensions that had been treated with , AMD3100 or
both and to CXCR4 receptors on the cell surface. The IC50 of PBS respectively were transferred into the upper chamber of a
was found to be 0.139 ± 0.003 μM, whereas the IC50 of was Transwell plate and the lower chamber was filled with CXCL12
1, 2
2
1
2
1
2
found to be 0.43 ± 0.03 μM; both are higher than AMD3100, in media. After 90 mins incubation, the number of cells
where the IC50 was measured to be 8.8 ± 0.7 nM under these present in the lower chamber was counted using flow
conditions. These data show that modification of the cytometry, to give a measure of the cells’ ability to move
bis(cyclam) core causes the biggest drop in affinity (16-fold) directionally through a barrier in response to CXCL12 following
compared to AMD3100, and addition of the cholesterol lipid CXCR4 blockade (Figure 3c).
anchor then further diminished the affinity of the key In the presence of CXCL12 alone, high levels of migration
intermediate
interaction with CXCR4 takes place through the cyclams, pre-incubation with
alterations to the spacer region between the cyclams are also migration, and was comparable to AMD3100 inhibition or nil
known to reduce the molecule’s efficacy and potency.20,22,26 stimulation. These results suggest that
disrupts the
Nevertheless, both and retain good efficacy and this CXCR4/CXCL12 signalling pathway, which leads to reduced
1
for CXCR4 by ca. 4-fold. Although the through the Transwell membrane were observed. However
2
(20 μM, 3 h) strongly reduced cell
2
1
2
potency is comparable to other molecules of its class. chemotaxis. This activity could therefore reduce cells’
Calculating the percentage antibody inhibition data under migration into chemoprotective niches in CLL, as well as
saturating conditions allows comparison with two non- metastasis in other implicated cancers.
metallated bis(cyclam) compounds reported by Poty et al.20
Bis(cyclam)
binding at 20 μM and
1
showed (93.2 ± 1.4)% inhibition of antibody
showed (95.4 ± 6.8)% at 10 μM. Both
Conclusions
2
values are higher than the reported inhibition values (ca. 45%
at 20 μM) for the non-metallated bis(cyclams) (substituted on
the aryl spacer), which were measured against a 12G-5 PE-
conjugated antibody.20
We have developed a rapid seven step synthesis to give a
cholesterol functionalised bis(cyclam), with an overall yield of
17% from 4-methoxyphenol
8 (or ca. 30% from the
commercially available advanced intermediate 3a). This
pathway provided access to >50 mg of the target compound,
Effect on CXCL12-induced downstream signalling: After
as well as the important bis(cyclam) intermediate
1 that has a
confirming that
2 specifically binds CXCR4 in an antibody
primary amine for functionalisation. Conditions were
competition assay, its antagonistic effect was confirmed. The
signalling cascade following CXCR4/CXCL12 complexation
involves the RAS/RAF/MEK/ERK pathway, where ERK
phosphorylation leads to chemotaxis, transcription and gene
expression.33 A Western Blot was performed to assess ERK
phosphorylation following incubation with CXCL12, carried out
developed that allowed the unprotected bis(cyclam)
selectively modified on this primary amine.
1 to be
The cholesterol tail on bis(cyclam) lipid
2 was hoped to
interact with the membranes of targeted cells, combining this
hydrophobic interaction with binding of the bis(cyclam) to the
cell surface CXCR4 receptor, and/or to drive self-assembly of
into nanoparticles that can be taken up by cells. Although the
hydrophobic tail led to self-assembly in water, giving both
micelles and non-micellar structures around 30 nm in
2
in the presence or absence of either
confirmed that CXCL12 strongly induces ERK phosphorylation
in the CLL lymphocytes. Neither nor AMD3100 demonstrated
any agonist effect, but each prevented the CXCL12-induced
ERK phosphorylation (Figure 3b). In control assay,
stimulation of the B-cell receptor also strongly induced ERK
phosphorylation, but this was not inhibited by or by
AMD3100, confirming that the inhibitory effects of and
2 or AMD3100. The blot
2
diameter, adding the cholesterol tail to
improve affinity for CXCR4 in primary CLL cells. Although sub-
micromolar affinities of for CXCR4 were measured (0.4 μM),
this was 4-fold weaker than the affinity of for CXCR4 and 50-
1 to give 2 did not
a
2
2
1
2
fold weaker than the affinity of the archetypical bis(cyclam)
drug AMD3100 for CXCR4. Despite this lower affinity for
AMD3100 are specific to CXCR4-CXCL12 interactions (see the
ESI).
CXCR4, bioconjugate
20 μM) in reducing chemotaxis along CXCL12 gradients,
showing that may be effective in disrupting migration of CLL
2 was as effective as AMD3100 (both at
Effect on chemotaxis along CXCL12 gradients: In addition to
pro-survival signalling, the ligand CXCL12 is known to play a
significant role in the emigration into and retention of CLL cells
within protective niches such as the bone marrow and
lymphoid organs. Production of CXCL12 by cells that are
present in these environments (such as macrophages and
mesenchymal stromal cells) causes CLL cells to move along the
2
cells into protective niches, such as the bone marrow and
lymphoid organs. This disruption has been observed for
AMD3100 during in vivo experiments and in clinical practice,1-5
but further work in vivo would be needed to confirm this effect
for 2. In future, transition metal ion complexation to 1 and 2
could be employed to boost affinities for CXCR4; metalation
has been shown to significantly increase the affinities of other
bis(cyclam) drugs for CXCR4.
concentration gradient and into the tissues. To confirm that
2
could effectively disrupt CLL cell migration into and residence
within these protective niches, it is important to observe that
2
6 | J. Name., 2012, 00, 1-3
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Page 7 of 12
Organic & Biomolecular Chemistry
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Journal Name
Although use of
ARTICLE
2
alone provided no advantage over column chromatography was carried out using Sigma-Aldrich
View Article Online
DOI: 10.1039/C8OB01439F
AMD3100, the lipid anchor on
2 provides several new silica 40-63 μm particle size, 60 Å pore size.
applications. It will allow decoration of liposomes with Semi-preparative high pressure liquid chromatography (HPLC)
bis(cyclam) motifs, where the cholesterol anchor mediates purification was performed on HPLC Agilent 1100 series
insertion of
2 into the membrane of drug-loaded liposomes. equipped with a semi-preparative C8 column Agilent eclipse
Such bis(cyclam) coated liposomes would have dual XDB-C8, 5 μm, 9.4 mm × 250 mm. Analytical HPLC was
functionality, delivering the liposomal cargo to cancer cells performed using the Agilent 1260 Infinity LC with an Agilent
over-expressing CXCR4 while retaining the anti-migration ZORBAX Eclipse XDB reverse phase C8 column (150 × 4.6 mm)
properties of 2. Such bis(cyclam)-coated liposomes would have with a 5 μm particle size. Preparative HPLC was on a DuPont
the potential for the addition of further targeting functionality, Instruments Zorbax C8 column, (21.2 mm × 25 cm) with 5 μm
such as glycolipids,34 and/or a triggered release mechanism.35 particle size.
It should also be noted that Linning et al. found that the tether Flow cytometry was performed using a BD FACSCantoTM II
length between their BACE1-inhibiting molecule and the lipid flow cytometer with two lasers (488 nm solid state and 633 nm
anchor was fundamental in the activity of their molecule;24c HeNe) and analysed using BD FACSDivaTM and Flowing
therefore, future work may include the alteration of the length Software v 2.5.
of the OEG tether in 2. Key intermediate 1 should also find
many other applications as the primary amine permits Synthetic procedures
bioimaging groups, such as fluorophores, MRI contrast agents
or PET imaging agents, to be ligated to the CXCR4-targeting
bis(cyclam). Further investigations into biomedical applications
Sodium
Method adapted from the literature,29 13C NMR data reported
here for the first time. 4-Methoxyphenol (4.00 g, 32.22
mmol) was dissolved in MeOH/Dioxane (1:1 vol/vol, 100 mL).
NaOH powder (1.30 g, 32.5 mmol) was added in portions. After
the NaOH was fully dissolved, 1,2-oxathiolane 2,2-dioxide (3.94
g, 32.2 mmol) was added. The reaction mixture was stirred
overnight, and the resulting white precipitate was filtered and
3-(4’-methoxyphenoxy)propane-1-sulfonate
9:
8
of both 1 and 2 are ongoing.
Experimental
Materials and methods
dried under vacuum, giving
9 as a white powder (7.31 g, 27.3
3-[2’,5’-Bis(chloromethyl)-4-methoxyphenoxy]-1-
1
mmol, 85%). H NMR (400 MHz, D2O, 298 K) δ ppm 2.04 - 2.15
(quin, J = 7.7 Hz, 2 H), 2.99 (t, J = 7.7 Hz, 2 H), 3.72 (s, 3 H), 4.05
(t, J = 6.2 Hz, 2 H), 6.86 - 6.95 (m, 4 H); 13C NMR (100 MHz,
D2O, 298 K) δ ppm 24.2, 47.8, 55.9, 67.6, 115.1, 116.4, 152.3,
153.4; HRMS (ESI): m/z calcd. for C10H13Na1O5S + K+: 307.0002,
found: 307.0013.
propanesulfonyl chloride, N,N-di-iso-propylethylamine (99.5%
biotech.
grade
with
Sure/SealTM),
1,4,8,11-
tetraazacyclotetradecane (98%), 2,2,2-trifluoroacetic acid and
trifluoroacetic anhydride, cholesteryl chloroformate and all
deuterated solvents were obtained from Sigma-Aldrich® Co.
(UK). N,N-Dimethylformamide (99.8% extra dry with AcroSeal),
acetonitrile (99.9%, extra dry with AcroSeal), chloroform,
petroleum ether, dichloromethane, acetonitrile, methanol,
diethyl ether and ethyl acetate (all Analytical Reagent Grade)
were obtained from Fisher Scientific. O-(2-Aminoethyl)-O′-[2-
(Boc-amino)ethyl]hexaethylene glycol was obtained from
Polypure AS, Norway.
3-(4’-Methoxyphenoxy)propane-1-sulfonyl chloride 10: Using
a method adapted from the literature.29 Sodium 3-(4’-
methoxyphenoxy)propane-1-sulfonate
9 (1.00 g, 3.73 mmol)
were suspended in dry DMF (3 mL). Thionyl chloride (1.5 mL,
20.65 mmol) was added dropwise at 0 °C and the resulting
yellow suspension was allowed to stir at room temperature for
2 h. Afterwards the reaction mixture was added to ice water
(50 mL) to quench the excess thionyl chloride (caution: toxic
vapours). The aqueous layer was extracted with diethyl ether
(2 × 20 mL). The organic layer was washed with cold water (20
mL) and dried over MgSO4. Filtration and evaporation of the
solvent afforded (0.93 g, 3.52 mmol, 94 %) of a pale yellow
liquid, which becomes a yellow solid after cooling to 4 °C. The
sulfonyl chloride 10 was used without further purification.
3-(2’,5’-bis(chloromethyl)-4’-methoxyphenoxy)propane-1-
sulfonyl chloride 3b: Method adapted from the literature,29
13C NMR data reported here for the first time. 4-
Nuclear Magnetic Resonance (NMR) spectra were recorded on
a Bruker Ultrashield 400 or 500 MHz spectrometer in CDCl3,
1
D2O or CD3OD. H and 13C spectra were referenced relative to
the solvent residual peaks and chemical shifts (δ) reported in
ppm downfield of tetramethylsilane. Coupling constants (J) are
reported in Hertz and rounded to 0.1 Hz. Splitting patterns are
abbreviated as follows: singlet (s), doublet (d), triplet (t),
quintet (quin), multiplet (m), broad (br) or a combination of
these.
Mass spectra were recorded by staff at the University of
Manchester. High resolution mass spectra (HRMS) were
recorded on a Thermo Finnigan MAT95XP mass spectrometer
and analysed using the in-built Agilent MassHunter
Workstation software; all HRMS values are accurate to
± 0.001 Da. Thin layer chromatography (TLC) was performed
Methoxyphenol
8 (4.00 g, 32.22 mmol) was dissolved in MeOH
To a stirred suspension of 3-(4’-methoxyphenoxy)propane-1-
sulfonyl chloride, 10 (0.5 g, 1.9 mmol) and paraformaldehyde
(0.153 g, 5.1 mmol) in glacial acetic acid (3 mL) was added
dropwise a mixture of concentrated hydrochloric acid (1 mL) in
glacial acetic acid (2.5 mL) at room temperature. Then the
reaction mixture was stirred at 85 °C for 4 h resulting in a clear
using
commercially
available
pre-coated
plates
(Macherey Nagel alugram. Sil G/UV254); phosphomolybdic
acid dip and UV light were used to visualise the products. Flash
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Organic & Biomolecular Chemistry
Page 8 of 12
ARTICLE
Journal Name
yellow liquid. After the clear liquid cooled to room the gradient - A (%): 80-60 (0-5 mins), 60-50 (5-40 mins), 50-0
View Article Online
temperature, it was poured into water (10 mL) and a fine (40-50 mins), 0 (50-60 mins). This providDeOdI: 10.1039/C8OB01439F
6
(9 mg, 5.5 μmol,
1
white solid was formed. The suspension was heated at 55 °C yield 9.8 %). H NMR spectra were recorded at 293 K and 345
for another 10-15 min to form a thick precipitate. The white K, with the increase in temperature providing sharper
solid was collected by filtration and dried under vacuum for resonances.24 1H NMR (400 MHz, CD3CN, 293 K) δ ppm 1.27 -
24 h (0.51 g, 1.4 mmol, 74 %). 1H NMR (400 MHz, CDCl3, 298 K) 1.49 (m, 64 H), 1.70 (br s, 4 H), 1.85 (br s, 4 H), 2.15 - 2.25 (m,
δ ppm 2.53 - 2.62 (m, 2 H), 3.87 (s, 3 H), 3.98 (t, J = 8.5 Hz, 2 H), 4 H), 2.41 (br s, 4 H), 2.54 - 2.65 (m, 3 H), 2.95 - 3.42 (m, 31 H),
4.21 (t, J = 5.7 Hz, 2 H), 4.63 (s, 2H), 4.65 (s, 2 H), 6.91 (s, 1 H), 3.47 (t, J = 5.6 Hz, 3 H), 3.52 (br s, 2 H), 3.53 - 3.61 (m, 28 H)
6.94 (s, 1 H); 13C NMR (100 MHz, CDCl3, 298 K) δ ppm 24.9, 3.76 (s, 3 H), 4.04 (br t, J = 5.9 Hz, 2 H), 5.29 - 5.53 (m, 1 H),
1
41.0, 41.5, 56.3, 62.4, 65.6, 113.5, 114.5, 127.1, 127.3, 149.8, 5.54 - 5.76 (m, 1 H), 6.87 (br s, 1 H), 6.93 (br s, 1 H). H NMR
151.6; HRMS (ESI): m/z calcd for C12H15Cl3O4S - H+: 358.9687, (500 MHz, CD3CN, 345 K) δ ppm 1.28 (d, J = 1.9 Hz, 17 H), 1.31 -
found: 358.9684
1.32 (m, 1 H), 1.32 (s, 17 H), 1.33 (s, 9 H), 1.35 (s, 19 H), 1.59
tert-Butyl
(23-((3-(5-(bromomethyl)-2-(chloromethyl)-4- (br s, 5 H), 1.79 (br dd, J = 14.0, 7.1 Hz, 6 H), 1.73 - 1.81 (m,
methoxyphenoxy)propyl)sulfonamido)-3,6,9,12,15,18,21-
1 H), 2.07 - 2.15 (m, 2 H), 2.33 (br d, J = 6.0 Hz, 4 H), 2.53 (dt,
heptaoxatricosyl)carbamate 5a: 3-(2,5-Bis(bromomethyl)-4- J = 14.9, 5.6 Hz, 4 H), 3.09 (q, J = 5.9 Hz, 2 H), 3.12 - 3.16 (m,
methoxyphenoxy)-1-propanesulfonyl chloride (450 mg, 1.03 4 H), 3.16 - 3.28 (m, 25 H), 3.38 (t, J = 5.5 Hz, 2 H), 3.44 (s, 2 H),
mmols)
3a
,
O-(2-aminoethyl)-O′-[2-(Boc- 3.45 - 3.48 (m, 23 H), 3.49 (s, 4 H), 3.67 (s, 3 H), 3.95 (t,
amino)ethyl]hexaethylene glycol
4 (390 mg, 0.86 mmols), N,N- J = 6.1 Hz, 2 H), 5.10 (br s, 1 H), 5.29 (br s, 1 H), 6.79 (s, 1 H),
di-iso-propylethylamine (DIPEA) (143 μL, 1.03 mmols) were 6.84 (s, 1 H); HRMS (ESI): m/z calcd. for C83H152N10O25S + H+:
added to dry CHCl3 under inert conditions and the reaction 1722.0729, found: 1722.0668.
was sonicated for 30 mins and then stirred for a further 30 tert-Butyl
(23-((3-(2,5-bis(chloromethyl)-4-
mins. The CHCl3 and DIPEA were then removed under reduced methoxyphenoxy)propyl)sulfonamido)-3,6,9,12,15,18,21-
pressure using a rotary evaporator. The crude mixture was re- heptaoxatricosyl)carbamate 5b: 3-[2,5-Bis(chloromethyl)-4-
dissolved in chloroform and purified using flash column methoxyphenoxy]-1-propanesulfonyl chloride (371 mg, 1.03
chromatography (10% (v/v) methanol in chloroform on silica), mmols)
giving 5a as a yellow oil (476 mg, 0.54 mmol, 63%). Rf: 0.78 amino)ethyl]hexaethylene glycol
3b
,
O-(2-aminoethyl)-O′-[2-(Boc-
(390 mg, 0.86 mmols), N,N-
4
(10% (v/v) methanol in chloroform on silica, silica pre-flushed diisopropylethylamine (DIPEA) (143 μL, 1.03 mmols) were
with 100% chloroform). For analytical purposes, the chloro- added to dry CHCl3 under inert conditions and the reaction
substituted arms were exchanged for bromines under was sonicated for 30 mins and then stirred for a further
Finkelstein conditions: to a solution of 5a (35 mg, 42 μmols) in 30 mins. The CHCl3 and DIPEA were then removed under
dry dimethylformamide (DMF) (5 mL) was added an excess of reduced pressure using a rotary evaporator. The crude mixture
sodium bromide (26 mg, 252 μmols); the mixture was kept was re-dissolved in chloroform and purified using flash column
stirring overnight. The DMF was then removed under reduced chromatography (10% (v/v) methanol in chloroform on silica),
pressure and the mixture redissolved in in CDCl3. Excess giving 5b as a yellow oil (610 mg, 0.77 mmol, 75%). Rf: 0.79
1
sodium salts were removed via filtration for analysis. H NMR (10% (v/v) methanol in chloroform on silica, silica pre-flushed
1
(400 MHz, CDCl3, 298 K) δ ppm 1.37 (s, 9 H), 2.29 (m, 2H), 3.26 with 100% chloroform). H NMR (400 MHz, CDCl3, 298 K) δ
(m, 6 H), 3.47 (t, J = 5 Hz, 2 H), 3.52-3.60 (m, 26 H), 3.80 (s, 3 ppm 1.43 (s, 9 H) 2.30-2.37 (m, 2 H) 3.26 - 3.34 (m, 6 H) 3.52 (t,
H), 4.07 (t, J = 5.74 Hz, 2H), 4.42 (s, 2 H), 4.45 (s, 2 H), 5.06 (br J = 5.1 Hz 2 H) 3.59 - 3.64 (m, 26 H) 3.85 (br s, 3 H) 4.11 - 4.14
s, 1 H), 5.59 (br s, 1 H), 6.78 (s, 1 H), 6.80 (s, 1 H); 13C NMR (m, 2 H) 4.60 (s, 2 H), 4.61 (s, 2 H) 5.09 (br s, 1 H), 5.44 (br s, 1
(125 MHz, CDCl3, 298 K) δ ppm 24.1, 28.4, 28.8, 40.36, 43.1, H), 6.89 (br s, 1 H) 6.92 (br s, 1 H); 13C NMR (400 MHz, CDCl3,
49.6, 56.2, 66.6, 70.06, 70.1, 70.3, 70.4, 79.2, 113.7, 114.7, 298 K) δ ppm 24.0, 28.4, 29.7, 40.4, 41.2, 41.5, 42.8, 43.1, 49.2,
127.4, 127.6, 150.2, 151.5, 156.1 HRMS (ESI): m/z calcd for 49.5, 56.3, 66.9, 69.8, 69.8, 70.0, 70.0, 70.1, 70.1, 70.1, 70.2,
C33H58Br2N2O13S + Na+: 903.1919, found: 903.1919.
tert-Butyl (23-((3-(2,5-bis((1',4',8'-tri(tert-butoxycarbonyl)- 156.0; HRMS (ESI): m/z calcd for C33H57Cl2N2O13S : 791.2978,
70.2, 70.4, 70.5, 70.5, 113.4, 114.5, 126.9, 127.3 150.2, 151.4,
-
1',4',8',11'-tetraazacyclotetradecan-11'-yl)methyl)-4-
methoxyphenoxy)propyl)sulfonamido)-3,6,9,12,15,18,21-
found: 791.2964.
tert-Butyl
(23-((3-(2,5-bis((1',4',8',11'-
heptaoxatricosyl)carbamate 6: Molecule 5a (50 mg, 56 μmol) tetraazacyclotetradecan-11'-yl)methyl)-4-
and tri-tert-butyl 1,4,8,11-tetraazacyclotetradecane-1,4,8- methoxyphenoxy)propyl)sulfonamido)-3,6,9,12,15,18,21-
tricarboxylate (67 mg, 134.4 μmol) were added to dry heptaoxatricosyl)carbamate 7: Molecule 5b (569 mg, 0.648
acetonitrile with sodium carbonate (12 mg, 112 μmol) and left mmol) and 1,4,8,11-tetraazacyclotetradecane (cyclam, 779 mg,
stirring for 48 h under reflux. The reaction mixture was 3.89 mmol) were added to dry acetonitrile (30 mL) with
filtered using fluted filter paper to remove the sodium sodium hydrogen carbonate (436 mg, 5.18 mmol) and left
carbonate and the solvent was removed under reduced stirring for 48 h under reflux. The reaction mixture was filtered
pressure using a rotary evaporator. The crude mixture was using fluted filter paper to remove the sodium hydrogen
directly purified using HPLC on a C8 reverse phase column carbonate and excess unreacted cyclam, and the solvent was
using two solvent systems, solvent A (0.1% (v/v) acetic acid in removed under reduced pressure using a rotary evaporator.
water) and solvent B (0.1% (v/v) acetic acid in acetonitrile), in The crude mixture was purified using reverse phase C8
8 | J. Name., 2012, 00, 1-3
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Page 9 of 12
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
preparative HPLC with a mobile phase composed of water and The acetonitrile was removed under reduced pressure. The
View Article Online
DOI: 10.1039/C8OB01439F
acetonitrile with 0.1% (v/v) acetic acid in the gradient – A (%): crude product was washed with diethyl ether (20 mL) to
80-60 (0-5 mins), 60-50 (5-40 mins), 50-0 (40-50 mins), 0 (50- remove unreacted cholesteryl-NHS carbamate. The compound
60 mins) to afford a yellow oil (327 mg, 0.292 mmol, 45%). Rt: was then dissolved in chloroform (60 mL) and the remaining
11.46 mins. 1H NMR (500 MHz, CD3OD, 348 K) δ ppm 1.34 (s, 9 NaHCO3 and
1 were removed through filtration, affording a
H), 1.70-1.78 (m, 4 H), 1.86-1.97 (m, 4 H), 2.18 (quin, J = 6.6 Hz, pale, yellow oil, which was purified using reverse phase C8
2 H), 2.56-2.66 (m, 8 H), 2.74-2.84 (m, 8 H), 2.90-2.96 (m, 4 H), preparative HPLC with a mobile phase composed of water (A)
2.95-3.06 (m, 12 H), 3.11 (t, J = 5.7 Hz, 2 H), 3.16 (t, J = 5.2 Hz, and acetonitrile (B) with 0.1% (v/v) acetic acid in the gradient –
2 H), 3.18-3.20 (m, 2 H), 3.40 (t, J = 5.5 Hz, 2 H), 3.45-3.59 (m, A (%): 95-80 (0-5 mins), 80-50 (5-10 mins), 50-0 (10-50 mins), 0
26 H), 3.69 (s, 2 H), 3.78 (s, 5 H), 4.09 (t, J = 6.1 Hz, 2 H), 6.87 (50-70 mins), 0-95 (70-80 mins). The final product was a pale
(s, 2 H); 13C NMR (100 MHz, D2O, 298 K) δ ppm 21.9, 22.1, 22.9, yellow oil (66 mg, 0.046 mmol, 73%). 1H NMR (400 MHz, CDCl3,
24.6, 27.7, 39.6, 42.1, 44.1, 44.3, 44.7, 44.8, 46.4, 46.5, 46.7, 298 K) δ ppm 0.61 (s, 3H), 0.79 (dd, J = 6.6, 1.9 Hz, 6H), 0.84 (d,
46.8, 48.2, 48.5, 49.2, 50.5, 50.70, 50.8, 52.1, 53.7, 57.1, 67.09, J = 6.5 Hz, 3H), 0.87 – 0.97 (m, 5H), 0.98 – 1.34 (m, 6H), 1.34 –
69.4, 69.45, 69.5, 80.9, 117.3, 118.2, 123.6, 124.1, 151.0, 1.54 (m, 6H), 1.71 – 1.84 (m, 6H), 1.85 – 1.98 (m, 6H), 2.17 –
152.3, 158.2; HRMS (ESI): m/z calcd for C53H104N10O13S + 2H+: 2.25 (m, 2H), 2.25 – 2.33 (m, 2H), 2.53 – 2.64 (m, 8H), 2.78 –
561.3825, found: 561.3836.
23-((3-(2,5-bis((1',4',8',11'-tetraazacyclotetradecan-11'-
yl)methyl)-4-methoxyphenoxy)propyl)sulfonamido)-
2.86 (m, 8H), 2.86 – 2.94 (m, 8H), 2.94 – 3.01 (m, 4H), 3.18 (br
t, J = 7.5 Hz, 2H), 3.23 (t, J = 5.5 Hz, 2H), 3.29 (dt, J = 5.1, 5.2 Hz,
2H), 3.45 – 3.51 (m, 4H), 3.51 – 3.61 (m, 24H), 3.69 (s, 3H),
3,6,9,12,15,18,21-heptaoxatricosylamine 1: Concentrated 3.70 (s, 2H), 3.84 (s, 2H), 4.01 (t, J = 5.9 Hz, 2H), 4.37 – 4.47 (m,
hydrochloric acid (37 %, 8 eq.) was added to (0.175 mmol, 1H), 5.18 (t, 2H), 5.27 – 5.32 (m, 1H), 6.68 (s, 1H), 6.75 (s, 1H);
7
196 mg) dissolved in dry acetonitrile and the mixture was 13C NMR (126 MHz, CDCl3) δ ppm36 11.9, 18.7, 19.4, 21.0,
stirred under an inert atmosphere at room temperature for 25 22.6, 22.8, 23.8, 23.9, 24.3, 24.6, 25.9, 27.8, 28.0, 28.2, 28.2,
minutes. The acetonitrile and HCl were removed under 28.4, 29.7, 31.9, 31.9, 35.8, 36.2, 36.6, 37.0, 38.6, 39.5, 39.7,
reduced pressure and the residue was kept under vacuum for 40.7, 42.3, 42.8, 45.9, 46.0, 46.1, 47.8, 48.2, 48.5, 49.0, 49.1,
a further 24 h to remove any residual solvent or acid. Crude 49.2, 49.7, 49.8, 50.0, 50.7, 53.1, 53.4, 56.1, 56.3, 56.7, 61.8,
white solid was purified on neutral aluminium oxide (activated, 66.9, 70.1, 70.3, 70.3, 70.3, 70.4, 70.5, 70.5, 70.6, 70.6, 70.6,
neutral, Brockmann 1, 150 Mesh) (90% (v/v) acetonitrile in 70.7, 70.7, 72.5, 74.3, 115.3, 116.5, 122.5, 124.1, 125.0, 139.9,
Water); Rf: 0.36. This afforded
1 as a waxy transparent solid 151.1, 152.3, 156.2; HRMS (ESI): m/z calcd for C76H140N10O13S +
that became pale yellow upon dissolution in solvents (161 mg, K+: 1471.9954, found: 1471.9998.
0.157 mmol, 90 %). 1H NMR (500 MHz, D2O, 298 K) δ ppm 1.80 Self-assembly assays
– 1.69 (m, 4H), 2.03 (s, 4H), 2.18 (quin, J = 6.4 Hz, 2H), 2.75 –
NMR spectroscopy of
performed using a 400 MHz spectrometer at 298 K, with
concentration of 13.9 mM in D2O.
2
in D2O: 1H NMR spectroscopy was
at a
Dynamic light
2.61 (m, 8H), 2.88 – 2.78 (m, 8H), 3.24 – 2.98 (m, 22H), 3.29 (t,
J = 7.6 Hz, 2H), 3.55 (t, J = 5.2 Hz, 2H), 3.65 – 3.57 (m, 24H),
3.68 (t, J = 4.8 Hz, 2H), 3.80 (s, 3H), 3.84 (s, 2H), 4.15 (t, J = 6.3
Hz, 2H), 6.94 (s, 1H), 6.96 (s, 1H); 13C NMR (125 MHz, D2O, 298
K) δ ppm 22.9, 23.2, 23.2, 23.3, 24.7, 24.7, 39.3, 42.1, 44.9,
45.1, 45.2, 45.3, 45.3, 46.1. 46.8, 47.0, 47.1, 48.5, 49.1, 49.2,
49.4, 49.6, 49.7, 51.0, 51.2, 53.2, 54.0, 57.1, 57.1, 58.8, 67.3,
68.3,68.3,68.4, 69.4, 69.5, 69.6, 69.6, 69.6, 116.5, 118.1, 124.9,
125.7, 150.8, 152.4. HRMS (ESI): m/z calcd for C48H96N10O11S +
H+: 1021.70535, found: 1021.7019
2
scattering (DLS): Aggregate sizes of 2 were assessed in MilliQ®
water at concentrations between 2.56 nM-1 mM using a Wyatt
DynaPro Plate Reader, with a 633 nm laser and a scattering
angle of 173°. Ten scans were completed for each sample,
where each scan was 5 s and each sample was prepared in
triplicate. All measurements were performed at 25 °C. Atomic
Force Microscopy (AFM): Suspensions of
2 in MilliQ® water
were drop-cast onto freshly cleaved mica for 120 s, after which
any excess moisture was wicked away and the surface washed
with MilliQ® water. The substrates were allowed to dry
Cholester-3”-yl
tetraazacyclotetradecan-11'-yl)methyl)-4-
(23-((3-(2,5-bis((1',4',8',11'-
methoxyphenoxy)propyl)sulfonamido)-3,6,9,12,15,18,21-
heptaoxatricosyl)carbamate 2: Cholesteryl chloroformate
(1.00 g, 2.23 mmol), NHS (307 mg, 2.67 mmol) and
triethylamine (74 µL, 5.34 mmol) were dissolved in CH2Cl2 (200
mL) and stirred overnight at 0 °C. The reaction mixture was
washed with 1 M HCl and brine. The organic layer was dried
over MgSO4 and filtered. The solvent was removed from the
filtrate under reduced pressure (0.82 g, 83 %) to obtain the
cholesteryl-NHS carbamate as a white solid, which was used
overnight. The surfaces were imaged using
a Bruker
Multimode in ScanAsyst® mode, and Nanoscope® and
Gwyddion 2.51 were used for image analysis. The AFM tip was
a ScanAsyst-Air, with tip radius 2 nm.
Biological assays
Cell culture: Primary CLL lymphocytes were cultured in RPMI
1640 (Sigma Aldrich, UK) supplemented with 10% foetal calf
serum (FCS), 1% L-glutamine, 1% penicillin streptomycin
(Thermofisher, UK). Primary CLL lymphocytes were taken from
liquid nitrogen storage; these had been collected from CLL
patients with full Research Ethics Committee (REC) approval
for use in evaluation of new treatment strategies (REC
reference 10/H1017/73). All samples had been collected with
without further purification. To a suspension of molecule
1 (64
mg, 0.063 mmol) and NaHCO3 (42 mg, 0.50 mmol) in dry
acetonitrile (60 mL) was added the cholesteryl-NHS carbamate
(68 mg, 0.126 mmol) in dry acetonitrile (25 mL) dropwise over
0.5 h. The resulting suspension was stirred at 75 °C overnight.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
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ARTICLE
Journal Name
full consent for research use and are stored in pseudo- and the well underneath was filled with 600 μL complete
View Article Online
anonymised form with identity traced only through the media with or without CXCL12 (carrier-frDeOe,I:R10&.1D03S9y/Cst8eOmB0s1,4U39KF)
collecting medical institution (Manchester Royal Infirmary, at 200 ng/mL; the cells were incubated for a further 90 mins at
custodian Dr John Burthem). There are no direct or indirect 37 °C. The bottom well was then agitated and 400 μL of the
medical implications for donors. AMD3100,
1 and 2 were used media removed for cell counting. Samples were passed
without pre-complexation to zinc(II) or other metal ions. It through a flow cytometer at a consistent flow rate for 30 s to
should be noted, however, that cells were cultured in media give a cell number relative to the positive control (CXCL12 with
with 10 % FCS; FCS contains ca. 40 μM zinc (II),37 so no antagonist).
adventitious complexation of some zinc(II) may occur in situ.
Flow cytometry: The antibody competition assay was
developed by modification of a published procedure.38 Primary
CLL cells were cultured for 24 h at 2 × 106 cells/mL in a 96-well
plate (300 μL) before incubation with antagonist in the
concentration range 0.2 nM – 20 μM for 3 h or PBS (-ve
control). An aliquot of each sample (100 μL) was harvested
and stained with fluorescent labelled anti-CXCR4 antibody (10
μL of the supplied solution of mouse anti-human PE-
conjugated monoclonal CXCR4 antibody (clone #44717), R&D
Systems®, UK) on ice for 30 mins, then fixed with
paraformaldehyde in PBS (final conc. 1 %). Samples were
diluted with PBS (300 μL) then analysed by flow cytometry (BD
FACS CantoTM II, analysis performed using BD FACSDivaTM and
Flowing Software v 2.5) with 10000 events recorded for each
sample, and all were illuminated using a laser of λex = 633 nm.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This work was supported by a University of Manchester EPSRC
Doctoral Prize Award, an EPSRC NOWNANO DTC studentship
(grant number EP/G03737X/1), funding from the Manchester
Pharmacy School (University of Manchester, UK) and a Colloid
Science PhD studentship from the School of Chemistry
(University of Manchester, UK). We thank Mrs. Rehana Sung
for her guidance in developing HPLC protocols and the School
of Chemistry's nuclear magnetic resonance (NMR)
spectroscopy service. We also thank Dr. Reynard Spiess and
the Mass Spectrometry Service in the School of Chemistry
(University of Manchester) for mass spectrometry support.
Finally, we thank Dr. Nigel Hodson (BioAFM Facility, University
of Manchester) for his help in both preparing the samples and
performing AFM imaging.
The IC50 values for 1, 2 and AMD3100 were determined using
non-linear regression with a 3 parameter Hill equation
(GraphPad Prism v 7.03).
Western Blot: Primary CLL cells were cultured overnight in a
24 well plate with 6 × 106 cells/well. Cells were then exposed
to
2 or AMD3100 at 20 μM, or PBS for 3 h before incubating
with CXCL12 at 200 ng/mL for 10 mins at 37 °C, before placing
on ice. A control blot where the B-cell receptor was activated
was also performed: cells were exposed to biotinylated anti-
IgM followed by avidin to cross-link the receptor in the
Abbreviations
BACE1, β-site APP cleaving enzyme 1; BM, bone marrow; Boc,
tert-butyloxycarbonyl; CLL, chronic lymphocytic leukaemia;
CXCR4, C-X-C chemokine receptor type 4; CXCL12, C-X-C motif
chemokine ligand 12; DLS, dynamic light scattering; DMF, N,N-
presence or absence of 2 or AMD3100 (see the ESI). Cells were
lysed and cytosolic proteins extracted with RIPA buffer (50 mM
Tris, 150 mM NaCl, 1 % (v/v) Triton-X-100, 0.25 % (w/v) sodium
deoxycholate, 1 mM EDTA, pH 7.4). The extracted protein in
Laemmli buffer with 5 % β-mercaptoethanol was loaded onto
a 10 % SDS-PAGE gel then blotted onto a PVDF membrane
(AmershamTM Hybond® P Western Blotting membranes, Sigma
Aldrich, UK). The membranes were incubated with phospho-
p44/42 MAPK (Erk1/2) rabbit monoclonal antibody (#9102 Cell
Signaling Technology®) at 1:1000 overnight at 4 °C, following
which they were exposed to the secondary antibody (NA934V,
ECLTM anti-rabbit IgG HRP-linked whole antibody, GE
Healthcare Life Sciences) at 1:10000 for an hour at room
temperature. Visualisation of bound antibody employed
Amersham ECL Prime Western Blotting Detection Reagent
using a Bio-RadTM Chemidoc XRS Molecular Imager and Image
LabTM software. A loading-control blot was performed for total
ERK using p44/42 MAPK (Erk1/2) (9102, Cell Signaling
Technology®).
dimethylformamide;
EC50,
half
maximal
effective
concentration; ESI, electrospray ionisation; FCS, foetal calf
serum; HIV, human immunodeficiency virus; HPLC, high
performance liquid chromatography; IC50, half maximal
inhibitory concentration; MALDI, matrix assisted laser
desorption/ionization; NHS, N-hydroxysuccinimide; OEG,
octa(ethylene glycol); PBS, phosphate buffered saline; PE, R-
phycoerythrin; TFA, trifluoroacetic acid; TFAA, trifluoroacetic
anhydride; TLC, thin layer chromatography.
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Organic & Biomolecular Chemistry
Page 12 of 12
View Article Online
DOI: 10.1039/C8OB01439F
Synthesis and biological activity of a CXCR4-targeting bis(cyclam) lipid
Anna D. Peters, Catriona McCallion, Andrew Booth, Julie A. Adams, Karen Rees-Unwin, Alain Pluen,
John Burthem, Simon J. Webb
A bis(cyclam)-capped lipid, obtained through a short synthetic pathway, binds cell surface CXCR4
and prevents migration of chronic lymphocytic leukaemia cells.