New AMD3100 derivatives for CXCR4 chemokine receptor targeted molecular imaging studies: Synthesis, anti-HIV-1 evaluation and binding affinities

Article abstract of DOI:10.1039/c4dt02972k

CXCR4 is a target of growing interest for the development of new therapeutic drugs and imaging agents as its role in multiple disease states has been demonstrated. AMD3100, a CXCR4 chemokine receptor antagonist that is in current clinical use as a haematopoietic stem cell mobilising drug, has been widely studied for its anti-HIV properties, potential to inhibit metastatic spread of certain cancers and, more recently, its ability to chelate radiometals for nuclear imaging. In this study, AMD3100 is functionalised on the phenyl moiety to investigate the influence of the structural modification on the anti-HIV-1 properties and receptor affinity in competition with anti-CXCR4 monoclonal antibodies and the natural ligand for CXCR4, CXCL12. The effect of complexation of nickel(ii) in the cyclam cavities has been investigated. Two amino derivatives were obtained and are suitable intermediates for conjugation reactions to obtain CXCR4 molecular imaging agents. A fluorescent probe (BODIPY) and a precursor for ¹⁸F (positron emitting isotope) radiolabelling were conjugated to validate this route to new CXCR4 imaging agents.

Full text of DOI:10.1039/c4dt02972k

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New AMD3100 derivatives for CXCR4 chemokine
receptor targeted molecular imaging studies:
synthesis, anti-HIV-1 evaluation and binding
affinities.
Sophie Poty,a Pauline Désogère,a Christine Goze,a* Frédéric Boschetti,b
Thomas D‘huys,c Dominique Schols,c Christopher Cawthorne,d Stephen J.
Archibald,d Helmut R. Maëcke,e and Franck Denata*
CXCR4 is a target of growing interest for the development of new therapeutic drugs
and imaging agents as its role in multiple disease states has been demonstrated.
AMD3100, a CXCR4 chemokine receptor antagonist that is in current clinical use as a
haematopoietic stem cell mobilising drug, has been widely studied for its antiꢀHIV
properties, potential to inhibit metastatic spread of certain cancers and, more recently,
its ability to chelate radiometals for nuclear imaging. In this study, AMD3100 is
functionalised on the phenyl moiety to investigate the influence of the structural
modification on the antiꢀHIVꢀ1 properties and receptor affinity in competition with antiꢀ
CXCR4 monoclonal antibodies and the natural ligand for CXCR4, CXCL12. The effect
of complexation of nickel(II) in the cyclam cavities has been investigated. Two amino
derivatives were obtained and are suitable intermediates for conjugation reactions to
obtain CXCR4 molecular imaging agents. A fluorescent probe (BODIPY) and a
precursor for 18F (positron emitting isotope) radiolabelling were conjugated to validate
this route to new CXCR4 imaging agents.
Introduction
The CXCR4 chemokine receptor is a seven transmembrane helix protein, member of the Gꢀproteinꢀcoupled receptor (GPCR)
superfamily.1 It has only one naturally occurring endogenous ligand known as SDFꢀ1 or CXCL12.2 Together with its natural
α
ligand, CXCR4 is a central part of the signalling system in the human body that results in a variety of normal physiological
responses, such as chemotaxis, cell survival and proliferation, intracellular calcium flux, and gene transcription. However CXCR4
is also involved in the pathogeneses of a wide range of diseases.3 Malignant cells from at least 23 different types of cancer express
the chemokine receptor CXCR4 and respond to its ligand CXCL12.4 Furthermore, CXCR4 is a coꢀreceptor for Tꢀcell tropic strains
of human immunodeficiency virusꢀ1 (HIVꢀ1) and allows fusion and entry of the virus into human white blood cells.5 Hence,
CXCR4 is a target of growing interest for the development of antiꢀHIV drugs and in the field of diagnostic oncology. Clinically
AMD3100 is used in combination with GCSF (granulocyte colony stimulating factor) to mobilise haematopoietic stem cells and
allow harvesting.
Inhibition of the CXCR4ꢀCXCL12 signalling was investigated as a therapeutic strategy using antagonists of the natural ligand.
Several CXCR4ꢀbinding agents, including antibodies and peptideꢀbased antagonists have been developed.6 In the search for new
antiꢀHIV agents, bicyclam compounds were discovered as CXCR4 antagonists with potent and selective antiꢀHIV activities.7
These molecules consist of two 14ꢀmember tetraaza macrocyclic rings, linked either by an aliphatic bridge or an aromatic bridge
such as AMD3100. The latter, unlike many other existing HIV drugs that target the virus after it has infected a healthy cell, blocks
the virus from entering the cell, inhibiting the replication of both HIVꢀ1 and HIVꢀ2.8 AMD3100 binds to the CXCR4 chemokine
receptor mainly via electrostatic interaction between the positively charged protonated amino nitrogens of the cyclam moieties and
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the negatively charged carboxylates of the aspartate and glutamate residues of the receptor.9 AMD3100 has also been
demonstrated to be a ligand for another chemokine receptor, CXCR7. In contrast to its antagonistic effect blocking the
CXCR4/CXCL12 interaction, AMD3100 positively modulates CXCL12 effects on binding to CXCR7, it is therefore qualified as
an allosteric agonist of CXCR7.10 AMD3100 was clinically tested as an antiꢀHIV drug but the trial was discontinued in favour of
an orally available CXCR4 antagonist.11 Nevertheless, AMD3100 demonstrated a great potential in hematopoietic stem cell
mobilisation and is now approved by the U.S. Food and Drug Administration for use in nonꢀHodgkin’s lymphoma and multiple
myeloma patients.12
With the aim of optimising the antiꢀHIV activity of bisꢀazamacrocycles, many parameters of AMD3100 have been studied to
determine the modifications that can be tolerated whilst maintaining high CXCR4 receptor affinity. Analogues were synthesised
either with a different linker, a macrocycle ring size varying from 12 to 16 ring members,13 the introduction of one or more
heteroatoms in the macrocycle ring or replacement of one amino group by a heteroaromatic, all analogues resulting in a reduced
antiꢀHIV potency.14
Because of the preorganisation and the flexibility of their macrocyclic framework, macrocyclic polyamines such as cyclam, are
known to bind to metal ions and form highly stable metal complexes. Complexes of AMD3100 with metal cations were prepared
and the incorporation of ZnII, NiII and CuII led to the enhancement of the binding affinity to CXCR4 receptor and a higher antiꢀ
HIV activity in most cases. The nickel(II) complex of AMD3100 in solution can adopt the folded (cisꢀV) and planar (transꢀIII)
configuration, rearranging to give the cisꢀV compound on binding to the protein aspartate residues.15 Studies with AMD3100
analogues complexes with configurationally restricted macrocycles, where ethyl bridges link adjacent and nonꢀadjacent nitrogen
atoms to give respectively side and cross bridged cyclams, demonstrated that the rigidification of the cyclic scaffold may be a
route toward the optimisation of the interactions of the antagonist with the receptor.16 Additionally, as metal ion incorporation was
shown to enhance the affinity of AMD3100 for its receptor, the ligand was used for direct radiolabeling with 62Zn, 64Cu, 67Ga and
99mTc, resulting in tracers suitable for PET and SPECT to image CXCR4 expression in human cancer xenografts in mice.17
Only two examples of optical CXCR4 imaging agents based on cyclam structures were studied despite the advantages of optical
imaging. A rhodamine conjugated to a reinforced cyclam competes with antiꢀCXCR4 antibodies during binding competition when
labelled with copper(II)18 and a fluorescent AMD3100 analogue with an anthracenyl moiety used as a spacer shows a significant
reduction of the affinity probably due to the lipophilic character of the anthracenyl group.19
Figure 1: AMD3100 and derivatives studied for their anti-HIV activities and CXCR4 affinities.
In this study, new CXCR4 antagonists are investigated as potent antiꢀHIV agents and platforms for conjugation in molecular
imaging agent design. The syntheses of new AMD3100 analogues functionalised on the phenyl moiety by an ester or an
ethylenediamine moiety are discussed and we report the influence of the functionalisation on the affinity toward the CXCR4
chemokine receptor and antiviral potency against an X4 HIVꢀ1 strain. Metal complex formation with nickel(II) and the influence
on the binding and antiviral potency will also be presented. Ni2+ was chosen because its incorporation in the biscyclam AMD3100
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enhances the binding to the chemokine receptor CXCR4 by 50ꢀfold. This is the highest affinity obtained by incorporation of a
transition metal.22 Finally, the functionalisation on the phenyl spacer allowed the introduction of an imaging component, i.e.
optical probe or PET imaging agent precursor, confirming the versatility of the modified AMD3100 derivatives.
Results and Discussion
Synthetic procedures
Several synthetic routes to produce AMD3100 are reported in the literature.20 A cyclam ring protected with tosyl, mesityl,
diethylphosphoramidate (Dep) or tertꢀbutoxycarbonyle (Boc) groups is used as a starting material. Indeed, the best synthetic
approach for bridging two macrocyclic units is to use a triprotected tetraazamacrocycle, in order to avoid the formation of
polymers. Boc protecting groups were chosen as they can be easily removed by acidic treatment. The reaction of the protected
macrocycle with
1).
α,αꢀdibromoꢀpꢀxylene and the deprotection in acidic conditions give access to AMD3100 in two steps (Scheme
Scheme 1: General synthesis of AMD3100.
To investigate the influence of a functionalisation of the phenyl moiety on AMD3100 properties, we considered the synthesis of a
ꢀdibromoxylyl spacer substituted with an ester group. The precursor is obtained in two steps (Scheme 2) starting from a
commercially available product, 2,5ꢀdimethylbenzoic acid.21 Esterification with methanol using sulfuric acid as a catalyst yields
compound in 92 % yield. is then prepared through NBSꢀbased bisꢀbromination of the aryl methyl groups of
p
1
2
1.
Scheme 2: Synthesis of a dibromo aromatic spacer bearing an ester group.
AMD3100 derivative
presence of a base (Scheme 3). Nucleophilic attack of ethylenediamine on the ester function gives access to
Compounds and are then deprotected in acidic condition yielding respectively and in good yields.
3
is obtained in 82 % yield, after condensation of two equivalents of tris(Boc)cyclam on compound
2 in the
4
after 6 days at 60 °C.
3
4
5
6
Scheme 3: Synthesis of new AMD3100 analogues functionalised with an ester and an ethylenediamine group.
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As it has been shown that the incorporation of Ni2+ in the biscyclam AMD3100 enhances the binding to the chemokine receptor by
50ꢀfold,22 we investigated the metal complex formation of our analogues. Additionally, Ni2+ꢀcyclam complexes are known to be
particularly inert due to their stability constants (log K) indicative of high thermodynamic stability (19.9ꢀ20.3)23 and their slow
kinetics of dissociation.24 The complexes persist almost indefinitely even in strong acidic solutions and the only reported method
to remove Ni2+ from cyclam involves cyanide at high temperature.
Thus, after deprotonation of
added to obtain the corresponding Ni2+ complexes. Purification by reverse phase chromatography to remove any trace of free
nickel (II) was performed to obtain and in 18% and 39% yield respectively. Yields are low in these cases due to the
purification process. In order to investigate if ethylenediamine pendant arm coordinates one of the nickel atoms in compound
5 and 6 with NaOH (16M) and extraction with chloroform, two equivalents of Ni(NO3)2.6H2O were
7
8
8
,
UVꢀVisible absorption spectra was recorded in PBS at room temperature (see supporting information). The latter shows a band at
460 cmꢀ1, characteristic of a lowꢀspin NiII tetramine complex. The amino pendant arm is therefore not involved in the coordination
sphere of nickel.25
Scheme 4: Complex formation with nickel(II) of the AMD3100 analogues
Biological assays: antibody competition, calcium signalling and anti-HIV activity
Cellular binding assays of our AMD3100 analogues to the CXCR4 receptor were investigated in a competition assay with an antiꢀ
CXCR4 monoclonal antibody (mAb) (12G5 conjugated to Phycoerythrin). A Tꢀlymphocyte cell line (Jurkat) that expresses high
levels of the CXCR4 receptor was used and the binding of compounds
The binding assay is carried out by saturation of the cells’ receptors with a high concentration of analogues (ca. 20
5
,
6
,
7
and
8
to the cells are analysed by flow cytometry.
M). After
ꢀ
incubating with a saturating concentration of the antagonist, cells are washed to remove the excess of unbound compound and the
specific fluorescent antibody is introduced. After a second incubation, cells are washed to remove the excess of antibody and
compound displaced by the antibody. Then, cells are analysed by flow cytometry. The Mean Fluorescent Intensity (MFI) is used as
a measure of binding and a quantitative way of calculating the inhibition percentage of mAb binding. AMD3100 is used as a
reference compound. The nonꢀmetallated ligands
AMD3100 with 45 % and 46 % respectively (Table 1). Incorporation of a metal ion into the macrocyclic rings of AMD3100
analogues increases binding affinity to CXCR4, complexes and show higher inhibition percentages, 73 % and 84 %
5 and 6 both show a significant decrease in the inhibition percentage compared to
7
8
respectively, than the nonꢀmetallated ligands (Table 1). This is in agreement with Bridger and coꢀworkers studies.22 A mutational
analysis of the CXCR4 binding site for AMD3100 has identified amino acid Asp171 and Asp262 as key residues involved in the
binding to the CXCR4 receptor.11 The carboxylate groups of the receptor residues form three (one strong, one intermediate and
one weak) hydrogen bonds with protonated cyclam of AMD3100. It was later determined that the increased binding affinity of the
metal ion substituted AMD3100 is obtained through enhanced interaction of one of the cyclam ring systems with carboxylate
group of Asp262 22
hydrogen bond to a nitrogen atom in the cyclam ring remains. Coordination interactions are stronger interactions than hydrogen
bonding. Hence they are thought to be the dominant interactions for metal complexes. Complexes and should have stronger
interactions with the CXCR4 receptor as aspartate residue coordinates nickel(II), resulting in higher inhibition percentages
compared to their unmetalated ligand and . Even if the functionalisation of our AMD3100 analogues disrupted the affinity
towards CXCR4, the incorporation of nickel(II) in the cyclam cavities has a positive effect resulting in inhibition percentages close
to those observed for the reference compound AMD3100. If we compare the different functionalisations of the complexes and 8,
. With metal complexes, coordination of the carboxylate oxygens to the metal centre can occur while one weaker
7
8
5
6
7
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the ethylenediamine analogue has a slightly higher inhibition percentage, which may be due to more flexible pendant arm that can
form hydrogen bonds more easily.
Table 1: Inhibition of antiꢀCXCR4 mAb (clone 12G5) binding to CXCR4+ jurkat cells
Compound
Control
MFIa
344.67
708.57
704.29
524.78
451.59
413.90
1005. 00
Inhibition percentage
Negative
5
45 %
46 %
73 %
84 %
90 %
6
7
8
AMD3100
Positive
[a] Mean Fluorescent Intensity used to calculate the amount of antibody bound to CXCR4.
Figure 2: Flow cytometric histogram plots for the binding of 12G5 mAb in competition with compound
5 (light green) and 6 (dark green) (A) and with compounds 7
(light blue) and 8 (dark blue) (B). Negative control (purple), positive control (red) and binding of 12G-5 mAb in competition with AMD3100 (orange) are shown.
Further biological characterisation was carried out to determine the impact of the antagonism of the receptor in signalling
processes and the use of the receptor for viral cell entry. Prevention of signal transduction by the natural ligand is a useful indicator
of cellular targeting and would inhibit activation of cell recruitment. Signal inhibition was validated by measurement of the
intracellular calcium concentration increase, which is a downstream effect of CXCR4 receptor activation by its specific chemokine
ligand CXCL12.26 AMD3100 and the copper(II) complex of AMD3100 were included as controls, see Table 1. The antiꢀHIV
assay was used to coꢀvalidate the signalling data, as this is a wellꢀestablished assay, which provides further information on the
antagonist characteristics. The antiꢀHIVꢀ1 (X4 HIVꢀ1 NL4.3) activity of these compounds was determined in CD4+CXCR4+
human T cells using the wellꢀdocumented HIVꢀ1 infection assay (see Table 2).27
Table 2: AntiꢀHIV X4 activity and inhibition of CXCL12ꢀinduced calcium signalling (in ꢁM)
Compound
AMD3100
CuAMD3100
Anti-HIV-1 (EC50)a
0.011
Ca2+ flux CXCR4 (IC50)b
0.120
0.113
0.340
0.929
1.20
0.016
ꢀ
0.062
0.150
0.295
0.710
0.095
0.845
4.50
0.800
5
6
7
8
10
11
12
ꢀ
ꢀ
[a] EC50 is the effective concentration (in µM) to reduce the cytopathic effect of the CXCR4ꢀusing (X4) HIVꢀ1 strain NL4.3 by 50% in MTꢀ4 cells. [b] IC50 is
the inhibitory concentration of the compound (in µM) required to inhibit 50% of the CXCLꢀ12 (SDFꢀ1) induced Ca2+ signalling in the CXCR4ꢀtransfected
U87 cell line.
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As already mentioned, the chelator units would be expected to interact with aspartate residues on the CXCR4 chemokine receptor
surface via Hꢀbonding and electrostatic interactions and the metal complexes would form coordinate bonds.22 In the signalling
assay we can compare compounds
our compounds for CXCR4 as was observed for the same compounds in the flow cytometry study. This result correlates with the
antiꢀHIV infection assay although both still demonstrate subꢀµM values. Compounds and show increased potency of the
nickel(II) complex in both calcium signalling and antiviral assays, which is consistent with antibody competition assay. With an
IC50 of 16 nM, presents a higher potency, compared to both AMD3100 and its copper(II) complex which both have IC50 values >
5 and 7 showing in this case that the coordination of nickel(II) does not increase the affinity of
6
8
8
100 nM. The presence of the appended amino group may be responsible for this bonding impact as it will be an effective hydrogen
bond donor for interaction with the aspartate/ glutamate rich surface of the CXCR4 receptor.
Overall, although the AMD3100 type structure has been modified by the introduction of functional groups on the phenyl moiety,
compounds
6 and 8 can be considered as highly effective CXCR4 antagonists and we can now consider the introduction of a probe
component for molecular imaging into these structures.
Toward molecular imaging: introduction of an imaging probe component
Efficient syntheses towards functionalised AMD3100 were developed whilst maintaining strong antiꢀHIV potency and CXCR4
affinity. Both amine analogues
6 and 8 can be used to conjugate the AMD3100 moiety to an imaging probe as the primary amine
enables coupling reactions with carboxylic acid, active ester or anhydride. Two different imaging probe components were
attached, a fluorescent moiety and a PET isotope radiolabelling precursor. The main advantage for PET imaging here is to
introduce the radiolabelling site by AMD3100 functionalisation rather than using the cyclam cavity to chelate a radiometal as has
already been described and shows low in vivo stability.17 Cyclam can form complexes that are thermodynamically stable in vitro
but it has been shown that transchelation of the radiometal to ceruloplasmin and ultimately superoxide dismutase (SOD) can occur
in vivo in the case of 64Cu,28 resulting in accumulation of the radioisotope in the blood and liver.17c)
INTRODUCTION OF A BODIPY DYE
Amongst the options for fluorescent imaging components, borodipyrromethane type dyes (4,4ꢀdifluoroꢀ4ꢀborataꢀ3aꢀazoniaꢀ4aꢀazaꢀ
sꢀindacene, abbreviation BODIPY ) exhibits high stability, high extinction coefficients, sharp emission bands and high quantum

yields.29 Additionally, BODIPYs emission can be shifted to the redꢀNIR region by extending the
πꢀconjugation of the central
core30 and water soluble dyes can be obtained by addition of sulfonic acid moieties31 or coupling to an azamacrocycle.32 With all
of these attractive properties, BODIPYs appear to be an excellent choice as an optical imaging component and a BODIPY carrying
an activated ester was prepared to couple to the amine AMD3100 analogue 6.
Starting from BODIPYꢀacid, the corresponding Nꢀhydroxysuccinimidyl ester was obtained upon reaction with NHS in the
presence of EDCI in dichloromethane at room temperature in 70 % yield.33 The subsequent coupling of the activated ester with
6
yields 10 in a 65 % yield after purification by column chromatography. Compound 10 was labelled with Ni2+ and Zn2+ by adding
two equivalents of appropriate metal salts. It has to be noted that, in this case, the zinc complex was also synthesised, in order to
study the influence of the metal on the fluorescence of the compound. Moreover, Zn2+ also strongly increases the affinity of
AMD3100 for the CXCR4 receptor. Both complexes 11 and 12 were isolated in almost quantitative yield. One important point is
that these BODIPYꢀAMD3100 analogues are waterꢀsoluble without the requirement for any solubilising agent (such as DMSO),
which is crucial for biological applications such as molecular imaging. Indeed, one main drawback for the use of BODIPY dyes in
medical imaging is their very poor solubility in water. It is still currently challenging to introduce, in a simple synthetic manner,
solubilising groups for biological applications.
AntiꢀHIV assays were carried out with compounds 10, 11 and 12. These experiments showed that the introduction of the
fluorescent probe induces a reduction of the antiꢀHIV activity, although 10 and 12 still maintained subꢀµM activity levels, see
Table 2. It was interesting to note that the nickel(II) complex (11) was again less active than the metalꢀfree chelator 10. This
suggests that the geometric preferences of the nickel(II) ion on binding to CXCR4 protein aspartate/glutamate side chains may be
disrupted by functionalisation of the central phenyl ring. Zinc(II) is d10 and is more flexible in its coordination geometry. This may
account for the higher activity of 12 compared to 11, which is more similar to the metalꢀfree chelating unit 10
.
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Scheme 5: Synthesis of Bodipy-AMD3100 analogues
The photophysical properties of the different BODIPYꢀAMD derivatives were studied in PBS at room temperature. The data are
collected in Table 1. All compounds exhibit two absorption bands characteristic of the BODIPY signature, which consists of the
S0−S1 feature placed near 525 nm and the S0−S2 one located near 380 nm, both readily assigned to spinꢀallowed π−π* transitions.
It is important to note that no aggregation can be observed in PBS, which is mainly characterized by a broadening of the
absorption band as well as a dual absorption. The ε (λ) values of the Ni(II) and Zn(II)ꢀAMDꢀBODIPY 11 and 12, are relatively
high, ranging from 30200 to 53900 Mꢀ1cmꢀ1, those of free ligand 10 is a little lower (16500 Mꢀ1cmꢀ1).
Table 3: Photophysical properties of the BODIPY derivatives in PBS at 298 K.
Compound
Brightness (M-1.cm-1)
6 6 6 6 a)
λ
abs (nm)
525
λ
em (nm)
539
ε (M-1.cm-1)
16452
Φ
10
11
12
5
7
822
3771
8145
524
540
53872
524
540
30167
27
a) The quantum yields, ΦF, were measured at 298 K, using rhodamine 6G (ΦF = 0.78 in water, λexc = 488 nm). b) Brightness = Φ x ε
As an example, figure 3 exhibits the absorption, emission and excitation spectra of compound 12 (see ESI for all spectra). The ΦF
value of the AMDꢀBODIPY 10 is relatively low, mainly because of the presence of several amine functions and a flexible linker
between the BODIPY and the AMD part (“loose bold effect”). Complexation of the BodipyꢀAMD3100 analogue with Zn2+ (which
naturally occurs when injecting AMD3100 in vivo), resulted in a consequent enhancement of the fluorescence, yielding to a
quantum yield of 27% and a brightness of 8200 Mꢀ1 cmꢀ1, which is sufficient for at least in vitro fluorescence imaging.34
Concerning the nickel complex 11, the fluorescence is still relatively low (Φ = 7%). Further investigations would be needed to
explain this phenomena, which may be due to a paramagnetic character of NiII. Nevertheless, the brightness of the compound
(almost 4000 Mꢀ1 cmꢀ1) is sufficient for biological applications.
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Figure 3: Absorption (plain line), emission (large dashed line) and excitation (small dashed line) spectra of compound 11
INTRODUCTION OF A PRECURSOR FOR RADIOFLUORINATION
Fluorineꢀ18 has gained prominence in positron emission tomography imaging in the last decades due to its widespread availability,
relatively low cost of production and its optimal nuclear and chemical properties, i.e. low positron energy (635 keV), high
abundance, and relatively long halfꢀlife (109.7 min) when compared to 11C.35 Nevertheless with a halfꢀlife of 109.7 min, the
synthesis of labelled imaging agents needs to be rapid and efficient. The BꢀF bond is one of the strongest bonds known, Ting et al.
developed a pinacol phenylboronate diester able to react with Fꢀ in a single, rapid and highꢀyielding step at pH 4ꢀ7 in aqueous
solvents and at temperatures that are unlikely to denature biomolecules.36 Inspired by this work, the pinacol phenylboronate diester
PET precursor was coupled to the functionalised compounds
6 and 8.
Starting from a commercially available product, 4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3ꢀ2ꢀdioxaborolanꢀ2ꢀyl)benzoic acid, an activated ester 13
of the pinacol phenylboronate diester is synthesised by reaction with N,N’ꢀdisuccinimidyl carbonate and triethylamine in a 56 %
yield. Nucleophilic attack of
6 and 8
gives the resulting AMD3100 analogues 14 and 15 that constitute two precursors for 18F
radiolabelling. It has to be noted that 15 was synthesised starting from compound
8 and not by metallation of 14, because the
pinacol boronate intermediate was subject to hydrolysis during the metallation step.
Scheme 6: Synthesis of AMD3100 pinacol phenyl boronate analogues as PET precursors and AMD3100 trifluoroborate.
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Fluoride treatment was performed on 14 to validate our concept. Following a previous reported protocol,35 14 was dissolved in a
small volume of deuterated methanol and an aqueous solution of KHF2 (3.3 eq.) was added. The solution turned cloudy
immediately and further deuterated methanol was added to dilute the solution. The reaction mixture was stirred for 30 min at room
temperature and the reaction was followed by 11B and 19F NMR. The 11B signal was shifted from 31.6 ppm to 0.4 ppm and
appeared as a quadruplet due to the coupling between boron and fluoride. In the 19F NMR, the consumption of KHF2 was
evidenced by the disappearance of the signal at ꢀ170 ppm and the presence of a signal at ꢀ142.7 ppm, characteristic of a ꢀBF3.
Fluorination was also confirmed by mass spectrometry. This experiment confirms the high reactivity of the pinacol phenylboronate
derivatives and their potential as precursors for the synthesis of 18F radiopharmaceuticals.
Conclusion
A series of novel CXCR4 antagonists have been synthesised in this work and it has been demonstrated that functionalisation of the
central bridging phenyl group in AMD3100 can be carried out without significant loss in the affinity for the CXCR4 receptor. The
compounds have been characterised as subꢀµM antagonists in a series of assays demonstrating effective binding to the receptor in
vitro. They have then been modified for imaging applications via addition of a prosthetic group for radiofluorination or a
fluorescent dye molecule to delineate the route to application. This design separates out the targeting and imaging components of
the molecule, which is in contrast to the previously described complexes of AMD3100 with radiometals. The modular approach is
advantageous in tuning for different modalities and optimum receptor ‘on’ and ‘off’ binding rates. There is also the potential for
multimodal imaging to allow evaluation of the compounds by a range of imaging techniques to more precisely characterise the
interactions from a cellular level up to in vivo studies. The next steps for this study are to select the optimal candidates, optimise
the radiolabelling conditions and carry out preliminary in vivo studies. In addition, the affinity towards CXCR7 of the novel
compounds is under evaluation to determine the binding profile with this important and related chemokine receptor target.
Acknowledgements
Support was provided by the CNRS, the University of Burgundy and the Conseil Régional de Bourgogne through the 3MIM
Project. S.P. and P.D. thank the Ministère de l’Enseignement Supérieur et de la Recherche for PhD grants. COST action TD 1007
PETꢀMRI is also acknowledged for financing STSM of S.P. This work, in part, was supported by funding of the K.U. Leuven
(GOA/10/014 and PF/10/018) and the Foundation of Scientific Research (FWO no. G.0485.08 and G.0528.12).
This journal is © The Royal Society of Chemistry 2014
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DOI: 10.1039/C4DT02972K
Experimental Section
Synthetic Procedures
General information
Additional experimental details, data for compounds
Information.
1ꢀ16 (NMR, MS spectra and HPLC chromatograms) are given in Supporting
Material TriBocCyclam was generously donated by CheMatech and all other chemicals were purchased from Acros and Aldrich
and were used without further purification. Organic solvents were removed under reduced pressure using rotary evaporator. Water
was removed by lyophilisation.
Methyl-2,5-dimethylbenzoate (1): A mixture of 2,5ꢀdimethylbenzoic acid (10.0 g, 66.6 mmol), sulphuric acid (14 mL, 13.3
mmol), and methanol (27 mL) was heated at reflux for 4 h. The mixture was poured into water (250 mL) and extracted with ethyl
acetate (2 x 200 mL). The pooled organics were washed with saturated sodium bicarbonate (2 x 50 mL) and brine (50 mL), dried
1
over MgSO4, and concentrated in vacuo to give
1
(10.1 g, 92 %) as a colorless oil. H NMR (300 MHz, CDCl3, 300 K): δ = 7.70
4
3
4
3
(d, J (H,H) = 1.7 Hz, 1 H; CH), 7.18 (dd, J (H,H) = 7.9 Hz, J (H,H) = 1.4 Hz, 1 H; CH), 7.10 (d, J (H,H) = 7.9 Hz, 1 H; CH),
3.86 (s, 3 H; OCH3), 2.53 (s, 3 H; CH3), 2.32 ppm (s, 3 H; CH3); 13C{1H} NMR (75 MHz, CDCl3, 300 K): δ = 168.4 (C=O), 137.2
(Car), 135.4 (Car), 132.9 (CHar), 131.8 (CHar), 131.2 (CHar), 129.6 (Car), 51.9 (OCH3), 21.4 (CH3), 20.9 ppm (CH3); ESIꢀMS:
m/z: 165.31 [M+H]+.
Methyl-2,5-bis(bromomethyl)benzoate (2): To a solution of
1 (10.1 g, 61.6 mmol) in carbon tetrachloride (300 mL) were added
N−bromosuccinimide (23.0 g, 129.3 mmol) and benzoyl peroxide (1.0 g, 4.1 mmol). The reaction mixture was heated at reflux for
4 h. The resulting suspension was filtered, and the residue was washed with chloroform (3 x 150 mL). The pooled organics were
1
concentrated in vacuo to give a mixture of product and succinimide as determined by H NMR. The mixture was dissolved in
dichloromethane (600 mL) and washed with water (3 x 150 mL). The dichloromethane phase was dried over MgSO4 and
concentrated in vacuo, and the resulting residue was purified by reverse phase flash chromatography on C18. After evaporation of
acetonitrile, the aqueous solution was freeze dried to give
2
(13.9 g, 70 %) as a white solid. 1H NMR (500 MHz, CDCl3, 300 K): δ
3
3
= 8.00 (d, 4J (H,H) = 1.8 Hz, 1 H; CH), 7.53 (dd, J (H,H) = 7.8 Hz, 4J (H,H) = 1.8 Hz, 1 H; CH), 7.46 (d, J (H,H) = 7.8 Hz, 1 H;
CH), 4.94 (s, 2 H; CH2), 4.49 (s, 2 H; CH2), 3.98 ppm (s, 3 H; OCH3); 13C{1H} NMR (75 MHz, CDCl3, 300 K): δ = 166.6 (C=O),
139.6 (Car), 138.5, 133.2 (CHar), 132.5, 132.0, 129.7 (Car), 52.6 (OCH3), 32.0 (CH2), 31.0 ppm ; ESIꢀMS: m/z: 322.54 [M+H]+.
AMD3100 Boc–ester (3): To a solution of trisꢀtertꢀbutylꢀ1,4,8,11ꢀtetraazacyclotetradecaneꢀ1,4,8ꢀtricarboxylate (2.00 g, 3.98
mmol) in acetonitrile (20 mL) was added K2CO3 (1.38 g, 9.99 mmol) and compound
2 (0.63 g, 1.99 mmol). The reaction mixture
was heated at 40 °C overnight. After cooling, the solution was filtered on celite. The solvent was evaporated and the resulting oil
was taken up in 200 mL of diethylether, washed with 2 x 50 mL of water, dried over MgSO4 and concentrated in vacuo. The
resulting residue was purified by flash chromatography (A: CH2Cl2, B: CH3OH, B 35%) to give
3 (1.89 g, 82 %) as a white foam.
1
3
3
m.p. 89 +/ꢀ 1°C; H NMR (300 MHz, CDCl3, 300 K): δ = 7.66 (s, 1 H; CH), 7.43 (d, J (H,H) = 7.7 Hz, 1 H; CH), 7.31 (d, J
(H,H) = 7.7 Hz, 1 H, CH), 3.85 (s, 3 H; OCH3), 3.81 (s, 2 H; CH2Ph), 3.51 (s, 2 H; CH2Ph), 3.45ꢀ3.14 (m, 23 H), 2.67ꢀ2.52 (m, 4
H), 2.45ꢀ2.28 (m, 4 H), 1.94ꢀ1.77 (m, 4 H; CH2β), 1.70ꢀ1.61 (m, 4 H; CH2β), 1.50ꢀ1.24 ppm (m, 54 H; CH3); 13C{1H} NMR (125
MHz, DMSO, 343 K): δ = 167.4 (C=O), 154.5 (*2), 154.4, 154.3 (*2), 154.2, 138.1 (Car), 137.5 (Car), 131.3 (Car), 130.3 (CHar),
129.7 (CHar), 129.6 (CHar), 78.4, 78.3, 78.2, 78.1, 78.0 (*2), 57.8, 56.2, 52.7, 52.5, 51.3, 51.1, 46.7, 46.6, 46.5(*3), 46.2, 46.0,
45.9 (*2), 45.8, 45.7, 45.5, 45.4, 27.8 (*12) (CH3), 27.7 (*3) (CH3), 27.6 (*3) (CH3), 25.9 (*2) (CH2β), 25.6 pm (*2) (CH2β); ESIꢀ
MS: m/z: 1183.76 [M+Na]+, 1199.73 [M+K]+; HRMS (ESI): m/z calcd for C60H104N8O14 + Na+: 1183.7571, found 1183.7564 ;
elemental analysis calcd (%) for C60H104N8O14 : C 62.04, H 9.02, N 9.65; found : C 61.62, H 8.98, N 9.55; HPLC : tr = 8.13 min,
purity 98 %.
AMD3100 Boc–ethylenediamine (4): Compound
3 (1.89 g, 1.63 mmol) was dissolved in distilled ethylenediamine (6.5 mL). The
mixture was stirred at 40 °C for 7 days. The mixture was concentrated by evaporation and the resulting oil was taken up in acetone
(200 mL). After filtration, the pooled organics were concentrated in vacuo to give a yellow foam. The resulting residue was
purified by flash chromatography on silica gel (A: CH2Cl2, B: CH3OH, B 15 %) to furnish
98 +/ꢀ 1 C; 1H NMR (500 MHz, CDCl3, 324 K): C=O), 7.49 (s, 1 H; CH), 7.25 (d, 3J (H,H) = 7.3 Hz, 1 H;
= 8.17 (br.s, 1 H; N
CH), 7.20 (d, 3J (H,H) = 7.6 Hz, 1 H; CH), 3.64 (s, 2 H, CH2Ph), 3.50 (s, 2 H, CH2Ph), 3.46 (q, 3J (H,H) = 6.0 Hz, 2 H;
H2NHC=O), 3.40ꢀ3.16 (m, 24 H), 2.93 (t, 3J (H,H) = 6.0 Hz, 2 H; CH2NH2), 2.66 (t, 3J (H,H) = 6.5 Hz, 2 H), 2.60ꢀ2.52 (m, 2 H),
2.46 (t, 3J (H,H) = 6.5 Hz, 2 H), 2.40 (t, 3J (H,H) = 5.7 Hz, 2 H), 2.10 (s, 2 H), 1.89ꢀ1.77 (m, 4 H; CH2β), 1.71ꢀ1.61 (m, 4 H;
CH2β), 1.44ꢀ1.39 (m, 36 H; CH3), 1.37ꢀ1.24 ppm (m, 18 H; CH3); 13C{1H} NMR (125 MHz, CDCl3, 324 K):
= 169.9 (C=O),
156.1, 156.0, 155.9 (*2), 155.7 (*2), 138.7 (Car), 137.4 (Car), 135.1 (Car), 131.2 (CHar), 130.5 (CHar), 129.3 (CHar), 80.0, 79.9 (*2),
4 (0.77 g, 40 %) as a white foam. m.p.
°
δ
H
C
δ
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79.8 (*2), 79.7, 59.6 (CH2), 58.0, 53.5, 52.5, 52.4, 52.1, 51.1, 50.9, 48.1 (*2), 48.0, 47.5, 47.3 (*2), 46.9, 46.5, 43.1 (*2), 42.1 (*2),
28.9 (*3) (CH3), 28.8 (*9) (CH3), 28.7 (*3) (CH3), 28.6 (*3) (CH3), 27.2 (*2) (CH2β), 26.1 ppm (*2) (CH2β); ESIꢀMS: m/z:
1189.82 [M+H]+; HRMS (ESI): m/z calcd for C61H108N10O13 + H+: 1189.8170, found 1189.8212; elemental analysis calcd (%) for
C61H108N10O13: C 61.59, H 9.15, N 11.77; found C 61.17, H 9.43, N 11.51; HPLC : tr = 8.48 min, purity 96 %
AMD3100-ester (5): A solution of 3 M HCl (3.0 mL) was added to
3
(100.0 mg, 0.08 mmol) and the reaction mixture was stirred
+ 6 HCl
for one hour. The resulting product was left one night at room temperature. The acid was evaporated in vacuo giving
5
1
+10 H2O. (60.7 mg, yield = 73%) as a white foam. H NMR (300 MHz, D2O, 300 K): δ = 8.32 (d, 4J (H,H) = 1.8 Hz, 1H), 7.88
(dd, 3J (H,H) = 7.8 Hz, 4J (H,H) = 1.8 Hz, 1H), 7.76 (d, 3J (H,H) = 7.8 Hz, 1H), 4.46 (br.s, 2H), 3.98 (s, 3H; OMe), 3.92ꢀ3.81 (m,
2H), 3.76ꢀ3.53 (m, 17H), 3.49ꢀ3.28 (m, 15H), 2.29ꢀ2.12 ppm (m, 8H); 13C{1H} NMR (75 MHz, D2O, 300 K):
δ = 168.1, 135.9,
134.9, 134.2, 133.4, 131.2, 131.0, 48.2, 47.9, 45.5, 44.8, 42.1, 41.6, 41.5, 40.8, 40.7, 38.44, 38.38, 38.0, 37.2, 36.8, 19.1 (CH2β),
19.0, 18.5, 17.9 ppm; HRMS (ESI): m/z calcd for C30H56N8O2 + H+: 561.4599, found 561.4573; elemental analysis calcd (%) for
C30H56N8O2 + 10 H2O + 6 HCl: C 37.54, H 8.61, N 11.68; found C 37.82, H 8.05, N 11.65; HPLC : tr = 5.53 min, purity 97%.
AMD3100–ethylenediamine (6): Compound
was stirred overnight at room temperature. The mixture was evaporated, taken up in acetone (50 mL) and then stirred overnight.
The precipitate was filtered, washed with acetone and ether and finally dried in vacuo. The compound + 9 HCl + 5 H2O was
4 (0.47 g, 0.40 mmol) was dissolved in a solution of 3 M HCl (10 mL). The mixture
6
1
obtained as a white solid (400 mg, yield = 100 %). H NMR (300 MHz, D2O, 300 K): δ = 7.93ꢀ7.91 (s, 1 H), 7.81ꢀ7.74 (m, 2 H),
4.51 (s, 2 H; CH2Ph), 4.29 (s, 2 H; CH2Ph), 3.82ꢀ3.78 (m, 2 H), 3.67ꢀ3.34 (m, 32 H), 3.18ꢀ3.14 (m, 2 H), 2.27ꢀ2.16 ppm (m, 8 H;
CH2β); 13C{1H} NMR (75 MHz, D2O, 300 K): δ = 171.0 (C=O), 135.7 (CHar), 134.5 (CHar), 134.4 (CHar), 133.4 (Car), 131.6
(Car), 130.2 (Car), 58.1, 57.5, 48.4, 47.9, 47.8, 45.6, 45.3, 45.0, 42.1, 41.8, 41.7, 41.2, 41.0, 38.9, 38.7, 38.5, 38.1, 37.8, 37.7, 37.6,
19.3 (CH2β), 19.2 (CH2β), 18.8 (CH2β), 18.5 ppm (CH2β); HRMS (ESI): m/z calcd for C31H60N10O + H+: 589.5024, found
589.5034; elemental analysis calcd (%) for C31H61N10O + 9 HCl + 5 H2O: C 36.97, H 7.91, N 13.91; found C 37.13, H 8.06, N
14.33.
Few drops of NaOH (16 mol.Lꢀ1) were added on compound
chloroform (100 mL) was added quickly. The two layers were separated and the organic layer was dried over magnesium sulfate.
6 + 9 HCl + 5 H2O in order to obtain a viscous solution and
1
After evaporation of the solvent,
6 + 9.5 H2O (276 mg, yield = 91 %) was obtained as a light yellow oil. H NMR (300 MHz,
3
3
4
CDCl3, 300 K): δ = 8.64 (t, J (H,H) = 5.4 Hz, 1 H; NHC=O), 7.41 (d, J (H,H) = 7.8 Hz, 1 H), 7.37 (d, J (H,H) = 1.7 Hz, 1 H),
7.12 (dd, J (H,H) = 7.8 Hz, 4J (H,H) = 1.7 Hz, 1 H), 3.51 (s, 2 H; CH2Ph), 3.37 (s, 2 H; CH2Ph), 3.32 (q, J (H,H) = 5.9 Hz, 2 H;
CH2NHC=O), 2.76 (t, J (H,H) = 6.2 Hz, 2 H; CH2NH2), 2.71ꢀ2.19 (m, 40 H), 1.72 (quint, J (H,H) = 5.5 Hz, 2H; CH2β), 1.63
(quint, J (H,H) = 5.5 Hz, 2H; CH2β), 1.55ꢀ1.45 ppm (m, 4 H; CH2β); 13C{1H} NMR (75 MHz, D2O, 300 K): δ = 170.5 (C=O),
3
3
3
3
3
137.9 (Car), 137.6 (Car), 135.7 (Car), 130.6 (CHar), 130.4 (CHar), 128.0 (CHar), 57.9 (CH2Ph), 55.8 (CH2Ph), 54.6, 54.2, 53.3,
53.2, 50.9, 50.5, 49.3, 49.1, 48.7, 48.6, 48.5, 48.3, 48.2, 47.8, 47.7, 47.3, 43.1, 41.9, 28.6 (CH2β), 28.4 (CH2β), 26.5 (CH2β), 26.1
ppm (CH2β); elemental analysis calcd (%) for C31H61N10O + 9.5 H2O: C 48.99, H 10.48, N 18.43; found C 49.00, H 10.57, N
18.33; HPLC : tr = 5.36 min, purity 99 %.
Ni2AMD3100-ester (7): 0.60 mL (0.06 mmol) of a titrated solution of Ni(NO3)2, 6 H2O (c = 0.106 mol.Lꢀ1) in water were added to
a solution of 125 (17.3 mg, 0.03 mmol) in a 3 mL mixture of MeOH/H2O (9:1). The reaction was stirred at 50 °C overnight. After
completion, reverse phase C8 semiꢀpreparative HPLC (A: H2O) was performed to furnish
7 (3.7 mg, 18 %) as a light orange foam.
HRMS (ESI): m/z calcd for C30H54N8Ni2O2 ꢀ 2H2+: 337.1533, found 337.1529; HPLC : tr = 5.88 min, purity 95 %.
Ni2AMD3100-ethylenediamine (8): 15.0 mL (1.59 mmol) of a titrated solution of Ni(NO3)2, 6 H2O (c = 0.106 mol.Lꢀ1) in water
were added to a solution of
6
+ 9,5 H2O (568 mg, 0.79 mmol) in a 110 mL mixture of MeOH/H2O (9:1). The reaction was stirred
+ 4 NO3 + 7
at 50 °C overnight. After completion, reverse phase C18 flash chromatography (A: H2O) was performed to furnish
8
H2O (332 mg, 39 %) as a light orange foam. UVꢀVis (PBS), λ (nm): 460; HRMS (ESI): m/z calcd for C31H58N10Ni2O ꢀ 2H2+:
351.1746, found 351.1740; elemental analysis calcd (%) for C31H58N10Ni2O + 4 NO3 + 9.5 H2O: C 34.59, H 6.56, N 18.22; found
C 34.24, H 6.30, N 18.88; HPLC : tr = 5.77 min, purity 88 %.
Bodipy-NHS-ester (9):30 54 mg of Nꢀhydroxysuccinimide (0.48 mmol), 58 mg of dimethylaminopyridine (DMAP) (0.48 mmol)
and 92 mg of 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀethylcarbodiimide hydrochloride (EDCI) (0.48 mmol) were added to a solution of 4,4ꢀ
difluoroꢀ8ꢀ(4ꢀcarboxyphenylꢀ)1,3,5,7ꢀtetramethylꢀ2,6ꢀdiethylꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindacene (100 mg, 0.24 mmol) in CH2Cl2 (50
mL). The reaction was stirred at 35 °C. After complete activation of the acide function (2h) followed by TLC, the mixture was
washed with 2 x 10 mL of water, the organic phase was dried over MgSO4 and the solvent was evaporated to give a red oil. The
crude product was purified by chromatography on silica gel (AcOEt/Hexane 50:50). Upon concentration of the pure fraction,
1
recrystallisation in a mixture of dichloromethane and hexane gave
9
as a red solid (88 mg, yield = 70 %). H NMR (300 MHz,
3
3
CDCl3, 300 K): δ = 8.23 (d, J (H,H) = 8.3 Hz, 2H; CHarBod), 7.47 (d, J (H,H) = 8.3 Hz, 2H; CHarBod), 2.92 (bs, 4H; CH2C=O),
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3
3
2.51 (s, 6H; CH3), 2.27 (q, J (H,H) = 7.5 Hz, 4H; CH2CH3), 1.25 (s, 6H; CH3), 0.96 ppm (t, 6H, J (H,H) = 7.5 Hz; CH2CH3);
13C{1H} NMR (75 MHz, CDCl3, 300 K): δ = 169.2 (*2) (NC=O), 161.4 (CO2), 154.6 (*2) (Cpyrrole), 143.0 (CarBod), 138.7 (CBod),
138.0 (*2) (Cpyrrole), 137.7 (*2) (Cpyrrole), 133.3 (*2) (CHarBod), 131.2 (*2) (Cpyrrole), 129.3 (*2) (CHarBod), 125.6 (CarBod), 25.7 (*2)
(CH2C=O), 17.3 (*2) (CH2CH3), 14.6 (*2) (CH2CH3), 12.7 (*2) (CH3), 12.1ppm (*2); 11B{1H} NMR (192.5 MHz, CDCl3, 300 K):
1
δ = 0.79 ppm (t, J (B,F) = 33.1 Hz); ESIꢀMS: m/z = 544.21 [M+Na]+; UVꢀVis (CH3CN), λ (nm) (ε, Mꢀ1cmꢀ1): 525 (73000), 492
(sh, 23200), 378 (7940); elemental analysis calcd (%) for C28H30BF2N3O4: C 68.50, H 6.67, N 6.39; found C 68.78, H 6.87, N
5.95.
AMD3100-bodipy (10): Diisopropylethylamine (172 ꢁL, 1.04 mmol) was added to a solution of
6 (408 mg, 0.69 mmol) in
acetonitrile (40 mL). A solution of bodipy (694 mg, 0.69 mmol) in acetonitrile (10 mL) was then added and the mixture was
9
stirred at room temperature during 6 h, until total consumption of the starting materials was observed by TLC. Solvent was
evaporated, and the resulting red oil was purified by column chromatography on silica gel (eluent: CH2Cl2/EtOH/NH4OH
20:60:20). Precipitation from CH2Cl2/hexane gave the desired compound 10 as a red solid (446 mg, yield = 65 %) Mp: > 200 °C;
3
3
1H NMR (300 MHz, CDCl3, 300 K): δ = 9.80ꢀ9.67 (bs, 1H), 8.62ꢀ8.49 (bs, 1H), 8.12 (d, J (H,H) = 8.2 Hz, 2H), 7.55 (d, J (H,H)
3
3
= 7.7 Hz, 1H), 7.43 (s, 1H), 7.33 (d, J (H,H) = 8.2 Hz, 2H), 7.20 (d, J (H,H) = 7.7 Hz, 1H), 3.73ꢀ3.46 (m, 12H), 2.79ꢀ2.31 (m,
3
3
37H), 2.26 (q, 4H, J (H,H) = 7.5 Hz; CH2CH3), 1.92ꢀ1.52 (m, 8H), 1.32ꢀ1.24 (m, 9H), 0.94 ppm (t, J (H,H) = 7.5 Hz, 6H;
CH2CH3); 13C{1H} NMR (75 MHz, CDCl3, 300 K): δ = 173.0 (NHC=O), 166.6, 154.3 (*2) (Cpyrrole), 139.8 (Car), 139.3 (CarBod),
139.2 (CBod), 138.3 (*2) (Cpyrrole), 138.2 (Car), 136.1, 134.5 (CarBod), 133.2 (*2) (Cpyrrole), 131.5 (CHar), 130.6 (*2) (Cpyrrole), 130.5
(CHar), 128.8 (*2) (CHarBod), 128.3 (*2), 127.2 (CHar), 58.3 (CH2), 55.2, 54.9, 54.6, 54.2, 53.8, 53.2, 51.3, 51.1, 49.9, 49.4 (*2),
48.8, 48.6, 48.5, 48.4, 48.1, 47.1, 42.8, 39.7, 28.7 (CH2β), 28.6, 26.6, 26.2, 17.2 (*2) (CH2CH3), 14.8 (*2) (CH2CH3), 12.7 (*2)
1
(CH3), 12.0 ppm(*2); 11B{1H} NMR (96 MHz, CDCl3, 300 K): δ = 0.76 ppm (t, J (B,F) = 33.6 Hz); ESIꢀMS: m/z = 995.7
[M+H]+, 1021.7 [M+Na]+, 1033.7 [M+K]+; HRMS (ESI): m/z calcd for C55H85BF2N12O2 + H+: 995.7062, found 995.7012.
Ni2AMD3100-bodipy (11): 3.1 mg of NiCl2 (0.023 mmol) was added to a solution of 10.4 mg of compound 10 (0.012 mmol) in 6
mL of methanol. The mixture was stirred at 50 °C for 2 h. After evaporation, compound 11 was obtained as a red solid (14.2 mg,
yield = 95 %). ESIꢀMS: m/z = 555.27 [Mꢀ2Hꢀ4Cl]2+; HRMS (ESI): m/z calcd for C55H85BCl4F2N12Ni2O2 ꢀ 2H ꢀ 4Cl 2+: 555.2740,
found 555.2699.
Zn2AMD3100-bodipy (12): 4.7 mg of Zn(OAc)2, 2 H2O (0.021 mmol) was added to a solution of 9.5 mg of compound 10
(0.011 mmol) in 6 mL of methanol. The mixture was stirred at 50 °C for 2 h. After evaporation, compound 12 was obtained as a
red solid (27.7 mg, yield = 97 %). ESIꢀMS: m/z = 592.28 [MꢀHꢀ3(OAc)]2+; HRMS (ESI): m/z calcd for C63H97BF2N12O10Zn2 ꢀ H ꢀ
3(OAc) 2+: 592.2783, found 592.2780.
Pinacol Phenylboronate activate ester (13): Triethylamine (340 µL, 2.43 mmol) and N,N’ꢀdisuccinimidyl carbonate (310 mg,
1.21 mmol) were added to a solution of 4ꢀ(4,4,5,5ꢀtetramethylꢀ1,3ꢀ2ꢀdioxaborolanꢀ2ꢀyl)benzoic acid (200 mg, 0.81 mmol) in
acetonitrile (20 mL). The reaction mixture was stirred at room temperature for 5 h. The solvent was evaporated in vacuo with a
bath temperature lower than 35 °C. The resulting product was dissolved in dichloromethane and washed with a saturated solution
of sodium bicarbonate. After drying over magnesium sulfate, the organic layer was evaporated in vacuo with a low temperature
bath >35 °C. Chromatography on silica gel (A: Pentane, B: EtOAc, B 50 %) gave 13 (0.157 g, 56 %) as a white powder. 1H NMR
(300 MHz, CDCl3, 300 K): δ = 8.09 (d, 3J (H,H) = 8.1 Hz, 2H; CHarBpinacol), 7.90 (d, 3J (H,H) = 8.1 Hz, 2H; CHarNHS), 2.86 (s, 4H;
CH2 NHS), 1.33 ppm (s, 12H; CH3); 13C{1H} NMR (75 MHz, CDCl3, 300 K): δ = 169.4 (*2) (CC=O NHS), 162.1 (C C=O), 135.1
(Cpinacol), 129.6 (*4) (CH), , 127.3 (CNHS), 84.6 (*2) (CMe2), 25.9 (*2) (CH2), 25.0 ppm (*4) (CH3); HRMS (ESI): m/z calcd for
C17H20BNO6 + Na+: 368.1279, found 368.1274.
AMD3100-pinacol phenylboronate (14): 6 (26 mg, 0.04 mmol) was dissolved in a mixture of acetonitrile (600 µL) and DMF (20
µL), triethylamine (77 µL) was then added. The solution was stirred at room temperature for 10 min and 13 (15 mg, 0.04 mmol)
was added to the reaction. After one night stirring at 40 °C, the solvent was evaporated under a nitrogen flux to give 14 which still
contains traces of DMF and was directly used without any further purification step. 1H NMR (500 MHz, CDCl3, 500 K): δ = 9.79ꢀ
3
4
3
9.63 (bs, 1H; NH), 8.77ꢀ8.65 (bs, 1H; NH), 8.07 (dd, J (H,H) = 5.2 Hz, J (H,H) = 1.5 Hz, 2H), 7.75 (d, J (H,H) = 8.1 Hz, 1H),
3
3
4
7.72ꢀ7.65 (m, 1H), 7.63 (d, J (H,H) = 8.1 Hz, 1H), 6.63 (dd, J (H,H) = 5.1 Hz, J (H,H) = 1.5 Hz, 2H), 3.70ꢀ3.31 (m, 7H), 2.90ꢀ
2.85 (m, 7H), 2.73ꢀ2.20 (m, 35H), 1.79ꢀ1.52 (m, 6H), 1.19 ppm (s, 12H); 11B (160 MHz, CDCl3, 300 K): δ = 31.6 ppm; ESIꢀMS:
m/z = 819.6 [M+H]+; MALDIꢀTOF: m/z = 819.63 [M+H]+.
Ni2AMD3100-pinacol phenylboronate (15): 8 (20 mg, 0.02 mmol) was dissolved in DMF (3 mL) and triethylamine (25 µL) was
added. The solution was stirred at room temperature for 10 min and 13 (8 mg, 0.02 mmol) was added to the reaction. After one
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night stirring at 40 °C, the solvent was evaporated in vacuo with a bath temperature lower than 35 °C to give 15. MALDIꢀTOF:
m/z = 931.36 [Mꢀ3H]+.
AMD3100-phenyltrifluoroboronate (16): 14 (0.04 mmol) was dissolved in methanolꢀd4 (150 µL) and a solution of KHF2 in D2O
(35 µL, c = 4M) was added. The solution immediately turned blurry and 1 mL of methanolꢀd4 was added. The mixture was stirred
1
3
for 30 min and NMR was recorded on the crude product. H NMR (500 MHz, CD3OD, 500 K): δ = 8.08 (d, J (H,H) = 7.5 Hz,
3
2H), 7.72ꢀ7.69 (m, 1H), 7.61ꢀ7.57 (m, 1H), 7.44 (bs, 1H), 6.87 (d, J (H,H) = 6.9 Hz, 2H), 3.80ꢀ3.61 (m, 6H), 3.23ꢀ3.15 (m, 7H),
3.14ꢀ3.03 (m, 7H), 2.99ꢀ2.87 (m, 10H), 2.76ꢀ2.54 (m, 12H), 2.02ꢀ1.76 ppm (m, 6H); 11B (160 MHz, CD3OD, 300 K): δ = 0.34
ppm (q, 1J (B,F) = 11.9 Hz); 19F (202 MHz, CD3OD, 300 K): δ = ꢀ142.7 ppm; ESIꢀMS: m/z =759.5 [MꢀH]ꢀ.
Chemokine-induced calcium signalling assay
Ca2+ mobilisation assays were performed by the use of a fluorometric imaging plate reader (FLIPR) (Molecular Devices,
Sunnyvale, USA) as described previously.26 Briefly, U87.CXCR4ꢀ and U87.CCR5ꢀtransfected cells were loaded with the
fluorescent calcium indicator Fluoꢀ3 acetoxymethyl (Molecular Probes, Leiden, The Netherlands) in the appropriate culture
medium for 45 min at 37°C, after which the cells were washed three times in Hanks balanced salt solution buffer containing 20
mM HEPES and 0.2% bovine serum albumin (pH 7.4). The cells were then incubated in the dark at 37 °C for 15 min with the
compounds. Changes in intracellular calcium concentration upon addition of CXCLꢀ12 (SDFꢀ1) the specific ligand for CXCR4
were simultaneously measured in all 96 wells in a blackꢀwall microtiter plate and in real time with the FLIPR. The data were
expressed as fluorescence units versus time and were analysed using the FLIPR Control Software (Molecular Devices) and IC50
values were calculated using GraphPad Prism 4.0 software (San Diego, CA).
Anti-viral assays
AntiꢀHIV activity and cytotoxicity measurements in MTꢀ4 cells were based on the viability of cells that had been infected or not
infected with the CXCR4ꢀusing (X4) HIVꢀ1 strain NL4.3 and exposed to various concentrations of the test compound. After the
MTꢀ4 cells were allowed to proliferate for 5 days, the number of viable cells was quantified by a tetrazoliumꢀbased colorimetric
method as described by Pauwels et al 27
.
Flow cell cytometry anti-CXCR4 antibody competition studies
Cell culture: Human leukaemia T cell lymphoblasts (Jurkat) were obtained from MRL (University of Hull, UK). These cells were
cultured in RPMI 1640 medium treated with sterile filtered fetal bovine serum (FBS) (10 %) and penicillin and streptomycin (100
units/ 5 mL) antibiotics. The cells cultures were maintained at 37 °C in a humidified, CO2 (5 %) controlled atmosphere with
subculturing done every 2ꢀ3 days as appropriate.
General procedure for binding assay with 12G-5 conjugated to phycoerythrin:
Phycoerythrin (PE)ꢀconjugated mouse monoclonal antiꢀhuman CXCRꢀ4 and Mouse IgG2A isotype controlꢀPE were purchased
from R&D systems Europe, Abingdon, UK.
Cells are harvested at about 75 % confluency, centrifuged, resuspended in 10 mL PBS and centrifuged again. Cells are kept on ice
to prevent receptor internalisation. Cells are resuspended in 1.0 mL of PBS/0.25 % BSA/0.01M NaN3 and viable cells are counted
using trypanꢀblue exclusion test. Cells at a density of 1ꢀ2.105 cells were aliquoted into polypropylene FACS tubes and
preincubated with 20 ꢀM of the compound (10 ꢀL in 18.2 mꢁ water) for one hour at 4 °C. A high concentration of compound was
used to ensure saturation of the receptors. Thereafter, cells were washed with 1mL of the buffer (PBS/ 0.25 % BSA/ 0.01M NaN3)
to remove the excess of compound that did not bind to the cells’ receptors. Cells were then incubated with the 12Gꢀ5 mAb
conjugated to phycoerythrin for a further 60 min. The cells were washed with 1 mL of the buffer and put in suspension in 300 ꢀL
of the buffer. Negative control was performed using the same protocol without the preincubation step. The binding of our
compound was analysed by flow cytometry on a FACScan flow cytometer (BD Biosciences Europe, Erembodegem, Belgium)
using the following FL channel FLꢀ2 575/26 nm (PE/PI).
The potency of compounds is reported as a concentration required to inhibit a specified amount (%) of the mABs. The Mean
Fluorescent Intensity (MFI) was used as a measure of binding and a quantitative way of calculating the inhibition percentage of
mAb.
ꢉ
ꢙ
ꢊꢋꢌ ꢍꢎꢏꢏꢐ ꢑꢃꢅℎ ꢍꢆꢀꢒꢆꢓꢄꢔ − ꢊꢋꢌ ꢄꢎꢕꢖꢅꢃꢗꢎ ꢍꢆꢄꢅꢘꢆꢏ
% ꢀꢁꢂ ꢃꢄℎꢃꢂꢃꢅꢃꢆꢄ = 100 − ꢇꢈ
ꢚ ꢛ 100ꢜ
ꢉ
ꢙ
ꢊꢋꢌ ꢒꢆꢐꢃꢅꢃꢗꢎ ꢍꢆꢄꢅꢘꢆꢏ − ꢊꢋꢌ ꢄꢎꢕꢖꢅꢃꢗꢎ ꢍꢆꢄꢅꢘꢆꢏ
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Notes and references
a ICMUB (UMR CNRS 6302), 9 Av. Alain Savary, BP 47870 21000 Dijon – France
b Chematech, 9 Av. Alain Savary, BP 47870 21000 Dijon – France
c Rega Institute for Medical Research, KU, Leuven, Bꢀ3000 Leuven – Belgium
d Positron Emission Tomography Research Centre, The University of Hull, Cottingham Road, Hull HU67RX – UK
e Universitätsklinikum Freiburg, IMSRobertꢀKochꢀStr. 1, Freiburg, Dꢀ79106 – Germany
Electronic Supplementary Information (ESI) available: spectra and analytical data for the compounds synthesised. See DOI: 10.1039/b000000x/
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