CXCR4 chemokine receptor antagonists: Nickel(ii) complexes of configurationally restricted macrocycles

Article abstract of DOI:10.1039/c2dt31137b

Tetraazamacrocyclic complexes of transition metals provide useful units for incorporating multiple coordination interactions into a single protein binding molecule. They can be designed with available sites for protein interactions via donor atom-containi

Full text of DOI:10.1039/c2dt31137b

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PAPER
CXCR4 chemokine receptor antagonists: nickel(II) complexes of
configurationally restricted macrocycles†
Rachel Smith,a Dana Huskens,b Dirk Daelemans,b Ryan E. Mewis,a Courtney D. Garcia,c Amy N. Cain,c
TaRynn N. Carder Freeman,c Christophe Pannecouque,b Erik De Clercq,b Dominique Schols,b
Timothy J. Hubin*c and Stephen J. Archibald*a
Received 25th May 2012, Accepted 11th July 2012
DOI: 10.1039/c2dt31137b
Tetraazamacrocyclic complexes of transition metals provide useful units for incorporating multiple
coordination interactions into a single protein binding molecule. They can be designed with available
sites for protein interactions via donor atom-containing amino acid side chains or labile ligands, such as
H2O, allowing facile exchange. Three configurationally restricted nickel(II) cyclam complexes with either
one or two macrocyclic rings were synthesised and their ability to abrogate the CXCR4 chemokine
receptor signalling process was assessed (IC50 = 8320, 194 and 14 nM). Analogues were characterised
crystallographically to determine the geometric parameters of the acetate binding as a model for aspartate.
The most active nickel(II) compound was tested in several anti-HIV assays against representative viral
strains showing highly potent EC50 values down to 13 nM against CXCR4 using viruses, with no
observed cytotoxicity (CC50 > 125 μM).
Introduction
Chemokine receptors are signalling proteins (G-protein coupled
receptors) located on the cellular surface, which have key phys-
iological roles including the recruitment of leukocytes to sites of
infection and the trafficking of stem cells for organ formation at
the embryonic stage. There are currently 19 known chemokine
receptors with a confirmed role1 and we have been particularly
interested in producing molecules to bind to the CXCR4 recep-
tor.2 There are only two clinically approved drugs targeting che-
mokine receptors: maraviroc, which targets the CCR5 receptor
and is used to treat HIV infection3 and plerixafor, which targets
the CXCR4 chemokine receptor4–7 and is used for haemato-
poietic stem cell mobilisation8,9 to allow harvesting in patients
with B-cell non-Hodgkin’s lymphoma and multiple myeloma
prior to immuno-compromising treatment. CXCR4 is also linked
with many other diseases such as arthritis, asthma, HIV infection
CXCL12.2 Our use of metal complexes shows that increased
and various types of cancer.10,11
activity and receptor residence time can be achieved.12–16 Plerix-
There is a current interest in high affinity compounds that
afor is a cyclam based compound that contains two macrocyclic
antagonise the chemokine receptor CXCR4 and block its inter-
rings with an ideal ring size cavity for binding to first row
actions with both HIV and its cognate signalling partner
transition metals. It was initially demonstrated that some of
the metal complexes show increased biological activity and the
key binding interactions were investigated by site directed
aDepartment of Chemistry, The University of Hull, Cottingham Road,
Hull, UK, HU6 7RX. E-mail: s.j.archibald@hull.ac.uk;
mutagenesis.17,18
Side and cross bridged macrocyclic chelators have been devel-
Fax: +44(0)1482466410; Tel: +44 (0)1482 465488
bRega Institute for Medical Research, KU, Leuven, B-3000 Leuven,
oped for a variety of biomedical applications including stable
complex formation with metal radioisotopes for in vivo use.19–24
Belgium
cDepartment of Chemistry and Physics, Southwestern Oklahoma State
They adopt a more rigid shape which impacts on the kinetics of
University, Weatherford, OK 73096, USA
complex formation and dissociation.25–27 They can also be used
†CCDC reference numbers 891843 and 891844. For crystallographic
data in CIF or other electronic format see DOI: 10.1039/c2dt31137b
to fix the configuration of the metal complexes. The unrestricted
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cyclam ring can adopt one of six configurations on complex for-
mation with a metal ion when binding to all four of the N-donor
atoms.28
two steps). Reduction of the cyano group was then carried out
using lithium aluminium hydride to give the desired product in
an 86% yield.
The only nickel(II) complex investigated as a CXCR4 antag-
onist is the plerixafor/AMD3100 complex.4 However, nickel(II)
is particularly interesting due to its varying coordination prefer-
ences. It can either form diamagnetic square planar complexes or
expand its coordination sphere to incorporate additional donors.
The anti-HIV data on [Ni2AMD3100]4+ shows that the com-
pound has a very similar potency to the free ligand and is lower
than the equivalent zinc(II) complex.17 Following on from our
previous studies on configurationally restricted macrocyclic
zinc(II) complexes that showed increased potency, relative to
both plerixafor and its complexes, we now extend this study to
nickel(II).29
The meta-substituted bis-macrocyclic derivative 3 was syn-
thesised according to a procedure developed for the analogous
para-derivative. The first step was again carried out using a
modified version of Handel and co-workers reaction and
the final two steps by our published procedure, which is based
on protocols developed by Weisman and co-workers.23,24,31
The final two steps (methylation and reduction) resulted in the
isolation of the desired product in an overall 59% yield.
Sadler and co-workers have studied the configuration of
the nickel(II) complex of plerixafor/AMD3100 and shown the
adoption of the folded (cis-V) and planar (trans-III) geo-
metries.30 They demonstrated that a configurational equilibrium
exists in solution and that the cis-V compound can form on
binding to protein aspartate residues. We selected three com-
pounds for our studies that are all configurationally fixed by
either steric interactions or a topological constraint. This is
provided via an ethyl bridge between adjacent or non-adjacent
nitrogens, respectively.
We have previously synthesised the piperazino side-bridged
cyclam chelator 1 and studied its copper(II) and zinc(II) com-
plexes.13,29 X-ray structural data revealed a trans-II macrocyclic
configuration with the metal ion displaced from the plane of
the macrocyclic nitrogen donors. NMR data showed that the
zinc(II) complex adopts this single configuration in aqueous
solution.29 Chelator 2 is a novel monomacrocyclic compound
with a single primary amino group in the position of the
second macrocyclic group. It was thought that this may
provide an H-bonding interaction with a second aspartate/gluta-
mate residue on binding to the protein. The third cyclam-based
compound 3 incorporates two rings at the meta positions on
the central phenyl ring. This shortens the distance between
the two metal centres on complex formation. Nickel(II) com-
plexes were formed and their interaction with the CXCR4
chemokine receptor probed through cellular signalling and anti-
viral assays.
Nickel(II) complexes were formed by stirring with an
appropriate metal salt at RT or heating in a methanolic solution.
The orange colour (λmax = ca. 480 nm) of the complexes
formed with chelators 1 and 2 indicates a square planar co-
ordination environment. However, the brown colour of [Ni23]4+
indicates a six coordinate geometry. To produce single crystals
of the related complexes, nickel(II) compounds were also syn-
thesised with known ligands (8, 9 and 10) using similar
procedures.
Single crystal X-ray crystallographic studies
X-ray crystallographic studies were carried out to determine the
exact geometric parameters of the nickel(II) coordination, see
Tables 1 and 2. We did not have any success in growing crystals
of sufficient quality for single crystal X-ray structural determi-
nation of the nickel(II) complexes of 1, 2 or 3 and so model
systems were studied using chelators 8, 9 and 10 that were more
amenable to crystallisation. Three single ring cross bridged
nickel(II) complexes were crystallised. The chelating ligands 9
and 10 were used to give an indication of the steric impact of
methyl vs. benzyl N-substitution on the acetate interaction as a
model for aspartate binding in the protein. A search of the crys-
tallographic database was carried out to find known structures
for comparison.
Results and discussion
Synthesis
There are currently no side bridged (piperazino) cyclam
nickel(II) complexes deposited in the Cambridge Structural
Database.33 We prepared the nickel(II) complex of the known
mono-N-methyl piperazino cyclam 8. A full structural refinement
was not possible due to the low data quality but the coordination
geometry around the nickel(II) and the ligand configuration
were clear, see Fig. 1. The trans-II configuration is consistent
with all the other X-ray structures known for piperazino methyl
cyclam-based chelators. It can be compared to the nickel(II)
perchlorate complex of the piperazino side bridged cyclen,
which was initially prepared by Ramasubbu and Wainwright
and the crystal structure reported later.34,35 The UV-vis spectrum
contains a band centred at approximately 480 nm, indicating a
Compound 1 was synthesised according to our previously pub-
lished route.29 The standard route to mono-substitution, required
to form compound 2, was carried out using a synthesis devel-
oped from the work of Handel and co-workers, see Scheme 1.31
In this case, a cyano derivative was used to later introduce a
primary amino group that offers additional H-bonding potential
with the protein target or a position for subsequent functionalisa-
tion (not used in this work). We have previously synthesised a
compound with an aniline amino group that has a shorter dis-
tance to the macrocycle and reduced flexibility for the orientation
of the primary amine.12 The substitution reaction and sodium
borohydride reaction proceed in high yields (71% overall for the
11370 | Dalton Trans., 2012, 41, 11369–11377
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Scheme 1 Synthetic routes to produce the novel chelators 2 and 3.
Table 1 Crystal data for the single crystal X-ray structures of Ni9 and
Ni10
Table 2 Selected bond lengths (Å) and angles (°) for the single crystal
X-ray structures of Ni9 and Ni10
[Ni9(OAc)OH2]+
[Ni10(OAc)OH2]+
Bond lengths/
angles Ni9
Bond lengths/
angles Ni10
Formula
Mr
C16H35N4O3F6NiP
535.16
C29H45N4O4F6NiP
717.37
Ni–N1/N22
2.091(3)
2.135(3)
Crystal system
Space group
Trigonal
R3c
Triclinic
P1
Ni–N2/N23
Ni–N3/N21
2.181(3)
2.103(3)
2.188(3)
2.128(3)
ˉ
a (Å)
b (Å)
c (Å)
α (°)
26.005(2)
26.005(2)
17.2968(11)
90
90
120
10 129.9(13)
18
1.579
1.008
150
2.52 to 34.73
18 505
8024 [0.0839]
99.5% (34.73)
0.681
0.0404, 0.0717
0.1165, 0.0815
0.035(12)
0.766 and −0.381
10.2997(14)
10.4483(13)
16.902
98.981(10)
104.607(10)
106.546(11)
1635.2(4)
2
1.457
0.717
150
2.57 to 34.87
37 739
13 881[0.0864]
99.8% (25.00)
0.907
0.0852, 0.2159
0.1642, 0.2680
0.161(9)
Ni–N4/N10
Ni–O1
Ni–O1W/O3
2.202(3)
2.064(2)
2.077(3)
2.177(3)
2.052(2)
2.109(3)
N1–Ni–N2/N22–Ni–N23
N1–Ni–N3/N22–Ni–N21
N1–Ni–N4/N22–Ni–N10
N2–Ni–N3/N23–Ni–N21
N2–Ni–N4/N23–Ni–N10
N3–Ni–N4/N21–Ni–N10
O1–Ni–N1/O1–Ni–N22
O1–Ni–N2/O1–Ni–N23
O1–Ni–N3/O1–Ni–N21
O1–Ni–N4/O1–Ni–N10
O1W–Ni–N1/O3–Ni–N22
O1W–Ni–N2/O3–Ni–N23
O1W–Ni–N3/O3–Ni–N21
O1W–Ni–N4/O3–Ni–N10
O1–Ni–O1W/O1–Ni–O3
84.15(10)
84.02(10)
91.29(11)
90.67(11)
173.41(11)
84.11(10)
91.19(10)
95.08(10)
172.12(10)
89.76(10)
170.63(10)
86.56(10)
97.39(10)
98.06(11)
88.35(10)
85.66(12)
85.50(12)
91.70(12)
91.74(11)
175.41(10)
84.30(11)
88.93(11)
93.31(10)
172.18(11)
90.39(11)
170.47(11)
85.44(11)
98.17(12)
97.39(11)
88.18(11)
β (°)
γ (°)
Volume (Å3)
Z
Density (calc.) (M gm−3
)
μMo–Kα (mm−1
T (K)
)
θ range (°)
Measured reflections
Unique reflections [Rint
Completeness (θo)
]
Goodness-of-fit on F2
R1, wR2 (I > 2σ(I))
R1, wR2 (all data)
Extinction coefficient
Largest diff. peak and hole
1.108 and −1.003
(e Å−3
)
The Hirshfeld surfaces clearly indicate the direction of these
interactions, see Fig. 2. The [Ni10OAc(OH2)]+ structure has the
same coordination sphere but an additional solvent molecule
involved in a H-bonding interaction, see Table 3 and Fig. 2. The
two structures have very similar bond lengths and angles, with
the nickel(II) atom sitting very slightly further into the cavity for
the complex with 9, as demonstrated by the comparison between
the bond lengths and angles for the ethyl linked N-donors N1
and N3.
There are two cross bridged nickel(II) cyclam structures depos-
ited in the crystallographic database (reference codes: AGIXIX
and AGIXUJ).36 However, of greater relevance to this study are
the nickel(II) complexes of cyclam-based macrocycles that have
a bound carboxylate ligand, with 103 such structures in the
square planar coordination geometry in aqueous solution.
The X-ray structure confirms the distorted square planar environ-
ment with nickel(II)–nitrogen bond lengths of ca. 1.85 to 1.88 Å.
The nickel(II) centre sits out of the mean nitrogen plane by 0.2 Å
with the piperazino unit in the expected boat conformation to
position the two nitrogen lone pairs into the cavity.
The structure of [Ni9OAc(OH2)]+ shows an octahedral
nickel(II) centre with a cis arrangement of a monodentate acetate
and a water molecule, giving a distorted octahedral coordination
geometry. The acetate and water molecules are involved in an
intramolecular hydrogen bonding interaction and both have inter-
molecular H-bonds with neighbouring complexes, see Table 3.
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cis-V macrocycle configuration and an identical coordination
sphere, including a bound water molecule and monodentate
acetate (CEBKOK).39 The most striking structure in the database
for comparison is one where two C-alkylated cyclam nickel(II)
complexes (tetA) are bridged by a bis-carboxylate (MASVOS).40
This contains one metal centre which is six coordinate with a
coordinated water molecule and a monodentate acetate ligand
and a second metal centre which is six coordinate with a biden-
tate acetate. The appearance of both binding modes in separate
identical macrocyclic species within the asymmetric unit
suggests a fine energetic balance between the two structural
types.
Fig. 1 X-ray structures of nickel(II) piperazino methylcyclam ([Ni8]2+
and the piperazinocyclen macrocyclic complexes. (The cyclam complex
from this work and the cyclen complex from Hancock and co-
workers).32
)
Biological assays: calcium signalling and anti-HIV activity
Table 3 Hydrogen bonds with H⋯A < r(A) + 2.0 Å and DHA > 110°
An initial screen of the compounds was carried out using a
calcium signalling assay in response to stimulation by the
chemokine protein binding partner of the CXCR4 receptor,
CXCL12. The concentration of intracellular calcium released
was measured and the abrogation of this flux by the antagonists
was recorded at a series of concentrations to give a value
whereby a 50% reduction was observed, see Table 4. The bis-
ring nickel(II) complexes show a higher potency than the mono
ring compound [Ni2]2+, which is only active at a micromolar
level. Due to its relatively low activity, this compound was
excluded from further studies. The para side bridged compound
[Ni21]4+ was significantly more active than the meta cross
bridged compound and 2-fold more active than plerixafor. The
cytotoxicity of the three nickel(II) complexes was measured in
MT-4 cells as a CC50 (a cytotoxic effect that reduces cell viabi-
lity by 50%) and in all cases this was greater than 125 μM, the
maximum value tested. It is anticipated that the metal free
macrocyclic compounds 1, 2 and 3 will have considerably lower
activity, consistent with the analysis of related ethyl bridged
macrocycles, due to the reduction in H-bond donors compared to
plerixafor.14,29
An initial anti-HIV assay was carried out with the two bis-
macrocyclic compounds using plerixafor as a comparison and
an X4 viral strain that utilises CXCR4 as a co-receptor for
viral entry rather than CCR5. In this assay, the meta compound
[Ni3]4+ was again less active than the para compound [Ni1]2+,
which shows a similar activity to plerixafor. On this basis,
[Ni1]2+ was selected for further studies of anti-viral activity
against a selected series of different viral strains: HIV-1,
HIV-2 and SIV. Activity was observed against both HIV-1 and
HIV-2 in two cell lines (MT-4 and Jurkat) and also in
peripheral blood mononuclear cells (PBMCs), as these are the
most relevant cellular model for evaluating HIV replication, see
Table 5.
d(D–H)
(Å)
d(H⋯A)
(Å)
<DHA
(°)
d(D⋯A)
(Å)
D–H⋯A
[Ni9OAc(OH2)]+
O1W– H1C⋯O2b
O1W–H1D⋯O2
O1W–H1D⋯O1
0.821
0.852
0.852
1.958
1.802
2.446
155.35
173.99
114.13
2.726
2.651
2.900
[Ni10OAc(OH2)MeOH]2+
O3–H1O3⋯O2
O3–H2O3⋯O4a
O4–H4C⋯O2
0.788
0.879
0.836
1.933
1.805
1.804
153.65
172.59
169.96
2.662
2.680
2.632
a [−x + 1, −y + 1, −z + 1]. b [−x + y − 1, −x + 1, z].
CSD. The large number appears to be due to the popularity of
this motif in crystal engineering, metal–organic frameworks and
coordination polymer formation.
This search was refined by limiting the hits to only the coordi-
nation to the carboxylate component of the additional ligand (i.e.
amino acids that also have N-donor binding were excluded). The
majority of the structures, including a large number of examples
showing coordination polymer formation using bis-carboxylates,
have the trans-III cyclam ring configuration28 with a six coordi-
nate octahedral nickel(II) centre. The four N-donors are planar
with further ligands above and below the ring plane to give an
octahedral geometry (typical example: NEXQAJ).30 Our
assumption is that a single carboxylate from the peptidic side
chain in the CXCR4 protein is bound to our compounds from
one side of the ring, due to the steric/topological constraint of
including an ethyl bridge, and so these structures were excluded
from further analysis. Seventeen structures with bound acetate
groups that are not planar were found, comprising fourteen
bidentate cis-V structures (including the structure determined by
Sadler and co-workers in their study of configuration)37 and
three structures containing monodentate acetate ligands, with
two of them including a bound water molecule.
High activity was observed against the wild type NL4.3,
which can be used as a reference for mutated viral strains. For
comparison, nevirapine, a non-nucleoside reverse transcriptase
inhibitor (NNRTI), that is currently used in clinical antiretroviral
drug combinations for treatment of HIV/AIDS, was also ana-
lysed against NL4.3. Nevirapine is ca. 50 times more active than
[Ni21]4+. However, nevirapine has a much higher cytotoxicity
value (CC50) of 2 μg mL−1 giving a lower selectivity index of
400 vs >5000 for [Ni21]4+.
One compound has a bulky acetate group forcing a five co-
ordinate geometry (distorted square based pyramidal) with an
unusual trans-IV ring configuration and a monodentate acetate
(ILOCUI).38 A cyclam derivative, with two of the nitrogens
cyclised to form a more rigid chelator, adopts a very similar geo-
metry to that observed for the compounds in this work, with a
11372 | Dalton Trans., 2012, 41, 11369–11377
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Fig. 2 ORTEP and Hirshfeld surface plots of [Ni9OAc(OH2)]+ and [Ni10OAc(OH2)]+.
Table 4 Calcium signalling inhibition and anti-HIV activity (nM)
CXCR4 for HIV cell entry: BaL and MAC251. As expected,
no activity was observed as the compounds synthesised are pro-
posed to be selective CXCR4 antagonists. To confirm this,
[Ni1]4+ was also tested in a CCR5 signalling abrogation assay
and shown to have no effect up to a concentration of 1 μM.
Ca2+ flux IC50
Anti-HIV-1b
Compound
CXCR4a (nM)
av. EC50c (nM)
[Ni21]4+
[Ni2]2+
14
74
—
398
11
8320
194
31
[Ni23]4+
Plerixafor
a U87.CXCR4 cell line. IC50 is the concentration of the compound
required to inhibit 50% of the CXCL12 (SDF-1) induced Ca2+
signalling. b The assay was carried out in a CD4+CXCR4+T cell line,
MT-4 cells, using the HIV-1 X4 strain IIIB. c Average effective
concentration to reduce the HIV-induced cytopathic effect by 50% in
MT-4 cells.
Conclusions
Two novel chelators and three novel nickel(II) complexes were
synthesised. X-ray crystal structures of related complexes were
used to infer possible binding modes when interacting with the
aspartate side chains of the CXCR4 protein. The para arrange-
ment of the macrocyclic components appears to be essential for
the high activity of the two ring compounds. The most active
compound showed highly potent anti-HIV activity (13 nM)
against the X4 NL4.3 viral strain and improved the blocking of
CXCL12/CXCR4 signalling compared to AMD3100/plerixafor.
There is potential for further development of configurationally
restricted nickel(II) macrocyclic compounds to optimise both the
arrangement of the units and binding with protein aspartate
residues. The X-ray structural data showed that a water molecule
can also be coordinated to the metal centre. This could be incor-
porated when bound to the protein but analysis of the related
structures in the crystallographic database suggested a fine
Resistant viral strains were also investigated. They were develo-
ped by repeat passaging and selection of the virus under chal-
lenge from AMD3100/plerixafor. It is assumed that the binding
site for [Ni21]2+ will be similar to plerixafor and so similar resis-
tance may be observed. This was the case, as a drop in activity
but not complete resistance was observed. Competition with
AMD3100/plerixafor for inhibition of the resistant viral strain
did not show a significant impact on the activity.
Experiments were carried out to determine the activity against
two viral strains that use the CCR5 co-receptor rather than
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Table 5 Anti-HIV activity (nM) of [Ni21]4+ in assays using three different cell lines
MT-4
EC50
PBMC
EC50
Jurkat
EC50
Viral straina
SIb
SIb
SIb
IIIB
ROD
NL4.3 WT
NL4.3 AMD3100res
74
450
13
>1698
>278
>9690
>52
273
—
24
>458
—
>5000
>40
537
4360
370
>233
>29
>338
>2.6
2400
3130
47 860
MAC251
BaL
—
—
—
—
>125 000
>125 000
1
1
—
—
—
—
a IIIB (HIV-1), ROD (HIV-2), NL4.3 WT (HIV-1), NL4.3 AMD3100res (AMD3100 resistant strain of HIV-1 NL4.3), MAC251 (R5 SIV strain) and
HIV-1 BaL (R5 HIV-1 strain). b Selectivity index based on the ratio of CC50 to EC50
.
Synthesis of 1-[4-cyanobenzyl]-1,4,8,11-tetraazabicyclo[10.2.2]-
hexadecane, 5
energetic balance between a bidentate acetate or a monodentate
acetate and a water molecule.
We have demonstrated that nickel(II) complexes can have low
cytotoxicity and high potency against HIV, offering excellent
selectivity indices. The flexible coordination sphere of nickel(II)
is a good match for introducing strong binding interactions with
the protein amino acid residues and offers significant scope for
the future development of metallodrugs.
3a-[4-Cyanobenzyl]-decahydro-3a,5a,8a,10a-tetraaza-pyrenium
bromide (4) (1.00 g, 2.4 mmol) was dissolved in ethanol
(100 mL). Sodium borohydride (2.27 g, 60.0 mmol) was added
in small portions and the mixture was stirred for 30 minutes then
heated to reflux for 2 hours. Water (100 mL) was added to
decompose any excess sodium borohydride and the solvents
were removed in vacuo. The residue was taken up into water
(150 mL) and made strongly basic (pH 14, KOH). The basic
solution was extracted with DCM (4 × 150 mL) and the com-
bined organic extracts were dried (Na2SO4), filtered and evapo-
Experimental
NMR spectra were obtained using a JEOL400 spectrometer.
UV-vis spectra were obtained using an Agilent 800 diode
spectrometer. Electrospray mass spectra were obtained using a
Finnigan LCQ spectrometer. Accurate mass measurements were
carried out by the EPSRC’s National Mass Spectrometry Service
at Swansea. All materials were purchased from Aldrich Chemical
Co. or Lancaster Synthesis Ltd, and used as supplied. Cyclam,41
glyoxal cyclam,31 6,31 8,42 9 and 1024 were synthesised in
accordance with literature procedures. Compounds 129 and 423
were synthesised by our previously reported methods. Solvents
were dried over molecular sieves prior to use.43
Diffraction data sets were collected on a Stoe IPDS-II image
plate diffractometer, using Mo–Kα (0.71073 Å) radiation. The
crystal was kept at a temperature of 150(1) K during data collec-
tion, controlled using the Oxford Cryosystems Cryostream
Cooler.44 Data sets were all collected to θ = 35° but truncated
where necessary. For weakly diffracting crystals, refinement was
attempted with both full and truncated data sets. All structures
were solved by direct methods (SHELXS45) and refined against
F2 (SHELXL46). Hydrogen atoms located on carbon atoms were
placed geometrically and refined against a riding model with
Uiso = 1.2 Ueq of the carrier atom or Uiso = 1.5 Ueq for methyl
groups. The WinGX package was used for the refinement and
production of data tables and ORTEP-3 was used for the struc-
ture visualisation.47 Hydrogen bonding was determined using
SHELXL and by the generation of Hirshfeld surface plots based
on dnorm using CrystalExplorer.48 All ORTEP representations
show ellipsoids at a 50% probability level.
1
rated in vacuo to yield a colourless oil. Yield = 93%. H NMR
(400 MHz, CDCl3, δ): 1.62–1.71 (m, N-β-CH2, 4H), 2.18 (m,
N-α-CH2, 2H), 2.45–2.61 (m, N-α-CH2,12H), 2.80 (t, N-α-CH2,
2H), 2.96 (m, N-α-CH2, 2H), 3.12 (m, N-α-CH2, 2H), 3.61 (s,
Ar CH2 and NH, 3H), 7.35 (d, Ar H, 2H), 7.54 (m, H–Ar, 2H).
13C{1H} NMR (CDCl3): δ 23.46 (N-β-CH2), 25.80 (N-β-CH2),
48.08 (N-α-CH2), 48.16 (N-α-CH2), 50.03 (N-α-CH2),
51.16 (N-α-CH2), 54.49 (N-α-CH2), 54.76 (N-α-CH2), 54.98
(N-α-CH2), 56.78 (N-α-CH2), 57.53 (Ar CH2), 110.67 (Ar C),
118.80 (CN), 129.71 (Ar CH), 131.80 (Ar CH), 144.41 (Ar C).
HRMS: [M + H]+ calcd for C20H31N5 m/z = 342.2658, found
m/z = 342.2652.
Synthesis of 1-[4-aminomethylbenzyl]-1,4,8,11-tetraazabicyclo-
[10.2.2]hexadecane, 2
Lithium aluminium hydride (0.63 g, 16.70 mmol) was sus-
pended in dry THF (20 mL). 1-[4-Cyanobenzyl]-1,4,8,11-tetra-
azabicyclo[10.2.2]hexadecane (6) (1.90 g, 5.50 mmol) in dry
THF (30 mL) was added dropwise under ice-cooling. After com-
plete addition, the mixture was stirred for 30 minutes and then
heated to reflux for 3 hours. The reaction was cooled in an ice-
bath, water (0.40 mL) was added dropwise followed by 15%
sodium hydroxide (0.40 mL) followed by a second portion of
water (1.20 mL). The resulting white precipitate was filtered
and washed with THF (2 × 20 mL) then water (2 × 10 mL).
The aqueous layer was made strongly basic (pH 14, KOH) and
extracted with THF (5 × 25 mL). The organic phases were dried
(Na2SO4), filtered and evaporated in vacuo to yield a yellow oil.
WARNING: Perchlorate salts are potentially explosive
when dry. Although no problems were encountered in these
preparations, care should be exercised when using these
compounds.
1
Yield = 86%. H NMR (400 MHz, CDCl3, δ): 1.74–1.86 (m,
N-β-CH2, 4H), 2.23–2.27 (m, N-α-CH2, 2H), 2.52–2.62 (br m,
11374 | Dalton Trans., 2012, 41, 11369–11377
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Synthesis of [Ni1](NO3)4
N-α-CH2 + NH, 12H), 2.92–3.00 (m, N-α-CH2, 3H), 3.25–3.28
(m, N-α-CH2, 2H), 3.65 (s, N-α CH2, 1H), 3.73–3.76 (m,
CH2-NH2, 2H), 3.86 (s, Ar CH2, 2H), 7.23–7.29 (m, Ar H, 4H).
13C{1H} NMR (100 MHz, CDCl3, δ): 23.37 (N-β-CH2),
25.51 (N-β-CH2), 26.07 (N-α-CH2), 30.22 (N-α-CH2), 46.11
(N-α-CH2), 48.02 (N-α-CH2), 50.45 (N-α-CH2), 51.03
(N-α-CH2), 54.59 (N-α-CH2), 54.90 (N-α-CH2), 55.32
(N-α-CH2), 56.21 (N-α-CH2), 56.85 (Ar CH2), 60.87 (CH2–
NH2), 126.59 (Ar C), 129.66 (Ar CH), 136.15 (Ar CH), 141.83
(Ar C). HRMS: [M + H]+ calcd for C20H36N5 m/z = 346.2971,
found m/z = 346.2969.
1 (200 mg, 0.36 mmol) was dissolved in methanol (20 mL) and
a methanolic (5 mL) solution of nickel(II) nitrate hexahydrate
(230 mg, 0.79 mmol) was added dropwise. The mixture was
heated to 60 °C for 30 minutes under nitrogen, then stirred at RT
overnight. The resulting solution was concentrated to ∼5 mL
in vacuo then purified via size exclusion chromatography (sepha-
dex LH20) to yield an orange solid. Yield = 87%. HRMS:
[M − 4NO3 − 2H]2+ calcd for C32H56N8Ni2 m/z = 334.1663,
found m/z = 334.1662. Anal. calcd for C32H56Ni2N12O12·
2CH3OH: C, 41.49; H, 6.76; N, 17.08; found C, 41.11; H, 6.70;
N, 16.78. UV-vis. (MeOH): λmax 483 nm, ε = 396 M−1 cm−1
.
Synthesis of 1,3-xylyl-bis(8a[methyl]-decahydro-3a,5a,8a,10a-
tetraazapyrenium) tetraiodide, 7
Synthesis of [Ni2](NO3)2
6 (2.60 g, 3.7 mmol) was suspended in dry acetonitrile (150 mL)
under nitrogen and methyl iodide (2.31 mL, 37 mmol) was
added in one portion. The resulting white suspension was stop-
pered and stirred at room temperature for 14 days under an
atmosphere of nitrogen. At the end of the reaction time, excess
methyl iodide was removed by bubbling nitrogen through the
suspension for 30 minutes. The white solid was filtered off,
washed with diethyl ether (200 mL) and dried in vacuo. Yield =
76%. 13C{1H} NMR (75 MHz, DMSO, δ): 16.94 (N-β-CH2),
17.12 (N-β-CH2), 44.83 (N-α-CH3), 45.08 (N-α-CH2), 46.39
(N-α-CH2), 47.52 (N-α-CH2), 49.15 (N-α-CH2), 50.29
(N-α-CH2), 58.58 (N-α-CH2), 59.18 (N-α-CH2), 61.77
(N-α-CH2), 62.73 (N-α-CH2), 73.93 (Caminal), 74.17 (Caminal),
125.88 (Caromatic), 129.03 (Caromatic), 134.74 (Caromatic), 135.02
(Caromatic). MS (ES): m/z = 343.0 [M − macrocycle]+, 237.0
[M − xylyl − macrocycle]+. Anal. calcd for C34H58N8I4: C,
37.59; H, 5.38; N, 10.31; found: C 37.84, H 5.25, N 10.15.
The same method was used as for the synthesis of [Ni1](NO3)4
with the following amounts: 1-[4-aminomethylbenzyl]-1,4,8,11-
tetraazabicyclo[10.2.2] hexadecane (2) (110 mg, 0.32 mmol)
was dissolved in methanol (10 mL) and nickel(II) nitrate hexa-
hydrate (102 mg, 0.35 mmol). Yield = 63%. HRMS: M2+ calcd
for C20H35N5Ni m/z = 201.6118, found m/z = 201.6117.
Anal. calcd for C20H34N7NiO6: C, 42.37; H, 7.11; N, 16.47;
found C, 42.51; H, 6.88; N, 16.64. UV-vis. (H2O): λmax 476 nm,
ε = 174 M−1 cm−1
.
Synthesis of [Ni3](CH3CO2)2(PF6)2
In an inert atmosphere glovebox, 3 (0.437 g, 0.75 mmol) was
dissolved in DMF (20 mL) and anhydrous Ni(CH3CO2)2
(0.265 g, 1.5 mmol) was added with stirring. The solution was
heated to ca. 60 °C overnight. The reaction mixture was removed
from the glovebox, filtered to remove trace solids, evaporated to
dryness and dried in vacuo. The brown solid was dissolved in
4 ml of dry methanol and NH4PF6 (1.22 g, 7.5 mmol), dissolved
in an additional 4 mL of dry methanol, was added dropwise to
the stirring complex solution. A fine, brown precipitate formed.
Completion of precipitation was achieved by storing the mixture
overnight at −5 °C. The brown solid product was collected by
filtration on a fine frit, washed with cold methanol and then ether
and dried in vacuo. Yield = 0.413 g (56%). The elemental analy-
sis is satisfactory as a mixed acetate/hexafluorophosphate salt,
[Ni23(CH3CO2)2.1](PF6)1.9. Anal. calcd for C38.2H68.3F5.4Ni2-
N8O4.2P1.9: C, 41.72; H, 6.26; N, 10.19; found C, 42.02; H,
6.37; N, 9.87. MS (ES): m/z = 961.2 [M − PF6]+. UV-vis.
(H2O): λmax 376sh, 561sh, 897 nm.
Synthesis of 1,3-xylyl-bis(8-methyl-1,4,8,11-tetraazabicyclo-
[6.6.2]hexadecane, 3
7 (3.03 g, 2.8 mmol) was dissolved in ethanol (150 mL).
Sodium borohydride (6.38 g, 168 mmol) was added in small
portions and the clear solution was stirred at room temperature
under N2 for 7 days and concentrated in vacuo. Water (100 mL)
was added then 6 M HCl was added to decompose any excess
sodium borohydride until a pH = 2 was reached. The mixture
was made strongly basic (pH 14, KOH) and extracted from
dichloromethane (5 × 100 mL). The combined organic fractions
were dried (MgSO4), filtered and evaporated in vacuo to yield a
yellow oil. The HCl salt was prepared by bubbling HCl gas into
an ethanolic solution of the ligand, collecting the resultant
yellow precipitate by filtration and washing it with ethanol fol-
lowed by diethyl ether. Yield = 77%. 13C{1H} NMR (75 MHz,
D2O, δ): 18.52 (N-β-CH2), 18.91 (N-β-CH2), 39.69 (N-α-CH3),
44.56 (N-α-CH2), 49.00 (N-α-CH2), 49.13 (N-α-CH2), 50.01
(N-α-CH2), 52.53 (N-α-CH2), 52.91 (N-α-CH2), 56.02 (N-
α-CH2), 57.36 (N-α-CH2), 58.41 (N-α-CH2), 59.16 (N-α-CH2),
127.09 (Caromatic), 130.23 (Caromatic), 133.98 (Caromatic), 135.62
(Caromatic). Anal. calcd for C34H62N8·8HCl·5.05H2O: C 42.29, H
8.36, N 11.60; found: C 42.66, H 8.64, N 11.21. MS (ES): m/z =
584 [M + H]+.
Synthesis of [Ni8](ClO4)2
8 (1.19 g, 2.4 mmol) was dissolved in acetonitrile (20 mL) and
Ni(ClO4)2·6H2O (0.91 g, 2.5 mmol) in acetonitrile (20 mL) was
added dropwise whilst stirring. The crude product was purified
using size exclusion chromatography (sephadex LH20). The
solvent was then removed from the orange solution, producing
an orange crystalline solid, and dried in vacuo. Yield = 71%.
1H NMR (400 MHz, CDCl3, δ): 1.31–1.38 (m, 2H, N-β-CH2),
1.64–1.75 (m, 2H, N-β-CH2) 2.08–2.15 (m, 8H, N-α-CH2),
2.21–2.31 (m, 2H, N-α-CH2), 2.38–2.55 (m, 2H, N-α-CH2),
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2.62–2.78 (m, 2H, N-α-CH2), 2.83–2.2.96 (m, 2H, N-α-CH2),
3.12–3.21 (m, 2H, N-α-CH2), 3.33–3.47 (m, 2H, N-α-CH2),
3.56 (s, 3H, N-CH3). 13C{1H} NMR (100 MHz, CDCl3, δ):
21.31 (N-β-CH2), 24.2 (N-β-CH2), 39.11 (N-CH3), 45.21 (N-
α-CH2), 47.44 (N-α-CH2), 51.88 (N-α-CH2), 52.31 (N-α-CH2),
55.13 (N-α-CH2), 58.13 (N-α-CH2), 59.11 (N-α-CH2), 59.97
(N-α-CH2). MS (ES): m/z = 297 M+.
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 minutes with the compounds. Changes in
the intracellular calcium concentration upon the addition of
CXCL12 (SDF-1), the specific ligand for CXCR4, was simul-
taneously measured in all 96 wells in a black-wall microtiter
plate and in real time with the FLIPR. The data was expressed as
fluorescence units versus time and was analyzed using the
Softmax PRO 4.0 program (Molecular Devices) and IC50 values
were calculated using the GraphPad Prism 4.0 software
(San Diego, CA).
Synthesis of [Ni9OAc(OH2)]PF6
In an inert atmosphere glovebox, 0.177 g (0.001 mol) of nickel
(II) acetate and 0.254 g (0.001 mol) of 9 were stirred in 25 mL of
anhydrous acetonitrile and heated to 50–60 °C overnight. The
reaction mixture was then removed from the glovebox, filtered
through filter paper and the acetonitrile evaporated. The remain-
ing nickel complex was dried in vacuo overnight to remove any
solvent, leaving a hygroscopic purple powder. This powder was
dissolved in 3 ml of anhydrous methanol. Five equivalents
(0.005 mol, 0.815 g) of NH4PF6 dissolved in 3 mL of anhydrous
methanol were added dropwise to the stirring metal complex,
causing precipitation of a pale purple powder. The reaction
mixture was stored overnight in a freezer to complete the precipi-
tation. A pale purple powder was collected on a fine glass frit,
washed with cold methanol and ether and dried in vacuo. Yield:
0.343 g (75%). X-ray quality crystals, pale purple plates, were
grown from an ether diffusion into a nitromethane solution.
Elemental analysis (%) calcd [NiC14H30N4(C2H3O2)](PF6)·1.5
H2O: C 35.32, H 6.67, N 10.30; Found C 35.46, H 6.68,
N 10.10. MS (ES) m/z = 371 [Ni9(OAc)]+, 331 [Ni9(H2O)]+.
Anti-viral assays
Anti-HIV activity and cytotoxicity measurements in MT-4 and
other cells were based on the viability of the cells that had been
infected or not infected with HIV-1, HIV-2 or SIV and exposed
to various concentrations of the test compound.50 After the cells
were allowed to proliferate for 5 days, the number of viable cells
was quantified by a tetrazolium-based colorimetric method, as
described by Pauwels et al.51,52 PBMCs were purified and
infected with HIV and SIV, as described in detail by Schols
et al.53
Acknowledgements
We would like to acknowledge the University of Hull for an 80th
Anniversary studentship awarded to RS and the EPSRC for
funds to purchase the diffractometer on which the X-ray data
were collected. SJA thanks the Yorkshire Forward for the award
of a Yorkshire Enterprise Fellowship in translational molecular
imaging technologies. We would also like to acknowledge the
use of the EPSRC’s National Mass Spectrometry Service Centre
at Swansea and the Chemical Database Service at Daresbury.
This project was supported by the National Center for Research
Resources and the National Institute of General Medical
Sciences of the National Institutes of Health through Grant
Number 8P20GM103447. This work was also supported by
the FWO (G.0528.12 N) and the KU Leuven (PF/10/018 and
GOA/10/014).
Synthesis of [Ni10OAc(OH2)]PF6·MeOH
In an inert atmosphere glovebox, 0.177 g (0.001 mol) of nickel
(II) acetate and 0.406 g (0.001 mol) of 10 were stirred in 25 mL
of anhydrous DMF at room temperature for 18 hours. The re-
action mixture was then removed from the glovebox, filtered to
remove any trace solids and evaporated to dryness. The pale blue
solid residue was dissolved in 10 mL of dry methanol to which a
5 mL dry methanol solution of 5 equivalents (0.815 g,
5.00 mmol) of NH4PF6 was added dropwise. A pale purple
powder precipitated, was collected on a fine glass frit, washed
with cold methanol and ether and dried in vacuo. Yield: 0.584 g
(91%). X-ray quality crystals, pale blue needles, were obtained
from a cooled methanol solution. The elemental analysis is satis-
factory as a mixed acetate/hexafluorophosphate salt. Elemental
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