3,5-Dibromophenyl-functionalised imidazolium salts and their corresponding [Au(NHC)2]+ complexes: synthesis, supramolecular chemistry and anti-cancer activity

Article abstract of DOI:10.1007/s10847-021-01082-6

The synthesis and spectroscopic and structural characterisation of new 3,5-dibromophenyl-functionalised imidazolium salts and their corresponding [Au(NHC)₂]⁺ complexes is reported. X-ray diffraction studies revealed intra- and interm

Full text of DOI:10.1007/s10847-021-01082-6

Journal of Inclusion Phenomena and Macrocyclic Chemistry
https://doi.org/10.1007/s10847-021-01082-6
ORIGINAL ARTICLE
3,5‑Dibromophenyl‑functionalised imidazolium salts and their
corresponding [Au(NHC)2]+ complexes: synthesis, supramolecular
chemistry and anti‑cancer activity
Hawraa S. Al‑Buthabhak1,3 · Yu Yu2,4 · Alexandre Sobolev3 · Hani Al‑Salami5 · Murray V. Baker3
Received: 31 March 2021 / Accepted: 18 May 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021
Abstract
The synthesis and spectroscopic and structural characterisation of new 3,5-dibromophenyl-functionalised imidazolium salts
and their corresponding [Au(NHC)2]+ complexes is reported. X-ray difraction studies revealed intra- and intermolecular
interactions involving the 3,5-dibromophenyl group, including Br…π interactions with imidazolyl and C6 arene rings. Au-
NHC complexes functionalised with 3,5-dibromophenyl substituents showed potent activity against OVCAR-8 (ovarian
cancer) cells at low micromolar concentrations.
Keywords 3,5-Dibromophenyl-functionalised compounds · Imidazolium salts · Au-NHC complexes · Anti-cancer activity ·
Lipophilic cations · X-ray structures · Supramolecular Br…π interactions
Introduction
cations (DLCs) [4–6] and cationic gold-phosphine com-
plexes [7] and gold-NHC complexes, can accumulate in the
mitochondria and subsequently trigger apoptosis [8].
Mitochondria play a central role in regulating programmed
cell death via apoptosis. Many cancers arise due to failures
in mediating the apoptotic processes, resulting in ability
to resist cell death and unrestricted proliferation of cancer
cells [1–3]. Consequently, many cancer treatments target
mitochondria as a way to promote apoptosis of cancer cells.
Various cationic compounds, such as delocalised lipophilic
Gold-based compounds, e.g., cationic gold-phosphine
complexes [7, 9–12] and gold-NHC complexes, [8, 13, 14]
are receiving much attention due to their promising anti-
cancer activity against a wide variety of cancer cell lines [4,
15, 16]. The dominant proposed mechanism for the activity
of Au-NHCs is that Au inhibits the activity of mitochondrial
thioredoxin reductase (TrxR) by binding to the soft Se and
S atoms in the cysteine and selenocysteine residues at the
active site [4, 8]. TrxR is an important enzyme that regulates
the cellular redox system and inhibition of TrxR can lead to
a build-up of reactive oxygen species, which sets of a cas-
cade of processes that leads to apoptosis [4, 16, 17].
* Murray V. Baker
murray.baker@uwa.edu.au
Hawraa S. Al-Buthabhak
hawraas.dawood@uokufa.edu.iq
1
Department of Chemistry, Faculty of Science, University
The activity of Au-based compounds is highly dependent
on their lipophilicity, with an increase in lipophilicity gener-
ally resulting in higher activity [4, 10]. Au complexes that
behave as delocalised lipophilic cations can target TrxR in
mitochondria. Since the mitochondrial membrane potential
is higher (more negative) in cancer cells compared to normal
cells, lipophilic, cationic Au-NHCs can selectively target
mitochondria in cancer cells over normal cells [4]. However,
for complexes that are too lipophilic, such as [Au(dppe)2]+
(dppe = 1,2-bis(diphenylphosphino)ethane), there can be
severe toxic efects, such as cardiotoxicity [18] and hepa-
totoxicity [4]. Understanding the role of lipophilicity and
of Kufa, P.O. Box 21, Najaf 54001, Iraq
2
Curtin Medical School, Curtin Health Innovation Research
Institute, Curtin University, Perth, WA 6102, Australia
3
School of Molecular Sciences M310, The University
of Western Australia, 35 Stirling Highway, Perth, WA 6009,
Australia
4
Division of Obstetrics & Gynaecology, The University
of Western Australia Medical School, Perth, WA 6009,
Australia
5
Biotechnology and Drug Development Research Laboratory,
Curtin Medical School, Curtin Health Innovation Research
Institute, Curtin University, Perth, WA 6102, Australia
Vol.:(0123456789)
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
structure–activity relationships of Au-NHC complexes is
essential for enhancing their anticancer activity and mini-
mising their undesirable side efects.
transfer catalyst), and potassium phosphate at 130 °C [24].
Using a modifcation of this method (NaOH as base, DMSO
as solvent, and no phase transfer catalyst), imidazole was
N-arylated with bromobenzene and 1,3,5-tribromoben-
zene to form 1-phenylimdazole 1 and 1-(3,5-dibromo-
phenyl)imidazole 2 respectively (Scheme 1). Subsequent
N-alkylation resulted in the phenylimidazolium salt 3 and
the 3,5-dibromophenylimidazolium salts 4–7, and the hex-
afuorophosphate salt 8 was prepared by metathesis of the
iodide salt 4 with KPF6 (Scheme 1).
To date, while there is much evidence that Au-NHC com-
plexes target mitochondria and trigger apoptosis, few studies
have clearly shown subcellular distribution of Au-NHCs.
One study of fuorescent dinuclear Au-NHCs by confocal
fuorescence microscopy showed that these compounds were
accumulated in lysosomes, not mitochondria [19]. Another
study (by transmission electron microscopy) showed that
when MDA-MB-231 human breast adenocarcinoma cells
were incubated with [Au(d2pype)2]Cl (d2pype=1,2-bis(2-
pyridylphosphinoethane), electron rich elements (assumed
to be Au) were accumulated in mitochondria [11]. The same
study also showed (by NanoSIMS) that Au became associ-
ated with sulfur rich regions of the nucleus and cytoplasm of
the cells [11]. Nevertheless, clear evidence concerning the
fate of Au-NHC complexes and the NHC ligands themselves
in cells remains elusive.
The new imidazolium salts 3 and 5–8 were converted into
the corresponding complexes of form [Au(NHC)2]X (9–13)
by reaction with (DMS)AuCl or (DMS)AuBr in the presence
of K2CO3 at room temperature for 24 h (Scheme 1). The
method used was a modifcation of the procedure reported by
Collado et al., for the synthesis of other Au-NHC complexes
[25]. The new complexes are soluble in a most organic sol-
vent but not soluble in hexane or water.
Results of 1H and 13C NMR studies of the new salts 3–8
and complexes 9–13 are consistent with their proposed
structures. During synthesis of the imidazolium salts 3–7,
formation of the imidazolium species was indicated by the
appearance of 1H NMR signals due to the azolyl H2 protons
in the range 9.8–10 ppm and disappearance of the signal
due to the H2 protons (~7.9 ppm) of the imidazole precur-
sors 1 and 2. In the 13C{1H} NMR spectrum, the signal due
to the C2 carbon for the imidazolium salts 3–7 occurred
near δ 137 ppm, which is consistent with the literature val-
ues for C2 in other imidazolium salts [8, 26, 27]. For the
[Au(NHC)2]X complexes 9–13, the 13C{1H} NMR spectrum
showed a signal for the carbene carbon near 181 ppm, which
is in the range reported for similar [Au(NHC)2]X [8, 28, 29].
With the above considerations in mind, this work focuses
on Au-NHC complexes containing 3,5-dibromophenyl sub-
stituents. These compounds are of interest for a number of
reasons. Firstly, the 3,5-dibromophenyl substituents are
large, lipophilic groups, and their inclusion in Au-NHC
complexes introduces a new moiety that can be used to fne-
tune lipophilicity and, hopefully, enhance biological activity.
Secondly, halogens, and bromine in particular, can play a
signifcant role in the activity of a drug due to the ability of
halogens to participate in binding interactions with target
molecules [20]. Thirdly, the 3,5-dibromophenyl substitu-
ents are a convenient moiety via which bromine atoms can
be introduced into an NHC ligand and its Au-NHC com-
plexes. Bromine atoms are convenient atomic labels that can
be exploited in cellular mapping studies by the NanoSIMS
technique.
X‑ray studies
In this study, Au-NHC complexes have been tested
against OVCAR-8 ovarian cancer cells, a cisplatin-resistant
cell line [21]. Previous genetic profling on OVCAR-8 sug-
gested that it resembles high-grade serous ovarian carcinoma
in patients [22] which is the most common histological sub-
type of ovarian cancer.
The new imidazolium salts 4, 5, and 8 and their complexes
10–13 have been characterised by X-ray difraction. Details
of solvents used in growth of crystals suitable for X-ray
difraction are recorded in the Experimental Section. Com-
pounds 10 and 11 were obtained as the solvates 10·EtOAc
and 11·CH3CN. X-ray data and key bond lengths and angles
summarised in Tables 1 and 2 respectively. The bond dis-
tances and angles are similar to corresponding values
reported previously for similar imidazolium salts and Au-
NHC complexes. Interesting features of the structures are
summarised below.
Results and discussion
Synthesis and NMR studies
The solid-state structure of 1-(3,5-dibromophenyl)-
3-methylimidazolium iodide 4 contained two independent
molecules (Fig. 1a). Interestingly, the 3,5-dibromophenyl
moieties of the two molecules are approximately parallel
and appear to be involved in a π–π interaction [30] (or Br-π
interactions). The distance between the centroids of the C6
rings is 4.70 Å and the distances between the centroid of
N-Arylation of heterocyclic compounds is typically achieved
via an Ullman-type coupling of the heterocyclic compound
with an aryl halide in the presence of a copper species as
catalyst [23]. N-Phenylimidazole has previously been syn-
thesized by reaction with iodobenzene in H2O in presence
of cuprous oxide, tetrabutylammonium bromide (as a phase
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Scheme 1 Synthesis of N-arylimidazoles 1 and 2, imidazolium salts 3–8, and Au-NHC complexes 9–13
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Table 1 Crystal data for salts 4, 5, and 8 and complexes 10–13
4
5
8
10
11
12
13
Empirical
formula
C
10H9Br2N2·I
C11H11Br3N2
C10H9Br2N2·F6P
C20H16AuBr4N4·F6P·C4H8O2 C22H20AuBr4N4·Br·C2H3N
C24H24AuBr4N4·Br C32H24AuBr4N4·Br
Mr
443.91
0.71073
410.95
1.54178
461.98
1.54178
1062.05
0.71073
977.99
0.71073
964.99
0.71073
1061.07
0.71073
Wave
length
[Å]
Crystal
Triclinic
Orthorhombic
Triclinic
Monoclinic
Triclinic
Monoclinic
Monoclinic
system
Space
group
Pnna
C2/c
C2/c
P21/n
P1
P1
P1
a [Å]
b [Å]
c [Å]
α [º]
β [º]
γ [º]
V [Å3]
Z
8.4901(2)
10.9180(3)
14.9044(4)
98.417(2)
105.241(2)
90.824(2)
1316.59(6)
4
9.8442(5)
12.7548(3)
24.1456(8)
7.9523(5)
13.1938(11)
14.7836(12)
97.900(7)
99.511(6)
104.142(7)
1457.6(2)
4
27.5547(5)
8.0827(1)
14.1653(3)
11.4138(5)
12.2346(5)
12.5493(5)
65.243(4)
68.992(4)
68.032(4)
1432.81(12)
2
22.7764(4)
16.0601(2)
10.4146(2)
12.2854(3)
22.8693(3)
12.3872(3)
99.878(2)
116.983(2)
114.445(3)
3031.7(2)
8
3108.07(10)
4
3394.86(11)
4
3168.31(14)
4
ρ
calc [Mg
m−3
µ [mm−1
2.240
1.801
2.105
2.270
2.267
1.888
2.224
]
]
8.47
9.70
8.71
9.99
12.13
10.24
10.98
Crystal size 0.29×0.21×0.08 0.21×0.14×0.11 0.44×0.11×0.03 0.35×0.18×0.12
0.33×0.22×0.10
0.19×0.13×0.11
0.22×0.18×0.04
[mm3]
2 θmax [°]
65.3
27,938
134.5
16,011
134.5
8840
58.5
24,060
74.8
51,397
64.6
35,134
65.6
39,864
Refs. col-
lected
Indep.
refec-
tions
8938
2708
8840
3973
14,450
5749
10,705
Rint
0.027
0.548/0.184
0.052
1.0/0.717
n/a (twin)
1.0/0.877
0.029
0.403/0.152
0.059
0.368/0.087
0.028
1.0/0.686
0.056
0.677/0.189
Max/min
trans
Restrains/
params
0/271
4/154
0/382
224/256
3/327
0/156
0/379
GooF on F2 1.000
1.000
1.003
1.003
1.001
1.000
1.001
R1
[I>2σ(I)]
wR2
[I>2σ(I)]
0.0247
0.0479
0.0816
0.0184
0.0488
0.0249
0.0370
0.0596
0.1296
0.1952
0.0474
0.0807
0.0674
0.0685
R1(all data) 0.0339
0.0601
0.1395
0.0917
0.2026
0.0222
0.0491
0.0942
0.1101
0.0294
0.0699
0.0626
0.0778
wR2(all
0.0637
data)
Δρmax/min [e 1.12/−1.05
1.35/−1.25
2074425
3.73/−1.44
2074426
0.65/−0.61
2074427
4.03/−5.74
2074428
2.01/−2.23
2074429
1.25/−0.98
2074430
Å−3
]
CCDC
2074424
number
the each C6 ring and the closest Br atom from the opposing
molecule are 3.508 and 3.776 Å (Fig. 1b). The Br…C6 (cen-
troid) distances are shorter than the mean values (4.11 Å or
3.95 Å, depending on the details of the analysis) reported for
C-Br…π interactions in the solid state structures of nucleic
acids [31].
distance between the centroids of the C6 rings (3.948 Å)
again suggests a π–π interaction (Fig. 2b).
The structure of 1-(3,5-dibromophenyl)-3-ethylimidazo-
lium bromide 5 showed one independent molecule but with
the ethylimidazolyl moiety disordered over two sites. In this
structure the 3,5-dibromophenyl moieties in adjacent mol-
ecules are approximately perpendicular, with a Br atom from
one molecule oriented toward the centroid of the C6 ring
of the next (Br…C6 centroid~3.547 Å) (Fig. 3b). In adja-
cent molecules the imidazolium moieties are approximately
−
In the structure of the corresponding PF6 salt 8, there
are again two independent molecules, the 3,5-dibromophe-
nyl moieties of the two molecules are "less parallel" than in
4 (Fig. 2a), but nevertheless they face one another and the
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Table 2 Selected bond distances (Å) and angles (°) for salts 4, 5, and
8 and complexes 10–13
contacts with the two imidazolyl moieties in an adjacent
cation, so that the cations are arranged into chains (Fig. 4b).
In the structure of 11·CH3CN, the cation is no longer
centrosymmetric. The NHC rings are inclined relative to
one another by approximately 18.3° about the C-Au-C axis
(approximating an anti arrangement around the Au centre,
Fig. 5a). The cations are arranged in pairs. In these pairs, the
intermolecular distance between imidazolyl ring centroids
is 4.234 Å and the Au…Au distance is 4.476 Å, too large to
be an aurophilic interaction. There are intermolecular Br…
C6 (centroid) contacts of 3.861 Å (Fig. 5b).
C1-N2
C1-N5
N2-C1-N5 Au1-C1 Au1-C2 C1-Au1-C2
N2-C2-N5
4 Mol(1) 1.337(3) 109(1)
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
1.32(3)
Mol(2)
1.338(3) 108.5(2)
1.32(1)
5
1.340(9) 108.0(7)
1.34(1)
8 Mol(1) 1.35(1)
109(1)
In the structure of 12, the cation adopts a similar con-
formation to the cation in 10·EtOAc, possessing an inver-
sion centre and having its imidazolyl moieties coplanar
(Fig. 6a). The cations are arranged into chains. The two Br
atoms (Br53 and Br55) in each 3,5-dibromophenyl group are
involved in diferent interactions. Br53 points toward an imi-
dazolyl moiety in one adjacent cation and the Br55 atom in
another adjacent cation. Br55 points toward the Br53 atom in
one adjacent cation and the centroid of the 3,5-dibromophe-
nyl C6 ring in another adjacent cation (Fig. 6b). In the struc-
ture of 12, the inter-cation Br53…C3N2 (centroid) distance
is 4.689 Å, the Br55…C6 (centroid) distance is 3.670 Å) and
the Br53…Br55 distance is 3.606 Å, similar to the sum of
the van der Waals radii, 3.66 Å as reported by Bondi [32].
In the structure of 13, the cation adopts a conformation
similar to the cation in 11·CH3CN with the two imidazolyl
ring planes inclined~8.68° with respect to each other. The
cations form pairs. Within the pairs, the inter-cation distance
between opposing imidazolyl rings is 3.717 Å, and the inter-
cation Au…Au distance is 3.896 Å, too large to be consid-
ered an aurophilic interaction. There is an inter-cation π–π
1.32(1)
Mol(2)
10
1.35(2)
1.32(1)
109(1)
1.342(3) 104.80(18) 2.023(2) 2.023(2) 180.00
1.359(3)
11
1.346(6) 104.6(4)
1.336(6)
1.359(3) 103.7(2)
1.368(3)
1.355 5) 104.6(3)
1.356(5)
2.020(4) 2.025(4) 177.5(2)
2.026(2) 2.026(2) 180.00(14)
2.028(4) 2.028(4) 178.26(15)
12
13
parallel with a distance between the centroids of adjacent
C3N5 rings being~4.052 Å.
In the structure of 10·EtOAc, the [Au(NHC)2]+ cation
has inversion symmetry, and the two imidazolium moieties
in the cation are coplanar (Fig. 4a). The two bromine atoms
of each 3,5-dibromophenyl moiety in one cation have close
Fig. 1 Crystal structure of the 4 (50% level for the displacement ellipsoids). a The two independent molecules. b Interactions between the
3,5-dibromophenyl moieties of the two independent molecules
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Fig. 2 Crystal structure of the 8 (50% level for the displacement ellipsoids). a The two independent cations. b The two independent cations and
anions, showing the π–π interaction between the 3,5-dibromophenyl moieties
Fig. 3 Crystal structure of the 5. a The major component of the cation (50% level for the displacement ellipsoids). b Three cations, showing Br-π
interactions between 3,5-dibromophenyl moieties and π–π interactions between imidazolyl moieties (H atoms omitted for clarity)
interaction between 3,5-dibromophenyl moieties (distance
between C6 centroids=3.817 Å) and an intra-cation Br…π
interaction (Br…C6 centroid distance=4.063 Å) (Fig. 7b).
Activity of the Au‑NHC complexes against OVCAR‑8
cells
The new Au-NHC complexes containing one or more
3,5-dibromophenyl substituents on the NHC group showed
notably high cytotoxic potency against OVCAR-8 cells
(Table 3 and Fig. 8). The new Au-NHC complexes 9–13
were two orders of magnitude more cytotoxic than the
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Fig. 4 Crystal structure of 10·EtOAc. a The cation, anion, and associated EtOAc molecule (50% level for the displacement ellipsoids). b Chain
of cations, showing close contact between Br in one cation and imidazolium moiety of the adjacent cation (H atoms omitted for clarity)
3,5-dibromophenyl-functionalised imidazolium salts 6
and 7. All the complexes 9–13 are slightly more active
than [(Pr2Im)2Au]Br 14 (Pr2Im = 1,3-dipropylimidazolin-
2-ylidene; IC50 1.0 0.2, not shown in Fig. 8). This result is
in accord with previous fndings that lipophilicity is impor-
tant in determining activity within the [Au(NHC)2]+ class
[8, 14, 33, 34]. A recent study suggested introducing halo-
gens into the NHC backbone can increase the activity of
Au-NHCs in inhibition of TrxR isolated from rat liver, thus
may afect the cytotoxicity of the compounds [34]. How-
ever, the N-phenyl-functionalised compound 9 showed very
similar activity to its N-(3,5-dibromophenyl) analogue 12,
suggesting that the presence of bromine does not necessar-
ily have a signifcant impact upon potency. Amongst the
series of compounds 9–13, the range of IC50 values is too
small to indicate signifcant or obvious trends in activity
with increasing lipophilicity.
It is important to mention that 1-(3,5-dibromophenyl)-
3 - i s o p r o p yl i m i d a z o l i u m b r o m i d e
6
a n d
1-(3,5-dibromophenyl)-3-benzylimidazolium bromide 7
also have some inhibition activity at high concentration
(IC50 ~ 47 µM and ~ 121 µM respectively), even though
the diisopropylimidazolium bromide and diisopropylb-
enzimidazolium bromide were both found to be inactive
(IC50 >100 µM; results not shown in Fig. 8). It may be that
the 3,5-dibromophenyl-functionalised imidazolium ions are
sufciently lipophilic to exert antimitochondrial activity as
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Fig. 5 Crystal structure of 11·CH3CN. a A cation, anion, and associated CH3CN molecule (50% level for the displacement ellipsoids). b A pair
of cations, showing inter-cation distances (H atoms omitted for clarity)
Fig. 6 Crystal structure of 12. a A cation and associated anion (50% level for the displacement ellipsoids). b Three adjacent cations, showing
inter-cation interactions (H atoms omitted for clarity)
delocalised lipophilic cations [4], whereas the other azolium
ions are insufciently lipophilic to exert activity.
group leads to higher activity against the OVCAR-8 cells.
Intuitively, the compounds with the benzyl substituent
should be more lipophilic than the compounds with the
isopropyl substituent, but, nevertheless the presence of the
It is interesting that within each of the pairs 12, 13 and
6, 7, the presence of an isopropyl group instead of a benzyl
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Fig. 7 Crystal structure of 13. a A cation and associated anion (50% level for the displacement ellipsoids). b Two adjacent cations, showing
inter-cation interactions (H atoms omitted for clarity)
Table 3 IC50 values for 3,5-dibromophenyl-functionalised Au-NHC
ligands about the Au-C axis on the NMR timescale, so that
the structure seen in the solid state does not remain rigid in
solution, so the relevance of the solid state structures of 12
and 13 to the biological results is unclear.
complexes against OVCAR-8 cells
Compound
IC50 (μM) SEM
n
6a
7a
9
10
11
12
13
47 16
3
2
3
3
3
3
3
121
6
Experimental
0.44 0.04
0.63 0.07
0.59 0.06
0.322 0.008
0.53 0.08
Nuclear magnetic resonance spectra were recorded using
Bruker Bruker ARX500 (500.13 MHz for 1H, 125.77 MHz
for 13C and 202.45 MHz for 31P), or Bruker ARX600
1
(600.13 MHz for H, 150.90 MHz for 13C) spectrometers
at ambient temperature unless otherwise indicated. 1H and
13C NMR chemical shifts were referenced to the residual sig-
nal of the solvent (DMSO-d6: 1H 2.50 ppm; 13C 39.52 ppm.
and 31P chemical shifts were referenced to an external 85%
H3PO4 solution. 1H-13C HSQC (heteronuclear single quan-
OVCAR-8 cells were incubated with test compounds for 72 h and
activity of the compounds on cell viability was evaluated using an
MTT assay. Data are shown as mean SEM of three independent
experiments (n) each of which used 2–4 replicates per concentration
for a test compound
aImidazolium salts 6 and 7 used as a negative control
1
tum coherence), H-13C HMBC (heteronuclear multiple
bond correlation) were used to assign signals, and micro-
analysis were performed by The School of Chemistry &
Molecular Biosciences, University of Queensland, Australia.
High resolution mass spectra were measured using Agilent
LCMS 6510 Q-TOF or Waters LCT Premier XE spectrom-
eters, using the ESI method, with CH3CN:H2O (9:1) as sol-
vent. All organometallic compounds were prepared under a
nitrogen atmosphere and in dark by using aluminium foil.
Chromatography was performed on silica (Davisil) unless
otherwise stated. Crystallographic data were measured at
100(2) K on either an Oxford Difraction Gemini or an
isopropyl group instead of the benzyl group appears to be
advantageous. In the solid state (Figs. 6 and 7), the isopro-
pyl-functionalised cation in 12 adopts a more planar con-
formation than its benzyl-functionalised counterpart in 13,
but whether the conformation of the cations is responsible
for the diference in activities of 12 and 13 requires further
investigation. We note that in the 1H NMR spectrum of 13,
the number of signals and their splitting patterns indicate
there is only a single benzyl environment. This result in
turn indicates that there must be some rotation of the NHC
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
Fig. 8 The inhibition activity of the 3,5-dibromophenyl-functional-
ised Au-NHC complexes 9–13 against OVCAR-8 cells, measured by
MTT assay after 72 h of treatment. The imidazolium salts 6 and 7
were used as negative controls. Data are shown as mean SEM of
n=3 independent experiments, each of which used 2 to 4 replicates
per concentration for a test compound
Oxford Difraction Xcalibur difractometer using Mo Kα or
Cu Kα radiation. Following analytical absorption correc-
tions and solution by direct methods, the structures were
refned against F2 with full-matrix least-squares using the
SHELX program suite [35]. Unless stated diferently below,
all hydrogen atoms were added at calculated positions and
refned by use of riding models. Except for those atoms
mentioned below, anisotropic displacement parameters were
employed throughout for the non-hydrogen atoms. Residual
electron density in the crystal of compounds 5 and 12, which
could not be interpreted as chemically reasonable moieties,
was efectively removed by use of the program SQUEEZE
[36]. Crystallographic data for the structures reported in
this paper have been deposited at the Cambridge Crystal-
lographic Data Centre. Copies of data with CCDC numbers
2074424-2074430 can be obtained free of charge via https://
www.ccdc.cam.ac.uk/structures/, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge
CB2 1EZ, UK (fax+441223336033; email deposit@ccdc.
cam.ac.uk).
Materials
The following compounds were used as received: potassium
carbonate (Analar Normapur), potassium hexafuorophos-
phate (Aldrich, 98%), imidazole (Fluka), 1,3,5-tribromoben-
zene (BDH), bromobenzene (Fluka), sodium hydroxide
(Chem Supply), magnesium sulfate anhydrous (Scharlau),
2-bromopropane (Acros Organics), bromoethane (Ajax),
methyl iodide, benzyl bromide (Merck). The solvents were
of analytical grade and used without further drying.
Synthesis of 1‑phenylimidazole (1)
Sodium hydroxide (950 mg, 23.7 mmol) was added to a
solution of imidazole (1.50 g, 22.0 mmol) in DMSO (10 mL)
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
and the mixture was stirred for 30 min at room tempera-
ture. Bromobenzene (2.3 mL, 22.0 mmol) was added, fol-
lowed by cuprous oxide (180 mg, 1.26 mmol). The mixture
was heated at 100 °C overnight. The mixture was diluted
with H2O (100 mL) and extracted with dichloromethane
(8 × 25 mL). The combined organic layer was evaporated
under reduced pressure. The green residue was suspended
in H2O (50 mL), the mixture was fltered, and the green
solid that was collected was washed with H2O (3×10 mL)
and dried, then dissolved in dichloromethane (200 mL). The
clear green solution was dried over MgSO4 and evaporated.
The resulting green oily residue was purifed by fash column
chromatography eluting with 3:2 ethyl acetate/cyclohexane.
The product was obtained as a yellow oil (1.70 g, 53%).
1H NMR (500 MHz, DMSO-d6): δ 8.25 (1H, apparent t,
splitting 1.1 Hz, imidazolyl H2), 7.74 (1H, apparent t, split-
ting 1.2 Hz, imidazolyl H4 or H5), 7.63–7.66 (2H, m, ArH
ortho), 7.53–7.49 (2H, m, ArH meta), 7.36 (1H, m, ArH
para), 7.10 (1H, apparent t, splitting 1.2 Hz, imidazolyl H5
or H4). NMR data are consistent with literature values [37].
The oily residue was triturated with diethyl ether (20 mL),
the diethyl ether was decanted of, and the trituration was
repeated twice more. Traces of ether were removed under
fow of N2, and the oily residue was purifed by column
chromatography, eluting with a mixture of dichloromethane
(100 mL) and acetonitrile (15 mL). The colourless product
was triturated with diethyl ether (3×20 mL), and removal
of residual ether under a fow N2 left 1-phenyl-3-isopro-
pylimidazolium bromide as a colourless hygroscopic solid
1
(320 mg, 69%). H NMR (500 MHz, DMSO-d6): δ 9.90
(1H, apparent t, splitting 1.5 Hz, imidazolyl H2), 8.38 (1H,
apparent t, splitting 1.7 Hz, imidazolyl H4), 8.20 (1H, appar-
ent t, splitting 1.7 Hz, m, imidazolyl H5), 7.8 (2H, m, ArH
ortho), 7.67 (2H, m, ArH meta), 7.60 (1H, m, ArH para),
3
4.74 (1H, sept, JH,H = 6.7 Hz, CH(CH3)2), 1.57 (6H, d,
3JH,H =6.7 Hz, CH(CH3)2). 13C NMR (125.7 MHz, DMSO-
d6): δ 134.85 (ArC ipso), 134.21 (imidazolyl C2) 130.11
(ArC meta), 129.68 (ArC para), 121.88 (ArC ortho), 121.53
(imidazolyl C5), 121.23 (imidazolyl C4), 52.94 (CH(CH3)2);
22.24 (CH(CH3)2). Microanalysis: Found: C, 52.19; H, 5.84;
N, 10.14% C12H15BrN2.(H2O)0.5 requires. C, 52.08; H, 5.73;
N, 10.06%.
Synthesis of 3,5‑dibromophenylimidazole (2)
Sodium hydroxide (480 mg, 12.0 mmol) was added to a solu-
tion of imidazole (681 mg, 10.0 mmol) in DMSO (20 mL)
and the mixture was stirred for 60 min at room temperature.
1,3,5-Tribromobenzene (3.50 g, 11.1 mmol) was added fol-
lowed by cuprous oxide (90.0 mg, 0.63 mmol). The mixture
was heated at 100 °C for 48 h. The mixture was diluted with
H2O (200 mL) and a dark grey precipitate formed, which
was fltered of and washed with H2O several times. The
solid dried then suspended in dichloromethane (250 mL) and
stirred at room temperature, then the dark brown solution
was fltered through a plug of Celite, and the orange fltrate
was evaporated to dryness. The orange residue was purifed
by fash column chromatography eluting with 2:1 ethyl ace-
tate/cyclohexane. The product was obtained as a pale yellow
solid (1.27 g, 42%). 1H NMR (500 MHz, CDCl3): δ 7.84 (br
apparent t, splitting 1.2 Hz, 1H, imidazolyl H2), 7.67 (1H, t,
4JH,H =1.6 Hz, ArH para), 7.51 (2H, d, 4JH,H =1.6 Hz, ArH
ortho), 7.25 (1H, apparent t, splitting 1.3 Hz, imidazolyl H4
or H5), 7.22 (1H, br apparent t, splitting 1.3 Hz, imidazolyl
H5 or H4). NMR data are consistent with literature values
[37].
Synthesis of 1‑(3,5‑Dibromophenyl)‑3‑methyllimi‑
dazolium iodide (4)
Methyl iodide (0.52 mL, 8.41 mmol) was added to a solu-
tion of 1-(3,5-dibromophenyl)imidazole (1.00 g, 3.31 mmol)
in ethyl acetate (40 mL). The mixture was refuxed over-
night. The solvent was evaporated by fow of N2, and the
product was stirred with diethyl ether (3×50 mL), then was
fltered of and dried in air to leave a beige powder (505 mg,
1
35%). H NMR (500 MHz, DMSO-d6): δ 9.84 (br s, 1H,
imidazolyl H2), 8.34 (1H, apparent t, splitting 1.7 Hz, imi-
4
dazolyl H5), 8.14 (2H, d, JH,H =1.7 Hz, ArH ortho), 8.11
4
(1H, t, JH,H = 1.7 Hz, ArH para), 7.94 (1H, br apparent
t, splitting 1.6 Hz, imidazolyl H4), 3.93 (s, 3H, CH3).13C
NMR (125.7 MHz, DMSO-d6): δ 136.70 (imidazolyl C2),
134.54 (ArC para), 124.38 (imidazolyl C4), 124.03 (ArC
ortho), 123.43 (ArC-Br), 121.00 (imidazolyl C5); 36.27
(CH3). Microanalysis: Found: C, 27.38; H, 2.05; N, 6.06%
C10H9Br2IN2 requires C, 27.06; H, 2.04; N, 6.31%. Crystals
suitable for X-ray difraction studies were grown by difu-
sion of vapours between diethyl ether and a solution of the
imidazolium salt (4) in acetonitrile.
Synthesis of 1‑phenyl‑3‑isopropylimidazolium
bromide (3)
Synthesis of 1‑(3,5‑Dibromophenyl)‑3‑ethyllimida‑
zolium bromide (5)
A mixture of 1-phenylimidazole (0.22 mL, 1.73 mmol) and
isopropyl bromide (26.2 mL, 279 mmol) were dissolved in
ethyl acetate (20 mL). The solution was refuxed for 12 days.
The mixture was cooled to room temperature and the solvent
and isopropyl bromide evaporated under reduced pressure.
1-(3,5-Dibromophenyl)imidazole (350 mg, 1.16 mmol)
and ethyl bromide (10.0 mL, 134 mmol) were combined
and the mixture was refuxed for 7 days. The mixture was
cooled to room temperature and the product was fltered
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
1
of and washed with diethyl ether (3 × 30 mL) and dried
as a white powder (650 mg, 83%). H NMR (600 MHz,
under flow of N2 to leave the product as a white solid
DMSO-d6): δ 10.0 (1H, apparent t, splitting 1.6 Hz, imida-
1
(320 mg, 67%). H NMR (600 MHz, DMSO-d6): δ 9.88
zolyl H2), 8.40 (1H, apparent t, splitting 1.8 Hz, imidazolyl
4
(1H, apparent t, splitting 1.6 Hz, imidazolyl H2), 8.37 (1H,
apparent t, splitting 1.8 Hz, imidazolyl H5), 8.18 (2H, d,
4JH,H =1.6 Hz, ArH ortho), 8.11 (1H, t, 4JH,H =1.6 Hz, ArH
para), 8.05 (1H, apparent t, splitting 1.8 Hz, imidazolyl
H5), 8.20 (2H, d, JH,H = 1.6 Hz, C6H3Br2 H ortho), 8.12
(1H, t, 4JH,H =1.6 Hz, C6H3Br2 H para), 8.03 (1H, apparent
t, splitting 1.8 Hz, imidazolyl H4), 7.51–7.41 (5H, m, Ph-H),
5.50 (s, 2H, CH2). 13C NMR (150.90 MHz, DMSO-d6): δ
136.7 (C6H3Br2 C ipso), 136.3 (imidazolyl C2) 134.5 (Ph
C ipso), 134.2 (C6H3Br2 C para), 129.0 (Ph C meta), 128.8
(Ph C para), 128.5 (Ph C ortho), 124.2 (C6H3Br2 C ortho),
123.3 (C6H3Br2 C-Br); 123.1 (imidazolyl C4); 121.7 (imi-
dazolyl C5), 52.54 (CH2). Microanalysis: Found: C, 40.93;
H, 2.58; N, 5.68% C16H13Br3N2. requires. C, 40.63; H, 2.77;
N, 5.92%. HRMS (ESI+): Calcd for C16H13Br2N2 [M-Br+],
m/z 390.9445. Found, m/z 390.9432.
3
H4), 4.27 (2H, q, JH,H = 7.3 Hz, CH2CH3), 1.50 (3H, t,
3JH,H =7.3 Hz, CH2CH3). 13C NMR (150.90 MHz, DMSO-
d6): δ 136.75 (ArC ipso), 135.84 (imidazolyl C2) 134.48
(ArC para), 124.00 (ArC ortho), 123.39 (ArC-Br), 123.03
(imidazolyl C4), 121.06 (imidazolyl C5), 44.94 (CH2CH3);
14.66 (CH2CH3). Microanalysis: Found: C, 32.48; H,
2.88; N, 6.44% C11H11Br2N2 requires C, 32.15; H, 2.70;
N, 6.82%. HRMS (ESI+): Calcd for C11H11Br2N2 [M-Br+],
328.9289 m/z. Found, m/z 328.9305. Crystals suitable for
X-ray difraction studies were grown by difusion of vapours
between diethyl ether and a solution of the imidazolium salt
(5) in methanol.
Synthesis of 1‑(3,5‑dibromophenyl)‑3‑methyllimida‑
zolium hexafuorophosphate (8)
Potassium hexafluorophosphate (230 mg, 1.26 mmol)
in (5 mL) H2O was added to a solution of
1-(3,5-dibromophenyl)-3-methylimidazolium iodide
(224 mg, 0.503 mmol) in 1:1 H2O:MeOH (4 mL each),
and a white precipitate formed immediately. The mixture
was stirred at room temperature overnight. The precipitate
was fltered of, washed with H2O (2 × 10 mL) and dried
under vacuum to leave a white powder (178 mg, 77%). 1H
NMR (500 MHz, DMSO-d6): δ 9.82 (1H, br s, imidazolyl
H2), 8.34 (1H, apparent t, splitting 1.8 Hz, imidazolyl
Synthesis of 1‑(3,5‑dibromophenyl)‑3‑isopropylimi‑
dazolium bromide (6)
1-(3,5-Dibromophenyl)imidazole (670 mg, 2.22 mmol)
and isopropyl bromide (22 mL, 234 mmol) were combined
and the mixture was refuxed for 14 days. The mixture was
cooled to room temperature and the product was fltered of
and washed with diethyl ether (3×30 mL) and dried under
a fow of N2 to leave the product as a beige solid (700 mg,
74%). 1H NMR (500 MHz, DMSO-d6): δ 9.88 (1H, appar-
ent t, splitting 1.6 Hz, imidazolyl H2), 8.39 (1H, apparent t,
splitting 1.9 Hz, imidazolyl H5), 8.20 (2H, d, 4JH,H =1.6 Hz,
ArH ortho), 8.15 (1H, apparent t, splitting 1.9 Hz, imidazolyl
H4), 8.10 (1H, t, 4JH,H =1.6 Hz, ArH para), 4.68 (1H, sept.,
4
H5), 8.14 (2H, d, JH,H = 1.7 Hz, ArH ortho), 8.11 (1H,
4
t, JH,H = 1.7 Hz, ArH para), 7.93 (1H, apparent t, split-
ting 1.8 Hz, imidazolyl H4), 3.92 (s, 3H, CH3). 31P NMR
(202.45 MHz, DMSO-d6): δ -144.2 (sept., 1JP,F =708 Hz, 1P,
−
PF6 ). Microanalysis: Found: C, 26.18; H, 2.04; N, 5.80%
3
3JH,H = 6.6 Hz, (CH(CH3)2), 1.55 (6H, d, JH,H = 6.6 Hz,
C10H9Br2F6N2P requires C, 26.00; H, 1.96; N, 6.06%. Crys-
tals suitable for X-ray difraction studies were grown by dif-
fusion of vapours between diethyl ether and a solution of the
imidazolium salt (8) in acetonitrile.
CH(CH3)2).13C NMR (125.7 MHz, DMSO-d6): δ 136.81
(ArC ipso), 135.89 (imidazolyl C2) 134.45 (ArC para),
124.04 (ArC ortho), 123.37 (ArC-Br), 121.68 (imidazolyl
C4), 121.18 (imidazolyl C5), 53.20 (CH(CH3)2); 22.16
(CH(CH3)2).Microanalysis: Found: C, 33.32; H, 3.00; N,
6.37% C12H13Br3N2 (H2O)0.2 requires. C, 33.63; H, 3.15; N,
6.54%. HRMS (ESI+): Calcd for C12H13Br2N2 [M-Br+], m/z
342.9445. Found, m/z 342.9426.
Bis(1‑phenyl‑3‑isopropylimidazolin‑2‑ylidene)
gold(I) bromide (9)
1-Phenyl-3-isopropylimidazolium bromide (72.7 mg,
0.27 mmol) and potassium carbonate (380 mg, 2.72 mmol)
were stirred in acetonitrile (10 mL) at room temperature
for 5 min. (Me2S)AuBr (44.6 mg, 0.131 mmol) was added
and the mixture was stirred for 24 h. The mixture was fl-
tered through Celite and the fltrate evaporated to dryness,
to leave the product as an of-white powder (71 mg, 82%).
1H NMR (500 MHz, DMSO-d6): δ 7.91–7.90 (4H, m, imida-
zolyl H5 and imidazolyl H4), 7.73–7.71 (4H, m, ArH ortho),
7.55–7.54 (6H, m, ArH meta and ArH para), 4.70 (2H, sept,
Synthesis of 1‑(3,5‑dibromophenyl)‑3‑benzylimida‑
zolium bromide (7)
1-(3,5-Dibromophenyl)imidazole (500 mg, 1.65 mmol)
and benzyl bromide (0.40 mL, 3.36 mmol) were dissolved
in toluene (30 mL) and the clear solution was refuxed for
2 days. The mixture was cooled to room temperature and
the product was fltered of and washed with diethyl ether
(3 × 20 mL) and dried under vacuum to leave the product
3
3JH,H = 6.7 Hz, CH(CH3)2), 1.42 (12H, d, JH,H = 6.7 Hz,
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
CH(CH3)2) 13C NMR (125.7 MHz, DMSO-d6): δ 180.06
(C2), 139.02 (ArC ipso) 129.50 (ArC meta), 128.96 (ArC
para), 124.94 (C ortho), 123.22 (C5), 119.60 (C4), 53.52
CH(CH3)2; 22.85 CH(CH3)2. Microanalysis: Found: C,
38.80; H, 4.86; N, 7.11% C24H28AuBrN4.(H2O)5 requires
C, 38.98; H, 5.18; N, 7.58%.
(H2O)0.2 requires C, 28.09; H, 2.19; N, 5.96%. HRMS
(ESI+): Calcd for C22H20Br4N4Au [M-Br+], m/z 852.8087.
Found, m/z 852.8118. Crystals suitable for X-ray difraction
studies were grown by difusion of vapours between diethyl
ether and a solution of complex 11 in acetonitrile.
Bis(1‑(3,5‑dibromophenyl)‑3‑methylimidazo‑
lin‑2‑ylidene)gold(I) hexafuorophosphate (10)
Bis(1‑(3,5‑dibromophenyl)‑3‑isopropylimidazo‑
lin‑2‑ylidene)gold(I) bromide (12)
1-(3,5-Dibromophenyl)-3-methylimidazolium hexafuoro-
phosphate (50.0 mg, 0.108 mmol) and potassium carbon-
ate (200 mg, 1.44 mmol) were stirred in acetonitrile (5 mL)
at room temperature for 5 min. (Me2S)AuCl (16.0 mg,
0.054 mmol) was added and the mixture was stirred for 24 h.
The mixture was fltered through Celite and the fltrate was
evaporated to dryness. The residue was recrystallised from
ethyl acetate to leave the product as light yellow crystals
(30 mg, 57%). 1H NMR (500 MHz, DMSO-d6): δ 8.03 (4H,
1-(3,5-Dibromophenyl)-3-isopropylimidazolium bromide
(100 mg, 0.235 mmol) and potassium carbonate (330 mg,
2.38 mmol) were stirred in acetonitrile (5 mL) at room tem-
perature for 5 min. (Me2S)AuBr (40.0 mg, 0.117 mmol) was
added and the mixture was stirred for 24 h. The mixture was
fltered through Celite and the fltrate evaporated to dry-
ness. The residue was recrystallised from acetonitrile/diethyl
ether and dried under vacuum to leave the product as an of-
white powder (85 mg, 75%). 1H NMR (500 MHz, DMSO-
4
4
4
d, JH,H = 1.7 Hz, ArH ortho), 7.97 (2H, t, JH,H = 1.7 Hz,
ArH para), 7.96 (2H, d, 3JH,H =1.8 Hz, imidazolyl H5), 7.74
(2H, d, 3JH,H =1.8 Hz, imidazolyl H4), 3.90 (s, 6H, CH3). 13C
NMR (125.7 MHz, DMSO-d6): δ 181.68 (imidazolyl C2),
140.63 (ArC ipso), 133.97 (ArC para), 126.87 (ArC ortho),
124.48 (imidazolyl C4), 122.88 (imidazolyl C5), 122.76
(ArC-Br); 37.94 (CH3). Microanalysis: Found: C, 26.97; H,
2.09; N 5.19% C20H16AuBr4F6N4P. (CH3CH2OCOCH3)0.8
requires C, 26.68; H, 2.16; N, 5.36%. HRMS (ESI+): Calcd
d6): δ 8.05 (2H, t, JH,H =1.6 Hz, ArH para), 8.04 (4H, d,
4JH,H =1.6 Hz, ArH ortho), 7.94 (2H, d, 3JH,H =1.9 Hz, imi-
3
dazolyl H5), 7.92 (2H, d, JH,H = 1.9 Hz, imidazolyl H4),
3
4.67 (2H, sept, JH,H = 6.7 Hz, CH(CH3)2), 1.44 (12H, d,
3JH,H =6.7 Hz, CH(CH3)2). 13C NMR (125.7 MHz, DMSO-
d6): δ 180.29 (C2), 141.11 (ArC ipso) 134.15 (ArC para),
127.56 (ArC ortho), 123.59 (imidazolyl C5), 122.83 (ArC-
Br), 119.79 (imidazolyl C4), 53.84 CH(CH3)2)); 22.93
CH(CH3)2). Microanalysis: Found: C, 29.50 H, 2.31;
N, 5.53% C24H24AuBr5N4 requires C, 29.87; H, 2.51; N,
5.81%. HRMS (ESI+): Calcd for C24H24Br4N4Au [M-Br+],
m/z 880.8400. Found, m/z 880.8420. Crystals suitable
for X-ray difraction studies were grown by difusion of
vapours between pentane and a solution of complex 12 in
dichloromethane.
+
for C20H16Br4N4Au [M-PF6 ], m/z 824.7775. Found, m/z
824.7774. Crystals suitable for X-ray difraction studies were
grown by recrystallisation from ethyl acetate.
Bis(1‑(3,5‑dibromophenyl)‑3‑ethylimidazo‑
lin‑2‑ylidene)gold(I) bromide (11)
1-(3,5-Dibromophenyl)-3-ethylimidazolium bromide
(83.7 mg, 0.20 mmol) and potassium carbonate (270 mg,
1.95 mmol) were stirred in acetonitrile (5 mL) at room tem-
perature for 5 min. (Me2S)AuBr (29.1 mg, 0.086 mmol) was
added and the mixture was stirred for 24 h. The mixture was
fltered through Celite and the fltrate removed to dryness.
The residue was recrystallised from acetonitrile/diethyl ether
and dried under vacuum to leave the product as a beige pow-
der (69 mg, 86%). 1HNMR (500 MHz, DMSO-d6): δ 8.03
(4H, d,4JH,H =1.7 Hz, ArH ortho), 8.01 (2H, t,4JH,H =1.7 Hz,
Bis(1‑(3,5‑dibromophenyl)‑3‑benzylimidazo‑
lin‑2‑ylidene)gold(I) bromide (13)
1-(3,5-Dibromophenyl)-3-benzylimidazolium bromide
(100 mg, 0.21 mmol) and potassium carbonate (290 mg,
2.09 mmol) were stirred in acetonitrile/ethanol (4:1 mL)
at room temperature for 5 min. (Me2S)AuBr (31.0 mg,
0.091 mmol) was added and the mixture was stirred for
24 h. The mixture was fltered through Celite and the fl-
trate evaporated to dryness. The residue was recrystalised
from acetonitrile/diethyl ether and dried under vacuum to
leave the product as a white powder (89 mg, 92%). 1H NMR
3
ArH para), 7.94 (2H, d, JH,H = 1.9 Hz, imidazolyl H5),
3
7.84 (2H, d, JH,H = 1.9 Hz, imidazolyl H4), 4.22 (4H, q,
3
3JH,H = 7.3 Hz, CH2(CH3)2), 1.4 (6H, t, JH,H = 7.3 Hz,
4
(500 MHz, DMSO-d6): 8.04 (4H, d, JH,H = 1.7 Hz, ArH
CH2(CH3)2). 13C NMR (125.7 MHz, DMSO-d6): δ 181.4
(imidazolyl C2), 141.3 (ArC ipso), 134.5 (ArC para), 127.6
(ArC ortho), 123.6 (imidazolyl C5), 123.3 (ArC-Br), 123.2
(imidazolyl C4), 46.6 CH2(CH3)2, 16.9 CH2(CH3)2. Microa-
nalysis: Found: C, 28.31; H, 2.28; N, 5.76% C22H20AuBr5N4.
ortho), 7.97 (2H, d, 3JH,H =1.9 Hz, imidazolyl H5), 7.89 (2H,
t, 4JH,H =1.7 Hz, ArH para), 7.85 (2H, m, imidazolyl H4),
7.31–7.27 (6H, m, Ph-H meta and Ph-H para), 7.19 (4H,
m, Ph-H ortho), 5.37 (s, 4H, CH2). 13C NMR (125.7 MHz,
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
DMSO-d6): δ 181.4 (imidazolyl C2), 140.7 (C5H3Br2 C
ipso), 135.8 (Ph C ipso) 134.1 (C5H3Br2 C para), 128.7
(Ph C meta), 128.3 (Ph C para), 127.6 (Ph C ortho), 127.2
(C5H3Br2 C ortho), 123.7 (imidazolyl C5); 123.3 (imida-
zolyl C4), 122.8 (C5H3Br2 C-Br), 53.99 (CH2). Microanaly-
sis: Found: C, 36.00; H, 2.35; N, 5.18% C32H24AuBr5N4
requires, C, 36.22; H, 2.28; N, 5.28%. HRMS (ESI+): Calcd
for C32H24Br4N4Au [M-Br+], m/z 976.8400. Found, m/z
976.8420. Crystals suitable for X-ray difraction studies were
grown by difusion of vapours between diethyl ether and a
solution of complex 13 in acetonitrile.
Cell viability assays
Stock solutions of Au-NHC compounds (10 mM) were pre-
pared in DMSO and were stored at − 20 °C until required.
These stock solutions were serially diluted in cell culture
medium. For cell viability assays, OVCAR-8 cells that
had reached 80% confuency in the tissue culture fasks
were used. For cell seeding, the cells were dissociated as
described above and cell number counted using a hemocy-
tometer and suspended in culture medium at a fnal concen-
tration of 2000 cells/100 µL.
Cell viability was determined using the MTT assay fol-
lowing the previously published protocol [38]. Briefly,
OVCAR-8 cells were seeded in polystyrene 96-well plates
(Costar; 2000 cells in 100 µL culture medium/well) and
incubated for 24 h. Aliquots of solutions of Au-NHC com-
pounds in cell culture medium (100 µL) were added to the
wells so that the fnal concentrations of Au-NHC complex
in wells was 0.39, 0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, or
100 µM. The cells with the Au-NHCs were then incubated
for 72 h. The medium was then carefully and completely
removed by multichannel pipette and replaced by fresh cell
culture medium (90 µL/well) and MTT solution (5 mg/mL
MTT dissolved in PBS, 10 µL), and the plates were agitated
to ensure homogeneity of solutions. The plates were incu-
bated at 37 °C for 2 h, then the solution was removed by
multichannel pipette and replaced by DMSO (200 µL/well)
to solubilise the formazan purple colour from the live cells.
The absorbance at 570 nm (A570) was measured using a plate
reader (Ensight Multimode Plate Reader, PerkinElmer).
Relative cell viability (%) was determined by comparison
of A570 for wells that had contained cells treated with Au-
NHC complexes with A570 for wells that contained cells in
culture medium alone. GraphPad Prism 8 software was used
to determine IC50 values from cell viability data and to plot
graphs of cell viability as a function of concentration of the
Au-NHC complexes. Some imidazolium salts (precursors of
NHC ligands) were used as negative controls for the cyto-
toxicity of the compounds to OVCAR-8 cells.
Cell culture reagents
Rosewell Park Memorial Institute (RPMI-1640) medium
(with l-glutamine and sodium bicarbonate), fetal bovine
serum (FBS), penicillin–streptomycin were obtained
from Sigma Aldrich. Cell culture medium consists of
RPMI-1640 medium (with l-glutamine and sodium bicar-
bonate) supplemented with 10% v/v FBS and 1% v/v
penicillin–streptomycin.
Cells were dissociated during passaging using trypsin
solution 0.05% in Hanks balanced salt solution (HBSS), and
ethylenediaminetetraacetic acid EDTA, without calcium or
magnesium (Hyclone GE Healthcare Life Sciences). MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) was obtained from Thermo Fisher. Phosphate bufered
saline (PBS) was obtained from Astral Scientifc. Stock solu-
tions (10 mM) of test compounds were prepared in DMSO
(cell culture grade), obtained from Bio-Strategy. DMSO (to
dissolve the MTT after incubation step in the MTT assay)
was obtained from Sigma Aldrich.
Cell culture
Human ovarian cancer cells (OVCAR-8, from ATCC)
were cultured in 75 cm2 tissue culture fasks in cell cul-
ture medium (10 mL) at 37 °C under air (5% humidity,
5% CO2). After being incubated for 2 days, the cells had
reached ~ 80% confuency and were then passaged as fol-
lows. The medium was poured of, the cells were detached
by incubation and agitation with trypsin (3 mL) for 3–5 min
at 37 °C. To neutralise trypsin solution, cell culture medium
(3 mL) was added, and the cell suspensions were transferred
to Falcon tubes and centrifuged for 5 min at 1500 rpm. The
supernatant was drawn of by pipette then cell pellet was
resuspended in culture medium (10 mL). The cells were re-
plated into three tissue culture fasks (2 mL cell suspension
and 8 mL culture medium per fask) and incubated for two
days. The cell culture medium was replaced after 1 day if
the cells had shown substantial proliferation and confuency.
Conclusion
A number of Au-NHC complexes functionalised with
3,5-dibromophenyl substituents were synthesised. The
syntheses were straightforward and their 1H and 13C NMR
spectra were as expected. X-Ray difraction showed that
in the solid state, the intramolecular bond distances and
bond angles were unexceptional, but the 3,5-dibromo-
phenyl groups appear to exert signifcant intermolecular
interactions. 3,5-Dibromphenyl groups were involved in
intermolecular Br…π interactions with imidazolyl, phenyl
1 3

Journal of Inclusion Phenomena and Macrocyclic Chemistry
lipophilicity in determining cellular uptake and antitumour activ-
ity of gold phosphine complexes. Cancer Chemother. Pharmacol.
46, 343–350 (2000)
or 3,5-dibromophenyl groups, with Br…π distances in
the range 3.6–4.7 Å. The 3,5-dibromophenyl-functional-
ised Au-NHC complexes showed potent activity against
OVCAR-8 cells, and proved to be slightly more active than
[Au(Pr2Im)2]Br reported previously. We attribute the higher
activity of the 3,5-dibromophenyl-functionalised complexes
to their greater lipophilicity compared to [Au(Pr2Im)2]Br.
11. Wedlock, L.E., Kilburn, M.R., Clif, J.B., Filgueira, L., Saun-
ders, M., Berners-Price, S.J.: Visualising gold inside tumour cells
following treatment with an antitumour gold(I) complex. Metal-
lomics 3, 917–925 (2011). https://doi.org/10.1039/c1mt00053e
12. Berners-Price, S.J., Mirabelli, C.K., Johnson, R.K., Mattern,
M.R., McCabe, F.L., Faucette, L.F., Sung, C.-M., Mong, S.M.,
Sadler, P.J., Crooke, S.T.: In vivo antitumor activity and in vitro
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Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s10847-021-01082-6.
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Acknowledgements The authors acknowledge the facilities and the
scientifc and technical assistance of the Australian Microscopy &
Microanalysis Research Facility at the Centre for Microscopy, Char-
acterisation & Analysis, The University of Western Australia. Hawraa
S. Al-Buthabhak thanks The Higher Committee for Education Devel-
opment in Iraq (HCED) for fnancial support. Al-Salami is supported
by the European Union’s Horizon 2020 research and innovation pro-
gramme under the Marie Skłodowska-Curie Grant Agreement No
872370. Al-Salami is also supported by Beijing Nat-Med Biotechnol-
ogy Co., Ltd, and Glanis Biotech Ltd.
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