Synthesis and cytotoxicity studies of novel NHC?-gold(I) complexes derived from lepidiline A

Article abstract of DOI:10.3390/molecules23082031

Ten novel N-heterocyclic carbene gold(I) complexes derived from lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride) are reported here with full characterisation and biological testing. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (NHC?-AuCl) (1) was modified by substituting the chloride for the following: cyanide (2), dithiocarbamates (3-5), p-mercaptobenzoate derivatives (12-14) and N-acetyl-L-cysteine derivatives (15-17). All complexes were synthesised in good yields of 57-78%. Complexes 2, 12, 13, and 14 were further characterised by X-ray crystallography. Initial evaluation of the biological activity was conducted on all ten complexes against the multidrug resistant MCF-7^topo breast cancer, HCT-116^wt, and p53 knockout mutant HCT-116^-/- colon carcinoma cell lines. Across the three cell lines tested, mainly single-digit micromolar IC₅₀ values were observed. Nanomolar activity was exhibited on the MCF-7^topo cell line with 3 displaying an IC₅₀ of 0.28 μM ± 0.03 μM. Complexes incorporating a Au-S bond resulted in higher cytotoxic activity when compared to complexes 1 and 2. Theoretical calculations, carried out at the MN15/6-311++G(2df,p) computational level, show that NHC? is the more favourable ligand for Au(I)-Cl when compared to PPh₃.

Full text of DOI:10.3390/molecules23082031

molecules
Article
Synthesis and Cytotoxicity Studies of Novel
NHC*-Gold(I) Complexes Derived from Lepidiline A
Danielle Curran 1, Oyinlola Dada 1, Helge Müller-Bunz 1, Matthias Rothemund 2,
Goar Sánchez-Sanz 1,3 ID , Rainer Schobert 2, Xiangming Zhu 1 and Matthias Tacke 1,
ID
*
1
School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland;
danielle.curran@ucdconnect.ie (D.C.); oyinlola.dada@ucdconnect.ie (O.D.);
helge.muellerbunz@ucd.ie (H.M.-B.); goar.sanchez@ichec.ie (G.S.-S.); xiangming.zhu@ucd.ie (X.Z.)
Organic Chemistry Laboratory, University of Bayreuth, Universitätsstr. 30, 95440 Bayreuth, Germany;
bt303719@uni-bayreuth.de (M.R.); rainer.schobert@uni-bayreuth.de (R.S.)
2
3
Irish Centre of High-End Computing, Grand Canal Quay, Dublin 2, Ireland
*
Correspondence: matthias.tacke@ucd.ie; Tel.: +353-1-7168428; Fax: +353-1-7162044
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 August 2018; Accepted: 13 August 2018; Published: 14 August 2018
Abstract: Ten novel N-heterocyclic carbene gold(I) complexes derived from lepidiline A
(1,3-dibenzyl-4,5-dimethylimidazolium chloride) are reported here with full characterisation and
biological testing. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (NHC*-AuCl) (
was modified by substituting the chloride for the following: cyanide ( ), dithiocarbamates (
p-mercaptobenzoate derivatives (12 14) and N-acetyl-L-cysteine derivatives (15 17). All complexes
were synthesised in good yields of 57–78%. Complexes 12 13, and 14 were further characterised
1)
2
3–5),
–
–
2,
,
by X-ray crystallography. Initial evaluation of the biological activity was conducted on all ten
complexes again−st/−the multidrug resistant MCF-7topo breast cancer, HCT-116wt, and p53 knockout
mutant HCT-116
colon carcinoma cell lines. Across the three cell lines tested, mainly single-digit
micromolar IC50 values were observed. Nanomolar activity was exhibited on the MCF-7topo cell line
with displaying an IC50 of 0.28 0.03 M. Complexes incorporating a Au–S bond resulted in
higher cytotoxic activity when compared to complexes and . Theoretical calculations, carried out
3
µM
±
µ
1
2
at the MN15/6–311++G(2df,p) computational level, show that NHC* is the more favourable ligand
for Au(I)-Cl when compared to PPh3.
Keywords: lepidiline A; N-heterocyclic carbene; gold anticancer drug; TrxR inhibition; MTT
cytotoxicity assay; DFT calculations
1. Introduction
Metal-based drugs are an important tool in the development of new therapeutic drugs.
Auranofin, the successful gold(I)-based drug, exhibits both high potency antiarthritic and antitumour
properties [1,2]. Auranofin analogues have since been investigated for their interesting coordination
to both a phosphine and a thioglucoside. In many cases, N-heterocyclic carbenes (NHCs) have been
utilized as an alternative to the phosphine ligand [
stabilizing the highly active gold(I) species, due to their good electron donating ability and their highly
stable carbene from -backbonding [ ]. As a result, several metal NHC complexes have reported
strong anticancer activity [8–10].
3–5]. NHCs have proved to be suitable ligands for
π
6,7
Lepidiline A (Figure 1), a naturally occurring imidazolium compound extracted from the root of
Lepidium meyenii, has presented many biological properties, including cytotoxicity [11]. Lepidiline A
exhibits activity against the human ovarian cancer cell line FDIGROV, with an ED50 of 7.39
µg/mL [11].
Furthermore, this biologically active imidazolium compound acts as a promising structural motif for
Molecules 2018, 23, 2031; doi:10.3390/molecules23082031
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2031
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NHC derivatives [12], and more effective applications of lepidiline A may lie in the development of
metal-based complexes with lepidiline A as the coordinating ligand.
Gold(I) complexes are an important class of anticancer drugs, due to their unique mechanism of
action. It has been shown that gold(I) complexes can elicit tumour cell death through targeting members of
the intracellular redox-homeostasis system, such as the mitochondria associated thioredoxin reductase
(TrxR), whose inhibition leads to reactive oxygen species formation [13–15]. A selenocysteine–cysteine
bridge at the C-terminal of the TrxR enzyme acts as the target for gold(I) [9,16]. Gold(I) has a high affinity
for thiols, due to their soft nature, resulting in strong Au–S bonds. However, gold(I) also binds strongly
to blood thiols such as serum albumin or glutathione, reducing the amount of drug arriving at cancer
cells [17]. Therefore, there is a desire to design a gold(I)-NHC complex that has a suitably strong Au–S
bond incorporated to lessen the chance of blood thiol conjugation.
The effectiveness of these gold(I)-NHC complexes are still restricted by cell selectivity. Introducing
targeting biomolecules to the complex could ensure the drug is delivered directly to the cancer cells,
thus minimizing the death of normal cells and increasing the drug’s efficacy [18]. Modifying the
coordinating ligand of the NHC-gold(I) complex to include a carboxylic acid would allow increased
functionality, such as esters or amides.
Figure 1. Structure of lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolum chloride) (
NHC*-Au(I)-Cl (1) (b).
a) and
Herein we present
(1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (NHC*-AuCl),
a
structural assessment of NHC-Au(I) complexes, based on
Figure 1.
(1)
The synthesis, characterisation, and biological testing of ten new NHC*-gold(I) complexes is
reported. The effect of altering the coordinating ligands of the NHC*-gold(I) on the cytotoxicity is
investigated via MTT-based proliferation assays. The cytotoxicity studies of these novel compounds
have been conducted in vitro against three different tumour cell lines: MCF-7topo (multidrug-resistant
breast cancer), HCT-116wt, and the p53 knockout mutant HCT-116−/− (colon cancer). These
cytotoxicity studies, compared to that of 1, can provide information on the ideal structures of future
gold(I) chemotherapeutic complexes. Additionally, a computational study of
advantages of employing an NHC ligand, as opposed to a phosphine.
1 can highlight the
2. Results and Discussion
2.1. Synthesis and Characterisation
The synthetic route for the ten NHC*-gold(I) complexes described in this paper are shown in
Schemes 1–4. NHC*-Au(I)-Cl ( ) was synthesised according to a procedure previously published [ ].
The preparation of , p-mercaptobenzoate derivatives and , and N-acetyl-L-cysteine (NAC) derivatives
10 and 11 (Scheme 3), were confirmed with 1H and 13C-NMR spectra. Novel complexes
and 12 17 were
1
3
1
7
8
2–5
–
characterised with elemental analysis, high resolution mass spectrometry, IR spectroscopy, and melting
point. See Supplementary Material for 1H and 13C spectra of complexes 2–5 and 12–17.

Molecules 2018, 23, 2031
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Complex
The precursor
produce complex
2
1
was formed in a 66% yield from the anion exchange of chloride to cyanide (Scheme 1).
was reacted with potassium cyanide in dry dichloromethane at reflux for 48 h to
. The reaction does not form the desired product when conducted in a biphasic
2
solvent system with ethyl acetate and water. Upon reaction in the presence of water, the carbene
is protonated to form the corresponding imidazolium dicyanoaurate(I), confirmed by a signal at
δ = 8.66 ppm, representing the protonated carbene.
Scheme 1. General reaction scheme for the synthesis of NHC*-Au-CN (2).
The 1H-NMR spectrum of
2
shows a slight shift of the CH2 protons of the benzyl groups, from
. The quaternary carbon of the cyanide
= 152.6 ppm. An absorption band at 2144 cm−1 in the IR
δ = 5.44 ppm to 5.37 ppm, when compared to the 1H-NMR of
1
ligand appears in the 13C-NMR spectra at
δ
spectra of 2 represents the C≡N stretch.
Complexes 3–5 were prepared by reacting complex 1 with the corresponding sodium carbamate
salt (Scheme 2). This was performed under biphasic conditions by stirring at room temperature in
ethyl acetate and water for 48 h, with relatively good yields of 61–69%. Complexes
synthesised in dichloromethane at reflux for 24 h, this, however, gave lower yields.
3–5 were also
Scheme 2. General reaction scheme for the synthesis of NHC*-Au(I) dithiocarbamate complexes 3–5.
1
Similar to
2
, the CH2 signal in the H-NMR of complexes
3
–
5
is shifted to
δ
= 5.57–5.55 ppm
= 3.51 ppm
) confirms its
coordination to the NHC*-Au(I). Similarly, the CH2 and CH3 peaks of the diethyldithiocarbamate
complex appear at = 3.96 ppm and 1.31 ppm, respectively. The pyrrolidine CH2 peaks of appear
at δ = 3.85 and 1.97 ppm, with a triplet and pentet distinguishing these two peaks.
Previous metal-dialkyldithiocarbamate complexes reported the IR stretch of the carbon sulphur
bond from 820–1050 cm−1
19 20]. The IR spectra of and show a medium band at 971, 910,
and 949 cm−1, respectively, corresponding to the C=S stretch. A nickel(II) dimethyldithiocarbamate
upon coordination to the dithiocarbamates. The addition of a new 1H-NMR singlet at
δ
corresponding to the two methyl groups of the dimethyldithiocarbamate moiety (
3
4
δ
5
[
,
3,
4
5
complex exhibited a carbon–sulphur bond stretch at 975 cm−1
dimethyldithiocarbamate complex . IR spectra of , and
[19
,
21], which correlates well with t−h1e
3
3,
4
5
show bands at 1447, 1411, and 1406 cm
,
respectively, which correspond to the carbon–nitrogen stretching of the carbamate. Interestingly,

Molecules 2018, 23, 2031
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these IR values account for an intermediate bond in the 1450–1550 cm−1 range [20]. This indicates
a resonance structure is present where the carbon–nitrogen bond exhibits more double bond character
than the carbon–sulphur bonds. Furthermore, the presence of only one band for the C=S bond implies
the molecule is symmetrical, and therefore, in the resonant structure shown in Figure 2 [19].
Figure 2. The dominant resonance form of a dithiocarbamate complex.
The synthetic route to ester formation is highlighted below in Scheme 3. The esters
were made with Fischer esterification, by refluxing 4-mercaptobenzoic acid ( ) and N-acetyl-L-cysteine
(NAC) ( ) (both commercially available) in methanol and ethanol with a catalytic amount of sulphuric acid,
to make their corresponding methyl and ethyl esters. Compounds 11 were conjugated with complex
under basic conditions, to obtain complexes 12–17, in relatively good yields of 57–78% (Scheme 4).
7, 8, 10, and 11
6
9
6–
1,
Scheme 3. General reaction scheme for the synthesis of esters 7, 8, 10, and 11.
Compounds
molecules 12 and 15. Esterification of the acid ends of 12 and 15 was unsucessful. Attempts were
made to synthesise complexes 13 14 16 17 by reacting 12 and 15 with methanol or ethanol; however,
6 and 9 were initially conjugated to 1 to make the corresponding NHC*-Au-S-linker
,
,
,
this also proved to be unsucessful. Due to the lack of success via the linear synthesis, we moved to
convergent synthesis, which was successful.
1
The most diagnostic feature in the H-NMR spectra of complexes 12
–
17 is the disappearance
= 3.64–2.48 ppm
= 1.33–1.31 ppm range for the NAC
17). In the NAC series, the acetyl protons on the nitrogen atom of compounds 10
and 11 are observed at = 2.07 and 2.09 ppm, respectively. However, once linked to the NHC*-Au(I)
centre, there is an observed upfield chemical shift of the acetyl protons to = 1.95 and 1.94 ppm in
of the SH signal of the thiols once coordinated to the gold. This appears in the
range for the p-mercaptobenzoate compounds (12 14) ,and
compounds (15
δ
–
δ
–
δ
δ
compounds 16 and 17, respectively. For complexes 13 and 16 there is a slight upfield shift of the CH3
singlet of the methyl compounds upon coordination to the gold; however, in the ethyl compounds,
a downfield shift is noted.

Molecules 2018, 23, 2031
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Scheme 4. General reaction scheme for the synthesis of NHC*-Au-S-linker (12–17).
2.2. Structural Discussion
X-ray crystallography data was obtained for four of the complexes synthesised. The crystal of
complex was developed from the slow diffusion of pentane into a saturated dichloromethane solution
at crystallised in the monoclinic space group P21/m (#11) (Figure 3). The crystals of
18 ◦C. Complex
12 and 13 were formed in a saturated solution of ethyl acetate with the slow infusion of pentane (Figures 4
and 5). Both crystallised in the triclinic space group P (#2), in the absence of any solvent molecules.
2
−
2
1
Crystal 14 was formed in a saturated solution of dichloromethane with slow infusion of diethyl ether
(Figure 6). Complex 14 crystallized in the monoclinic space group C2/c (#15), also in the absence of any
solvent molecules. The X-ray crystal data and structure refinement of complexes 2, 12, 13, and 14 are
found in Table 1, with the selected bond lengths and bond angles compiled in Tables 2 and 3.
C4
C3
C2
C5
C6
C1
C14
C11
C13
C15
C7
C12
C10
N1
C8
C9
C30
N3
Au
N2
C16
C17
C22
C21
C23
C29
C18
C19
C24
C25
C28
C27
C20
C26
Figure 3. X-ray diffraction structure of NHC*-Au-CN (2); thermal ellipsoids are drawn on the 50%
probability level.

Molecules 2018, 23, 2031
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O2
C3
C4
C2
C1
C36
O1
C5
C33
C12
C32
C11
C34
C6
C13
C31
C7
C10
C35
C9
C30
C14
N1
C15
C17
C16
C22
C8
Au
S
N2
C21
C23
C24
C18
C20
C19
C25
C29
C26
C28
C27
Figure 4. X-ray diffraction structure of 12; thermal ellipsoids are drawn on the 50% probability level.
The Au–C(8) bond lengths of 2.031(8) Å for 2, 2.012(3) Å for 12, 2.008(2) Å for 13 and 2.008(3)
Å for 14 suggest that the gold is strongly bound to the carbene in all four complexes. Additionally,
the Au–S bond distance of 2.2856(7) Å in 12, 2.2851(6) Å in 13 and 2.3012(8) Å in 14 is within the
range of reported Au–S bond lengths [22,23]. The C(30)–N(3) bond of 2 of 1.113 Å is indicative of the
triple bond of the cyanide ligand [24]. The X-ray structures of all four compounds show an almost
linear bond angle of 179.6(4)◦ for
C(8)–Au–C(30) and C(8)–Au–S angles, respectively. Bond angles of 108.40(10) for 12, 109.44(8) for 13
and 108.83(12)◦ for 14 are observed for the Au–S–C(30) angle. These values are in good agreement
2
, 177.48(8)◦ for 12, 175.20(6)◦ for 13, and◦173.45(9)◦ for 14◦for the
,
with similar compounds reported earlier by the Tacke group [25,26].
C37
C4
C3
C2
O1
C5
C6
O2
C13
C36
C34
C33
C1
C12
C11
C32
C31
C14
C7
C15
C10
N1
C35
C9
C30
C8
Au
S
C16
N2
C22
C17
C21
C23
C25
C18A
C19A
C26
C24
C20
C27
C29
C28
Figure 5. X-ray diffraction structure of 13; thermal ellipsoids are drawn on the 50% level,
disorder neglected.

Molecules 2018, 23, 2031
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C12
C13
C14
C3
C11
C2
C10
C9
C1
C7
C15
C4
C19
C18
C20
C5
N1
C6
C17
C21
C16
C22
N2
C8
C23
Au
C32
C29
C38
C31
C30
C24
O2
C28
C33
C34
S
C37
C25
C36
C27
C35
O1
C26
Figure 6. X-ray diffraction structure of 14; thermal ellipsoids are drawn on the 50% probability level.
Table 1. Crystal data and structure refinement for complexes 2, 12, 13, and 14.
2
12
13
14
Empirical Formula
Formula Weight (g·mol
Temperature (K)
Crystal system
C30H24AuN3
623.49
100(2)
Monoclinic
C36H29N2O2SAu
750.54
C37H31N2O2SAu
764.66
C38H33N2O2SAu
778.69
100(2)
Monoclinic
−1
)
100(2)
Triclinic
100(2)
Triclinic
Space group
P21/m (#11)
C2/c (#15)
P1 (#2)
P1 (#2)
Unit cell dimensions
12.8150(7)
6.4797(3)
15.6802(8)
90
112.268(6)
90
1204.94(12)
2
9.3234(3)
10.4210(3)
16.0388(5)
75.663(3)
85.553(2)
83.264(3)
1497.46(8)
2
1.665
5.018
740
8.89830(6)
12.12378(8)
15.5000(1)
103.1637(6)
105.3420(6)
98.8340(6)
26.1234(3)
10.2154(1)
23.9038(3)
90
100.387(1)
90
6274.45(12)
8
1.649
a (Å)
b (Å)
c (Å)
◦
α ( )
◦
β ( )
◦
γ ( )
Volume (Å3)
1528.942(19)
2
Z
Density (calcd) (mg/m3)
1.718
11.641
608
1.661
9.964
756
−1
9.723
3088
Absorption coefficient (mm
F (000)
)
Crystal size (mm3)
0.255 × 0.034 × 0.026
3.727 to 77.196
−16 ≤ h ≤ 15
−8 ≤ k ≤ 8
−19 ≤ l ≤ 19
24,420
0.194 × 0.121 × 0.082
2.90 to 29.59
−12 ≤ h ≤ 12
−13 ≤ k ≤ 13
−21 ≤ l ≤ 21
20,276
0.248 × 0.193 × 0.120
3.845 to 76.876
−11 ≤ h ≤ 11
−15 ≤ k ≤ 15
−19 ≤ l ≤ 19
34,268
0.113 × 0.035 × 0.010
3.44 to 76.91
−32 ≤ h ≤ 32
−12 ≤ k ≤ 12
−30 ≤ l ≤ 28
39,269
◦
θ ( )
Index ranges
Reflections collected
Independent reflections Rint
Completeness to θ (%)
2760(0.1335)
99.8
7306(0.0341)
99.2
6409(0.0268)
100.0
6584(0.0339)
99.4
max
Absorption correction
Gaussian
Gaussian
Gaussian
Gaussian
Max and min transmission
0.788 and 0.291
Full-matrix
Least-squares on F2
2760/0/147
1.139
0.714 and 0.472
Full-matrix
Least-squares on F2
7306/0/380
1.046
0.455 and 0.227
Full-matrix
Least-squares on F2
6409/0/389
1.085
0.913 and 0.516
Full-matrix
Least-squares on F2
6584/0/398
1.041
Refinement method
Data/restraints/parameters
Goodness-of-fit on F2
R1 = 0.0442,
wR2 = 0.0983
R1 = 0.0464,
wR2 = 0.0998
1.982 and −1.411
R1 = 0.0255,
wR2 = 0.0437
R1 = 0.0324,
wR2 = 0.0465
1.009 and −0.762
R1 = 0.0178,
wR2 = 0.0439
R1 = 0.0187,
wR2 = 0.0442
0.639 and −0.859
R1 = 0.0266,
wR2 = 0.0674
R1 = 0.0305,
wR2 = 0.0701
1.508 and −1.500
Final R indices [I > 2σ(I)]
R indices (all data)
Largest diff. peak and hole

Molecules 2018, 23, 2031
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Table 2. Selected bond angles for 2, 12, 13, and 14.
2
12
13
14
Au–C(8)
Au–C(30)
C(30)–N(3)
Au–S(1)
S(1)–C(30)
C(36)–O(1)
C(36)–O(2)
O(2)–C(37)
2.031(8)
2.026(9)
1.113(12)
2.012(3)
2.008(2)
2.008(3)
2.2856(7)
1.751(3)
1.249(3)
1.304(3)
2.2851(6)
1.755(2)
1.211(3)
1.349(3)
1.442(3)
2.3012(8)
1.735(3)
1.215(5)
1.337(5)
1.450(5)
Bond lengths (Å).
Table 3. Selected bond angles for 2, 12, 13, and 14.
2
12
13
14
C(8)–Au–S
C(8)–Au–C(30)
Au–S–C(30)
Au–C(30)–N(3)
S(1)–C(30)–S(2)
O(1)–C(36)–O(2)
O(1)–C(36)–C(33)
O(2)–C(36)–C(33)
C(36)–O(2)–C(37)
177.48(8)
175.20(6)
173.45(9)
179.6(4)
177.0(8)
108.40(10)
109.44(8)
108.83(12)
122.8(3)
120.8(3)
116.4(3)
122.9(2)
125.2(2)
111.9(2)
115.4(2)
123.4(3)
124.2(4)
112.4(3)
117.0(3)
Bond angle (◦).
2.3. Biological Evaluation
The in vitro anticancer activity of
against the human colon carcinoma cell line HCT-116wt, the p53 knockout mutant HCT-116−/−
and the multidrug-resistant (mdr) human breast cancer cell line MCF-7topo (Table 4). Bar
and 13
2–5 and 12–17 was tested via MTT-based proliferation assays
,
,
2
all complexes reached low single-digit micromolar IC50 values against the tested cell lines after 72
h of treatment. These two complexes exhibit only moderate toxicities with IC50 values up to 20 M.
While the IC50 values of the dithiocarbamate complexes and the p-mercaptobenzoate complexes
12 14 vary depending on the nitrogen substitution, and the respective esterification, the complexes
carrying NAC, 15 17, show single-digit IC50 values in the low micromolar range for all tested cell
µ
3–5
–
–
lines, with almost similar cytotoxic activities throughout. Esterification of NAC with methanol or
ethanol slightly increased the antitumor activity against all three cell lines. Amongst the three types
of thiolated complexes, the dithiocarbamate complexes
3
–
5
showed the highest activity against the
being the most active complex in total, with IC50
0.03
M against the MCF-7topo cells. To test
mdr MCF-7topo breast cancer cells, with complex
3
values of 1.5 ± 0.1 µM against the HCT-116wt or 0.28
±
µ
the complexes for their dependency on fully functional p53, one activator of the apoptotic cascade, the
complexes were tested for their toxicity against a HCT-116 p53 knockout mutant. Surprisingly, only
a few of the tested complexes showed similar or higher IC50 values against the knockout mutant than
against the wildtype cells. Complexes 4, 5,
12, and 13 exert a higher toxicity against the HCT-116−/−
than against the wildtype HCT-116wt. Overall, the herein presented complexes exhibit high to moderate
antitumoral activity against colon carcinoma cells and a mdr breast cancer cell line. Dithiocarbamate
complex 3 shows the overall highest activity in all tested cell lines.

Molecules 2018, 23, 2031
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Table 4. IC50 values (
µ
M) of compounds 2, 3, and 12
–
17 against MCF-7topo, HCT-116wt, and HCT-116
−/−
p53
cells after 72 h of incubation.
HCT-116wt
MCF-7topo
−/−
HCT-116 p53
2
3
4
14.8 ± 1.9
1.5 ± 0.1
8.0 ± 0.1
6.2 ± 0.3
5.5 ± 0.1
18.1 ± 6.5
6.8 ± 0.2
4.5 ± 1.2
2.8 ± 0.1
2.9 ± 0.1
-
-
10.8 ± 0.9
0.28 ± 0.03
0.36 ± 0.03
1.5 ± 0.3
5.4 ± 0.5
21.3 ± 3.4
13.2 ± 3.7
7.1 ± 0.3
6.3 ± 0.5
5.4 ± 0.5
3.8 ± 0.4
2.0 ± 0.6
2.7 ± 0.2
9.5 ± 0.6
7.9 ± 0.2
6.6 ± 0.3
4.5 ± 0.6
3.7 ± 0.2
5
12
13
14
15
16
17
2.4. Computational Results
The enthalpy of formation has been obtained at the MN15/6-311++G(2df,p)/LANL2TZ(f) level
for both NHC*-AuCl and Ph3P-AuCl compounds (Figure 7). The results show that NHC*-AuCl
presents a more negative enthalpy (
that the formation of NHC*-AuCl is more favourable. Natural bond orbital (NBO) analysis shows
that the AuCl unit in NHC*-AuCl is slightly less negative (
0.32e−) than in Ph3P-AuCl ( 0.35e−),
and displays slightly shorter Au–Cl distances in NHC*-AuCl (2.291 Å) than in Ph3P-AuCl (2.299 Å).
−
315.0 kJ/mol) than Ph3P-AuCl (
−
274.3 kJ/mol), which indicates
−
−
This is indicative of a stronger bond between the gold and the carbene due to the higher
effect of the nucleophilic NHC* ligand.
σ-donating
Figure 7. Molecular orbital corresponding to the σ-C–Au and σ-P–Au bonds of NHC*-AuCl and
Ph3P-AuCl respectively.
Also, for NHC*-AuCl, two backbonding donations from the gold into the
orbitals are observed, E(2) = 15.9 and 16.1 kJ/mol; while in Ph3P-AuCl, three backbonding donations
are observed from the Au atom into the * P–C antibonding orbitals with E(2) = 16.2, 16.0, and 14.9
π* C–N antibonding
π
kJ/mol. The additional backbonding in the Ph3P-AuCl molecule reduces its bond strength, resulting
in a weaker donating ligand. Conclusively, these results give credence to NHCs being the more
favourable ligand than phosphines.

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3. Materials and Methods
3.1. General Conditions
All chemicals were purchased and used as received, unless otherwise stated. Solvents were
1
dried according to the standard procedures, when necessary. H and 13C spectra were recorded on
either a 300 or 400 MHz Varian spectrometer at room temperature (rt). Both chloroform (CDCl3)
and dimethyl sulfoxide (DMSO) were used as deuterated solvents. The residual solvent peak or
tetramethylsilane (TMS) were used as the internal standard. All chemical shifts are reported as
δ
values in parts per million (ppm). Infrared spectra were recorded on a Bruker ALPHA PLATINUM
ATR spectrometer (Millerica, MA, USA). High resolution accurate mass data were obtained on
a Waters/Micromass LCT TOF spectrometer (Milford, MA, USA). under electrospray ionisation
technique. Melting points were measured on a Stuart
™ (Stone, UK). melting point apparatus SMP10.
Elemental analysis was conducted on an Exeter Analytical CE-440 elemental analyser (Coventry, UK).
X-ray crystallography data was collected on a Rigaku Oxford Diffraction (Chalgrove, UK) SuperNova
A diffractometer. Absorbance measurements were done with a TECAN (Männedorf, Switzerland)
Infinite F200 plate reader.
3.2. Synthesis
3.2.1. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) Chloride (1)
Prepared according to literature method [
2H, CHbenzyl), 7.25–7.16 (m, 10H, CHbenzyl + CHphenyl), 7.06–6.92 (m, 8H, CHphenyl), 5.44 (s, 4H, CH2).
13C-NMR (101 MHz, CDCl3,
ppm): 171.6 (NCN), 135.8, 132.2, 130.9, 129.5, 128.8, 128.7, 128.2, 127.6,
3 δ ppm): 7.30 (t, J = 7.4 Hz,
]. 1H-NMR (300 MHz, CDCl3,
δ
127.4 (CHimidazol + CHphenyl + CHbenzyl), 53.2 (CH2).
3.2.2. (1,3-Dibenzyl-4,5-diphenyl-2-ylidene)gold(I) Cyanide (2)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (65 mg, 0.10 mmol) and potassium
cyanide (7.5 mg, 0.12 mmol) were stirred in dichloromethane (15 mL) under reflux for 48 h. The reaction
was washed with deionised water (2
anhydrous MgSO4. This was filtered, and the excess solvent reduced under pressure to 3 mL. Pentane
×
10 mL). The organic solution was extracted and dried over
(40 mL) was added to precipitate a white solid. The product was filtered, washed with pentane (15 mL),
1
and dried in vacuo. Yield: 40.8 mg, 66%. H-NMR (400 MHz, CDCl3,
δ
ppm): 7.32 (t, J = 7.4 Hz, 2H,
CHbenzyl), 7.25–7.20 (m, 10H, CHbenzyl + CHphenyl), 6.99 (t, J = 5.9 Hz, 8H, CHphenyl), 5.37 (s, 4H, CH2).
13C-NMR (101 MHz, CDCl3,
δ
ppm): 182.9 (NCN), 152.6 (CN), 135.7, 132.6, 130.9, 129.7, 128.9, 128.8,
128.4, 127.6, 127.2 (Cimidazol + Cphenyl + Cbenzyl), 53.0 (CH2). IR (ATR): 3058 (w), 3030 (w), 2143 (w),
1594 (w), 1488 (m), 1447 (m), 1348 (m), 1026 (m), 758 (m), 696 (s). MS (ESI+) m/z: 624.2 [M + H]+.
◦
Melting point range: 264–268 C. Anal. calcd for C30H24AuN3 (623.51): C, 57.79; H, 3.88; N, 6.74.
Found: C, 61.08; H, 4.04; N, 6.90. Although these elemental results are outside the acceptable range to
establish purity, they demonstrate the best results yet obtained.
3.2.3. General Procedure for NHC-Au(I) Complexes 3–5
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (65 mg, 0.10 mmol) and the
corresponding sodium carbamate salt (0.12 mmol) were stirred in a biphasic solution of ethyl acetate
(7 mL) and deionised water (6 mL) at rt for 48 h. The reaction mixture was washed with deionised water
(2
×
10 mL) and an aqueous saturated solution of NaCl (10 mL). The combined organic phase was
dried over anhydrous MgSO4, filtered, and reduced to approximately 3 mL under reduced pressure.
Pentane (40 mL) was added to precipitate a solid. The product was filtered, washed with pentane
(15 mL), and dried in vacuo.

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(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) Dimethyldithiocarbamate (3)
1
A white solid was formed. Yield: 50.4 mg, 69%. H-NMR (300 MHz, CDCl3,
δ ppm): 7.32–7.27 (m,
2H, CH), 7.24–7.16 (m, 10H, CH), 7.10–7.04 (m, 4H, CH), 6.95 (d, J = 7.1 Hz, 4H, CH), 5.55 (s, 4H, CH2),
3.51 (s, 6H, CH3). 13C-NMR (101 MHz, CDCl3,
ppm): 136.9, 132.4, 131.2, 131.1, 129.6, 129.1, 128.9,
δ
128.3, 127.1 (Cimidazol + Cphenyl + Cbenzyl), 53.0 (CH2), 45.4 (CH3). MS (ESI+) m/z: 718.2 [M + H]+. IR
(ATR): 3025 (w), 2910 (w), 1603 (w), 1496 (m), 1447 (m), 1248 (m), 1140 (m), 971 (m), 726 (m), 695 (s).
Melting point range: 186–187 ◦C. Anal. calcd for C32H30N3S2Au (717.71): C, 53.55; H, 4.21; N, 5.85; S,
8.94. Found: C, 53.50; H, 4.17; N, 5.77; S, 8.64.
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) Diethyldithiocarbamate (4)
A white solid was formed. Yield: 49.5 mg, 65%. 1H-NMR (300 MHz, CDCl3,
δ ppm): 7.27 (t,
J = 7.3 Hz, 2H, CH), 7.25–7.14 (m, 15H, CH), 7.08–7.04 (m, 3H, CH), 6.93 (d, J = 7.2 Hz, 3H, CH), 5.57
(s, 4H, CH2-Bz), 3.96 (q, J = 7.1 Hz, 4H, CH2-ethyl), 1.31 (t, J = 7.0 Hz, 6H, CH3). 13C-NMR (101 MHz,
CDCl3,
δ ppm): 205.8 (SCS), 180.1 (NCN), 136.3, 132.1, 130.9, 129.2, 128.6, 128.5, 127.9, 127.8 (CHimidazol
+ CHphenyl + CHbenzyl), 53.1 (CH2-benzyl), 49.3 (CH2-ethyl), 12.4 (CH3). MS (ESI+) m/z: 746.2 [M + H]+.
IR (ATR): 3025 (w), 2925 (w), 1603 (w), 1495 (w), 1411 (m), 1260 (m), 1133 (m), 981 (m), 910 (m), 733 (s),
694 (s). Melting point range: 187–188 ◦C. Anal. calcd for C34H34N3S2Au (745.75): C, 54.76; H, 4.60; N,
5.63; S, 8.60. Found: C, 54.58; H, 4.52; N, 5.53; S, 8.72.
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) Pyrrolidinedithiocarbamate (5)
1
A white solid was formed. Yield: 46.6 mg, 61%. H-NMR (300 MHz, CDCl3,
δ ppm): 7.29 (d,
J = 7.5 Hz, 1H, CH), 7.24–7.14 (m, 10H, CH), 7.12–7.03 (m, 4H, CH), 6.95 (d, J = 7.0 Hz, 4H, CH),
5.56 (s, 4H, CH2-Bz), 3.85 (t, 4H, CH2), 1.97 (p, 4H, CH2). 13C-NMR (101 MHz, CDCl3,
ppm): 202.8
δ
(SCS), 180.2 (NCN), 136.3, 132.1, 130.9, 129.3, 128.6, 128.5, 128.0, 127.9, 127.8 (CHimidazol + CHphenyl
+ CHbenzyl), 54.43 (CH2), 53.08 (CH2-benzyl), 26.30 (CH2). MS (ESI+) m/z: 744.2 [M + H]+. IR (ATR):
3027 (w), 2961 ◦(w), 1602 (w), 1494 (w), 1406 (m), 1165 (m), 949 (m), 733 (m), 695 (s). Melting point
range: 188–189 C. Anal. calcd for C34H32N3S2Au (743.73): C, 54.91; H, 4.34; N, 5.65; S, 8.63. Found: C,
54.48; H, 4.26; N, 5.52; S, 8.85.
3.2.4. General Procedure for 7–8, 10–11
Esters
7
–
8
and 10
–
11 were prepared according to modified literature methods [27
,28].
The carboxylic acid (
6
or
9) was dissolved in either methanol (30 mL) or ethanol (30 mL), with 2 drops
of concentrated sulfuric acid added to the solution before refluxing at 90 ◦C for 24 h. The reaction
progress was monitored by TLC (cyclohexane-ethyl acetate; 1:1). The reaction was concentrated under
reduced pressure to yield a white solid.
Methyl-p-mercaptobenzoate (7)
The residue was purified with column chromatography (cyclohexane-ethyl acetate; 3:1) to produce
a white solid. Yield: 319 mg, 95%. The NMR data were in agreement with those reported in
1
literature [27,28]. H-NMR (400 MHz, CDCl3, δ ppm): 7.88 (d, J = 8.6 Hz, 2H, CH), 7.27 (d, J = 8.6 Hz,
2H, CH), 3.89 (s, 3H, CH3), 3.60 (s, 1H, SH). 13C-NMR (101 MHz, CDCl3,
(CH), 130.2 (CH), 128.1 (CH), 127.1 (CH), 52.0 (CH3).
δ ppm): 166.9 (C=O), 138.3
Ethyl-p-mercaptobenzoate (8)
The residue was purified with column chromatography (cyclohexane-ethyl acetate; 3:1) to produce
a white solid. Yield: 319 mg, 95%. The NMR data were in agreement with those reported in
1
literature [29]. H-NMR (400 MHz, DMSO-d6,
δ ppm): 7.77 (d, J = 8.5 Hz, 2H, CH), 7.40 (d, J = 8.5 Hz,
2H, CH), 4.26 (q, J = 7.1 Hz, 2H, CH2), 2.48 (s, 2H, SH), 1.28 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (101 MHz,
DMSO-d6, ppm): 165.4 (C=O), 141.7 (CH), 130.5 (CH), 129.1 (CH), 126.5 (CH), 61.2 (CH2), 14.5 (CH3).
δ

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N-Acetyl-L-cysteine Methyl Ester (10)
The crude product was used without further purification as a white solid. Yield: 618 mg, 88%.
1
The NMR data were in agreement with those reported in literature [30]. H-NMR (400 MHz, CDCl3,
δ
ppm): 4.89 (dt, J = 7.8, 4.1 Hz, 1H, CH), 3.79 (s, 3H, OCH3), 3.01 (ddd, J = 9.0, 4.1, 2.7 Hz, 2H, CH2),
2.07 (s, 3H, CH3), 1.33 (t, J = 9.0 Hz, 1H, SH). 13C-NMR (101 MHz, CDCl3,
δ ppm): 170.5 (C=O), 170.0
(C=O), 53.5 (CH), 52.8 (OCH3), 26.8 (CH2), 23.1 (CH3).
N-acetyl-L-cysteine Ethyl Ester (11)
The crude product used without further purification as a white solid. Yield: 650 mg, 85%.
1
The NMR data were in agreement with those reported in literature [31]. H-NMR (400 MHz, DMSO-d6,
δ
ppm): 4.91–4.82 (m, 2H, CH2), 4.32–4.19 (m, 2H, OCH2), 3.15–2.94 (m, 3H, CH3), 2.09 (s, 3H, CH3),
1.31 (t, J = 7.1 Hz, 1H, SH). 13C-NMR (101 MHz, DMSO-d6,
δ ppm): 172.1 (C=O), 169.8 (C=O), 61.9
(OCH2), 54.7 (CH), 51.7 (CH3), 26.0 (CH2), 22.8 (CH3).
3.2.5. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) p-Mercaptobenzoic Acid (12)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (63 mg, 0.10 mmol) and
p-mercaptobenzoic acid (31 mg, 0.20 mmol) were dissolved in ethyl acetate (5 mL), and K2CO3
(27 mg, 0.20 mmol) was dissolved in water (5 mL). Both solutions were mixed and stirred vigorously
at rt for 24 h. The two phases were separated, and the aqueous phase was re-extracted twice with ethyl
acetate (10 mL). The combined organic phase was washed with 8% HCl (2
over MgSO4 and filtered. The filtrate was concentrated to approximately 3 mL before the addition of
pentane (40 mL). The solution was cooled down to
26 ◦C to allow the product to precipitate out of
×
10 mL), before drying
−
the solution before filtering and drying in vacuo. An off-white product was isolated. Yield: 55 mg,
1
70%. H-NMR (400 MHz, CDCl3,
δ ppm): 7.63 (d, J = 8.4 Hz, 2H, Hb), 7.47 (d, J = 8.4 Hz, 2H, Ha),
7.34–7.18 (m, 12H, CH), 7.09–6.96 (m, 8H, CH), 5.45 (s, 4H, CH2-benzyl). 13C-NMR (101 MHz, CDCl3,
ppm): 182.3 (NCN), 171.7 (C=O), 153.7, 135.8, 132.0, 131.8, 130.6, 129.4, 129.3, 128.6, 128.6, 128.1, 127.3,
δ
127.2, 123.1 (CHimidazol + CHphenyl + CHbenzyl), 52.6 (CH2). MS (QMS-MS/MS) m/z: 773.15 [M + Na]+.
IR (ATR): 3056 (w), 1668 (w), 1580 (w), 1487 (m), 1446 (w), 1025 (m), 764 (m), 729 (s), 694 (s), 628(w),
518 (w). Melting point range: 177–179 ◦C. Anal. calcd for C36H29N2O2SAu (750.70): C, 57.59; H, 3.90;
N, 3.73; S, 4.27; Found: C, 57.33; H, 3.72; N, 3.60; S, 4.59.
3.2.6. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I)-methyl-p-mercaptobenzoate (13)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (253 mg, 0.40 mmol) and
methyl-p-mercaptobenzoate (218 mg, 1.60 mmol) were dissolved in ethyl acetate (20 mL),
and potassium carbonate (222 mg, 1.60 mmol) was dissolved in water (20 mL). Both solutions were
mixed and stirred vigorously at rt for 24 h. The two phases were separated, and the aqueous phase
was washed with ethyl acetate (2
HCl (20 mL), an aqueous saturated solution of NaHCO3 (20 mL) and an aqueous saturated solution of
NaCl (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated to approximately
5 mL before the addition of pentane (40 mL). The solution was cooled down to
product to precipitate out of the solution before filtering and drying in vacuo. An off-white product
×
20 mL). The organic phases were combined and washed with 8%
−
20 ◦C to allow the
1
was isolated. Yield: 202 mg, 65%. H-NMR (400 MHz, CDCl3,
δ ppm): 7.62–7.57 (m, 2H, Hb), 7.51–7.46
(m, 2H, Ha), 7.34–7.28 (m, 2H, CH), 7.27–7.21 (m, 10H, CH), 7.08–7.04 (m, 4H, CH), 7.03–6.98 (m,
4H, CH), 5.46 (s, 4H, CH2), 3.84 (s, 3H, OCH3). 13C-NMR (101 MHz, CDCl3,
ppm): 182.3 (NCN),
167.9 (C=O), 135.8, 132.0, 131.8, 130.6, 129.3, 128.8, 128.6, 128.6, 128.1, 127.3, 127.2, 124.1 (CHimidazol
δ
+
CHphenyl + CHbenzyl), 52.6 (CH2), 51.6 (OCH3). MS (QMS-MS/MS) m/z: 765.18 [M + H]+. IR (ATR):
3057 (w), 1705 (s), 1584 (s), 1432 (m), 1279 (s), 1270 (s), 1172 (w), 1107 (m), 1085 (m), 1021 (w), 760 (s),
696 (s), 526 (w). Melting point range: 149–152 ◦C. Anal. calcd for C37H31N2O2AuS (764.73): C, 58.11;
H, 4.09; N, 3.66; S, 4.19. Found: C, 58.28; H, 4.02; N, 3.41; S, 4.20.

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3.2.7. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I)-ethyl-p-mercaptobenzoate (14)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (254 mg, 0.40 mmol) and
ethyl-p-mercaptobenzoate (291 mg, 1.60 mmol) were dissolved in ethyl acetate (20 mL), and potassium
carbonate (221 mg, 1.60 mmol) was dissolved in water (20 mL). Both solutions were mixed and stirred
vigorously at rt for 24 h. The two phases were separated, and the aqueous phase was re-extracted twice
with ethyl acetate (20 mL). The organic phases were combined and washed with 8% HCl (20 mL), an
aqueous saturated solution of NaHCO3 (20 mL), and an aqueous saturated solution of NaCl (20 mL).
The organic phase was dried over MgSO4, filtered, and concentrated to approximately 5 mL before
the addition of pentane (40 mL). The solution was cooled down to
−
20 ◦C to allow the product to
precipitate out of the solution before filtering and drying in vacuo. An off-white product was isolated.
1
Yield: 178 mg, 57%. H-NMR (300 MHz, CDCl3,
δ
ppm): 7.61 (d, J = 8.4 Hz, 2H, Hb), 7.48 (d, J = 8.4,
2H, Ha), 7.35–7.27 (m, 2H, CH), 7.25–7.17 (m, 10H, CH), 7.09–6.93 (m, 8H, CH), 5.44 (s, 4H, CH2-benzyl),
4.30 (q, J = 7.1 Hz, 2H, CH2-ethyl), 1.35 (t, J = 7.1 Hz, 3H, CH3). 13C-NMR (101 MHz, CDCl3),
ppm):
166.9 (C=O), 135.6, 132.0, 131.8, 130.6, 129.3, 128.8, 128.5, 128.0, 127.4, 127.2 (CHimidazol + CHphenyl
δ
+
CHbenzyl), 60.3 (CH2-ethyl), 52.7 (CH2-benzyl), 14.3 (CH3). MS (QMS-MS/MS) m/z: 779.20 [M + H]+. IR
(ATR): 3056 (w), 1698 (m), 1585 (m), 1445 (m), 1277 (m), 1267 (m), 1105 (m), 1092 (m), 763 (m), 729 (s),
694 (s), 527 (m). Melting point range: 163–166 ◦C. Anal. calcd for C38H33N2O2AuS (778.76): C, 58.60;
H, 4.28; N, 3.60; S, 4.11. Found: C, 58.45; H, 4.01; N, 3.74; S, 4.32.
3.2.8. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I)-N-acetyl-L-cysteine (15)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (63 mg, 0.10 mmol) and
N-acetyl-L-cysteine (18 mg, 0.11 mmol) were dissolved in ethyl acetate (5 mL), and potassium carbonate
(15 mg, 0.11 mmol) was dissolved in water (5 mL). Both solutions were mixed and stirred vigorously
at rt for 24 h. The two phases were separated, and the aqueous phase was washed with ethyl acetate
(
2 × 10 mL). The combined organic phase was washed with 8% HCl (2
MgSO4 and filtered. The filtrate was concentrated to approximately 3 mL before the addition of
pentane (40 mL). The solution was cooled down to
20 ◦C to allow the product to precipitate out of
×
10 mL), then dried over
−
the solution before filtering and drying in vacuo. An off-white product was isolated. Yield: 45 mg,
60%. H-NMR (300 MHz, CDCl3,
(m, 4H, CH2-benzyl), 4.51 (q, J = 4.1 Hz, 1H, CH), 3.91 (s, 2H, CH2-NAC), 1.88 (s, 3H, CH3). 13C-NMR
1
δ
ppm): 7.35–7.08 (m, 12H, CH), 7.04–6.88 (m, 8H, CH), 5.44–5.28
(101 MHz, CDCl3, ppm): 178.0 (NCN), 172.1 (C=O), 169.6 (C=O), 136.2, 132.0, 130.6, 129.2, 128.5,
δ
128.4, 127.8, 127.5, 127.2 (CHimidazol + CHphenyl + CHbenzyl), 56.8 (CH2-NAC), 52.5 (CH2-benzyl), 23.6
(CH3). MS (QMS-MS/MS) m/z: 760.59 [M + H]+. IR (ATR): 3057 (w), 3030 (w), 1665 (m), 1495 (m),
1447 (w), 1075 (w), 1022 (), 764 (m), 730 (m), 696 (s), 518 (w). Melting point range: 102–105 ◦C. Anal.
calcd for C34H32N3O3AuS (759.72): C, 53.75; H, 4.25; N, 5.53; S, 4.22. Found: C, 53.42; H, 4.34; N, 5.13;
S, 3.94.
3.2.9. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I)-N-acetyl-L-cysteine Methyl Ester (16)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (253 mg, 0.40 mmol) and
N-acetyl-L-cysteine methyl ester (285 mg, 1.60 mmol) were dissolved in ethyl acetate (20 mL),
and potassium carbonate (223 mg, 1.60 mmol) was dissolved in water (20 mL). Both solutions were
mixed and stirred vigorously at rt for 24 h. The two phases were separated, and the aqueous phase
was washed with ethyl acetate (2
HCl (20 mL), an aqueous saturated solution of NaHCO3 (20 mL), and an aqueous saturated solution of
NaCl (20 mL). The organic phase was dried over MgSO4, filtered, and concentrated to approximately
5 mL before the addition of pentane (40 mL). The solution was cooled down to
product to precipitate out of the solution before filtering and drying in vacuo. An off-white product
×
20 mL). The organic phases were combined and washed with 8%
−
20 ◦C to allow the
1
was isolated. Yield: 242 mg, 78%. H-NMR (300 MHz, CDCl3,
δ
ppm): 7.34–7.26 (m, 2H, CH), 7.25–7.18
(m, 10H, CH), 7.06–6.93 (m, 8H, CH), 5.43 (s, 4H, CH2-benzyl), 4.73 (dt, J = 7.5, 4.7 Hz, 1H, CH), 3.65

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(s, 3H, OCH3), 3.36 (dd, J = 13.1, 5.5 Hz, 1H, CH2), 3.23 (dd, J = 13.1, 4.7 Hz, 1H, CH2), 1.95 (s, 3H,
CH3-NAC). 13C-NMR (101 MHz, CDCl3,
ppm): 171.8 (C=O), 169.9 (C=O), 135.9, 131.9, 130.6, 129.2,
δ
128.7, 128.5, 128.0, 127.4, 127.3, 126.5 (CHimidazol + CHphenyl + CHbenzyl), 55.0 (CH), 52.6 (CH2-benzyl),
52.1 (CH3), 30.0 (CH2-NAC), 23.1 (CH3-NAC). MS (QMS-MS/MS) m/z: 774.20 [M + H]+. IR (ATR): 3058
(w), 1740 (m), 1670 (m), 1496 (m), 1447 (m), 1207 (w), 1022 (w), 764 (m), 733 (m), 698 (s), 518 (w).
Melting point range: 77–78 ◦C. Anal. calcd for C35H34N3O3AuS (773.75): C, 54.33; H, 4.44; N, 5.43; S,
4.14. Found: C, 54.61; H, 4.26; N, 5.21; S, 4.44.
3.2.10. (1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I)-N-acetyl-L-cysteine Ethyl Ester (17)
(1,3-Dibenzyl-4,5-diphenylimidazol-2-ylidene)gold(I) chloride (254 mg, 0.40 mmol) and
N-acetyl-L-cysteine ethyl ester (306 mg, 1.60 mmol) were dissolved in ethyl acetate (20 mL),
and potassium carbonate (221 mg, 1.60 mmol) was dissolved in water (20 mL). Both solutions were
mixed and stirred vigorously at rt for 24 h. The two phases were separated, and the aqueous phase was
washed with ethyl acetate (2
×
20 mL). The organic phases were combined and washed with 8% HCl
(20 mL), an aqueous saturated solution of NaHCO3 (20 mL), and an aqueous saturated solution of NaCl
(20 mL). The organic phase was dried over MgSO4, filtered, and concentrated◦to approximately 5 mL
before the addition of pentane (40 mL). The solution was cooled down to
−
20 C to allow the product
to precipitate out of the solution before filtering and drying in vacuo. An off-white product was
isolated. Yield: 245 mg, 78%. H-NMR (400 MHz, CDCl3,
1
δ ppm): 7.32–7.26 (m, 2H, CH), 7.24–7.18 (m,
10H, CH), 7.05–6.94 (m, 8H, CH), 5.43 (s, 4H, CH2-benzyl), 4.69 (dd, J = 8.4, 4.0 Hz, 1H, CH), 4.18–4.07 (m,
2H, CH2), 3.29–3.19 (m, 2H, CH2), 1.94 (s, 3H, CH3-NAC), 1.21 (t, J = 7.1 Hz, 3H, CH3-methyl). 13C-NMR
(101 MHz, CDCl3,
δ ppm): 171.3 (NCN), 170.3 (C=O), 169.9 (C=O), 135.9, 131.9, 130.6, 129.2, 128.7,
128.5, 128.5, 127.9, 127.4, 127.4 (CHimidazol + CHphenyl + CHbenzyl), 61.0 (CH2-ethyl), 55.1 (CH), 52.9
(CH2-benzyl), 40.9 (CH2-NAC), 23.1 (CH3-NAC), 14.2 (CH3-ethyl). MS (QMS-MS/MS) m/z: 788.21 [M + H]+
IR (ATR): 3030 (w), 1743 (m), 1660 (m), 1496 (m), 1446 (m), 1202 (m), 1178 (m), 1022 (m), 764 (m),
732 (m), 697 (s), 517 (m). Melting point range: 65–68 ◦C. Anal. calcd for C36H36N3O3AuS (787.78): C,
54.88; H, 4.62; N, 5.33; S, 4.07. Found: C, 54.83; H, 4.61; N, 5.27; S, 4.15.
.
3.3. Structure Determination
X-ray crystallography data was collected on a Rigaku Oxford Diffraction SuperNova A
diffractometer. Complex 12 was measured with Mo-Kα (0.71073 Å), while complexes 2, 13, and 14
were measured with Cu-Kα (1.54184 Å). A complete dataset was collected, assuming that the Friedel
pairs are not equivalent. An analytical absorption correction based on the shape of the crystal was
performed [32]. The structures were solved by direct methods using SHELXS [33] and refined by full
matrix least-squares on F2 for all data using SHELXL [33]. Hydrogen atoms were added at calculated
positions and refined using a riding model. Their isotropic temperature factors were fixed to 1.2 times
(1.5 times for methyl and OH groups) the equivalent isotropic displacement parameters of the parent
atom. Anisotropic thermal displacement parameters were used for all non-hydrogen atoms. CCDC
1854008 (2), CCDC 1850909 (12), CCDC 1850910 (13), CCDC 1850908 (14) contain the supplementary
crystallographic data for this paper, available free of charge from the Cambridge Crystallographic Data
Centre via www.ccdc.cam.ac.uk/structures.
3.4. MTT-Based Proliferation Assay
The cytotoxic activity of al gold complexes was determined via MTT-based proliferation
assays for the colon carcinoma cell line HCT-116wt, its p53 knockout mutant HCT-116−/−, and the
multidrug-resistant MCF-7topo breast cancer cell line. The cells, kept in Dulbecco’s Modified Eagle
Medium (1% anti-anti, 10% FBS), were seeded into the wells of a clear 96 well plate (5
and incubated for 24 h at standard cell culture conditions (37 C, 5% CO2, 95% humidity). Appropriate
×
104 cells/well)
◦
pre-dilutions of freshly made stock solutions (10 mM in DMSO) of
2–5, 12–15, and DMSO as negative
control, were added into the wells of the pre-incubated cells. After 72 h, the medium was exchanged

Molecules 2018, 23, 2031
15 of 17
for a MTT solution (0.05% in PBS) and the cells were further incubated for 2 h. Thereupon, the MTT
solution was again discarded, and the cells and violet formazan were dissolved in an SDS/DMSO
◦
solution (1% SDS, 0.6% AcOH). After another incubation time of 1 h at 37 C, the absorbance of
formazan at 570 nm, and the background at 630 nm, were measured. Means and SDs are calculated
from four independent measurements.
3.5. Computational Details
All compounds have been optimized at the MN15 [34] computational level with the
6–311++G(2df,p) basis set [35] applied to the lighter elements inclusive chlorine. The LANL2TZ(f) basis
set [36] is used throughout for the gold atoms. Frequency calculations have been performed at the same
level in order to confirm that the structures obtained correspond to energetic minima. The effect of
water solvation was then accounted for using the SMD approach implemented in the Gaussian16 [37
]
package including dispersing, repulsing, and cavitation energy terms of the solvent in the optimisation.
Orbitals have been calculates using NBO 6.0 [38] and plotted using Jmol software [39].
4. Conclusions
In summary, a novel NHC*-Au(I)-cyanide complex (2), three NHC*-Au(I)-dithiocarbamates (
3–5),
three NHC*-Au(I)-p-mercaptobenzoates (12
–
14), and three NHC*-Au(I)-NAC (15 17) complexes, were
–
synthesised and characterised.
Complexes
been shown to be stronger
MN15/6-311++G(2df,p)/LANL2TZ(f) level, show the formation of NHC*-AuCl is more desired
than the phosphine alternative, Ph3P-AuCl. A more negative H and concurrent NBO analysis favours
2–
5
and 12
–
17 were based on the NHC* ligand system, as NHCs have
σ
-donors than phosphines. DFT calculations, carried out at the
∆
the NHC* ligand. Furthermore, calculated Au–Cl bond distances reveal the bond is shorter in the
NHC*-AuCl compound, and therefore, stronger than in the phosphine compound.
Cytotoxicity studies conducted against the human colon carcinoma cell lines HCT-116wt, its
p53 knockout mutant HCT-116−/−, and the mdr human breast cancer cell line MCF-7topo, show low
micromolar and even nanomolar activity. Complex 3 exhibited the best activity with IC50 values of
1.5
±
0.1
µ
M and 0.28
±
0.03 µ
M, against HCT-116wt and MCF-7topo cell lines, respectively. Overall,
the NHC*-Au(I)-thiolates proved to be more biologically active than complex
influential Au–S bond.
1 or 2, which lack the
Furthermore, the series of complexes with the NAC derivative (15–17) were the most successful
series of compounds tested. Complexes 15 17 displayed consistently high cytotoxic activity when
–
compared to the other sets, strongly suggesting the benefit of conjugating the NHC*-Au(I) to
a biological vector. These encouraging results may be valuable in the development of new anticancer
drugs that incorporate amino acid derivatives.
Supplementary Materials: See attached for 1H and 13C-NMR spectra of all novel compounds.
Author Contributions: Conceptualization, M.T. and X.Z.; Software, G.S.-S.; Formal Analysis, H.M.-B. and G.S.-S.;
Investigation, D.C., O.D. and M.R.; Resources, R.S.; Writing-Original Draft Preparation, D.C.; Writing-Review &
Editing, M.T., X.Z., G.S.-S., H.M.-B., M.R., and R.S.; Supervision, M.T., X.Z. and R.S.
Funding: We thank the University College Dublin College of Science and the School of Chemistry [R16002] for
funding this research.
Acknowledgments: We thank Ibon Alkorta for helping with the NBO 6.0 calculations and many useful discussions
and to the Irish Centre for High-End Computing (ICHEC) for the provision of computational facilities.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.

Molecules 2018, 23, 2031
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References
1.
Brown, D.H.; Smith, W.E. The chemistry of the gold drugs used in the treatment of rheumatoid arthritis.
Chem. Soc. Rev. 1980, 9, 217–240. [CrossRef]
2.
3.
Eisler, R. Chrysotherapy: A synoptic review. Inflamm. Res. 2003, 52, 487–501. [CrossRef] [PubMed]
Hackenberg, F.; Muller-Bunz, H.; Smith, R.; Streciwilk, W.; Zhu, X.; Tacke, M. Novel ruthenium(II) and gold(I)
NHC complexes: Synthesis, characterization, and evaluation of their anticancer properties. Organometallics
2013, 32, 5551–5560. [CrossRef]
4.
Baker, M.V.; Barnard, P.J.; Berners-Price, S.J.; Brayshaw, S.K.; Hickey, J.L.; Skelton, B.W.; White, A.H. Synthesis
and structural characterisation of linear Au(I) N-Heterocyclic carbene complexes: New analogues of the
Au(I) phosphine drug auranofin. J. Organomet. Chem. 2005, 690, 24–25. [CrossRef]
Zhao, W.; Ferro, V.; Baker, M.V. Carbohydrate-N-heterocyclic carbene metal complexes: Synthesis, catalysis
and biological studies. Coord. Chem. Rev. 2007, 339, 1–16. [CrossRef]
5.
6.
7.
Hopkinson, M.N.; Richter, C.; Schedler, M.; Glorius, F. An overview of N-heterocyclic carbenes. Nature 2014
510, 485–496. [CrossRef] [PubMed]
,
Patil, S.A.; Patil, S.A.; Patil, R.; Keri, R.S.; Balakrishna, G.R.; Tacke, M. N-heterocylic carbene metal complexes
as bio-organometallic antimicrobial and anticancer drugs. Future Med. Chem. 2015, 7, 1305–1333. [CrossRef]
[PubMed]
8.
9.
Marinelli, M.; Santini, C.; Pellei, M. Recent advances in medicinal applications of coinage-metal (Cu and Ag)
N-heterocyclic carbene complexes. Curr. Top. Med. Chem. 2016, 16, 2995–3017. [CrossRef] [PubMed]
Rubbiani, R.; Kitanovic, I.; Alborzinia, H.; Can, S.; Kitanovic, A.; Onambele, L.A.; Stefanopoulou, M.;
Geldmacher, Y.; Sheldrick, W.S.; Wolber, G.; et al. Benzimidazol-2-ylidene gold(I) complexes are thioredoxin
reductase inhibitors with multiple antitumor properties. J. Med. Chem. 2010, 53, 8608–8618. [CrossRef]
[PubMed]
10. Muenzner, J.K.; Biersack, B.; Albrecht, A.; Rehm, T.; Lacher, U.; Milius, W.; Casini, A.; Zhang, J.; Ott, I.
Ferrocenyl-coupled N-heterocyclic carbene complexes of gold (I): A successful approach to multinuclear
anticancer drugs. Chem. Eur. J. 2016, 22, 18953–18962. [CrossRef] [PubMed]
11. Cui, B.; Zheng, B.L.; He, K.; Zheng, Q.Y. Imidazole alkaloids from lepidium meyenii. J. Nat. Prod. 2003, 66,
1101–1103. [CrossRef] [PubMed]
12. Riduan, S.N.; Zhang, Y. Imidazolium salts and their polymeric materials for biological applications.
Chem. Soc. Rev. 2013, 42, 9055–9070. [CrossRef] [PubMed]
13. Rubbiani, R.; Can, S.; Kitanovic, I.; Alborzinia, H.; Stefanopoulou, M.; Kokoschka, M.; Mönchgesang, S.;
Sheldrick, W.S.; Wölfl, S.; Ott, I. Comparative in vitro evaluation of N-heterocyclic carbene gold(I) complexes
of the benzimidazolylidene type. J. Med. Chem. 2011, 54, 8646–8657. [CrossRef] [PubMed]
14. Liu, W.; Gust, R. Update on metal N-heterocyclic carbene complexes as potential anti-tumor metallodrugs.
Coord. Chem. Rev. 2016, 329, 191–213. [CrossRef]
15. Ott, I. On the Medicinal chemistry of gold complexes as anticancer drugs. Coord. Chem. Rev. 2009, 253,
1670–1681. [CrossRef]
16. Zou, T.; Lum, C.T.; Lok, C.N.; To, W.P.; Low, K.H.; Che, C.M. A binuclear gold(I) complex with mixed
bridging diphosphine and bis(N-heterocyclic carbene) ligands shows favorable thiol reactivity and inhibits
tumor growth and angiogenesis in vivo. Angew. Chem. Int. Ed. 2014, 53, 5810–5814. [CrossRef] [PubMed]
17. Zou, T.; Lum, C.T.; Lok, C.; Zhang, J.; Che, C. Chemical biology of anticancer gold(III) and gold(I) complexes.
Chem. Soc. Rev. 2015, 44, 8786–8801. [CrossRef] [PubMed]
18. Gutiérrez, A.; Marzo, I.; Cativiela, C.; Laguna, A.; Gimeno, M.C. Highly cytotoxic bioconjugated gold(I)
complexes with cysteine-containing dipeptides. Chem. Eur. J. 2015, 21, 11088–11095. [CrossRef] [PubMed]
19. Sathyanarayana, D.N. Vibrational Spectroscopy: Theory and Applications, 1st ed.; New Age International (P)
Ltd.: New Delhi, India, 2004.
20. Jamaluddin, N.A.; Baba, I. Synthesis and structural characterization of new dithiocarbamate complexes from
Sb(III) and Bi(III). AIP Conf. Proc. 2013, 1571, 789–794.
21. Durgaprasad, G.; Sathyanarayana, D.N.; Patel, C.C. Normal coordinate analysis of dialkylditlhioearbamate
and its selenium analogue. Can. J. Chem. 1969, 47, 631–635. [CrossRef]
22. Pakiari, A.H.; Jamshidi, Z. Nature and strength of M
−
S Bonds (M = Au, Ag, and Cu) in binary alloy gold
clusters. J. Phys. Chem. A 2010, 114, 9212–9221. [CrossRef] [PubMed]

Molecules 2018, 23, 2031
17 of 17
23. Kokkin, D.L.; Zhang, R.; Steimle, T.C.; Wyse, I.A.; Pearlman, B.W.; Varberg, T.D. Au–S bonding revealed
from the characterization of diatomic gold sulfide, AuS. J. Phys. Chem. A 2015, 119, 11659–11667. [CrossRef]
[PubMed]
24. Hormann, A.L.; Bennett, D.W.; Shaw, C.F.; Reiff, W.M. Solid-state structure and solution equilibria of
cyano(Triethylphosphine)gold(I). Inorg. Chem. 1986, 25, 3953–3957. [CrossRef]
25. Tacke, M.; Dada, O.; O’Beirne, C.; Zhu, X.; Müller-Bunz, H. The non-isomorphous crystal structures of
NHC–Au–Cl and NHC–Au–Br (NHC is 1,3-dibenzyl-4,5-diphenylimidazol-2-ylidene). Acta Crystallogr. Sect.
C Struct. Chem. 2016, 72, 857–860. [CrossRef] [PubMed]
26. Dada, O.; Curran, D.; O’Beirne, C.; Müller-Bunz, H.; Zhu, X.; Tacke, M. Synthesis and cytotoxicity studies of
novel NHC–gold(I) pseudohalides and thiolates. J. Organomet. Chem. 2017, 840, 30–37. [CrossRef]
27. Novoa, A.; Eierhoff, T.; Topin, J.; Varrot, A.; Barluenga, S.; Imberty, A.; Roemer, W.; Winssinger, N. A LecA
ligand identified from a galactoside-conjugate array inhibits host cell invasion by Pseudomonas aeruginosa.
Angew. Chem. 2014, 53, 8885–8889. [CrossRef] [PubMed]
28. Martin, D.P.; Cohen, S.M. Nucleophile recognition as an alternative inhibition mode for benzoic acid based
carbonic anhydrase inhibitors. Chem. Commun. 2012, 48, 5259–5261. [CrossRef] [PubMed]
29. Sala, O.; Santschi, N.; Jungen, S.; Lüthi, H.P.; Iannuzzi, M.; Hauser, N.; Togni, A. S-trifluoromethylation of
thiols by hypervalent iodine reagents: A joint experimental and computational study. Chem. Eur. J. 2016, 22,
1704–1713. [CrossRef] [PubMed]
30. Bernardes, G.J.L.; Grayson, E.J.; Thompson, S.; Chalker, J.M.; Errey, J.C.; El Oualid, F.; Claridge, T.D.W.;
Davis, B.G. From disulfide-to thioether-linked glycoproteins. Angew. Chem. 2008, 47, 2244–2247. [CrossRef]
[PubMed]
31. Uhrig, R.K.; Picard, M.A.; Beyreuther, K.; Wiessler, M. Synthesis of antioxidative and anti-inflammatory
drugs glucoconjugates. Carbohydr. Res. 2000, 325, 72–80. [CrossRef]
32. Clark, R.C.; Reid, J.S. The analytical calculation of absorption in multifaceted crystals. Acta Crystallogr. Sect. A
1995, 51, 887–897. [CrossRef]
33. Sheldrick, G.M. A short history of SHELX. Acta Crystallogr. Sect. A 2008, A64, 112–122. [CrossRef] [PubMed]
34. Yu, H.S.; He, X.; Li, S.L.; Truhlar, D.G. MN15: A Kohn-Sham global-hybrid exchange-correlation density
functional with broad accuracy for multi-reference and single-reference systems and noncovalent interactions.
Chem. Sci. 2016, 7, 5032–5051. [CrossRef]
35. Frisch, M.J.; Pople, J.A.; Binkley, J.S. Self-consistent molecular orbital methods 25. supplementary functions
for gaussian basis sets. J. Chem. Phys. 1984, 80, 3265–3269. [CrossRef]
36. Hay, P.J.; Wadt, W.R. Ab Initio effective core potentials for molecular calculations: Potentials for the transition
metal atoms Sc to Hg. J. Chem. Phys. 1985, 82, 270–283. [CrossRef]
37. Frisch, M.J.; Trucks, G.W.; Schlegel, H.B.; Scuseria, G.E.; Robb, M.A.; Cheeseman, J.R.; Scalmani, G.; Barone, V.;
Petersson, G.A.; Nakatsuji, H.; et al. Gaussian 16, Revision B.01; Gassian, Inc.: Wallingford, CT, USA, 2016.
38. Glendening, E.D.; Badenhoop, J.K.; Reed, A.E.; Carpenter, J.E.; Bohmann, J.A.; Morales, C.M.; Landis, C.R.;
Weinhold, F. NBO 6.0; Theoretical Chemistry Institute, University of Wisconsin: Madison, WI, USA, 2013.
39. Jmol: An Open-Source Java Viewer for Chemical Structures in 3D. Available online: http://www.jmol.org/
(accessed on 15 April 2018).
Sample Availability: Samples of the compounds 1–5 and 12–17 are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).