Synthesis, structural characterisation and anti-proliferative activity of NHC gold amino acid and peptide conjugates

Article abstract of DOI:10.1039/b906140a

We report the synthesis of new NHC gold(i) and NHC gold(iii) halide, amino acid and dipeptide complexes. Transmetallation of the N-phenylalanine- substituted NHC silver complex 3 with Me₂SAuCl yields the phenylalanine-NHC gold(i) conjugate 4a. Halide exchange with LiBr and oxidation of 4a with Br₂ in CH₂Cl₂ yields the phenylalanine-NHC Au(i) and Au(iii) bromides 4b and 4c, respectively. Reaction of N-Boc protected cysteine methyl ester (Boc-Cys-OMe) or the dipeptide N-Boc-Leu-Cys-OMe with the NHC gold chloride 6a yields the (NHC)Au-S complexed amino acid and dipeptide derivatives 8 and 9. The NHC gold(iii) complexes 4c and 6c were characterised by single crystal X-ray analysis. All of the tested gold carbene complexes showed significant anti-tumor activity on the HeLa, HepG2 and HT-29 cancer cell lines. The best compounds show activity comparable to the well-known anti-cancer drug cisplatin. There seems to be no clear cut structure-activity relationship in the compounds tested, nor did we observe a dependence on the metal oxidation state or the different halide substituents. Given the ease of preparation, stability and high activity of the compounds described herein, it may be possible to design tumor-specific anti-cancer agents based on NHC gold amino acid conjugates in the future.

Full text of DOI:10.1039/b906140a

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PAPER
www.rsc.org/dalton | Dalton Transactions
Synthesis, structural characterisation and anti-proliferative activity
of NHC gold amino acid and peptide conjugates†
Jessica Lemke, Antonio Pinto, Philip Niehoff, Vera Vasylyeva and Nils Metzler-Nolte*
Received 27th March 2009, Accepted 28th May 2009
First published as an Advance Article on the web 16th July 2009
DOI: 10.1039/b906140a
We report the synthesis of new NHC gold(I) and NHC gold(III) halide, amino acid and dipeptide
complexes. Transmetallation of the N-phenylalanine-substituted NHC silver complex 3 with Me
yields the phenylalanine–NHC gold(I) conjugate 4a. Halide exchange with LiBr and oxidation of 4a
with Br in CH Cl yields the phenylalanine–NHC Au(I) and Au(III) bromides 4b and 4c, respectively.
2
SAuCl
2
2
2
Reaction of N-Boc protected cysteine methyl ester (Boc–Cys–OMe) or the dipeptide N-Boc–Leu–Cys–
OMe with the NHC gold chloride 6a yields the (NHC)Au–S complexed amino acid and dipeptide
derivatives 8 and 9. The NHC gold(III) complexes 4c and 6c were characterised by single crystal X-ray
analysis. All of the tested gold carbene complexes showed significant anti-tumor activity on the HeLa,
HepG2 and HT-29 cancer cell lines. The best compounds show activity comparable to the well-known
anti-cancer drug cisplatin. There seems to be no clear cut structure–activity relationship in the
compounds tested, nor did we observe a dependence on the metal oxidation state or the different halide
substituents. Given the ease of preparation, stability and high activity of the compounds described
herein, it may be possible to design tumor-specific anti-cancer agents based on NHC gold amino acid
conjugates in the future.
11
Introduction
been described. However, none of these compounds were ever
functionalised with biomolecules.
In the last decade, research on N-heterocyclic carbenes (NHCs)
has developed strongly with applications mainly in organometallic
catalysis, metathesis and lately, medicinal applications, as docu-
A small number of reports appeared in the literature, in which
NHCs or NHC metal complexes were linked to biomolecules
12
13–15
such as carbohydrates and peptides.
In our group, we
1–3
mented in this issue. Strong interest exists in late transition metal
NHC complexes, such as the coin metals silver and gold. Synthesis
of NHC gold complexes allowed the testing of their application
have synthesised a variety of organometal–peptide bioconjugates,
16–24
25
mostly with metallocenes,
scorpionate or cobalt–carbonyl
26
27
complexes, as well as with ruthenium NHC complexes. The
application of gold–phosphine peptide conjugates for the synthesis
on solid phase has successfully been applied as well, using the
4
in fields ranging from luminescent devices to potential drug
5
candidates. Furthermore, important general features regarding
28
6
the nature of the NHC–metal bond were revealed.
aurophilic behaviour of sulfur to couple a thiol-functionalised
biomolecule to the gold centre.
Two examples of NHC gold conjugates with biomolecules were
reported in the literature. The first is a carbene analogue of the
anti-arthritic drug auranofin, which is a gold triethylphosphine
It is widely accepted that the replacement of phosphine ligands
by the isolobal NHC ligands frequently improves the properties
of the new compounds for practical applications, which are water
and air-stable and easier to handle. Moreover, the imidazolium
salts as the ligand precursor can be functionalised with almost
any substituent, a rarely accessible feature in phosphines.
complex coupled to 2¢,3¢,4¢,6¢-tetra-O-acetyl-b-D-glucopyranosyl-
29
1
-thiolate as an ancillary ligand, and the second a gold carbene
In medicinal applications, the focus is mostly concentrated on
cationic NHC gold complexes, which are known to possess anti-
tumor activity. Cationic gold carbenes are lipophilic compounds
which are able to induce mitochondrial membrane permeabilisa-
30
complex of saccharin. The ability of these compounds to act
as potential chemotherapeutic agents due to their DNA-binding
capacity is currently under study. To the best of our knowledge, no
NHC gold conjugates with amino acids or peptides were reported
to the present day.
7–9
tion of rat liver mitochondria, as shown by Berners-Price et al.
Raubenheimer et al. reported a promising ferrocenyl bis(carbene)
Given the promise of NHC gold complexes for medicinal ap-
plications, we decided to extend our efforts in metal–bioconjugate
systems to NHC gold complexes. In this paper, the synthesis of
NHC gold(I) chloride and bromide, as well as the synthesis of
NHC gold(III) bromide complexes are described, using symmetric
or asymmetric NHC carbene precursors, where an amino acid is
attached to a nitrogen atom of the imidazole moiety. Furthermore,
NHC gold complexes, in which the metal atom is coordinated to
the thiol group of cysteine are reported. Finally, a preliminary
study of the cytotoxic activity of the NHC gold(I) and gold(III)
gold(I) complex with an IC50 value in the range of cisplatin against
10
Jurkat, CoLo320 DMand MCF 7cells. Also, a few simple neutral
NHC gold halide complexes with anti-proliferative activity have
Lehrstuhl f u¨ r Anorganische Chemie I - Bioanorganische Chemie, Fakult a¨ t f u¨ r
Chemie und Biochemie, Ruhr-Universit a¨ t Bochum, Universit a¨ tsstrasse 150,
D-44801 Bochum, Germany. E-mail: nils.metzler-nolte@rub.de; Fax: +49
(
0)234 321 4378; Tel: +49 (0)234 322 8152
†
CCDC reference numbers 725410 and 725411. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/b906140a
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7063–7070 | 7063

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Scheme 1 (i) THF, N
CH Cl , 3 h.
2 2 2 2 2 2 2 2 2
, reflux, over night, (ii) CH Cl , Ag O, N , MS, over night, (iii) CH Cl , N , DMSAuCl, 6 h, (iv) acetone, LiBr 24 h, (v) Br ,
2
2
complexes, with halide ligands or thio-derivatives, has been
undertaken and the results are reported.
In order to achieve the halide exchange, the metathesis reaction
described by Nolan was used without further modification,
33
yielding the desired NHC gold(I) bromide 4b. The oxidation of
this Au(I) complex to the desired Au(III) complex was performed
with bromine in acetone at room temperature, resulting in the
formation of 4c as an orange powder.
Results and discussion
Syntheses
For comparison, we also prepared symmetric NHC
3
4
gold derivatives from the 1,3-di-tert-butyl imidazolium salt
A set of neutral amino acid derived NHC gold complexes, namely
t
29,30,35
(
( Bu
2
NHC)AuX, with X = Cl (5a), Br (5b), Br
3
(5c)),
as well
1
-tert-butyl-3-(methylenecarbonyl-phenylalaninemethylester)-
as the chloro (1-methyl-3-diphenylmethane-imidazole-2-ylidene)
imidazol-2-ylidene gold halides, were designed to investigate
their anti-tumor activity, and comparisons were made to their
unfunctionalised neutral NHC gold analogues.
gold(I) complex 6a. Compound 6a was synthesised with little
36
variation of the literature procedure, using Me SAuCl instead
2
of tetrahydrothiophene gold(I) chloride (thtAuCl). The new NHC
gold(I) bromide 6b and NHC gold(III) bromide 6c were obtained
using the above described methods (Scheme 2). All prepared
NHC gold complexes are air- and water-stable. Only 4a shows
a slight colour change when exposed to light, probably due to
degradation.
To exclude the formation of linear cationic bis(NHC) gold
complexes, bulky tert-butyl substituents were placed on the
nitrogen atoms of the imidazole ring. The imidazolium salt
2
was easily synthesised by reacting tert-butylimidazole with
bromoacetyl-L-phenylalanine methylester 1, which in turn was
obtained using the mixed anhydride method where bromoacetic
acid was first activated with isobutyl chloroformiate and
31
N-methylmorpholine. The imidazolium salt 2 was reacted with
Ag O in CH Cl to yield the Ag(I) bis(carbene) complex, which
2
2
2
was then used as a transfer agent to synthesise the desired Au(I)
complex in analogy to the reported procedure by Wang and Lin
32
(
Scheme 1). The NHC silver complex 3 was obtained as a white,
light-sensitive powder. Treatment of 3 with dimethylsulfide gold(I)
chloride (Me SAuCl) in CH Cl afforded the NHC gold(I) chloride
a as a white powder, along with a precipitate of AgBr, which was
filtered off.
2
2
2
4
2 2 2
Scheme 2 (iv) Acetone, LiBr 24 h, (v) Br , CH Cl , 3 h.
7
064 | Dalton Trans., 2009, 7063–7070
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For peptide attachment to the NHC gold complexes via the
gold atom, the dipeptide Boc–cysteine–leucine methylester 7 was
prepared. The NHC gold(I) chloride 6a was stirred with Boc–
a further downfield shift of the C(Au) carbene carbon atom from
172.9 ppm to 185.4 ppm, and a shift to 185.1 ppm in the case
35
of 9.
cysteine ethylester or 7 in CH
desired complexes 8 and 9 as white powders after filtration through
2
Cl
2
, containing NEt , to obtain the
3
Single crystal X-ray structural studies†
Celite (Scheme 3).
Crystals of 4c could be obtained by covering CH
2
2
Cl with a layer
of pentane. The crystal structure of this amino acid derivative is
shown in Fig. 1. Complex 4c has a four-coordinate gold atom,
8
in a square-planar environment, as expected for d metals. The
C(1)–Au(1)–Br(1) and Br(2)–Au(1)–Br(3) bonds are nearly linear,
◦
with angles of 176.1(4) and 177.84(7) . The C(1)–Au(1) distance is
˚
2
.03(2) A, which is in accordance with published NHC–Au(III)
38
bromide complexes. The Br–Au distances were found to be
˚
between 2.408(2) and 2.443(2) A, with the Au–Br bond in trans
being slightly longer than the other Au–Br bonds (Fig. 1).
3 2 2 2
Scheme 3 (i) NEt , CH Cl , N , over night.
Fig. 1 ORTEP plot of 4c with thermal ellipsoids drawn at 50% probability
NMR spectroscopy
level. Hydrogen atoms were omitted for clarity. Selected bond lengths ( A˚ )
◦
The imidazolium salt 2 was fully characterised. As a typical
spectroscopic feature, the acidic proton (NCHN) of 2 appears
as a broad downfield shifted singlet at 9.2 ppm. The two vicinal
protons of the imidazolium group show pseudo-triplets at 7.7 and
and angles ( ): N1–C1 1.35(2), N2–C1 1.32(2), Au1–C1 2.02(2), Au1–Br3
2
.408(2), Au1–Br2 2.418(2), Au1–Br1 2.443(2), C1–Au1–Br1 176.1(4),
C1–Au1–Br2 88.0(5), C1–Au1–Br3 89.9(5), Br1–Au1–Br2 90.76(7),
Br1–Au1–Br3 91.39(8), Br2–Au1–Br3 177.84(7).
3
4
8
.1 ppm (NCH=CHN), due to similar J- and J-coupling in
An X-ray structure of 6c could be obtained using the same
deuterated DMSO, which is common for imidazolium salts.
The NHC silver(I) bromide 3 showed the absence of the acidic
imidazole proton at 9.2 ppm due to complex formation. Further,
the two remaining ring protons of the imidazole moiety were
˚
method. The C(1)–Au(1) bond shows a distance of 2.016(8) A and
the Br–Au distances were as well found in the region of 2.4151(8) to
˚
2
.39(1) A, with the longest Au–Br bond again being opposite to the
carbene ligand. The angles between C(1)–Au(1)–Br(4) and Br(2)–
3
monitored as doublets at 7.1 and 7.3 ppm with a J coupling
◦
◦
Au(1)–Br(3) are also between 178.7(2) and 176.25(4) (Fig. 2).
constant of 2 Hz, in accordance with reported similar carbene
37
13
complexes. The C NMR spectrum showed the expected silver-
Biological studies
bound carbene signal at 180.1 ppm (NC(Ag)N).
1
3
The C NMR of 4a showed the gold–carbene (NC(Au)N) signal
at 171.4 ppm. In contrast to 4a, the carbene signal of 4b appears
In vitro cytotoxicity assays were performed to obtain an insight
into the anti-tumor activity of the neutral gold carbenes. All
NHC gold(I) chloride and NHC gold(III) bromide complexes,
as well as complexes 8 and 9 were screened against the human
cell lines HeLa (human cervix epitheloid carcinoma cell line),
HT-29 (human caucasian colon adenocarcinoma grade II cell line)
and HepG2 (human hepatocellular liver carcinoma cell line). Cell
viability, which correlates with the metabolic activity of a cell, was
4
.1 ppm downfield shifted at 175.1 ppm. This correlates with the
lower Lewis acidity of the gold center, probably afforded by the
3
5
13
lower electronegativity of bromine compared to chloride. The C
NMR resonance of 4c is shifted upfield by 39.3 ppm compared to
the NHC gold(I) bromide 4b. As expected, this shift arises from
the increased acidity of Au(III) due to an increase in the oxidation
30
39
state.
determined by the resazurin assay. Additionally, absolute cell
40
Noteworthy for the complex series 6 is the downfield shift
of the C(Au) resonance of the NHC gold(I) bromide 6b from
numbers were determined by the crystal violet (CV) assay, which
can be applied after elution of resazurin. Both assays gave similar
results (Table 1).
1
72.9 ppm (NHC gold(I) chloride 6a) to 176.3 ppm. In case of
the NHC gold(III) bromide 6c, a highfield shift of -35.0 ppm to
41.3 ppm could be observed. After functionalising 6a with the
A known number of cells were exposed to increasing concentra-
tions of the gold complexes on a 96-well tissue culture plate and
incubated for a given period of time. Due to the poor solubility of
the substances, DMSO stock solutions had to be used. Because of
1
1
cysteine-derivatives, the signal of the thiol proton in the H NMR
disappeared, proving the complexation. The C NMR of 8 shows
1
3
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Table 1 Cytotoxicity expressed as IC50 values (mM)
HeLa
HepG2
CV
HT-29
CV
a
CV
Resazurin
Resazurin
Resazurin
4
4
5
5
6
6
8
9
a
c
a
c
a
c
45.5 ± 4.8
41.3 ± 10.4
10.0 ± 3.7
4.5 ± 0.9
3.3 ± 0.9
27.3 ± 4.8
3.4 ± 1.3
17.3 ± 3
52.7 ± 5.0
36.2 ± 12.9
12.2 ± 5.1
8.0 ± 1.3
5.8 ± 1.9
12.4 ± 3.0
8.3 ± 1.4
29.4 ± 1.8
7.3 ± 0.4
61.4 ± 7.4
71.3 ± 5.9
63.8 ± 6.7
125.8 ± 49.7
37.8 ± 11.4
13.5 ± 5.8
2.8 ± 1.7
12.7 ± 1.2
10.5 ± 1.9
26.8 ± 2.1
7.9 ± 1.2
58.9 ± 4.3
282.5 ± 41.8
62.7 ±10.3
48.5 ± 6.2
5.2 ± 3.0
16.2 ± 1.6
16.9 ± 1.7
34.6 ± 1.8
12.3 ± 2.1
b
b
n.d.
n.d.
119.0 ± 17.4
37.9 ± 11.5
4.7 ± 2.5
28.5 ± 5.3
15.2 ± 1.7
28.1 ± 4.5
1.1 ± 0.1
186.9 ± 34.8
b
n.d.
n.d.
b
65.3 ± 6.2
20.4 ± 0.9
30.0 ± 2.6
0.9 ± 0.1
Cisplatin
3.5 ± 2.5
a
b
CV = crystal violet. n.d. = not determinable.
For the amino acid and peptide conjugates 8 and 9, an increase
of activity compared to 6a could not be detected. Compound
8, with the additional amino acid Boc–cysteine, has IC50 values
almost comparable to 6a. Compound 9, where the dipeptide is
coupled to the gold complex, already exhibits a decrease of anti-
tumor activity. Finally, of all compounds tested herein, compound
5a exhibits the most pronounced cell line dependence, with
excellent activity in HeLa cells, but more than 10-fold decreased
activity in the HepG2 cell line.
Conclusions
Herein we have reported the synthesis of new NHC gold(I) and
NHC gold(III) halide and amino acid complexes. Two of those
gold(III) complexes with NHC ligands were characterised by single
crystal X-ray analysis. All of the tested gold carbene complexes
showed acceptable anti-tumor activity compared to the well-
known anti-cancer drug cisplatin. Increased activity of the NHC
gold(III) bromides 4c and 5c on some cell lines cannot be due to
the presence of bromine, because the NHC gold(III) complex 6c is
Fig. 2 ORTEP plot of 6c with thermal ellipsoids drawn at 50%
probability level. Hydrogen atoms were omitted for clarity. Selected bond
◦
lengths ( A˚ ) and angles ( ): N1–C1 1.350(9), N2–C1 1.317(8), Au1–C1
2
.016(8), Au1–Br2 2.4151(8), Au1–Br3 2.4220(9), Au1–Br4 2.4399(10),
C1–Au1–Br4 178.71(19), C1–Au1–Br2 87.43(18), Br2–Au1–Br4 91.29(3),
Br3–Au1–Br4 91.95(3), C1–Au1–Br3 89.34(18), Br2–Au1–Br3 176.25(4).
3
–8 times less active than the chloride 6a in all tested cell lines.
Nevertheless, introducing an amino acid to the imidazole moiety
of the gold complexes leads to a decrease in biological activity. On
the other hand, amino acid transporters are over-expressed on
some tumor cell lines. By hijacking such amino acid transporters,
these amino acid derivatives could be specific for tumor cells over
the cytotoxicity of DMSO in higher concentrations, final DMSO
concentrations were limited to 0.5% in all samples. In Table 1, IC50
data are reported. Drug-free solvent controls were carried out,
and data for cisplatin, a well-established anti-cancer drug, were
42
normal, healthy cells. In the case of the NHC gold complexes
which were functionalised via a cysteine thiol group, a decrease of
activity was observed for the dipeptide conjugate but not for the
simple amino acid derivative. Comparing 6a, 8 and 9, the results
41
included for comparison.
Generally, all compounds show at least promising anti-
proliferaitve activity, with IC50 values between ca. 100 to very
low mM values. HeLa cells were the most sensitive, while the
HT-29 cells were the least sensitive cells to the carbene complexes.
Compounds 6a and 8 showed the best activity, with consistently
low mM values. The effect of the oxidation state of the gold
atom could not be clarified. For the amino acid labelled gold
complexes 4a and 4c, a decrease in activity could be detected
for HepG2 and HT-29 cells. The opposite trend of anti-tumor
activity was observed for the complexes 5a and 5c, where the
gold(III) complex showed higher activity. In the cases of 6a and 6c,
the anti-proliferative activity was higher for the gold(I) complex,
which again opposes the results for 5a and 5c. As 6a proved to be
the most active gold complex, it was chosen for the syntheses of 8
and 9.
+
show that the [NHC–Au] -fragment alone cannot be responsible
for the cytotoxicity. Whether differences in cytotoxicity are due to
differential uptake or different intracellular interactions requires
further investigation. Clearly, the combination of very stable NHC
metal complexes with biomolecule functionalisation holds great
promise for further investigations.
Experimental section
General remarks
All reagents were purchased from commercial sources and used
as received. L-Amino acids were purchased from IRIS Biotech
or Novabiochem. Antibiotics, colour dyes and cell culture media
7
066 | Dalton Trans., 2009, 7063–7070
This journal is © The Royal Society of Chemistry 2009

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1
◦
were purchased from PAA, Riedel-de-Ha e¨ n and Sigma-Aldrich.
Solvents were distilled over CaCl (CH Cl ) or taken from the
solvent purification system MBraun SPS (THF). Glassware was
powder (580 mg, 46%). H NMR (DMSO-d
6
, 250 MHz, 25 C): d
3
9.37 (1H, br. s, N–CH=N), 9.18 (1H, d, JH,H = 7.41 Hz, NHPhe),
2
2
2
3
3
8.05 (1H, t, JH,H = 1.77 Hz, N–CH=CH–N), 7.69 (1H, t, JH,H
1.77 Hz, N–CH=CH–N), 7.37–7.14 (5H, m, CAr
PheH), 5.1 (2H, s,
N–CH –CO), 4.60–4.48 (1H, m, Ca,PheH), 3.62 (3H, s, CO CH ),
=
used oven dry. NMR spectra were recorded on a Bruker DPX
,
1
2
50 ( H at 250 MHz) spectrometer. The NMR chemical shifts
2
2
3
2
3
(
d) are reported in ppm relative to the residual proton chemical
3.02 (2H, dq, JH,H = 13.75 Hz, JH,H = 7.41 Hz, Cb,PheH), 1.60
1
3
◦
shifts of the deuterated solvent set relative to external TMS.
Microanalysis was performed on a Analytik Jena Multi EA 3100.
Electrospray ionisation mass spectra (ESI) were recorded with a
Bruker Esquire 6000 instrument. FAB mass spectra were recorded
on a Finnigan VG Autospec (glycerol or 3-nitrobenzylalcohol
(9H, s, N–C(CH
171.4 (CO CH
3
)
3
). C NMR (DMSO-d
6
, 63 MHz, 25 C): d
), 165.2 (CO), 136.7 (N–CH=N), 135.3 (Cq,Phe),
2
3
129.2 (CAr,Phe), 128.6 (CAr,Phe), 127.3 (CAr,Phe), 123.9 (N–CH=CH–
N), 119.6 (N–CH=CH–N), 59.6 (N–C(CH
3
)
3
), 54.2 (N–CH –
2
CO), 51.8 (Ca,Phe), 50.2 (CO
2
CH
3
), 36.8 (Cb,Phe), 29.0 (N–C(CH
3
3
) ).
3
4
+
+
(
NBA) as matrix). Di-tert-buylimidazolium chloride, tert-butyl-
MS (ESI ): m/z = 344.16 [M - Br] . Anal. calc. for C19
H
26BrN
3
O
3
43
imidazol, chloro-(1,3,-di-tert-butylimidazol-2-ylidene) gold(I)
a, bromo-(1,3,-di-tert-butylimidazol-2-ylidene) gold(III) 5b,
trisbromo-(1,3,-di-tert-butylimidazol-2-ylidene) gold(III) 5c, 1-
methyl-3-diphenylmethyl-imidazolium chloride and chloro-(1-
methyl-3-diphenylmethyl-imidazol-2-ylidene) gold(I) 6a and
Me S)AuCl, were prepared according to the literature. X-Ray
(m = 424.34): C, 53.78; H, 6.18; N, 9.90. Found: C, 54.39; H, 5.85;
29
35
5
N, 9.56.
33
36
{
[1-tert-Butyl-3-(2-(2-acetamido)-3-phenylalanine methylester)
3
6
imidazol-2-ylidene)] AgBr} (3)
2
44
(
2
˚
data were collected using a Bruker-axs SMART 1000 CCD
diffractometer. Structure solution was performed with direct
To a dried Schlenk tube charged with molecular sieves (4 A, 50 mg)
was added 2 (258 mg, 0.61 mmol) and Ag O (74 mg, 0.32 mmol).
The mixture was backflashed three times with N and then dry
CH Cl was added (30 mL). The flask was closed and shaken over
2
45
2
methods (SHELXS-97), and refined against F with all measured
2
45
46
reflections (SHELXL-97, Platon-Squeeze).
2
2
night in the dark. The solution was filtered through Celite and
the solvent was removed under reduced pressure. The residue was
recrystallised from THF–hexane (10 mL : 30 mL) at 4 C to yield a
Methyl 2-(2-bromoacetamido)-3-phenyl-methylpropanoate (1)
◦
1
◦
To a stirred solution of bromoacetic acid (1.4 g, 10 mmol) in THF
was added N-methylmorpholine (1.12 mL, 10 mmol), followed
by the addition of isobutyl chloroformiate (1.32 mL, 10 mmol),
resulting in a precipitation of a white solid. In a second flask
L-phenylalanine-methylester hydrochloride (2.16 g, 10 mmol) was
suspended in THF (50 mL) containing triethylamine (1.38 mL,
white powder (173 mg, 54%). H NMR (CD
3
CN, 250 MHz, 25 C):
3
d = 7.32 (1H, d, JH,H = 1.7 Hz, N–CH=CH–N), 7.27–7.19 (6H,
3
m, CAr,PheH, NHPhe), 7.14 (1H, d, JH,H = 1.7 Hz, N–CH=CH–
N), 4.87 (2H, s, N–CH –CO), 4.71–4.58 (1H, m, Ca,PheH), 3.62
2
2
3
(3H, s, CO
2
CH
3
), 3.05 (2H, dq, JH,H = 13.80 Hz, JH,H = 6.87 Hz,
). C NMR (CD CN, 63 MHz,
25 C): d 180.1 (NCN), 167.8 (CO CH ), 156.4 (CONH), 138.1
1
3
C
b,PheH), 1.68 (9H, s, N–C(CH
3
)
3
3
◦
1
0 mmol). Both suspensions were mixed and stirred for 1 h at
2
3
room temperature. After removal of the white precipitates by
filtration, the solvent was removed under reduced pressure and the
(Cq,Phe), 129.9 (CAr,Phe), 129.5 (CAr,Phe), 127.8 (CAr,Phe), 122.5 (N–
CH=CH–N), 120.3 (N–CH=CH–N), 58.8 (N–C(CH
) ), 55.3
), 38.2 (Cb,Phe), 29.8 (N–
3
3
residual oil dissolved in CHCl
with H O (50 mL) and the aqueous solution was back-extracted
with CHCl (3 ¥ 50 mL). The combined CHCl solutions were
dried over Na SO . Evaporation of the solvent under reduced
3
(100 mL). The solution was washed
(N–CH
C(CH
2
–CO), 54.4 (Ca,Phe), 52.9 (CO
2
CH
3
+
+
2
3
)
3
). MS (Fab ): 794.4 [M - AgBr
2
]
(100%). Anal. calc.
3
3
for C38
H
50Ag
2
Br
2
N
6
O
6
(m = 1062.39): C, 42.96; H 4.74; N, 7.91.
2
4
Found: C, 44.45; H, 5.19; N, 8.40.
1
pressure yielded the product as a white solid (2.51 g, 84%). H
◦
NMR (DMSO-d
6
, 200 MHz, 25 C): d 7.34–7.16 (3H, m, HAr,Phe),
(
1-tert-Butyl-3-(2-(2-acetamido)-3-phenylalanine methylester)
3
7
4
3
7
.09–7.05 (2H, m, HAr,Phe), 6.83 (1H, d, JH,H = 7.1 Hz, NH), 4.87–
imidazol-2-ylidene) gold(I) chloride (4a)
2
.72 (1H, m, Ca,PheH), 3.79 (1H, d, JH,H = 1.4 Hz, Br–CH
2
3
–CO),
2
.68 (3H, s, CO
2
CH
3
), 3.10 (2H, dq, JH,H = 13.75 Hz, JH,H
=
To a dry Schlenk tube was added 3 (258 mg, 0.09 mmol) and
1
3
◦
.25 Hz, Cb,PheH). C NMR (DMSO-d
6
, 50 MHz, 25 C): d 171.3
(Me
2
S)AuCl (54 mg, 0.18 mmol). The mixture was backflashed
and then dry CH Cl was added (20 mL). The
(
CO
2
CH
3
), 165.1 (CO), 135.3 (Cq,Phe), 129.2 (CAr,Phe), 128.6 (CAr,Phe),
three times with N
2
2
2
1
CH
C
27.3 (CAr,Phe), 53.7 (Ca,Phe), 52.5 (CO
2
CH
3
), 37.6 (Cb,Phe), 28.6 (Br–
suspension was stirred over night in the dark, during which the
colour changed to grey-violet. The mixture was filtered through
Celite and the solvent was removed under reduced pressure. The
+
+
2
–CO). MS (FAB ): m/z = 300.0, 302.0 [M ]. Anal. calc. for
12
H
14BrNO
3
(m = 300.15): C, 48.02; H, 4.70; N, 4.67. Found: C,
◦
50.44; H, 5.12; N, 4.74.
residue was recrystallised from CH
to yield 4a as a white powder (215 mg, 74%). H NMR (CD
2
Cl
2
–pentane (1 : 5) at -20 C
1
2
Cl ,
2
◦
2
7
7
50 MHz, 25 C): d 7.36–7.08 (6H, m, N–CH=CH–N, CAr,PheH),
(
1-tert-Butyl-3-(2-(2-acetamido)-3-phenylalanine methylester)
3
3
.01 (1H, d, JH,H = 2.0 Hz, N–CH=CH–N), 6.44 (1H, d, JH,H
=
imidazolium bromide (2)
3
.0 Hz, NHPhe), 4.95 (1H, d, JH,H = 6.1 Hz, N–CH
2
–CO), 4.87–
2
To a solution of tert-butylimidazol (372 mg, 3 mmol) in dry THF
4.79 (1H, m, Ca,PheH), 3.72 (3H, s, CO
2
CH
3
), 3.12 (2H, dq, JH,H
=
3
13
(
10 mL) was added dropwise a solution of 1 (897 mg, 3 mmol) in
13.80 Hz, JH,H = 5.8 Hz, Cb,PheH), 1.82 (9H, s, N–C(CH
3
)
3
).
C
◦
THF (10 mL). After complete addition, the mixture was refluxed
for 24 h, during which the solution turned pale orange. The solvent
was removed under reduced pressure and the residue was washed
with pentane and dried in vacuo to give a white-orange hygroscopic
NMR (CD
2
Cl
2
, 63 MHz, 25 C): d 172.0 (CO
2
CH ), 171.4 (NCN),
3
165.8 (CONH), 136.3 (Cq,Phe), 129.9 (CAr,Phe), 129.2 (CAr,Phe), 127.6
(CAr,Phe), 122.7 (N–CH=CH–N), 119.5 (N–CH=CH–N), 59.7 (N–
C(CH
3
)
3
, 55.4 (N–CH
2
–CO), 54.1 (Ca,Phe), 53.0 (CO
2
CH ), 38.2
3
This journal is © The Royal Society of Chemistry 2009
Dalton Trans., 2009, 7063–7070 | 7067

View Article Online
+
+
1
◦
(
4
C
b,Phe), 31.8 (N–C(CH
3
)
3
). MS (FAB ): 540.1 [M - Cl] (95%),
white powder (25 mg, 65%). H NMR (CD
2
Cl
2
, 250 MHz, 25 C):
+
+
84.1 [M - Cl-tert-butyl + H] (20), 344.2 [M - AuCl] (100).
Anal. calc. for C19
(m = 575.85): C, 39.63; H, 4.38;
N 7.30. Found: C, 39.9; H, 4.21; N, 7.34.
d 7.40–7.35 (6H, m, CArH), 7.30 (1H, s, NCH(Ph)
2
), 7.19–7.13
3
H
25AuClN
3
O
3
(4H, m, CArH), 6.97 (1H, d, JH,H = 1.8 Hz, N–CH=CH–N), 6.83
3
13
(1H, d, JH,H = 1.8 Hz, N–CH=CH–N), 3.85 (3H, s, N–CH
). C
3
◦
NMR (CD
2
Cl
2
, 63 MHz, 25 C): d 176.3 (NCN), 138.8 (Cq,Phe),
(
1-tert-Butyl-3-(2-(2-acetamido)-3-phenylalanine methylester)
imidazol-2-ylidene) gold(I) bromide (4b)
129.4 (CAr,Phe), 129.1 (CAr,Phe), 128.9 (CAr,Phe), 122.5 (N–CH=CH–
N), 120.2 (N–CH=CH–N), 68.5 (N–CH(Ph)
), 49.0 (N–CH ).
2
3
+
+
+
MS (Fab ): 693.1 [M + carbene] (60%), 445.0 [M - Br] (25).
Anal. calc. for C17
(m = 525.20): C, 38.88; H, 3.07; N,
.33. Found: C, 38.10; H, 3.24; N, 4.99.
Compound 4a (215 mg, 0.37 mmol) was dissolved in acetone
H
16AuBrN
2
(
1 mL) and LiBr (273 mg, 3.18 mmol) was added. The reaction
was stirred for 24 h in the dark. The solvent was removed under
reduced pressure. The residue was dissolved in CH Cl (2 mL) and
MgSO was added. The salts were filtered off using a short plug
5
2
2
(
1-Diphenylmethyl-3-(methyl imdazol-2-ylidene) gold(III)
4
bromide (6c)
of silica and the solvent was removed under reduced pressure. The
◦
residue was recrystallised from CH
4
2
2
Cl
2
–pentane at -20 C to yield
To a solution of 6b (15 mg, 28.6 mmol) in CH
2
Cl (2 mL) was
2
1
b as a white powder (70 mg, 30%). H NMR (CD
2
Cl , 250 MHz,
2
added bromine (4 mg, 34.4 mmol). The reaction was stirred for 3 h
in the dark and the solvent was removed under reduced pressure,
as well as the excess of bromine. The residue was recrystallised
◦
5 C): d 7.36–7.12 (6H, m, N–CH=CH–N, CAr,PheH), 7.05 (1H,
3
3
d, JH,H = 2.1 Hz, N–CH=CH–N), 6.55 (1H, d, JH,H = 7.0 Hz,
3
NHPhe), 4.99 (1H, d, JH,H = 1.9 Hz, N–CH
2
–CO), 4.88–4.81 (1H,
◦
from CH
2
Cl
2
–pentane (1 : 5) at -20 C to yield 6c as an orange
m, Ca,PheH), 3.72 (3H, s, CO
1
d 175.1 (NCN), 172.0 (CO
1
2
CH
3
), 3.21–3.01 (2H, m, Cb,PheH),
1
◦
powder (10 mg, 51%). H NMR (CD
.44–7.39 (6H, m, CArH), 7.28–7.18 (5H, m, NCH(Ph)
2
Cl
2
, 250 MHz, 25 C): d
1
3
◦
.81 (9H, s, N–C(CH
3
)
3
). C NMR (CD
2
Cl
2
, 63 MHz, 25 C):
7
7
2
6
1
2
, CArH),
2
CH
3
), 166.2 (CONH), 136.2 (Cq,Phe),
3
3
.16 (1H, d, JH,H = 2.0 Hz, N–CH=CH–N), 6.95 (1H, d, JH,H
=
29.9 (CAr,Phe), 129.2 (CAr,Phe), 127.6 (CAr,Phe), 120.7 (N–CH=CH–
13
.0 Hz, N–CH=CH–N), 3.92 (3H, s, N–CH
). C NMR (CD Cl ,
3
2
2
N), 119.5 (N–CH=CH–N), 59.8 (N–C(CH
)
), 55.3 (N–CH
–
◦
3
3
2
3 MHz, 25 C): d 141.3 (NCN), 137.0 (Cq,Phe), 129.6 (CAr,Phe),
CO), 54.1 (Ca,Phe), 53.1 (CO
2
+
CH
3
), 38.1 (Cb,Phe), 31.9 (N–C(CH
3
3
) ).
29.6 (CAr,Phe), 129.3 (CAr,Phe), 125.1 (N–CH=CH–N), 123.2 (N–
+
+
MS (FAB ): 540.0 [M - Br] (100%), 344.2 [M - AuBr] (30). Anal.
+
CH=CH–N), 68.5 (N–CH(Ph)
), 38.9 (N–CH
). MS (Fab ):
2
3
calc. for C19
Found: C, 36.11; H, 4.24; N 6.98.
H
25AuBrN
3
O
3
(m = 620.30): C, 36.79; H, 4.06; N 6.77.
+
4
6
45.0 [M - 3Br] (100%). Anal. calc. for C17
H
16AuBr
3
N
2
(m =
85.01): C, 29.81; H, 2.35; N, 4.09. Found: C, 27.93; H, 2.37;
N, 3.50.
(
1-tert-Butyl-3-(2-(2-acetamido)-3-phenylalanine methylester)
imidazol-2-ylidene) gold(III) bromide (4c)
Boc–leucine–cysteine–OEt (7)
To a solution of 4b (36 mg, 58 mmol) in CH
bromine (11 mg, 70 mmol). The reaction was stirred for 3 h in
the dark and the solvent was removed under reduced pressure,
2
Cl (2 mL) was added
2
To a stirred solution of Boc–leucine (0.72 g, 2 mmol) in THF
(
20 mL) was added N-methylmorpholine (0.22 mL, 2 mmol)
followed by the addition of isobutyl chloroformiate (0.26 mL,
2 mmol), resulting in a precipitation of a white solid. In a second
flask, cysteine ethylester hydrochloride (0.37 g, 2 mmol) was
suspended in THF (20 mL) containing triethylamine (0.28 mL,
2 mmol). Both suspensions were mixed and stirred for 1 h at room
temperature. After removal of the white precipitates by filtration,
the solvent was removed under reduced pressure and the residual
as well as the excess of bromine. The residue was recrystallised
◦
from CH
2
Cl
2
–pentane (1 : 5) at -20 C to yield 4c as an orange
1
◦
powder (41 mg, 90%). H NMR (CD
2
Cl
2
, 250 MHz, 25 C): d
7
.40–7.14 (7H, m, N–CH=CH–N, N–CH=CH–N, CAr,PheH), 6.62
3
(
1H, d, JH,H = 7.0 Hz, NHPhe), 5.09–4.87 (3H, q + m, N–CH
2
–
CO, Ca,PheH), 3.74 (3H, s, CO
2
CH
3
), 3.22–3.01 (2H, m, Cb,PheH),
1
3
◦
1
.85 (9H, s, N–C(CH
3
)
3
). C NMR (CD
3
CN, 63 MHz, 25 C):
oil dissolved in CHCl
water (30 mL) and the aqueous solution was back-extracted with
CHCl (3 ¥ 30 mL). The combined CHCl solutions were dried
over Na SO . Evaporation of the solvent under reduced pressure
yielded the product as a white solid (0.58 g, 80%). H NMR
3
(50 mL). The solution was washed with
d 172.1 (CO CH
1
2
3
), 165.1 (CONH), 137.4 (Cq,Phe), 135.8 (NCN),
30.6 (CAr,Phe), 129.6 (CAr,Phe), 128.0 (CAr,Phe), 127.1 (N–CH=CH–
3
3
N), 124.0 (N–CH=CH–N), 62.7 (N–C(CH
3
)
3
), 55.0 (N–CH –
2
2
4
CO), 53.7 (Ca,Phe), 52.9 (CO
2
CH
3
), 38.3 (Cb,Phe), 31.7 (N–C(CH
3
3
) ).
1
+
+
+
MS (FAB ): 540.0 [M - 3Br] (100%), 344.1 [M - AuBr] (20).
◦
3
(DMSO-d
6
, 250 MHz, 25 C): d 8.17 (1H, d, JH,H = 7.8 Hz,
Anal. calc. for C19
N, 5.39. Found: C, 28.72, H, 3.45; N, 6.00.
H
25AuBr
3
N
3
O
3
(m = 780.10): C, 29.25; H, 3.23;
3
NHLeu), 6.93 (1H, d, JH,H = 8.2 Hz, NHBoc), 4.55–4.39 (1H,
m, Ca,CysH), 4.14–4.09 (3H, m, CO
2
–CH
2
–CH3, Ca,LeuH), 2.91–
2
.65 (2H, m, Cb,CysH), 1.68–1.52 (3H, m, Cb,LeuH, Cg,LeuH), 1.43
(
1-Diphenylmethyl-3-(methyl imdazole-2-ylidene) gold(I)
3
(
0
10H, s, Boc, SCysH), 1.30 (3H, t, JH,H = 6.8 Hz, CO
2
–CH
2
–CH ),
3
bromide (6b)
3
2
13
.86 (6H, dd, JH,H = 6.8 Hz, JH,H = 4.1 Hz, Cd,LeuH). C NMR
◦
Compound 6a (35 mg, 73 mmol) was dissolved in acetone (1 mL)
and LiBr (53 mg, 620 mmol) was added. The reaction was stirred
for 24 h in the dark. The solvent was removed under reduced
pressure. The residue was dissolved in CH
was added. The salts were filtered off through a short plug of silica
(DMSO-d
6
, 63 MHz, 25 C): d 172.9 (CO
2
Et), 170.1 (COLeu),
155.4 (COBoc), 78.1 (C(CH
52.4 (Ca,Leu), 40.6 (Cb,Leu), 27.8 (C(CH
(Cd,Leu), 21.3 (Cg,Leu), 14.0 (CO
[M + Na] , 334.14 [M - C
3
)
3
), 60.9 (CO
2
–CH
2
–CH ), 54.0 (Ca,Cys),
3
3
)
3
), 25.1 (Cb,Cys), 24.0, 22.6
+
2
Cl
2
(2 mL) and MgSO
4
2
+
–CH
2
–CH
3
). MS (ESI ): 385.13
+
+
2
H ] , 307.10 [M - tert-butyl] . Anal.
5
and the solvent was removed under reduced pressure. The residue
calc. for C16
H
30
N
2
O
5
S (m = 362.49): C, 53.01; H, 8.34; N, 7.73; S,
◦
was recrystallised from CH
2
Cl
2
–pentane at -20 C to yield 6b as a
8.85. Found: C, 53.19; H, 8.36; N, 7.67; S, 8.88.
7
068 | Dalton Trans., 2009, 7063–7070
This journal is © The Royal Society of Chemistry 2009

View Article Online
-
1
(
1-Diphenylmethyl-3-(methyl imdazol-2-ylidene) gold(I)
262 parameters, m = 10.919 mm , 6721 measured reflections, 3852
unique (Rint = 0.0497) which were used in all calculations. The final
Boc–cysteine (8)
R
1
= 0.0594 for 3540 observed reflections (I > 2s(I)) and wR =
2
To a solution of 6a (40 mg, 83 mmol) and Boc–cysteine (18.4 mg,
3
9
Celite and recrystallised from CH
to yield 8 as a white powder (33 mg, 60%). H NMR (CD
2
NHCys, N–CH(PH)
(
1
1
0
.1619 for all data.
mmol) in dry CH
2
2
Cl (5 mL) was added triethylamine (13 mL,
Crystal data of 6c.
C
17
H
16AuBr
3
N
2
, M = 685.01, monoclinic,
◦
1.7 mmol) and stirred over night. The mixture was filtered through
◦
˚
a = 9.6826(5), b = 12.2416(4), c = 20.3950(8) A, b = 91.488(4) ,
2
Cl
2
–pentane (1 : 5) at -20 C
3
◦
1
˚
V = 2416.6(2) A , T = -80 C, space group P2
1
/n, Z = 4, 2qmax
=
2
Cl ,
2
◦
-3
-1
◦
5
1
0.2 , r(calc) = 1.883 mg m , 208 parameters, m = 11.048 mm ,
4 411 measured reflections, 4284 unique (Rint = 0.062) which were
50 MHz, 25 C): d 7.41–6.76 (15H, m, NCH(Ph)
2
, NHBoc,
, N–CH=CH–N, N–CH=CH–N), 4.40–4.26
1H, m, Ca,CysH), 3.86 (2H, m, Cb,CysH), 3.04 (3H, s, N–CH ),
, 63 MHz, 25 C): d
H), 155.8 (COBoc), 139.1 (Cq,Ar), 129.5
2
used in all calculations. The final R
1
= 0.0307 for 2497 observed
3
1
3
◦
reflections (I > 2s(I)) and wR
2
= 0.0472 for all data.
.37 (9H, s, C(CH
85.4 (NCN), 174.1 (CO
3
)
3
). C NMR (CD
2
Cl
2
2
Ar), 129.3 (CAr), 128.7 (CAr), 122.6 (N–CH=CH–N), 120.0 (N–
Cell culture and IC50 values
(
C
CH=CH–N), 79.3 (C(CH
)
), 68.36 (CH(Phe)
), 57.6 (Cb,Cys), 46.2
3
3
2
HeLa, HepG2 and HT-29 cells were kept in culture with RPMI
640 medium supplemented with 10% FCS (fetal calf serum),
-
(
6
4
C
a,Cys), 38.7 (N–CH
64.03 [M - H] . Anal. calc. for C25
3
), 28.6 (C(CH
3
)
3
). MS (ESI , acetonitrile):
1
2
-
H
30AuN
3
O
4
S (m = 665.56): C,
-
1
mM L-glutamin and the antibiotic penicillin (100 U mL )
5.12; H, 4.54; N, 6.31; S, 4.82. Found: C, 46.02; H, 5.89; N, 6.62;
-
1
and streptomycin (100 mg mL ) in 5% CO -atmosphere. In vitro
2
S, 3.84.
cytotoxicity of gold compounds was studied on HeLa, HT-29 and
HepG2 cells. Cell viability, which correlates with the metabolic
activity of a cell, was determined by the resazurin assay. In ad-
(
1-Diphenylmethyl-3-(methyl imdazol-2-ylidene) gold(I)
39
cysteine-leucine ethylester (9)
dition to the cell viability, absolute cell numbers were determined
40
by the crystal violet assay, which can be applied after elution
of resazurin. The cells were seeded in 96-well microtiterplates
To a solution of 6a (40 mg, 83 mmol) and 7 (31.5 mg, 87 mmol) in
dry CH
and stirred over night. The mixture was filtered through Celite and
recrystallised from CH
white powder (17 mg, 25%). H NMR (CD
d 7.47–7.04 (13H, m, NCH(Ph) , NHBoc, NHCys, N–CH(PH)
6
1
2
2
Cl (5 mL) was added triethylamine (13 mL, 91.7 mmol)
(MTP) coated with 0.2% of gelatine. After seeding, the cells were
◦
grown for 24 h under standard conditions. The compounds were
dissolved in culture medium with 0.5% DMSO and applied to the
cells in various concentrations for 48 h. Every concentration was
tested six-fold. Before resazurin was added to the cells they were
washed three times with phenol red-free RPMI-1640 medium.
Then, phenol red-free RPMI-medium with 10% resazurin was
added. Absorbance at 600 nm was directly measured with a Tecan
2
Cl
2
–pentane (1 : 5) at -20 C to yield 9 as a
1
◦
2
Cl
2
, 250 MHz, 25 C):
2
2
),
=
3
3
.97 (1H, d, JH,H = 1.9 Hz, N–CH=CH–N), 6.81 (1H, d, JH,H
.9 Hz, N–CH=CH–N), 4.61–4.50 (1H, m, Ca,CysH), 4.22–3.97
–CH –CH ), 3.84 (3H, s, N–CH ), 3.35–3.12
(
(
3H, m, Ca,LeuH, CO
2
2
3
3
2H, m, Cb,CysH), 1.75–1.56 (3H, m, Cg,Leu
H Cb,LeuH), 1.40 (9H, s,
,
2
◦
3
Sapphire microplate reader (Tecan, Germany) at 37 C. After
C(CH
3
)
3
), 1.36 (3H, t, JH,H = 7.3 Hz, CO
2
–CH
2
–CH ), 0.88 (6H,
3
◦
3
2
13
2 h of incubation at 37 C and 5% CO
2
, the measurement was
dd, JH,H = 6.1 Hz, JH,H = 10.5 Hz, Cd,LeuH). C NMR (CD
2
Cl ,
2
◦
repeated. The decrease in absorbance gave the viability. Resazurin
was removed and the cells were fixated with 4% paraformaldehyde
6
1
1
6
4
3 MHz, 25 C): d 185.1 (NCN), 172.1 (CO
2
Et), 171.0 (COLeu),
55.3 (COBoc), 138.8 (Cq,Ar), 129.9 (CAr), 128.9 (CAr), 128.3 (CAr),
22.2 (N–CH=CH–N), 119.6 (N–CH=CH–N), 79.4 (C(CH
),
8.3 (CH(Phe) ), 68.0 (CO –CH –CH
2.3 (Ca,Leu), 38.2 (N–CH ), 28.1 (C(CH
d,Leu), 21.8 (Cg,Leu), 14.1 (CO –CH –CH
(
PFA) in PBS (phosphate buffered saline) for 15 min at room
3 3
)
temperature. PFA was eluted twice with PBS and membranes were
permeabilised by 0.1% Triton-X100 in PBS for 10 min. Afterwards,
an aqueous 0.04% crystal violet solution was added to the cells and
the MTP was mechanically shaken for 1 h. The cells were washed
2
2
2
3
), 55.0 (Cb,Cys), 45.8 (Ca,Cys),
3
3
)
3
), 24.7 (Cb,Leu), 22.9
+
(
C
2
2
+
3
). MS (Fab ): 693.2
- Au] (85%), 830.3 [M + Na] , 1251.2 [M + NHC–Au] .
+
+
[
NHC
2
seven times with H O, and crystal violet was eluted with 96%
2
Anal. calc. for C33
N, 6.94; S, 3.97. Found: C, 45.7; H, 6.52; N, 7.32; S 3.06.
H
45AuN
4
O
5
S (m = 806.78): C, 49.13; H, 5.62;
ethanol for 4 h. The absorbance was determined at 570 nm, after
subtraction of 24 h pre-substance incubation absorbance values,
cell biomass could be calculated. In case the compound showed
a promising cytotoxic activity, the inhibitory concentration at
Crystal structure determination†
5
0% growth (IC50) for both assays was calculated. Therefore, a
Crystals of 4c and 6c were mounted on a glass capillary in per-
fluorinated oil and measured in a cold gas stream. The intensities
were measured with a Bruker-axs-SMART diffractometer (Mo
series of concentration suitable for the estimated IC50 was applied
to the cells according to the procedure described above. The
obtained viability or cytotoxicity data were plotted against the
concentration in half-logarithmical scale and a sigmoidal function
fit was performed with Origin 7 (Originlab, Northampton, USA)
until the fit converged. IC50-values were directly calculated from
the fit function.
˚
Ka radiation, l = 0.7170 A, w scan). The structure was solved
2
by direct methods (SHELXS-97), and refined against F with all
measured reflections (SHELXL-97). All non-hydrogen atoms were
refined anisotropically and the hydrogen atoms were included in
calculated positions.
Crystal data of 4c.
C
19
H
25AuBr
3
N
3
O
3
, M = 780.12, mono-
◦
Acknowledgements
˚
clinic, a = 8.452(4), b = 9.212(4), c = 15.756(7) A, b = 92.894(9) ,
3
◦
˚
V = 1225.2(9) A , T = -60 C, space group P2
1
, Z = 2,
Financial support from the Ruhr-University Research School
to J. L. is gratefully acknowledged. This work is supported by
◦
-3
2
q
max = 50 , Flack-parameter = 0.00(2), r(calc) = 2.115 mg m ,
This journal is © The Royal Society of Chemistry 2009
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the Research Units “Biological Function of Organometallic Com-
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