Synthesis, molecular structure and evaluation of new organometallic ruthenium anticancer agents

Article abstract of DOI:10.1039/b918902e

A number of new ruthenium compounds have been synthesised, isolated and characterised, which exhibit excellent cytotoxicity against a number of different human tumour cell lines including a defined cisplatin resistant cell line and colon cancer cell lines

Full text of DOI:10.1039/b918902e

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Metal Anticancer Compounds
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PAPER
www.rsc.org/dalton | Dalton Transactions
Synthesis, molecular structure and evaluation of new organometallic
ruthenium anticancer agents†
Kenneth D. Camm,a Ahmed El-Sokkary,a Andrew L. Gott,a Peter G. Stockley,b Tamara Belyaevab and
Patrick C. McGowan*a
Received 14th September 2009, Accepted 29th October 2009
First published as an Advance Article on the web 16th November 2009
DOI: 10.1039/b918902e
A number of new ruthenium compounds have been synthesised, isolated and characterised, which
exhibit excellent cytotoxicity against a number of different human tumour cell lines including a defined
cisplatin resistant cell line and colon cancer cell lines. Addition of hydrophobic groups to the ruthenium
molecules has a positive effect on the cytotoxicity values. Evidence is provided that, after incubation of
a ruthenium compound with a 46 mer oligonucleotide duplex and subsequent nuclease treatment,
ruthenium is bound to a guanine residue.
strands and deriving the fate of the ruthenium species after
incubation with nuclease enzymes.
Introduction
Ruthenium compounds have shown promising anticancer activity
and have been evaluated in vitro and in vivo1-5 and two RuIII
complexes have entered clinical trials.6-8 The anticancer potential
Experimental
of RuII “half-sandwich” arene complexes have also been evaluated
and exhibit both in vitro and in vivo activity.2-5,9 It has been
Preparation of ligands was carried out under air using wet solvents.
Unless otherwise stated all manipulations were conducted using
standard Schlenk line techniques, under an inert atmosphere of
di-nitrogen or argon, using a modified dual vacuum/di-nitrogen
line or in a Braun Labmaster 100 glove box under an atmosphere
of di-nitrogen. Diethyl ether, petroleum ether (bp 40–60 ◦C),
THF and toluene were pre-dried with Na metal and distilled
over Na or Na/benzophenone under di-nitrogen. DCM, CH3CN
and methanol were pre-dried and distilled over CaH2 under N2.
All solvents were subsequently stored in ampoules under N2.
shown that analogs containing the heavier congener osmium can
be potently cytotoxic towards cancer cells with activity that is
comparable to the clinical drugs carboplatin and cisplatin.10-13
Modifications have been carried out to ruthenium arene complexes
and the cytotoxic effects examined. Sadler et al. have synthesised
6
RuII arene complexes of the type [(h -arene)RuII(en)X][PF6] (en =
ethylenediamine, X = halogen) which show in vitro and in vivo
activity.14-19 Dyson et al. have reported ruthenium arene systems
with phosphine ligands and metallarectangles that have high
anticancer activity.20-22 Ruthenium arene complexes have also
been prepared which incorporate bio-ligands in aqueous solution
and comparative binding study investigations carried out.23-28
Sheldrick and co-workers postulate that the binding of the Ru
arene fragment to DNA will not only be affected by the shape of
the intercalating ligand, but also the co-ligands.
As well as our interest in early transition metal anticancer
complexes,29-31 we have been interested in developing a range of
ruthenium complexes incorporating ligands which have a high
degree of flexibility, both sterically and electronically, in order to
evaluate effects on cytotoxic behaviour. To this end we explored
synthesising a range of novel ruthenium compounds, which also
incorporated fragments for H-bonding and aryl groups to allow
potential intercalation into DNA as well as affecting increased
hydrophobic nature of the compounds. In addition to evaluating
the ruthenium compounds in vitro, we were also interested in
the mechanism of action and binding to duplex oligonucleotide
6
The preparation of [Ru(h -p-cymene)Cl2]2 was achieved using
literature methods.32 Pyridine-2-carboxylic acid-(4-nitrophenyl)-
amide was prepared as previously reported by Dutta et al.33 1H and
13C NMR spectra were recorded on Bruker 250 MHz, 300 MHz
and 500 MHz spectrometers. High resolution mass spectrometry
was performed by the University of Leeds mass spectrometry
service. Elemental analyses were performed by the University of
Leeds microanalytical services.
Crystallographic data collection and structure solution
X-ray crystallography. Data for compounds 1–7 were collected
on a Nonius KappaCCD area-detector diffractometer using
˚
graphite monochromated Mo-Ka radiation (l = 0.71073 A)
using 1.0 f rotation frames. Pertinent crystallographic details are
given in Table 1. The crystal was cooled to 150 K by an Oxford
Cryosystems low temperature device34 before data collection using
1.0◦ j rotation frames. The images were processed using the
DENZO and SCALEPACK programs35 followed by structure
solution by direct methods via one of the SHELXS8636 SIR92,37
or SIR9738 programs. All structures were refined by full-matrix
least squares on F2 using SHELXL9739 Molecular graphics were
plotted using POVray40 via XSeed.41 All non-hydrogen atoms were
refined anisotropically. Hydrogen atoms were constrained with a
aDepartment of Chemistry, University of Leeds, Leeds, LS2 9JT, UK.
E-mail: p.c.mcgowan@leeds.ac.uk
bAstbury Centre for Structural Molecular Biology, University of Leeds,
Leeds, LS2 9JT, UK
† Electronic supplementary information (ESI) available: Experimental
details. CCDC reference numbers 752541–752546. For ESI and crystallo-
graphic data in CIF or other electronic format see DOI: 10.1039/b918902e
10914 | Dalton Trans., 2009, 10914–10925
This journal is
The Royal Society of Chemistry 2009
©

Table 1 Crystallographic data for 1–6
1
2
3
4
5
6
Formula
Mol. wt.
Temperature (K)
Wavelength [Mo-Ka] (A)
Crystal system
C16H20Cl2N2ORu C23H22ClN3O3Ru C22H23ClN3O3Ru C26H24ClN3O3Ru C29H27Cl10N3O3Ru
C12H10ClNO2Ru
336.73
150(2)
0.71073
Triclinic
¯
P1
6.3800(2)
8.5720(3)
10.8540(3)
75.937(2)
77.251(2)
79.785(2
556.79(3)
2
2.008
1.633
0.07 ¥ 0.03 ¥ 0.03
9665
428.31
150(2)
524.96
150(2)
513.95
150(2)
563.11
150(2)
921.11
150(2)
˚
0.71073
Monoclinic
P21/n
0.71073
Monoclinic
C2/c
0.71073
Monoclinic
C2/c
0.71073
Monoclinic
P21/n
0.71073
Monoclinic
P21/c
Space group
˚
a (A)
9.8470(4)
11.8300(6)
14.8190(7)
90
29.8887(4)
8.69930(10)
15.9600(3)
90
100.24(3)
90
31.444(6)
8.5608(17)
16.642(3)
90
113.19(3)
90
9.1994(2)
18.2026(4)
14.1456(3)
90
102.5280(9)
90
12.5540(2)
15.2770(3)
19.4440(3)
90
104.3070(10)
90
˚
B (A)
˚
C (A)
a (◦)
b (◦)
g (◦)
92.679(2)◦
90
3
˚
Volume (A )
1724.38(14)
4
1.65
4083.66(11)
8
1.281
4118.0(14)
8
1.2433
0.691
2312.32(9)
4
1.617
3613.46(11)
4
1.693
Z
Density (calcd) (Mg m-3)
m (mm-1)
1.221
0.698
0.828
1.21
Crystal size (mm)
Reflections collected
Independent reflections
0.13 ¥ 0.03 ¥ 0.01 0.26 ¥ 0.13 ¥ 0.07 0.2 ¥ 0.2¥ 0.2
0.2 ¥ 0.13 ¥ 0.12
0.42 ¥ 0.04 ¥ 0.04
15832
3934
18405
4009
109847
4002
34376
54562
4544
8300
2499
[R(int) = 0.0868] [R(int) = 0.0856] [R(int) = 0.0534] [R(int) = 0.1118]
[R(int) = 0.1409]
[R(int) = 0.0815]
Data/restraints/
parameters
3934/0/205
4009/0/274
4002/0/274
4544/0/307
8300/0/419
2499/0/154
Goodness-of-fit on F2
1.029
1.073
1.006
1.063
1.033
1.065
Final R indices [I > 2s(I)] R1 = 0.0414,
R1 = 0.0356,
wR2 = 0.0895
R1 = 0.0474,
wR2 = 0.0956
R1 = 0.0387,
wR2 = 0.0892
R1 = 0.0586,
wR2 = 0.0991
R1 = 0.0418,
wR2 = 0.1062
R1 = 0.0571,
wR2 = 0.1154
R1 = 0.0431,
wR2 = 0.0982
R1 = 0.0724,
wR2 = 0.1111
0.701 and -1.123
R1 = 0.0323,
wR2 = 0.0815
R1 = 0.0348,
wR2 = 0.0833
0.625 and -1.12
wR2 = 0.0788
R indices (all data)
R1 = 0.0726,
wR2 = 0.0881
Largest diff. peak and
0.727 and -1.181 0.842 and -1.894 0.628 and -0.902 1.12 and -1.357
-3
˚
hole e A
riding model; U(H) was set at 1.2 ¥ (1.5 ¥ for methyl groups) Ueq
RuC22H22N3O3Cl 2
for the parent atom.
6
[Ru(h -p-cymene)Cl2]2 (0.1 g, 0.16 mmol) was dissolved in dry
ethanol (50 mL). N-3-nitro-Ph-picolinamide (0.1 g) was added
with stirring. The mixture was heated under reflux (1 h), cooled
and filtered over NH4PF6 (0.1 g). The mixture was heated under
reflux overnight to afford an orange solution. The volume of
solvent was reduced and petroleum ether (bp 40–60 ◦C) added
(50 mL) to afford an orange precipitate which was isolated via
filtration, washed in petroleum ether (bp 40–60 ◦C) and dried
in vacuo to obtain an orange powder (0.0908 g, 0.15 mmol, 92%).
Crystals suitable for X-ray analysis were obtained from methanol.
Quadrupolar ES MS (+): m/z 478.2. Found C 51.0; H 4.25; N
8.0% Calc. for RuC22H22N3O3Cl C 51.51; H 4.32; 8.19% 1H NMR
RuC16H18NO2Cl 1
6
To a Schlenk tube charged with [Ru(h -p-cymene)Cl2]2 (0.25 g,
0.4 mmol) in THF (150 mL) was added C6H4NO2K (0.13 g,
0.8 mmol) with stirring. The mixture was allowed to stir overnight
to yield an orange solution with a white precipitate of KCl.
The solvent was removed in vacuo and the product extracted in
dichloromethane (3 ¥ 10 mL). Diethyl ether was added (10 mL) to
afford a yellow precipitate which was washed in diethyl ether (3 ¥
5 mL) and dried in vacuo to afford a yellow powder. Crystals of the
product suitable for X-ray crystallographic analysis were obtained
via vapour diffusion from dichloromethane and petroleum ether
(bp 40–60 ◦C). (0.2 g, 0.5 mmol, 62.5%). Found: C 48.35; H 4.61;
(CDCl3, 300.1 MHz, 300 K) d 9.01 [d, 1H, J(1H-1H) = 6 Hz,
3
CH of C5H4N], 8.62 [d, 1H, 3J(1H-1H) = 6 Hz, CH of C6H4NO2],
8.11 [m, 2H, CH of C5H4N and CH of C6H4NO2], 7.99 [m, 2H,
C5H4N and CH of C6H4NO2], 7.49 [m, 2H, C5H4N and CH of
C6H4NO2], 5.30 [d, 1H, 3J(1H-1H) = 6 Hz, CH of C6H4], 5.22 [d,
1
N 3.55%. Calc. for RuC16H18NO2Cl : C 48.92; H 4.62; 3.57%. H
NMR (CDCl3, 500.13 MHz, 300 K) d 8.87 [br. d, 1H, 3J(1H-1H) =
7.7 Hz, C5H4N], 7.95 [d, 1H 3J(1H-1H) = 7.7 Hz, C5H4N], 7.87 [t,
1H, J(1H-1H) = 6 Hz, CH of C6H4], 5.16 [d, 1H, J(1H-1H) =
6 Hz], 4.79 [d, 1H, 3J(1H-1H) = 6 Hz, CH of C6H4], 2.56 [m, 1H,
CH of CH(CH3)2], 2.25 [s, 3H, CH3 of C6H4CH3], 1.08 [d, 6H,
3
3
3
1H, J(1H-1H) = 6.7 Hz, C5H4N], 7.50 [br. t, 1H, C5H4N], 5.55
3
3
[d, 1H, J(1H-1H) = 5.8 Hz, CH of C6H4], 5.53 [d, 1H, J(1H-
3J(1H-1H) = 6 Hz, 2 ¥ CH3 of CH(CH3)2]; 13C{ H} NMR (CDCl3,
1
1H) = 5.8 Hz, CH of C6H4], 5.39 [d, 1H, J(1H-1H) = 5.8 Hz,
3
CH of C6H4], 5.31 [d, 1H, J(1H-1H) = 5.8 Hz, CH of C6H4],
3
75.5 MHz, 300 K) d 155.8 [C of CONR], 153.6 [C of C5H4N], 153.5
[CH of C5H4N], 151.8 [C of C6H4NO2], 148.8 [C of C6H4NO2],
139.2 [CH of C5H4N], 133.5 [CH of C5H4N], 129.3 [CH of C5H4N],
127.3 [CH of C6H4NO2], 126.7 [CH of C6H4NO2], 122.2 [CH of
C6H4NO2], 119.4 [CH of C6H4NO2], 102.9 [C of C6H4], 101.5 [C
of C6H4], 84.9 [CH of C6H4], 84.7 [CH of C6H4], 84.3 [CH of
C6H4], 84.0 [CH of C6H4], 31.4 [CH of CH(CH3)2], 22.8 [CH3 of
CH(CH3)2], 22.3 [CH3 of CH(CH3)2], 19.2 CH3 of CH3C6H4].
2.81 [sept, 1H, CH(CH3)2], 2.23 [s, 3H, C6H4CH3], 1.17 [m, 6H,
1
CH(CH3)2]. 13C{ H} NMR (CDCl3), 125.76 MHz, 300 K) d 171.1
[CCO2], 152.3 [CH of C5H4N], 151.3 [C5H4NCCO2], 139.2 [CH
of C5H4N], 127.9 [CH of C5H4N], 126.9 [CH of C5H4N], 102.6 [C
of C6H4], 98.7 [C of C6H4], 82.8 [CH of C6H4], 82.5 [CH of C6H4],
81.5 [CH of C6H4], 80.9 [CH of C6H4], 31.1 [CH(CH3)2], 22.3 22.2
[2 ¥ CH(CH3)2], 18.7 [C6H4CH3].
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 10914–10925 | 10915
©

RuC22H22N3O3Cl 3
C6H4], 31.26 [CH(CH3)2], 22.46 [CH(CH3)2], 22.40 [CH(CH3)2],
19.18 [CH3].
6
[Ru(h -p-cymene)Cl2]2 (0.1 g, 0.16 mol) was dissolved in dry
ethanol (50 mL). N-4-nitro-Ph-picolinamide (0.1 g) was added
with stirring. The mixture was heated under reflux (1 h), cooled
and filtered over NH4PF6 (0.1 g). The mixture was allowed to stir
(1 h) to afford a yellow precipitate. The precipitate was isolated
via filtration, washed in petroleum ether (bp 40–60 ◦C) and dried
in vacuo to afford a yellow powder (0.07 g, 0.14 mmol, 85%).
Crystals suitable for X-ray analysis were obtained from the mother
liquor. Found C 51.45; H 4.25; N 8.3% Calc. for RuC22H22N3O3Cl
C 51.51; H 4.32; 8.19% 1H NMR (CDCl3, 300.1 MHz, 300 K) d
RuC26H24ClN3O3 5
6
To a Schlenk tube charged with a suspension of [Ru(h -p-
cymene)Cl2]2, 0.08 mmol) in dry ethanol (20 mL) was added
N-4-nitro-Ph-quinlinamide (0.047 g, 0.16 mmol). The mixture was
then heated until dissolution occurred and filtered over NH4PF6
(0.05 g, excess). The volume of solvent was reduced in vacuo to
afford an orange precipitate, which was isolated via filtration,
washed in diethyl ether (30 mL) and dried in vacuo to afford
an orange powder. Crystals suitable for X-ray crystallographic
analysis were obtained from CDCl3. (0.075 g, 0.1 mmol, 79%).
Analysis for RuC26H24N3O3Cl·C2H5OH. Found C 54.25; H 4.31;
3
8.9 [d, 1H, J(1H-1H) = 6 Hz, CH of C5H4N], 8.2 [m, 2H, CH
3
of C6H4NO2], 8.1 [d, 1H, J(1H-1H) = 6 Hz, CH of C5H4N], 7.9
[m, 3H, CH of C5H4N, 2 ¥ CH of C6H4NO2], 7.6 [m, 1H, CH of
C5H4N], 5.3 [d, CH of C6H4], 5.2 [d, CH of C6H4], 5.1 [d, CH
1
N 7.33%. Calc. C 54.50; H 4.74; N 7.06%. H NMR (CDCl3,
3
of C6H4], 4.8 [d, CH of C6H4], 2.5 [sept., 1H, J(1H-1H) = 6 Hz,
300.13 MHz, 300 K) d 8.95 [d, 1H, J(1H-1H) = 9 Hz, CH of
3
C9H6N], 8.43 [d, 1H, 3J(1H-1H) = 6 Hz, CH of C9H6N], 8.27 [m,
3H, 2 ¥ CH of C6H4NO2, CH of C9H6N], 8.23 [d, 2H, 2 ¥ CH
of C6H4NO2], 8.01 [m, 1H, CH of C9H6N], 8.00 [m, 1H, C9H6N],
7.80 [t, 1H, 3J(1H-1H) = 6 Hz, C9H6N], 5.47 [d, 1H, 3J(1H-1H) =
6 Hz, CH of C6H4], 5.34 [d, 1H, 3J(1H-1H) = 6 Hz, CH of C6H4],
5.24 [d, 1H, 3J(1H-1H) = 6 Hz, CH of C6H4], 4.77 [d, 1H, 3J(1H-
1H) = 6 Hz, CH of C6H4], 2.28 [m, 4H, CH3 of C6H4CH3, CH of
CH of (CH3)2CH], 2.3 [s, 3H, C6H4CH3], 1.1 [m, 6H, CH(CH3)2];
1
13C{ H} NMR (CDCl3, 75.5 MHz, 300 K) d 19.3 [CH(CH3)2],
22.4 [CH(CH3)2], 22.7 [CH(CH3)2], 31.4 [C6H4CH3], 83.8 [C6H4],
84.3 [C6H4], 84.6 [C6H4], 85.2 [C6H4], 101.9 [C of C6H4], 102.9 [C
of C6H4], 119.6 [2 ¥ CH of C6H4NO2], 124.5 [2 ¥ CH of C6H4NO2],
125.6 [CH of C5H4N], 126.9 [CH of C5H4N], 127.5 [CH of C5H4N],
127.6 [CH of C5H4N], 138.4 [C of C5H4N], 139.3 [C of C6H4NO2],
153.5 [C of C6H4NO2], 159.3 [CONAr]. ES MS(+): m/z 478.0786
[M+]-Cl.
CH(CH3)2], 1.07 [d, 3H, J(1H-1H) = 9 Hz, CH3 of CH(CH3)2],
3
0.90 [d, 3H, 3J(1H-1H) = 9 Hz, CH3 of CH(CH3)2];13C-{ H} NMR
1
(CDCl3, 75.47 MHz, 300 K) d 167.40 [C of CONR]; 159.08 [C of
C6H4NO2]; 157.59 [C of C6H4NO2]; 148.69 [C of C9H6N]; 144.04
[C of C9H6N]; 139.99 [CH of C9H6N]; 131.82 [CH of C9H6N];
130.75 [C of C9H6N]; 129.40 [CH of C9H6N]; 129.30 [CH of
C9H6N]; 129.08 [CH of C9H6N]; 127.42 [2 ¥ CH of C6H4NO2];
124.47 [2 ¥ CH of C6H4NO2]; 122.68 [CH of C9H6N]; 103.27 [C
of C6H4]; 102.31 [C of C6H4]; 85.81 [CH of C6H4]; 84.69 [CH
of C6H4]; 84.57 [CH of C6H4]; 84.52 [CH of C6H4]; 31.22 [CH3];
22.47 [CH3 of CH(CH3)2]; 22.40 [CH3 of CH(CH3)2]; 19.17 [CH
of CH(CH3)2]; ES MS(+): m/z 528.0859 [M+]-Cl-.
RuC26H24ClN3O3 4
6
To a Schlenk tube charged with [Ru(h -p-cymene)Cl2]2 (0.1 g,
0.16 mmol) in dry ethanol (20 mL) was added N-3-nitro-Ph-
quinlinamide (0.096 g, 0.33 mmol). The mixture was then heated
until dissolution occurred and filtered over NH4PF6 (0.1 g, excess).
The volume of solvent was reduced in vacuo to afford an orange
precipitate, which was isolated via filtration, washed in diethyl
ether (3 ¥ 10 mL) and dried in vacuo to afford an orange powder.
Crystals suitable for X-ray crystallographic analysis were obtained
from the mother liquor, (0.15 g, 0.27 mmol, 84%). Found C
55.30; H 4.45; N 7.55%. Calc. C 55.47; H 4.30; N 7.46% for
RuC26H24N3O3Cl. 1H NMR (CDCl3, 300.13 MHz, 300 K) d 8.95
RuC12H10NO2Cl 6
6
To a Schlenk tube charged with a suspension of [Ru(h -
benzene)Cl2]2 (0.05 g, 0.1 mmol) in THF (50 mL) was added
potassium picolinate (0.03 g, 0.2 mmol) with stirring. The mixture
was then heated under reflux (24 h) to afford a yellow suspension.
The solvent was removed in vacuo, the product extracted in
methanol (50 mL) and stored at -20 ◦C. Orange crystals suitable
for X-ray crystallographic analysis were obtained from the mother
liquor. (0.03 g, 0.1 mmol, 45%). Analysis for RuC12H10NO2Cl.
Found: C 42.60; H 3.11; N 4.25%. Calc.: C 42.80; H 2.99; N
4.16%. 1H NMR (d6-DMSO, 300.13 MHz, 300 K) d 9.43 [d, 1H,
3
3
[d, 1H, J(1H-1H) = 8.34 Hz, CH of C9H6N], 8.90 [t, 1H, J(1H-
1H) = 1.74 Hz, CH of C6H4NO2], 8.42 [d, 1H, 3J(1H-1H) = 8.10 Hz,
CH of C9H6N & CH of C6H4NO2], 8.30 [d, 1H, J(1H-1H) =
3
8.40 Hz, CH of C9H6N], 8.00 [m, 3H, 2 ¥ CH of C9H6N & CH
of C6H4NO2], 7.79 [t, 1H, J(1H-1H) = 6.0 Hz, CH of C9H6N],
3
7.54 [t, 1H, 3J(1H-1H) = 8.10 Hz, CH of C6H4NO2], 5.49 [d, 1H,
3J(1H-1H) = 6.0 Hz, CH of C6H4], 5.36 [d, 1H, 3J(1H-1H) = 5.7 Hz,
CH of C6H4], 5.25 [d, 1H, 3J(1H-1H) = 5.7 Hz, CH of C6H4], 4.75
[d, 1H, 3J(1H-1H) = 6.0 Hz, CH of C6H4], 2.31 [m, 4H, CH(CH3)2
3
3J(1H-1H) = 6.6 Hz, CH of C5H4N], 8.11 [t, 1H, J(1H-1H) =
& CH3], 1.06 [d, 3H, J(1H-1H) = 6.9 Hz, CH(CH3)2], 0.88 [d,
3
6.6 Hz, CH of C5H4N], 7.78 [m, 2H, 2 ¥ CH of C5H4N], 5.95 [s,
3
1
3H, J(1H-1H) = 6.9 Hz, CH(CH3)2]; 13C-{ H} NMR (CDCl3,
75.47 MHz, 300 K) d 167.45 [C=O], 157.70 [C of C9H6N], 153.42
[C of C6H4NO2], 148.73 [C of C6H4NO2], 148.67 [C of C9H6N],
139.88 [CH of C9H6N], 133.26 [CH of C6H4NO2], 131.74 [CH of
C9H6N], 130.70 [C of C9H6N], 129.37 [CH of C6H4NO2], 129.34
[CH of C6H4NO2], 129.29 [CH of C9H6N], 128.98 [CH of C9H6N],
122.69 [CH of C9H6N], 122.05 [CH of C6H4NO2], 119.35 [CH of
C9H6N], 103.28 [C of C6H4], 102.06 [C of C6H4], 85.50 [CH
of C6H4], 84.88 [CH of C6H4], 84.58 [CH of C6H4], 84.42 [CH of
1
6H, C6H6];13C{ H} NMR (d6-DMSO, 62.90 MHz, 300 K) d 170.8
[C of CON], 154.5 [CH of C5H4N], 150.8 [C of C5H4N], 140.1 [CH
of C5H4N], 128.3 [CH of C5H4N], 125.8 [CH of C5H4N], 83.7 [CH
of C6H6].
RuC18H14N3O3Cl 7
6
To a Schlenk tube charged with a suspension of [Ru(h -
benzene)Cl2]2 (0.1 g, 0.19 mmol) in methanol (50 mL) was added
10916 | Dalton Trans., 2009, 10914–10925
This journal is
The Royal Society of Chemistry 2009
©

N-4-nitro-Ph-picolinamide (0.1 g, 0.28 mmol) with stirring. The
mixture was then heated under reflux (ca. 3 h.) and the cooled
mixture filtered over NH4PF6 (0.1 g, excess) and heated under
reflux overnight. On cooling the mixture an orange precipitate
was formed. The precipitate was isolated via filtration, washed
in petroleum ether (bp 40–60 ◦C) and dried in vacuo to afford
an orange powder. Crystals suitable for X-ray crystallographic
analysis were obtained via vapour diffusion from acetone and
diethyl ether. (0.08 g, 0.175 mmol, 92%). Found C 47.13.; H 2.84.;
N 8.97.% Calc. for RuC18H14N3O3Cl C 47.32; H 3.09; N 9.20%
1H NMR (d6-DMSO, 300.13 MHz, 300 K) d 9.41 [d, 1H, 3J(1H-
with cold trichloroacetic acid (TCA) to fix the cells to the bottom
of the plates. These were then washed with water to ensure the
removal of all TCA. The plates were then stained with a solution
of 0.02% (w/v) SRB in 1.0% (v/v) acetic acid for 30 min. The
plates were washed thoroughly with acetic acid and allowed to dry
overnight. 100 ml of 10 mM unbuffered tris was added to each
well to re-suspend the SRB dye. The plates were then read using
a plate reader which records the mean absorbance at 570 nm of
each concentration and using a linear interpolation the IC50 is
calculated.
3
1H) = 6.2 Hz, CH of C5H4N], 8.23 [d, 2H, J(1H-1H) = 9.0 Hz,
DNA nuclease treatment
3
2 ¥ CH of C6H4NO2], 8.10 [1H, t, J(1H-1H) = 3.3 Hz, CH of
10 ml DNA was incubated with 10 ml compound 3 or cisplatin for
45 min at room temperature. The reaction mixture was then treated
with 8 ml (0.02 units) snake venom Phosphodiesterase I (purchased
from Sigma-Aldrich Company Ltd.) for 45 min at room tempera-
ture to produce single nucleotides-5¢-monophosphate. 2 ml (2 units)
Shrimp Alkaline Phosphatase (purchased from Sigma-Aldrich
Company Ltd.) was added to the nuclease reaction mixture, which
was made up to 100 ml with nuclease buffer (32 mM Tris HCl,
pH 7.4, 25 mM MgCl2 in nuclease-free water) and incubated
overnight at 37 ◦C in a DNA Engine PTC200 Peltier Thermal
Cycler PCR following the method of Eadie et al.42 The reaction
mixture was purified by adding 10 ml NaCl (5 mM), together
C5H4N], 7.89 [3H, m, 1 ¥ CH of C5H4N, 2 ¥ CH of C6H4NO2],
3
7.70 [1H, t, J(1H-1H) = 3.3 Hz, CH of C5H4N], 5.68 [6H, s, 6 ¥
1
CH of C6H6];13C-{ H} (d6-DMSO, 62.90 MHz, 300 K) d 167.1
[C of CON], 159.9 [C of C5H4N], 155.3 [CH of C5H4N], 154.6
[C of C6H4NO2], 143.0 [C of C6H4NO2], 139.7 [CH of C5H4N],
128.7 [CH of C5H4N], 127.7 [2 ¥ CH of C6H4NO2], 125.5 [CH of
C5H4N], 124.0 [2 ¥ CH of C6H4NO2], 85.8 [6 ¥ CH of C6H6]. ES
MS(+): m/z 422.0161 [M+]-Cl-.
[RuC26H31N6O7][PF6] 8
Guanosine hydrate (0.02 g, 0.08 mmol.) was added to a stirred
solution of 1 (0.03 g, 0.08 mmol.) in distilled water. The mixture
was allowed to stir at 65 ◦C (5 days) and filtered over NH4PF6
(0.1 g, excess). A green precipitate was immediately formed which
was isolated via filtration, washed in diethyl ether and dried to
afford a pale green powder. (0.025 g, 0.03 mmol, 40%) Analysis
for RuC26H31N6O7PF6·2.5H2O Found: C 37.05; H 4.50; N 10.70%.
Calc.: C 37.64; H 4.25; N 10.12%. 1H NMR (D2O, 300.13 MHz,
300 K) d 9.49 [br. d, 1H, CH of C5H4N], 7.90 [t, 1H, 3J(1H-1H) =
◦
with 200 ml absolute ethanol at 0 C, then chilled on dry ice for
30 min, and centrifuged for 30 min at 4 ◦C in a Biofuge fresco
(top speed 13,000 r.p.m.). The supernatant was collected, and
the total volume was made up to 1 ml with absolute ethanol.
At that time the mixture was chilled for a further 30 min in dry
ice, then centrifuged as before. The supernatant obtained from
the second step was evaporated to dryness via lyophilisation in a
speed vacuum lyophiliser to concentrate the samples which were
◦
3
1
1
dissolved in ultra pure nuclease free water and stored at -80 C
=
9.0 Hz, CH of C5H4N], 7.80 [d, 1H J( H- H) = 9.0 Hz, N CH-N],
7.63 [m, 2H, 2 ¥ CH of C5H4N], 5.96 [m, 1H, CH of C6H4], 5.75 [m,
1H, CH of C6H4], 5.67 [m, 2H, 1 ¥ CH of C6H4 1 ¥ CH of C5H9O4],
for mass spectrometry analyses.
5.50 [d, 1H, J(1H-1H) = 3.3 Hz, CH of C6H4], 4.42 [d of t, 1H,
3
Results and discussion
CH of C5H9O4], 4.20 [d of t, 1H, CH of C5H9O4], 4.11 [m, 1H, CH
of C5H9O4], 3.72 [m, 2H, CH2OH], 2.50 [m, 1H, CH(CH3)2], 1.86
[s, 3H, CH3], 1.05 [t, 3H, 1 ¥ CH(CH3)2], [d, 3H, 1 ¥ CH(CH3)2];
Compound 1 was synthesised by the reaction of potassium
6
picolinate with [Ru(h -p-cymene)Cl2]2. Compound 1 was recrys-
13C{ H} NMR (D2O, 75.47 MHz, 300 K) d 174.5 [C5H4NCO2],
tallised from a mixture of ethanol/petroleum ether (bp 40–60 ◦C)
and was characterised by NMR spectroscopy, microanalysis and
X-ray crystallography. The molecular structure of compound 1
is shown in Fig. 1 and shows binding of the ligand through the
pyridinyl nitrogen and the carbonyl oxygen (N/O). Selected bond
lengths and angles are shown in Table 2. Compound 1 crystallised
in a monoclinic cell and the structural solution was performed
in the space group P21/n. The asymmetric unit comprises one
molecule of 1. Compound 1 (and all subsequent ruthenium arene
complexes synthesised in this work) possess the characteristic
1
=
=
156.5 [Guanine C O], 155.6 [C5H4N], 154.8 [Guanine N C-
=
NH2], 151.7 [Guanine N-C-C O], 148.0 [Guanine N-C-N], 141.2
[C5H4N], 139.3 [N C N], 129.9 [C5H4N], 127.1 [C5H4N], 116.9
= =
[C of C5H4NCO2], 104.8 [C of C6H4], 102.4 [C of C6H4], 88.9
[Ribose N-CH], 86.0/85.7 [2 ¥ CH of C6H4], 85.5 [Ribose HOCH2-
CH], 82.8/82.4 [2 ¥ CH of C6H4], 74.5 [Ribose N-CH-CHOH],
70.4 [Ribose CHOH-CH-CH2OH], 61.4 [Ribose CH2OH], 31.0
[(CH3)2CH], 21.9 [(CH3)2CH], 21.18 [(CH3)2CH], 17.7 [CH3].31P
NMR (D2O, 101.26 MHz, 300 K) d -60.76 [septet, J(31P-19F)
1
-
6
708 Hz, PF6 ]. ES-MS: m/z 641.1271 [M+].
pseudo-tetrahedral geometry exhibited by all h -ruthenium arene
6
complexes. The h p-bonded arene ring occupying one vertex of the
tetrahedron, with the three other ligands occupying the remaining
three sites. An alternative term for the molecular structure of
ruthenium arene complexes is given by the “piano stool” analogy,
Typical SRB assay
The assay was carried out in 96 well plates with an average of
0.5 ¥ 104 cells per mL with 100 ml in each well. The cells were
then treated with varying concentrations of drug for 6 days and
then incubated for 6 days in an atmosphere of 5% CO2/95% air.
After 6 days the cells were removed from the incubator and washed
6
whereby the h p-bonded arene ligand forms the seat and the three
remaining ligands form the legs of the stool.43
Compound 1 crystallised in the RRu configuration (R configu-
ration at the ruthenium metal centre). The bond lengths observed
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 10914–10925 | 10917
©

◦
◦
˚
˚
Table 2 Selected bond distances (A) and bond angles ( ) for compound
1. Estimated standard deviations are given in parentheses
Table 3 Selected bond distances (A) and bond angles ( ) for compound
2. Estimated standard deviations are given in parentheses
Ru(1)–O(1)
Ru(1)–Cl(1)
Ru(1)–C(5)
Ru(1)–C(9)
Ru(1)–C(10)
O(2)–C(11)
C(5)–C(6)
C(9)–C(7)
C(10)–C(9)
2.0809(17)
2.4133(6)
2.173(2)
2.186(2)
2.175(3)
1.228(3)
1.402(4)
1.401(4)
1.425(4)
2.186(3)
Ru(1)–N(1)
Ru(1)–C(4)
Ru(1)–C(6)
Ru(1)–C(7)
O(1)–C(11)
C(4)–C(5)
C(6)–C(7)
C(4)–C(10)
2.096(2)
2.190(3)
2.175(3)
2.216(3)
1.290(3)
1.436(4)
1.438(4)
1.410(4)
Ru(1)–N(1)
Ru(1)–C(9)
Ru(1)–C(4)
2.077(2)
2.163(3)
2.207(3)
2.210(3)
2.4067(8)
1.343(3)
1.345(4)
1.225(4)
1.399(4)
1.383(4)
1.388(4)
1.377(4)
1.385(4)
1.381(4)
76.59(9)
87.13(7)
Ru(1)–N(2)
Ru(1)–C(5)
Ru(1)–C(8)
Ru(1)–C(7)
O(1)–C(17)
N(1)–C(22)
O(2)–N(3)
N(2)–C(11)
N(3)–C(15)
C(11)–C(12)
C(12)–C(13)
C(14)–C(15)
C(17)–C(18)
C(21)–C(22)
N(1)–Ru(1)–Cl(1)
2.107(2)
2.183(3)
2.210(3)
2.239(3)
1.241(3)
1.347(4)
1.225(3)
1.416(3)
1.469(4)
1.392(4)
1.392(4)
1.380(4)
1.504(4)
1.375(4)
84.33(7)
Ru(1)–C(6)
Ru(1)–Cl(1)
N(1)–C(18)
N(2)–C(17)
O(3)–N(3)
C(11)–C(16)
C(13)–C(14)
C(15)–C(16)
C(18)–C(19)
C(20)–C(21)
C(19)–C(20)
N(1)–Ru(1)–N(2)
N(2)–Ru(1)–Cl(1)
Ru(1)–C(Arene)
Average
O(1)–Ru(1)–N(1)
O(1)–Ru(1)–Cl(1)
C(4)–C(10)–C(9)
O(2)–C(11)–O(1)
C(4)–C(10)–C(9)
C(5)–C(6)–C(7)
C(9)–C(7)–C(6)
77.95(7)
85.56(5)
120.9(2)
124.4(2)
120.9(2)
121.1(2)
117.1(2)
N(1)–Ru(1)–Cl(1)
O(2)–C(11)–O(1)
C(6)–C(5)–C(4)
84.04(6)
124.4(2)
121.5(2)
C(6)–C(5)–C(4)
C(7)–C(9)–C(10)
C(10)–C(4)–C(5)
121.5(2)
122.1(2)
117.3(2)
◦
˚
Table 4 Selected bond distances (A) and bond angles ( ) for compound
3. Estimated standard deviations are given in parentheses
N(1)–Ru(1)
Cl(1)–Ru(1)
C(5)–Ru(1)
C(9)–Ru(1)
C(6)–Ru(1)
O(2)–N(3)
C(4)–C(5)
C(7)–C(8)
C(5)–C(6)
C(16)–O(1)
Ru(1)–C(Arene)
Average
2.083(3)
2.4173(10)
2.161(4)
2.187(3)
2.197(4)
1.211(5)
1.402(5)
1.425(5)
1.420(5)
1.242(4)
2.194(4)
N(2)–Ru(1)
C(4)–Ru(1)
C(7)–Ru(1)
C(8)–Ru(1)
C(20)–N(3)
O(3)–N(3)
C(7)–C(6)
C(9)–C(8)
C(9)–C(4)
2.093(3)
2.191(4)
2.228(3)
2.200(4)
1.471(5)
1.235(5)
1.404(5)
1.392(5)
1.425(5)
Cg(2)–Ru(1)a
Cg(2)–Ru(1)–Cl(1)a
Cg(2)–Ru(1)–N(2)a
N(1)–Ru(1)–Cl(1)
O(2)–N(3)–O(3)
C(5)–C(4)–C(9)
C(4)–C(5)–C(6)
C(9)–C(8)–C(7)
1.6801
127.68
131.31
85.12(8)
124.3(4)
117.0(3)
121.4(3)
120.4(3)
Cg(2)–Ru(1)–N(1)a
N(1)–Ru(1)–N(2)
N(2)–Ru(1)–Cl(1)
131.36
76.45(11)
87.15(9)
C(6)–C(7)–C(8)
C(7)–C(6)–C(5)
C(8)–C(9)–C(4)
118.0(3)
120.9(3)
122.2(3)
a Cg(2) denotes centroid of the C6 ring C(4)–C(9).
Compounds 2 and 3 were synthesised by the reaction of the
N-3-nitro-Ph-picolinamide and N-4-nitro-Ph-picolinamide with
6
[Ru(h -p-cymene)Cl2]2. Compounds 2 and 3 were recrystallised
from ethanol and methanol respectively. Both compounds were
characterised by NMR spectroscopy, microanalysis and X-ray
crystallography. Selected bond lengths and angles are shown in
Tables 3 and 4 respectively. The molecular structures are shown in
Fig. 2 and 3. The ligands are bound through the pyridyl nitrogen
and the amidinato nitrogen atoms (N/N) similar to their osmium
analogues.47 Thus, for compounds 2 and 3, substituting a NO2
group in the p- and m-position makes the ligand bind through the
pyridyl N and amidinato N atoms, whilst the unsubstituted/alkyl
substituted derivative tends to N/O binding. The increased acidity
of the proton is caused by the electron withdrawing groups on
the phenyl ring. Studies carried out for the p-substituted phenol,
systems show that p-nitro has the largest s- (enhanced s-value due
to electron withdrawing resonance effects) value of 1.27.48 This is
reinforced by the fact that for the p-nitro derivative, the reaction
proceeds smoothly at room temperature, whereas for the m-nitro
derivative, heat is needed to ensure that the reaction proceeds to
completion.
Fig. 1 The molecular structure of compound 1, with 50% probability
thermal ellipsoids.
˚
in compound 1: Ru(1)–O(1) of 2.0809(17) A and Ru(1)–Cl(1) of
˚
2.4133(6) A are within the ranges observed for other ruthenium
44
˚
˚
arene a-amino acidate complexes, of 2.079(11) A–2.392(7) A.
The Ru(1)–N(1) bond length of 2.096(2) A is within the range of
analogous ruthenium arene pyridyl complexes.45
˚
˚
The Ru–C(arene) average bond distance of 2.186(3) A is within
the range of ruthenium arene complexes of this type (as are the
Ru–C(arene) bond lengths in all subsequent ruthenium arene
complexes synthesised in this work).46 As reported in previous
works,43 the bond length between the ruthenium centre and the
substituted carbon atoms of the p-cymene ligand are longer by
˚
ca. 0.017–0.043 A. This feature is observed in all subsequent
ruthenium arene complexes reported in this work.
10918 | Dalton Trans., 2009, 10914–10925
This journal is
The Royal Society of Chemistry 2009
©

Fig. 2 The molecular structure of compound 2, with 50% probability thermal ellipsoids.
Fig. 3 The molecular structure of compound 3, with 50% probability thermal ellipsoids.
Studies have revealed that increasing the hydrophobic character
hydrophobic character than the less substituted pyridine analogue.
There is also the possibility for p–p stacking. In order to
investigate these effects on the cytotoxic activity of ruthenium
arene complexes, a series of quinaldic acid derivatives (analogues
of the picolinic acid complexes) (vide supra) were synthesised.
Synthetic routes to a range of quinaldamide derivatives were
originally developed by Davis et al. for studies of the potential
of a complex resulted in a concomitant increase in the activity
of that complex against a range of tumour types.16 Quinoline-2-
carboxylic acid (quinaldic acid) is structurally similar to picolinic
acid, and the quinoline residue is abundant in naturally occurring
molecules. Quinaldic acid possesses an extra four-membered ring
relative to picolinic acid and as a result may possess more
This journal is
The Royal Society of Chemistry 2009
Dalton Trans., 2009, 10914–10925 | 10919
©

◦
˚
application of quinaldic acid for the identification and isolation of
amino acids, peptides and proteins.49 Initially quinaldic acid was
converted to the acid chloride via treatment with excess thionyl
chloride. Condensation of quinaldic acid with the appropriate ani-
line in the presence of base afforded quinaldamide ligands in mod-
erate to good yields (they are also commercially available). Few
examples of quinaldic acid and quinaldamide species with metal
ions have been reported in the literature. Amongst these, there have
been studies on the coordination modes of ruthenium complexes
containing the phosphine ligand, bis(diphenylphosphino)methane
(DPPM).50 The interaction and coordination modes of the
ligands, N-(2-aminophenyl)quinoline-2¢-carboxamide and N-(3-
aminophenyl)quinoline-2¢-carboxamide with cobalt, copper, zinc
and nickel metal ions has been investigated.51
Table 5 Selected bond distances (A) and bond angles ( ) for compound
4. Estimated standard deviations are given in parentheses
Ru(1)–N(13)
Ru(1)–Cl(1)
Ru(1)–C(21)
Ru(1)–C(23)
Ru(1)–C(25)
O(16A)–N(16)
C(20)–C(25)
C(25)–C(24)
C(23)–C(22)
2.088(3)
Ru(1)–N(2)
2.112(3)
2.220(4)
2.249(4)
2.184(3)
1.232(4)
1.217(5)
1.419(5)
1.424(5)
1.416(5)
1.7003(15)
2.4091(9) Ru(1)–C(20)
2.181(4)
2.261(3)
2.165(3)
1.215(4)
1.407(5)
1.408(5)
1.399(5)
2.188(2)
Ru(1)–C(22)
Ru(1)–C(24)
O(12)–C(12)
N(16)–O(16B)
C(20)–C(21)
C(24)–C(23)
C(21)–C(22)
Cg(4)–Ru(1)a
Ru(1)–C(Arene)
Average
Cg(4)–Ru(1)–Cl(1)a
Cg(4)–Ru(1)–N(13)a
N(13)–Ru(1)–Cl(1)
129.70(6) Cg(4)–Ru(1)–N(2)a
128.66(9) N(13)–Ru(1)–N(2)
132.18(9)
77.21(10)
84.57(7)
86.43(8)
N(2)–Ru(1)–Cl(1)
O(12)–C(12)–N(13) 128.3(3)
C(20)–C(25)–C(24)
C(22)–C(23)–C(24)
C(23)–C(22)–C(21)
O(16A)–N(16)–O(16B) 123.1(3)
Compounds 4 and 5 were synthesised according to Scheme 1.
C(25)–C(20)–C(21)
C(25)–C(24)–C(23)
C(22)–C(21)–C(20)
116.4(3)
120.2(3)
121.8(3)
122.4(3)
118.2(3)
120.7(3)
6
[Ru(h -p-cymene)Cl2]2 was heated with a slight excess of N-3-nitro-
Ph-quinlinamide or N-4-nitro-Ph-quinlinamide in dry ethanol.
The mixture was then filtered over an excess of NH4PF6 to
afford [RuC26H24N3O3Cl] 4 and [RuC26H24N3O3Cl] 5. As ob-
served in the ruthenium arene picolinic acid complexes (2 &
3) the quinaldamide derivatives incorporating a nitro-functional
group exhibit preference for the formation of a neutral species
in which the ligand is bound through the pyridyl nitrogen
and the amidato nitrogen (vide supra). Compounds 4 and 5
were obtained as analytically pure orange powders in 84%
and 79% yields respectively as air stable racemic mixtures
([a]2D5 = 0◦). Both complexes were characterised by NMR
spectroscopy, mass spectrometry, elemental analyses and X-ray
crystallography.
a Cg(4) denotes centroid of the C6 ring C(20)–C(25).
Orange single crystals of 4 suitable for X-ray crystallographic
analysis were obtained from CDCl3 in an NMR tube. The
molecular structure is given in Fig. 4. Selected bond lengths
and angles are given in Table 5. Compound 4 crystallised in
a monoclinic cell and the structural solution was performed
in the space group P21/n. The asymmetric unit comprises one
molecule of 4. Compound 4 crystallised in the SRu configuration
(S configuration at the ruthenium metal centre).
Scheme 1 (i) potassium picolinate, THF; (ii) N-3-nitro-Ph-picolinamide and N-4-nitro-Ph-picolinamide, THF; NH4PF6 (iii) N-3-nitro-Ph-quinlinamide
and N-4-nitro-Ph-quinlinamide, THF; NH4PF6.
10920 | Dalton Trans., 2009, 10914–10925
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The Royal Society of Chemistry 2009
©

Fig. 4 The molecular structure of compound 4, with 50% probability thermal ellipsoids.
◦
˚
synthesis of these complexes, the effect of the p-cymene ligands
Table 6 Selected bond distances (A) and bond angles ( ) for compound
5. Estimated standard deviations are given in parentheses
on the anticancer activity of the complexes synthesised in this
6
work may be elucidated. The half-sandwich h -benzene ruthenium
Ru(1)–N(2)
Ru(1)–Cl(1)
Ru(1)–C(21)
Ru(1)–C(23)
Ru(1)–C(26)
O(2)–N(3)
C(20)–C(21)
C(22)–C(23)
2.092(2)
2.4081(8)
2.182(3)
2.230(3)
2.169(3)
1.241(4)
1.425(4)
1.426(5)
1.504(4)
2.200(3)
Ru(1)–N(1)
Ru(1)–C(20)
Ru(1)–C(22)
Ru(1)–C(25)
O(1)–C(10)
O(3)–N(3)
C(21)–C(22)
C(23)–C(25)
C(25)–C(26)
2.119(2)
2.206(3)
2.215(3)
2.200(3)
1.237(4)
1.231(4)
1.396(5)
1.408(5)
1.425(4)
complexes were synthesised using analogous methods to those
employed for the synthesis of ruthenium arene complexes 1–5.
Compound 6 was synthesised according to Scheme 2. Reaction
6
of [Ru(h -benzene)Cl2]2 with two equivalents of potassium picol-
inate in THF solution afforded [RuC12H10NO2Cl] 6. Compound
6 was characterised by NMR spectroscopy, microanalysis, mass
spectrometry and X-ray crystallography. In contrast to all other
complexes synthesised in this work, only one signal, with an
C(23)–C(24)
Ru(1)–C(Arene)
Average
1
Cg(4)–Ru(1)a
1.6837
integration corresponding to six protons is observed in the H
Cg(4)–Ru(1)–Cl(1)a
Cg(4)–Ru(1)–N(2)a
N(2)–Ru(1)–Cl(1)
O(3)–N(3)–O(2)
C(21)–C(22)–C(23)
C(23)–C(25)–C(26)
C(22)–C(21)–C(20)
128.95
128.72
87.54(7)
122.7(3)
120.5(3)
120.9(3)
122.4(3)
Cg(4)–Ru(1)–N(1)a
N(2)–Ru(1)–N(1)
N(1)–Ru(1)–Cl(1)
O(1)–C(10)–N(2)
C(25)–C(23)–C(22)
C(20)–C(26)–C(25)
C(21)–C(22)–C(23)
132.46
1
and 13C{ H} NMR spectra for the aromatic protons of the
77.26(9)
84.16(7)
127.5(3)
118.1(3)
121.3(3)
120.5(3)
a Cg(4) denotes centroid of the C6 ring C(20)–C(26).
Orange single crystals of 5 suitable for X-ray crystallographic
analysis were obtained from CDCl3 in a NMR tube. The molecular
structure is given in Fig. 5. Selected bond lengths and angles are
given in Table 6. Compound 5 crystallised in a monoclinic cell and
the structural solution was performed in the space group P21/c.
The asymmetric unit comprises one molecule of 5. Compound
5 crystallised in the SRu configuration (S configuration at the
ruthenium metal centre).
In order to determine the effect of substituents on the arene
ring on the anticancer activity of the ruthenium arene complexes
studied in this work, a selection of analogous half-sandwich
Scheme 2 (i) potassium picolinate, THF; (ii) N-4-nitro-Ph-picolinamide,
THF.
6
h -benzene ruthenium complexes were synthesised. From the
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©

Fig. 5 The molecular structure of compound 5, with 50% probability thermal ellipsoids.
6
◦
˚
h -bound arene ring; with chemical shifts of d 5.95 ppm and
83.7 ppm respectively.
Table 7 Selected bond distances (A) and bond angles ( ) for compound
6. Estimated standard deviations are given in parentheses
Orange single crystals of 6 suitable for X-ray crystallographic
analysis were obtained from a saturated methanol solution of
6 at 4 ◦C, over a prolonged period of time in an environment
free from disturbance and vibration. The molecular structure is
shown in Fig. 6. Selected bond lengths and angles are given
in Table 7. Compound 6 crystallised in a triclinic cell and the
Ru(1)–N(14)
Ru(1)–O(1)
Ru(1)–Cl(1)
2.046(10)
2.068(9)
2.413(3)
1.414(10)
2.178(11)
Ru(2)–N(15)
Ru(2)–O(3)
Ru(2)–Cl(2)
2.117(7)
2.085(8)
2.413(3)
1.414(10)
2.179(13)
Arene C–C Average
Ru(1)–C(Arene)
Average
Arene C–C Average
Ru(2)–C(Arene)
Average
Cg(3)–Ru(1)a
1.660(5)
130.3(2)
131.4(3)
127.6(3)
78.2(4)
84.3(3)
87.5(3)
Cg(4)–Ru(2)a
1.665(6)
127.5(2)
129.6(4)
133.8(3)
77.1(4)
84.2(3)
86.8(3)
Cg(3)–Ru(1)–Cl(1)a
Cg(3)–Ru(1)–N(14)a
Cg(3)–Ru(1)–O(1)a
N(14)–Ru(1)–O(1)
N(14)–Ru(1)–Cl(1)
O(1)–Ru(1)–Cl(1)
Cg(4)–Ru(2)–Cl(2)a
Cg(4)–Ru(2)–O(3)a
Cg(4)–Ru(2)–N(15)a
O(3)–Ru(2)–N(15)
N(15)–Ru(2)–Cl(2)
O(3)–Ru(2)–Cl(2)
¯
structural solution was performed in the space group P1. The
bond lengths and angles observed for 6 are comparable to those
observed for compound 1. The average Ru–C(arene) bond lengths
for 6 are within the range observed for similar ruthenium benzene
complexes.52
a Cg(3) & Cg(4) (denote centroids of the C6 rings C(1)–C(6) & C(13)–C(18)
respectively).
the nitro functionalized ligand binds with the pyridyl and amidato
nitrogen atoms. Compound 7 was obtained as an analytically
pure orange powder in 92% yield as a racemic mixture ([a]D25
=
0◦). Compound 7 was characterised by NMR spectroscopy,
mass spectrometry, elemental analysis and X-ray crystallography.
Orange single crystals of 7 suitable for X-ray crystallographic
analysis were obtained via vapour diffusion of diethyl ether into a
saturated acetone solution of 7. (The molecular structure is given
in Fig. S1. Selected bond lengths and angles are given in Table
S1—ESI.†)
The reaction of potential anticancer agents with guanine
residues has led to the synthesis of model complexes which provide
an insight into the mechanisms of activity of the agents in vitro and
in vivo.15,44 In double stranded nucleic acids, some of the potential
binding sites of nucleobases are occupied in hydrogen bonding to
the complementary strand.53 The N7 sites of both adenine and
guanine residues are free, and are thus found to be the major sites
for binding to metal ions in DNA. Furthermore, N7 of guanine
is more basic than N7 of adenine and thus coordination of metal
ions to the guanine residues is favoured over the other available
binding sites.54
Fig. 6 (a) The molecular structure of compound 6, with 50% probability
thermal ellipsoids.
6
Compound 7 was synthesised (Scheme 2) when [Ru(h -
benzene)Cl2]2was heated with two equivalents of N-4-nitro-Ph-
picolinamide in dry methanol until dissolution occurred. The
mixture was then filtered over an excess of NH4PF6 and heated to
afford [RuC18H14N3O3Cl] (7). As observed previously in this work,
10922 | Dalton Trans., 2009, 10914–10925
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Scheme 3 (i) H2O, D (ii) NH4PF6.
6
Table 8 In vitro cytotoxicity of complexes 1, 2, 3, 5, 6 and 7 for a range
of human tumour cell lines (6 day exposure) IC50 (mmol L-1)
The binding capability of a h -arene ruthenium complex syn-
thesised in this work was investigated by reaction with guanosine
hydrate (Guo). The guanine adducts developed in previous
investigations were synthesised by heating the complex with the
guanine residue for a period of time, ranging from one hour44 to
four days.15 The reaction conditions employed by Sadler et al.15
were modified and applied here. Compound 8 was synthesised
according to Scheme 3. Compound 1 was reacted with Guo at
338 K in distilled water for 5 days. The resulting green solution
was concentrated and filtered over an excess of ammonium
hexafluorophosphate (NH4PF6) to afford [RuC26H31N6O5][PF6]
(8) Scheme 3. Compound 8 was obtained as a green powder in
40% yield as a racemic mixture ([a]2D5 = 0◦) and was characterised
by mass spectrometry, NMR spectroscopy and microanalysis.
Study of the NMR spectra of 8 reveal that one guanosine residue
is bound to the ruthenium metal centre to afford the cationic
species, 8. 31P NMR spectroscopy revealed the presence of the PF6
counter anion as a septet, with a coupling constant of 1J(31P-19F)
708 Hz at a chemical shift of d -60.76 ppm. The 1H NMR signals
observed are similar to those found for other guanine residues44
Compound A2780
A2780cis
LS174T
MCF7
LoVo
Cisplatin
0.8
42.5
7.6
4.6
6.1
>50
>50
3.3
4.6
2
0.7
1
2
3
5
6
7
36.1
10.7
5.6
6.4
>50
>50
N/A
22.5
18.4
7.7
>50
>50
49.9
11.5
14.4
3.4
>50
>50
78.3
16.4
9.1
8.1
>50
>50
comparisons with respect to the breast and ovarian cancer cell
line testing as the osmium congener [(h -p-cym)Os(N-4-nitro-
6
Ph-picolinamide)Cl].47 Compound 5 exhibited excellent cytotoxic
activity against all the human tumour cell lines investigated, with
values in the same order of magnitude as either cisplatin or
carboplatin. Significantly, compounds 2, 3 and 5 exhibited activity
against the cisplatin resistant cell line A2780cis; which eventually
develops resistance to treatment with cisplatin. The activity of
compound 5 against the colon adenocarcinomas, LS174T and
LoVo is of significance owing to the fact that colorectal tumours
are at present intractable.55
1
and the H NMR signals assigned to the ribose unit of 8 are
comparable to those observed for other literature examples.15 The
NMR spectroscopic evidence for 8 suggests that one guanosine
residue is bound to the ruthenium centre via the N7 site.
Comparison of the anticancer activity of compound 1 with the
analogous compound 6 reveal that, upon removal of the lipophilic
substituents on the arene functionality (p-cymene to benzene)
the activity of the complex is reduced. Furthermore, the most
promising compound in the series, compound 5, possesses the
most hydrophobic ligand system (quinaldic acid as opposed to
picolinic acid). These observations suggest that an increase in the
hydrophobic character and possible p–p stacking character of a
complex may result in a concomitant increase in the anticancer
activity of that complex.
Biological results
Cancer cell cytotoxicity
The cytotoxicity of a selection of the complexes was tested towards
SRB assays of ovarian A2780; ovarian cisplatin resistant A2780cis;
colon adenocarcinoma LS174T; breast adenocarcinoma MCF7
and colon adenocarcinoma LoVo. Each compound was incubated
over a period of 6 days. Complexes 6 and 7 (Table 8) were
found to be non-toxic in all three cell lines up to the highest
test concentration of 50 mM. Their IC50 values (concentration at
which 50% of the cell growth is inhibited) are therefore likely
to be > 100 mM and the compounds can therefore be described
as inactive. Compound 1 shows moderate activity, however,
extremely promising activity was observed for the neutral N,N-
coordinated compounds 2, 3, and 5 as shown in Table 8.
Nuclease treatment assay
We were interested in the DNA binding ability and mechanistic
implications of the most cytotoxic molecule, compound 5. In
order to investigate the DNA binding nature and subsequent
breakdown of the ruthenated DNA fragments, nuclease cleavage
experiments were performed. Two polynucleotides (Fig. 7) were
annealed together in the DNA Engine PTC200 Peltier Thermal
Cycler PCR together to yield the double stranded section of
DNA. Compound 4 was incubated with the double stranded DNA
fragment for 45 min. The incubated material was treated with
Compounds 2 and 3 exhibited IC50 values in the same order of
magnitude as carboplatin. It is interesting to note that compound
3 shows a promising cell cytotoxicity profile and has favourable
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Dalton Trans., 2009, 10914–10925 | 10923
©

Fig. 7 Two 46-mer oligonucleotides used for the double stranded DNA fragment incubation experiment.
two enzymes, phosphodiesterase I, which cleaves DNA at 3¢ ends,
13 A. F. A. Peacock, A. Habtemariam, S. A. Moggach, A. Prescimone, S.
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15 H. M. Chen, J. A. Parkinson, S. Parsons, R. A. Coxall, R. O. Gould
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17 R. E. Aird, J. Cummings, R. E. Morris, A. A. Ritchie, P. J. Sadler and
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and alkaline phosphatase, which releases the phosphate from the
5¢-end. Mass spectrometry analyses were carried out at the end
of the experiment, which showed that, compound 4 formed an
adduct with guanine nucleotides with a peak at 795.97.
Conclusion
6
A series of h -arene ruthenium complexes containing ligands
with variable hydrophobic characteristics were synthesised and
characterised. Some of the ruthenium compounds have exhibited
very promising cytotoxic activity against a series of human tumour
cell lines with IC50 values within the same order of magnitude as
cisplatin and the cisplatin derivative carboplatin. The most active
complexes tested were found to be those in which the ligand
system incorporates a nitro group, either in the meta or para
positions. The excellent activity of a functionalised quinaldimide
against the colon adenocarcinomas, LS174T and LoVo is of
significance owing to the fact that colorectal tumours are at present
intractable.55 It has been shown that addition of hydrophobic
groups to the arene moiety of the ruthenium molecules has a
positive effect on the cytotoxicity values. From a mechanistic
aspect, we have proven that after incubation of a functionalised
quinaldimide ruthenium compound with a 46 mer oligonucleotide
duplex and subsequent nuclease treatment, ruthenium is bound to
a guanine residue.
19 F. Wang, H. Chen, J. A. Parkinson, P.d.S. Murdoch and P. J. Sadler,
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20 WO Pat., 0240494, 2002.
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Acknowledgements
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Mary and Alice Smith Memorial Scholarship from the University
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©