Discovery and investigation of anticancer ruthenium-arene Schiff-base complexes via water-promoted combinatorial three-component assembly

Article abstract of DOI:10.1021/jm500455p

The structural diversity of metal scaffolds makes them a viable alternative to traditional organic scaffolds for drug design. Combinatorial chemistry and multicomponent reactions, coupled with high-throughput screening, are useful techniques in drug discovery, but they are rarely used in metal-based drug design. We report the optimization and validation of a new combinatorial, metal-based, three-component assembly reaction for the synthesis of a library of 442 Ru-arene Schiff-base (RAS) complexes. These RAS complexes were synthesized in a one-pot, on-a-plate format using commercially available starting materials under aqueous conditions. The library was screened for their anticancer activity, and several cytotoxic lead compounds were identified. In particular, [(η⁶-1,3,5-triisopropylbenzene)RuCl(4-methoxy-N-(2- quinolinylmethylene)aniline)]Cl (4) displayed low micromolar IC₅₀ values in ovarian cancers (A2780, A2780cisR), breast cancer (MCF7), and colorectal cancer (HCT116, SW480). The absence of p53 activation or changes in IC₅₀ value between p53^+/+ and p53^-/- cells suggests that 4 and possibly the other lead compounds may act independently of the p53 tumor suppressor gene frequently mutated in cancer.

Full text of DOI:10.1021/jm500455p

Article
pubs.acs.org/jmc
Discovery and Investigation of Anticancer Ruthenium−Arene Schiff-
Base Complexes via Water-Promoted Combinatorial Three-
Component Assembly
Mun Juinn Chow,†,‡ Cynthia Licona,§,∥ Daniel Yuan Qiang Wong,† Giorgia Pastorin,‡,⊥
Christian Gaiddon,§,∥ and Wee Han Ang*,†,‡
†Department of Chemistry, National University of Singapore, 3 Science Drive 3, 117543 Singapore
‡NUS Graduate School of Integrative Sciences and Engineering, Singapore
§U1113 INSERM, 3 Avenue Molier
∥Universite
de Strasbourg, Strasbourg, France
̀
e, Strasbourg 67200, France
́
⊥Department of Pharmacy, National University of Singapore, 18 Science Drive 4, 117543 Singapore
S
* Supporting Information
ABSTRACT: The structural diversity of metal scaffolds makes
them a viable alternative to traditional organic scaffolds for drug
design. Combinatorial chemistry and multicomponent reac-
tions, coupled with high-throughput screening, are useful
techniques in drug discovery, but they are rarely used in
metal-based drug design. We report the optimization and
validation of a new combinatorial, metal-based, three-
component assembly reaction for the synthesis of a library of
442 Ru−arene Schiff-base (RAS) complexes. These RAS
complexes were synthesized in a one-pot, on-a-plate format
using commercially available starting materials under aqueous
conditions. The library was screened for their anticancer
activity, and several cytotoxic lead compounds were identified.
In particular, [(η6-1,3,5-triisopropylbenzene)RuCl(4-methoxy-N-(2-quinolinylmethylene)aniline)]Cl (4) displayed low micro-
molar IC50 values in ovarian cancers (A2780, A2780cisR), breast cancer (MCF7), and colorectal cancer (HCT116, SW480). The
absence of p53 activation or changes in IC50 value between p53+/+ and p53−/− cells suggests that 4 and possibly the other lead
compounds may act independently of the p53 tumor suppressor gene frequently mutated in cancer.
Transition-metal scaffolds could therefore be uniquely suited
for the development of potential drug candidates. They can
readily accommodate higher coordination numbers and thus
access different molecular geometries not possible with a purely
organic framework.6 Transition-metal complexes are also
endowed with tunable properties, such as variable oxidation
states that are stable under room conditions and predictable
reactivity based on metal−ligand bonding interactions. Since
the first successful platinum-containing drug cisplatin was
discovered and approved by Food and Drug Administration
(FDA) for the treatment of testicular and ovarian cancer in
1978,7 much research has been directed at uncovering new
metallodrugs that are more effective than cisplatin.8,9 This
research is beginning to bear fruit, with Ru-containing NAMI-A
receiving favorable evaluations in clinical trials and KP1019
showing promising results in preclinical studies.10,11 More
recently, metal-based compounds have been designed and
INTRODUCTION
■
Traditionally, drug discovery research has focused on the
development of organic molecules as pharmacophores.
However, their limited structural diversity makes it difficult
for them to access other scaffolds that span the entirety of the
biologically relevant chemical space,1−3 some of which may
contain promising drug candidates. Out of the 24 million
organic molecules registered under the Chemical Abstract
Service (CAS) Registry, half could be classified into 143 unique
motifs, a small number compared to the vast quantities of
molecules being classified.4 In addition, of the 5000 compounds
in the Comprehensive Medicine Chemistry (CMC) database
that were analyzed for diversity in topological shapes, half of
them belong to only 32 shape classifications,5 leaving large
areas of this chemical space containing structurally and
chemically unique motifs unexplored. In order to tap into
this substantial and potentially valuable unexplored chemical
space, scaffolds that are not accessible with organic chemistry
need to be investigated.
Received: March 23, 2014
© XXXX American Chemical Society
A
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tested against biological therapeutic targets, which are known to
regulate processes involved in the development of cancer.12
One class of Ru compound in particular, namely, half-
sandwiched Ru−arene complexes, has been garnering much
interest, with a large body of evidence demonstrating their
therapeutic properties against cancer13−15 or their inhibitory
activity toward enzyme targets linked to cancer.16−19 A few
examples include the antimetastatic RAPTA complexes20 and
antitumor [(η6-biphenyl)Ru(en)Cl]+ (RM175) scaffold,21 as
well as organoruthenium-based paullone complexes22 (Figure
1). These studies pave the way for the development of Ru−
arene complexes as the next generation of metal-based
anticancer drugs.
pyridyl−Ru complex capable of strongly inducing apoptosis in
cancer cells.25 Another group made use of automated solid-
phase combinatorial synthesis to synthesize a library of peptide-
tethered Pt(II) complexes for screening and identification of
cytotoxic leads.26,27 There have also been a number of reports
on one-pot multicomponent reactions for the preparation of
metal complexes,28,29 although, to the best of our knowledge,
none have been used for the purpose of drug discovery.
To that end, we designed a water-promoted multicomponent
reaction for the combinatorial assembly of organoruthenium−
arene complexes in aqueous solutions. Using a water/DMSO
system to carry out the assembly, the reaction directly “selects”
for water-stable metallocomplexes of moderate solubility. We
report the application of this metal-based three-component
assembly (3CA) reaction methodology to prepare a library of
water-soluble Ru−arene Schiff-base (RAS) complexes (Figure
2). We perform extensive functional groups variation and
screen the compound library for anticancer activity. The
identified lead compounds were tested against a variety of
cancer cell types and their p53-dependence was also
investigated.
RESULTS AND DISCUSSION
■
Optimization and Validation of a Novel One-Step
Three-Component Assembly Reaction in Aqueous
Solvents. Our approach was to design and optimize a one-
pot three-component reaction that can be used to synthesize a
large library of Ru−arene complexes with ease, requiring
minimal purification prior to characterization and testing. The
reaction comprises three components, namely, a Ru−arene
complex, a picolinaldehyde, and an aniline. Imine condensation
between the picolinaldehyde and aniline forms a Schiff-base
ligand that is predisposed for metal chelation. Without metal
chelation, such ligands are typically poorly stable in aqueous
environment due to hydrolysis of the imine bond. Incorpo-
ration of the Ru−arene component yields an organoruthenium
complex that is stabilized by the electrodonating Schiff-base
chelate. Similar Ru−arene Schiff-base complexes have been
reported, but as yet they have not been prepared in a manner
suitable for library synthesis.30 Thus, 3CA comprises Ru−arene
percursor A with at least two accessible coordination sites (via
labile ligands) that could chelate with Schiff-base ligand formed
from condensation between picolinaldehyde B and aniline C,
both of which are widely available from commerical sources
(Figure 3a). Components A, B, and C with different functional
Figure 1. Examples of anticancer organoruthenium compounds.
Yet, discovery of new organoruthenium complexes with the
necessary prerequisites for therapy is a laborious process
involving iterative steps of synthesis, characterization, function-
alization, and testing. For example, good aqueous stability and
solubility are essential to ensure biocompatibility, but these
attributes are not easily incorporated into the scaffolds.
Combinatorial coordination chemistry and multicomponent
reactions are potential tools that can generate large libraries of
diverse compounds23,24 for drug screening, but they have
seldom been applied to metal-based drug discovery. One report
employed a two-step combinatorial synthesis approach coupled
with high-throughput screening (HTS) to identify a poly-
Figure 2. Concept of RAS complex library synthesis, screening, and lead identification.
B
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Figure 3. (a) Combinatorial, three-component assembly of RAS complexes. (b−d) The different functional groups on components A, B, and C used
for the library synthesis.
Figure 4. (a) 1H NMR spectrum of crude reaction mixture in D2O of representative RAS complex obtained using A3, B1, and C1 (Figure 3) without
any workup or purification. (b) Molecular structure and calculated molecular weight of cation. (c) Parent molecular peak observed in the ESI-MS
spectrum corresponding to the expected m/z.
C
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a
groups could be mixed in a combinatorial fashion in a one-pot
reaction to give a library of ruthenium−arene Schiff-base (RAS)
complexes (Figure 3). Because the components were initially
insoluble in water, complete dissolution provided an indication
of a successful assembly. Reactions that yielded insoluble
products were assumed to have failed or have led to products
that were insufficiently soluble to warrant further studies and
were eliminated from the library. An initial library of 48 RAS
complexes were synthesized in D2O and directly characterized
with 1H NMR and ESI-MS without prior workup or
purification. Several complexes were analyzed by RP-HPLC
to approximate the levels of purity achieved.
Table 1. Selected X-ray Crystallographic Data
Complex
formula
[RAS-A3/B1/C8]PF6
RuC23H25N3OClPF6
640.96
formula weight
temp (K)
wavelength (Å)
crystal size (mm3)
crystal system
space group
a (Å)
100(2)
0.710 73
0.24 × 0.12 × 0.10
monoclinic
P21/n
9.887(2)
b (Å)
18.473(4)
14.002(2)
90
c (Å)
The 1H NMR spectra typically presented resonances
corresponding to the expected products with minor observable
byproducts, indicating that RAS complexes were formed almost
exclusively (Figure 4a). The RAS complexes exhibited two
characteristic features, namely, the disappearance of the singlet
peak corresponding to the aldehyde proton at ca. 10 ppm and
the appearance of a singlet attributable to the imine proton at
8−9 ppm (a downfield shift from ca. 6 ppm for uncoordinated
ligands). In addition, resonances of the η6-arene protons (for A
= toluene, cymene, and 1,3,5-triisopropylbenzene) were
resolved to distinct peaks due to desymmetrization of the
arene ligand upon complexation of the Schiff-base at the Ru
center. ESI-MS typically revealed only one m/z peak
corresponding to the molecular [M]+ ions with the character-
istic Ru isotopic pattern, further confirming the formation of
RAS complexes in the 3CA reaction (Figure 4c). RP-HPLC
analysis of seven selected RAS complexes from the initial library
showed purity levels between 70 and 90% of the crude mixture,
which is sufficiently pure for drug screening [see Supporting
Information (SI)]. The main cause of the lower purity is the
presence of excess reaction components that were added
inadvertently and the formation of aquation products.
α (deg)
β (deg)
104.589(3)
90
γ (deg)
V (Å3)
2474.8(9)
4
Z
Dc (Mg/m3)
1.720
μ (mm−1
)
0.874
θ range (deg)
1.86−27.50
17 246
no. of unique data
max., min. transmn
final R indices
0.9177 and 0.8176
R1 = 0.0525
wR2 = 0.1214
R1 = 0.0823
wR2 = 0.1308
1.050
[I > 2σ(I)]
R indices (all data)
goodness-of-fit on F2
peak/hole (e Å−3
)
2.822 and −0.880
a
R = ∑∥Fo| − |Fc∥/∑|Fo|, wR2 = [∑[w(Fo2 − Fc2)2]/∑[w(Fo )2]]1/2
.
2
Goodness-of-fit (GOF) = [∑[w(Fo − Fc2)2]/(n − p)]1/2, where n is
2
the number of data and p is the number of parameters refined.
Table 2. Selected Bond Lengths (Å) and Angles (deg)
Ru(1)−N(1)
Ru(1)−N(2)
Ru(1)−Cl(1)
N(2)−C(6)
2.088(4)
2.101(4)
Structural information was obtained via single crystal X-ray
diffraction studies of the RAS complex synthesized from A3,
−
2.3976(13)
1.293(6)
B1, and C8, with PF6 as the counteranion (Figure 5). The
N(2)−C(7)
1.425(6)
N(1)−Ru(1)−N(2)
N(1)−Ru(1)−Cl(1)
N(2)−Ru(1)−Cl(1)
76.98(15)
84.50(11)
86.81(11)
At this stage of the methodology development, all complexes
were prepared and characterized in D2O and thus assumed to
be sufficiently water-stable for at least 72 h, which encompassed
the time of experiment from the start of synthesis to the end of
characterization. Upon optimization, we noted that a solvent
mixture comprising 1:1 v/v DMSO-d6/D2O to be a more
suitable solvent system for 3CA for preparation of less water-
soluble RAS complexes, which were otherwise insoluble in pure
D2O.
Figure 5. Molecular representation of a RAS complex formed from
A3, B1, and C8 (Figure 3). There are four cationic centers with
corresponding anionic PF6− in each unit cell. PF6− anion was omitted
for clarity.
Mechanistic Investigations into Formation of RAS
Complexes. We performed a mechanistic investigation of the
3CA reaction to understand the factors affecting assembly of
the target complexes in order to be able to fully exploit its
versatility. Several experiments were designed to probe the
reaction mechanism and to understand how RAS complexes
were formed. These experiments were performed in deuterated
complex adopted an expected pseudo-“piano stool” structure,
with the cymene ligand bonded to the Ru atom as the “stool
top”. The Schiff-base ligand rotated slightly around N2−C7 to
give a “twisted” conformation, presumably to reduce the steric
interaction with the cymene ligand. Selected cystallographic
and structural data are given in Tables 1 and 2.
1
solvents to facilitate immediate H NMR analysis (see SI).
From the experimental results, the factors affecting the
formation of RAS complexes were inferred.
D
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Scheme 1. Postulated Reactive Intermediates under Different Solvent Systems
Scheme 2. Putative Reactive Intermediates Formed under Different pH Conditions
Ru precursor A exists as a dimer in solid state or when
dissolved in noncoordinating solvents such as toluene or
CHCl3. However, in coordinating solvents such as DMSO,
MeOH, or H2O the dimers are cleaved to form reactive
[Ru(arene)(solvent)x+1Cl2‑x] monomers.31 We theorized that
3CA occurred via these reactive intermediates and coordinating
solvents were necessary to activate A. Thus, 3CA was carried
out in the noncoordinating solvent CDCl3, as well as the
coordinating solvent systems DMSO-d6, MeOD-d4, D2O, 1:1
v/v DMSO-d6/D2O. Components that were soluble in each of
the solvents were chosen so as to eliminate any possible
complications arising from a difference in solubility. As
expected, RAS complexes were not formed at all in CDCl3.
However, the 3CA reaction was also unsuccessful in the
coordinating solvents DMSO-d6 and MeOD-d4, producing
many unexpected side products. Indeed, assembly was only
successful in solvent systems containing D2O.
The unexpected observation could be explained by the
difference in the reactive intermediate formed in DMSO and
MeOH compared to that in H2O (Scheme 1).31 In DMSO and
MeOH, monosolvated species [Ru(arene)XCl2] is formed as
the only species (X = DMSO, MeOH). In H2O, an additional
chloride ligand is displaced from the monosolvated [Ru-
(arene)(H2O)Cl2] to form the disolvated species [Ru(arene)-
(H2O)2Cl]+. The presence of two labile solvent ligand in the
disolvated species allowed rapid reaction with the ligand
chelate, giving rise to fewer side reactions and leading to the
formation of RAS complexes exclusively. The presence of water
in DMSO/H2O solvent mixture presumably replaced the
second chloride to yield the reactive intermediate [Ru(arene)-
(DMSO)(H2O)Cl]+. This hypothesis was further validated by
performing the 3CA reaction at different pH conditions
(Scheme 2). We hypothesized that at acidic to neutral pH,
the disolvated [Ru(arene)(H2O)2Cl]+ would be formed
primarily. At basic pH, the kinetically inert μ-OH bridged
dimer [Ru2(arene)2(μ-OH)3]+ would form preferentially, which
would hinder 3CA.32,33 Thus, 3CA was carried out at pH 4.5
and 10.0 using deuterated reagents in D2O and analyzed by
1
both H NMR and ESI-MS. Indeed, the results showed that
3CA was hindered in basic pH medium.
Next, we attempted to understand why little or no side
products were obtained in 3CA by studying the reactivity of the
individual components after they had been added sequentially
(Scheme 3). We first carried out the reactions in the absence of
one of the components, i.e., (i) B and C, (ii) A and B, and (iii)
A and C, for 24 h and then supplemented each of the reaction
mixtures with the missing component for a further 24 h. Like
previous experiments, reactions i, ii, and iii were done in a
1
deuterated solvent, D2O, so that immediate analysis by H
NMR was possible. For reaction i, the expected imine product
was observed after 1 h, and most of the starting material had
been converted to the imine product after 24 h. Reaction ii
E
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Scheme 3. Influence of Reagent Addition Sequence on Product Formation
Scheme 4. Postulated Reaction Mechanism for Three-Component Assembly Reaction
yielded multiple products after 24 h. Even after C was added,
there was further reaction. Reaction iii gave a mixture of starting
materials and products after 24 h. However, after B was added
for 24 h, the reaction mixture resolved to yield a clean 1H NMR
spectrum with RAS complex being the only product. This
suggested that A reacted reversibly with C and subsequent
addition of B would still drive 3CA reaction to completion.
Taken together, these experiments explained why the 3CA
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Figure 6. Analyses of cell viability data from the 442-compound library. (a) Plot of percent cell viability (CV%) against hydrophobicity index (HI).
CV% decreases significantly after HI = 3, with the most cytotoxic compounds in the range 4 < HI < 6 (shaded area). (b, c) Plot of CV% against H-
bond acceptor and H-bond donor, respectively. CV% increases as the number of H-bond acceptors or donors on a molecule increases, with most
cytotoxic compounds having two or less H-bond acceptors or donors (shaded area). (d) Plot of CV% against molecular weight. Most cytotoxic
compounds appear to have MW between 530 and 660 g/mol (shaded area).
worked despite the possibility of forming many different side
products. First, RAS complexes are thermodynamically the
most stable product due to the “chelate effect”. Second, RAS
complexes are kinetically favored. At room temperature, the
reaction between [Ru(arene)(H2O)2Cl]+ and B or [Ru(arene)-
(H2O)2Cl]+ and C is slower compared to the reaction between
[Ru(arene)(H2O)2Cl]+ and the imine ligand (B + C) or the
formation of the imine ligand (Scheme 4).
To validate this hypothesis, we compared 3CA using the
more basic aliphatic amine, 4-aminomethylbenzenesulfona-
mide, in place of aniline, 4-aminobenzamidine, as component
C. Although they are both primary amines, aniline is poorly
basic by virtue of the delocalization of the N-atom lone pair
into the aromatic ring. We reasoned that aniline would not
undergo dative bonding with [Ru(arene)(H2O)2Cl]+ and
would not directly compete with 3CA. Indeed, 1H NMR
analysis revealed that, unlike 4-aminobenzamidine, 4-amino-
methylbenzenesulfonamide reacted irreversibily with A, pre-
maturely arresting 3CA. We further tested the functional group
tolerance using analogues of B and C and found that functional
groups that competed for direct Ru-binding, such as thiol and
imidazoles, also directly interfered with 3CA. Nevertheless, a
wide variety of functional groups were tolerated (Figure 3).
Plate Format Synthesis and Direct Screening of
Cytotoxicity. To demonstrate the versatility of 3CA, we
carried out extensive synthesis of RAS complexes using the
possible combinations shown in Figure 3 on a plate format. A
total of 450 compounds were prepared and characterized.
Synthesis were performed on 96-well plates, in a high-
throughput manner with 1:1 v/v DMSO/H2O used as the
solvent system (see SI) to maximize the solubility of the
products while maintaining some degree of aqueous solubility.
DMSO stock solutions of all the starting reagents were
prepared and mixed in a predetermined order in wells
containing an equal volume of H2O. Each well gave a different
RAS complex at 5 mM in 1:1 v/v DMSO/H2O by the end of
the reaction, and they were analyzed by ESI-MS. Successful
assembly was indicated by the molecular [M]+ ion as the
predominant m/z peak. Out of the 450 complexes synthesized,
only one was insoluble and eliminated as a candidate for
screening. All complexes synthesized were used without further
purification.
G
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Figure 7. Lead compounds identified during the screening and their HI and CV% data.
Scheme 5. Synthetic Route for the Preparation of 1−6
For the screening of cytotoxicity, A2780 ovarian cancer cells
were used because the cells are sensitive to cisplatin treatment
and can be transplated to in vivo models as xenografts.
Cytotoxicity of the assembled RAS complexes were tested at a
single concentration by using the MTT assay.34 The complexes
were first diluted on a separate 96-well plate with RPMI 1640
H
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medium to give a final concentration of 25 μM in medium
containing 0.5% (v/v) DMSO. Seven more compounds were
insoluble under this condition and were removed from the
screen. The remaining 442 RAS complexes were tested against
A2780 cells in quintuplicate, and the percentage of cell viability
(CV%) after treatment of each drug was then determined.
Using this mass screening method, we identified 20 lead
compounds with CV% below 10% at 25 μM concentration; out
of these 20 lead compounds, 16 had CV% below 5%.
Figure 8. Steric hindrance between bulky isopropyl and naphthalene
groups creates a barrier of rotation of the C−N bond, resulting in
pseudo-E/Z isomerism.
To determine if there was any correlation between the
hydrophobicity and the cytotoxicity of the complexes, CV%
could be compared with their clogP. Because there are currently
no available software to calculate the clogP of these
organometallic complexes, we adopted an alternative standard
of measurement based on the sum of clogP values of the arene
and the chelate ligand. We assumed that differences in their
hydrophobicities were largely due the different R, R′, and R″
groups (Figure 3), since all 442 complexes were based on the
same core structure. RAS complex [RAS-A1/B1/C1] (R = R′ =
R″ = H) was assigned an arbitrary hydrophobicity index (HI)
of 0 and the remaining complexes were assigned HI values
relative to this, calculated from the clogP of their ligands.
Similar to clogP, the more positive the HI, the greater the
hydrophobicity. The CV% value of each compound was then
plotted against their HI value (Figure 6a). When HI > 3, CV%
decreased significantly. The most cytotoxic RAS complexes
were relatively hydrophobic, having HI between 4 and 6,
suggesting that cellular uptake might be via passive diffusion.35
We further analyzed the cell viability data against additional
measures of druglikeness from “Lipinski’s rule of five,”36 such as
hydrogen bond (H-bond) acceptors, H-bond donors, and
molecular weight, to see if there were any additional observable
trends (Figure 6b−d). In general, CV% increased with
increasing number of H-bond acceptors and donors with a
majority of the cytotoxic RAS complexes having less than two
H-bond acceptors or donors. In addition, the most cytotoxic of
these complexes tends to have a molecular weight between 530
and 660 Da.
compounds. All six compounds were shown to be at least 95%
pure, either by EA or RP-HPLC analysis.
Determination of Aqueous Stability, Solubility and
log POW. The aqueous stability, solubility, and log POW are
important factors that affect a potential drug’s bioavailability
and cell permeability.37 The aqueous stability of lead
compounds 1−6 were investigated by monitoring their UV
profiles over a period of 24 h at every 1 h interval. Each
compound was first dissolved in DMSO to make up a 5 mM
stock solution which was then diluted with water to 50 μM (1%
v/v DMSO). All compounds except 6 demonstrated good
stability in water with negligible variation to their UV peak
profiles after 24 h. A significant change in the UV peak profile
of 6 was observed with two isosbestic points at 290 and 454 nm
(see SI). When the experiment was repeated for 6 in the
presence of 0.6% w/v NaCl, no significant change in its UV
peak profile was observed. This suggests that the chloride
ligand of 6 was significantly more labile as compared to those of
1−5 and that aquation could be suppressed with the addition of
chloride ions.
The thermodynamic solubilities of 1−5 were determined at
saturation in ddH2O for 24 h. After 24 h, undissolved solid was
removed by centrifugation, and Ru concentration of the
resulting saturated solutions was determined via ICP-OES.
The lead compounds showed moderate solubilities in water,
with 3 having solubility of 131.6 mg/mL, on the same order of
magnitude as common inorganic salts. The solubility of 6 in
0.6% w/v NaCl was too low to warrant further analyses.
The log POW of 1−6 was determined using the shake-flask
method.38 1−5 were partitioned between n-octanol and
ddH2O, and the amounts of compounds in the aqueous
fractions before and after the partitioning were measured using
UV−vis spectroscopy at their respective λmax. The same was
done for 6 using 0.6% w/v NaCl instead of ddH2O. 1−5 were
relatively hydrophilic, as shown by their negative log POW
values. The ionic nature of the complexes endowed them with
hydrophilic character, despite having hydrophobic surface
groups. In general, 1−6 were considered “druglike” based on
Lipinski’s rule of five.36 However, there was no observable
correlation between the hydrophilicity (log POW) and the
aqueous solubility. In fact, the more hydrophilic 1 and 2 were
much less soluble compared to hydrophobic 3 and 4. This
could be due to the ability of 1 and 2 to lose a H+ ion from the
phenol functional group to form an overall charge-neutral
zwitterion, which may have reduced solubility.
Synthesis and Characterization of Lead Compounds.
In order to validate the cytotoxic mechanisms of the lead
compounds, we synthesize them on a preparative scale for
detailed in vitro evaluation. This would also help us eliminate
false positives, which are relatively common in high-throughput
screening. We selected six out of the 20 lead compounds
(Figure 7) with low CV% and resynthesized them individually
according to Scheme 5, including two outliers (1 and 2) that
did not fit into the general cytotoxicity trend. The identity and
1
purity of compound 1−6 were ascertained by H NMR, ESI-
1
MS, and RP-HPLC or elemental analysis (EA). The H NMR
spectra of the 1, 2, 4, 5, and 6 were as expected, with the imine
singlet peak appearing around 9.0−9.4 ppm and additional
splitting of the arene proton peaks. However, 1H NMR
spectrum of 3 showed two sets of compounds instead of one
(see SI). This was presumably due to the restriction of free
rotation of the C−N bond caused by the steric repulsion
between the bulky napthalene group and the triisopropyl
groups, which resulted in the formation of pseudo-E and -Z
stereoisomers (Figure 8). By increasing the temperature of
NMR data acquisition to 343 K, the barrier of rotation could be
overcome and the peaks coalesced, giving rise to only one set of
resonances. Similar E/Z isomerism was previously reported for
another class of Ru−arene Schiff-base complexes.22 The ESI-
MS spectra showed the expected molecular ion peaks for all six
In Vitro Evaluation of Lead Compounds against
Cancer Cell Lines. The efficacy values of RAS complexes
1−6 in the inhibition of cell viability were evaluated against a
panel of human cancer cell lines (Table 3). All experiments
were carried out in at least triplicates, and IC50 was determined
from the average of three separate experiments. Ru
concentrations of stock solutions were also determined using
ICP-OES and the IC50 values were adjusted to the actual
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Table 3. Properties of Lead Compounds 1−6
d
IC50 (μM)
a
c
e
complex
log Pow
solubility (mg/mL)
MCF7
>50
A2780
A2780cisR
>50
resistance factor
1
−1.78 0.07
−1.92 0.24
−0.81 0.01
−0.79 0.03
−1.03 0.01
2.69 0.01
7.34 0.01
131.6 1.6
35.0 1.6
2.16 0.03
ND
>50
>50
NA
NA
1.0
1.1
1.3
0.7
3.6
2
>50
>50
3
9.00 0.62
2.93 0.21
6.47 0.45
13.13 1.35
>50
9.88 2.13
2.76 0.55
7.58 0.98
11.19 1.41
9.75 3.47
3.17 0.29
9.78 2.14
7.59 1.24
34.30 4.01
4
5
b
6
0.57 0.07
cisplatin
−2.03 0.4751
3.37 0.0551
9.54 2.36
b
a
log Pow values determined via the shake-flask method with 1:1 n-octanol:H2O partitioning. log Pow for 6 was determined in 1:1 n-octanol: 0.6% w/
v NaCl. Compounds were dissolved in 0.5 mL of water to saturation at 24 h at rt and [Ru] was determined using ICP-OES. ND = not determined.
IC50 values are the concentration of Ru complexes required to inhibit 50% of cell growth with respect to control groups, measured by MTT assay
c
d
after 6 h of drug exposure and 66 h of incubation. Data obtained are based on the average of at least three independent trials, and the reported errors
e
are the corresponding standard deviations. The IC50 were corrected using actual [Ru] determined using ICP-OES. On the basis of the ratio of
IC50[A2780cisR] to IC50[A2780]. NA = not applicable.
Figure 9. Dose-dependent drug efficacy studies for 3−6 and oxaliplatin on colon cancer cell lines HCT116 and SW480.
concentration. Contrary to what was expected, 1 and 2 did not
show significant cytotoxicity for any of the cell lines. We
concluded that 1 and 2 were false positives and removed them
from further testing.
oxalato complexes,41,42 suggesting that the resistance mecha-
nisms induced by platinum anticancer drugs do not impact the
biological activity of ruthenium compounds significantly.
We further evaluated the lead compounds against colorectal
carcinoma cell lines known to be resistant to treatment by
cisplatin. This intrinsic resistance to cisplatin had previously
been linked to overexpression of organic cation transporter
(OCT) proteins in colorectal carcinoma cells. On the other
hand, oxaliplatin, by virtue of its hydrophobic R,R-diaminocy-
clohexane motif (DACH), can undergo aquation to yield
cationic species that were postulated to be OCT1/2
substrates.43 As RAS complexes are persistently cationic and
contained hydrophobic ligands, they could potentially be
substrates of OCT1/2 and effective against colorectal
carcinoma cell lines. 1−6 were tested against two such cell
lines, HCT116 and SW480, and their activity was compared
against that of oxaliplatin (Figure 9).
RAS complexes 3−6 showed good killing efficacies against
the cancer cell lines tested. Against A2780, the complexes
exhibited efficacies comparable to or better than cisplatin, with
4 achieving IC50 values 3 times lower than that of cisplatin. In
addition, they did not exhibit any cross-resistance toward
cisplatin-resistant A2780cisR, with 6 achieving a resistance
factor as low as 0.7 and 4 having IC50 value 10 times lower than
that of cisplatin. Furthermore, 3−6 also demonstrated good
toxicity toward MCF7, whereas cisplatin showed negligible
cytotoxicity. Other classes of Ru−arene complexes have also
demonstrated activity against cell lineages with reduced
susceptibility to cisplatin or other platinum anticancer
drugs.39,40 In particular, the class of Ru−arene complexes
(RM175) reported by Sadler et al. exhibited resistance factors
between 0.7 and 1.0 in A2780cisR, similar to RAS complexes
3−6.41 This was in contrast to the cross-resistance exhibited by
platinum anticancer drugs such as carboplatin or platinum(II)−
With the exception of 6, RAS complexes were more cytotoxic
toward HCT116 than SW480. 3 and 5 demonstrated modest
toxicity toward HCT116 but displayed poor IC50 values above
20 μM in SW480. 4 displayed good cytotoxicity in both cell
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Figure 10. Dot plots of HCT116 cells after 24 h exposure to 4 at various concentrations: (a) untreated control, (b) 2.5 μM, and (c) 10 μM. Viable
cells are shown in the lower left quadrant, early apoptotic cells are in the lower right quadrant, late-stage apoptotic cells are in the upper right
quadrant, and necrotic cells are in the upper left quadrant. Numbers shown indicate the percentage of cells in each quadrant.
Figure 11. (a) Western blot for detection of p53 expression in HCT116 cells treated with 4 and oxaliplatin for different exposure times.
Homogeneous protein loading determined with reference to actin. (b, c) Inhibition of cell viability on BJwt and BJshp53 cells treated with varying
concentrations of 4 and DOX, respectively. (d) A comparison of IC50 values of 4 and DOX in BJwt and BJshp53 cells.
lines, achieving IC50 of 1.0 μM in HCT116 and 7.3 μM in
SW480, compared to oxaliplatin, which was equally efficacious
against both cell lines. Nevertheless, 4 demonstrated greater
efficacies toward HCT116 compared to oxaliplatin.
Cell Cycle Analysis and Apoptosis Assay. The ability of
4 to cause cell cycle arrest and apoptosis was investigated.
HCT116 cells were treated with 4 for 24 h at 2.5 μM and 10
μM. The cell cycle profiles of treated cells stained with
propidium iodide (PI) were obtained by flow cytometry
analysis and compared with that of an untreated control.
Significant cell growth arrest at the G2/M phase was induced
by 4 only at the higher concentration (Figure S5, SI). However,
at 2.5 μM a modest G0/G1 growth arrest was observed. Flow
cytometry analysis using annexin V−EGFP/PI apoptosis
K
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detection assay revealed that 4 induced apoptosis in a dose-
dependent manner (Figure 10). The percentage of total
apoptotic cells increased from 14% to 50% when concentration
increased from 2.5 to 10 μM. The percentage of nercotic cells
remains low at both concentrations (<5.0%).
of RM175 and related compounds and showed that it was
strongly correlated to their cytotoxicity; highly cytotoxic
compounds undergo aquation, while the inactive compounds
were resistant to aquation. In contrast, RAS complexes did not
exhibit a similar trend, suggesting that DNA may not be the
molecular target. RAS compounds 3−5 displayed good
cytotoxicity toward the examined panel of cancer cells, even
though they are resistant to aquation. On the other hand, the
ability to undergo aquation did not confer improved cytotoxic
efficacies to 6. To further rule out aquation and subsequent
DNA binding as the activation mechanism, we incubated lead
complex 4 with DNA nucleoside dGMP and biological
nucleophile GSH and monitored its UV−vis profile over a 24
h period. No change in its UV−vis profile was observed in the
presence of either dGMP (2 molar equiv) or GSH (10 mM).
Furthermore, no new adducts were observed in ESI-MS,
indicating that 4 did not interact with the model DNA base and
that the chloride ligand on 4 was stable toward nucleophilic
substitution (see SI). Coupled with earlier results indicating
that 4 operated via a p53-independent pathway, we
hypothesized that DNA may not be a target of our RAS
complexes, unlike other DNA-binding Ru agents such as
RM175.
Mechanistic Considerations: RAS Complexes Exhibit
p53-Independent Cytotoxicity. p53 is a key tumor
suppressor gene often mutated in human cancers and is
involved in the mediation of cellular DNA repair, apoptosis
induction, and cell cycle arrest.44 p53 activation also mediates
the neurotoxicity of anticancer drugs.45 The chemoresistance of
some cancer cell lines toward certain anticancer drugs such as
oxaliplatin or doxorubicin (DOX) have been shown to be due
to mutated p53-encoding genes or deficient p53 levels, which
are less effective in inducing apoptosis even when significant
DNA damage has occurred.46,47 Anticancer agents that are p53-
independent could be promising drug candidates against drug-
resistant cancers. However, previous reports indicated that
several ruthenium complexes of various structure, such as
RAPTA-C, RM175, and RDC11 (Figure 1), induced p53 and
p53 target genes at various degrees.48−50 To investigate if lead
RAS complexes inhibit cell viability via a p53-dependent or
-independent mechanism, we determined the expression of p53
in HCT116 cells and AGS human adenocarcinoma cells. They
were treated with varying concentration of 3−5 and their p53
levels were compared with that of cells treated with either
oxaliplatin or cisplatin as controls. In addition, we measured the
p53 expression in HCT116 cells after different exposure times
(2, 6, 12, and 24 h) to 4 and oxaliplatin. We also tested the p53-
dependence of 4 by measuring its cytotoxicity against wild-type
human foreskin cells BJwt and the p53 knockdown BJshp53.51
As a control, DOX was also tested against both cell lines, and
the results were compared.
As expected, time- and concentration-dependent upregula-
tion of p53 was observed in cells treated with oxaliplatin
(Figure 11a). In contrast, p53 expression in HCT116 cells
remained at basal levels, regardless of the duration or
concentration of exposure to 4. HCT116 cells did not show
any increase in expression of p53 even at concentrations much
higher than their IC50 values after 24 h of treatment (Figure S4,
SI). These results were confirmed in the gastric cancer cell line
AGS, in which cisplatin treatment induced p53 protein levels
while 3−5 had no significant effect. Furthermore, 4
demonstrated similar efficacies in both BJwt and BJshp53,
while DOX was 7 times more cytotoxic against BJwt than p53-
deficient BJshp53 (Figure 11b−d). Taken together, these
results suggest that 3−5 exert their antiproliferation activity via
a p53-independent mechanism, unlike RAPTA-C, RM175, or
RDC11. Recent studies by Sadler and co-workers on a class of
Ru−arene complexes bearing similar structural features also
demonstrated a p53-independent mechanism of action.15
Intriguingly, they reported that the halide ligand affected p53-
dependence and that p53-independent activity was only
observed for iodido complexes and not for chlorido analogues.
Our preliminary results were dissimilar and more extensive
studies involving the several target genes of p53 are being
performed to validate these findings.
CONCLUSION
■
A one-pot combinatorial metal three-component reaction was
validated as a methodology to prepare RAS complexes in a
large-scale format. The aqueous reaction conditions for
compound synthesis directly selects for complexes that exhibit
water stability and solubility. Screening of these complexes
against A2780 cells led to the identification of several lead
compounds, most of which displaying low micromolar IC50
values in the inhibition of cell viability against the tested panel
of cancer cell lines. Further investigations revealed RAS
complex 4 to be highly efficacious and induced apoptosis via
a p53-independent mechanism, suggesting that DNA was not
its primary biological target. This results hold promise for
treatment of cancers that are drug-resistant due to their
dysfunctional p53 status.
EXPERIMENTAL SECTION
■
Materials. All experimental procedures were carried out without
additional precautions to exclude air or moisture, unless otherwise
specified. All chemicals and solvents were used as received. RuCl3·
xH2O was purchased from both Precious Metals Online. [(η6-
benzene)RuCl2]2, [(η6-toluene)RuCl2]2, [(η6-cymene)RuCl2]2, [(η6-
hexamethylbenzene)RuCl2]2, and [(η6-1,3,5-triisopropylbenzene)-
RuCl2]2 were synthesized according to previously reported proto-
cols.31,55−57 All other chemicals used were purchased from Sigma-
Aldrich (Singapore). Streptomycin and thiazolyl blue tetrazolium
bromide (MTT) were obtained from Sigma Chemical Co (St. Louis,
MO). PBS buffer (10×, pH 7.4) was purchased from Vivantis Inc..
Hyclone RPMI 1640, DMEM medium, fetal bovine serum (FBS), and
Pierce ECL Western blotting substrate were purchased from Thermo
Fisher Scientific Inc. (Logan, UT). Annexin V-EGFP apoptosis
detection kit (ab14153) was purchased from Abcam (Singapore).
Calbiochem anti-human p53 (pAb421) was purchased from Millipore
(Molsheim, France), and anti-actin was kindly provided by Dr. Aunis
(Strasbourg, France). Ultrapure water used was purified by a Milli-Q
UV purification system (Sartorius Stedim Biotech SA, Aubagne Cedex,
France). The human ovarian carcinoma cells A2780 and A2780cisR
were received with gratitude from Prof. Paul Dyson (EPFL). The
human breast adenocarcinoma cells MCF7, human colorectal
carcinoma cells HCT116, human colorectal adenocarcinoma cells
SW480, and human gastric cancer cells AGS were acquired from
Previous studies have shown that cytotoxic Ru−arene
complexes that target DNA are activated by aquation52−54 in
a similar fashion to cisplatin. Hydrolysis of the chloride ligands
yields a reactive aqua species that binds DNA, forming metal−
DNA adducts that directly interfere with cell proliferation. An
extensive study was carried out on the mechanism of aquation
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ATCC (Manassa,VA). The wild-type human foreskin fibroblast cells
BJwt and the p53-knockdown cells BJshp53 were generously provided
by Prof. David Leong Tai Wei (NUS). Final compounds tested for
biological activities have been shown to be >95% pure either by
elemental analysis or HPLC analysis.
NMR and ESI-MS without any workup or purification steps (1H NMR
and ESI-MS data in SI).
Studies on the Effects of Sequential Reagent Addition on
3CA. 3CA was performed sequentially in two separate steps using
[(η6-cymene)RuCl2]2 (10 μmol), 2-picolinaldehyde (20 μmol), and 4-
aminobenzamidine dihydrochloride (20 μmol). Briefly, two reagent
components were vigorously shaken in D2O (1 mL) at rt for 24 h,
before the third component was added. The reaction mixture was
analyzed by 1H NMR following 24 h after addition of the third reagent
component (1H NMR data in SI).
1
Instrumentation. H NMR spectra were obtained using either a
Bruker AMX 300, Avance 400, or AMX 500 spectrometer and the
chemical shifts (δ) are reported in parts per million with reference to
residual solvent peaks. Electrospray-ionization mass spectrometry
(ESI-MS) spectra were obtained using a Thermo Finnigan MAT ESI-
MS System. UV−vis spectra were obtained using the Shimadzu UV-
1800 UV spectrophotometer with a TCC-240A temperate controlled
cell holder. Ru concentrations were determined using the Optima ICP-
OES (PerkinElmer) operated by CMMAC, NUS. Elemental analyses
of selected Ru complexes were carried out using a PerkinElmer PE
2400 elemental analyzer by CMMAS, NUS. Cells were analyzed using
a BD LSRFortressa cell analyzer.
HPLC Analysis of Compound Purity. Determination of the
purity of a sample batch of seven RAS complexes was done using
analytical HPLC on a Shimadzu Prominence system equipped with a
DGU-20A3 degasser, two LC-20AD liquid chromatography pumps, a
SPD-20A UV/vis detector, and a Shim Pack GVP-ODS 2.0 mm 18
column (5 μM, 120 Å, 250 mm × 4.60 mm i.d.) at rt at a flow rate of
1.0 mL/min with detection at 254 nm. The gradient elution condition
were as follows: 10−30% solvent B for first 10 min, followed by 30−
80% over the next 20 min, where solvent A is water and solvent B is
MeOH. Determination of the purity of the Ru(II) complexes 3, 5, and
6 was done using the same analytical HPLC system with the same
column at rt at a flow rate of 1.0 mL/min with detection at both 214
and 254 nm. The gradient elution conditions were as follows: 20−80%
solvent B over 30 min, where solvent A is 10 mM NH4OAC pH 7.0
and solvent B is CH3CN.
Solid-State Structural Determination. X-ray diffraction data
were collected with a Bruker AXS SMART APEX diffractometer using
Mo Kα radiation at 223(2) K with the SMART suite of programs.58
Data were processed and corrected for Lorentz and polarization effects
using SAINT software59 and for absorption effects using the SADABS
software.60 Structural solution and refinement were then carried out
using the SHELXTL suite of programs.61 The structure was solved by
direct methods. Non-hydrogen atoms were located using difference
maps and were given anisotropic displacement parameters in the final
refinement. All H atoms were put at calculated positions using the
riding model.
General Procedure for the 450-Compound Library Synthesis
on a Plate for Screening. Separate stock solutions containing A (10
mM), B (40 mM), and C (40 mM) were prepared in DMSO. The
reactions were then carried out on a Microlon 200 96-well flat-bottom
plate (Greiner Bio-One) with A (50 μL), B (25 μL), and C (25 μL)
and ddH2O (100 μL) added to each well in one portion, yielding RAS
complexes (5 mM) in 1:1 v/v DMSO/H2O (200 μL). Wells
containing 1:1 v/v DMSO/H2O were included as controls for
subsequent experiments. The plates were sealed and incubated with
shaking at rt for 48 h. This procedure was repeated until all
combinations of the three components were achieved. Only one out of
the 450 products was insoluble and was eliminated from later
screening. The remaining 449 products were analyzed by ESI-MS.
Synthesis of 2-Quinolinylmethylene-4-aminophenol (L1). 2-
Quinolinecarboxaldehyde (157 mg, 1 mmol) and 4-aminophenol (109
mg, 1 mmol) were added to MeOH (50 mL) and refluxed for 2 h. The
reaction mixture was reduced to 1/10 of its original volume and cooled
in an ice bath. The resulting crystalline precipitate that formed was
separated by suction filtration, washed with cold methanol (3 × 10
mL), and dried in vacuo for 1 h. Yield: 136 mg (54%). 1H NMR (400
MHz, DMSO-d6): δ 9.72 (s, 1H, OH), 8.79 (s, 1H, PhNCH), 8.46
(d, 3JHH = 9 Hz, 1H, quinolinyl), 8.27 (d, 3JHH = 9 Hz, 1H, quinolinyl),
3
3
8.10 (d, JHH = 8 Hz, 1H, quinolinyl), 8.05 (d, JHH = 8 Hz, 1H,
quinolinyl), 7.83 (t, 3JHH = 8 Hz, 1H, quinolinyl), 7.67 (t, 3JHH = 7 Hz,
1H, quinolinyl), 7.40 (d, 3JHH = 9 Hz, 2H, CH4OH), 6.86 (d, 3JHH = 9
Hz, 2H, CH4OH) ppm. ESI-MS: (+ mode) m/z = 249 [M + H]+, (−
mode) m/z = 247 [M − H]−.
Synthesis of N-(2-Pyridylmethylene)-1-naphthylamine (L2).
2-Picolinaldehyde (95.1 μL, 1 mmol) and 1-naphthylamine (143 mg, 1
mmol) were added to dry EtOH (15 mL) and stirred at rt over 72 h.
The solvent was removed in vacuo and dried. The final product was
used for the next step without any further purification. 1H NMR (400
3
MHz, CDCl3): δ 8.75 (d, JHH = 4 Hz, 1H, py), 8.71 (s, 1H, PhN
General Procedure for the Synthesis of RAS Complexes. A
8.16 μmol portion of A (Ru−arene dimer), 16.3 μmol of B
(picolinaldehyde analogues), and 16.3 μmol of C (anilines derivatives)
were added in one portion and suspended in 1 mL of D2O in a 2 mL
graduated microtube with vigorous shaking at rt for 36 h. The
sparingly soluble reactants dissolved at the end of the reaction, giving
colored solutions. This procedure was repeated with different
combinations of the three components. Not all combinations were
attempted and some of the attempted combinations gave insoluble
suspensions, which were excluded from the library. An initial library of
48 RAS complexes was synthesized, and they were characterized
immediately by 1H NMR and ESI-MS without any workup or
purification steps. Distinctive signals corresponding to the expected
CH), 8.41 (d, 3JHH = 8 Hz, 1H, naphthyl), 8.36 (m, 1H, py), 7.88 (m,
1H, py), 7.87 (d, 3JHH = 7 Hz, 1H, naphthyl), 7.77 (t, 3JHH = 8 Hz, 1H,
3
naphthyl), 7.53 (m, 2H, naphthyl), 7.48 (d, JHH = 8 Hz, 1H,
naphthyl), 7.41 (m, 1H, py), 7.15 (d, 3JHH = 7 Hz, 1H, naphthyl) ppm.
ESI-MS: (+ mode) m/z = 233 [M + H]+, 255 [M + Na]+, 487 [2M +
Na]+.
Synthesis of 4-Methoxy-N-(2-quinolinylmethylene)aniline
(L3). 2-Quinolinecarboxaldehyde (157 mg, 1 mmol) and p-anisidine
(123 mg, 1 mmol) were added to a minimal amount of dry EtOH (5
mL) and stirred at rt for 72 h. The solvent was removed in vacuo and
dried. The resulting crude yellowish brown solid was used for the next
step without any further purification. 1H NMR (500 MHz, CDCl3): δ
3
8.82 (s, 1H, PhNCH), 8.37 (d, JHH = 8 Hz, 1H, quinolinyl), 8.24
(d, 3JHH = 8 Hz, 1H, quinolinyl), 8.16 (d, 3JHH = 9 Hz, 1H, quinolinyl),
1
product were observed in the H NMR spectrum, in addition to a
3
3
relatively small amount of impurities (1H NMR and ESI-MS data in
SI).
7.87 (d, JHH = 8 Hz, 1H, quinolinyl), 7.76 (t, JHH = 7 Hz, 1H,
quinolinyl), 7.60 (t, 3JHH = 7 Hz, 1H, quinolinyl), 7.41 (d, 3JHH = 9 Hz,
3
2H, CH4OMe), 6.98 (d, JHH = 9 Hz, 2H, CH4OMe) ppm.
Studies on the Effects of Solvent on 3CA. 3CA was carried out
in five different solvent systems (1 mL), namely, DMSO-d6, MeOD-d4,
CDCl3, D2O, and 1:1 v/v DMSO-d6/D2O, using [(η6-cymene)RuCl2]2
(10 μmol), 2-picolinaldehyde (20 μmol), and aniline (20 μmol) with
vigorous shaking at rt for 24 h. Reaction mixtures were characterized
Synthesis of [(η6-Benzene)RuCl(L1)]Cl (1). [(η6-Benzene)-
RuCl2]2 (50.4 mg, 0.101 mmol) and L1 (50.0 mg, 0.202 mmol)
were added to MeOH (25 mL) and stirred at rt over 12 h. The solvent
was removed in vacuo and the resulting solid was washed with ether (3
× 10 mL). The final product was dried in vacuo for 1 h to yield a
1
immediately by H NMR without any workup or purification steps.
1
Studies on the Effects of pH on 3CA. [(η6-cymene)RuCl2]2 (10
μmol), 2-picolinaldehyde (20 μmol), and aniline (20 μmol) of were
suspended in either NaOAc/DOAc/D2O (1 mL, pH 4.5) or ND4Cl/
ND4OD/D2O buffer (1 mL, pH 10.0) with vigorous shaking at rt for
orange solid. Yield: 98.0 mg (98%). H NMR (400 MHz, MeOH): δ
3
8.91 (s, 1H, PhNCH), 8.88 (d, JHH = 9 Hz, 1H, quinolinyl), 8.77
(d, 3JHH = 8 Hz, 1H, quinolinyl), 8.21 (d, 3JHH = 8 Hz, 2H, quinolinyl),
3
3
8.15 (t, JHH = 8 Hz, 1H, quinolinyl), 7.96 (t, JHH = 8 Hz, 1H,
1
24 h. The reaction mixtures were characterized immediately by H
quinolinyl), 7.85 (d, 3JHH = 9 Hz, 2H, CH4OH), 7.02 (d, 3JHH = 9 Hz,
M
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2H, CH4OH), 5.94 (s, 6H, C6H6) ppm. ESI-MS: (+ mode) m/z = 463
[M]+. Purity of the complex was determined to be >95% pure by
elemental analysis. Anal. Calcd for RuC22H18N2Cl2O (%): C 53.02, H
3.64, N 5.62. Found: C 52.81, H 3.44, N 5.62.
[M]+. Purity (HPLC): 97.4% at 214 nm and 95.6% at 254 nm; tR =
20.3 min. Purity of the complex was determined to be >95% pure by
HPLC.
Synthesis of [(η6-Hexamethylbenzene)RuCl(N-(2-quinolinyl-
methylene)-8-hydroxy-1-napthalenamine)]Cl (6). [(η6-
Hexamethylbenzene)RuCl2]2 (66.85 mg, 0.1 mmol), 2-quinolinecar-
boxaldehyde (31.4 mg, 0.1 mmol), and 8-hydroxy-1-napthalenamine
(31.8 mg, 0.1 mmol) were added to 1:1 v/v DMSO/H2O (10 mL)
and stirred at rt over 48 h. MeOH was then added to the resulting
suspension until a clear dark red solution was formed. The solution
was left to stir at rt for 24 h. The solvent mixture was removed in
vacuo and the resulting solid purified by gradient elution column
chromatography (1:4 v/v EtOH/CHCl3, Rf = 0.2; 1:1 v/v EtOH/
CHCl3, Rf = 0.6). The final product was dried in vacuo for 1 h to give a
dark red solid. Yield: 86.0 mg (69%). 1H NMR (300 MHz, MeOD): δ
9.11 (s, 1H, PhNCH), 8.75 (d, 3JHH = 8 Hz, 1H), 8.53 (d, 3JHH = 9
Synthesis of [(η6-Toluene)RuCl(L1)]Cl (2). Complex 2 was
obtained with [(η6-toluene)RuCl2]2 (53.2 mg, 0.101 mmol) and L1
(50.0 mg, 0.202 mmol) using the same protocol as used for 1 as a red
solid. Yield: 100 mg (97%). 1H NMR (400 MHz, DMSO): δ 10.42 (s,
3
1H, OH), 9.11 (s, 1H, PhNCH), 8.87 (d, JHH = 8 Hz, 1H,
quinolinyl), 8.75 (d, 3JHH = 9 Hz, 1H, quinolinyl), 8.29 (t, 3JHH = 8 Hz,
2H, quinolinyl), 8.14 (t, 3JHH = 7 Hz, 1H, quinolinyl), 7.98 (t, 3JHH = 8
Hz, 1H, quinolinyl), 7.87 (d, 3JHH = 9 Hz, 2H, CH4OH), 7.03 (d, 3JHH
= 9 Hz, 2H, CH4OH), 6.06 (t, 3JHH = 6 Hz, 1H, C6H5), 5.98 (d, 3JHH
=
3
3
6 Hz, 1H, C6H5), 5.81 (t, JHH = 6 Hz, 1H, C6H5), 5.72 (t, JHH = 6
3
Hz, 1H, C6H5), 5.39 (d, JHH = 8 Hz, 1H, C6H5), 2.16 (s, 3H, CH3)
ppm. ESI-MS: (+ mode) m/z = 477 [M]+. Purity of the complex was
determined to be >95% pure by elemental analysis. Anal. Calcd for
RuC23H20N2Cl2O (%): C 53.91, H 3.93, N 5.47. Found: C 53.57, H
4.23, N 5.27.
3
3
Hz, 1H), 8.48 (d, JHH = 8 Hz, 1H), 8.44 (bs, 1H), 8.29 (d, JHH = 8
3
3
Hz, 1H), 8.25 (d, JHH = 8 Hz, 1H), 8.14 (m, 1H), 7.98 (t, JHH = 7
Hz, 2H), 7.62 (t, 3JHH = 8 Hz, 2H), 7.05 (d, 3JHH = 8 Hz, 1H), 1.83 (s,
18H) ppm. ESI-MS: (+ mode) m/z = 565 [M]+. Purity (HPLC):
96.1% at 214 nm and 95.1% at 254 nm; tR = 19.5 min. Purity of the
complex was determined to be >95% pure by HPLC.
Synthesis of [(η6-1,3,5-Triisopropybenzene)RuCl(L2)]Cl (3).
[(η6-1,3,5-Triisopropylbenzene)RuCl2]2 (87.1 mg, 0.108 mmol) and
L2 (50.0 mg, 0.215 mmol) were added to MeOH (15 mL) and stirred
at rt over 12 h. The solvent was removed in vacuo and the resulting
solid was washed with ether (3 × 10 mL). The final product was dried
in vacuo for 1 h to give a yellow solid. Yield: 126 mg (96%). 1H NMR
Tissue Culture. The human ovarian carcinoma cells A2780 and
A2780cisR and gastric adenocarcinoma cells AGS were cultured in
RPMI 1640 medium containing 10% fetal bovine serum (complete
RPMI) and grown at 37 °C in a humidified atmosphere of 95% air and
5% CO2. Cisplatin (1 μM) was added to A2780cisR every three
passages to maintain its resistance. The human breast carcinoma cells
MCF7 and colon cancer cell lines HCT116 and SW480 were cultured
in DMEM medium containing 10% fetal bovine serum (complete
DMEM) and grown at 37 °C in a humidified atmosphere of 95% air
and 5% CO2. The medium used for the colon cancer cell lines also
contained 1% streptomycin. Experiments were performed on cells
within 20 passages.
Inhibition of Cell Viability Assay (Single Concentration
Screen). The antiproliferation activity of the 442 library of RAS
complexes on exponentially growing cancer cells was screened using a
MTT assay modified from one described previously.34 A2780 cells
were seeded at 8000 cells/100 μL per well in Cellstar 96-well plates
(Greiner Bio-One) and incubated for 24 h. PBS buffer (200 μL) was
added to the wells at the perimeter to prevent evaporation of the
media from the enclosed wells. After that, cancer cells were exposed to
the library of RAS complexes in a predetermined order, at a single
concentration of 25 μM for 6 h. The complex solutions used for the
exposure were made by serial dilution of the synthesized RAS
complexes with RPMI 1640 medium, resulting in a final concentration
of 25 μM with 0.5% (v/v) DMSO. After the 6 h, the drug-containing
medium was removed, new drug-free complete RPMI was added, and
the cells were incubated for additional 66 h. At 72 h after drug
addition, the medium was removed and replaced with MTT solution
in PBS (100 μL, 0.5 mg/mL) and incubated for an additional 4 h at 37
°C, 5% CO2. Subsequently, the medium was aspirated, and the purple
formazan crystals were dissolved in DMSO (100 μL). The absorbance
due to the dissolved purple formazan was then obtained at 595 nm. A
negative control (cancer cells not exposed to any RAS complexes) and
a positive control (cancer cells exposed to cisplatin) were also included
in the experiment. The experiments were performed in quintuplicate
for each RAS complex and control, and the cell viabilities associated
with each RAS complex were calculated using the measured
absorbance value. The cell viability of these complexes was further
analyzed using the software Prism Graphpad 5.
3
(500 MHz, CDCl3, 70 °C): δ 9.59 (d, JHH = 4 Hz, 1H, py), 9.02 (s,
1H, PhNCH), 8.35 (m, 2H, py), 8.15 (bs, 1H, naphthyl), 8.12 (d,
3JHH = 8 Hz, 1H, naphthyl), 7.95 (m, 1H, py), 7.71 (bs, 5H, naphthyl),
3
5.85 (s, 3H, C6H3), 2.19 (sept, JHH = 7 Hz, 3H, CHMe2), 1.06 (d,
3JHH = 7 Hz, 9H, C(CH3)2), 0.93 (bs, 9H, C(CH3)2) ppm. ESI-MS: (+
mode) m/z = 573 [M]+. Purity (HPLC): 95.5% at 214 nm and 92.4%
at 254 nm; tR = 25.6 min. Purity of the complex was determined to be
>95% pure by HPLC.
Synthesis of [(η6-1,3,5-Triisopropybenzene)RuCl(L3)]Cl (4).
[(η6-1,3,5-Triisopropylbenzene)RuCl2]2 (244 mg, 0.324 mmol) and
crude L3 (167 mg, 0.648 mmol) were added to MeOH (40 mL) and
stirred at rt over 12 h. The solvent was removed in vacuo and
reconstituted in a minimum amount of MeOH (2 mL). Diethyl ether
(40 mL) was than added to precipitate the product. The suspension
was filtered and the resulting solid washed with ether (3 × 10 mL).
The final product was then dried in vacuo for 1 h to give a reddish
1
brown solid. Yield: 290 mg (71%). H NMR (400 MHz, DMSO): δ
3
9.09 (s, 1H, PhNCH), 8.96 (d, JHH = 9 Hz, 1H, quinolinyl), 8.85
(d, 3JHH = 8 Hz, 1H, quinolinyl), 8.25 (d, 3JHH = 8 Hz, 2H, quinolinyl),
8.12 (m, 1H, quinolinyl), 8.11 (d, 3JHH = 9 Hz, 2H, CH4OMe), 7.97 (t,
3
3JHH = 8 Hz, 1H, quinolinyl), 7.19 (d, JHH = 9 Hz, 2H, CH4OMe),
3
5.54 (s, 3H, C6H3), 3.90 (s, 3H, CH3), 2.44 (sept, JHH = 7 Hz, 3H,
3
3
CHMe2), 1.16 (d, JHH = 7 Hz, 9H, C(CH3)2), 0.85 (d, JHH = 7 Hz,
9H, C(CH3)2) ppm. ESI-MS: (+ mode) m/z = 603 [M]+. Purity of the
complex was determined to be >95% pure by elemental analysis. Anal.
Calcd for RuC32H38N2Cl2O (%): C 60.16, H 6.00, N 4.39. Found: C
60.26, H 5.61, N 4.45.
Synthesis of [(η6-Hexamethylbenzene)RuCl(3-chloro-N-(2-
quinolinylmethylene)aniline)]Cl (5). 2-Quinolinecarboxaldehyde
(78.6 mg, 0.5 mmol) and 3-chloroaniline (52.9 μL, 0.5 mmol) were
added to dry EtOH (8 mL) and stirred at rt over 72 h. The solvent was
removed in vacuo and dried. Crude 3-chloro-N-(2-
quinolinylmethylene)aniline (135 mg) was treated with [(η6-
hexamethylbenzene)RuCl2]2 (167 mg, 0.25 mmol) in MeOH (20
mL) and stirred at rt over 12 h. The solvent was removed in vacuo and
the resulting solid purified by gradient elution column chromatography
(1:4 v/v EtOH/CHCl3, Rf = 0.2; 1:1 v/v EtOH/CHCl3, Rf = 0.6). The
final product was then dried in vacuo for 1 h to give a red solid. Yield:
Aqueous Stability Studies. Stock solutions of 1−6 (5 mM) were
each diluted with ddH2O to a final concentration of 50 μM with 1%
DMSO. The UV−vis peak profile of the sample was then monitored
for 24 at 1 h intervals. The temperature was kept constant at 25 °C
throughout and ddH2O with 1% DMSO was used as the blank sample.
Determination of Thermodynamic Aqueous Solubility.
Approximately 5 mg of 1−5 was added separately to a minimal
amount of ddH2O until a small amount of each compound was left in
a saturated solution of the respective compounds. The separate
1
182 mg (61%). H NMR (400 MHz, CDCl3): δ 9.37 (s, 1H, PhN
3
3
CH), 8.68 (d, J= 8 Hz, 1H, quinolinyl), 8.52 (d, JHH = 9 Hz, 1H,
3
3
quinolinyl), 8.48 (d, JHH = 8 Hz, 1H, quinolinyl), 8.40 (d, JHH = 8
3
Hz, 1H, quinolinyl), 8.34 (s, 1H, C6H4), 7.99 (d, JHH = 8 Hz, 1H,
C6H4), 7.88 (t, 3JHH = 7 Hz, 1H, quinolinyl), 7.79 (t, 3JHH = 7 Hz, 1H,
quinolinyl), 7.54 (t, 3JHH = 8 Hz, 1H, C6H4), 7.44 (d, 3JHH = 8 Hz, 1H,
C6H4), 1.90 (s, 18H, C6(CH3)6) ppm. ESI-MS: (+ mode) m/z = 565
N
dx.doi.org/10.1021/jm500455p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
mixtures were than left to stand with gentle agitation at rt for 24 h.
The individual mixtures were than centrifuged at 10 000 rpm for 2
min, and the supernatant liquid was collected and analyzed by ICP-
OES to determine the maximum solubility of each compound. The
same procedure was repeated for 6 but with 0.6% w/v NaCl solution
instead of ddH2O.
fluorescence was immediately analyzed by flow cytometry. The
resulting dot plots were acquired from 10 000 events and quantified
by software BD FACSDiva. The experiment was performed
independently twice.
Protein Extraction and Western Blot for p53. HCT116 cells
were grown on Cellstar six-well plates (Greiner Bio-One) and treated
with 4 at concentrations of 1 μM (IC50), 2.5 μM (IC25), and 10 μM
and at 2.5 and 10 μM for the control drug oxaliplatin and incubated at
37 °C, 5% CO2. Different sets of cells were treated for different
durations of 2, 6, 12, and 24 h. The cells were lysed with lysis buffer
[100 μL, 1% NP40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0),
protease inhibitor]. The cell lysate were transferred to separate 2 mL
tubes and sonicated for 10 s. The samples were then centrifuged at 13
000 rpm, 4 °C for 15 min. The supernatant liquid containing the
proteins was collected and the total protein content of each sample
was quantified via Bradford’s assay. Thirty micrograms of proteins
from each sample was reconstituted in loading buffer (5% DDT, 1×
protein loading dye) and heated at 95 °C for 5 min. The protein
mixtures were resolved on a 10% SDS−PAGE gel by electrophoresis
and transferred to a nitrocellulose membrane. Equal loading of protein
was confirmed by comparison with actin. Immunoblotting for p53 was
done using anti-human p53 antibodies. The proteins bands were
visualized via enhanced chemiluminescence imaging (PXi, Syngene).
Determination of log POW. log POW of 1−6 were determined
using the shake flask method.38 1−6 were predissolved in ddH2O that
was presaturated with n-octanol (for 24 h and left to stand until phase
separation occurs). The UV−vis spectrum for each sample was
obtained, and the absorbances at the λmax of each compound were
determined. An equal volume of n-octanol was added to each sample
solution, and the heterogeneous mixtures were shook for 96 h before
centrifuging at 7000 rpm for 1 min to achieve phase separation. The
final absorbance of the aqueous phase at the λmax of each compound
was determined, and their water−octanol partition coefficients were
calculated. The experiment was repeated for 6 in 0.6% w/v NaCl
solution instead of ddH2O. All experiments were done in triplicate.
Inhibition of Cell Viability Assay. The antiproliferation activity
of 1−6 on exponentially growing cancer cells was determined using
MTT assay as described previously.34 A2780 cells, A2780cisR, and
MCF7 were seeded at 8000, 6000, and 4000 cells, respectively, per
well (100 μL) in Cellstar 96-well plates (Greiner Bio-One) and
incubated for 24 h. PBS buffer (200 μL) was added to the wells at the
perimeter to prevent evaporation of the media from the enclosed wells.
Thereafter, cancer cells were exposed to drugs at different
concentration in RPMI medium (A2780/A2780cisR) or DMEM
medium (MCF7) without FBS or antibiotics and incubated at 37 °C,
5% CO2 for 6 h. The compounds were predissolved as DMSO stock
solutions and serially diluted with DMSO to a range of concentrations.
The final concentration of DMSO in medium was 1% (v/v), at which
cell viability was not significantly inhibited. After the 6 h, the drug-
containing medium was removed, and new drug-free complete RPMI
(A2780/A2780cisR) or DMEM (MCF7) was added, and the cells
were incubated for an additional 66 h. At 72 h after initial drug
addition, the medium was removed and replaced with MTT solution
(100 μL, 0.5 mg/mL) in PBS and incubated for an additional 4 h at 37
°C, 5% CO2. Subsequently, the medium was aspirated, and the purple
formazan crystals were dissolved in DMSO (100 μL). The absorbance
due to the dissolved purple formazan was than obtained at 595 nm.
The experiments were performed in six replicates for each drug
concentration and were carried out at least three times independently.
Inhibition to cell viability was evaluated with reference to the IC50
value, which is defined as the concentration needed for a 50%
reduction of survival based on the survival curves. IC50 values were
calculated from the dose−response curves (cell viability vs drug
concentration) obtained in repeated experiments and adjusted to the
actual Ru concentration administered, which was determined using
ICP-OES. For HCT116 and SW480 cells, the protocol was applied as
before except that cells were plated at 20 000 cells per well (100 μL) in
Cellstar 96-well plates (Greiner Bio-One) for 24 h and exposed to
varying concentration of RAS complexes continuously for 48 h in
DMEM complete medium containing <0.5% v/v DMSO. Similarly for
BJwt and BJshp53, cells were plated at 2000 cells per well (100 μL) in
Cellstar 96-well plates (Greiner Bio-One) for 24 h and then incubated
with varying concentration of 4 and DOX continuously for 48 h in
medium containing <0.8% v/v DMSO. The experiments were
performed in eight replicates for each drug concentration and were
carried out at least three times independently.
ASSOCIATED CONTENT
* Supporting Information
■
S
1H NMR and UV−vis spectra for lead compounds 1−6, UV−
vis and ESI-MS data for 4 with dGMP and GSH, 1H NMR data
and ESI-MS data of 63 compounds from the initial library,
HPLC chromatogram of selected RAS complexes, and X-ray
crystallographic data in CIF format for [RAS-A3/B1/C8]PF6.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: +65 6516 5131. E-mail: chmawh@nus.edu.sg.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Financial support from Ministry of Education and the National
University of Singapore (R143-000-512-112 and R143-000-
572-112) and Ligue contre le Cancer, COST CM1105 is
gratefully acknowledged. The authors thanks Paul J. Dyson
(EPFL) and David T. W. Leong (NUS) for their gift of cell
lines; Lip Lin Koh (NUS), Geok Kheng Tan (NUS), and
Yimian Hong (NUS) for performing X-ray crystallographic
analysis; and Ong Wei Fang Wendy for helping with flow
cytometry analysis.
Annexin V/Propidium Iodide Assay. The ability of 4 to induce
apoptosis in HCT116 cells was investigated via the annexin V/
propidium iodide assay previously described.62 HCT116 cells were
seeded at 200 000 cells per well (1 mL) in Cellstar 12-well plates
(Greiner Bio-One) and incubated for 24 h. Thereafter, cancer cells
were exposed to 2.5 and 10 μM of 4 in complete DMEM medium and
incubated at 37 °C, 5% CO2 for 24 h. The supernatant solution and
cells detached by trypsinization were collected in separate vials and
centrifuged at 100 rpm for 10 min to pellet the cells. Cells were
resuspended in 500 μL of binding buffer containing annexin V−EGFP
and propidium iodide and of final concentration 1 μg/mL. The
ABBREVIATIONS USED
■
D2O, deuterium oxide; NMR, nuclear magnetic resonance; ESI-
MS, electrospray ionization-mass spectrometry; RP-HPLC,
reverse-phase high-performance liquid chromatography;
DMSO-d6, deuterated dimethyl sulfoxide; MeOH-d4, deuter-
ated methanol; ddH2O, ultrapure water; ICP-OES, inductively
couple-optical emission spectrometry; GSH, L-glutathione;
dGMP, 2′-deoxyguanosine 5′-monophosphate sodium salt
hydrate.
O
dx.doi.org/10.1021/jm500455p | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
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Chemotherapy. J. Med. Chem. 2007, 50, 6343−6355.
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