Novel bis-C,N-cyclometalated iridium(III) thiosemicarbazide antitumor complexes: Interactions with human serum albumin and DNA, and inhibition of cathepsin B

Article abstract of DOI:10.1021/ic302219v

A series of new organoiridium(III) complexes [Ir(N-C)₂(N-S)]Cl (HN-C = 2-phenylpyridine (Hppy), N-S = methyl thiosemicarbazide (1), phenyl thiosemicarbazide (2) and naphtyl thiosemicarbazide (3)) have been synthesized and characterized. The crystal structure of (1) has been established by X-ray diffraction, showing the thiosemicarbazide ligand bound to the iridium atom as N,S-chelate. The cytotoxicity studies show that they are more active than cisplatin (about 5-fold) in T47D (breast cancer) at 48 h incubation time. On the other hand, very low resistance factors (RF) of 1-3 in A2780cisR (cisplatin-resistant ovarian carcinoma) at 48 h were observed (RF ≈ 1). Ir accumulation in T47D cell line after 48 h continuous exposure for complexes 1-3 are higher than that corresponding to cisplatin (about 10 times). The complexes 1-3 bind strongly to HSA with binding constants of about 10⁴ M ^-1 at 296 K, binding occurring at the warfarin site I for 2. Complexes 2 and 3 are also capable of binding in the minor groove of DNA as shown by Hoechst 33258 displacement experiments. Furthermore, complex 2 is also a good cathepsin B inhibitor (an enzyme implicated in a number of cancer related events), being the enzyme reactivated by cysteine.

Full text of DOI:10.1021/ic302219v

Article
pubs.acs.org/IC
Novel Bis-C,N-Cyclometalated Iridium(III) Thiosemicarbazide
Antitumor Complexes: Interactions with Human Serum Albumin and
DNA, and Inhibition of Cathepsin B
Jose Ruiz,*,† Consuelo Vicente,† Concepcion de Haro,† and Delia Bautista‡
́
́
†Departamento de Química Inorgan
́
ica and Regional Campus of International Excellence “Campus Mare Nostrum”, Universidad de
́
Murcia and Instituto Murciano de Investigacion Biosanitaria (IMIB), E-30071- Murcia, Spain
‡SAI, Universidad de Murcia, E-30100-Murcia, Spain
S
* Supporting Information
ABSTRACT: A series of new organoiridium(III) complexes [Ir(N−C)2(N−S)]Cl (HN−C =
2-phenylpyridine (Hppy), N−S = methyl thiosemicarbazide (1), phenyl thiosemicarbazide (2)
and naphtyl thiosemicarbazide (3)) have been synthesized and characterized. The crystal
structure of (1) has been established by X-ray diffraction, showing the thiosemicarbazide ligand
bound to the iridium atom as N,S-chelate. The cytotoxicity studies show that they are more
active than cisplatin (about 5-fold) in T47D (breast cancer) at 48 h incubation time. On the
other hand, very low resistance factors (RF) of 1−3 in A2780cisR (cisplatin-resistant ovarian
carcinoma) at 48 h were observed (RF ≈ 1). Ir accumulation in T47D cell line after 48 h
continuous exposure for complexes 1−3 are higher than that corresponding to cisplatin (about
10 times). The complexes 1−3 bind strongly to HSA with binding constants of about 104 M−1
at 296 K, binding occurring at the warfarin site I for 2. Complexes 2 and 3 are also capable of
binding in the minor groove of DNA as shown by Hoechst 33258 displacement experiments.
Furthermore, complex 2 is also a good cathepsin B inhibitor (an enzyme implicated in a
number of cancer related events), being the enzyme reactivated by cysteine.
highly cytotoxic toward HeLa cells.20 Furthemore, the synthesis
INTRODUCTION
■
of some cyclometalated pyridylnaphthalimide iridium(III)
complexes as protein kinase inhibitors has been also recently
reported.21 A number of C,N-cyclometalated antitumor
compounds of Ru, Rh, Ir, and Pt have been prepared recently
by our group.22−26
We report herein the synthesis and cytotoxic activity of a
series of new nonconventional organoiridium(III) complexes of
the type [Ir(N−C)2(N−S)]Cl (HN−C = 2-phenylpyridine
(Hppy), N−S = thiosemicarbazide). Values of IC50 were
studied for the new organoiridium(III) complexes against a
panel of human tumor cell lines representative of ovarian
(A2780 and A2780cisR), and breast cancer (T47D, cisplatin
resistant). The interaction of the new Ir(III) thiosemicarbazide
complexes toward human serum albumin (HSA, which is a
transport carrier for drugs) and DNA, and also their behavior as
cathepsin B inhibitors (a type of enzymes that are overex-
pressed in many cancer cell lines) have also been studied.
Cisplatin is one of the antitumoral agents most used in clinical
therapy.1−6 However, it has a high general toxicity, and some
tumors develop resistance to the drug. This limits the use of
cisplatin to the treatment of ovarian, head and neck, bladder,
and testicular tumors. For these reasons, the potential of other
transition metal-based drugs as anticancer agents is being
actively explored.7−13
Organometallic complexes offer enormous scope for the
design of anticancer drug candidates, in spite of such
compounds having long been considered to be unstable
under physiological conditions. Thus, investigations of the
cytotoxic properties of arene ruthenium(II) complexes such as
[Ru(η6-arene)Cl2(PTA)] (RAPTA; PTA = 1,3,5-triaza-7-
phosphatricyclo-[3.3.1.1]decane) and [Ru(η6-arene)(en)Cl]+
(en = ethylenediamine) have established promising anticancer
activity.12−16 On the other hand, Sadler et al. have recently
found that replacement of the neutral N,N-bound chelating
ligand bpy by the negatively charged C,N-bound 2-phenyl-
pyridine (ppy) ligand in an iridium(III) chlorido complex can
also switch on biological activity.17,18 In general iridium(III)
complexes are usually thought to be relatively inert and can be
used as biomolecular and cellular probes.19 Thus for example,
to design new biological probes for bovine serum albumin
(BSA), Lo et al. prepared a series of luminescent cyclo-
metalated Ir(III) complexes containing an indole derivative
(indole is known to bind to BSA) which were found to be
RESULTS AND DISCUSSION
■
Synthesis of the New Iridium Compounds [Ir(N−
C)2(N−S)]Cl. The organoiridium(III) complexes were prepared
from the reaction of [Ir2(N−C)4Cl2] (HN−C = Hppy) with
the corresponding thiosemicarbazide in 1:2 molar ratio in a
Received: October 11, 2012
© XXXX American Chemical Society
A
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mixture of EtOH and CH2Cl2 (Chart 1). All the complexes
were characterized by 1H NMR spectroscopy, positive-ion ESI-
Chart 1
Figure 2. Schematic showing weak S···H−C contacts in 1.
MS, and IR spectroscopy and gave satisfactory elemental
analyses. The assignments were confirmed by COSY, NOESY,
DEPT, and HSQC. No attempts to isolate enantiomerically
pure complexes 1−3 have been undertaken (Ir(III) center
being stereogenic).
X-ray Crystal Structure of Complex 1. The crystal
structure of 1 has been established by X-ray diffraction (Figure
1). The iridium(III) center adopts a distorted octahedral
Table 1. IC50(μM) and Resistance Factors for Cisplatin, and
Compounds 1−3
a
complex
T47D 48 h
15
A2780 48 h
11
A2780cisR 48 h (RF)
1
1
1
9.27 0.20 (0.84)
4.38 0.19 (0.9)
5.71 0.06 (1.04)
16 1 (10.39)
2
9.95 0.04
9.93 0.07
4.93 0.02
5.50 0.07
1.54 0.11
3
cisplatin
53
3
a
The numbers in parentheses are the resistance factors RF (IC50
resistant/IC50 sensitive).
resistant) and epithelial ovarian carcinoma cells A2780 and
A2780cisR (acquired resistance to cisplatin). Because of low
aqueous solubility of 1−3, the test compounds were dissolved
in DMSO first and then serially diluted in complete culture
medium such that the effective DMSO content did not exceed
1%. Complexes 1−3 were more active than cisplatin in T47D
(about 5-fold).
On the other hand, A2780cisR encompasses all of the known
major mechanisms of resistance to cisplatin: reduced drug
transport,31 enhanced DNA repair/tolerance,32 and elevated
GSH levels.33 The ability of complexes 1−3 to circumvent
cisplatin acquired resistance was determined from the resistance
factor (RF) defined as the ratio of IC50 resistant line to IC50
parent line, very low RF values being observed at 48 h (RF =
0.9−1.0, Table 1), an RF of <2 was considered to denote
noncross-resistance.34 The IC50 value for the free thiosemi-
carbazide ligands was higher than 100 μM in all the cancer cell
lines studied.
Figure 1. ORTEP plot (ellipsoids drawn at 50% probability level) of
complex 1. Selected bond lengths (Å) and angles (deg): Ir1−C1 =
2.025(3), Ir1−C12 = 2.013(3), Ir1−N1 = 2.055(3), Ir1−N2 =
2.043(3), Ir1−N3 = 2.180(3), Ir1−S1 = 2.4132(8), C1−Ir1−N1 =
80.05(12), C12−Ir1−N2 = 80.71(13), S1−Ir1−N3 = 81.75(18).
geometry. The thiosemicarbazide ligand binds the iridium atom
as N,S-chelate. The coordinating nitrogen atoms of the two ppy
rings are in a trans arrangement, as found typically in the crystal
structure of cyclometalated iridium complexes.27,28 The values
of the Ir−C and Ir−N bond distances of the bonded ppy
ligands are within the normal ranges expected for such
cyclometalated complexes.29,30 C−Ir−N bite angles are about
80°. The trans-influence of the carbon donors renders a longer
Ir−N bond length in the thiosemicarbazide ligand (Ir−N3 =
2.180(3) Å) than in the cyclometalating ligand. Supramolecular
interactions such as S···H−C (Figure 2), Cl···H−C and
Cl···H−N contacts are present in 1 (Supporting Information,
Figure S1).
The stability of the cytotoxic compounds 1−3 has been
tested in a mixture DMSO-d6/D2O (1:1) in the presence of an
1
excess of NaCl (100 mM) by H NMR, remaining unaltered
after 24 h in solution at 37 °C.
Iridium Cellular Accumulation Studies. We have
determined the Ir accumulation in a T47D cell line after 48
h continuous exposure with 20 μM of complexes 1−3 (Table
2), showing higher values (about 10 times) than that
corresponding to cisplatin. A direct relationship between
metal accumulation and activity has not been described
yet,35,36 and it is not clearly observed in our measurements.
Biological Assays: Gel Electrophoresis of Compound−
pBR322 Complexes. The influence of the compounds on the
tertiary structure of DNA was determined by their ability to
Biological Activity: Cytotoxicity Studies. The cytotox-
icity of the iridium(III) complexes 1−3 was evaluated (Table 1)
toward the T47D human breast cancer cell line (cisplatin
B
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Here F0 and F are the fluorescence intensity of the EB−DNA
adduct before and after the addition of the complex, KSV is the
Stern−Volmer quenching constant, and [Q] is the concen-
tration of the corresponding metal complex. The good linearity
of the Stern−Volmer plots (Supporting Information, Figure
S4) suggests a singular mode of quenching. The values for the
Table 2. Cellular Accumulation of Iridium Compounds 1−3
and Cisplatin in Human Breast Carcinoma T47D Cells
a
compound
cellular accumulation (pmol Ir/106 cells) 48 h
1
22.376 0.045
24.174 0.246
18.518 0.087
2.231 0.005
2
K
SV are given in Table 3. These values are in the 103−104 M−1
3
cisplatin
a
Drug-treatment period with 20 μM Ir complexes.
Table 3. Quenching and Binding Parameters for the
Interaction of 1−3 with ct-DNA
compound
10−3KSV (M−1
)
10−3Kapp (M−1
)
modify the electrophoretic mobility of the covalently closed
circular (ccc) and open (oc) forms of pBR322 plasmid DNA.
The compounds 1−3 were incubated at the molar ratio ri =
0.50 with pBR322 plasmid DNA at 37 °C for 24 h.
Representative gel obtained for the new compounds 1−3 are
shown in Supporting Information, Figure S2. The behavior of
the gel electrophoretic mobility of both forms, ccc and oc, of
pBR322 plasmid and DNA:cisplatin adducts is consistent with
previous reports.37 Noteworthy, the free thiosemicarbazide
ligands and their iridium(III) derivatives 1−3 do not seem to
cause important conformational changes of DNA.
Competitive Binding Experiments. To further inves-
tigate the binding mode between the new metal complexes and
DNA, fluorescence competition experiments with ethidium
bromide (EB) and Hoechst 33258 were employed. EB is a
planar cationic dye well-known to intercalate into the DNA
helix. While EB is only weakly fluorescent, the EB−DNA
adduct is a strong emitter on excitation near 520 nm.
Quenching of the fluorescence may be used to determine the
extent of the binding between the quencher (1−3) and DNA.
One reason for the quenching is the reduction in the number of
binding sites on the DNA that is available to the EB presumably
because of competition with complexes which is nonemissive
under the experimental conditions.38,39 As seen in Figure 3
1
2
3
5.47
5.49
17.5
3.33
4.05
7.72
range which makes them very weak intercalators. The apparent
binding constant for complexes was calculated using the eq 2.
Kapp = KEB[EB]/[Q]50%
(2)
Here KEB = 1.2 × 106 M−1.40 KEB is the binding constant of EB
to DNA and [Q]50% is the concentration of 1−3 at 50% of the
initial fluorescence. These Kapp values are also given in Table 3
and are on the same order of magnitude as the KSV values.
Furthermore, we carried out a similar competition experi-
ment using Hoechst 33258. This dye binds to DNA in two
concentration dependent ways. The first type of binding occurs
in the minor groove at low dye-to DNA ratios.41,42 The
fluorescence yield of Hoechst 33258 increases significantly in
presence of DNA.43 The displacement of bound Hoechst
33258 from its binding site on ct-DNA is implicated from a
decrease in its fluorescence intensity on addition of the
complexes. When the complexes are added to Hoechst-DNA
solution we observed a decrease (∼14% for 1 and ∼55% for 2
and ∼81% for 3, respectively) in the fluorescence (Figure 4).
This suggests that especially complexes 2 and 3 are capable of
binding in the minor groove of DNA.
Figure 4. Fluorescence spectra of the Hoechst-bound ct-DNA in
aqueous buffer in the absence and presence of increasing amounts of
complexes 1 (a), 2 (b), and 3 (c). λex = 338 nm, [Hoechst] = 2 μM,
[DNA] = 20 μM, [complexes] (μM): 0−30 in 2.5 μM increments. T =
298 K.
Figure 3. Fluorescence spectra of the EB-bound ct-DNA in aqueous
buffer in the absence and presence of increasing amounts of complex
3. λex = 520 nm, [EB] = 0.33 μM. [DNA] = 10 μM, [complexes]
(μM): 0−70 in 5 μM increments. T = 298 K.
Reactions of the Iridium(III) Complexes 1−3 with 9-
Ethylguanine. The reactions were carried out in D2O and
DMSO-d6 (1:1) as solvents. The model nucleobase 9-EtG was
incubated with the corresponding complex in a ratio 5:1 at 37
°C. After 24 h no reaction of 1−3 with 9-ethylguanine by H
NMR was observed in the assayed conditions.
In Vitro Biological Evaluation for Activity against
Bovine Cathepsin B. Cathepsin B (cat B) is an abundant and
ubiquitously expressed cysteine peptidase of the papain family.
complex 3 causes a moderate decrease in the fluorescence at
602 nm as the amount of 3 is increased. For complexes 1 and 2
decrease in the fluorescence at 602 nm is smaller (Supporting
Information, Figure S3).
To quantitatively assess the magnitude of this interaction
between the complexes and DNA, the Stern−Volmer eq 1 is
used.
1
F /F = 1 + KSV[Q]
(1)
0
C
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Increased expression and secretion of cat B have been shown to
be causally involved in immigration and invasion of numerous
human and experimental tumors44 and is a possible therapeutic
target for the control of tumor progression, and in this respect,
it is not surprising that the use of cat B inhibitors reduces both
tumor cell mobility and invasiveness in vitro.45 Recently, some
metal complexes were shown to be effective inhibitors of cat
B.46 Complexes 1−3 were evaluated for activity against bovine
cat B, and the in vitro IC50 data are reported in Table 4. These
structure consisting of a single polypeptide chain. Of the amino
acid residues in the chain, the single tryptophan (Trp 214) is
responsible for the majority of the intrinsic fluorescence of the
protein. HSA has a strong fluorescence emission with a peak
near 350 nm upon excitation at 295 nm. The emission is
sensitive to the changes in the local environment of the
tryptophan and so can be attenuated by binding of a small
molecule at or near this residue. Thus, the emission spectra of
HSA in the presence of different concentrations of Ir(III)
complexes were recorded in the wavelength range 300−580 nm
by exciting the protein at 295 nm. As seen in Figure 7, in all
cases the fluorescence intensities of the protein are decreased
regularly with increasing concentration of the probe com-
pounds, indicating the binding of complex to the protein. A
decrease in the fluorescence intensity of HSA at 348 nm
accompanies the appearance of one peak at about 490 nm,
which can be attributed to the fluorescence spectra of the
Ir(III) complexes bound to the protein.
Furthermore, the fluorescence quenching mechanism can be
described again by the Stern−Volmer eq 1. For a homoge-
neously emitting solution eq 1 predicts a linear plot of F0/F vs
[Q], but for many systems the plots have been found to curve
upward. This is the case for the new complexes herein reported
(Supporting Information, Figure S6). It sometimes has been
proposed as explanation that in addition to the dynamic
quenching which governs the Stern−Volmer plot, a second
mechanism, static quenching occurs.49,50
Table 4. In Vitro Biological Data for 1−3 against Bovine Cat
B
compound
IC50/μM vs cat B
1
2
3
7.9 1.3
6.1 1.4
17.7 3.3
values indicate that 1 and 2 are good cathepsin B inhibitors,
showing values in the same order of magnitude than RAPTA-
C.46 Figure 5 shows the reduction of enzyme activity as a
A common method to distinguish between static and
dynamic quenching is by careful examination of the absorption
spectra of the HSA in the presence of the metal complexes.
Figure 8 shows the absorption spectra of HSA before and
after the addition of Ir(III) complexes. The influence of the
absorbance of Ir(III) complexes were eliminated by adding in
the reference cells the solutions of Ir complexes of the same
concentrations as in the sample solution. As can be seen, the
protein possess two absorption peaks at 206 and 279 nm,
where that at 206 nm represents the content of α-helix in the
protein and the weak absorption peak at 279 nm arises from the
phenyl rings in Trp (tryptophan), Tyr (tyrosine), and Phe
(phenylalanine) residues. A dramatic decrease in the 206 nm
absorbance peak of the protein is observed upon addition of
complexes to the protein. This can be attributed to the induced
perturbation of α-helix of protein by a specific interaction with
the ligands. Furthermore, an obvious red shift in the position of
the absorbance peak at 206 nm (i.e., 228 nm for complex 1, 229
nm for complex 2, and 230 nm for complex 3) could also be
observed with the addition of complexes. Meanwhile, a subtle
change in the intensity and a blue shift in the position of the
absorbance peak at 279 nm (i.e., 277 nm for complex 1, 266 nm
for complex 2, and 273 nm for complex 3) could also be
observed indicating that more aromatic acid residues were
extended into the aqueous environment.51,52 Trp-214 in HSA,
which are originally buried in a hydrophobic pocket, were
exposed to an aqueous milieu to a certain degree.
Figure 5. Cat B activity inhibition curves for complex 2.
function of inhibitor concentration for compound 2 displayed
moderate cat B activity possibly because of the steric hindrance
of the larger arene ring, which might decrease the accessibility
of the iridium center in the active site.46
In addition, the cysteine reactivation properties were
evaluated for 2 to characterize the reversibility of inhibition
(Figure 6). It was found that the addition of 1 mM cysteine
Figure 6. Cysteine reactivation of cat B data inhibited by 12 μM of 2.
results in full recovery of activity within 2 h. These data support
the hypothesis that loss of enzyme activity is due to specific
interactions of the compound with the cat B active site instead
of metal complex induced enzyme denaturing.47
Reaction with HSA. The interaction of Ir(III) complexes
with HSA was monitored by studying the quenching of the
fluorescence of HSA with increasing concentration of Ir(III)
complexes. No correction for inner filter effect was applied
since the Ir(III) complexes represented very low absorbance
(less than 0.04) at the excitation and emission wavelengths,
Supporting Information, Figure S5.48 HSA has a well-known
This result indicated that the microenvironment of the three
aromatic acid residues was altered and the tertiary structure of
HSA was destroyed. Overall, the changes in the absorbance
spectra for HSA + complexes show that the interaction between
Ir(III) complexes and HSA was mainly a static quenching
process.
For a static quenching process, the association constant (KA)
and the number of binding sites (n) can be calculated using eq
3.
D
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Figure 7. Emission spectra of the HSA in presence of increasing amounts of complexes 1−3. λex = 295 nm, [HSA] = 5.4 μM, and [complexes]: 0−35
μM (top-to-bottom in 2.7 μM increments). Temperature = 296 K and pH = 7.4.
F − F
To investigate if complex 2 can also bind to the site II of
albumin, an ibuprofen competitive experiment was conducted.
Ibuprofen was gradually added to the solution containing
equimolar concentrations of HSA and 2, and the change in
fluorescence intensity of 2 (λex = 320 nm, λem = 520 nm) was
monitored (Supporting Information, Figure S9). The binding
constant of ibuprofen to HSA is 3.6 × 106 M−1, which is higher
than that of complex 2. So, if 2 binds to site II of albumin, it
would be substituted by ibuprofen. The unbound Ir complex
has a weaker fluorescence emission than the albumin-bound
complex. Thus a decrease in the fluorescence intensity of 2
upon addition of ibuprofen is expected if 2 binds to site II of
albumin. However, it was found that the fluorescence intensities
of albumin-bound 2 complex increases upon addition of
ibuprofen. Therefore, this seems to indicate that 2 is not able to
bind to the site II of albumin.
0
log
= n log KA − n
F
⎧
⎫
⎬
⎭
1
⎨
log
[Q ] − (F − F)[P]/F
⎩
0
t
0
(3)
t
Here F0 and F are the fluorescence intensities in the absence
and presence of complexes, respectively; [Qt] and [Pt] are the
total concentrations of complexes and HSA, respectively.
Supporting Information, Figure S7 shows the plots of log (F0
− F)/F versus log {1/([Qt] − (F0− F)[Pt]/F0)} for the
interaction between HSA and complexes 1−3. KA and n
obtained from the plots and Ksv are listed in Table 5.
The binding constant is on the order of 104 M−1 for all
complexes, indicating strong binding to the protein, and the
number of binding sites in HSA approximates to 2 for
complexes 2 and 3, indicating that two sites in HSA are reactive
to both complexes. For the compound 1 a single site in HSA is
reactive to complex.
CONCLUSIONS
■
Three new organometallic iridium(III) thiosemicarbazide
complexes [Ir(N−C)2(N−S)]Cl (HN−C = 2-phenylpyridine)
1−3 have been prepared, where the thiosemicarbazide ligand
binds the iridium atom as N,S-chelate, as confirmed for 1 by X-
ray diffraction. 1−3 were more cytotoxic than cisplatin in T47D
human brest cancer cell line (about 5-fold). Especially
noteworthy is the very low resistance factor (RF) of 2 and 3
at 48 h (RF = 0.9 and 1.1, respectively) against an A2780 cell
line which has acquired resistance to cisplatin indicating
efficient circumvention of cisplatin resistance. Ir accumulation
in T47D cell line after 48 h continuous exposure for complexes
1−3 are higher than that corresponding to cisplatin (about 10
times). Complexes 1 and 2 are good cathepsin B inhibitors (an
enzyme implicated in a number of cancer related events), being
the enzyme reactivated by cysteine. The binding constant
toward HSA is on the order of 104 M−1 at 296 K for all
complexes, indicating strong binding to the protein, and the
number of binding sites in HSA approximates to 2 for
complexes 2 and 3, indicating that two sites in HSA are reactive
to both complexes, binding occurring at the warfarin site I as
shown for 2. For the compound 1 a single site in HSA is
reactive to complex. Furthermore, as shown by Hoechst 33258
displacement experiments, 2 and 3 are able to bind to DNA in
Site-Selective Binding of Ir Complexes on HSA. To
identify the binding site location of Ir(III) complexes on the
region of HSA, competitive binding experiments were carried
out using warfarin, a characteristic marker for site I(subdomain
IIA), and ibuprofen as one for site II(subdomain IIIA).48 Thus
information about complex 2 binding site can be obtained by
monitoring the changes in the fluorescence spectra of Ir(III)
complex bound to HSA. The weak fluorescence intensity of
warfarin is enhanced upon binding with HSA because of its
interaction with Trp 214 in HSA when excited at 320 nm. The
fluorescence intensity of warfarin, in its bound state to HSA,
decreases if a second ligand competes for the site occupied by
it. Thus, the accessibility of Trp 214 in HSA by ligands can be
confirmed by monitoring the displacement of warfarin by 2 in
the HSA−warfarin complex. As shown in Supporting
Information, Figure S8, a decrease in the emission intensity
of HSA−warfarin (in the wavelength region of 348−450 nm,
excited at 320 nm) is observed when 2 is added. The quenching
of warfarin bound to protein upon addition of 2 reflects a
reduction in the warfarin binding capacity at the binding site of
HSA and preferential accessibility of Trp 214 in HSA by 2.
Therefore it is very likely that the 2 binding occurs at the
warfarin site I.
E
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1
spectrometer using KBr pellets. The H and 13C NMR spectra were
recorded on a Bruker AC 300E or Bruker AV 400 spectrometer, using
SiMe4 as standard. ESI mass (positive mode) analyses were performed
on a HPLC/MS TOF 6220. UV/vis spectroscopy was carried out on a
Perkin-Elmer Lambda 750 S spectrometer with operating software.
Fluorescence measurements were carried out with a Perkin-Elmer LS
55 50 Hz Fluorescence Spectrometer.
Materials. Solvents were dried by the usual methods. The starting
complex [Ir2(N−C)4Cl2] (HN−C = Hppy) was prepared by a
procedure described elsewhere.53 The thiosemicarbazide ligands were
synthesized by using procedures adapted from the literature.54,55
Sodium salt of calf thymus DNA, ethidium bromide (EB), Hoechst
33258, serumalbumin (HSA) were obtained from Sigma-Aldrich
(Madrid, Spain); pBR322 plasmid DNA used in the studies were
obtained from Boehringer-Mannheim (Mannheim, Germany).
Synthesis of Cyclometalated Iridium(III) Complexes. A
mixture of [Ir2(N−C)4Cl2] (100 mg, 0.145 mmol) and the
corresponding thiosemicarbazide (0.290 mmol) in 20 mL of EtOH/
CH2Cl2 (3:1, v/v) was stirred for 72 h at room temperature to yield a
solution, which was partially evaporated under vacuum, and ethyl ether
and hexane was added to precipitate the complexes as yellow solids,
which were collected by filtration and air-dried.
Complex 1. Yield: 62%. Anal. Calcd for 1 C24H23N5ClSIr: C,
44.96; H, 3.62; N, 10.92; S, 5.00 Found: C, 44.57; H, 3.89; N, 10.76; S,
5.01. Mp: 253 °C (dec). IR (cm−1): ν(NH2, NH) 3404, 3205, 3039;
ν(CN) 1580; ν(CS) 756. 1H NMR (300 MHz, DMSO-d6,
TMS): 10.87 (m, 1 H, NH), 9.24 (m, 1 H ppy), 8.84 (m, 1H,
NHCH3), 8.73 (m, 1 H ppy), 8.46 (m, 2H, NH2), 8.16 (m, 2H ppy),
7.97 (m, 2H ppy), 7.73 (m, 2 H ppy), 7.43 (m, 2 H ppy), 6.73 (m, 4 H
ppy), 6.21 (m, 1H ppy), 6.06 (m, 1 H ppy), 2.88 (m, 3H, CH3).
Positive-ion ESI mass spectra ion cluster (DMSO) at m/z 604
{[Ir(ppy)2(methylthiosemicarbazide)]+}+.
Complex 2. Yield: 65%. Anal. Calcd for 2 C28H25N5ClSIr: C,
48.65; H, 3.65; N, 10.13; S, 4.64. Found: C, 48.53; H, 3.87; N, 9.85; S,
4.63. Mp: 237 °C (dec). IR (cm−1): ν(NH2, NH) 3412, 3144, 3035;
ν(CN) 1496 ; ν(CS) 756. 1H NMR (300 MHz, DMSO-d6,
TMS): 11.27 (m, 2H, NH2), 9.25 (d, 1H ppy, J = 6 Hz), 8.78 (d, 1H
ppy, J = 6 Hz), 8.66 (m, 1H, NH), 8.19(m, 2H ppy), 7.99 (m, 3H, 2H
ppy +1H NH), 7.79 (d, 1H ppy, J = 6 Hz), 7.71 (d, 1H ppy, J = 6 Hz),
7.50−7.32 (m, 6H, 2H0 + 2Hm phenylthiosemicarbazide + 2H ppy),
7.19 (m, 1H, Hp phenylthiosemicarbazide), 6.89 (m, 1H ppy), 6.77
(m, 2H ppy), 6.23 (m, 1H ppy), 6.20 (d, 1H ppy, J = 6 Hz), 6.05 (d,
́
1H ppy, J = 6 Hz). Positive-ion ESI mass spectra ion cluster (DMSO)
at m/z 656 {[Ir(ppy)2(phenylthiosemicarbazide)]+}+.
Complex 3. Yield: 57%. Anal. Calcd for 3 C33H27N5ClSIr: C,
52.61; H, 3.61; N, 9.30; S, 4.26. Found: C, 52.42; H, 3.85; N, 9.12; S,
4.35. Mp: 223 °C (dec). IR (cm−1): ν(NH2, NH) 3434, 3159, 3040;
ν(CN) 1550; ν(CS) 756. 1H NMR (400 MHz, DMSO-d6,
TMS): 11.07 (s, 1H, NH), 9.24 (d, 1 H ppy, J = 7 Hz), 8.82 (d, 1
H, ppy, J = 7 Hz), 8.62 (d, 1 H, NHNH2), 8.20 (d, 1 H ppy, J = 7 Hz),
8.15 (d, 1 H ppy, J = 7 Hz), 8.04 (m, 4 H ppy), 7.91 (m, 2 H,
NHNH2), 7.78 (d, 1H ppy, J = 7 Hz), 7.73 (m, 1 H naphtyl
thiosemicarbazide), 7.66 (d, 1 H ppy, J = 7 Hz), 7.6 (m, 3 H
naphtylthiosemicarbazide), 7.5 (m, 3 H ppy), 6.87 (m, 1 H ppy), 6.7
(m, 2 H ppy), 6.6 (m, 1 H ppy), 6.22 (d, 1 H ppy, J = 7 Hz), 5.97 (d, 1
Figure 8. Absorption spectra of HSA = (10−6 M) (red), HSA +
complex 1, 2, or 3 = 10−6 M and 10−5 M respectively (blue).
Table 5. Quenching and Binding Parameters for the
Interaction of Complexes with HSA
a
a
T (K) 10−4 Ksv (M−1
)
R
10−4 KA (M−1
)
n
complex 1
complex 2
complex 3
296
296
296
9.35
4.24
4.26
0.993
0.989
0.980
5.34
3.48
3.92
1.32
2.18
1.91
́
H ppy, J = 7 Hz). Positive-ion ESI mass spectra ion cluster (DMSO) at
m/z 716 {[Ir(ppy)2(naphtylthiosemicarbazide)]+}+.
a
Reactions of the Iridium(III) Complexes with 9-Ethylguanine
Followed by 1H NMR. The reaction was carried out in an NMR tube
containing D2O and DMSO-d6 (1:1 in volume) as solvents. 9-
Ethylguanine was incubated with the complexes in a ratio 5:1 in the
above solvents mixture at 37 °C. The concentration of complexes was
1.0 mM.
X-ray Crystal Structure Analysis. Suitable crystals of 1 were
grown from dichloromethane/hexane. The crystal and molecular
structure of 1 have been determined by X-ray diffraction studies
(Table 6). Crystals were mounted on glass fibers and transferred to the
cold gas stream of the diffractometer Bruker Smart APEX. Data were
recorded with Mo Kα radiation (λ = 0.71073 Å) in ω scan mode.
Absorption correction for the compound was based on multiscans.
Calculated using only the data in the linear range.
the minor groove. No reaction of 1−3 with 9-EtG was observed
1
by H NMR in the assayed conditions.
EXPERIMENTAL SECTION
■
Instrumental Measurements. The C, H, N, and S analyses were
performed with a Carlo Erba model EA 1108 microanalyzer.
Decomposition temperatures were determined with a SDT 2960
simultaneous DSC-TGA of TA Instruments at a heating rate of 5 °C
min−1 and the solid samples under nitrogen flow (100 mL min−1).
Infrared spectra were recorded on a Perkin-Elmer 100 FT-IR
F
dx.doi.org/10.1021/ic302219v | Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
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The inhibitory potential of compounds was measured by calculating
concentration-percentage inhibition curves; these curves were adjusted
to the following equation:
Table 6. Crystal Structure Determination Details of 1
1
chemical formula
fw [g/mol]
cryst system
a [Å]
C24H23ClIrN5S
641.18
E = Emax/[1 + (IC50)/C)n]
monoclinic
15.5488(12)
9.3007(7)
20.6588(17)
90.836(2)
4049.4(3)
100(2) K
P2(1)/n
4
were E is the percentage inhibition observed, Emax is the maximal
effect, IC50 is the concentration that inhibits 50% of maximal growth, C
is the concentration of compounds tested, and n is the slope of the
semilogarithmic dose−response sigmoid curves. This nonlinear fitting
was performed using GraphPad Prism 2.01, 1996 software (GraphPad
Software Inc.).
For comparison purposes, the cytotoxicity of cisplatin was evaluated
under the same experimental conditions. All compounds were tested
in two independent studies with triplicate points. The in vitro studies
were performed in the USEF platform of the University of Santiago de
Compostela (Spain).
b [Å]
c [Å]
β [deg]
V [Å3]
temp (K)
space group
Z
μ [mm−1
]
4.646
Cellular Iridium Complexes Accumulation. T-47D cells were
plated in a 12-well cell culture plate at a density of 750000 cells/well
and maintained at 37 °C in a 5% CO2 atmosphere. Compounds to 20
μM were added to cells for 48 h, and after this time, cells were
detached from the plate by using EDTA-trypsin. The cell suspension
from each well was transferred to Eppendorf tubes and centrifuged at
400 g for 5 min at room temperature. The supernatant was discarded,
and the pellet was suspended in a cell culture medium and washed by
two additional centrifugations. The final pellet was resuspended in 0.5
mL of pure HNO3 and diluted with 5 mL of Milli-Q water for ICP-MS
analysis. A Varian 820-MS inductively coupled plasma-mass
spectrometer was used to determine Ir concentration. Samples were
introduced via a concentric glass nebulizer with a free aspiration rate of
1 mL/min, a Peltier-cooled double pass glass spray chamber, and a
quartz torch. A peristaltic pump carried samples from a SPS3
autosampler (Varian) to the nebulizer. Ir standards were prepared by
serial dilution of a solution containing 1000 mg/L of Ir in 5% HCl
(Trace Cert. Fluka). A nine-point calibration curve was made over a
concentration range of 0.2−100 μg/L of Ir.
reflcns collcd
indpndnt reflcns
R(int)
19904
6549
0.0304
a
R1 [I > 2σ (I) ]
0.0269
b
wR2 (all data)
0.0634
a
R1 = ∑||Fo| − |Fc||/∑|Fo|, wR2 = [∑[w(Fo − Fc2)2]/∑w(Fo )2]0.5.
2
2
b
2
2
w = 1/[σ2(Fo ) + (aP)2 + bP], where P(2Fc2 + Fo )/3, and a and b are
constants set by the program.
The structure was solved by direct method, and refined anisotropically
on F2.56 The hydrogens at N were located in the Fourier difference
maps and refined freely with DFIX. Methyl groups were refined using
rigid groups, and other hydrogens were refined using a riding method.
Special Features for 1. The structure contains a poorly resolved
region of residual electron density; this could not be adequately
modeled and so was “removed” using the program SQUEEZE,57
which is part of the PLATON58 system. The void volume per cell was
888.6 Å3, with a void electron count per cell of 187. This additional
solvent was NOT taken account of when calculating derived
parameters such as the formula weight because the nature of the
solvent was uncertain.
Cell Line and Culture. The T-47D human mammary
adenocarcinoma cell line used in this study was grown in RPMI-
1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS)
and 0.2 unit/mL bovine insulin in an atmosphere of 5% CO2 at 37 °C.
The human ovarian carcinoma cell lines (A2780 and A2780cisR) used
in this study were grown in RPMI 1640 medium supplemented with
10% (v/v) fetal bovine serum (FBS) and 2 mM L-glutamine in an
atmosphere of 5% CO2 at 37 °C.
Cytotoxicity Assay. Cell proliferation was evaluated by assay of
crystal violet. T-47D cells plated in 96-well sterile plates at a density of
5 × 103 cells/well with 100 μL of medium and were then incubated for
48 h. After attachment to the culture surface the cells were incubated
with various concentrations of the compounds tested freshly dissolved
in DMSO and diluted in the culture medium (DMSO final
concentration 1%) for 48 h at 37 °C. The cells were fixed by adding
10 μL of 11% glutaraldehyde. The plates were stirred for 15 min at
room temperature and then washed three-four times with distilled
water. The cells were stained with 100 μL of 1% crystal violet. The
plates were stirred for 15 min and then washed three-four times with
distilled water and dried. A 100 μL portion of 10% acetic acid was
added, and it was stirred for 15 min at room temperature.
193Ir and 191Ir were monitored, and 159Tb was used as the internal
standard. Data acquisition was done using peak hopping with a dwell
time of 30 ms, 60 scans/replicate, and three replicates per sample from
two independent experiments.
Electrophoretic Mobility Study. pBR322 plasmid DNA of 0.25
μg/μL concentration was used for the experiments. Four microliters of
charge maker (Lambda−pUC Mix marker, 4) were added to aliquots
parts of 20 μL of the drug−DNA complex. The iridium complexes
were incubated at the molar ratio ri = 0.50 with pBR322 plasmid DNA
at 37 °C for 24 h. The mixtures underwent electrophoresis in agarose
gel 1% in 1 × TBE buffer (45 mM Tris-borate, 1 mM EDTA, pH 8.0)
for 5 h at 30 V. Gel was subsequently stained in the same buffer
containing ethidium bromide (1 μg/mL) for 20 min. The DNA bands
were visualized with an AlphaImager EC (Alpha Innotech).
Ethidium Bromide and Hoechst 33258 Displacement
Experiments. In the ethidium bromide (EB) fluorescence displace-
ment experiment 3 mL of a solution, that is, 10 μM DNA and 0.33 μM
EB (saturated binding levels59), in Tris buffer was titrated with aliquots
of a concentrated solution of the complex producing solutions with
varied mole ratios of complex to ct-DNA. After each addition the
solution was stirred at the appropriate temperature for 5 min before
measurement. The fluorescence spectra of the solution were obtained
by exciting at 520 nm and measuring the emission spectra from 530−
700 nm using 5 nm slits. The procedure was the same for the Hoechst
33258 reactions using the following conditions: working solutions
were 20 μM DNA and 2 μM Hoechst 33258; λex = 338 nm and λem
350−650 nm (with λmax ∼ 600 nm).
=
Absorbance was measured at 595 nm in a Tecan Ultra Evolution
spectrophotometer.
The effects of complexes were expressed as corrected percentage
Cathepsin B Inhibition Assay. Crude bovine spleen cat B was
purchased from Sigma (C6286) and used without further purification.
The colorimetric cat B assay was performed in 100 mM sodium
phosphate, 1 mM EDTA, 0.025% polyoxyethylene (23) lauryl ether
(BRIJ), pH = 6 using Z-L-Lys-ONp hydrochloride (Sigma C3637) as
substrate. For the enzyme to be catalytically functional, the active site
cysteine needs to be in a reduced form. Therefore, before using, cat B
was prereduced with dithiothreitol (DTT) to ensure that the majority
inhibition values according to the following equation,
(%) inhibition = [1 − (T/C)] × 100
where T is the mean absorbance of the treated cells and C the mean
absorbance in the controls.
G
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Inorganic Chemistry
Article
of the enzyme is in a catalytically active form. Thus, cat B was
activated, before dilution, in the presence of excess DTT for 1 h at 30
°C.
IC50 determinations were performed in duplicate using a fixed
enzyme concentration of 1 μM, and a fixed substrate concentration of
0.08 mM. Inhibitor concentrations ranged from 1 μM to 50 μM. The
enzyme and inhibitor were co-incubated at 25 °C over a period of 24 h
prior to the addition of substrate. Activity was measured over 1 min at
326 nm.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jruiz@um.es. Fax: +34 868 884148. Tel: +34 868
887455.
Notes
The authors declare no competing financial interest.
Cysteine reactivation was evaluated using an inhibitor concentration
corresponding to 2 × IC50. The enzyme was preincubated with an
excess of DTT (Sigma D0632) for 1 h at 30 °C. After the activation,
the enzyme and the compound were incubated at 25 °C for 24 h.
Then, 1 mM L-cysteine was added and incubated at different times 1,
2, 3, 4, 6, and 24 h at 25 °C. Following incubation, the substrate was
added, and activity was assessed.
Reaction with Human Serum Albumin (HSA). Procedures
Fluorescence Quenching Studies. The stock solutions of proteins
(5.4 × 10−6 mol L−1) were prepared by dissolving the solid HSA in 50
mM Tris-HCl, 100 mM NaCl buffer of pH 7.4 and stored at 0−4 °C
in the dark for about a week The concentrations of HSA were
determined from optical density measurements, using the values of
molar absorptivity of ε278 = 36 000 M−1 cm−1.48
Quantitative analyses of the interaction between Ir(III) complexes
1−3 and biomacromolecules were performed by fluorimetric titration.
A 3.0 mL portion of aqueous solution of protein (5.4 × 10−6 mol L−1)
was titrated by successive additions of Ir(III) complexes solution (to
give a final concentration of 35 × 10−6 mol L−1). Titrations were done
manually by using a trace syringe. For every addition, the mixture
solution was shaken and allowed to stand for 5 min at the
corresponding temperature, and then the fluorescence intensities
were measured with an excitation wavelength of 295 nm and emission
wavelengths in the interval 300−550 nm. No correction for inner filter
effect was applied since the Ir(III) complexes represented very low
absorbance (less than 0.04) at the excitation and emission wave-
lengths, Supporting Information, Figure S7. The width of the
excitation and emission slit was set to 5 nm.
ACKNOWLEDGMENTS
■
́
This work was supported by the Spanish Ministerio de Economıa
y Competitividad and FEDER (Project SAF2011-26611) and
́ ́
also by Fundacion Seneca-CARM (Project 08666/PI/08).
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ASSOCIATED CONTENT
■
S
* Supporting Information
Structures, energies and Cartesian coordinates for all computed
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
H
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