Cytotoxicity and hydrolysis of trans -Ti(IV) complexes of salen ligands: Structure-activity relationship studies

Article abstract of DOI:10.1021/ic202092u

Eleven bis(dimethylphenolato) Ti(IV) complexes of salen ligands with different steric and electronic properties due to different aromatic substituents at the ortho and para positions are reported, and their cytotoxicity toward HT-29 and OVCAR-1 cells and

Full text of DOI:10.1021/ic202092u

Article
pubs.acs.org/IC
Cytotoxicity and Hydrolysis of trans-Ti(IV) Complexes of Salen
Ligands: Structure−Activity Relationship Studies
Avia Tzubery and Edit Y. Tshuva*
Institute of Chemistry, The Hebrew University of Jerusalem, 91904, Jerusalem, Israel
S
* Supporting Information
ABSTRACT: Eleven bis(dimethylphenolato) Ti(IV) com-
plexes of salen ligands with different steric and electronic
properties due to different aromatic substituents at the ortho
and para positions are reported, and their cytotoxicity toward
HT-29 and OVCAR-1 cells and its dependence on hydrolytic
behavior are discussed. Eight complexes of this series were
analyzed by X-ray crystallography, confirming the trans
geometry of the labile ligands with otherwise relatively similar
coordination features to those of cis-salan analogues. Relatively
high and similar hydrolytic stability is observed for all complexes, with t1/2 values for labile ligand hydrolysis of 2−11 h in 10%
D2O solutions. In contrast, varying cytotoxicities were achieved, identifying selected members as the first trans-Ti(IV) complexes
reported as anticancer agents. Steric bulk all around the complex diminished the activity, where a complex with no aromatic
substitutions is especially active and complexes substituted particularly at the ortho positions are mostly inactive, including ortho-
halogenated and ortho-tert-butylated, with one exception of the ortho-methoxylated complex demonstrating appreciable activity.
In contrast, para-halogenation provided the complexes of highest cytotoxic activity in this series (IC50 as low as 1.0 0.3 μM),
with activity exceeding that of cisplatin by up to 15-fold. Reaction of a representative complex with ortho-catechol yielded a “cis”-
Ti(IV) complex following rearrangement of the salen ligand on the metal center, with highly similar coordination features and
geometry to those of the catecholato salan analogues, suggesting that the complexes operate by similar mechanisms and
rearrangement of the salen ligand may occur upon introduction of a suitable chelating target. In additional cytotoxicity
measurements, a salen complex was preincubated in the biological medium for varying periods prior to cell addition, revealing
that marked cytotoxicity of the salen complex is retained for longer preincubation periods relative to known Ti(IV) complexes,
suggesting that the hydrolysis products may also induce cytotoxic effects, thus reducing stability concerns.
cisplatin-sensitive and -resistant cells with relatively minor
toxicity.16−25 The main disadvantage of the Ti(IV) complexes
is, however, their relatively rapid hydrolysis in biological environ-
ment,19,21,26,27 where the labile ligands of Cp2TiCl2 and (bzac)2-
Ti(OEt)2 (Cl, OEt) dissociate within seconds to minutes while
the inert ligands (Cp, diketonato) hydrolyze within hours to give
undefined aggregates, thus hampering mechanistic investigations
and applicability.
We previously introduced the family of cytotoxic Ti(IV)
complexes based on diamino bis(phenolato), salan ligands
(Scheme 1c).28−32 Complexes of this family have demonstrated
high cytotoxic activity along with exceptional hydrolytic
stability. Structure−activity relationship studies have revealed
that the ligand and its particular substitutions play an important
role in determining the complex performance, where the
hydrolytic behavior of the complexes and their cytotoxicity are
closely related.28,29 All such titanium(IV) complexes inves-
tigated thus far as cytotoxic agents feature two labile ligands in a
cis configuration and were designed as such under the
assumption that dissociation of the labile ligands enables
INTRODUCTION
■
Cisplatin, the first inorganic compound approved as an
anticancer agent, is widely used worldwide along with its
derivatives.1−5 Nevertheless, its disadvantages relating to high
toxicity and limited activity range encourage extensive research
aimed at finding new inorganic complexes of other metal
centers that may lead to different and improved anticancer
drugs.6−15 Among others, titanium(IV) complexes have shown
high antitumor activity, where in particular titanocene dichloride
(Cp2TiCl2, Scheme 1, a) and budotitane ((bzac)2Ti(OEt)2,
Scheme 1. Titanocene Dichloride (a), Budotitane (b), and
Salan Complexes (c)
Scheme 1, b), followed by their improved derivatives with
different substitutions, demonstrated promising activity toward
Received: September 26, 2011
Published: December 29, 2011
© 2011 American Chemical Society
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Chart 1. Salen Complexes Investigated
potential chelate binding to a biological target, as occurs for
cisplatin.1−4 In particular, transplatin lacks meaningful activity
due to the inability to form a 1,2-intrastrand cross-link to two
adjacent bases in DNA, although other trans-platinum
complexes exhibit cytotoxic properties by different mecha-
nisms.33 As the mechanism of cytotoxic activity of the
titanium(IV) complexes and their binding form to the cellular
target remain unknown, the requirement of this structural
feature is yet undetermined. In our recent communication34 we
reported on the first family of highly cytotoxic trans-Ti(IV)
complexes based on salen ligands, which include similar donor
atoms to those of the salan ligands, only known to prefer
equatorial binding due to the planar imine moiety leading to
trans-labile ligands (Chart 1).35,36 We employed the salophen
ligands that include a planar phenylenediamine bridge,37−39
found to more conveniently yield pure products. Herein we
elaborate on this family of complexes, with detailed structure−
activity relationship investigations that reveal the parameters of
influence on the cytotoxic activity of these complexes toward
colon HT-29 and ovarian OVCAR-1 cells and their hydrolytic
stability while shedding light on their possible binding
mechanism.
RESULTS AND DISCUSSION
Synthesis and Characterization. Ligands H2Lig1−11 were
■
prepared according to known procedures via a single step of a
condensation reaction between the substituted salicyladehyde
compounds and phenylenediamine.40,41 The ligand structure was
determined mainly by 1H and 13C NMR. The complexes Lig1−11
-
Ti(OArMe2)2 were prepared in analogy to known compounds by
stirring the ligands H2Lig1−11 with 1 equiv of Ti(OArMe2)4, pre-
pared as previously described from Ti(OiPr)4,42 at room temper-
ature in THF under an inert atmosphere to give the Ti(IV)
complexes in high yields.34 The precursor Ti(OArMe2)4 was
selected due to difficulties in isolating pure complexes when start-
ing from Ti(OiPr)4 and previous reports on lesser influence of
the labile ligands on cytotoxicity.21 The complexes were analyzed
by NMR, featuring a single set of signals for the phenolato moiety
of the salen ligand and a single type of a labile ligand.
Further support for the complexes structures came from
X-ray crystallography. Lig1−4,7−10Ti(OArMe2)2 were crystallized
to produce red single crystals that were crystallographically
analyzed.34 Lig4Ti(OArMe2)2 was crystallized from hexane,
while the rest were crystallized from diethyl ether. The ORTEP
drawings of these complexes along with lists of selected bond
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Figure 1. ORTEP drawing of Lig1−4,7−10Ti(OArMe2)2, depicted as 1−4,7−10, with 50% probability ellipsoids. H atoms were omitted for clarity. For
Lig1Ti(OArMe2)2, one molecule of the two found in the asymmetric unit is presented. For Lig3Ti(OArMe2)2, disorder in the 2,6-dimethylphenolato
ligand was omitted for clarity to include only the atoms of 70% occupancy. For Lig4Ti(OArMe2)2, disorder in one t-Bu group was omitted for clarity.
For Lig9,10Ti(OArMe2)2, dimethylphenol molecule was omitted for clarity.
lengths and angles are provided in Figure 1 and Table S1,
Supporting Information, respectively. Compound Lig1Ti-
(OArMe2)2 crystallized with two different molecules in the
asymmetric unit, which share similar coordination features.
Lig3Ti(OArMe2)2 and Lig4Ti(OArMe2)2 show disorder in one
2,6-dimethylphenolato ligand and one tert-butyl group,
respectively, where for the former the main set occurs with
70% occupancy and for the latter 50−50 occupancy is obtained.
Lig9,10Ti(OArMe2)2 crystallized with an accompanying single
molecule of 2,6-dimethylphenol each, a product of the reaction
of the ligands H2Lig9,10 with the precursor Ti(OArMe2)4.
The X-ray structures indicate a similar general geometry of C2v
symmetrical complexes with two trans-2,6-dimethylphenoxo
ligands and with the salophen ligand binding in an equatorial
fashion to the octahedral metal center. The Ti−O distances for the
phenolato groups are in the range of 1.90−1.94 Å similarly to the
analogous values obtained for salan complexes,28,29,43−46 while the
Ti−N bonds are between 2.15 and 2.17 Å, shorter than the values
obtained for the salan analogues due to the sp2 amine donor. The
O−Ti−O angles obtained for all salophen ligands are between
171° and 177°, and O−Ti−O angles for the labile ligands range
between 113° and 118°. Thus, it appears that the steric and
electronic features of the different substituents induce a relatively
small influence on the solid-state structure of the complexes.
Hydrolysis. To estimate and compare the hydrolytic
stability of the complexes, 1H NMR measurements were
preformed upon addition of 10% D2O (>1000 equiv relative to
Ti) to THF-d8 solutions of the complexes, as previously
described for related salan complexes.28,29 These measure-
ments, although not reflecting the exact biological environment,
provided a comparative tool to assess the relative stability of the
different complexes. The spectrum was measured every 5−10
min for up to 15 h, and integration of selected signals was
measured to gain insight on the complexes decomposition
process (Figure S1, Supporting Information). In particular, the
half-life of dissociation of the 2,6-dimethylphenoxide ligands to
give the free 2,6-dimethylphenol was measured by monitoring
the integration of the doublet and triplet signals of the aromatic
region and the singlet of the methyl substituents. The results
are presented in Table 1 (see also Figure S2 and Table S2,
Table 1. t1/2 values for Hydrolysis of the Labile Groups from
Lig1‑11Ti(OArMe2)2 at 1:9 D2O/THF-d8 Solution at Room
Temperature Based on Pseudo-First-Order Fit
complex
aromatic substitution
t1/2 [h]
Lig1Ti(OArMe2)2
Lig2Ti(OArMe2)2
Lig3Ti(OArMe2)2
Lig4Ti(OArMe2)2
Lig5Ti(OArMe2)2
Lig6Ti(OArMe2)2
Lig7Ti(OArMe2)2
Lig8Ti(OArMe2)2
Lig9Ti(OArMe2)2
Lig10Ti(OArMe2)2
Lig11Ti(OArMe2)2
(bzac)2Ti(OiPr)2
none
2
p-Me
3
p-t-Bu
o,p-di-t-Bu
p-OMe
p-Br
7
5
3
4
p-Cl
4
o-OMe
o-Br
3
2
o- Cl
11
4
o,p-di-Cl
0.5
Supporting Information). Interestingly, no signals correspond-
ing to free salen ligand were detected in the spectra of the
hydrolysis products (Figures S1 and S3, Supporting Informa-
tion), implying that the main product of hydrolysis is a salen-
bound oxo-bridged polynuclear compound, as also supported
by the crystallographic characterization of such dimeric
products obtained for related complexes (Figure S4, Supporting
Information).47
Complexes Lig1−2,5−9,11Ti(OArMe2)2 all showed similar
stability, with t1/2 values for hydrolysis of the labile groups
ranging between 2 and 4 h. This stability exceeds that of the
known Ti(IV) complex (bzac)2Ti(OiPr)2 and is comparable to
that of some members of the analogous salan family of Ti(IV)
complexes.29 Lig3Ti(OArMe2)2 and Lig10Ti(OArMe2)2 dem-
onstrated slightly improved hydrolytic stability with t1/2 values
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of 7 and 11 h, respectively, the reason of which is not exactly
clear as no particular steric or electronic effect stand out for
these complexes when compared to other derivatives. Never-
theless, it is obvious that the variation in hydrolytic stability for
this series of complexes is relatively minor, supporting the basic
chelating salen ligand core as the main contributor to the
complexes enhanced stability relative to known compounds.
Additionally, this suggests that any variation in the cytotoxicity
of these complexes cannot be attributed to hydrolytic stability
differences.
Cytotoxicity. Cytotoxic activity was measured on two types
of cancer cell lines: colon HT-29 and ovarian OVCAR-1. Analysis
was carried out by the methylthiazolyldiphenyl-tetrazolium
bromide (MTT) assay following 3 days of incubation with the
investigated complexes at different concentrations.28 Relative IC50
and maximal cell growth inhibition values are shown in Table 2.
Representative cytotoxicity plots are given in Figure 2.
Table 2. IC50 [μM] and Maximal Cell Growth Inhibition [%]
Values of Lig1‑11Ti(OArMe2)2 and Known Reference
Compounds towards HT-29 and OVCAR-1 Cell Lines
aromatic
complex
substitution
HT-29 [μM]
3.5 0.6 (98%) 3.3 0.5 (98%)
10 2 (95%) 1 (93%)
4.8 1.1 (60%) 5.2 0.8 (65%)
OVCAR-1 [μM]
Lig1Ti(OArMe2)2
Lig2Ti(OArMe2)2
Lig3Ti(OArMe2)2
Lig4Ti(OArMe2)2
Lig5Ti(OArMe2)2
Lig6Ti(OArMe2)2
Lig7Ti(OArMe2)2
Lig8Ti(OArMe2)2
Lig9Ti(OArMe2)2
Lig10Ti(OArMe2)2
Lig11Ti(OArMe2)2
reference
none
p-Me
9
p-t-Bu
o,p-di-t-Bu
p-OMe
p-Br
inactive
inactive
14 4 (83%)
14 3 (80%)
2.3 0.6 (96%) 2.1 0.6 (99%)
1.2 0.3 (99%) 1.0 0.3 (100%)
3.9 0.9 (98%) 3.6 0.4 (97%)
p-Cl
o-OMe
o-Br
Figure 2. Dependence of HT-29 cell viability based on the MTT
assay following a 3-day incubation period on added concentration of
Lig1−4Ti(OArMe2)2 (top), Lig1,5−7Ti(OArMe2)2 (middle), and
Lig1,8−11Ti(OArMe2)2 (bottom) presented on a logarithmic scale.
inactive
inactive
inactive
inactive
inactive
o- Cl
o,p-di-Cl
inactive
HT-29 [μM]
OVCAR-1 [μM]
cisplatin
20 2 (97%)
517 87 (100%)
11.6 0.8 (99%)
13 1 (100%)
554 120 (100%)
11.5 0.2 (100%)
that para-halogenation increases the cytotoxic activity, where
the analogous ortho-halogenated complexes Lig9,10Ti-
(OArMe2)2 are completely inactive. The para-halogenated
complexes Lig6,7Ti(OArMe2)2 are among the most active
members in this series, where the improved performance of the
chlorinated one is probably due to reduced steric bulk. In fact,
the negative effect of these substitutions at the ortho position is
more pronounced than their positive effect when located at the
para position, as may be observed in the inactivity of the ortho-
para-dichlorinated complex Lig11Ti(OArMe2)2. Thus, when
adding the inactivity of Lig4Ti(OArMe2)2 to the discussion, one
may argue that ortho substitutions abolish activity, which may
be attributed to (a) interference to the interaction with the
biological target, (b) interference to some rearrangement that
needs to occur on the metal center upon such interactions, or
(c) inhibition to formation of the active species that may be, for
instance, some polynuclear hydrolysis product formed in the
biological environment. One exception is the ortho-methoxy-
lated complex Lig8Ti(OArMe2)2, demonstrating appreciable
cytotoxic activity. It is yet to be determined the reason for
which the methoxy substitution leads to different behavior
when compared to other derivatives when located both at the
ortho and at the para positions. Possible explanations may
relate to the solubility of the complex or conformational
changes of the substituent affecting its steric influence.
Cp2TiCl2
(bzac)2Ti(OiPr)2
When comparing complexes Lig1−4Ti(OArMe2)2 differing by
steric bulk it is clear that reduced steric bulk is preferred for
achieving high cytotoxic activity, even where the alkyl
substituents are located at the para positions. Whereas the
ortho,para-tert-butylated complex Lig4Ti(OArMe2)2 is com-
pletely inactive, the para-tert-butylated complex Lig3Ti-
(OArMe2)2 demonstrates mild activity where maximal cell
growth inhibition does not reach over 70%. Maximal inhibition
is increased to >70% when decreasing the size of the para
substituent in the methylated analogue Lig2Ti(OArMe2)2, while
a further improved performance is detected for Lig1Ti-
(OArMe2)2, the complex lacking any aromatic alkyl sub-
stitutions. This negative influence of general steric bulk, as also
observed for analogues,28,29 may relate to cell penetration
inhibition and/or general hydrophobicity, negatively affecting
solubility in biological environment.
Having established that increasing the substituents bulk at
various positions negatively affects the cytotoxicity, when
examining complexes Lig1,5−11Ti(OArMe2)2, a combination of
steric and electronic influences at all positions should be taken
into account. Complexes Lig1,5−7Ti(OArMe2)2 clearly evince
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It is therefore obvious that trans-Ti(IV) complexes may
indeed be cytotoxic, with activity that exceeds even that of
cisplatin by up to ∼15-fold on the cell lines analyzed. This
raises the question whether the two labile positions interact
with the cellular target in an unrelated fashion, rearrangement
of the salen ligand occurs on the metal center upon this
interaction, or these positions are not at all involved in the
activity mechanism.
Table 3. Selected Bond Lengths [Angstroms] and Angles
[degrees] for Lig2Ti(O2Ar)
atoms
lengths
value
atoms
value
O(1)−Ti
O(2)−Ti
O(3)−Ti
O(4)−Ti
1.886(2)
1.880(2)
1.937(2)
1.938(2)
N(1)−Ti
N(2)−Ti
2.146(2)
2.184(2)
Analysis of Binding Options. To evaluate whether
reorganization on the metal center may occur upon the first
interaction of the complexes with a suitable target to form a
chelate binding, we reacted a representative complex with a
strong chelating bidentate ligand. Lig2Ti(OArMe2)2 was reacted
with 1 equiv of ortho-catechol in THF at room temperature
under an inert atmosphere. Complex Lig2Ti(O2Ar) thus
angles
O(1)−Ti−O(2)
O(1)−Ti−O(3)
O(1)−Ti−O(4)
O(2)−Ti−O(3)
O(2)−Ti−O(4)
O(3)−Ti−O(4)
N(1)−Ti−N(2)
92.19(7)
84.17(6)
162.22(6)
109.97(7)
98.88(7)
79.01(6)
72.87(7)
O(1)−Ti−N(1)
O(2)−Ti−N(1)
O(3)−Ti−N(1)
O(4)−Ti−N(1)
O(1)−Ti−N(2)
O(2)−Ti−N(2)
O(3)−Ti−N(2)
O(4)−Ti−N(2)
82.06(7)
149.76(7)
99.00(7)
94.87(7)
115.00(6)
83.02(7)
157.05(6)
80.34(6)
1
formed (Scheme 2), for which the H and 13C NMR analyses
Scheme 2
changed from a trans to a cis orientation.29 This result is in
agreement with the notion that reorganization of the ligand on
the metal center may occur where a strong chelating target is
introduced and also supports similar interaction for both the
salan and the salen families of complexes due to relatively high
flexibility of the octahedral metal center. Interestingly, as
occurred for the salan analogue29 and despite the reduced
lability, the catecholato complex exhibits appreciable cytotoxic
activity as well, with IC50 values of 16 5 and 15 5 μM for
HT-29 and OVCAR-1 cell lines, respectively, that are similar to
the values of the trans analog Lig2Ti(OArMe2)2. This raises the
additional possibility that the labile positions do not participate
directly as such in the cytotoxicity mechanism if, for instance,
an oxo-bridged polynuclear hydrolysis product is involved as the
active species. The catecholato complex also demonstrates, as
expected,29 increased hydrolytic stability where no substantial
catecholato hydrolysis is observed for days in 10% D2O solutions.
Hydrolysis Effect on Cytotoxicity. To further analyze the
relationship between hydrolysis and cytotoxicity and especially
as a similar hydrolysis rate was detected for complexes of
varying activities, we reinvestigated the cytotoxic activity of the
most active complex Lig7Ti(OArMe2)2 following varying
periods of preincubation in the biological medium without
cells. Thus, Lig7Ti(OArMe2)2 was exposed to the medium for
0, 1, 3, and 24 h of incubation, following which cells were added
for an additional 3 days of incubation and the viability was
measured. Figure 4 depicts the results obtained for Lig7Ti-
(OArMe2)2 toward HT-29 cells, while Figure 5 depicts the cell
viability recorded following 3 h of preincubation in comparison
to the results obtained for (bzac)2Ti(OiPr)2 and for an
analogous meta-para-alkylated salan complex of similar stability
in 10% D2O48 (t1/2 for isopropoxo hydrolysis of 5 h29). As no
activity was recorded for (bzac)2Ti(OiPr)2 following 24 h of
medium preincubation, Figure 6 depicts the results for this
measurements for the salen and salan complexes only. IC50
values are provided in Table 4.
revealed a single type of aromatic phenolato system and a single
set of the ortho-catechol ligand signals. Crystallization from
dichloromethane at room temperature yielded single red
crystals suitable for X-ray crystallography. An ORTEP drawing
of the complex Lig2Ti(O2Ar) is presented in Figure 3, and a list
of selected bond lengths and angles is given in Table 3.
Figure 3. ORTEP drawing of Lig2Ti(O2Ar) with 50% probability
ellipsoids. H atoms and dichloromethane solvent were omitted for
clarity.
The X-ray structure evinces that the catecholato ligand binds
to the metal in a bidentate chelating fashion, where
rearrangement of the salen ligand occurred to enable the cis
configuration of what used to be the labile ligands, while the
salen ligand is no longer equatorial, adopting a cis configuration
of the phenolato moieties. In fact, this C1-symmetrical structure
highly resembles that obtained for an analogous catecholato
salan complex following reaction of the bis(isopropoxo)
precursor with o-catechol, where the phenolato binding
The results depicted in Figure 4 reveal that although
cytotoxicity markedly decreases following preincubation in
biological medium which should probably be attributed to
some dissociation of the complex, appreciable activity is
retained even after 24 h of medium preincubation. Additionally,
higher sustainability of the activity is observed for the salen
complex in comparison to known Ti(IV) complexes: (bzac)2Ti-
(OiPr)2 loses all activity already following 3 h of medium
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inactive following 24 h of medium preincubation (Figure 6).
Assuming that a polynuclear hydrolysis product should readily
form under these conditions, it appears that such an oxo-
bridged salen cluster might itself be cytotoxic, where activity
decrease may be attributed to reduced solubility and
precipitation and/or to impaired cellular penetration capability
of the larger molecule.
CONCLUSIONS
■
In this study we discussed the first family of anticancer trans-
Ti(IV) complexes to be reported, which is based on salophen
ligands. Such ligands have previously been shown to lead to
highly cytotoxic complexes of other transition metals as well,
such as iron(III) and manganese(III).49−52 Additionally,
complexes of the Ti(IV)−salen family demonstrate hydrolytic
stability comparable to that of some alkylated analogues of the
salan family,29 with cytotoxic activity markedly higher than that
of cisplatin. Structure−activity relationship investigations
revealed that while aromatic substitutions have a minor effect
on the hydrolytic stability, they strongly influence the cytotoxic
properties of the complexes. Overall, we observed that reduced
steric bulk is favored, ortho-substitution mostly abolished the
activity, while para-halogenation produced the most active
complexes of this family.
Figure 4. Dependence of HT-29 cell viability on administered
concentration of Lig7Ti(OArMe2)2 following preincubation in aqueous
medium for varying periods (given in hours) prior to cell addition and
3 days incubation with cells. Presented on a logarithmic scale.
When compared to analogous salan complexes,28,29 the
effects of geometry, substitutions, and hydrolysis are quite
intriguing. It appears that a similar mechanism may involve
the activity of the two groups of complexes, as reaction with
catechol of both the bis(isopropoxo) salan−Ti(IV) and
the bis(dimethylphenolato) salen−Ti(IV) precursors yielded
catecholato complexes of highly similar geometry and cytotoxic
activity.29 Thus, similar reorganizations may occur upon
interaction with a chelating cellular target due to the higher
flexibility of the octahedral metal center than that of, for
instance, the square planar platinum compounds. It is possible
that for this reason ortho-halogenation affects differently the
salan and salen families of complexes: whereas salan complexes
become more active and stable,28 the salen compounds are
inactive, perhaps due to interference to the required
reorganization. An alternative mechanistic route may relate to
the activity and properties of the hydrolysis products, presumed
to be oxo-bridged salen-bound polynuclear compounds. As the
salen complexes retain notable cytotoxic activity after periods
of preincubation in water that largely exceed their t1/2 of
labile ligand hydrolysis in 10% D2O solution, it is certainly
plausible that polynuclear hydrolysis products might not only
be somewhat active themselves but may also exhibit some
membrane penetration ability. Such characteristics seem to be
lacking for the salan analogues,29,53,54 as the isolated clusters
lack cytotoxicity altogether, although their involvement in the
cytotoxicity mechanism when formed inside the cells has
previously been proposed.53−55 One possible reason for this
difference may relate to different sizes of the clusters, if indeed
dimeric, rather than trimeric salen-bound clusters are involved.
The activity of the stable hydrolysis products is certainly
advantageous since reduced hydrolysis concerns should allow
for longer shelf-lives of the compounds. Nevertheless, as the
activity of the salens clearly decreases following water exposure,
additional derivatives with increased solubility should be
analyzed in order to make such clusters of potential medicinal
use. Additional advantages of the salen complexes relate to the
lack of chirality that abolished the need for chiral separation for
applicability as occurs for the salan complexes53,55,56 and in
Figure 5. Dependence of HT-29 cell viability on administered
concentration of Lig7Ti(OArMe2)2, (bzac)2Ti(OiPr)2,48 and a
meta,para-alkylated salan analogue48 following 3 h preincubation in
aqueous medium prior to cell addition and 3 days incubation with
cells. Presented on a logarithmic scale.
Figure 6. Dependence of HT-29 cell viability on administered
concentration of Lig7Ti(OArMe2)2 and a meta,para-alkylated salan
analogue48 following 24 h preincubation in aqueous medium prior to
cell addition and 3 days incubation with cells. Presented on a
logarithmic scale.
Table 4. IC50 [μM] and Maximal Cell Growth Inhibition [%]
Values of Lig7Ti(OArMe2)2 Following Preincubation in
Aqueous Medium for Varying Periods [hours] Prior to Cell
Addition and 3 Days Incubation with Cells
preincubation [h]
HT-29 [μM]
OVCAR-1 [μM]
0
1.7 0.4 (93%)
3.9 0.5 (92%)
9.5 0.9 (88%)
43 15 (74%)
1.7 0.3 (91%)
3.9 0.2 (91%)
9.2 0.6 (89%)
45 18 (70%)
1
3
24
preincubation where both the salan and salen complexes are
still notably active (Figure 5), while the salan analogue is
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a
Table 5. Crystal Data for Lig1‑4,8,9Ti(OArMe2)2 and Lig2Ti(O2Ar)
parameter/
a
compound
1
2
3
4
8
9
Lig2Ti(O2Ar)
C36H32N2O4Ti C38H36N2O4Ti C44H48N2O4Ti C52H64N2O4Ti C38H36N2O6Ti C36H30Br2N2O4Ti·C8H10O C28H22N2O4Ti·CH2Cl2
formula
Mw
604.54
632.59
716.74
828.95
664.59
884.50
583.30
P-1
space group
a [Å]
P21/n
P21/n
P21/c
P21/c
Cc
P21/c
22.886(4)
9.457(2)
29.041(6)
13.1517(9)
14.281(1)
16.989(1)
9.104(4)
26.33(1)
17.051(7)
13.656(1)
18.749(1)
18.542(1)
13.372(1)
23.313(4)
12.053(2)
11.673(1)
19.951(2)
17.293(2)
8.877(3)
9.769(3)
16.796(5)
93.968(5)
106.103(3)
109.979(5)
1307.6(7)
173(1)
2
b [Å]
c [Å]
α [deg]
β [deg]
γ [deg]
V [Å3]
T [K]
Z
100.420(3)
100.437(1)
101.481(7)
99.331(1)
117.448(3)
105.846(2)
6181(2)
173(1)
8
3138.0(4)
173(1)
4
4004(3)
123(1)
4
4684.9(6)
173(1)
4
3334.2(9)
173(2)
4
3874.3(6)
173(1)
4
μ(Mo Kα) [mm−1] 0.319
no. of reflns measd 66 071
no. of reflns unique 13 460
0.317
0.256
44 124
8748
0.228
0.306
2.333
0.571
34 149
6828
50 477
10 217
0.0960
0.1028
19 307
7797
44 147
9220
14 979
6024
Rint
0.1312
0.1397
0.0766
0.0707
0.0498
0.0837
0.0500
0.0625
0.0418
0.0375
0.0246
2
R(Fo ) for [I >
2σ(I)]
0.0483
Rw for [I > 2σ(I)] 0.2368
0.1399
0.2176
0.1846
0.1135
0.0845
0.1186
a
Compounds 1−4, 8, and 9 represent Lig1−4,8,9Ti(OArMe2)2, respectively. For Lig7,10Ti(OArMe2)2 see ref 34.
medium exposure were conducted similarly, following preincubation of
the complex with the medium at 37 °C for 1, 3, and 24 h, where the
compound Lig7Ti(OArMe2)2 was administered in DMSO. Kinetic
studies by NMR to monitor hydrolysis of dimethylphenolato groups to
give polynuclear products (Figure S1, Suporting Information), for
establishment of relative water resistance, were performed as
previously described28,29 using ca. 6 mM of the complex solution in
d8-THF and adding D2O to give a final solution of 1:9 D2O/d8-THF
with added D2O being >1000 equiv relative to Ti(IV). The t1/2 value is
based on a pseudo-first-order fit for each compound (Figure S2,
Suporting Information, all R2 fit values ≥ 0.98), as the average of
values obtained for several signals of the labile ligands. The results
were verified by including p-dinitro benzene as an internal standard.
The sum of integration of (CH3)2C6H3O−Ti and (CH3)2C6H3OH in
the first measurement following D2O addition was assigned as
integration 1. Characterization of hydrolysis products was further
conducted by reacting selected complexes with 10 000 equiv of water
for 3 days, removing the volatiles, washing with hexane, and dissolving
their enhanced solubility in biologically relevant solvents, such
as DMSO. Studies currently underway with these highly
promising anticancer complexes include the detailed inves-
tigation of their mechanism of action to identify the active
species and its relation to the hydrolysis processes.
EXPERIMENTAL SECTION
■
For H2Lig1,2,7,10 and Lig1,2,7,10Ti(OArMe2)2 see our previous
communication.34 Syntheses of ligands H2Lig3−6,8,11 were achieved as
previously described starting from the commercially available
substituted salicylaldehyde.57−62 1,2-Diaminobenzene (99.5%), 3-
bromo-2-hydroxybenzaldehyde (97%), and pyrocatechol (99%) were
purchased from Sigma Aldrich Chemical Co. Inc. and used without
further purification. Ti(OArMe2)4 was synthesized as previously
described.42 All solvents used for procedures requiring an inert
atmosphere were either distilled from potassium or potassium/
benzophenone under nitrogen or dried over alumina columns on a
M. Braun SPS-800 solvent purification system. All experiments requiring
dry atmosphere were preformed in an M. Braun drybox under nitrogen
atmosphere or using Schlenk line techniques. NMR data were
recorded using an AMX-500 MHz Bruker spectrometer. CDCl3
(99.8%) was purchased from Cambridge Isotope Laboratories Inc.
and used without further purification. X-ray diffraction data were
obtained with a Bruker Smart Apex diffractometer, running the
SMART software package. After collection, the raw data frames
were integrated by the SAINT software package. The structures were
solved and refined using the SHELXTL software package. Crystal
data for Lig1−4,8,9Ti(OArMe2)2 and Lig2Ti(O2Ar) are summarized in
Table 5.34
1
the product in DMSO-d8 for H NMR analysis (Figure S3, Suporting
Information).
H2Lig9. 1,2-Diaminophenol (0.27 g, 2.50 mmol) dissolved in
methanol was added to 3-bromosalicylaldehyde (1.00 g, 5.0 mmol)
dissolved in methanol. The solution was stirred at reflux for 3 h. The
solution was then cooled to give an orange precipitate, which was
collected by vacuum filtration and dried in vacuo, giving H2Lig9 (84%)
Anal. Calcd for C20H14Br2N2O2: C, 50.66; H, 2.98; N 5.91. Found: C,
1
50.69; H, 2.64; N, 5.93. H NMR (500 MHz; CDCl3) 13.71 (2H, s,
OH), 8.59 (2H, s, CH), 7.63 (2H, dd, J = 7.5, 1.5 Hz, Ar), 7.36 (4H,
m, Ar), 7.22 (2H, m, Ar), 6.83 (2H, t, J = 7.5 Hz Ar). 13C NMR (125
MHz; CDCl3) 163.8, 158.2, 141.9, 136.9, 131.8, 128.2, 120.7, 120.2,
119.9, 111.5.
Elemental analyses were preformed in the microanalytical laboratory
in our institute. Accurate-Mass Q-TOF LC/MS measurements were
carried on an Agilent Technologies 6520. Cytotoxicity was measured
on HT-29 colon and OVCAR-1 ovarian cancer cells obtained from
ATCC Inc. using the methylthiazolyldiphenyltetrazolium bromide
(MTT) assay as previously described,28 starting from 0.9 × 106 cells,
incubated with the complexes at different concentrations for 3 days,
where each measurement was repeated at least 3 × 3 times.
(Bzac)2Ti(OiPr)2 was administered in THF as were all salen
complexes under general study, while the reference compounds
cisplatin and Cp2TiCl2 were administered in DMSO. Relative IC50
values were determined by a nonlinear regression of a variable slope
(four parameters) model. Measurements relating to the effect of
Lig3Ti(OArMe2)2. Lig3Ti(OArMe2)2 was synthesized in analogy to
Lig1,2,4,10Ti(OArMe2)234 from Ti(OArMe2)4 (100 mg, 0.19 mmol) and
H2Lig3 (81 mg, 0.19 mmol) (73%). Anal. Calcd for C44H48N2O4Ti: C,
1
73.73; H 6.75; N, 3.91. Found: C, 73.22; H, 6.47; N, 3.78. H NMR
(500 MHz; CDCl3) 8.64 (2H, s, CH), 7.57 (2H, m, Ar), 7.41 (2H, dd,
J = 9.0, 2.5 Hz, Ar), 7.39 (2H, m, Ar), 7.27 (2H, d, J = 2.5 Hz, Ar),
6.67 (2H, d, J = 9.0 Hz, Ar), 6.60 (4H, d, J = 7.5 Hz, Ar), 6.33
(2H, t, J = 7.5 Hz, Ar), 1.97 (12H, s, CH3), 1.26 (18H, s, CH3).
13C NMR (125 MHz; CDCl3) 164.4, 164.3, 143.1, 141.1, 135.5,
131.1, 129.2, 128.8, 127.6, 126.0, 122.3, 118.5, 117.5, 117.1, 34.1,
31.3, 17.4.
1802
dx.doi.org/10.1021/ic202092u | Inorg. Chem. 2012, 51, 1796−1804

Inorganic Chemistry
Article
Lig4Ti(OArMe2)2. Lig4Ti(OArMe2)2 was synthesized similarly from
Ti(OArMe2)4 (100 mg, 0.19 mmol) and H2Lig4 (102 mg, 0.19 mmol).
The crude product was washed several times with dry hexane to give
the red product (58%). Anal. Calcd for C52H64N2O4Ti: C, 75.34; H,
7.78; N, 3.38. Found: C, 74.74; H, 7.56; N, 3.27. ESI-HRMS
(C52H64N2O4Ti + Na) m/z Calcd: 851.4238. [M+Na+] Found:
851.4242. 1H NMR (500 MHz; CDCl3) 8.62 (2H, s, CH), 7.57
(2H, d, J = 2.5 Hz, Ar), 7.48 (2H, m, Ar), 7.30 (2H, m, Ar), 7.17 (2H,
d, J = 2.5 Hz, Ar), 6.58 (4H, d, J = 7.5 Hz, Ar), 6.35 (2H, t, J = 7.5 Hz,
13C NMR (125 MHz; CDCl3) 162.5, 160.4, 160.0, 143.9, 138.5, 133.6,
129.9, 129.0, 122.6, 119.3, 118.6, 117.0, 110.7, 20.6. Following solution
of the X-ray structure, remeasuring the unit cell parameters of
additional crystals, and redetermining their H NMR confirmed that
the solved structure corresponds to the main product.
1
ASSOCIATED CONTENT
■
S
* Supporting Information
Ar), 1.78 (12H, s, CH3), 1.46 (18H, s, CH3), 1.32 (18H, s, CH3). 13
C
Crystallographic data, representative spectra and plots of
hydrolysis, spectra of representative hydrolysis products and
related structures, and plots of cytotoxicity toward OVCAR-1
cells. This material is available free of charge via the Internet at
http://pubs.acs.org.
NMR (125 MHz; CDCl3) 164.5, 163.8, 159.5, 142.9, 140.6, 137.3,
132.8, 129.5, 128.9, 127.7, 126.3, 123.7, 118.2, 116.7, 35.7, 34.3, 31.5,
30.2, 17.8.
Lig5Ti(OArMe2)2. Lig5Ti(OArMe2)2 was synthesized similarly from
Ti(OArMe2)4 (100 mg, 0.19 mmol) and H2Lig5 (71 mg, 0.19 mmol)
(78%). 1H NMR (500 MHz; CDCl3) 8.59 (2H, s, CH), 7.54 (2H, m,
Ar), 7.39 (2H, m, Ar), 7.02 (2H, dd, J = 9.0, 3.0 Hz, Ar), 6.80 (2H, d,
J = 3.0 Hz, Ar), 6.69 (2H, d, J = 9.0 Hz, Ar), 6.62 (4H, d, J = 7.5 Hz,
Ar), 6.36 (2H, t, J = 7.5 Hz, Ar), 3.74 (6H, s, CH3), 1.97 (12H, s,
CH3). 13C NMR (125 MHz; CDCl3) 164.3, 161.6, 159.1, 151.9, 129.5,
128.8, 127.6, 126.5, 126.0, 122.2, 118.9, 118.5, 117.1, 116.2, 56.1, 17.4.
Lig6Ti(OArMe2)2. Lig6Ti(OArMe2)2 was synthesized similarly from
Ti(OArMe2)4 (100 mg, 0.19 mmol) and H2Lig6 (89 mg, 0.19 mmol)
(72%). Anal. Calcd for C36H30Br2N2O4Ti: C, 56.72; H 3.97; N, 3.67.
Found: C, 56.42; H, 3.94; N, 3.59. 1H NMR (500 MHz; CDCl3) 8.57
(2H, s, CH), 7.55 (2H, m, Ar), 7.49 (2H, d, J = 3.0 Hz, Ar), 7.45 (2H,
m, Ar), 7.41 (2H, dd, J = 9.0, 2.5 Hz, Ar), 6.66 (2H, d, J = 9.0 Hz, Ar),
6.64 (4H, d, J = 7.5 Hz, Ar), 6.41 (2H, t, J = 7.5 Hz, Ar), 1.95 (12H, s,
CH3). 13C NMR (125 MHz; CDCl3) 165.1, 164.1, 158.6, 142.8, 140.0,
136.8, 130.1, 127.8, 126.0, 124.1, 120.0, 119.3, 117.3, 109.9, 17.3.
Lig8Ti(OArMe2)2. Lig8Ti(OArMe2)2 was synthesized similarly from
Ti(OArMe2)4 (100 mg, 0.19 mmol) and H2Lig8 (71 mg, 0.19 mmol)
(75%). Anal. Calcd for C38H36N2O6Ti: C, 68.68; H, 5.46; N, 4.22.
Found: C, 68.74; H, 5.24; N, 4.17. 1H NMR (500 MHz; CDCl3) 8.64
(2H, s, CH), 7.53 (2H, m, Ar), 7.39 (2H, m, Ar), 6.98 (2H, dd, J = 8.0,
1.5 Hz, Ar), 6.91 (2H, dd, J = 8.0, 1.5 Hz, Ar), 6.63 (2H, t, J = 7.5 Hz,
Ar), 6.61 (4H, d, J = 7.5 Hz, Ar), 6.35 (2H, t, J = 7.5 Hz, Ar). 3.83
(6H, s, CH3), 2.01 (12H, s, CH3). 13C NMR (125 MHz; CDCl3)
164.4, 159.8, 157.8, 149.0, 143.1, 129.4, 127.4, 126.8, 126.5, 123.1,
118.8, 118.6, 118.0, 117.1, 56.6, 17.2.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: tshuva@chem.ch.huji.ac.il.
ACKNOWLEDGMENTS
■
We thank Dr. Shmuel Cohen for crystallographic analyses.
This research received funding from the European Research
Council under the European Community’s Seventh Framework
Programme (FP7/2007-2013)/ERC Grant agreement no.
239603.
REFERENCES
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Lig9Ti(OArMe2)2. Lig9Ti(OArMe2)2 was synthesized similarly from
Ti(OArMe2)4 (100 mg, 0.19 mmol) and H2Lig9 (89 mg, 0.19 mmol)
(81%). Anal. Calcd for C36H30Br2N2O4Ti: C, 56.72; H, 3.97; N, 3.67.
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143.1, 140.5, 134.7, 130.0, 127.7, 126.3, 123.1, 119.2, 119.1, 117.4,
113.2, 17.7.
Lig11Ti(OArMe2)2. Lig11Ti(OArMe2)2 was synthesized similarly
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mmol) (71%). Anal. Calcd for C36H28Cl4N2O41Ti: C, 58.25; H, 3.80;
N, 3.77. Found: C, 58.25; H, 3.40; N, 3.75. H NMR (500 MHz;
CDCl3) 8.62 (2H, s, CH), 7.60 (2H, m, Ar), 7.57 (2H, m, Ar), 7.45
(2H, d, J = 2.5 Hz, Ar), 7.27 (2H, d, J = 2.5 Hz, Ar), 6.68 (4H, d, J =
7.5 Hz, Ar), 6.45 (2H, t, J = 7.5 Hz, Ar), 2.02 (12H, s, CH3). 13C NMR
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128.8, 127.9, 126.2, 124.5, 123.3, 122.3, 119.7, 17.4.
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