Specific Design of Titanium(IV) Phenolato Chelates Yields Stable and Accessible, Effective and Selective Anticancer Agents

Article abstract of DOI:10.1002/chem.201601389

Octahedral titanium(IV) complexes of phenolato hexadentate ligands were developed and showed very high stability for days in water solutions. In vitro cytotoxicity studies showed that, whereas tetrakis(phenolato) systems are generally of low activity pres

Full text of DOI:10.1002/chem.201601389

DOI: 10.1002/chem.201601389
Full Paper
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Drug Design |Hot Paper|
Specific Design of Titanium(IV) Phenolato Chelates Yields Stable
and Accessible, Effective and Selective Anticancer Agents
Sigalit Meker,[a] Ori Braitbard,[b] Matthew D. Hall,[c] Jacob Hochman,[b] and Edit Y. Tshuva*[a]
Abstract: Octahedral titanium(IV) complexes of phenolato
hexadentate ligands were developed and showed very high
stability for days in water solutions. In vitro cytotoxicity stud-
ies showed that, whereas tetrakis(phenolato) systems are
generally of low activity presumably due to inaccessibility,
smaller bis(phenolato)bis(alkoxo) complexes feature high an-
ticancer activity and accessibility even without formulations,
also toward a cisplatin-resistant cell line. An all-aliphatic con-
trol complex was unstable and inactive. A leading phenolato
complex also revealed: 1) high durability in fully aqueous
solutions; accordingly, negligible loss of activity after prein-
cubation for three days in medium or in serum; 2) maximal
cellular accumulation and induction of apoptosis following
24–48 h of administration; 3) reduced impact on noncancer-
ous fibroblast cells; 4) in vivo efficacy toward lymphoma
cells in murine model; 5) high activity in NCI-60 panel, with
average GI50 of 4.6Æ2 mm. This newly developed family of
TiIV complexes is thus of great potential for anticancer thera-
py.
of these complexes remains unclear,[1i,6] partly due to difficul-
ties in investigating unstable complexes that rapidly hydrolyze
in vitro and in vivo.
Introduction
The ever-growing number of studies on titanium(IV) complexes
as anticancer agents has repeatedly revealed their high poten-
tial as effective alternatives for the drug cisplatin and its Pt-
based derivatives.[1] Titanium exhibits low intrinsic toxicity
when compared with platinum and other second and third pe-
riodic metals;[1b,d,h,2] moreover, TiIV complexes demonstrate
high cytotoxicity toward a wide range of cancer cells and, so
far, without reports on drug resistance development.[1b,c,g,h,2–3]
TiIV complexes that have reached clinical trials include budo-
titane [(bzac)2Ti(OEt)2] and titanocene dichloride [Cp2TiCl2],
which are based on diketonato (bzac) or cyclopentadienyl (Cp)
ligands, respectively.[4] They have failed the trials because of in-
sufficient solubility and rapid hydrolysis that led to the forma-
tion of inactive and unidentified oxo-bridged polynuclear spe-
cies, with low overall efficiency.[1c,e,i,5] The mechanism of action
Employing inert phenolato ligands, particularly, diamino bi-
s(phenolato) “salan” ligands (Scheme 1), produced TiIV com-
plexes with increased hydrolytic stability compared with budo-
titane and titanocene dichloride, as well as enhanced cytotox-
icity.[7] Salan TiIV complexes were effective toward various cell
lines, with strong dependence of activity on ligand structure;[8]
in particular, bulky substitutions diminished the cytotoxici-
ty.[8c,d,9] Formulated salan based complexes demonstrated in
vivo activity toward colon and cervical cancer.[10]
Scheme 1. Bis(isopropoxo) salan TiIV complexes.
[a] S. Meker, Prof. E. Y. Tshuva
The Institute of Chemistry, The Hebrew University of Jerusalem
Jerusalem 9190401 (Israel)
Fax: (+972)2-6584282
The salan TiIV complexes eventually undergo hydrolysis,
within hours in water solutions, to give oxo-bridged polynuc-
lear products.[7b,8c,9b,11] Later studies reported that these de-
fined clusters act as active cellular species.[12] Importantly, be-
cause these active species lacked labile ligands, their activity
proved that labile groups are not a prerequisite for cytotoxicity
of TiiIV complexes. Additional studies demonstrated that a mon-
onuclear TiIV complex without labile ligands was also cytotoxic.
Nevertheless, cytotoxicity was recorded only when these com-
pounds were formulated into nanoparticles, because their
steric bulk had rendered them biologically inaccessible.[12–13]
Recent work has also presented mixed-ligated complexes with
E-mail: edit.tshuva@mail.huji.ac.il
[b] Dr. O. Braitbard, Prof. J. Hochman
Department of Cell and Developmental Biology
Alexander Silberman Institute of Life Science
The Hebrew University of Jerusalem
Jerusalem 9190401 (Israel)
[c] Dr. M. D. Hall
NCATS Chemical Genomics Center
National Center for Advancing Translational Sciences
National Institutes of Health
9800 Medical Center Drive, Rockville, MD, 20850 (USA)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201601389.
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one salan ligand and an additional polydentate group, which
has also assisted in obtaining stable and active compounds,[10a]
as well as a polydentate ligand system that partially dissociat-
ed in water.[14]
rakis(alkoxo) system with the smallest size of all, was analyzed
for comparison. The different substitutions on the aromatic
rings also enabled fine tuning of the structural effects.
Ligands L1–6H4 were prepared by using a range of proce-
dures that included standard Mannich and other condensation
and substitution reactions, as detailed in the Supporting Infor-
mation (Scheme S1). Ligands were characterized by NMR,
HRMS, and elemental analyses. Preparation of L1–6Ti complexes
was achieved through a convenient single-step synthesis by
mixing Ti(OiPr)4 with one equivalent of the ligand in tetrahy-
drofuran (THF) for at least two hours at room temperature
(Scheme 2). Complexes L1,3Ti were isolated by evaporation of
the solvent to produce the complexes in quantitative yield.
Complex L2Ti was purified by recrystallization from diethyl
ether. Complexes L4–6Ti precipitated from THF solution and
were isolated by decantation without further purification. Due
to the water sensitivity of the precursor Ti(OiPr)4, all syntheses
were conducted under an inert atmosphere in a glove box,
but all other manipulations including purification and charac-
terization of the complexes were performed in air. Complex
L7Ti is commercially available and was used without further pu-
rification.
This paper presents a ligand structural design that optimizes
the TiIV properties that are relevant to anticancer therapy. Com-
plexes were obtained by employing tetra(anionic) hexadentate
phenolato ligands, based on the potent “salan” skeleton, and
demonstrated superior biological accessibility independently
of the formulation, durability for days in water, and high in
vitro and in vivo anticancer activity and selectivity. These com-
plexes operate in the cell as administered, without undergoing
any hydrolysis steps, and preliminary mechanistic studies point
to the involvement of ordered apoptotic pathways.
Results and Discussion
Synthesis and Characterization
To develop highly potent TiIV agents with increased water dura-
bility and biological accessibility, ligands were designed as hex-
adentate and tetra(anionic) to solely accommodate a coordina-
tively saturated octahedral geometry of neutral TiIV centers,
and to enable diffusion through the hydrophobic cell mem-
brane. Thus, suitable ligands that were variable in structure,
connectivity, substitutions, and donor type were synthesized
(see the Supporting Information). The complexes L1–6Ti synthe-
sized were therefore all octahedral with a single chelating
ligand (Scheme 2). Given that phenolato ligands presumably
contribute to stability but also increase the steric bulk and
thus hampers accessibility compared with aliphatic alkoxo li-
gands,[12a] varied number of phenolato moieties were em-
ployed: four in L1–3Ti, two in L4–6Ti, and none in L7Ti. Complexes
L1,2Ti are differently substituted derivatives of the chlorinated
tetrakis(phenolato) compound, which was reported in the pre-
ceding communication to exhibit high hydrolytic stability and
appreciable cytotoxicity but only in a nanoformulated form be-
cause of its poor accessibility.[12b] Complex L3Ti, with a different
connectivity, gives a more compact wrapping of the tetraki-
s(phenolato) ligand around the metal. Complexes L4–6Ti, with
substitution of two bulky aromatic systems with aliphatic li-
gands, are markedly smaller relative to L1,2Ti, giving a bis(phe-
nolato)bis(alkoxo) system. Complex L7Ti, as an all-aliphatic tet-
1
The complexes were characterized by H NMR spectroscopic
analysis, with the spectra featuring a single set of signals for
the phenolato moieties of the ligand in L4–6Ti with a fitting set
of signals for the aliphatic region, and two sets of phenolato
signals for L1–3Ti. The number of signals confirmed that no ad-
ditional ligand was bound to the TiIV center. Additionally, repre-
sentative complexes were characterized by single-crystal X-ray
crystallography. Complexes L2–5Ti were crystallized by slow
evaporation of solvents under air to produce yellow or orange
single crystals that were suitable for crystallographic analysis.
The ORTEP drawings of these complexes together with lists of
selected bond lengths are provided in Figure 1 and Table 1, re-
spectively.
All X-ray structures exhibited octahedral geometry with C2-
symmetry and were consistent with the NMR features. Bond
lengths and angles for these complexes were generally compa-
rable with those previously reported for bis(isopropoxo) salan
complexes:[7a,8c,d,15] NÀTi and OÀTi bond lengths were mostly
2.24–2.27 ꢁ, and 1.85–1.89 ꢁ, respectively; exceptions include
a relatively short NÀTi bond of 2.19 ꢁ in L3Ti, presumably be-
cause of the smaller five-membered ring, and the OÀTi bonds
Scheme 2. TiIV complexes of bis- and tetrakis-phenolato ligands L1–6Ti, and their aliphatic analogue L7Ti.
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Figure 1. Ortep drawing with 50% probability ellipsoids of a) L2Ti; b) L3Ti; c) L4Ti; and d) L5Ti. Hydrogen atoms and solvent molecules were omitted for clarity.
Activity towards Human Cancer Cell Lines
Table 1. Selected[a] bond lengths (ꢁ) for L2–5Ti.
Cytotoxicity of the TiIV complexes was tested toward human
Bond
Bond length (ꢁ)
L4Ti
L2Ti
L3Ti
L5Ti
colon HT-29 and human ovarian A2780 cancer cell lines. The
complexes were administered to the cells without surfactant-
based formulations in 0.5% dimethyl sulfoxide (DMSO) or THF
solutions and were incubated with the cells for 3 days, follow-
ing which, cell viability was determined by the methylthiazolyl-
diphenyl-tetrazolium bromide (MTT) assay, as previously pub-
lished (Figure 2, Table 2).[16] Tetrakis(phenolato) complexes L1,3Ti
were inactive toward HT-29 and A2780, presumably because of
the poor biological accessibility of the bulky tetrakis(phenola-
N(1)ÀTi
N(2)ÀTi
O(1)ÀTi
O(2)ÀTi
O(3)ÀTi
O(4)ÀTi
2.268(3)
2.239(3)
1.955(3)
1.827(3)
1.928(3)
1.839(3)
1.851(2)
2.249(3)
2.237(3)
1.865(2)
1.884(3)
1.887(3)
1.867(2)
2.238(3)
2.234(3)
1.864(2)
1.871(2)
1.866(2)
1.882(2)
1.917(2)
2.192(2)
[a] For additional bond lengths and angles see Table 1 in the Supporting
Information.
in L2Ti varied between 1.83 and 1.95 ꢁ, which is comparable to
its previously characterized analogue.[12b] The shorter bonds to
the two O-donors of the unsubstituted rings O2 and O4 than
those to O1 and O3 of the brominated rings probably reflect
the steric hindrance of the substituents. Interestingly, in com-
plexes L3–5Ti, the OAr-N-N-OAr moiety, that is, the ‘salan moiety’
of the ligand, was bound with a cis-configuration of the O-
donors, thus enabling trans-configuration for the two remain-
ing O-donors either aromatic, as in L3Ti, or aliphatic, as in
L4À5Ti; that is, in a mer-mer configuration. This binding mode
differs from that observed in the structures of the bis(isopro-
poxo) salan complexes, for which the two O-donors of the iso-
propoxo groups were in cis-configuration, giving a fac-fac geo-
metry.[7a,8c,d,15] However, this cis-configuration of the phenolato
O-donors was observed in the X-ray structures of polynuclear,
oxo-bridged hydrolysis products of the salan complexes.[7b,8c,9
b,11a]
Taking into account that these hydrolysis products were
suggested as the cellular active species,[12] a similar mechanism
of action for L1–6Ti and the bis(isopropoxo) salan complexes is
structurally plausible.
Figure 2. Dependence of a) HT-29 and b) A2780 cell viability on adminis-
tered concentration of L1–7Ti following a standard incubation period of 3
days.
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Hydrolytic Stability in Biologically-Relevant Environments
Table 2. Relative IC50 (mm) values toward HT-29, A2780, and A2780-cp
human cancer cells.
Hydrolytic stability is a key factor in the utilization of TiIV com-
plexes as anticancer agents. Moreover, kinetically stable com-
plexes may act as the active species, without mandatory hy-
drolytic activation in the biological environment. The hydrolyt-
ic stabilities of the complexes were measured by using
the comparative tool previously described, which involves
1H NMR analyses of 10% D2O solutions in [D8]THF or
[D6]DMSO.[7b,8d,9b,10a,19] Complexes L1–5Ti all demonstrated very
high stability, with less than 20% hydrolysis during the course
of three days following D2O addition (Table S2). Due to the in-
sufficient solubility of L6–7Ti in the aforementioned mixed aque-
Complex
HT-29
A2780
A2780-cp
RF[a]
L1Ti
>100[b]
>100[b]
>100[b]
5.7Æ0.4
6.4Æ0.9
0.74Æ0.08[c]
>100[b]
13Æ3
>100[b]
1.5Æ0.5
>100[b]
6.4Æ0.7
1.5Æ0.8
0.43Æ0.03
>100[b]
2Æ2
>100[b]
4Æ2
–
3
–
0.7
3
1
–
16
30
L2Ti
L3Ti
>100[b]
4.8Æ0.2
5Æ1
L4Ti
L5Ti
L6Ti
0.47Æ0.05
>100[b]
33Æ2[13]
210Æ90[13]
L7Ti
cisplatin
carboplatin
220Æ60
7Æ3
[a] Resistance factor: the ratio between IC50 value toward the resistant
cell line, A2780-cp, and IC50 value toward the sensitive cell line, A2780;
[b] Inhibition of cells growth at concentration >100 mm was under 50%
and therefore IC50 values were not determined; [c] Maximal inhibition
was 60% of cell growth.
ous–organic solution, the stability of L6Ti was not evaluated,
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1
and L Ti was analyzed in 90% [D4]MeOD. Interestingly, t = for
2
the hydrolysis of L7Ti was 3 h, which, even considering the dif-
ferent solvent, indicates substantially reduced stability for this
complex despite the chelating nature of its ligand. It is thus
clear that the strong binding of phenolato O-donors to the ti-
tanium metal is essential for the stability of the complexes
and, consequently, for their cytotoxicity, given that L7Ti was
inactive (see above).[7a] Importantly, L4Ti was identified as
a leading complex in terms of both its high cytotoxicity
(Table 2) and high hydrolytic stability (Table S2) and relative
solubility; it was thus selected as a representative derivative
for further testing.
to) system.[12] Interestingly, L2Ti, with two unsubstituted pheno-
lato rings, was a borderline case, with minor activity toward
HT-29 and significantly higher activity toward A2780 cells,
which are generally more sensitive. Its activity implies that
steric and electronic effects can improve the biological activity
and accessibility of the complex.[7b,8b,9,11b,12a,15a] Most important-
ly, L4–6Ti, with the markedly smaller bis(phenolato)bis(alkoxo)
system, demonstrated high activity toward both cell lines, sup-
porting their higher accessibility. Nevertheless, L7Ti, which is
a water-soluble and a compact complex, was inactive toward
both cell lines, evidencing that steric size and solubility are not
the only factors that determine cytotoxicity (see below). These
findings overall indicate that reducing steric hindrance caused
by the aromatic systems is beneficial for increasing accessibili-
ty, and consequently cytotoxicity, but nevertheless, that the
phenolato donors play an important role in the performance
of the complexes.[7a]
To evaluate the hydrolytic stability under conditions that
mimic the in vitro testing, L4Ti, at 54 mm concentration giving
maximal cytotoxicity, was incubated for 3 days at 378C in 0.5%
DMSO in water solution; thus, the high solubility of this com-
plex enabled analysis of a cytotoxic TiIV complex in nearly fully
aqueous solution, with no precipitation under the aforemen-
tioned conditions. Complex L4Ti showed no change in the
1H NMR spectrum following this incubation period (Figure S3).
The complex was also stable for 3 days in 0.5% DMSO aqueous
solution of pH 5.5, which is characteristic of the environment
of some tumor tissue. These studies overall attest to the high
stability and durability of this complex in aqueous solutions
under both neutral and acidic pH, relevant to biological condi-
tions. Additionally, these results imply that L4Ti itself may act as
the cellular active species, without ligand exchange or preced-
ing hydrolysis steps.
The activities of the complexes were also evaluated on a cis-
platin-resistant human ovarian A2780-cp cell line and the re-
sults were compared with those obtained with its sensitive an-
alogue A2780; the results were also compared with the analo-
gous results obtained with cisplatin and carboplatin. The
A2780-cp cell line features an increased ability to repair DNA
damage as well as cytogenetic abnormalities, and therefore
has cross-resistance toward the alkylating agent Melphalan, in-
tercalating DNA agent Doxorubicin, and irradiation, in addition
to cisplatin.[17]
To further challenge the hydrolytic stability of the com-
plexes, L4Ti was pre-incubated in cell culture growth media so-
lution: 0.5% DMSO and 10% Fetal Bovine Serum (FBS) in
RPMI-1640 medium solution, for different periods, following
which its cytotoxicity toward HT-29 was measured based on
the crystal violet assay (Figure 3, Table 3).[20] The complex re-
tained most of its cytotoxicity even following 72 h of pre-incu-
bation, with only a slight increase in IC50 from 6.3Æ0.6 to 10Æ
1 mm. Complex L4Ti demonstrated similar activity even follow-
ing 1 week of exposure to medium without any additional ac-
tivity loss, with IC50 of 10Æ2 mm (Figure S4), which emphasizes
the durability of the complex in aqueous solutions. This result
is exceptional considering that related previously reported TiIV
compounds demonstrated activity loss following 5–24 h in me-
dium.[7a,8a,21]
Whereas cisplatin and carboplatin exhibited a dramatic de-
crease in their cytotoxicity toward the A2780-cp resistant line,
with resistance factors of 16 and 30, respectively, L4Ti exhibited
the lowest resistance factor (<1), with similar cytotoxicity
toward both lines (Table 2, Figure S1). All other complexes
L1–3,5–7Ti also demonstrated similar cytotoxicity pattern toward
the resistant and the sensitive cell lines, with resistance factors
less than 3 (Table 2, Figure S2). This similar activity suggests
that different mechanistic pathways occur for the titanium
complexes than those involving the platinum drugs and the in-
tercalators and alkylating agents; possibly, these mechanisms
are not affected by mismatch repair (MMR) pathways.[18]
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Figure 3. Dependence of HT-29 cell viability on administered concentration
of L4Ti following pre-incubation in 0.5% DMSO cell growth medium for vary-
ing periods prior to administration to the cells for additional standard 3 day
incubation period.
Table 3. Relative IC50 (mm) values of L4Ti following pre-incubation in 0.5%
DMSO cell culture growth medium or blood serum for varying periods
prior to cell addition and 72 h incubation with HT-29 cells.
Figure 4. a) Dependence of HT-29 cell viability on administered concentra-
tions of L4Ti following varying periods of incubation with cells. b) Cellular
uptake of L4Ti, 54 mm, in HT-29 cells, presented as dependence of L4Ti influx
on time. c) Dependence of HT-29 cell viability on administered concentra-
tions of L4Ti following varying periods of exposure to cells, after which, incu-
bation in fresh medium continued to complete a standard 3 day incubation
period.
0 h
24 h
72 h
medium
serum
6.3Æ0.6
5Æ1
7Æ1
9Æ2
10Æ1
12Æ3
In an additional experiment, aiming to assess the resilience
of the complex to blood serum as is often required for treat-
ment, L4Ti was pre-incubated in 100% serum for varying peri-
ods before cytotoxicity was measured similarly (Table 3, Fig-
ure S5). Again, the cytotoxicity of L4Ti decreased only slightly
as a consequence of prolonged pre-incubation, with IC50 of
12Æ3 mm after 72 h. Notably, the complex activity was not in-
fluenced by the presence of serum proteins such as albumin,
which may imply that such proteins do not take active part in
the complex mechanism of action.[7a,14,22] Nevertheless, the re-
sults overall support the potential high effectiveness of the
complex for future utility.
clearly showed an increase of Ti influx with time for up to ca.
20 h, following which, the influx rate reached a plateau.[22] In
a complementary experiment, the cytotoxicity of L4Ti was mea-
sured following relatively short periods of exposure to the
cells, after which the medium was replaced to remove excess
complex that had not penetrated the cell, and the experimen-
tal setup was allowed to further incubate to reach a total stan-
dard 3 days incubation time (Figure 4c). Maximal effect of the
complex was observed after 16–24 h incubation with the cells,
which is in good agreement with the direct cellular uptake re-
sults. Given that maximal activity was recorded following 48 h
incubation with cells, it is certainly likely that parameters other
than uptake rate control the cell growth inhibition, such as
controlled cellular pathways. Moreover, the kinetic aspects that
were revealed emphasize the importance of employing hydro-
lytically stable TiIV complexes, which retain their structure and
remain active in the biological system for sufficiently long peri-
ods.
Kinetic Aspects of Cytotoxicity and Cellular Uptake
To assess the kinetic aspects of the biological activity that may
be relevant to drug mechanism and toxicity, the cytotoxicity of
L4Ti was measured following varying incubation periods with
the cells. Specifically, the cytotoxicity was measured after 0, 24,
48 h and compared with the cytotoxicity obtained after a stan-
dard 72 h incubation period as described above (Figure 4a).
Complex L4Ti promoted significant cell growth inhibition
after 24 h with IC50 of 30Æ10 mm, and maximal activity after
48 h with IC50 of 9Æ2 mm. The long period between the com-
plex administration and the growth inhibition suggests a con-
trolled death pathway or slow cellular uptake. To gain insight
on the kinetic aspects of cell entry, cellular uptake of L4Ti was
investigated by measuring the absorbance of the complex in
medium solution in the extracellular environment following
periods of incubation with HT-29 cells (Figure 4b). Control
measurements with no cells confirmed that no change in ab-
sorbance (<2%) occurred for more than 30 h. The results
Mechanistic Insights
Apoptotic cell death induced by exposure to L4Ti was evaluat-
ed by using an Annexin V-FITC kit (Figure 5a–d, Figure S8). HT-
29 cells were exposed to L4Ti at two different concentrations:
13 mm (ca. 2ꢂIC50) and 54 mm (inflecting maximal inhibition),
and were analyzed in comparison with control cells that were
treated with medium only for similar incubation periods. At
both concentrations, the treated cells showed gradual increase
of early and late apoptotic signs with longer exposure time to
the complex, with a more pronounced effect under the higher
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Figure 7. L4Ti protects mice against a challenge of malignant lymphoma
cells: mice inoculated IP with 1.5ꢂ106 T-65 lymphoma cells were subjected
to IP injections of L4Ti (2 mg) every other day for 4 weeks; control mice were
injected with PBS.
TiIV complex without the necessity of formulation. It is note-
worthy that no clinical signs of toxicity were observed in the
treated mice; the mice appeared healthy, with no weight or
hair loss, no grooming or behavioral changes, no swellings or
eye infections, and no mortality occurring during the period of
the experiment. These findings overall agree with the above
finding on tumor selectivity, confirming the advantage of the
titanium bio-friendly metal.
Figure 5. Annexin V-FITC apoptosis assay for HT-29 cells in early or late
apoptotic stages following exposure to L4Ti, 13 mm a) control, b) 9 h, c) 24 h,
d) 48 h.
concentration (Figure S8). These findings support the conclu-
sion that controlled cell death is induced by the titanium com-
plex, which may be beneficial for reduced toxicity. To further
assess the aspect of toxicity, the selectivity of L4Ti to cancerous
cells was evaluated by measuring its cytotoxicity toward non-
cancerous MRC-5 human fibroblast lung cells (Figure 6).[23]
Complex L4Ti showed reduced impact toward these cells, im-
plying a degree of cancer selectivity.
Activity toward the NCI-60 Panel: Toward Applicability
The range of activity of L4Ti was evaluated toward the NCI-60
panel of the Developmental Therapeutics Program (DTP) of
the National Cancer Institute (NCI).[24] The NCI-60 panel consists
of 57 cell lines of 9 distinct tumor types. L4Ti exhibited a wide
range of activity and significant cytotoxicity toward all cell
lines tested, which is manifested by its low mean growth inhib-
ition of 50% (GI50) value toward all cell lines: 4.6Æ2 mm
(Table 4, Figure S9). Particularly noteworthy is the high activity
of the complex toward multidrug resistant (MDR) cell lines,
NCI/ADR-RES ovarian, HCT-15 colon, and UO-31 renal.[24] This
high activity implies the ability of L4Ti to overpass such resist-
ance mechanism in the three different tumor types tested, as
also observed for cisplatin.[25] Together with the activity toward
cisplatin-resistant cells as described above, these findings em-
phasize the high potential of this complex for utility as a drug
in a wide range of cancer diseases.
The relative sensitivity of each cell line to L4Ti was calculated
by noting the difference between GI50 toward the particular
line and the mean GI50 toward all cell lines. A negative differ-
ence represents particularly sensitive lines (Figure 8). Most
cancer types included lines particularly sensitive to L4Ti; excep-
tions include leukemia and prostate cancer, which demonstrat-
ed relative resistance to the complex, with GI50 between 4 and
11 mm. Other TiIV complexes based on salan ligands previously
showed significantly lower activity toward the NCI-60 panel,
but, interestingly, showed similar sensitivity pattern, with leu-
kemia cell lines being more resistant and melanoma cell lines
being more sensitive.[10b] These results imply that a similar
mechanism for titanium complexes in general is likely. When
comparing the results with those obtained for cisplatin,[25] it is
Figure 6. Dependence of HT-29, A2780, and MRC-5 cell viability on adminis-
tered concentration of L4Ti following a standard 3 day incubation period;
MRC-5: IC50 =11 Æ1 mm.
In Vivo Analysis
The in vivo efficacy of L4Ti was evaluated on mice subjected to
the aggressive T-65 lymphoma cells as representative model.
Notably, L4Ti was administered to the animals without any ve-
hicle formulation. Of 10 mice treated, 8 survived beyond 60
days. In the control group 5/8 mice did not survive the lym-
phoma challenge (Figure 7). These findings demonstrate an an-
titumorigenic effect of L4Ti; furthermore, they emphasize the
stability and high accessibility of the complex in vivo that ena-
bled first determination of antitumoral activity of phenolato
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Table 4. NCI-60 panel, 57 human tumor cell lines screening data of L4Ti, presented as growth inhibition of 50% (GI50) values.
Panel/Cell Line
Leukemia
GI50 (mm)
Panel/Cell Line
melanoma
GI50 (mm)
CCRF-CEM
K-562
MOLT-4
RPMI-8226
SR
5.89
5.43
6.19
9.39
4.01
7.02
4.04
5.21
3.11
3.63
3.57
3.37
3.45
2.65
4.69
5.48
3.59
4.22
3.82
4.69
4.98
5.24
4.58
5.92
2.41
4.75
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
UO-31
PC-3
DU-145
MCF7
3.88
1.90
4.27
4.28
4.16
5.50
2.75
11.80
4.39
4.53
4.30
2.29
3.84
7.66
3.37
4.99
5.17
2.04
4.65
4.48
1.38
7.06
2.79
10.90
5.20
3.33
0.64
5.76
6.37
3.72
2.80
non-small cell lung
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
ovarian cancer
colon cancer
renal cancer
CNS cancer
SNB-19
SNB-75
U251
prostate cancer
breast cancer
MDA-MB-231/ATCC 0.7
HS 578T
BT-549
T-47D
MDA-MB-468
Figure 8. Relative sensitivity of cell lines of the NCI-60 to L4Ti. The delta between the mean GI50 value toward all cell lines and the GI50 value toward each cell
line (mm) is presented on the y-axis. Data for cell lines that were particularly sensitive to L4Ti appear above the x-axis, and appear bellow for relatively resistant
cell lines.
notable that some differences appear in sensitivities, with the
most pronounced being the sensitivity of non-small-lung
cancer to L4Ti, while being relatively resistant to cisplatin,[25]
emphasizing an additional advantage of the TiIV compound.
The screening results of L4Ti were compared to the activity
of additional standard anticancer agents toward the NCI-60 to
seek correlative information about L4Ti and its potential mech-
anism. Correlations were calculated by using the publicly avail-
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able NCI COMPARE algorithm.[26] There were no strong (R>0.6)
correlations between the activity of L4Ti and the standard anti-
cancer agents. The five strongest correlations found (Table S3)
represent diverse mechanisms of action. Two of these (alkylat-
ing agent/nucleoside analogue) interfere with DNA synthesis/
replication/fidelity. This suggests that the mechanism of action
of cell killing by the titanium complex is distinct, and is not re-
flected in the standard anticancer agents. Furthermore, this
distinct action also suggests that L4Ti might be used effectively
in combination with different chemotherapeutic drugs.
60 results that show very high general activity and identify par-
ticularly sensitive cancer types including those resistant to cis-
platin, all contribute to the high medicinal potential of the
leading complex L4Ti and its derivatives. The marked selectivity
to cancer tissue, as is also demonstrated by the lack of ob-
served toxicity to treated animals, further emphasizes the ad-
vantage of the titanium bio-friendly metal. Accordingly, pre-
liminary mechanistic insights imply that apoptotic pathways
are slowly activated following the secure insertion of the resil-
ient complexes in the cell, through an apparently distinct
mechanism.
To conclude, L4Ti and its derivatives represent highly attrac-
tive alternatives for anticancer therapy that are potentially
more widely active and selective than currently used drugs,
and may be administered either solely or in combination with
other drugs operating by different mechanisms. The com-
pounds may also afford technical advantages such as pro-
longed shelf life and durability under biologically relevant envi-
ronments. Additionally, their stability in water and high biologi-
cal accessibility independently of formulations enable launch-
ing a more profound mechanistic study in biological environ-
ment to finally elucidate the cellular target and interactions
with it. Such studies are ongoing in our laboratory.
Conclusion
This paper presents the design and development of highly ef-
fective anticancer TiIV complexes of hexadentate phenolato
chelates. The previously reported generations of TiIV anticancer
compounds, starting from titanocene dichloride and budoti-
tane, have taught us that water stability is of great significance
and certainly achievable, and, moreover, that labile ligands
that were responsible for reducing water resistance are not es-
sential for the activity.[12b] Additionally, an inert system without
rapidly hydrolyzing ligands may be potent without preceding
hydrolysis steps, avoiding undesired release of potentially toxic
side products such as the free dissociated labile ligands. Thus,
the simplest system presented herein includes a single cos-
tume designed chelating ligand of the proper number of cova-
lent and coordinative bonds to saturate the metal center with-
out the need for additional donors of any kind. The highly con-
venient synthesis makes this system particularly attractive, and
the ability to incorporate various substitutions to tune the
complex properties, and, in particular, the hydrophilicity/hydro-
phobicity balance, enabled producing highly effective com-
pounds that are both able and stable, and can be administered
both in vitro and in vivo without formulations and still reach
their target and effectively inhibit cancer cell growth.
Experimental Section
Full experimental details are given in the Supporting Information.
Ligands L1,4H4 were synthesized according to published proce-
dures.[27] Ligands L2,3,5,6H4 and TiIV complexes L1–3,5,6Ti were synthe-
sized as detailed in the Supporting Information. The synthesis of
L4Ti as a representative complex is detailed below. L7Ti is commer-
cially available. All compounds were obtained with >95% purity.
Hydrolysis studies by 1H NMR spectroscopic analysis were per-
formed first in accordance with published procedures,[9b] recording
spectra immediately upon addition of 10% D2O to a solution of
the L1–7Ti in [D8]THF or [D6]DMSO and then following 3 days of in-
cubation at 378C. L7Ti was analyzed in 90% [D4]MeOD because of
solubility limitations. Hydrolysis for L4Ti was also measured by dis-
solving the complex in [D6]DMSO and diluting the solution with
H2O to give final solutions of 99.5% H2O and 0.5% organic solvent.
The complex concentration was chosen as that giving maximal ac-
A marked advantage of the leading system presented herein
is in the combination of high water-resistance with sufficient
solubility and accessibility. This is achieved by the incorpora-
tion of the proper balance between phenolato and aliphatic
alkoxo donors. As all-phenolato based systems are highly
stable yet mostly inactive without formulations,[12b] and the all-
aliphatic alkoxo system is water soluble yet of mediocre stabili-
ty and thus inactive, the bis(phenolato)bis(alkoxo) system is
identified as optimal for achieving the desired features, with
not only stability for days in pure water solution, but also resil-
ience in biological environments, including media and sera, for
prolonged periods. Actually, the high stability of these com-
plexes enabled direct analysis in nearly fully aqueous solutions
of different pH conditions, which further emphasize their dura-
bility in biologically relevant environments.
The potency of the leading compound L4Ti is reflected by
high activity and marked selectivity, whereas mechanistic in-
sights point to programmed rather than instant cell death. Ac-
tivity, which is often higher even than that of cisplatin, along
with in vivo efficacy, constitute highly effective anticancer
drugs. A wide activity range toward both cisplatin-sensitive
and cisplatin-resistant lines, as well as MDR lines, and the NCI-
1
tivity (54 mm). H NMR spectrum was recorded at point 0, and the
aqueous solution was incubated at 378C for 3 days, followed by
evaporation of solvents with a lyophilizer. The sample was redis-
solved in [D6]DMSO and the 1H NMR spectrum was re-recorded.
This experiment was repeated by using acetic acid buffer solution
(pH 5.5).
Cytotoxicity was measured on human colon HT-29, noncancerous
human fibroblast lung cells MRC-5 (purchased in 2015 from ATCC
Inc), human ovarian cisplatin-sensitive A2780, and human ovarian
cisplatin-resistant A2780-cp (purchased in 2014 from ECACC Inc.).
Cytotoxicity was measured by using either the MTT assay as previ-
ously described,[16] or the crystal violet assay.[20] Cells (0.6ꢂ106) in
medium (1% penicillin/streptomycin antibiotics; 1% l-glutamine;
10% fetal bovine serum (FBS) and 88% medium RPMI-1640) were
seeded into a 96-well plate and allowed to attach for one day. The
cells were subsequently treated with the reagent tested at 10 dif-
ferent concentrations to give a final concentration of the com-
plexes up to 200 mgLÀ1, in 0.5% organic solvent solutions (see the
Supporting Information for details). L7Ti was dissolved directly in
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medium, without THF or DMSO, because of its high solubility in
water at concentrations of up to 1000 mgLÀ1. Control wells were
treated with similar amounts of THF or DMSO. MTT assay: After
a standard of 3 days incubation at 378C in 5% CO2 atmosphere,
MTT (0.1 mg in 20 mL) was added and the cells were incubated for
an additional 3 h. The MTT solution was removed, and the cells
were dissolved in 200 mL isopropanol. The absorbance at 550 nm
was measured with a Bio-Tek EL-800 microplate reader spectropho-
tometer. Crystal violet assay: Following 24, 48, or 72 h incubation,
the medium was removed and the cells were fixated in 200 mL
formaldehyde 37%, at RT, for 5 min. The formaldehyde was re-
moved and 100 mL of 1% crystal violet aqueous solution was
added. Following 20 min incubation of the cells with crystal violet
at RT, the crystal violet solution was removed and the cells were
washed twice with water. The cells were dissolved in 200 mL meth-
anol and the absorbance at 570 nm was measured.
iodide and their manipulation was conducted according to the
procedure detailed in the user protocol.
In vivo analysis was conducted as follows: Balb/C mice (6–8 weeks
old females) were obtained from Harlan (Israel), held in a SPF facili-
ty (AAALAC accreditation #1285) and treated in accordance with
NIH guidelines with approval of the institutional committee for
ethics in animal experimentation. Mice were inoculated IP with
1.5ꢂ106 T-65 lymphoma cells, derived from the T-25 murine lym-
phoma.[29] Starting 24 h after the challenge, mice were subjected
to IP injections of L4Ti dissolved in 0.5% DMSO saline solution
every other day for 4 weeks. Mice were followed up to 60 days
post inoculation.
Complex L4Ti
The complex was synthesized by stirring Ti(OiPr)4 (0.050 g,
0.18 mmol) and L4H4 (75 mg, 0.18 mmol) for 2 h in THF (5 mL)
under an inert atmosphere. The yellow precipitant that formed
spontaneously was isolated by decantation in quantitative yield.
Slow evaporation of the solvent from a chlorobenzene solution of
the complex under air afforded single crystals that were suitable
Measurements following preincubation in medium or serum were
performed similarly, following 24, 72, or 168 h preincubation and
analyzed by using the crystal violet assay.
Measurements following short exposure periods to complexes
were performed similarly, whereby after 2, 4, 6, 8, 16, or 24 h of in-
cubation with complexes, the medium was replaced with fresh
medium for completion of 72 h total incubation period. The analy-
ses were performed by using the MTT assay.
1
for X-ray crystallography. H NMR (500 MHz, [D6]DMSO): d=6.87 (s,
2H; Ar), 6.40 (s, 2H; Ar), 4.32 (d, J=13.5 Hz, 4H; CH2), 3.81 (t, J=
9.5 Hz, 2H; CH2), 3.71 (d, J=13.5 Hz, 2H; CH2), 3.50 (d, J=9.0 Hz,
2H; CH2), 3.24 (t, J=9.5 Hz, 2H; CH2), 3.11 (d, J=9.0 Hz, 2H; CH2),
2.82 (d, J=10.0 Hz, 2H; CH2), 2.13 (s, 6H; CH3), 2.12 ppm (s, 6H;
CH3); 13C NMR (125 MHz, [D6]DMSO): d=158.9, 136.5, 130.5, 126.1,
122.4, 116.9, 68.6, 63.0, 60.1, 59.9, 19.3, 18.4 ppm; HRMS: m/z calcd
for C24H32N2O4Ti: 458.1888; found: 458.1880; elemental analysis
calcd (%) for C24H32N2O4Ti: C, 62.61; H, 7.01; N, 6.08; found: C,
63.09; H, 6.81; N, 6.18.
Each measurement was repeated at least 3ꢂ3 times, namely, three
repeats per plate, all repeated at least 3 times on different days (9
repeats altogether). Relative IC50 values were determined by non-
linear regression of a variable slope (four parameters) model. IC50
values are reported as the mean ÆSD of at least three independ-
ent experiments.
Complex L4Ti was supplied to the Developmental Theraputic Pro-
gram of National Cancer Institute for evaluation on the 57 cell lines
in the NCI-60 panel. GI50 values reported by the NCI-60 program
represent growth inhibition of 50% as calculated from [(TiÀTz)/
(CÀTz)]ꢂ100=GI=50, where Ti is treated cells, Tz is cells at time 0,
C is control cells; this corresponds to concentration of the drug
that inflicts reduction of 50% in the net protein increase (mea-
sured by SRB staining) during incubation with the drug.[28] The
COMPARE analysis was based on the publicly available COMPARE
algorithm.[26]
Crystal data for L4Ti
C24H32N2O4Ti·(C6H5Cl); M=572.97; monoclinic; a=6.6037(9), b=
33.141(5), c=13.069(2) ꢁ, a=g=908, b=96.827(2)8; V=
2840.0(7) ꢁ3; T=173(1) K; space group P21/c; Z=4; m(MoKa)=
0.433 mmÀ1
; 30550 reflections measured, 6201 unique (Rint =
0.0810), R(F82) for [I>2s(I)]=0.0834, Rw for [I>2s(I)]=0.1645.
CCDC 1436310 (L4Ti) contains the supplementary crystallographic
data for this paper. These data are provided free of charge by The
Cambridge Crystallographic Data Centre.
Cellular uptake measurements of L4Ti were performed by growing
HT-29 cells in a 75 cm2 flask until 70% confluency was reached;
then growth cell medium was replaced with 40 mL of fresh
medium without phenol red. To the flask, 200 mL of L4Ti dissolved
in DMSO was added to give a final concentration of 54 mm in 0.5%
DMSO (1 mg per flask). The cells were incubated at 378C in 5%
CO2. Samples (1.5 mL) were collected after 0, 2, 4, 6, 8, 13, 16, 24,
26, 28, and 30 h; the absorbance at 330 nm was measured with
a UV/Vis spectrophotometer (Genesys 10S, Thermo Scientific); VI-
SIONlite program. Control experiments showed a negligible de-
crease of the complex absorbance (<2%) after 30 h in the afore-
mentioned solutions. The calibration curve for L4Ti at the relevant
range of concentrations and spectra of complex are given in the
Supporting Information (Figure S6–S7). Reported values represent
mean ÆSD of three independent experiments.
Apoptotic cell death assay was performed on HT-29 cells with an
Annexin V-FITC Apoptosis Detection Kit purchased from Merck Mil-
lipore. Cells were seeded (0.1ꢂ106) in 6-well plate. Each well was
referred to as one sample. Cells were incubated with 13 or 54 mm
of L4Ti in 0.5% DMSO solution for different periods of time. Control
wells were treated with only 0.5% DMSO for the same exposure
periods. Staining of the cells with Annexin V-FITC and propidium
Acknowledgements
We thank Dr. Benny Bogoslavsky for crystallographic analyses,
and Dr. Karina Hazan for elemental analyses and MS measure-
ments. Funding was received from the European Council
under the European Community’s Seventh Framework Pro-
gramme (FP7/2007-2013)/ERC Grant agreement (No. 239603).
Keywords: antitumor agents · drug design · ligand design ·
N,O ligands · titanium
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Received: March 23, 2016
Published online on && &&, 0000
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FULL PAPER
&
Drug Design
S. Meker, O. Braitbard, M. D. Hall,
J. Hochman, E. Y. Tshuva*
&& – &&
Specific Design of Titanium(IV)
Phenolato Chelates Yields Stable and
Accessible, Effective and Selective
Anticancer Agents
Titanium can have it all: Costume
design of octahedral titanium(IV) com-
plexes of singly chelating phenolato li-
gands produced anticancer agents with
the most desired combination of fea-
tures (see figure). A leading bis(phenola-
to)bis(alkoxo) complex is highly and
widely effective and selective both in
vitro and in vivo, it is highly water
stable, accessible without formulation,
and operates through apoptotic path-
ways ca. 24 h following administration.
Overall, the compounds have immense
medicinal potential.
Chem. Eur. J. 2016, 22, 1 – 11
www.chemeurj.org
11
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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These are not the final page numbers! ÞÞ