Cytotoxic titanium salan complexes: Surprising interaction of salan and alkoxy ligands

FULL PAPER

DOI: 10.1002/chem.200902312

Cytotoxic Titanium Salan Complexes: Surprising Interaction of Salan and

Alkoxy Ligands

Timo A. Immel, Ulrich Groth, and Thomas Huhn*^[a]

Abstract: The synthesis, biochemical

evaluation, and hydrolysis studies of a

wide selection of alkyl- and halogen-

substituted titanium salan alkoxides are

presented herein. A systematic change

in the employed alkoxides revealed

that both the bulk of the salan ligands

and the steric demand of the labile lig-

between the cytotoxicity and the steric

demand of the whole complex is

merely based on an altered hydrolysis

or on the interaction with biomole-

cules, the behavior of selected com-

plexes under hydrolytic conditions and

the influence of transferrin were inves-

tigated. Complexes differing only in

their labile alkoxy ligands gave the

same hydrolysis products with similar

hydrolysis rates but displayed cytotox-

icities that differed in the range of one

order of magnitude. Thus, it seems that

the hydrolysis product is not the active

species but rather that the unhydro-

lysed complex is important for the first

interaction with a biomolecule. This

promoted the idea of hydrolysis being

a detoxification pathway. In accordance

with the above conclusion, chloro-sub-

A

stituted complex [Ti

displayed high cytotoxicity (IC₅₀

(Ph^ClN^Me(O^iPr)₂]

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

obtained biological activity. Surprising-

ly, these two factors are not indepen-

ACHTUNGTRENNUNGdent from each other; lowering the

steric demand of the alkoxide of a hith-

erto nontoxic complex renders it cyto-

toxic. Therefore, our data suggest that

the overall size of the complex exerts a

strong influence on its biological activi-

ty. To decide whether the correlation

a

ꢀ5 mm) and surprisingly high hydrolytic

stability (t_1/2=108 h). These findings,

together with the observed cytotoxicity

in a cisplatin-resistant cell line, make

halo-substituted salan complexes an in-

teresting target for further studies.

Keywords: antitumor agents · cyto-

toxicity · hydrolysis · structure–ac-

tivity relationships · titanium salan

complexes

Introduction

fortunately the spectrum of activity of these platinum com-

plexes is limited and they are associated with significant

dose-limiting toxicities.^[1–3]Therefore, much research has

been devoted to the identification of new metal complexes

with lower toxicity, similar antitumor activity, and reduced

tumor resistance.^[4–14]Titanium(IV) complexes showed en-

couraging antitumor activity in various cell lines and little

cross resistance to cisplatin was observed.^[15–17]Although

much effort was undertaken to identify completely new tita-

nium complexes that show antitumor properties, nearly all

investigated complexes were derivatives of either titanocene

Today, metal complexes, such as cisplatin, carboplatin, and

oxaliplatin, play an important role in cancer therapy, but un-

[a] T. A. Immel, Prof. Dr. U. Groth, Dr. T. Huhn

Fachbereich Chemie and

Konstanz Research School Chemical Biology

Universitꢀt Konstanz

Universitꢀtsstrasse 10

Fach 720, 78457 Konstanz (Germany)

Fax : (+49)7531-884424

E-mail: thomas.huhn@uni-konstanz.de

dichloride ([Ti

(OEt)₂]; Hbzac=phenylbutane-1,3-dione).^[23–25]

One characteristic of the above-mentioned complexes is

ACHTUGNTRNEU(NGN Cl₂)ACHTUNGTRENNUNG ACHTUNGTRENNUNG(bzac)₂-

(Cp₂)])^[18–22]or budotitane ([Ti

AHCTUNGTRENNUNG

Supporting information for this article is available on the WWW

under http://dx.doi.org/10.1002/chem.200902312, and contains synthet-

ic procedures for the preparation of ligands and titanium complexes;

detailed spectroscopic and analytical data for all new compounds;

their very fast rate of hydrolysis that leads to unidentified

products. In an aqueous environment, the loss of the labile

groups occurs within seconds, whereas the more inert lig-

¹H NMR spectra of complexes [Ti

(O^iPr)₂], [Ti(Ph^FN^Me(O^iPr)₂], [Ti(Ph^ClN^Me

₂A

(O^iPr)₂] at their respective t_1/2values with overlaid spectra of the cor-

T

)₂ACTHUNGTRENNUG ACTHUNGTRENNUNG

(O^iPr)₂], [Ti

AHCTUNGTRENNUNG

ands hydrolyze on a timescale of hours or days.^[26,27]On the

A

E

)

U

A

)

G

ACHTUNGTRENNUNG

one hand, this hampers the identification of the active spe-

cies as well as the biological target and the mechanism of

action. It was found that titanocene dichloride is enriched in

responding salans; details of the cytotoxicity assays and viability plots

in Hep G2 for all compounds; color graphics for Figures 2–8 and Fig-

ures 11–12.

Chem. Eur. J. 2010, 16, 2775 – 2789

ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

2775

areas near the nuclear chromatin and covalently binds to

DNA and inhibits DNA synthesis.^[28–31]Titanocene dichlor-

moieties. Finally, employing four different titanium alkox-

ides ([Ti(OEt)₄], [Ti(OiPr)₄], [Ti(OnBu)₄], [Ti(OtBu)₄]) in

the complex formation allowed us to fine-tune the steric

demand of the titanium-bound labile ligands as well.

G

E

R

ACHTUNGTRENNUNG

ide was also reported to inhibit human topo

A

On the other hand, it is assumed that some ligand lability is

necessary for the biological effect and that ligand hydrolysis

leads to formation of the active species.

The o,p-disubstituted salans were accessible by a Mannich

condensation. Simple reflux of the appropriate substituted

phenol, N,N’-dialkylethylenediamine, and formaldehyde in

Concerning the cellular uptake, the role of ligand lability

is controversial. Although it is necessary to strip off the lig-

methanol gave ligands (Ph^b–fN^a–c

) in moderate yield as

2

ands when Ti^IVis trafficked via transferrin,^[33–37]a route via

shown in Scheme 1.^[43–45]In the case of aryl unsubstituted

ligand (Ph^HN^Me)₂, salicylaldehyde was reacted with ethylene-

albumin could leave the complexes intact. An adduct of the

complex stabilized by albumin might enter the cell, which

would promote a more active role for these drugs in con-

trast to the prodrug role proposed for the transferrin deliv-

ery mechanism.^[38]

Consistent with these findings, in 2007 two methyl substi-

tuted titanium salan complexes were reported to be cytotox-

ic independent of transferrin.^[39]By using a small collection

of six alkyl-substituted titanium salan complexes, Tshuva

and co-workers showed an influence due to the steric

demand of the ligand and proposed a connection between

the hydrolytic behavior of these complexes and their biolog-

ical activity.^[40–42]Exploring this third class of titanium com-

plexes with antitumor activity, we could demonstrate that

complexes of halogen-substituted salans in particular reveal

promising biological properties. Their IC₅₀values are com-

parable to cisplatin and, in contrast to their alkyl-substituted

congeners, they almost exclusively induce apoptotic cell

death.^[43]

Scheme 1. Synthesis of salan ligands by Mannich condensation.^[43–45]

[*] Salans with R¹=H were not accessible by this synthesis and were in-

stead synthesized according to a published procedure.^[46]

diamine, then the resulting imine was reduced with sodium

borohydride and subsequently methylated by reductive ami-

nation to give the desired salan.^[46]Finally, metalation of

Herein we describe the synthesis, biological evaluation,

and hydrolytic behavior of a library of more than 40 alkyl-

and halogen-substituted titanium salan complexes. The ob-

tained data are discussed with

these salans with different titanium alkoxides Ti

shown in Scheme 2, gave titanium(IV) salan complexes

(O^a–d)₄, as

ACHTUNGTRENNUNG

respect to structure–activity re-

lations and the influence of hy-

drolysis on cytotoxicity.

Results and Discussion

To elucidate the impact of both

steric and electronic properties

of the ligands on the antitumor

activity and also the hydrolysis

Scheme 2. Synthesis of titanium salan complexes by metalation with different titanium alkoxides.^{[43, 46,47]}

behavior, the synthesis of

a

small library of more than 40

different salan complexes was envisaged. For a systematic

study, the influence of substituents on the aromatic rings

and at the bridging nitrogen atoms of the salan as well as

the role of the labile alkoxy ligands had to be addressed in-

dependently. To differentiate between electronic and steric

influences, we decided to utilize a series of halogen-substi-

tuted phenols in the ligand preparation alongside different

alkyl-substituted ones. Coupling these phenols with three

different amino linkers allowed the systematic construction

of amino-phenolato-ligands (salans). Thus, those ligands fea-

ture different steric bulk at the bridging nitrogen atoms and

cover a broad range of electronic influences at the phenol

[Ti(Ph^a–fN^a–c(O^a–d)₂] in nearly quantitative yield as single

)₂ACHTUGNRTENNNUG

(racemic) isomers.^[43,47]To remove trace impurities, all com-

pounds were recrystallized at least once prior to cytotoxicity

studies. Purities were proven by combustion analysis.

All of the complexes described above were tested for an-

titumor behavior by using the well-established AlamarBlue

assay, which is known to be highly reproducible and more

sensitive than the MTT assay.^[48]The cytotoxicity was studied

in two different cell lines: the human cervix carcinoma cell

line HeLa S3 and the Hep G2 cell line, an established

human hepatocarcinoma cell line with epithelial morpholo-

gy.^[43]Cisplatin was used as a reference substance in all

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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

Table 1. Selected bond lengths [ꢂ] and angles [8] for a series of complexes with different alkoxides.

(Ph^MeN^Me(O^Et)₂] (Ph^MeN^Me(O^nBu)₂] (Ph^MeN^Me(O^tBu)₂]

[Ti ₂A [Ti ₂A [Ti )₂ACHTUNGTRENNUNG

assays. It showed an IC₅₀of

(1.2ꢁ0.4) mm in HeLa S3 and

(3.0ꢁ1.3) mm in Hep G2 cells.

G

)

N

G

)

E

G

ꢂ

O1 Ti1

1.8964(18)

1.8977(18)

1.8300(19)

1.8328(19)

2.342(2)

2.336(2)

104.60(9)

94.96(9)

92.50(8)

92.71(8)

95.97(8)

166.78(8)

164.54(8)

90.29(8)

88.41(8)

81.43(8)

90.20(8)

164.27(9)

80.72(8)

88.51(8)

75.43(8)

1.8937(13)

1.9086(13)

1.8209(13)

1.8226(13)

2.3392(16)

2.3214(14)

105.95(6)

97.03(6)

1.923(3)

1.903(3)

ꢂ

All IC₅₀values are given as O2 Ti1

ꢂ

O3 Ti1

1.780(2)

1.779(3)

2.371(3)

2.365(3)

mean values from at least three

independent experiments each

done in four replicates.

Selected complexes were sub-

ject to time-resolved hydrolysis

studies monitored by NMR

spectroscopy to assess their sta-

bility under biologically rele-

ꢂ

O4 Ti1

ꢂ

N1 Ti1

ꢂ

N2 Ti1

O3-Ti1-O4

O3-Ti1-O1

O4-Ti1-O1

O3-Ti1-O2

O4-Ti1-O2

O1-Ti1-O2

108.78(12)

95.90(11)

92.34(12)

93.18(11)

96.99(12)

164.16(11)

162.30(13)

88.50(12)

86.94(11)

80.57(11)

89.31(11)

161.01(12)

79.70(11)

87.48(11)

73.98(11)

93.58(6)

89.45(5)

96.06(6)

166.46(6)

164.25(6)

88.13(6)

88.92(5)

81.93(5)

90.36(6)

163.21(6)

80.21(5)

87.90(5)

76.24(5)

vant conditions. We were espe- O3-Ti1-N2

O4-Ti1-N2

cially interested in the influence

O1-Ti1-N2

of electronic features at the

O2-Ti1-N2

salan ligand (halogen vs. alkyl)

O3-Ti1-N1

on the speed and product for-

mation of hydrolysis. Further-

more, the impact of steric

crowding around the metal

center and its effect on hydroly-

O4-Ti1-N1

O1-Ti1-N1

O2-Ti1-N1

N2-Ti1-N1

sis was paid special attention by utilizing alkoxides with dif-

fering steric demands.

As revealed by the H NMR spectra, the geometrical fea-

were refined as described above, which resulted in a higher

R-factor of 6.97. A summary of experimental crystal data

for all determined structures is given in the Experimental

Section (Table 9).

1

tures of the tetradentate [ONNO]-type ligands in all of our

complexes are in general very similar. The N-CH₂CH₂-N

methylene and the benzylic protons showed the familiar AB

pattern consistent with C₂symmetry and a fac–fac wrapping

mode.^[49]To compare the influence of the two monodentate

ligands on complex geometry, a series of complexes with dif-

Titanium-bound alkoxides

Influence on cytotoxicity in alkylsalans: Cytotoxicity studies

done in the late 1980s by Kçpf-Maier et al. with titanocenes

with different labile ligand substitutions showed the negligi-

ble influence on cytotoxicity.^[50]The question as to whether

the influence of labile ligands is more pronounced in the

case of titanium salan complexes still remains open. Recent

studies by Tshuva and co-workers that utilized catechol as a

bidentate substitute for the isopropoxy groups showed a

two-fold decrease in the cytotoxicity of the resulting com-

plex.^[40]Although the observed effect is significant, an ex-

planation is not evident because not only is the steric influ-

ence altered, but also the exchange of the two monodentate

isopropoxy groups for one bidentate catecholato ligand

leads to a different complex geometry. In the starting com-

plex the isopropoxy groups are oriented in a cis fashion, but

the two phenolato substituents of the salan are now occupy-

ing their position, which results in a loss of symmetry. We

decided to tackle the question of the labile ligandsꢃ influ-

ence on cytotoxicity by a more systematic approach. There-

fore, in the first series of experiments the salan backbone

was kept unchanged and instead the labile alkoxy ligands

were permutated, in the hopes of keeping the complex sym-

metry unchanged.

ferent labile alkoxides ([TiACTHNUTRGENNUG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^a,c,d)₂]) was closely

investigated. Suitable crystals for X-ray structure determina-

tion were either grown by slow diffusion of hexane into a sa-

turated solution in toluene ([Ti

ly from hexane solutions at room temperature by slow evap-

oration ([Ti ₂A ₂A

(Ph^MeN^Me(O^Et)₂], [Ti(Ph^MeN^Me(O^tBu)₂]). The

(Ph^MeN^Me(O^nBu)₂]) or direct-

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

A

)

E

N

) CHTUNGTRENNUNG

X-ray structures of this complex series again revealed the

typical geometry of C₂-symmetrical complexes with very

similar structural features. With the labile alkoxy ligands

bound at the equatorial plane in a cis fashion and both phe-

nolates occupying the bis-trans-axial positions, the com-

plexes exhibit only minor differences in bond length and

ꢂ

angles. With a slightly shorter Ti alkoxide bond (1.78 ꢂ)

compared with complexes with unbranched alkoxides, [Ti-

ACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^tBu)₂] is different to the others members of this

ꢂ

series (1.82–1.83 ꢂ). This is compensated by a longer Ti N

bond of 2.37 ꢂ as compared with 2.32–2.34 ꢂ for the other

series members, which results in a smaller phenolate-Ti-phe-

nolate (O-Ti-O) angle of 164.168 compared with 166.46-

166.788 for the other members of this class of complexes

(Table 1, Figure 1). For [TiACTHNUTRGENNUG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^Et)₂], the H atoms

of all methyl groups were found to be disordered by a rota-

To keep the probable steric influence as low as possible,

the first set of salan complexes evaluated in the AlamarBlue

assay consisted of the aryl-unsubstituted N-methylated lig-

tion of 608 and were, therefore, refined to fix the occupan-

cies at 0.5 each. In [TiACTHNURGTENNUG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^tBu)₂], one of the tert-

butoxy groups was found to be disordered over two sites

with occupancies of 0.675 and 0.325. The methyl groups of

the second tert-butoxy group showed rotational disorder and

A

)

metalated with titanium alkoxides

(O^a–c)₄. The dose-response curves obtained with the

HeLa S3 cell line for these three complexes ([Ti

(Ph^HN^Me)₂-

2

TiACHTUGNTRENNNUG

AHCTUNGTRENNUNG

Chem. Eur. J. 2010, 16, 2775 – 2789

ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

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2777

T. Huhn et al.

Figure 2. Viability of Hela S3 cells after incubation for 48 h with top: [Ti-

(Ph^HN^Me

)

T

N

)

G

(O^nBu)₂] (

)₂ACHTUNRGTNEGNU )

!

*

(Ph^MeN^Me

)

U

), [Ti

G

)

E

(

*

), [Ti-

)

U

were measured by using an AlamarBlue assay and show the dependence

on the steric bulk of labile ligands in complexes with an unchanged

[ONNO]-backbone.

Figure 1. ORTEP diagram of the molecular structures of complexes [Ti-

A

)

₂A

(O^tBu)₂] (bottom). Displacement ellipsoids are drawn at the

E

CHTUNGTRENNUNG

In the third set of the alkyl series, the 2,4-di-tert-butyl-sub-

50% probability level. Main occupancy is shown for disordered groups;

hydrogen atoms are omitted for clarity.

stituted salan was employed. Its titanium isopropoxy com-

plex, [Ti

(Ph^tBuN^Me(O^iPr)₂], is known to be nontoxic. Based

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

on the above results of the [TiACHTNUGTRENNNUG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^a–d)₂] complexes,

A

we initially hoped to regain cytotoxicity by reducing the

steric demand of the employed alkoxide. But even when the

smaller, non-branched ethoxide was used as the labile

E

) CHTUNGTRENNUNG

ligand, the resulting [Ti

nontoxic. An attempt to synthesize [Ti

failed, possibly due to steric interference of the labile lig-

ands and the salan backbone.

Table 2 summarizes the IC₅₀values of these titanium salan

(Ph^tBuN^Me

ACHUTGTNRENNUG )₂ACHTUNGTRENNUNG

(O^Et)₂] complex remained

G

)

G

A

)₂ACHTUNGTRENNUNG

Unexpectedly, we did observe a clear influence due to the

labile ligands when testing the second set of four complexes

AHCTUNGTRENNUNG

([TiACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^a–d)₂]). With methyl groups at positions two

and four of the phenolato substituents of the salan back-

bone, they feature slightly increased steric crowding around

the metal center and an enlarged overall steric demand.

This influence on cytotoxicity is especially pronounced when

comparing the bulky tert-butoxy-substituted [Ti

ACHTUNGTRENNUNG

complexes in both HeLa S3 and Hep G2 cells.

Influence on cytotoxicity in halogen-substituted salans: It is

known that halogen-aryl-substituted salan complexes of tita-

nium show a favorable biological profile.^[43]For this reason

as well as the gradual change in electronic and steric influ-

ences, a group of complexes that have fluoro, chloro and

bromo substituents at the aromatic moiety was examined.

(Ph^MeN^Me)₂-

ACHTUNGTRENNUNG

thy 10-fold decrease in cytotoxicity was observed (Fig-

ure 2b).

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Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

Table 2. IC₅₀[mm] values in Hela S3 and Hep G2 cells after incubation

for 48 h with complexes [Ti

AlamarBlue assay.

(Ph^a–cN^Me(O^a–d)₂], as measured by using an

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

Hela S3

R¹=Me R¹=tBu R¹=H

Hep G2

R¹=H

R¹=Me R¹=tBu

R³=Et

2.5ꢁ0.4 2.0ꢁ0.3 nontoxic 5.2ꢁ1.3 1.6ꢁ0.3 nontoxic

R³=iPr 4.5ꢁ1.3 2.3ꢁ0.1 nontoxic 5.5ꢁ1.9 2.1ꢁ0.1 nontoxic

R³=nBu 2.7ꢁ0.7 5.6ꢁ0.5 nontoxic 4.4ꢁ2.6 5.2ꢁ0.5 nontoxic

R³=tBu

–

19.8ꢁ2.4

–

26.9ꢁ8.6

–

Again, changing the alkoxide while keeping the backbone

unchanged should reveal the influence of steric interference

on the IC₅₀. This may offer the possibility of fine-tuning the

biological properties of such complexes.

In the case of the fluoro-substituted salan [Ti

(O^a–c)₂] complexes, changing the alkoxy ligands had virtually

no effect on the activity (Figure 3a) and resembles the [Ti-

(Ph^HN^Me(O^a–c)₂] case in this respect. All three complexes

₂A

(Ph^FN^Me

2

ACHTUNGTRENNUNG

A

) CHTUNGTRENNUNG

had their IC₅₀value within a very narrow margin of 1.3 to

1.6 mm.

In the chloro-substituted salan set of complexes, a strong

influence on the observed cytotoxicity due to the bulkiness

of the labile alkoxy ligand was observed (Figure 3b). The

complex with an ethoxy group had a more than 40-fold

higher cytotoxicity in HeLa S3 cells as compared with its

tert-butoxy congener, which shows that the effect was even

more pronounced than in the case of complexes [Ti-

ACHTUNGTRENNUNG ) CHTUNGTRENNUNG

(Ph^MeN^Me(O^a–d)₂].

₂A

For the most sterically demanding bromo-substituted

salan backbone (Ph^BrN^Me)₂, a tremendous decrease in cyto-

toxicity was observed for complexes with the more sterically

demanding alkoxides (Figure 3c), with the tert-butoxide

complex being completely nontoxic.

The IC₅₀values of all halogen-bearing titanium salan com-

plexes [TiACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^d–fN^Me(O^a–d)₂] are summarized in Table 3.

Table 3. IC₅₀[mm] values obtained for the [TiACTHNUGTRENNGU )₂ACHTUNGTRENNNUG

(Ph^d–fN^Me(O^a–

^d)₂]complexes by using the AlamarBlue assay with Hela S3 and Hep G2

cells. The cells were incubated with the complexes for 48 h prior mea-

surement.

Figure 3. Viability of Hela S3 cells after incubation for 48 h with top: [Ti-

(Ph^FN^Me(O^Et)₂] ( ), [Ti(Ph^FN^Me(O^iPr)₂] ( ), [Ti(Ph^FN^Me

*

ACTHNUGTERNNUNG )₂AHCTNUGTRENNUGN AHCTNUGRTENNGNU ) CTHUGNTNERNUNG ACHTUTGNRENNUNG ) CTHUNGTRENNNUG

₂A

(O^iPr)₂] ( ), [Ti

(O^tBu)₂] ( ); and bottom: [Ti(Ph^BrN^Me

) CHTUNGTRENNUNG

!

*

middle: [Ti

(Ph^ClN^Me

)

G

(Ph^ClN^Me

)

E

ACHTUNGTRENNUNG

(O^nBu)₂] ( ), [Ti

(Ph^ClN^Me

A

!

(

(Ph^BrN^Me(O^iPr)₂] ( ), [Ti(Ph^BrN^Me

*

), [TiACHUTNRGENNGU )₂AHCTNUGTRENUNNG ACHTUNEGRTNNGNU )₂ACTHUNGTRENNNUG

!

*

curves were obtained by using an AlamarBlue assay.

To elucidate the influence of transferrin on the cytotoxici-

ty of the halogen-substituted salan complexes, human apo-

transferrin was added to the medium prior to incubation of

selected complexes. For alkyl-substituted salans, it has al-

ready been shown that the cell penetration mechanism is in-

dependent of transferrin.^[40]Remarkably, even though the

mechanism of action seems to be different for halogen-sub-

Hela S3

R¹=F R¹=Cl

Hep G2

R¹=F R¹=Cl

R¹=Br

R³=Et 1.3ꢁ0.4 1.0ꢁ0.2 1.3ꢁ0.4 1.8ꢁ0.3 1.9ꢁ0.3

1.5ꢁ0.6

40ꢁ6

R³=iPr 1.6ꢁ0.1 5.3ꢁ0.2 13ꢁ1

2.2ꢁ0.1 4.0ꢁ0.2

R³=nBu 1.4ꢁ0.6 8.4ꢁ2.0 45.5ꢁ5.8 1.5ꢁ0.4 14.6ꢁ6.0 83.6ꢁ33.5

R³=tBu

–

41.4ꢁ6.6 nontoxic

–

53.2ꢁ14.5 nontoxic

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2779

T. Huhn et al.

stituted complexes,^[43]their cytotoxicity was not altered upon

addition of human apo-transferrin (Figure 4).

*

Figure 5. Viability of cisplatin-resistant (MGH-U1; ) and cisplatin-hy-

(Ph^FN^Me)₂-

*

persensitive (833K; ) cells after incubation for 48 h with [Ti

AHCTUNGTRENNUNG

alkoxide is the most toxic one. Moreover, it seems that

these two factors are not independent from each other. The

cytotoxicity of the sterically more demanding complexes of

(Ph^Cl,BrN^Me

) is more strongly affected by an additional

2

bulky labile ligand than is the complex of (Ph^FN^Me)₂with

only small fluoro substituents at the salan backbone. It is

evident that the influence of the steric demand of the labile

alkoxy ligands increases with the size of the halogen at the

backbone.

This result also gives an explanation as to why the cyto-

toxicity of [TiACTHNUTRGENNUG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^a–d)₂] is dependent on the alkoxy

ligands in the earlier set of alkyl-substituted titanium salan

complexes, whereas it is not for the unsubstituted [Ti-

ACHUTNGRENNUG ) CHTUNGTRENNUNG

(Ph^HN^Me(O^a–c)₂] complexes.

It seems that cytotoxicity does not depend solely on

₂A

Figure 4. Viability of Hela S3 cells (top) and Hep G2 cells (bottom) after

incubation for 48 h with [Ti

(Ph^FN^Me

)

(O^iPr)₂] ( ) and [Ti

(Ph^ClN^Me

)

(O^iPr)₂]

*

!

(

) with (c) and without (b) added human apo-transferrin

either the steric demand of the salan backbone or the bulki-

ness of the alkoxide used. In fact, quite the contrary seems

to hold true. Complexes with small substituents at the aro-

(10 mgmL^ꢂ1).^[51]Dose-response curves were obtained by using an Ala-

marBlue assay.

matic rings ([TiACTHNUTRGENNUG )₂ACHTUNGTRENNUNG

(Ph^H,FN^Me(O^a–d)₂]) show no decreased IC₅₀

Concerning the mechanism of action of salan complexes,

we were interested in whether they bear a resemblance to

the well-explored mechanism of cisplatin. Therefore, [Ti-

even with the bulky tert-butoxide as the labile ligand. The

smallest increase in steric demand on the side of the back-

bone leads to a stepwise decreased cytotoxicity of the com-

A

)₂A

(O^iPr)₂] was incubated with two different cell lines,

plex ([Ti(Ph^Me,Cl,BrN^Me(O^a–d)₂]), which depends on the bulk-

)₂ACHTUGNRTENNNUG

one of which was cisplatin resistant (human bladder carcino-

ma, MGH-U1) and one of which was cisplatin hypersensi-

tive (testicular germ-cell tumor, 833K). With sub-micromo-

lar IC₅₀values of 0.5ꢁ0.1 mm for MGH-U1 and 0.4ꢁ0.1 mm

for 833K, both cell lines showed a comparable sensitivity

(Figure 5). This is an indication that titanium salan com-

plexes and cisplatin differ in their mode of action.

iness of the alkoxide used. Being independent from the

nature of the employed substituent, alkyl or halogen, this

effect appears not to be of electronic origin but rather based

on the sheer size of the involved groups. Seemingly, there is

something like an upper threshold, from where on the

whole complex loses its cytotoxicity. To verify this conclu-

sion, we decided to synthesize another series of complexes

with additional steric hindrance at the bridging nitrogen

atoms of the salan backbone.

The results of the halogen-substituted [Ti

ACHTUNGTERNNUNG )

2

(O^a–d)₂] complexes with N-methylated salans show that both

the bulk of the salan ligands and the size of the labile li-

gands are of great importance for the obtained biological ac-

tivity.

Steric influence at the bridging nitrogen atoms

In each series of complexes in which the salan is fixed and

the alkoxide changed, the complex featuring the smallest

Cytotoxicity of N-ethylated complexes: Recently it has been

reported that in the case of a salan ligand with two methyl

2780

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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

groups at each aromatic ring, exchange of the methyl at the

bridging nitrogen atoms by an ethyl group resulted in a

complete loss of toxicity of the corresponding bis(isopro-

ACHTUNGTRENNUNG

poxy)titanium complex.^[40]Therefore, we were interested in

restoring the cytotoxicity of similar complexes just by less-

ening the steric demand of the alkoxide groups.

A first set of N-ethylated complexes was, therefore, pre-

pared by metalating the dimethyl salan ligand (Ph^MeN^Et)₂

with four different titanium alkoxides Ti

ent steric demands. In accordance with results published for

a very similar complex,^[40]we found [Ti(Ph^MeN^Et) (O^iPr)₂] to

₂A

be nontoxic. As expected, in the case of complexes [Ti-

₂A ₂A

(Ph^MeN^Et) (O^nBu)₂] and [Ti(Ph^MeN^Et) (O^tBu)₂] cytotoxicity was

(O^a–d)₄with differ-

ACHTUNGTRENNUNG

A

CHTUNGTRENNUNG

A

N

G

CHTUNGTRENNUNG

also not observed, presumably due to the even bulkier labile

alkoxides adding to the overall size. In the much more inter-

esting case of the [TiACTHUNTRGENNUG ₂ACHTUNGTRENNUGN

(Ph^MeN^Et) (O^Et)₂] complex with reduced

steric demand from the alkoxide, we could indeed restore

cytotoxicity in both cell lines used. With an IC₅₀value of

ꢀ25 mm in HeLa S3 and Hep G2 cells it showed a mediocre

biological activity, being one order of magnitude less than

the most toxic complexes (Table 4).

Table 4. IC₅₀[mm] values obtained for the [TiACTHNUGTRENNGU )₂ACHTUNGTRENNNUG

(Ph^d–fN^Et(O^a–d)₂] complexes

in Hela S3 and Hep G2 cells. The cells were incubated with the complexes

for 48 h prior to measurement.

Figure 6. Viability of Hela S3 cells after incubation for 48 h with fluoro-

*

aryl N-ethyl-bridged complexes [Ti

(Ph^FN^Et)

(O^a–c)₂] (top;

: [Ti-

Hela S3

Hep G2

)₂ACTHNGUTERNNUG

(O^nBu)₂]) and

R¹=Me R¹=F R¹=Cl

!

*

: [Ti

G

)

R

G

)

U

R

G

) CHTUNGTRENNUNG

R³=Et 25.0ꢁ6.7 2.7ꢁ0.6 2.9ꢁ0.8

24.4ꢁ8.7 4.9ꢁ0.9 3.4ꢁ0.9

nontoxic 2.5ꢁ1.1 14.3ꢁ4.0

(Ph^ClN^Et

)

E

(Ph^ClN^Et

)

N

A

CHTUNGTRENNUNG

!

*

: [Ti

*

R³=iPr nontoxic 2.3ꢁ0.4 7.1ꢁ1.7

which demonstrate the dependence on the steric demand of the labile

ligand in complexes with an unchanged N-ethyl backbone. Dose-re-

sponse curves were obtained by using an AlamarBlue assay. The [Ti-

R³=nBu nontoxic 4.9ꢁ0.9 239.0ꢁ41.1 nontoxic 5.7ꢁ1.6 256.6ꢁ82.7

R³=tBu nontoxic

–

nontoxic

–

nontoxic

AHCTUNGTRENNUGN )₂ACHTUNGTRENNUNG

(Ph^ClN^Et(O^tBu)₂] complex was nontoxic.

Encouraged by this finding, we turned our attention to-

wards the electron-deficient fluoro- and chloro-substituted

(O^nBu)₂] already showed a biological activity that was de-

ACHTUNGTRENNUNG

creased by more than two orders of magnitude. Finally, the

tert-butoxy complex is completely nontoxic. This effect is

even more pronounced than for the series of corresponding

complexes with N-ethyl groups [TiACHTNUTRGENNUG ₂ACHTUNGTRENNUGN

(Ph^d,eN^Et) (O^a–d)₂]. As

shown in Figure 6a, all complexes resulting from metalation

of the sterically less demanding fluoro-substituted ligand

(Ph^FN^Et)₂showed similar IC₅₀values in the range of 2.3 to

4.9 mm, even with the bulkiest titanium alkoxide. It seems

that the critical overall size for a significant decrease in tox-

icity cannot be reached within this specific set of ligands. A

completely different situation revealed a series of chloro-

N-methylated complexes [TiACTHNUTRGENNUG )₂ACHTUNGTRENNUNG

(Ph^ClN^Me(O^a–d)₂] (Table 3).

The observed dependence of cytotoxicity on the alkoxides

employed in the N-ethylated complexes [Ti(Ph^b,d,eN^Et)₂

AHCTUNGTRENNUNG

(O^a–d)₂] and the intensification of this effect compared with

the corresponding N-methylated complexes [Ti(Ph^b,d,eN^Me

)

2

AHCTUNGTRENNUNG

(O^a–d)₂] supports our hypothesis: The combined steric hin-

substituted salan complexes, [Ti

G

N

drance of salan and labile ligands is crucial for the anti-

cancer activity of this class of titanium complexes.

dous effect on cytotoxicity that depended on the size of the

alkoxide used was observable (Figure 6b). With an IC₅₀

On comparing both groups, we deduced the existence of a

lower steric threshold; below this threshold, alteration of

the overall steric demand either at the salan backbone or at

the labile ligands has only a minor influence on cytotoxicity.

This can be exemplified by varying the smallest of our com-

value of 2.9 mm, complex [TiACHTNUTRGENNUG ₂ACHTUNGTRENNUGN

(Ph^ClN^Et) (O^Et)₂] with a labile

ethoxy ligand is highly toxic in HeLa S3 cells. With the in-

creasing size of the utilized alkoxide, the respective com-

plexes showed a stepwise decrease in cytotoxicity in both

cell lines (Table 4). Whereas the isopropoxy complex is

plexes ([Ti

backbone, either at the aromatic rings (F vs. Cl) or at the

(Ph^FN^Me(O^Et)₂]). Minor variations of its salan

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

slightly less toxic with an IC₅₀value of 7.1 mm, [TiACTHNUTRGNEUNG

(Ph^ClN^Et)₂-

Chem. Eur. J. 2010, 16, 2775 – 2789

ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

2781

T. Huhn et al.

bridging nitrogens (Me vs. Et) has virtually no influence on

measured cytotoxicity. The same is true for variations of the

labile ligands.

In all the above cases, a further reduction in size does not

enhance cytotoxicity. The minimum IC₅₀values for such

salan complexes seem to be in the region of 1 mm. When ex-

ceeding the lower steric threshold, even minor changes in

steric demand have a tremendous effect on biological activi-

ty. This can be impressively demonstrated by comparing

complexes [TiACHTUNGTRENNUNG ₂ACHUTNGTNERNUG ACHTUNGERTNNUNG ₂ACHTUNGTRENNGUN

(Ph^FN^Et) (O^nBu)₂] and [Ti(Ph^ClN^Et) (O^nBu)₂].

Replacing the small fluoro by a larger chloro substituent led

to a 50-fold decrease in cytotoxicity. Exchanging methyl for

ethyl at the bridging nitrogens of the highly toxic [Ti-

A

)₂ACHTUNGTRENNUNG

(O^iPr)₂] has an even more drastic effect because it

ACHUTNGRENNUG ₂CAHTUNGTRENNUGN

(Ph^MeN^Et) (O^iPr)₂].

Figure 7. Viability of Hela S3 cells after incubation for 48 h with [Ti-

Concerning the influence of the alkoxy ligands on cyto-

(Ph^FN^Me(O^Et)₂] (Ph^FN^Et(O^Et)₂] (Ph^FN^Bn(O^Et)₂]

*

ACHUTGNERTNUNG ) HCUTNTGRENUNG ( ), [TiACHUTGNRENTNUG )₂ACHTNUGRTENNUG ( ), and [TiAHCUTNGERTNNUNG )₂ACHTUNGTRENNUNG

₂A

!

toxicity, a simple change from ethoxy to n-butoxy led to a

*

(

), showing the dependence on steric demand of the residue at the

nearly 100-fold reduced toxicity for complexes [Ti

(O^a,c)₂].

Toxicity is completely lost when a certain overall size is

(Ph^ClN^Et)₂-

ACHTUNGTRENNUNG

bridging nitrogens. Dose-response curves were obtained by using an Ala-

marBlue assay.

ACHTUNGTRENNUNG

exceeded. For the tert-butoxy-substituted salan complexes,

this upper steric threshold is already reached; even with

small labile ligands, such as ethoxy ([Ti

no toxicity was achieved.

An analysis of all the measured biological data and com-

parison with the corresponding chemical structures elucidat-

ed interesting structure–activity relationships, that is, the ob-

tained biological activity is not only dependent on the bulk

of the salan ligands alone, but also on the steric hindrance

of the labile ligands. Moreover, it seems that these two fac-

tors are not independent from each other because for bulky

salan ligands an additional sterically hindered labile ligand

is not tolerated. In fact, it leads to a strong decrease in or

even a complete loss of cytotoxicity, whereas the same ex-

change of labile ligands does not affect the cytotoxicity of

complexes with less sterically demanding salans.

(Ph^tBuN^Me(O^Et)₂]),

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

Cytotoxicity of N-benzylated complexes: To explore the

utmost tolerable steric demand at the bridging nitrogen

atoms, we decided to introduce N-benzyl groups. For ease of

comparison, we used the same phenols as in the N-Et case

for the Mannich coupling. When tested for their cytotoxicity,

only the least sterically hindered complex [Ti

(O^Et)₂] was cytotoxic at all, with an IC₅₀value of ꢀ10 mm in

both cell lines (Table 5).

(Ph^FN^Bn)₂-

ACHTUNGTRENNUNG

The impact of this minute interplay of salan and labile

ligand on cytotoxicity pointed us to the conclusion that the

complex has to be intact at the particular moment when the

steric bulk determines the cytotoxicity.

Table 5. IC₅₀[mm] values obtained for N-benzylated complexes measured

in Hela S3 and Hep G2 cells after incubation for 48 h. Only the least

bulky complex, [TiACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^FN^Bn(O^Et)₂], displayed any cytotoxicity.

In contrast to titanocene complexes, not much is known

about the specific mechanism of action of titanium salan

complexes. Therefore, the question of at which point of in-

teraction with its biological target the complex size matters

still remains unanswered. In principle, the steric shielding

around the titanium center influences stability, either against

water alone or in conjunction with biomolecules, that is,

transferrin or albumin. Considering the reaction with water,

one might argue that the reduced or even absent toxicity of

the bulky complexes might be explained by the following

two scenarios: If the complex itself is toxic, then a higher

steric demand at the titanium center might lead to a faster

hydrolysis, thus generating nontoxic byproducts (detoxifica-

tion mechanism). If the starting complex acts like a pro-

drug and is activated by hydrolysis, slower activation should

lead to diminished or even absent toxicity (activation mech-

anism).

Hela S3

R¹=Me R¹=F

Hep G2

R¹=Me R¹=F

R¹=Cl

R³=Et

nontoxic 10.8ꢁ3.4 nontoxic nontoxic 9.86ꢁ5.7 nontoxic

R³=iPr nontoxic nontoxic nontoxic nontoxic nontoxic nontoxic

R³=nBu nontoxic

R³=tBu

–

nontoxic nontoxic

nontoxic

–

nontoxic

–

As demonstrated above, the overall steric demand is the

most important factor in determining biological activity. In a

comparison of [TiACHTUNGTRENNUNG ₂ACHTUNGTRENNUGN

(Ph^FN^Bn) (O^Et)₂] (the only N-benzylated

complex to show any cytotoxicity) with its N-Me and N-Et

congeners, the dose-response chart shows a stepwise de-

crease in cytotoxicity that again depends on the overall size

of the respective complex (Figure 7).

The decreased cytotoxicity of sterically demanding com-

plexes might originate from inhibition of a specific interac-

tion with biomolecules. This may affect their transport into

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Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

the cell, either by active transport via transferrin or by

simple passive diffusion, the transfer to or the interaction

with the cellular target. Experiments with added transferrin

showed no significant influence on cytotoxicity, making an

active cellular uptake via transferrin doubtable. If the

uptake is based on diffusion, one might expect a faster

transport of complexes featuring sterically demanding and,

therefore, lipophilic side groups (tBu, N-Bn, O-tBu). Be-

cause those complexes show a diminished cytotoxicity in

contrast to the ones with smaller and, therefore, more hy-

drophilic groups (F, Me, N-Me, O-Et), transport phenomena

do not explain the size-dependent alteration of cytotoxicity.

drolytic behavior of the halogen-substituted complexes [Ti-

AHCTUNGTRENNUGN )₂ACHTUNGTRENNUNG

(Ph^d–fN^Me(O^iPr)₂] because these show favorable biological

properties. In strong contrast to the alkylated complexes,

they induce cell death by a purely apoptotic pathway. This

might be due to a difference in hydrolysis, in respect to the

rate or obtained products. For comparison, the highly cyto-

toxic alkyl complex [Ti

N

(O^iPr)₂] and the nontoxic

) CHTUNGTRENNUNG

tert-butyl complex [Ti

A

(Figure 8).

Comparison of the hydrolytic half-life (t_1/2) of the investi-

gated complexes (Table 6) revealed an interesting correla-

tion: Within the alkyl-containing complexes [TiACTHNUTRGNEUNG

(Ph^b,cN^Me)₂-

Hydrolysis—Activation or deactivation mechanism? To elu-

cidate whether the correlation of cytotoxicity and steric

demand is merely based on an altered hydrolysis or depends

on the interaction with biomolecules, we investigated the

stability of several complexes upon the addition of a certain

amount of water. We were interested in the influence the

complex structure exerts on the speed of hydrolysis because

this determines, besides the solubility, the applicability of

such complexes in aqueous media. A systematic investiga-

tion of the life-time of such complexes under hydrolytic con-

ditions with respect to their structure might, therefore, help

to find substances with an improved pharmacological pro-

file. Given that the fast formation of hydrolysis products has

hampered mechanistic research for all titanium complexes

found so far, stable and at the same time highly cytotoxic

complexes would be valuable tools.

On the other hand, investigation of the hydrolysis process

and the formed products might give insights into the role of

hydrolysis in biological systems. Here it is interesting to ask

whether hydrolysis is an activating or deactivating process.

To obtain not only the rate of hydrolysis but also structural

information about the products formed in aqueous media,

we decided to follow the hydrolysis of our complexes by

using time-resolved ¹H NMR spectroscopy in [D₈]THF/

D₂O.^[40]

The experiments were conducted in a mixture of [D₈]THF

(95%), D₂O (4.8%), and DMSO (0.2%) at 378C. To ana-

lyze the data, the decrease in at least two distinct signals of

the titanium-bound salan backbone and the increase in the

evolving signals of the free alkoxy ligands were monitored.

The integrals were then normalized by using the DMSO

signal as the internal standard and plotted against the hy-

drolysis time. Control measurements done in the absence of

DMSO showed no significant differences in the hydrolysis

rate and products formed. A complexation of the titanium

center by the added standard could therefore be excluded.

Keeping in mind that hydrolysis under in vivo conditions

follows different rules, our impetus was merely to gather rel-

ative rates of hydrolysis. This allows us to determine which

of our complexes is more and which is less stable in compar-

ison to each other under given conditions.

Table 6. Half-life of complexes [Ti

conditions determined by time-resolved ¹H NMR spectroscopy at 378C.

(Ph^b–fN^Me(O^iPr)₂] under hydrolytic

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

Complex

t_1/2[h]

Complex

t_1/2[h]

[Ti

N

)

G

10

2

[Ti

G

)

G

6

108

>115

N

E

N

AHCTUNGTRENNUNG

A

) CHTUNGTRENNUNG

A

) CHTUNGTRENNUNG

as in the tert-butyl case half of the complex had already de-

composed 2 h after the initial water addition.

Under the chosen hydrolytic conditions, the bromo-substi-

tuted salan complex [Ti

sive t_1/2value of more than 115 h. It outruns its fluoro coun-

terpart [Ti ₂A

(Ph^FN^Me(O^iPr)₂], which has a t_1/2value of only

(Ph^BrN^Me(O^iPr)₂] showed an impres-

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

A

) CHTUNGTRENNUNG

6 h, by a 20-fold increased hydrolytic stability. Unfortunate-

ly, this enormously stable complex displays only moderate

biological activity. Surprisingly, the chloro-substituted salan

complex showed a rather similar hydrolytic stability with a

t_1/2value of 108 h. This is remarkable because even though

the stability is in the same range as the bromo counterpart,

the biological activity is not. Compared with the most stable

cytotoxic complex of this class known so far ([Ti

(Ph^MeN^Me)₂-

ACHTUNGTRENNUNG

AHCTUNGTRENNUNG

ty that was more than 10 times higher. A rather high toxicity

(IC₅₀ꢀ5 mm) and the fact that this complex exclusively in-

AHCTUNGTRENNUNG

duces apoptosis^[43]makes it one of the most interesting sub-

stances with respect to mechanistic studies, and a promising

candidate for further biological studies in the field of anti-

cancer titanium complexes.

It has been observed by Peri et al. that upon hydrolysis

[Ti

(Ph^tBuN^Me(O^iPr)₂] releases its salan ligand as well as the

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

labile alkoxides. Hydrolysis of [TiACTHNUGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^iPr)₂] results

in the formation of new species that possibly contain more

than one titanium center. Careful examination of our data

of the halogen-substituted complexes showed no liberation

1

of free salan ligand upon hydrolysis. Instead, the H NMR

In a first set of experiments, the influence of alterations at

the aromatic moieties on the hydrolysis of our complexes

was investigated. We were especially interested in the hy-

spectra showed the formation of new species with signals

that indicated lower symmetry compared with the starting

complex.^[52]

Chem. Eur. J. 2010, 16, 2775 – 2789

ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

2783

T. Huhn et al.

(Ph^tBuN^Me

)

(O^iPr)₂] ( ), [Ti

E

)

(O^iPr)₂] ( ), [Ti

E

)

G

(Ph^ClN^Me

)

G

~

&

^

Figure 8. Plots of hydrolysis vs. reaction time for [Ti

[Ti

(Ph^BrN^Me

)

(O^iPr)₂] ( ). Data acquired by time-resolved H NMR spectroscopy at 378C. Data gathered by monitoring the decrease in isolated signals of

1

~

the titanium-bound salan backbone and the increase in the signals from the liberated alkoxy ligands. Integrals are normalized against the internal stan-

dard.

(Ph^FN^Me

(Ph^ClN^Me

)

₂A

(O^iPr)₂]

It is worth noting that X-ray structure determination re-

vealed remarkably little difference between the chloro and

Table 7. Selected bond lengths [ꢂ] and angles [8] for [Ti

and [TiACHTNUTRGENNUG )₂ACHTUNGTRENNUNG

(Ph^ClN^Me

(O^iPr)₂].

the fluoro complexes ([Ti(Ph^F,ClN^Me(O^iPr)₂]). These com-

)₂AHCUTNGERNNUG

[Ti

H

)

C

[TiC

)

CHTUNGTRENNUNG

plexes have comparable angles and distances around the ti-

ꢂ

Ti1 O1

1.9081(13)

1.9315(13)

1.8204(12)

1.7989(12)

2.3336(14)

2.3425(14)

106.04(6)

97.17(5)

92.82(5)

92.45(5)

95.72(5)

164.87(5)

163.06(5)

90.36(5)

86.01(5)

81.47(5)

1.904(2)

1.930(2)

1.8031(19)

1.7945(18)

2.339(2)

2.336(2)

106.76(9)

97.49(9)

93.19(9)

91.80(9)

96.36(9)

164.19(8)

162.89(8)

89.58(8)

86.40(8)

81.11(8)

88.15(8)

164.46(8)

80.23(8)

87.34(8)

76.04(8)

ꢂ

tanium center (Table 7, Figure 9) and differ by only a d=

Ti1 O2

1

ꢂ

Ti1 O3

0.3 ppm shift of the aromatic protons in the H NMR spec-

ꢂ

Ti1 O4

tra, so the reason for the enormous difference in hydrolytic

stability remains unclear.

ꢂ

Ti1 N1

ꢂ

Ti1 N2

In a second set of experiments, we investigated the influ-

ence of different alkoxy ligands on the rate of hydrolysis.

We supposed the alkoxides to have a rather big effect on

the hydrolysis behavior compared with the substituents at

the aromatic rings because they are in the vicinity of the

O4-Ti1-O3

O4-Ti1-O1

O3-Ti1-O1

O4-Ti1-O2

O3-Ti1-O2

O1-Ti1-O2

O4-Ti1-N1

O3-Ti1-N1

O1-Ti1-N1

O2-Ti1-N1

O4-Ti1-N2

O3-Ti1-N2

O1-Ti1-N2

O2-Ti1-N2

N1-Ti1-N2

metal center. Therefore, complexes [TiACHTNURTGENNUG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^a–d)₂]

were exposed to the same hydrolysis conditions described

above.

Keeping the different cytotoxicities of these complexes in

mind, we were surprised to find that all four complexes re-

sulted in the formation of the same degradation products

upon hydrolysis (Figure 10). Interestingly, virtually no free

ligand was observed (Figure 11).

87.87(5)

165.49(5)

81.13(5)

87.61(5)

76.14(5)

1

Time-resolved H NMR spectroscopy revealed that in the

process of hydrolysis the distribution of the evolving prod-

ucts does not change over time. Figure 11 shows NMR spec-

tra of the aromatic region of [Ti

at different times.

To estimate the influence of the different labile ligands on

the half-life of the complexes, the change in intensity of dis-

tinct signals was monitored. Signals were chosen so that

(Ph^MeN^Me(O^Et)₂] recorded

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

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Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

their integral ratios stayed constant relative to each other

during the process of hydrolysis. This measure effectively ex-

cluded a possible overlay with signals of newly formed spe-

cies.

The decreasing integrals of both the titanium-bound alk-

oxides and salan backbone and the increase in the evolving

signals of the free alkoxy ligands were then normalized by

using the DMSO signal as the internal standard and plotted

against the hydrolysis time. Results for complexes [Ti-

A

)₂ACTHNGUTERNNUG

(O^a–d)₂] are given in Figure 12.

dependence on the steric demand of the labile ligand was

observed (Table 8). In particular, complexes with alkoxides

branched at C1, such as isoproACTHNUTRGENUGNpanolate and tert-butanolate,

displayed significantly faster hydrolysis than complexes with

linear alkoxides, such as ethanolate or n-butanolate. When

the cytotoxicity of the two complexes with branched alkox-

ides was compared, a decrease of one order of magnitude

was observed despite very similar rates of hydrolysis. For

complexes with similar IC₅₀values, the difference in rate of

hydrolysis is, in the case of [Ti

(Ph^MeN^Me(O^iPr)₂], quite pronounced.

₂A

However, the difference in the rate of hydrolysis between

[Ti ₂A

(Ph^MeN^Me(O^iPr)₂] and its tert-butoxy counterpart is

(Ph^MeN^Me(O^Et)₂] and [Ti-

ACHTUNGTRENNUGN )₂ACHTUNGTRENNUNG

N

) CHTUNGTRENNUNG

Figure 9. ORTEP diagrams of the molecular structures of [TiACTHNUTRGNEUNG

(Ph^FN^Me)₂-

ACHTUNGTRENNUNG ACHTUNGTRENNUNG )₂ACHTNUGTRENNUGN

(O^iPr)₂] (top) and [Ti(Ph^ClN^Me(O^iPr)₂] (bottom). Displacement ellipsoids

G

) CHTUNGTRENNUNG

are drawn at the 50% probability level. Hydrogen atoms are omitted for

clarity.

ACHTNUTRGNEUNG )₂ACHTUNGRETNNUGN CAHTUTNGRENNUG )₂CAHTUNGTNERNUGN AHCTUNGTRENNUNG )₂ACHTUNGTRENNUNG

Figure 10. Superimposed ¹H NMR spectra of the aromatic region of [Ti(Ph^MeN^Me(O^Et)₂] (c), [Ti(Ph^MeN^Me(O^iPr)₂] (c), [Ti(Ph^MeN^Me(O^nBu)₂]

(c), and [Ti

ACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^tBu)₂] (c) at their respective t_1/2values in the hydrolysis studies. Large signals arise from unhydrolysed complexes, small sig-

nals arise from hydrolysis products. No difference in the obtained products was observed.

Chem. Eur. J. 2010, 16, 2775 – 2789

ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

2785

T. Huhn et al.

1

Figure 11. Multiple H NMR spectra of the aromatic region of [Ti

G

)

G

position shows the unchanged distribution of hydrolysis products over time. The spectrum of the (Ph^MeN^Me)₂ligand (bold) is also shown for comparison.

Table 8. Comparison of half-life and IC₅₀values for complexes [Ti

[Ti

(Ph^MeN^Me(O^Et)₂] [Ti(Ph^MeN^Me(O^iPr)₂] [Ti

18 10

G

)₂ACHTUNTGRENNUNG

(O^a–d)₂].^[a]

ACTHNGUTRENNUG )ACHTUNGTRENNUNG

(O^nBu)₂] [Ti(Ph^MeN^Me(O^tBu)₂]

rather small at 3 h. This does

not reflect the rather drastic

change in biological activity. In-

G

)

G

E

)

G

E

)

G

t_1/2[h]

16

7

terestingly, the X-ray structure Hela S3 IC₅₀[mm] 2.0ꢁ0.3

2.3ꢁ0.1

2.1ꢁ0.1

1

5.6ꢁ0.5

19.8ꢁ2.4

ACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^MeN^Me(O^tBu)₂] showed

Hep G2 IC₅₀[mm] 1.6ꢁ0.3

5.2ꢁ0.5

26.9ꢁ8.6

of [Ti

alkoxy–titanium bond of

[a] Hydrolysis was followed by time-resolved H NMR spectroscopy at 378C, and cytotoxicities in Hela S3 and

Hep G2 cells were determined by using an AlamarBlue assay after incubation for 48 h.

a

1.78 ꢂ, which is shorter than

complexes with unbranched

alkoxides ([Ti

G

₂A

(O^nBu)₂] 1.82 ꢂ). Nevertheless, this

1.83 ꢂ, [Ti

N

)

U

biomolecule. This does not rule out the possibility of hydro-

lytic deactivation of very unstable complexes.

ꢂ

stronger O Ti bond does not result in enhanced hydrolytic

stability. Furthermore, the formation of the same degrada-

tion products indicates a similar mechanism of hydrolysis.

Taking into account the fact that there is a difference of one

order of magnitude in the cytotoxicity of these complexes, it

seems implausible that hydrolysis functions as an activation

mechanism. Activation via hydrolysis would require ex-

tremely different hydrolysis rates, which were not observed.

It seems that complexes with low IC₅₀values do not share

a common hydrolytic profile and neither do nontoxic com-

plexes. A certain rate of hydrolysis neither grants nor cir-

cumvents cytotoxicity in our experiments. Taken together

with the earlier observed cross-relevance of the steric

demand of salan and alkoxy ligand on cytotoxicity, our hy-

drolysis studies therefore strongly indicate that the unhydro-

lysed complex is important for the first interaction with a

Seemingly, the determination of cytotoxicity by the steric

demand of whole complexes and by inactivation via hydrol-

ysis interferes with each other, but hydrolysis only plays a

minor part within our complexes. This might be due to a

slow hydrolysis compared with a much faster cellular

uptake.

Conclusion

Herein, we present a library of more than 40 titanium salan

complexes in which the steric demand of both the salan and

the labile alkoxy ligands has been systematically altered. To

enlighten the interconnection between steric demand, cyto-

toxicity, and hydrolytic behavior, all complexes were tested

2786

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Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

(Ph^MeN^Me

)

N

(Ph^MeN^Me

)

E

(Ph^MeN^Me

)

G

(Ph^MeN^Me

)

N

~

^

*

Figure 12. Plots of hydrolysis vs. reaction time for [Ti

(^&). Data acquired by time-resolved NMR spectroscopy at 378C.

on their biological activity and the hydrolysis of a selected

set was followed by time-resolved H NMR spectroscopy. It

same hydrolysis products and displayed only slightly varying

t_1/2values. Moreover, the distribution of the evolving prod-

ucts does not change over time. Therefore, we concluded

that the active species is not a hydrolysis product. This sup-

ports our hypothesis of an important role for the intact com-

plex and leads to the conclusion that hydrolysis is not an ac-

tivation but a deactivation mechanism. The unhydrolyzed

complex seems to be important for the first interaction with

a biomolecule. Whether this is essential to transport the

complex into the cell, for the portage to the as-yet unknown

cellular target inside the cell, or the interaction at the site of

action remains an open question for further studies.

Because our results promote the idea of hydrolysis being

a detoxification pathway, we also screened our library for

bioactive and highly stable complexes. Hydrolysis and the

fast formation of unidentifiable byproducts have hampered

the investigation of the mechanism of action of all titanium

complexes found so far. Thus, such complexes might not

only have an improved pharmacological profile, but also

would be valuable tools for biochemical research. Therefore,

we were happy to find a chloro-substituted complex that dis-

played rather high toxicity (IC₅₀ꢀ5 mm) and surprisingly

high hydrolytic stability. With a hydrolytic half-life of 108 h,

this complex showed a stability over 10 times higher than

the most stable cytotoxic complex of this class known so far.

When combined with its enhanced selectivity in inducing

apoptosis,^[43]the stability of this complex in aqueous media

1

was already known that bulkier salan ligands result in less

toxic complexes. Interestingly, we found that, in contrast to

studies on titanocene derivatives, the exchange of the labile

monodentate ligands can also have an enormous effect on

cytotoxicity. However, not only did we find that the size of

the alkoxy ligands is crucial for the cytotoxicity, but we also

showed an interdependence of the steric demand of salan

and alkoxy ligands. Very small salan ligands tolerated even

the bulkiest of the alkoxides, tert-butoxide, without any loss

of cytotoxicity. Complexes of medium-sized salans were ex-

tremely sensitive to even minor changes in the steric

demand of the alkoxides. In this case, the respective com-

plexes showed a stepwise decrease in cytotoxicity as the size

of the alkoxide was increased. Finally, complexes of very

bulky salans were nontoxic, even with the smallest labile

ligand. We concluded that the overall size of the complexes

determines their biological activity. This ligand–ligand inter-

dependence also implies that the complex should be intact

at the particular moment when the steric bulk determines

the cytotoxicity of the complex. This result is especially un-

expected because partial hydrolysis is believed to be an acti-

vation mechanism for many metal complexes. To investigate

whether the overall steric demand itself alters the hydrolytic

behavior of our complexes and, therefore, the cytotoxicity,

hydrolysis experiments were conducted. We showed that

complexes that only differed in their labile alkoxy ligands

despite having very different cytotoxicities resulted in the

makes it one of the most promising sub

of anticancer titanium complexes.

ACHTUNGTRENsNUGN tances in the field

Chem. Eur. J. 2010, 16, 2775 – 2789

ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemeurj.org

2787

T. Huhn et al.

bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_

request/cif. A summary of the relevant crystallographic data for this

paper is shown in Table 9.

Experimental Section

[43,44]

Materials and Methods: Syntheses of ligands (Ph^a–fN^Me

)

and

2

[45]

(Ph^b,eN^Bn

)

were performed as described previously. Complexes [Ti-

Hydrolysis experiments followed by NMR spectroscopy were conducted

in a [D₈]THF (95%)/D₂O (4.8%)/DMSO (0.2%) mixture. The DMSO

signal was used as the internal standard for calibrating the integrals.

Spectra were recorded in 4 min intervals. Elemental analyses were per-

formed at the microanalytical laboratory of the University of Konstanz.

Cytotoxicity was measured on HeLa S3 and Hep G2 cells by using the

AlamarBlue assay. The cells were cultivated at 378C under humidified

5% CO₂in Dulbeccoꢃs modified Eagle medium (Invitrogen) that con-

tained 10% foetal calf serum, 1% penicillin, and 1% streptomycin, and

were tested for mycoplasma infections by using a mycoplasma detection

kit (Roche Applied Science, Mannheim, Germany) prior to use.

2

ACHTUNGTRENNUNG )₂ACHTUNGTRENNUNG

(Ph^a–fN^Me(O^iPr)₂] were synthesized according to literature proce-

dures.^{[43, 46,47]}

Titanium tetra(isopropoxide) (97%) and N,N’-dibenzylethylenediamine

(97%) were purchased from Aldrich. Titanium tetraACHTNUTRGNEUNG(ethoxide) (99%), ti-

tanium tetra(n-butoxide) (98%) and N,N’-diethylethylenediamine (96%)

were purchased from ABCR (Karlsruhe, Germany); titanium tetra(tert-

butoxide) (99%) and N,N’-dimethylethylenediamine (85%) were pur-

chased from Acros Organics (Geel, Belgium). Deuterated solvents where

purchased from Euriso-Top (Saarbrꢄcken, Germany) in sealed ampoules

and dried if necessary; other solvents were purified according to standard

procedures.^[53]All experiments requiring a dry atmosphere were carried

out under nitrogen by using Schlenk techniques. NMR spectra were mea-

sured by using JEOL Eclipse 400 and Bruker Avance DRX 600 spec-

trometers. Structure assignments were done based on 2D NMR experi-

ments (COSY, HMBC, HSQC).

Peri et al. showed recently that the toxicity of the uncomplexed alkyl-

substituted salans plays only a minor role in estimating complex toxici-

ty.^[40]By using flow cytometry (PI/annexin-FITC staining), we demon-

strated the negligible cytotoxicity of the halo-substituted salans case as

well.^[43]

General procedure for synthesis of the ligands: 2,4-disubstituted phenol

(10 mmol) was suspended or dissolved in methanol (5 mL), then formal-

dehyde (7.5 mL, 36% in water) and N,N’-disubstituted ethylenediamine

(5 mmol) were added immediately. The mixture was heated at reflux for

24 h. Upon cooling, the ligands crystallized as colorless solids (47–61%

yield).

The data collection for the X-ray structures was performed by using a

STOE IPDS-II diffractometer equipped with a graphite monochromated

radiation source (l=0.71073 ꢂ), an image plate detection system, and an

Oxford Cryostream 700 with nitrogen as the coolant gas. The selection,

integration, and averaging procedure of the measured reflection intensi-

ties, the determination of the unit cell by a least-squares fit of the 2q

values, data reduction, LP correction, and the space group determination

were performed by using the X-Area software package delivered with

the diffractometer. A semiempirical absorption correction method was

performed after indexing of the crystal faces. The structures were solved

by direct methods (SHELXS-97)^[54]and refined by standard Fourier tech-

niques against F²with a full-matrix least squares algorithm by using

SHELXL-97^[54]and the WinGX (1.80.05)^[55]software package. All nonhy-

drogen atoms were refined anisotropically. Hydrogen atoms were placed

in calculated positions and refined with a riding model. Graphical repre-

sentations were prepared with ORTEP-III.^[56]CCDC-739047 ([Ti-

General procedure for synthesis of the complexes: The ligand (1.5 mmol)

was dissolved in toluene (10 mL), then titanium alkoxide (1.5 mmol) was

added over a 10 min period under a nitrogen atmosphere. The resultant

yellow reaction mixture was stirred overnight at RT. The solvent was re-

moved under reduced pressure and the complex was obtained as a yellow

solid (>90% yield). The crude product was recrystallized by using the

procedure given in the Supporting Information.

Characterization data for new compounds and details of cell cultivation

and cytotoxicity studies in HeLa S3 and Hep G2 cells are given in the

Supporting Information.

A

)

₂A

G

)₂ACTHNGUTERNNUG

(O^tBu)₂)], -739049 ([Ti-

R

(O^nBu)₂]),-739050 ([Ti

N

) CHTUNGTRENNUNG

H

)

this paper. These data can be obtained free of charge from The Cam-

Table 9. Crystallographic data for complexes [Ti

(Ph^MeN^Me

)

E

R

)

G

)₂ACHTUNTGRENNUNG

(O^iPr)₂].

[Ti

(Ph^FN^Me

[Ti ₂A

A

)

U

[Ti

A

)

E

G

)

N

U

)

G

[TiACHTGNUTRENNUG )₂ACHTUNGTRENNUNG

(Ph^ClN^Me(O^iPr)₂]

formula

M_r

C₂₆H₄₀N₂O₄Ti

492.50

0.55ꢅ0.35ꢅ0.1

monoclinic

P21/c

11.7789(7)

16.9074(10)

13.1780(8)

90.00

C₃₀H₄₈N₂O₄Ti

548.60

0.55ꢅ0.4ꢅ0.05

monoclinic

P21/c

11.4262(7)

15.4592(6)

17.2942(10)

90.00

C₃₀H₄₈N₂O₄Ti

548.60

0.6ꢅ0.5ꢅ0.2

monoclinic

P21/c

17.5177(18)

9.9728(6)

18.3729(19)

90.00

C₂₄H₃₂F₄N₂O₄Ti

536.39

0.5ꢅ0.35ꢅ0.15

monoclinic

P21/c

8.2933(4)

12.6602(7)

23.7332(10)

90.00

C₂₄H₃₂Cl₄N₂O₄Ti

602.22

0.3ꢅ0.3ꢅ0.3

monoclinic

P21/c

9.270(4)

12.783(4)

23.673(6)

90.00

crystal size [mm³]

crystal system

space group

a [ꢂ]

b [ꢂ]

c [ꢂ]

a [8]

b [8]

105.661(5)

90.00

2527.0(3)

4

1.295

100(2)

107.064(5)

90.00

2920.4(3)

4

1.248

100(2)

109.273(8)

90.00

3029.9(5)

4

1.203

100(2)

95.308(3)

90.00

2481.2(2)

4

1.436

100(2)

95.31(2)

90.00

2793.3(16)

4

1.432

373(2)

g [8]

V [ꢂ³]

Z

1_calc[gcm^ꢂ1

T [K]

]

m

N

]

0.372

0.329

0.317

0.408

0.721

F

G

1056

1184

1120

1248

1.80–25.81

32883

1.3–28.4

37408

2.26–26.21

26501

1.72–26.88

35513

1.2–15.13

6483

)

4870 (0.0986)

4784/0/298

1.023

8771 (0.0719)

8688/0/342

0.975

6080 (0.1301)

6077/54/368

0.988

5363 (0.0661)

5273/0/316

1.063

6103 (0.0322)

6103/0/322

1.085

data/restraints/param

GOF on F²

R₁, wR₂[I>2s(I)]

R₁, wR₂(all data)

4.86, 10.88

7.63, 11.74

0.412/ꢂ0.365

5.25, 10.24

9.75, 11.45

0.444/ꢂ0.289

6.97, 13.42

14.17, 15.92

0.339/ꢂ0.388

3.46, 8.74

4.69, 9.61

0.274/ꢂ0.525

4.00, 9.05

6.34, 11.29

0.694/ꢂ0.332

largest diff. peak/hole [eꢂ^ꢂ3

]

2788

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ꢁ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Chem. Eur. J. 2010, 16, 2775 – 2789

Titanium Salan Complexes

FULL PAPER

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Published online: January 26, 2010

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Cytotoxic titanium salan complexes: Surprising interaction of salan and alkoxy ligands

DOI: 10.1002/chem.200902312

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