7.4.1 Chemistry and anti-cancer activity

The [TiIV(bzac)2(OEt)2] complex (budotitane, bzac = 1-phenylbutane-1,3-dionate = benzoylacetonate) was the first non-platinum complex tested in clinical trials.18 Budotitane and related bis(^-diketonato) metal complexes can be synthesized from the corresponding metal tetrahalogenides and the diketonates in an anhydrous organic solvent. Budotitane was selected from about 300 related derivatives for further development. The structure activity relation of complexes of the type M(^-diketonate)2X2 has been extensively tested by variation of the ^-diketonate ligand, of the leaving group X and by variation of the central metal atom M, using the animal sarcoma 180 ascitic tumor model. The main results of these tests may be summarized as follows.19

The [Ti(acac)2(OEt)2] complex, which bears a symmetric acetylacetonate ligand, does not produce any anti-tumor activity, whereas the introduction of a phenyl group into the diketonate ligand may turn an inactive complex into a highly active substance. Unsubstituted aromatic ring systems in the periphery of the molecule have significant positive effects on the anti-tumor activity of such complexes, while substitutions at the aromatic ring system may be considered to be detrimental to the activity. It appears that a planar aromatic group in the ^-diketonate ligand is responsible for the anti-tumor activity of the titanium(IV) complexes of the type M(^-diketonate)2X2. This situation leads to the suggestion that the activity of the complexes is determined by a DNA intercalating mechanism. It is interesting to note that the organometallic compound titanocene dichloride shows similar aspects exhibiting two ^-bonded aromatic cyclopentadienyl rings, which also could be involved in a DNA intercalating mechanism of action.

The nature of the cis-configurated hydrolizable group X does not seem to contribute much to the anti-tumor activity of the diketonate complexes. However, the galenic behavior is considerably influenced by X because stability in water clearly increases in the order I— < Br— < Cl— <F— < OR—. The bromine and fluorine complexes in addition have disadvantages over ethoxide as to the way the hydrolizable group is physiologically tolerated.

The activities of the titanium and zirconium complexes with the benzoylace-tonate ligand are relatively similar, but they then decrease markedly in the order Hf > Mo > Sn > Ge, the germanium compound being virtually inactive even at higher doses.

7.4.2 Isomer abundance of budotitane

In any case, the spatial arrangement of the monomeric budotitane and/or the resulting hydrolyzed species seems to be of importance for the understanding of its anti-cancer activity. Complexes of the general formula [Ti(^-diketonato)2X2] can occur with a cis- or a trans- arrangement of the X ligands, the cis configuration being more stable. According to NMR data and the results of force field calculations, only those benzoyl acetonato complexes with the extremely bulky substituents iodide or p-dimethylaminophenoxy as the hydrolyzable group X have the trans form.18 Steric interactions are thought to be a key feature for the relative stabilities of the ^-diketonato complexes, but electronic effects may also be important.33

As the ,3-diketonate ligand in [Ti(bzac)2X2] complexes is unsymmetrical, there are eight possible isomers of the six-coordinate octahedrally configured complexes (Figure 7.2). Six of these have the X ligands cis, forming three enantiomeric pairs, which are diastereomers to each other, whereas the two trans-isomers are achiral. The isomeric configuration has been defined by three cis or trans prefixes which specify first the relative position of the X ligands, then the relative orientation of the benzoyl groups and finally the relative orientation of the acetyl ends of the benzoylacetonate ligands.34 It is not known which one of these isomers exhibit anti-cancer activity. Isomer distributions have so far only been determined in CDCl3 solutions by interpretation of NMR data,33 but not in the galenic CremophorEL/propyleneglycol formulations used for the in vivo tests.

Force field calculations lead to calculated isomer distributions that are in satisfactory agreement with the experimental data derived from

Figure 7.2 Isomers of budotitane [TiIV(bzac)2(OEt)2]. The three cis-isomers form three enantiomeric pairs (A and A configuration) which are diastereomers to each other. The two trans-isomers are achiral. In the crystal structure of budotitane, all molecules exhibit the cis-cis-trans configuration trans-trans-trans trans-cis-cis

Figure 7.2 Isomers of budotitane [TiIV(bzac)2(OEt)2]. The three cis-isomers form three enantiomeric pairs (A and A configuration) which are diastereomers to each other. The two trans-isomers are achiral. In the crystal structure of budotitane, all molecules exhibit the cis-cis-trans configuration temperature-dependent 1H-NMR spectroscopy in CDCl3 solution.18,33 In budotitane [TiIV(bzac)2(OEt)2], the observed/calculated isomer distributions (Figure 7.2) are cis-cis-cis 60%/57%, cis-cis-trans 19%/17% and cis-trans-cis 21%/26%, respectively.

Probably due to the fact that no isomerically pure fractions may be isolated from the isomeric mixture that exists in solution, budotitane usually does not give diffraction-quality crystals. Nevertheless, we have recently been able to crystallize budotitane [TiIV(bzac)2(OEt)2] and its dichloro-derivative [TiIV(bzac)2Cl2] from ethanol and toluene solutions, respectively.35 Budotitane exhibits two crystallographically independent molecules involving Ti(1) and Ti(2) centers, both of which are octahedrally coordinated by six oxygen atoms with the two ethoxy ligands cis. Both molecules adopt the cis-cis-trans configuration with the acetyl ends of the benzoylacetonate ligands trans (Figure 7.2). The molecule involving Ti(1) exists in the A configuration, whereas the molecule involving Ti(2) shows the enantiomeric A configuration. An analysis of the crystal packing of budotitane shows that there are no intra- or inter-molecular ^-^-stackings of the phenyl moieties of the benzoylacetonate ligands.35 According to the spectroscopic data mentioned above, the cis-cis-trans configuration uniquely observed in the solid state occurs at only 19% in CDCl3 solutions.

In the dichloro-derivative of budotitane, [TiIV(bzac)2Cl2], the titanium atom is octahedrally coordinated within a Cl2O4 donor set with the two chloride atoms cis. In contrast to budotitane, the corresponding dichloro-derivative shows a cis-trans-cis arrangement with the benzoyl groups trans. The centro-symmetric space group accomodates equal numbers of A and A enantiomers within the unit cell. In contrast to budotitane, its dichloro-derivative clearly exhibits inter-molecular ^-^-stackings of neighboring phenyl moieties.35

The phenyl groups in budotitane and in its dichloro-derivative are in approximately coplanar conjugation to the metal enolate rings. This is evidence of a certain aromaticity of the system which could support DNA intercalating mechanisms of the compounds.

7.4.3 Reaction with biomolecules

Because of the hardness of Ti(IV) and its affinity toward oxygen, many bio-molecules offering oxygen donors are of interest in the context of possible metabolic pathways. For this reason, numerous steroid, sugar and nucleoside derivatives of budotitane have been synthesized and characterized mainly by spectroscopic methods, and in each case coordination by oxygen has been found.20 Synthesis was possible with steroids like cholesterol or stigmasterol, with the bile acid dehydrocholic acid, as well as with steroid hormones like testosterone. Stable adducts of the nucleosides cytidine, uridine and thymidine were also synthesized. In these adducts, relatively fast binding to the hydroxy functions of the sugar moieties of the nucleosides took place, whereas coordination to the basic nitrogen of the nucleobases was not observed. These findings support the assumption that titanium-oxygen bonding involving the sugar or the phosphate moieties of DNA has to be considered in discussing the mode of action of budotitane.20

7.4.4 Animal studies

A preclinical study of budotitane in human tumor xenografts in nude mice has been reported in 1984.36 In this investigation, the anti-tumor activities of budotitane and of the established anti-tumor drug cisplatin have been shown to be comparable.

Anti-tumor activity in transplantable animal tumor models in mice has been extensively studied by Keppler etal.19 The results are presented in terms of T/C values, T/C (%) = (median survival time or medium tumor weight of treated animals vs median survival time or medium tumor weight of control animals) x 100. T/C values >300%, indicating that a high percentage of animals are cured, have been found as a result of the budotitane therapy in the Stockholm ascitic tumor, the Ehrlich ascitic tumor and in the MAC 15A colon tumor, a transplantable colon adenocarcinoma. In contrast to cisplatin, activity against the quick-growing P338 and L1210 leukemias was marginal.

These studies were performed using transplantable tumors, which can be transplanted from one animal to another. An important disadvantage of such models is that they cannot ultimately define the organ tumors against which a new compound will be active in humans. Therefore, the evaluation of antitumor activity has also been performed using autochthonous tumors, which are mainly induced by a carcinogen, and which mimic the human situation fairly closely.37 When comparing the activity of the established anti-tumor drugs 5-fluorouracil and cisplatin with that of budotitane, using AMMN (acetoxy-methyl-methyl-nitrosamine)-induced colorectal tumors, it was observed that budotitane is the most active drug. It reduces tumor volume to about 20% of the initial value. 5-Fluorouracil effects a tumor remission to about 40% of tumor volume, whereas cisplatin, with a value of about 120%, stimulates tumor growth a little. In addition, budotitane is the only compound to increase the lifespan of adenotumor-bearing animals, from 25 weeks in the control group to 36 weeks in the budotitane-treated group.19

7.4.5 Clinical investigations

For budotitane, a galenic formulation19 had to be found which guarantees some water solubility and insensitivity to hydrolysis. The use of CremophorEL, a glycerine-polyethylene-glycolericin-oleate, was successful. Addition of 1,2-propyleneglycol resulted in a co-precipitate of the drug. Budotitane, CremophorEL and propyleneglycol are dissolved separately in water-free etha-nol in the weight ratio of 1:9:1. The solutions are then mixed and evaporated at 30^0 °C. Within the resulting co-precipitate, budotitane is enveloped between layers of the solubilizer and is protected against hydrolysis. This co-precipitate can be dissolved in water under the formation of micelles. The co-precipitate solutions used in the clinic have favorable concentrations of 100 200 mg of budotitane per 100 ml. While such formulated solutions are stable for hours, the components possibly cannot be sufficiently well characterized to advance to further clinical trials.38

Budotitane has been extensively investigated in numerous preclinical studies.20 It entered phase I clinical trials in 1986.39 In 1996, a clinical phase I study and pharmacokinetic trial with budotitane administered as iv infusion twice weekly was reported.40 The trial was performed on 18 patients refractory to all other known treatment. No objective tumor response was observed. The maximum tolerated dose of budotitane administered twice weekly was 230 mg/m2, with cardiac arrhythmia as dose-limiting toxicity. Fifteen patients reported loss of taste at the day of infusion, reversible within 24 h. Budotitane, unlike cisplatin, did not cause vomiting. From these studies, the recommended dose for further clinical trials was 180mg/m2. However, subsequent clinical evaluations of budotitane have been stopped because its galenic formulation did not meet modern standards, although new formulations are being developed.41

The use of isomerically pure crystals of budotitane in the galenic formulation of the drug could eventually help to overcome these difficulties and may also increase the activity and/or bioavailability of budotitane.

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