James Claffey

Oxali‐Titanocene Y: A Potent Anticancer Drug

Outside of platinum and ruthenium anticancer drugs, there is significant unexplored space for further metal-based drugs that target cancer, such as titanocene dichloride. One of these promising drug candidates is bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) dichloride (titanocene Y, 1), which is accessible via the hydridolithiation reaction of 6-anisylfulvene and subsequent transmetallation with titanium tetrachloride. Compound 1 exhibits an IC50 value of 21 mm toward the LLC-PK cell line, which has proven to be a good mimic of a kidney carcinoma cell line and a reliable tool for the optimisation of titanocenes against this type of cancer. Additionally, the antiproliferative activity of 1 and other titanocenes has been studied in 36 human tumour cell lines and against explanted human tumours. 6] These in vitro and ex vivo experiments showed that renal cell cancer is the prime target for this novel class of titanocenes, but there is significant activity against ovarian, prostate, cervical, lung, colon, and breast cancers as well. Furthermore, titanocene derivatives give a positive immune response by up-regulating the number of natural killer (NK) cells in mice. These results were underscored by initial mechanistic studies of the effects of these titanocenes on apoptosis and the apoptotic pathway in prostate and cervical cancer cells. Recently, animal studies demonstrated the successful treatment of mice bearing xenografted A431, Caki-1, and MCF-7 tumours with 1. Herein the synthesis and initial cytotoxicity studies of titanocene oxalate and oxali-titanocene Y (2) are presented. The structures and syntheses of 1 and 2 are shown in Scheme 1. A simple anion-exchange reaction in THF employing silver oxalate eliminates insoluble silver chloride and produces 2 or titanocene oxalate. An X-ray crystallographic study established the molecular structure of 2 (Figure 1). Despite the addition of the oxalate bidentate ligand in replacement of the two chlorides on the titanium centre, there is almost no apparent variance in the molecular structures of 1 and 2. The length of the bond between the titanium centre and the carbon atoms of the cyclopentadienide rings are very similar for both 1 and 2. They vary from 2.34 to 2.41 C for 1 and from 2.33 to 2.40 C for 2. Very slight differences can be observed in comparing the titanium centroid distances in 1 (2.06 C) and 2 (2.04 C). The centroid–titanium–centroid angle is 130.78 for 1, whereas the corresponding angle in 2 is 134.88. This widening goes nicely with the more blade-like shape of the oxalate anion, compared with the spherical chloride anions. As expected, the average titanium–chloride bond in 1 is considerably longer (2.37 C) than the average titanium–oxygen bond in 2 (1.99 C). The average carbon–oxygen single and double bonds in 2 are, with 1.30 C and 1.21 C, quite close to those found in free carboxylic acids, showing that there is very little, if any, negative charge located on the oxygen atoms that are not bonded to titanium. In 1 the chloride–titanium–chloride angle is 95.98, which is in the range of other titanocene dichlorides. In 1, however, the oxygen atoms are part of a chelate ring which forces the oxygen–titanium–oxygen angle to 79.08. Despite the addition of the bidentate oxalate ligand. minimal structural change incurred. In tests against LLC-PK cells, which have proven to be a valuable in vitro model for kidney cancer, titanocene oxalate shows an IC50 value of 140 mm, which is a significant improvement (14-fold) relative to titanocene dichloride, with an IC50 value of 2000 mm. Similar behaviour is observed for the p-dimethoxybenzyl-substituted species; 2 is 13-fold more cytotoxic against LLC-PK than 1 and exhibits an IC50 value of 1.6 mm (see Figure 2), which is twice as cytotoxic as cisplatin (3.3 mm). The Scheme 1. Structures and syntheses of titanocene Y (1) and oxali-titanocene Y (2): a) 2LiBEt3H, Et2O, 2BEt3 ; b) TiCl4, THF, 2LiCl(s) ; c) Ag2C2O4, THF, 2AgCl(s).

Titanocene Difluorides with Improved Cytotoxic Activity

Titanocene difluorides can be obtained by halide metathesis of the respective titanocene dichlorides with trimethyltin fluoride (Me(3)SnF), giving access to a new class of cytotoxic active substances. Furthermore, an improved method for the synthesis of diaryl-substituted titanocene dichlorides is presented.

Antitumor activity of Titanocene Y in xenografted PC3 tumors in mice

Chemotherapeutic options for androgen-independent prostate cancer are extremely limited with minimum survival advantage. The benzyl-substituted unbridged titanocene bis-[(p-methoxybenzyl) cyclopentadienyl] titanium(IV) dichloride (Titanocene Y) was tested in vitro against the human prostate cancer androgen-independent cell, PC-3, which demonstrated an IC50 value of 56 x 10(-6) mol/L compared to 5.6 x 10(-6) mol/L for cisplatin. Then Titanocene Y was given at the maximum tolerable dose of 40 mg/kg/d on five consecutive days to one cohort of eight PC3 tumor-bearing male NMRI: nu/nu mice, while a second cohort was treated similarly with 3 mg/kg/d of cisplatin. Both of these mouse cohorts showed a statistically significant tumor growth reduction with respect to the third solvent-treated control group, which led to T/C values of 42% for cisplatin and 52% for Titanocene Y at the end of the experiment. This encouraging activity of Titanocene Y against prostate tumors in vivo, which is almost comparable with respect to cisplatin hopefully leads to further development of Titanocene Y in the future.

Binding and hydrolysis studies of antitumoural titanocene dichloride and Titanocene Y with phosphate diesters.

The interaction of the antitumoural metallocene dihalides, titanocene dichloride (Cp(2)TiCl(2)) and Titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) chloride), with bis(4-nitrophenyl) phosphate (BNPP), which is a widely used model for the phosphate diester linkages in DNA, has been studied. Cp(2)TiCl(2) has been shown to promote the cleavage of the phosphate diester in weakly acidic solution. At pH 4, 37 degrees C, a 10(6)-fold rate acceleration over the uncatalysed reaction was observed under pseudo-first-order conditions, when freshly prepared solutions of Cp(2)TiCl(2) were applied. The activity of aged solutions dropped significantly due to the formation of insoluble precipitates of hydrolysed Ti species. The precipitates isolated from aged solutions were shown to act as moderately active, heterogeneous catalysts for BNPP cleavage. By contrast, no hydrolysis of the phosphate diester could be observed in the presence of Titanocene Y. Implications for the mode of action of the apoptosis-inducing metallocene dihalides are discussed.

The role of the intrinsic FAS pathway in Titanocene Y apoptosis: The mechanism of overcoming multiple drug resistance in malignant leukemia cells.

Despite the advantages in the outcome of patients with acute lymphoblastic leukemia, 25% of the affected children suffer relapses. As the response to chemotherapy is essentially determined by the development of cellular drug resistance, new drugs that are capable to overcome resistance to conventional chemotherapeutics are urgently needed. With regard to this demand, we investigated the titanium-based anticancer drug Titanocene Y. Treatment with Titanocene Y leads to inhibition of tumour cell proliferation and induces apoptosis in established cell lines of leukemia, lymphoma and melanoma. The extrinsic pathway appears to be responsible, at least in part, for the effect: cell death is partly inhibited in BJAB cells overexpressing a dominant negative Fas-associated death domain (FADD) mutant and via real time PCR we found a significant up-regulation of Fas ligand in the affected cells. Apoptosis is triggered regardless of the expression of anti-apoptotic Bcl-2 and pro-apoptotic smac and the agent is also effective on cells that are multidrug resistant due to overexpression of P-gp. In combination with vincristine impressive synergistic effects could be observed, exposing Titanocene Y as a possible component for polychemotherapy. Taken together, Titanocene Y turns out to be a promising candidate for anti-tumour therapy, especially for the treatment of multidrug resistant malignancies.

Effects of titanocene dichloride derivatives on prostate cancer cells, specifically DNA damage-induced apoptosis.

While locally advanced prostate cancer is initially treatable with androgen ablation, eventually cells develop a castrate‐resistant phenotype. Currently, there are no effective treatments for this form of the disease with Docetaxel only providing a small survival advantage. In this study, the effects of novel derivatives of titanocene dichloride on prostate cancer cell lines has been investigated.

The Antiangiogenic and Antitumoral Activity of Titanocene Y* In Vivo

The 4-diethylaminomethylbenzyl-substituted titanocene dichloride (Titanocene Y*), which is completely water- soluble and showed nanomolar activity against the human renal cancer cells CAKI-1, was tested in vitro in an antiangiogenesis assay against human umbilical vein endothelial cells, HUVEC, delivering an IC50 value of 23 +/- 17 {Mu}M. Titanocene Y* was then given at 25, 50 and 75 mg/kg/d, on five consecutive days per week for up to three weeks to one cohort of six CAKI-1 tumor-bearing female NMRI:nu/nu mice, while a further cohort was treated with solvent only. At the two higher dosages Titanocene Y* showed high toxicity leading to mortality, while the titanocene-treated mouse cohort treated with the lowest dosage showed a moderate but statistically significant tumor growth reduction with respect to the solvent-treated control group, with an optimal T/C value of 76% at the end of the experiment. Immunohistological analysis revealed that the expression of the proliferation marker Ki-67 was reduced by 21%. Furthermore, anti-angiogenic activity was identified by CD31 staining; the number of micro vessels in a defined tumor area decreased by 23% due to Titanocene Y* treatment. The substance caused dose-dependent body weight loss but did not reduce the number of white blood cells at doses of 25 and 50 mg/kg/d.

Synthesis and Cytotoxicity Studies of New Morpholino-Functionalised and N-Heteroaryl-Substituted Titanocene Anticancer Drugs

From the carbolithiation of 6-morpholino fulvene (3) and different lithiated nitrogen containing heterocycles (2-N-methylimidazolyl, 2-N-(N,N-dimethylamino)methyl-imidazolyl, and 2-N-methylindolyl), the corresponding lithium cyclopentadienide intermediate (4a-c) was formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl(4) resulting in morpholino-functionalised titanocenes 5a-c. When these titanocenes were tested against LLC-PK cells, the IC(50) values obtained were of 24, 36, and 41 microM respectively. The most cytotoxic titanocene in this paper (5a) with an IC(50) value of 24 microM is found to be almost ten times less cytotoxic than cis-platin, which showed an IC(50) value of 3.3 microM when tested on the epithelial pig kidney LLC-PK cell line, and approximately 2 times less cytotoxic than its dimethylamino-functionalised analogue. Encouragingly however, the IC(50) value obtained for titanocene 5a is approximately 100 times better than titanocene dichloride itself.

Pseudo-halide derivatives of titanocene: synthesis and cytotoxicity studies.

The well-known anticancer drug candidate bis-[(p-methoxybenzyl)cyclopentadienyl] titanium(IV) dichloride (Titanocene ) was reacted with sodium azide or potassium cyanate, thiocyanate or selenocyanate in order to give pseudo-halide analogues of Titanocene . and were characterised by single crystal X-ray diffraction, which confirmed the expected nitrogen binding of the cyanate and thiocyanate to the titanium centre. All four titanocenes had their cytotoxicity investigated through preliminary in vitro testing on the LLC-PK (pig kidney epithelial) cell line in an MTT based assay in order to determine their IC50 values. Titanocenes were found to have IC50 values of 24 (± 8) μM, 101 (± 14) μM, 54 (± 21) μM and 27 (± 4) μM respectively. All four titanocene derivatives show significant cytotoxicity improvement when compared to unsubstituted titanocene dichloride and and showed similiar cytotoxic behaviour to Titanocene in vitro.

Structures of 6-(substituted-phenyl)fulvenes

The syntheses and crystal structures of a series of 6-(substituted-phenyl) fulvenes are reported. In three instances the molecular structures determined by X-Ray crystallography were compared to the results of DFT calculations. In these cases only minor differences were found between the optimised DFT calculation and the molecule found in the crystal structure. This is of particular interest in the case of 6-(p-[N-acetyl]aminophenyl)fulvene which shows intermolecular hydrogen bonding.