Novel enantiopure cyclopentadienyl Ti(IV) oximato compounds as potential anticancer agents

Abstract

The synthesis and characterization of new enantiopure cyclopentadienyl titanium oximato compounds (S,R)-[(η(5)-C5H5)Ti{к(2)NO,(R)NH·HCl}Cl2] (R=Ph (phenyl) 1a·HCl, Bn (benzyl) 1b·HCl, 2-pic (2-picolyl) 1c·HCl), (S,R)-[(η(5)-C5H5)TiCl2{к(2)NO,(Ph)NH}] (1a) and (S,R)-[(η(5)-C5H5)2TiCl{к(2)NO,(R)NH}] (R=Ph 2a, Bn 2b, 2-pic 2c), along with studies on their behavior in D2O at different pD values are reported. The structure of previously described ammonium-oxime (2S,5R)-{NOH,(Bn)NH·HCl} (b·HCl) and novel titanium derivative 1a have been determined by single crystal X-ray crystallography. The effect of the compounds on cytotoxicity, cell adhesion and migration of the androgen-independent prostate cancer PC-3 cells has been assessed. Compounds 2b and 2c are more cytotoxic than additive doses of titanocene dichloride and free oxime proligand, probing the synergistic effect of these novel compounds. The cytotoxicity of 2b and 2c has been further evaluated against human renal Caki-1, colon DLD-1 and triple negative breast MDA-MB-231 cancer cell lines. The activity found for 2c on PC-3 and Caki-1 is higher than that of highly active Titanocene Y (bis-[(p-methoxybenzyl)cyclopentadienyl]titanium(IV) dichloride), while showing selectivity against renal cancer when compared to a non-tumorigenic human renal (HEK-293T) cell line. Compounds 2b and especially 2c are apoptotic in Caki-1 cancer cell lines. Cell adhesion and wound-healing assays confirmed that derivatives 1c·HCl, 2b and 2c affect the adhesion and migration patterns of the PC-3 cell line. Interactions of the novel compounds with plasmid (pBR322) DNA have also been studied, showing that the oximato Ti(IV) derivatives have a weak or no interaction with DNA at physiological pH.

Keywords: Cell adhesion; Cell migration; Chirality; Cyclopentadienyl titanium compounds; DNA; PC-3 and Caki-1 cell lines.

Fig. 1

Selected titanium compounds with relevant antitumor properties

Fig. 2

Enantiomerically pure amino-oxime ligands and related Ti(IV) compounds

Fig. 3

Synthesis of monocyclopentadienyl oximato titanium compounds

Fig. 4

ORTEP drawing of compound 1a with 30% probability ellipsoids. Hydrogen bonded to carbon atoms have been omitted for clarity. Representative bond lengths (Å) and angles (deg): Ti(1)-O(1) 1.8860(19); Ti(1)-N(1) 2.102(2); N(1)-O(1) 1.401(3); N(1)-C(1) 1.281(3); O(1)-Ti(1)-N(1) 40.71(9); N(1)-O(1)-Ti(1) 77.91(14); O(1)-N(1)-Ti(1) 61.34(11); C(1)-N(1)-Ti(1) 178.9(2); C(6)-N(2)-C(21) 124.3(3).

Fig. 5

Synthesis of titanocene amino-oximato compounds

Fig. 6

ORTEP drawing of compound b·HCl with 30% probability ellipsoids. Hydrogen bonded to carbon atoms has been omitted for clarity. Representative bond lengths (Å) and angles (deg): N(1)-O(1) 1.400(3); N(1)-C(6) 1.271(4); O(1)-N(1)-C(6) 112.9(2); C(10)-N(2)-C(4) 114.9(2).

Fig. 7

Compounds 2b, 2c, cisplatin and Titanocene Y impaired the viability of Caki-1 cells by inducing apoptosis. Following a 72 h incubation period, the compounds (5 μM) 2b or 2c or (30 μM) Cisplatin or Titanocene Y reduced Caki-1 viability by 61% (cisplatin), 48% (Titanocene Y), 56% (2b) and 84% (2c); induced necrosis in 9% (cisplatin), 5% (Titanocene Y), 17% (2b) and 6% (2c), and induced apoptosis in 38% (cisplatin), 33% (Titanocene Y), 38 % (2b) and 76 % (2c) of treated cells. The effect of cisplatin and Titanocene Y, 2b and 2c on viability, necrosis and apoptosis was assessed by measuring protease activity using the non-cell-permeable substrates, cell-permeable substrates and by measuring the total caspase-3 and -7 activities with the ApoTox-Glo Triplex Assay. The effect of each treatment was determined by comparing treated and untreated cells. From the Apotox assay a cell population that was neither necrotic nor apoptotic was detected and their mode of death could not be assigned. Those cells were not included in the graph.

Fig. 8

Effect of [(η⁵-C₅H₅)TiCl₃], [(η⁵-C₅H₅)₂TiCl₂], c·HCl, 1c·HCl, 2b and 2c derivatives on adhesion of PC-3 cells to type-I collagen was studied after treatment with organic and organometallic compounds for 40 min. Data are the mean ± S.E.M of at least four experiments. ^*, P < 0.05; ^***, P < 0.001 versus Control.

Fig. 9

Effect of [(η⁵-C₅H₅)TiCl₃], [(η⁵-C₅H₅)₂TiCl₂] c·HCl, 1c·HCl, 2b and 2c on cell migration was studied in human androgen-independent prostate cancer PC-3 cell line. Microscopic analysis of the cell-free area was carried out at the indicated time (6 h) after the addition of the complexes and the width of the area invaded by prostate cells was estimated.

Fig. 10

Electrophoresis mobility shift assays for cisplatin and compounds [(η⁵-C₅H₅)TiCl₃], [(η⁵-C₅H₅)₂TiCl₂], a·HCl-c·HCl, 1a·HCl-1c·HCl and 2a–2c (see Experimental Section for details). DNA refers to untreated plasmid pBR322. Lanes a, b, c and d correspond to metal/DNAbp ratios of 0.25, 0.5, 1.0 and 2.0 respectively. OC open circular or relaxed form (Form II), CCC covalently closed or supercoiled form, (Form I).