Synthesis, structure, docking and cytotoxic studies of ferrocene-hormone conjugates for hormone-dependent breast cancer application.

Previously, ferrocene incorporation into the principal structural component of biologically active molecules resulted in enhanced cytotoxic activity against hormone-dependent MCF-7 and T-47D and hormone-independent MDA-MB-231 breast-cancer cell lines. Here we explore 4 new ferrocene estrogen conjugates at position 16 of the estrogen hormone and compared them to the previously reported ferrocene estrogen conjugate 3-ferrocenyl-estra-1,3,5(10)-triene-17ß-ol. The ferrocene conjugate 16-ferrocenylidene-3-hydroxyestra-1,3,5(10)-trien-17-one was synthesized using estrone and ferrocene carboxaldehyde as starting materials in 86% yield. This ferrocene complex was used as the starting material for the synthesis of new ferrocene estrogen conjugates by a short linker group at position 16 of the estrogen hormone. The position and stereochemistry of the linker was verified by its crystal structure. The ferrocene redox behavior, in vitro studies on breast-cancer cell lines and docking studies on ERα are presented. The data suggest that the ferrocene conjugates presented, either at position 3 or 16 of the estrogen, could serve as vectors and can be recognized by ERα as a delivery mechanism into the cell. These new ferrocene hormone conjugates showed cytotoxic activity comparable to that of conventional therapeutic drugs such as tamoxifen and cisplatin.

A molecular docking study of the interactions between human transferrin and seven metallocene dichlorides.

Human Transferrin (hTf) is a metal-binding protein found in blood plasma and is well known for its role in iron delivery. With only a 30% of its capacity for Fe+3 binding, this protein has the potential ability to transport other metal ions or organometallic compounds from the blood stream to all cell tissues. In this perspective, recent studies have described seven metallocene dichlorides (Cp2M(IV)Cl2, M(IV)=V, Mo, W, Nb, Ti, Zr, Hf) suitable as anticancer drugs and less secondary effects than cisplatin. However, these studies have not provided enough data to clearly explain how hTf binds and transports these organometallic compounds into the cells. Thus, a computational docking study with native apo-hTf using Sybyl-X 2.0 program was conducted to explore the binding modes of these seven Cp2M(IV)Cl2 after their optimization and minimization using Gaussian 09. Our model showed that the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) can interact with apo-hTf on a common binding site with the amino acid residues Leu-46, Ile-49, Arg-50, Leu-66, Asp-69, Ala-70, Leu-72, Ala-73, Pro-74 and Asn-75, while the next four Cp2M(IV)Cl2 (M(IV)=Nb, Ti, Zr, Hf) showed different binding sites, unknown until now. A decreasing order in the total score (equal to -log Kd) was observed from these docking studies: W (5.4356), Mo (5.2692), Nb (5.1672), V (4.5973), Ti (3.6529), Zr (2.0054) and Hf (1.8811). High and significant correlation between the affinity of these seven ligands (metallocenes) for apo-hTf and their bond angles CpMCp (r=0.94, p<0.01) and Cl-M-Cl (r=0.95, p<0.01) were observed, thus indicating the important role that these bond angles can play in ligand-protein interactions. Fluorescence spectra of apo-hTf, measured at pH 7.4, had a decrease in the fluorescence emission spectrum with increasing concentration of Cp2M(IV)Cl2. Experimental data has a good correlation between KA (r=0.84, p=0.027) and Kd (r=0.94, p=0.0014) values and the calculated total scores obtained from our docking experiments. In conclusion, these results suggest that the seven Cp2M(IV)Cl2 used for this study can interact with apo-hTf, and their affinity was directly and inversely proportional to their bond angles CpMCp and ClMCl, respectively. Our docking studies also suggest that the binding of the first three Cp2M(IV)Cl2 (M(IV)=V, Mo, W) to hTf could abrogate the formation of the hTf-receptor complex, and as a consequence the metallocene-hTf complex might require another transport mechanism in order to get into the cell.

Crystal structure of 16-ferrocenylmethyl-3ß-hydroxy-estra-1,3,5(10)-trien-17-one: a potential chemotherapeutic drug.

A new ferrocene complex, 16-ferrocenylmethyl-3ß-hy-droxy-estra-1,3,5(10)-trien-17-one dimethyl sulfoxide monosolvate, [Fe(C5H5)(C24H27O2)]·C2H6OS, has been synthesized and structurally characterized by single-crystal X-ray diffraction techniques. The mol-ecule crystallizes in the space group P21 with one mol-ecule of dimethyl sulfoxide. A hydrogen bond links the phenol group and the dimethyl sulfoxide O atom, with an O⋯O distance of 2.655 (5) Å. The ferrocene group is positioned in the ß face of the estrone moiety, with an O-C-C-C torsion angle of 44.1 (5)°, and the carbonyl bond length of the hormone moiety is 1.216 (5) Å, typical of a C=O double bond. The average Fe-C bond length of the substituted Cp ring [Fe-C(Cp*)] is similar to that of the unsubstituted one [Fe-C(Cp)], i.e. 2.048 (3) versus 2.040 (12) Å. The structure of the complex is compared with those of estrone and eth-oxy-methyl-estrone.

New platinum(II) complexes with benzo-thia-zole ligands.

Four new platinum(II) complexes, namely tetra-ethyl-ammonium tri-bromido-(2-methyl-1,3-benzo-thia-zole-κN)platinate(II), [NEt4][PtBr3(C8H7NS)] (1), tetra-ethyl-ammonium tri-bromido-(6-meth-oxy-2-methyl-1,3-benzo-thia-zole-κN)platinate(II), [NEt4][PtBr3(C9H9NOS)] (2), tetra-ethyl-ammonium tri-bromido-(2,5,6-trimethyl-1,3-benzo-thia-zole-κN)platinate(II), [NEt4][PtBr3(C10H11NS)] (3), and tetra-ethyl-ammonium tri-bromido-(2-methyl-5-nitro-1,3-benzo-thia-zole-κN)platinate(II), [NEt4][PtBr3(C8H6N2O2S)] (4), have been synthesized and structurally characterized by single-crystal X-ray diffraction techniques. These species are precursors of compounds with potential application in cancer chemotherapy. All four platinum(II) complexes adopt the expected square-planar coordination geometry, and the benzo-thia-zole ligand is engaged in bonding to the metal atom through the imine N atom (Pt-N). The Pt-N bond lengths are normal: 2.035 (5), 2.025 (4), 2.027 (5) and 2.041 (4) Šfor complexes 1, 2, 3 and 4, respectively. The benzo-thia-zole ligands are positioned out of the square plane, with dihedral angles ranging from 76.4 (4) to 88.1 (4)°. The NEt4 cation in 3 is disordered with 0.57/0.43 occupancies.

Functionalized ferrocenes: The role of the para substituent on the phenoxy pendant group.

Six ferrocenecarboxylates with phenyl, 4-(1H-pyrrol-1-yl)phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-bromophenyl, 4-iodophenyl as pendant groups were synthesized and fully characterized by spectroscopic, electrochemical and X-ray diffraction methods. The anti-proliferative activity of these complexes were investigated in hormone dependent MCF-7 breast cancer and MCF-10A normal breast cell lines, to determine the role of the para substituent on the phenoxy pendant group. The 4-fluorophenyl ferrocenecarboxylate is inactive in both cell lines while 4-(1H-pyrrol-1-yl)phenyl ferrocenecarboxylate is highly cytotoxic in both cell lines. 4-chlorophenyl and 4-bromophenyl ferrocenecarboxylates have moderate to good anti-proliferative activity in MCF-7 and low anti-proliferative activity on normal breast cell line, MCF-10A whereas the 4-iodophenyl analog is highly toxic on normal breast cell line. The phenyl ferrocenecarboxylate has proliferative effects on MCF-7 and is inactive in MCF-10A. Docking studies between the complexes and the alpha-estrogen receptor (ERα) were performed to search for key interactions which may explain the anti-proliferative activity of 4-bromophenyl ferrocenecarboxylate. Docking studies suggest the anti-proliferative activity of these ferrocenecarboxylates is attributed to the cytotoxic effects of the ferrocene group and not to anti-estrogenic effects.

Water soluble molybdenocene complexes: synthesis, cytotoxic activity and binding studies to ubiquitin by fluorescence spectroscopy, circular dichroism and molecular modeling.

Four new molybdenocene complexes, Cp2Mo(l-ascorbato), Cp2Mo(6-O-palmitoyl-l-ascorbato), [Cp2Mo(ethyl maltolato)]Cl and Cp2Mo((2S)-2-amino-3-methyl-3-thiolato-butanoato), were synthesized and structurally characterized by standard analytical methods. The cytotoxicity of these complexes was assessed on colon HT-29 and breast MCF-7 cancer cell lines using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A higher cytotoxic activity was shown by all the new complexes on the MCF-7 cells over the Cp2MoCl2 complex. The complexes Cp2Mo(l-ascorbato), Cp2Mo(6-O-palmitoyl-l-ascorbato) and [Cp2Mo(ethyl maltolato)]Cl displayed a stronger cytotoxic activity on colon cancer HT-29 cell line, over the molybdenocene dichloride (Cp2MoCl2). In contrast, Cp2Mo((2S)-2-amino-3-methyl-3-thiolato-butanoato) exhibited proliferative properties on this cell line. Ubiquitin (Ub)-molybdenocene interactions were investigated using cyclic voltammetry, fluorescence quenching spectroscopy, circular dichroism (CD) and molecular modeling. The thermodynamic parameters (ΔH and ΔS) obtained using fluorescence quenching spectra and van't Hoff plot indicate the Ub-molybdenocene interactions are mainly hydrophobic. The CD data also support hydrophobic interactions with conformational changes in the Ub protein. Docking studies using molecular modeling revealed the amino acids involved in the Ub-molybdenocene interactions and corroborated the hydrophobic nature of the binding combined with hydrogen bonding.

Titanium(IV) complexes: cytotoxicity and cellular uptake of titanium(IV) complexes on caco-2 cell line.

Replacement of the ancillary ligand in titanocene dichloride by amino acids provides titanocene species with high water solubility. As part of our research efforts in the area of titanium-based antitumor agents, we have investigated the cytotoxic activity of Cp(2)TiCl(2) and three water soluble titanocene-amino acid complexes - [Cp(2)Ti(aa)(2)]Cl(2) (aa=L-cysteine, L-methionine, and D-penicillamine) and one water soluble coordination compound, [Ti(4)(maltolato)(8)(micro-O)(4)] on the human colon adenocarcinoma cell line, Caco-2. At pH of 7.4 all titanocene species decompose extensively while [Ti(4)(maltolato)(8)(micro-O)(4)] is stable for over seven days. In terms of cytotoxicity, the [Cp(2)Ti(aa)(2)]Cl(2) and [Ti(4)(maltolato)(8)(micro-O)(4)] complexes exhibited slightly higher toxicity than titanocene dichloride at 24h, but at 72h titanocene dichloride and [Ti(4)(maltolato)(8)(micro-O)(4)] have higher cytotoxic activity. Cellular titanium uptake was quantified at various time intervals to investigate the possible relationship between Ti uptake and cellular toxicity. Results indicated that there was not a clear relationship between Ti uptake and cytotoxicity. A structure-activity relationship is discussed.

Synthesis, Ti(IV) intake by apotransferrin and cytotoxic properties of functionalized titanocene dichlorides.

Functionalization of cyclopentadienyl (Cp) ligands and incorporation of these into a Ti(IV) center require careful design and selection of the appropriate synthetic routes to obtain the desired product in reasonably good yields. As part of our research efforts in the area of titanocene antitumor agents, we have revisited the synthesis of Cp rings with electron-withdrawing groups and their corresponding titanocene dichlorides, (Cp-R)(2)TiCl(2) and (Cp-R)CpTiCl(2), where R is CO(2)CH(3) and CO(2)CH(2)CH(3). These complexes were characterized by elemental analysis and (1)H and (13)C NMR and IR spectroscopies. This report presents the first detailed synthetic route for (Cp-CO(2)CH(2)CH(3))CpTiCl(2) and provides an alternate route for synthesis of (Cp-R)(2)TiCl(2) complexes. The ability of these complexes to deliver Ti(IV) to apotransferrin was investigated to elucidate how the functionalized Cp ligands affect the titanium intake by apotransferrin. The subject complexes transfer Ti(IV) to human apotransferrin, loading both N- and C-lobes. The antitumor activity of these complexes against HT-29 cancer colon cells was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Carboethoxy Cp functionalization results in complexes with a toxicity comparable to that of titanocene dichloride. The carbomethoxy-functionalized complexes proved to be nonactive at the time intervals studied here, regardless of their ability to donate the titanium atom to human apotransferrin.

Determination of the titanium content of human transferrin by inductively-coupled plasma-atomic-emission spectroscopy.

We report a simple method that combines dialysis, as a purification method, with the multielement capability of ICP to determine the titanium-to-transferrin mole ratio at physiological pH, under buffer conditions. The method, by means of which titanium and transferrin are determined simultaneously, enabled us to assess the binding capacities of different titanocene complexes.

Molybdenocene-oligonucleotide binding study at physiological pH using NMR spectroscopy and cyclic voltammetry.

The self-complementary oligonucleotide CGCATATATGCG was used as a model to establish the binding interactions of antitumor molybdenocene dichloride and DNA. The free dodecamer was first characterized using (1)H, NOESY, and DQF-COSY NMR experiments, which enable to pinpoint the guanines and adenines as well as the cytosines and thymines signals in the aromatic region. Molybdenocene dichloride was characterized in saline and buffer solutions as function of pH by (1)H NMR spectroscopy. In 10mM NaCl/D(2)O solution at pH of 6.5 and above, Cp(2)Mo(OD)(D(2)O)(+) is in equilibrium with its dimeric species, [Cp(2)Mo(mu-OH)(2)MoCp(2)](2+). In 25mM Tris/4mM NaCl/D(2)O at physiological pH, a new stable species is formed, coordinated by the buffer, Tris(hydroxymethyl)aminomethane. The interactions of molybdenocene dichloride species with CGCATATATGCG were studied at different pH. At pH 6.5, in 4mM NaCl/D(2)O solution, (1)H NMR spectra of CGCATATATGCG exhibit downfield shifts in the signals associated mainly to adenines and guanines, upon addition of molybdenocene dichloride. At pH 7.4, in 25mM Tris/4mM NaCl/D(2)O, molybdenocene species causes broadening and small downfield shifts to the purines and pyrimidine signals, suggesting that molybdenocene dichloride can get engaged in binding interactions with the oligonucleotide in a weak manner. (31)P NMR spectra of these interactions at pH 7.4 showed no changes associated to Mo(IV)-OP coordination, indicating that molybdenocene-oligonucleotide binding interactions are centered, most likely, on the bases. Cyclic voltammetry titration showed a 4.9% of molybdenocene-oligonucleotide interaction. This implicates that possible binding interactions with DNA are weak.

Study of titanocene-DNA and molybdenocene-DNA interactions by inductively coupled plasma-atomic emission spectroscopy.

Titanocene and molybdenocene dichlorides belong to a new class of organometallic antitumor agent. Although these complexes are isostructural, they behave differently under physiological conditions and hence have different mechanisms of action. It was initially proposed that these species interact with DNA, inhibiting the cell cycle. Recent studies using nucleotides and oligonucleotides suggest that molybdenocene does not bind DNA constituents at physiological pH whereas titanocene apparently interacts weakly with nucleotides through the phosphoesters. The evidence for this was, however, obtained under non-physiological conditions. Herein we report an analytical method that enables determination of the amount of metal bound to DNA under physiological conditions (pH 7.4 and buffer solution) and with sample preparation (dialysis) that resembles the cell environment. It was found that more than 90% titanium was bound to DNA after 46 h whereas binding of molybdenum was no more than 5%.

Leishmaniasis tegumentaria urbana en Barquisimeto, Venezuela / Urban cutaneous leishmaniasis in Barquisimeto, Venezuela

Se realizó un estudio clínico, parásitológico, epidemiológico y ecológico, y a la vez se adoptaron las medidas para combinar un brote epidémico urbano de leishmaniasís tegumentaria en la ciudad de Barquisimeto, Estado Lara, Venezuela. Las variedades clínicas encontradas en 30 casos fueron: clásica ulcerosa en 20 (66,7%), nodular en 7 (23,3%) y ulceronodular en 3 (10,0%). Las localizaciones más frecuentes de las lesiones fueron: la cara 13 casos (43,3%) y las extremidades superiores 10 casos (33, 3%). La enfermedad se presentó en ambos sexos y en todos los grupos de edad estudiados, pero fue más frecuente en los hombres que en las mujeres así como en los menores de 15 años. El agente causal es semejante a Leishmania mexicana venezuelensis, parásito sensible a la acción terapéutica de los antimoniales pentavalentes. El vector más probable es Lutzomyia olmeca bicolor

Leishmaniasis tegumentaria urbana en Barquisimeto, Venezuela

Since 1974 significant numbers of cutaneous leishmaniasis cases have turned up in the area of Barquisimeto, Venezuela's third-largest city and the capital of Lara State. One case was found in 1974, three more in 1980, and 89 more during the course of surveys conducted in 1982-1984. All 93 of these patients received parasitologic examinations and 32 were given the Montenegro test. In addition, suspensions of tissues from their lesions were inoculated into hamsters, and the infecting were isolated by culturing sera obtained from the hamster's resulting lesions. The Montenegro test produced responses with diameters from 5 to 9 mm in 23 patients and from 10 to 13 mm in nine. Parasite strains isolated from 88 of the patients were morphologically and biologically similar to Leishmania mexicana venezuelensis, and parasite strains isolated from five patients were morphologically and biologically similar to L. braziliensis ssp. The authors suggest that Lutzomyia olmeca bicolor is the most likely vector of L. m. venezuelensis. All of the patients studied were given a course of treatment with antimonial medication and all recovered