Peptide Bioconjugates of Electron-Poor Metallocenes: Synthesis, Characterization, and Anti-Proliferative Activity.

We report the synthesis of metallocene compounds Cp2 M with two different electron-withdrawing substituents on both cyclopentadienyl rings (hexafluoroacetone (HFA) and chlorobenzoyl (1-5); HFA and COOH (6 and 7), M=Fe or Ru). The COOH-containing derivatives were used to synthesize peptide bioconjugates with enkephalin (8 and 9) and neurotensin (10 and 11) as well as fluorescein-labeled neurotensin (12). All the molecules were fully characterized, including X-ray structures for 6 and 7. The physicochemical properties (lipophilicity and electrochemistry) and cytotoxicity on MCF-7, HT-29, and PT-45 cancer cells were evaluated for selected compounds. Electrochemical investigation by cyclic voltammetry revealed that all bis-substituted metallocenes are up to 300 mV harder to oxidize compared to the monosubstituted 2-ferrocenylhexafluoropropan-2-ol (FcHFA: Δ${E{{{\rm f}\hfill \atop 0\hfill}}}$=214 mV; disubstituted derivatives: up to Δ${E{{{\rm f}\hfill \atop 0\hfill}}}$=512 mV; both vs. FcH(0/+) ). For the bis-substituted compounds, log P determinations by RP-HPLC showed increased lipophilicity in comparison to the monosubstituted FcHFA and RcHFA. Cellular uptake was investigated by fluorescence microcopy, and this revealed endosomal entrapment for 12.

Structure-activity relationship of trifluoromethyl-containing metallocenes: electrochemistry, lipophilicity, cytotoxicity, and ROS production.

We report the synthesis of trifluoromethylated metallocenes (M=Fe, Ru) and related metal-free compounds for comparison of their biological properties with the aim to establish structure-activity relationships toward the anti-proliferative activity of this compound class. All new compounds were comprehensively characterized by NMR spectroscopy ((1) H, (13) C, (19) F), mass spectrometry, IR spectroscopy, and elemental analysis. A single-crystal X-ray structure was obtained on the Ru derivative, 1-(1-hydroxy-1-hexafluoromethylethyl)ruthenocene (3). The cytotoxicity of all compounds was tested on MCF-7, HT-29, and PT-45 cells, and IC50 values as low as 12 µM were observed. Both the metallocene moiety and the hydroxy function are crucial for cytotoxicity. In addition, the activity decreased sharply even if only one trifluoromethyl group was replaced with a methyl group. Electrochemical investigations by cyclic voltammetry revealed that all CF3 -containing compounds are harder to oxidize than the unsubstituted metallocenes. Moreover, log P determination by RP-HPLC showed the fluorinated derivatives to have higher lipophilicity, with log P values up to 4.6. At the same time, the generation of reactive oxygen species (ROS) in Jurkat cells by these compounds was investigated by flow cytometry. Strong ROS production was shown exclusively for the bis-CF3 derivative 1-(1-hydroxy-1-hexafluoromethylethyl)ferrocene (1) after 6 and 24 h. Also on the Jurkat cell line, only compound 1 strongly induces necrosis after 24 and 48 h, as shown by annexin V/propidium iodide staining. No induction of apoptosis was observed. We propose that compound 1 is more efficiently incorporated into cancer cells relative to all other derivatives, causing significant induction of oxidative stress within the cell, which ultimately leads to cell death.

Towards perfunctionalized dodecahedranes--en route to C20 fullerene.

"One-pot" substitution of the twenty hydrogen atoms in pentagonal dodecahedrane (C(20)H(20)) by OH, F, Cl, and Br atoms is explored. Electrophilic insertion of oxygen atoms with DMDO and TFMDO as oxidizing reagents ended, far off the desired C(20)(OH)(20), in complex polyol mixtures (up to C(20)H(10)(OH)(10) decols, a trace of C(20)H(OH)(19)?). Perfluorination was successful in a NaF matrix but (nearly pure) C(20)F(20) could be secured only in very low yield. "Brute-force" photochlorination (heat, light, pressure, time) provided a mixture of hydrogen-free, barely soluble C(20)Cl(16) dienes in high yield and C(20)Cl(20) as a trace component. Upon electron-impact ionization of the C(20)Cl(16) material sequential loss of the chlorine atoms was the major fragmentation pathway furnishing, however, only minor amounts of chlorine-free C(20) (+) ions. "Brute-force" photobrominations delivered an extremely complex mixture of polybromides with C(20)HBr(13) trienes as the highest masses. The MS spectra exhibited exclusive loss of the Br substituents ending in rather intense singly, doubly, and triply charged C(20)H(4-0) (+(2+)(3+)) ions. The insoluble approximately C(20)HBr(13) fraction (C(20)Br(14) trienes as highest masses) obtained along a modified bromination protocol, ultimately allowed the neat mass selection of C(20) (-) ions. The C(20)Cl(16) dienes and C(20)H(0-3)Br(14-12) tri-/tetraenes, in spite of their very high olefinic pyramidalization, proved resistant to oxygen and dimerization (polymerization) but added CH(2)N(2) smoothly. Dehalogenation of the respective cycloaddition products through electron-impact ionization resulted in C(22-24)H(4-8) (+(2+)) ions possibly constituting bis-/tris-/tetrakis-methano-C(20) fullerenes or partly hydrogenated C(22), C(23), and C(24) cages.