Triazol-substituted titanocenes by strain-driven 1,3-dipolar cycloadditions

Summary An operationally simple, convenient, and mild strategy for the synthesis of triazole-substituted titanocenes via strain-driven 1,3-dipolar cycloadditions between azide-functionalized titanocenes and cyclooctyne has been developed. It features the first synthesis of titanocenes containing azide groups. These compounds constitute ‘second-generation’ functionalized titanocene building blocks for further synthetic elaboration. Our synthesis is modular and large numbers of the complexes can in principle be prepared in short periods of time. Some of the triazole-substituted titanocenes display high cyctotoxic activity against BJAB cells. Comparison of the most active complexes allows the identification of structural features essential for biological activity.


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H NMR and 13 C NMR spectra were recorded on a DPX 300 and DPX 400 Bruker spectrometer; the chemical shifts (in ppm) are reported relative to nondeuterated solvent residual as reference. EI mass spectra were recorded on a MS 50 spectrometer from Kratos as well as on a MAT 95 spectrometer from Thermoquest. ESI mass spectra were recorded on a micrOTOF-Q spectrometer from Bruker Daltonik and IR spectra were recorded on an ATR Nicolet 380 spectrometer from Thermo Electron. Melting Points were measured on a Büchi 530 Melting Point and are uncorrected.

Determination of cell concentration and cell viability:
In a similar manner as described in [5], cell viability was determined by CASY® Cell Counter + Analyzer System of Schaerfe System GmbH (Reutlingen, Germany). Settings were specifically defined for the requirements of the used cells. With this system the cell concentration is analyzed simultaneously in three different size ranges: cell debris, dead cells, and viable cells were determined in one measurement. BJAB cells were seeded at a density of 1 Å~ 105 cells/mL and treated with different concentrations of a titanocene-derivate, non treated cells served as controls. After 24 h of incubation at 37 °C, 5% CO 2 , cells were resuspended properly and 100 μL of each well was diluted in 10 mL CASYton (ready-to-use isotonic saline solution) for an immediate automated count of the cells.

Measurement of DNA fragmentation:
In a similar manner as described in [6], apoptotic cell death was determined by a modified cell cycle analysis, which detects DNA fragmentation on the single cell level. For measurement of DNA fragmentation cells were seeded at a density of 1 Å~ 105 cells/mL and treated with different concentrations of a titanocene-derivate. After 72 h of incubation at 37 °C, 5% CO 2 , cells were collected by centrifugation at 1500 rpm for 5 min, washed with PBS at 4 °C, and fixed in PBS/2% (v/v) formaldehyde on ice for 30 min. After fixation, cells were incubated with ethanol/PBS (2:1, v/v) for 15 min, pelleted, and resuspended in PBS containing 50 μg/mL RNase A. After incubation for 30 min at 37 °C, cells were pelleted again and finally resuspended in PBS containing 50 μg/mL propidium iodide. Nuclear DNA fragmentation was then quantified by flow cytometric determination of hypodiploid DNA. Data were collected and analyzed using a FACScan (Becton Dickinson, Heidelberg, Germany) equipped with the CELLQuest software. Data are given in percentage of hypoploidy (subG1), which reflects the number of apoptotic cells.

AnnexinV-propidium iodide binding assay:
In a similar manner as described in [7], early apoptotic rates were assessed with flow s3 cytometry using the annexin V-fluorescein isothiocyanate/propidium iodide (PI) kit (BD Pharmingen, San Diego, CA, USA), in which annexin Vbound to exposed phosphatidylserine of the early apoptotic cells, whereas PI stained the cells that had an increased membrane permeability, i.e., the late apoptotic cells. Samples were prepared according to the manufacturer's instructions. Flow cytometry analysis was performed using a FACS-Calibur cytometer (Becton Dickinson, Heidelberg, Germany). The annexin-V+/PI-cells were defined as early apoptotic cells.

General procedure for the synthesis of azide-functionalized titanocenes from carboxylates
To a solution of the carboxylate (1 equiv.) in CH 2 Cl 2 (3 mL/mmol) was added SOCl 2 (3 mL/mmol). After stirring for 3 h at r.t. excess SOCl 2 and solvent was removed in vacuo for 6 h at 45 °C. The resulting acid chloride was dissolved in CH 2 Cl 2 (6 mL/mmol) and transferred via syringe to a suspension of NaH (10 equiv.) and amino azide (2 equiv.) in CH 2 Cl 2 (10 mL/mmol). Stirring was continued for 16 h at r.t.. After filtration through Celite the volatiles were removed under reduced pressure and the residue was chromatographed on BioBeads S-X3 to yield the desired product.

General Procedure for the Synthesis of Triazoles from Azides
To a solution of the carboxylate (1 equiv.) in CH 2 Cl 2 (10 mL/mmol) was added cyclooctyne (5 equiv.). After stirring for 16 h at r.t. the solvent was removed and the residue washed with cyclohexane and chromatographed on BioBeads S-X3 to yield the desired product.