Direct Regioselective Synthesis of Tetrazolium Salts by Activation of Secondary Amides under Mild Conditions

Tetrazolium salts are biologically active molecules that have found broad applications in biochemical assays. A regioselective synthesis of tetrazolium salts is described through a formal (3 + 2) cycloaddition. The possibility of employing simple amides and azides as starting material and the mild conditions allow a broad functional group tolerance.


General information
All glassware was oven dried at 100 ºC before use. All solvents were distilled from appropriate drying agents prior to use. All reagents were used as received from commercial suppliers unless otherwise stated.
Triflic anhydride was freshly distilled over P 2 O 5 before use. Neat infra-red spectra were recorded using a Perkin-Elmer Spectrum 100 FT-IR spectrometer. Wavenumbers (υ ) are reported in cm-1. Mass spectra were obtained using a Finnigan MAT 8200 or (70 eV) or an Agilent 5973 (70 eV) spectrometer, using electrospray ionization (ESI) All 1 H-NMR and 13 C-NMR experiments were recorded using Bruker AV-400, spectrometers at 300 K. Chemical shifts (δ) are quoted in ppm and coupling constants (J) are quoted in Hz. The 7.27 ppm resonance of residual CHCl 3 for proton spectra and 77.16 ppm resonance for carbon spectra were used as internal references. 1

General procedure for the synthesis of the starting materials Amides synthesis
Amides were synthetized from the corresponding chloride or acid according to the procedure A or B. Spectroscopic data of known compounds are according to the literature. [1] General procedure A: To a solution of Et 3 N (3 eq.) and amine (1.5 eq.) in dichloromethane (0.2 M) at 0°C was slowly added the corresponding acyl chloride (1 eq.) and the reaction was allowed to warm to r.t. overnight. The reaction was quenched by addition of NH 4 Cl, extracted with dichloromethane, dried over Na 2 SO 4 and evaporated. The crude product was purified by column chromatography (0 to 50% heptane/ethyl acetate) to afford the pure amide.

General procedure B:
To a solution of Et 3 N (2.4 eq.), amine (1.2 eq.) and carboxylic acid (1 eq.) in DMF (0.2 M) was added HATU (1.2 eq.) and the reaction was stirred at r.t. overnight. The reaction was quenched with NaOH 1M, extracted with dichloromethane, dried over Na 2 SO 4 and evaporated. The crude product was purified by column chromatography (0 to 50% heptane/ethyl acetate) to afford the pure amide.

General procedure for the synthesis of tetrazolium salts
To a mixture of amide (0.2 mmol) and 2-fluoropyridine (0.4 mmol, 2 equiv., 38.8 mg, 34.4 µl) in dichloromethane (0.6 mL) triflic anhydride was added dropwise (0.2 mmol, 1 equiv., 56.4 mg, 33.6 μL.) at 0 °C under Ar. The mixture was stirred for 15 minutes at this temperature. Then a solution of azide (0.4 mmol, 2 equiv.) in 0.5 ml of dichloromethane was added and the mixture was brought to room temperature ad heated to 40°C. After 16 hours, the solvent was removed under reduced pressure. Purification through column chromatography on Al 2 O 3 with 0 to 100% dichloromethane /DMA (DMA = dichloromethane/methanol/NH 4 OH mixture 9:1:0.75) afforded the desired products.

Computational details
All geometries were optimized at the B3LYP-D3/6-31+G(d,p) level of theory. [3][4][5] The nature of all stationary points (minima and transition states) was verified through computation of the vibrational frequencies. The thermal corrections to the Gibbs free energy were combined with single point energies calculated at the RI-MP2/def2-TZVP//B3LYP-D3/6-31+G(d,p) level [6] to yield Gibbs free energies (G 298 ) at 298.15 K (all energies are reported in kcal mol -1 ). The densitybased solvation model SMD [7] (for geometry optimization) and Conductor-like screening model COSMO [8] (for RI-MP2 single-point calculations) were applied to consider solvent effects. The DFT calculations have been performed with the Gaussian09 program package, [9] while for the RI-MP2 single point calculations the Turbomole V7.0 program package [10] was used. Computed structures were visualized using the Chemcraft software. [11]