Easy-to-Implement Hydrogen Isotope Exchange for the Labeling of N-Heterocycles, Alkylkamines, Benzylic Scaffolds, and Pharmaceuticals

Facilitating access to deuterated and tritiated complex molecules is of paramount importance due to the fundamental role of isotopically labeled compounds in drug discovery and development. Deuterated analogues of drugs are extensively used as internal standards for quantification purposes or as active pharmaceutical ingredients, whereas tritiated drugs are essential for preclinical ADME studies. In this report, we describe the labeling of prevalent substructures in FDA-approved drugs such as azines, indoles, alkylamine moieties, or benzylic carbons by the in situ generation of Rh nanoparticles able to catalyze both C(sp2)–H and C(sp3)–H activation processes. In this easy-to-implement labeling process, Rh nanocatalysts are formed by decomposition of a commercially available rhodium dimer under a deuterium or tritium gas atmosphere (1 bar or less), using the substrate itself as a surface ligand to control the aggregation state of the resulting metallic clusters. It is noteworthy that the size of the nanoparticles observed is surprisingly independent of the substrate used and is homogeneous, as evidenced by transmission electron microscopy experiments. This method has been successfully applied to the one-step synthesis of (1) deuterated pharmaceuticals usable as internal standards for MS quantification and (2) tritiated drug analogues with very high molar activities (up to 113 Ci/mmol).


I-1. Materials
All substrates were purchased and used without further purification. When substrates have been received as salts (dextromethorphan hydrochloride or chloroquine hydrochloride for instance), they were solubilized in water and free bases were recovered by addition of K2CO3 until basic pH and extracted with CH2Cl2. THF was dried by distillation over sodium and benzophenone. CH2Cl2 was dried by distillation over calcium hydride. Anhydrous and inhibitor-free 2-MeTHF and a solution of HCl/Diethyl ether (1M) were purchased from Sigma-Aldrich. 1 H NMR (400 MHz), 2 H NMR (61 MHz) and 3 H NMR (427 MHz) spectra were recorded on a Bruker Avance 400 MHz spectrometer. Chemical shifts are reported in parts per million (ppm) downfield from residual solvent peaks and coupling constants are reported in Hertz (Hz). Splitting patterns are designated as singlet (s), doublet (d), triplet (t), broad signal (brs). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m). Electrospray mass spectra were recorded using either an ESI/TOF Mariner Mass Spectrometer or a Q-TOF Premier Waters Mass Spectrometer. LC-MS analyses for tritium experiments were performed using a Waters Alliance 2695 system (Column: Waters BEH XBridge C18 100 × 4.6 mm, 3.5 µm). Liquid scintillation countings were performed using a PerkinElmer Tricarb 2910 TR Liquid Scintillation analyser equipped with automatic external standardization. The samples for liquid scintillation counting were prepared with a 9 mL Ultima Gold TM LLT scintillator in 10 mL PerkinElmer super polyethylene vials. For each measurement, 3 countings have been realized and the results given correspond to an average of these 3 values. Electron microscopy observations were carried out on a Philips CM12 microscope operated at 80 kV.
[α]D 20 measurements were realized on a Jasco P-2000 polarimeter equipped with sodium lamp (589 nm) at 20 °C and using a 1.5 mL cell. Centrifugations were performed on a VWR Mini Star Silverline.

I-2. H/D exchange quantification
Deuterium incorporation was quantified by the decrease of 1 H NMR integral intensities at the specified positions compared to the starting material. Integral intensities were calibrated against hydrogen signals that do not undergo H/D-exchange. Mass spectrometry quantification was performed by subtraction of the mean molecular masses of the product and substrate isotopologue clusters in order to eliminate the contribution of the natural isotope abundance to the total mass. When all positions are deuterated, the isotopic enrichment was evaluated by 1 H NMR and corrected either with an internal standard (for compound 10) or using mass spectrometry analysis (for compound 17).

I-3. General Procedure for H/D exchanges
A 100 mL Fischer-Porter glassware equipped with a magnetic stirrer was charged with the corresponding substrate (0.02 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.5 or 5 mol%). Then, distilled THF or 2-MeTHF (C = 0.1 M) was added under argon. The solution was stirred under vacuum until bubbling and then pressurized with D2 gas (1 bar) for 5 min. This operation was repeated twice. The reaction mixture was stirred at the corresponding temperature under pressure of D2 (1 bar) for 24 h or 48 h. The final black solution was cooled down to room temperature and filtered through a nylon syringe filter (0.2 µm) before being evaporated under reduced pressure to give the desired product.

Indole 1
Following general procedure with indole (23.4 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of distilled THF. The reaction mixture was stirred at room temperature under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 1 as a white solid with a quantitative yield.

Harmane 2
Following general procedure with harmane (36.4 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of distilled THF. The reaction mixture was stirred at room temperature under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 2 as a yellow oil with a quantitative yield.

Dodecylamine 12
Following general procedure with dodecylamine (37 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of dry 2-MeTHF. The reaction mixture was stirred at 80 °C under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 12 as a pale yellow oil with a quantitative yield.

Piperidine 16
Following general procedure with piperidine (19.8 µL, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (4.8 mg, 0.01 mmol, 5 mol%) in 2 mL of dry 2-MeTHF. The reaction mixture was stirred at 80°C under pressure of D2 (1 bar) for 48 h. Then the crude reaction mixture was filtered over a nylon syringe filter (0.2 µm) and 0.5 mL of an HCl/Et2O (1 M) solution were added. The solvent was removed under vacuum and the desired product 16 was obtained as a white solid (hydrochloride salt) with a quantitative yield.

Nicotine 18
Following general procedure with nicotine (32.4 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of distilled THF. The reaction mixture was stirred at room temperature under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 18 as a yellow oil with a quantitative yield.

Papaverine 20
Following general procedure with papaverine (67.9 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of distilled THF. The reaction mixture was stirred at room temperature under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 20 as a white solid with a quantitative yield.

Zolmitriptan 22
Following general procedure with zolmitriptan (57.4 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of distilled THF. The reaction mixture was stirred at room temperature under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 22 as a yellow oil with a quantitative yield.

Lidocaïne 26
Following general procedure with lidocaine (46.9 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of dry 2-MeTHF. The reaction mixture was stirred at 80°C under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was purified according to the general procedure and gave the desired product 26 as a white solid with a quantitative yield.

Dextromethorphan-T
A 5 mL Fischer-Porter glassware was equipped with a magnetic stirrer and charged with dextromethorphan (0.02 mmol, 5.4 mg, 1 equiv.) and 0.96 mg of [Rh(COD)OMe]2 (10 mol%) in distilled THF (0.2 mL). The solution was frozen in liquid nitrogen at the liquid/gas interface and the reaction vessel was evacuated. The Fisher Porter tube was maintained under static vacuum for 5 min to check for leaks. Then the Fisher Porter tube was charged with tritium gas, Pfrozen = 458 mbar. The reaction mixture was then warmed to room temperature, Prt = 637 mbar. As soon as the gas pressure achieved a stable value, the stirring was launched and the mixture was heated to 55 °C for 3 h. The final black solution was cooled down to room temperature and the solution was filtered through a nylon syringe filter (0.2 µm). The Fisher-Porter tube was washed with dichloromethane (3 × 0.5 mL) and the filtrate was evaporated under reduced pressure. 2 × 20 mL of MeOH were added and the solution was then evaporated. The desired product was obtained with a molar activity of 40.4 Ci mmol -1 and a total activity of 496 mCi.

Nefiracetam-T
A 5 mL Fischer-Porter glassware equipped with a magnetic stirrer was charged with nefiracetam (0.02 mmol, 4.9 mg, 1 equiv.) and 0.96 mg of [Rh(COD)OMe]2 (10 mol%) in distilled THF (0.2 mL). The solution was frozen in liquid nitrogen at the liquid/gas interface and the reaction vessel was evacuated. The Fisher Porter tube was maintained under static vacuum for 5 min to check for leaks. Then the Fisher Porter tube was charged with tritium gas, Pfrozen = 542 mbar. The reaction mixture was warmed to room temperature, Prt = 710 mbar. As soon as the gas pressure achieved a stable value, the stirring was launched and the mixture was heated to 55 °C for 2 h. The final black solution was cooled down to room temperature and the solution was filtered through a nylon syringe filter (0.2 µm). The Fisher-Porter tube was washed with dichloromethane (3 × 0.5 mL) and the filtrate was evaporated under reduced pressure. 2 × 20 mL of MeOH was added and the solution was

II-3. TEM analyses
Procedure for TEM grids preparation: 4 µL of the corresponding solution was deposited on a carbon film 200 Mesh Copper (50) and analyzed by TEM (samples containing substrates 3 and 13 were diluted 10 times). III. Recyclability studies III-1. General procedure Following general procedure with dodecylamine (37 mg, 0.2 mmol, 1 equiv.) and [Rh(COD)OMe]2 (2.4 mg, 0.005 mmol, 2.5 mol%) in 2 mL of dry 2-MeTHF. The reaction mixture was stirred at 80 °C under pressure of D2 (1 bar) for 24 h. Then the crude reaction mixture was concentrated under vacuum and distilled THF and water were added followed by addition of the additive (for DANTA: 1.5 mL of dry THF, 0.5 mL H2O and 40 mg of DANTA; for PDAMAC: 2 mL of dry THF, 0.5 mL of H2O, 0.1 mL of PDAMAC 20 wt% in water; for sodium oleate: 2 mL of a stock solution (0.441 mL of oleic acid in 10.2 mL of THF with 55.99 mg of NaOH in 3.4 mL of H2O)). The solution was centrifuged and the supernatant was removed and analyzed. The black solid obtained was reused for a new run on dodecylamine or lidocaine.