Programmable Deuteration of Indoles via Reverse Deuterium Exchange

Methods for selective deuterium incorporation into drug-like molecules have become extremely valuable due to the commercial, mechanistic, and biological importance of deuterated compounds. Herein, we report a programmable labeling platform that allows access to C2, C3, or C2- and C3-deuterated indoles under mild, user-friendly conditions. The C2-deuterated indoles are accessed using a reverse hydrogen isotope exchange strategy which represents the first non-directed C2-deuteration of indoles.


GENERAL INFORMATION
All solvents and chemicals were used as purchased unless stated otherwise; all solvents were dried according to conventional methods. Anhydrous 1,4-dioxane was purchased from Sigma Aldrich and further dried over molecular sieves. NaOAc was dried at room temperature under vacuum for 4 hours prior to use. CD3CO2D was purchased from Deutero. All reactions were performed in oven dried apparatus with magnetic stirring under an inert atmosphere of argon or nitrogen. The reactions were followed by thin layer chromatography (TLC) carried out on aluminium-foil backed plates coated with silica gel (Merck Kieselgel 60 F 254). The products were visualized using UV fluorescence (254 nm) or potassium permanganate stain. Silica flash column chromatography was performed over Merck silica gel C60 (40-60 μm) using eluent systems as described for each experiment.
All NMR spectra were recorded on Varian VNMRS 500 MHz, Jeol 400 MHz, or Bruker AV 400 MHz spectrometers. NMR data were processed using MNova 12.0.4 software. Proton and carbon-13 NMR spectra are reported as chemical shifts (δ) in parts per million (ppm) relative to residual undeuterated solvent peak or TMS. Coupling constants (J) are reported in units of hertz (Hz) and are rounded to the nearest 0.5 Hz for 1 H NMR and the nearest 1 Hz for 13 C NMR. The following abbreviations are used to describe multiplets: s (singlet), d (doublet), ad (apparent doublet), q (quartet), p (pentet), m (multiplet), br (broad). Structural assignments were made with additional information from gCOSY, gHSQC, and gHMBC experiments. HRMS measurements were carried out on Agilent 6530 Accurate-Mass Q-TOF LC/MS and Jeol Accutof GCX EI-TOF instruments. IR spectra were measured on a Perkin Elmer Spectrum One FT-IR with ATR attachment. Known compounds have been checked against literature references and only relevant analytical data are given.
Determination of Deuteration Incorporation. Deuterium incorporation was quantified by comparing the 1 H NMR integral intensity at the deuterated position with the starting material (see Fig. S1). 1 H NMR experiments were run with T1 relaxation times of 1 second and integral intensities were calibrated against hydrogen signals that did not undergo H/D-exchange. Figure S1: 1 H NMR spectra of unlabelled (top) and labelled/deuterated (bottom) indole in CDCl3. Integration of the signal at 6.47 ppm decreases from 100% (1a) to 5% intensity (2a), indicating 95% deuterium incorporation. The use of CH3CO2D significantly reduced deuteration at C2 compared with CD3CO2D (entry 5 cf. entry 4)presumably due to proton exchange between the methyl and hydroxyl positions in acetic acid. Low C2-deuteration was also observed with D2O (entry 9), and efforts to generate a viable deuterium source in situ from cheaper D2O and CH3CO2H (entry 10) or pivalic anhydride (entry 11) were unsuccessful.  As the palladium loading is decreased, the uncatalyzed acid-base background reaction becomes dominant. Conditions: 1 (0.4 mmol), Pd(OAc)2 (10 mol%), NaOAc (1.5 equiv.), CD3CO2D/dioxane (1.2 mL/3 mL), 120 °C, 16 h. Grey circles show the labelling positions, with values in brackets denoting isotope incorporation, as determined by 1 H NMR, before purification on silica.

S6
The use of anhydrous 1,4-dioxane and rigorous drying of NaOAc proved instrumental to avoid acid/base background reactions causing isotopic dilution at C3 (Table S5). When an older bottle of dioxane was used, deuterium incorporation at C3 was markedly lower than with a new bottle of anhydrous 1,4-dioxane (stored over molecular sieves). Pleasingly, the addition of 4Å molecular sieves to a reaction with the older dioxane largely succeeded in suppressing isotopic dilution (4f in Table S5).
Step 2: Treatment with base. d2-Deuterated indole 4 (0.2 mmol) was dissolved in a solution of MeOH (1.2 ml) and H2O (0.4 ml). K2CO3 (28 mg, 0.2 mmol) was added. The reaction was placed in an oil bath pre-heated to 80 °C and stirred for 16 h, after which it was allowed to cool to rt. The reaction was diluted with H2O and extracted 3 times with DCM. The organic layer was dried using MgSO4 and solvent was removed under reduced pressure to provide the pure product. In some cases, further purification by silica flash column chromatography was required.
Method B: C3-Deuteration. To a mixture of indole 1 (0.2 mmol) in anhydrous 1,4-dioxane (0.6 mL, 0.3M) was added deuterated acetic acid (CD3CO2D) (1.2 mL, 10 mmol). The reaction was placed in an oil bath pre-heated to 80 °C and stirred for 16 h, after which it was cooled to room temperature and solvents were removed under vacuum. No further purification was required, unless stated otherwise.
When the synthesis of 5a was scaled up to 119 mg (1.0 mmol) following method A, step 2, 84 mg (71% yield, 60% deuterium incorporation) of the title compound were isolated.
Analytical data is provided for the 0.2 mmol scale reaction:

C2-and-C3-Deuterated Indoles
Note: While every effort was made to keep the compounds on silica for the minimum amount of time possible to avoid protonation (isotopic dilution) at C3, all compounds in this section showed a reduction in deuteration during column chromatography (compared to the crude product). E.g. for compound 4a, deuterium incorporation was 70% at C3 prior to purification, but dropped to 45% after silica flash column chromatography (see Table S5 above for more C3 deuteration values before silica flash column chromatography). -2,3-d2 (4a) 4a was synthesized from indole (47 mg, 0.4 mmol) according to method A, step 1. It was purified by silica flash column chromatography (gradient: cyclohexane to 7:3 cyclohexane:ethyl acetate) to afford 40 mg (83% yield, 81% deuterium incorporation at C2, 45% deuterium incorporation at C3) of the title compound as an orange solid.
Analytical data is provided for the 0.4 mmol reaction: