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Deuterium- and tritium-labelled compounds find widespread application in the pharmaceutical industry because of their ability to alter metabolism7,8, to exploit kinetic isotope effects9 and most commonly to perform ADME studies required for registration1,2,3,4,5,6. The most common methods for preparation of 3H-radiolabelled drug molecules include reduction of a functional group to a C–3H bond using stoichiometric reagents or by catalytic methods involving direct hydrogen isotope exchange (HIE)2,4,6. With the latter approach, hydrogen atoms in the drug molecule or an advanced intermediate undergo exchange with tritium gas (3H2) or tritiated water (3H2O) in the presence of a transition metal catalyst. Whereas both 3H2 and 3H2O have been applied in drug discovery and ADME studies, 3H2 is preferred over 3H2O for three reasons: the latter is prepared from tritium gas, is known to undergo decomposition to its constituent elements by autoradiolysis, and presents challenges to safe handling owing to a relatively high radioactivity-to-volume ratio requiring commercial sources to be diluted with natural abundance water6. The higher isotopic purity and atom efficiency associated with 3H2 enables the preparation of radiolabelled drug compounds with higher specific activity and further demonstrates the preference for tritium gas for HIE. Because many known precious metal catalysts used for HIE of organic substrates and drug molecules rely on water6,10,11 or organic molecules12,13 as the source of the hydrogen isotope, the discovery of new catalyst technology that enables HIE using 3H2 gas would be transformative for ADME studies6.

Among precious metal catalysts used for isotopic labelling of pharmaceuticals, Crabtree’s iridium catalyst, [(COD)Ir(py)PCy3]PF6 (COD = 1,5-cyclooctadiene; py = pyridine), a compound initially developed for alkene hydrogenation14, and Kerr’s iridium-carbene catalysts15 are the most widely used (Fig. 1)15,16,17,18. These catalysts and other Ir(I) variants typically operate in a limited range of solvents, and over the past two decades efforts have been focused on improving their stability and compatibility with functional groups commonly present in pharmacueticals12,15,16,18,19. The site selectivity of C–H exchange is predictable and relies on directing groups to enable reactivity of the ortho positions in aromatic and heteroaromatic rings. Depending on the specific target and purpose of the metabolic studies, isotopologues and isotopomers of the radiolabelled drug molecules—where the radiolabels are introduced at different locations in the molecule and in different amounts—may be required2,6. Accordingly, new catalysts that introduce tritium at sites orthogonal to those accessed by iridium and with high degrees of incorporation and hence specific activities are valuable in augmenting existing technologies and in providing new diagnostics for ADME studies.

Figure 1: Homogeneous transition metal-catalysed hydrogen/tritium exchange using tritium gas.
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a, Ir catalysts are widely used for the tritium labelling of pharmaceuticals; these catalysts operate through directed ortho exchange in the presence of a directing group (DG); Crabtree’s catalyst (right) is the most popular and only operates in CH2Cl2. More recently, Kerr and co-workers developed a class of Ir-carbene variants that exhibit superior catalyst stability, efficiency and functional group compatibility15,18. b, Complementary technology: iron catalyst (1) as an alternative tritiation catalyst that exhibits orthogonal C–H bond selectivity that is determined by steric accessibility and C–H bond acidity. Directing groups are not required and the catalyst operates in a range of solvents including THF, DMF and NMP.

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The bis(arylimdazol-2-ylidene)pyridine iron bis(dinitrogen) complex20 has recently been shown by our laboratory to be an exceptionally active base metal catalyst for the hydrogenation of unactivated, essentially unfunctionalized alkenes21,22. In situ monitoring of the variant bearing saturated N-heterocyclic carbenes, (H4-iPrCNC)Fe(N2)2 (H4-iPrCNC = 2,6-(2,6-iPr2-C6H3-4,5-H2-imidazol-2-ylidene)2C5H3N), 1, during the course of alkene hydrogenation in perdeuterated benzene demonstrated C–H isotopic exchange between the sp2 C–2H bonds of the solvent and free H2 gas. Quantitative 13C{1H} NMR spectroscopy (126 MHz) established formation of C6Hx2H6−x isotopologues with diagnostic chemical shifts (Δδ = 37–65 Hz) and multiplicity (δbenzene-d6 = 128.06 p.p.m., 2JC-2H = 24 Hz, see Supplementary Information for representative spectrum). To explore the generality and the potential for tritium exchange reactions, additional representative substrates were evaluated with the iron catalyst.

Catalyst evaluation studies were initially conducted with 2H2 gas as a more convenient and readily handled 3H2 surrogate. The iron-catalysed HIE of arenes and heteroarenes was initially examined (Fig. 2). All of the catalytic reactions were conducted under standard conditions employing 1 mol% of 1, 3.02 M substrate in THF with 4 atm of 2H2 at 45 °C. The extent of isotopic exchange was determined after 24 h using a combination of quantitative 1H and quantitative 13C{1H} NMR spectroscopies. The latter analytical technique has been especially informative for substrates where multiple aromatic C–H signals are poorly resolved in 1H NMR spectra. Iron pre-catalyst 1 has proven particularly effective for the 1H/2H exchange with many important substructures in pharmaceuticals, including substituted arenes (2–7), pyridine derivatives (8–12) as well as other nitrogen-, oxygen- and sulphur-containing heteroarenes (13–21)23,24. Examination of the site selectivity of the iron-catalysed deuteration of arenes (2–7) established two salient trends—namely, electron-poor substrates (6, 7) undergo C–H isotopic exchange faster than electron-rich ones (for example, 3–5), and the site selectivity of the C–H bond activation occurs at the most sterically accessible C–H bonds. The iron-catalysed method is also versatile within various classes of nitrogen heterocycles. With (−)-nicotine (12) exclusive deuteration of the sterically accessible positions of the pyridine occur in the presence of the N-methyl pyrrolidine. With 2,6-lutidine (10), the 4-position is the only sp2 hybridized C–H bond to undergo isotopic exchange. Notably, significant (40%) deuterium incorporation was observed in the sp3 benzylic positions, a feature absent in 2-methylpyridine (8) and 2-ethylpyridine (9). In the case of N-methyl-indole (20) and imidazo[1,2-α]pyridine (21), exchange occurred at positions adjacent to the ring junctions.

Figure 2: Fe-catalysed deuterium labelling of representative arenes and heteroarenes.
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a, Here selectivity for isotopic exchange is governed by steric accessibility; percentages in parentheses correspond to the extent of deuterium incorporation at the designated positions; for 12 and 19, 3 mol% catalyst loading was employed. b, Selectivity comparison between 1 and Crabtree’s iridium catalyst. c, Inverse pressure dependence exhibited by 1.

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The complementary selectivity of 1 as compared to Crabtree’s catalyst is highlighted by the deuteration of N,N-dimethylbenzamide (22). As shown in Fig. 2b, the iridium catalyst promoted isotopic exchange exclusively at the ortho positions as expected for an amide-directed C–H activation. In contrast, the iron catalyst enabled deuterium incorporation statistically at the sterically accessible meta and para positions. Precious metal catalysts exhibiting sterically preferred C–H selectivity are well documented, although in most cases 2H2O or organic solvents serve as the source of the isotopic label10,11,25. An iridium example, [(η5-C5Me5)(Me3P)Ir(CH3)CH2Cl2][BArF4] (where [BArF4] = tetrakis(3,5-bis(trifluoromethyl)phenyl)borate)) has been reported whose selectivity is determined by sterically accessible C–H bonds and employs 2H2 or 3H2 gas but requires a stoichiometric rather than catalytic amount of metal complex25. Because practical tritiation of pharmaceuticals are typically conducted with a modest excess of tritium gas (that is, at subatmospheric pressure)6, the deuteration of toluene catalysed by 1 was evaluated at both 1 atm and 0.35 atm of 2H2 gas. Higher activity was observed at the lower pressures of 2H2 gas, as 42% and 52% deuterium incorporation was observed in the para and meta positions after 6 h. These values were reduced to 17% and 22%, respectively, when the pressure was increased to 1 atm (Fig. 2c).

The promising functional group compatibility and the favourable inverse pressure dependence observed with 1H/2H exchange with 1 prompted evaluation of the deuteration and tritiation of pharmaceuticals (Fig. 3). Although THF was used for catalyst evaluation studies with representative arenes and heteroarenes, the limited solubility of more functionalized pharmaceuticals in this solvent inspired use of other media for catalytic HIE. Iron catalyst 1 has proven robust and operated efficiently in a range of polar aprotic solvents including dimethylacetamide (DMA), dimethylformamide (DMF) and N-methylpyrrolidinone (NMP)—the last being used for all subsequent drug labelling studies. With 10 mol% 1, selective deuteration of papaverine (27), an opium alkaloid antispasmodic26, was achieved where exclusive deuterium incorporation was observed at the 3-position of the isoquinoline fragment. This site selectivity was as expected on the basis of the small molecule studies—this C–H bond is the only sterically accessible position for the iron catalyst. Other commercially available pharmaceuticals, namely varenicline, loratadine, cinacalcet and flumenzil, underwent iron-catalysed deuteration at the predicted sterically accessible sites. With loratadine, isotopic exchange was preferred at the 3-position over the 2-position of the ring, contrary to typical C–H functionalization preferences. With the experimental orexin receptor antagonist, MK-6096 (30)27, quantitative deuterium incorporation occurred at the 3-position of the 1,2,5-trisubstituted benzene subunit, probably through ortho-directed C–H activation from the nitrogen atom of the proximal pyrimidine ring. This preferred selectivity demonstrates that in the presence of appropriate ligands, directed C–H activation can be preferential to steric site selectivity. Pharmaceuticals such as the CRTH2 antagonist, MK-7246 (34)28, and the anti-viral drug candidate, MK-822829, proved incompatible with 1, probably as a result of the carboxylic acid functional group. Preparation of the NMP soluble conjugate sodium carboxylate salt of MK-7246, 35, readily obtained by addition of NaH to the free acid, overcame this limitation. Following acidic workup after iron-catalysed deuteration provided the isotopically enriched product, 36, in high yield with deuterium selectively incorporated into the C–H bonds ortho to the fluorine. As illustrated with MK-8228 (37), a Merck developmental drug candidate for treatment of Human Cytomegalovirus (HCMV) infections in transplant patients currently undergoing Phase III clinical trials, the carboxylate salt was generated in situ and used in the HIE reaction without isolation or further purification.

Figure 3: Fe-catalysed deuterium labelling of drug molecules.
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a, Direct labelling of drug substrates, general catalytic reaction conditions as follows: 10 mol% 1, 0.154 mmol substrate, 0.5 ml NMP solution, 1 atm 2H2, 45 °C, 24 h. b, Deprotonation-protection strategy of carboxylic acids present in 34 and 37 for compatibility with the iron catalyst. For 37, the same deprotonation strategy was employed, but the carboxylate salt was generated in situ and used without further isolation.

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To demonstrate the utility and to further highlight the complementarity of the iron catalyst with existing iridium-catalysed methods, a series of representative, commercially available pharmaceuticals were tritiated using both 1 (Fig. 4a) and Crabtree’s iridium catalyst (Fig. 4b). Use of 3H2 gas was particularly informative, to not only determine site selectivity but to also establish the relative specific activity of the final product between the base and precious metal C–H exchange catalysts. In all cases, tritium exchange reactions were performed using subatmospheric pressures (~0.15 atm, approximately 12 equiv. 3H atoms with respect to substrate) of 3H2 gas at room temperature at higher (25 mol%) catalyst loading, and the positions of labelling were confirmed by 3H NMR spectroscopy. With MK-6096 ([3H]30a), flumazenil ([3H]32a) and suvorexant ([3H]33a), tritium was detected in trace quantities from iron-catalysed isotopic exchange in positions previously unobserved in the deuteration experiments (Fig. 4a). These positions are adjacent to arene substitution such as methyl groups or ring junctions where C–H activation has a higher barrier due to steric inaccessibility. The high sensitivity of 3H NMR spectroscopy as compared to the corresponding 1H and 13C experiments allowed detection of the small quantities of isotopic label introduced into these positions. For suvorexant ([3H]33a), the iron and iridium exchange methods are complementary; the iron catalyst prefers isotopic exchange at the relatively electron deficient triazole, whereas iridium favours directed C–H functionalization and tritium incorporation in the aryl ring at the site ortho to the triazole subunit. Similar orthogonal yet complementary site selectivity was observed with MK-7246 where after carboxylate deprotonation, the iron catalyst enables sterically driven tritiation of the fluorinated arene ring while iridium promoted directed C–H exchange exclusively in the saturated nitrogen heterocycle.

Figure 4: Tritium labelling of drug molecules.
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a, Using 1, reaction conditions as follows: 25 mol% catalyst loading, 7 μmol substrate, 1.2 Ci 3H2 (0.15 atm), 0.2 ml NMP, 23 °C, 16 h. b, Using Crabtree’s catalyst, reaction conditions as follows: 25 mol% catalyst loading, 7 μmol substrate, 1.2 Ci tritium (0.15 atm), 0.5 ml CH2Cl2, 23 °C, 16 h. For [3H]34a, the sodium salt conjugate base, 35 was used in place of 34 owing to incompatibility of the carboxylic acid functionality with 1 (see Supplementary Information for details). Specific activities for each compound are given in Ci mmol−1. *A comparison using another Ir-based catalyst, [(COD)Ir(IMes)PPh3]PF6 (see Supplementary Information for catalyst representation), originally developed by Kerr and co-workers15,18, was performed using MK-6096 as the model substrate; the Kerr catalyst labelled the same position as Crabtree’s catalyst with a lower specific activity of 8.8 Ci mmol−1 under identical conditions.

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The tritiation of flumazenil highlights the beneficial reactivity enabled by 1. Sterically accessible C–H bonds ortho to fluorine in the arene fragment resulted in a relatively high specific activity of 16.1 Ci mmol−1 ([3H]32a). Typically, specific activity values in the range 10–20 Ci mmol−1 are acceptable for ADME studies30. With Crabtree’s iridium catalyst, no tritiation of flumazenil was observed under similar conditions (Fig. 4b, [3H]32b). Although amide and ester functionalities are present that could potentially serve as directing groups and hence enable isotopic exchange, conformational effects probably inhibit the accessibility of proximal C–H bonds required for tritiation. Significantly higher specific activity was observed with the iron-catalysed tritiation of MK-6096. This difference may be traced to the abundance of sterically accessible C–H bonds where the iridium catalyst only introduces the isotopic label at one site (Fig. 4b, [3H]30b).

In summary, an iron catalyst for the tritiation of pharmaceuticals has been discovered with selectivity for C–H bonds that is orthogonal and complementary to existing precious metal catalyst technology. This catalyst is compatible with a range of pharmaceutically relevant functional groups and operates efficiently in polar aprotic solvents at low pressures of tritium gas. The ability to introduce radiolabels into unique positions provides a new diagnostic for ADME studies, a critical component of the drug approval process. Current efforts are devoted to elucidating the mechanism of action and to improve the stability and handling of the iron precursor.