Hydrogen Isotope Exchange Catalyzed by Ru Nanocatalysts: Labelling of Complex Molecules Containing N-Heterocycles and Reaction Mechanism Insights.

Ruthenium nanocatalysis can provide effective deuteration and tritiation of oxazole, imidazole, triazole and carbazole substructures in complex molecules using D 2 or T 2 gas as isotopic sources. Depending on the substructure considered, this approach does not only represent a significant step forward in practice, with notably higher isotope uptakes, a broader substrate scope and a higher solvent applicability compared to existing procedures, but also the unique way to label important heterocycles using hydrogen isotope exchange. In terms of applications, the high incorporation of deuterium atoms, allows the synthesis of internal standards for LC-MS quantification. Moreover, the efficacy of the catalyst permits, even under subatmospheric pressure of T 2 gas, the preparation of complex radiolabeled drugs owning high molar activities. From a fundamental point of view, a detailed DFT-based mechanistic study identifying undisclosed key intermediates, allowed a deeper understanding of C-H (and N-H) activation processes occurring at the surface of metallic nanoclusters.


Introduction
Nitrogen-based heterocycless uch as imidazoles, benzimidazoles and triazoles rank on the top of the most frequently approveds mallm olecule drugs by US FDA. [1] For example,1 ,2,4triazoles are prominents tructural motifsi nm any commercial antifungal drugs, [2] anti-tumor-, [3] anti-migraine agents [4] and many others. [5] Other heterocycless uch as oxazoles and carbazoles are also commonly encountered substructures in drug development, [6] biologically active natural products [7] and in materials cience as fluorescent molecules where deuterium incorporation can be interestingf or the enhancement of fluorescencep roperties. [8] Due to the occurrence of N-heterocycles in many types of usefulm olecules, the development of efficient methodsf or isotopicl abelling, particularly with hydrogen isotopes, is of paramount importance.I ndeed, tritiated analogues of drug candidates are considered as essential tools for studying the in vivo fate of drug candidates during absorption, distribution,m etabolism ande xcretion (ADME) studies. [9] In this context,t he development of robustm ethods allowingt he incorporation of at least 0.5 T( corresponding to am olar activity of 15 Ci mmol À1 )i nc omplexm olecules using mild reaction conditions (ideally with as ubatmosphericp ressure of T 2 gas) is of particular interest.D euterated compounds are also widely employed in various life-science applications, such as metabolomicsa nd proteomics as internal standards for quantitative LC-and GC-MSa nalyses. [10] To facilitate the synthetica ccess to these valuable isotopically labelled compounds, the discovery of new approaches enabling the selective incorporationo fa t least three deuterium atoms in smallc omplexm olecules, is appealing. [11] In the case of molecules containing oxazole and imidazole substructures, high isotopic enrichments can be obtainedu sing Ir I catalysts, but only for compounds owning an aromatic ring in position 2( see Figure 1a). In addition, only C(sp 2 )ÀHb onds in the g-position relative to the directing atom (DA) can be activated efficiently, and as ac onsequence only two deuterium atoms can be incorporated (per DA). [12] Concerning the deuterium and tritium labelling of some other Nheterocyclic cores such as 1,2,3-triazole, remarkable results have been described recently by Chirik and co-workerse mploying an air sensitiveb is(arylimidazol-2-ylidene)pyridine iron bis(dinitrogen) [13] (see Figure 1b)a nd ad imeric nickel hydride complex. [14] Even if the iron catalyzed hydrogen isotopee xchange (HIE) procedure possesses the advantage to work at low pressure of D 2 (1 bar), it afforded moderate deuterium incorporation on the 1,2,3-triazolem oiety of suvorexant (0.8D incorporated). For 1,2,4-triazole-a nd carbazole substructures (see Figure 1c), no broadly applicable HIE methods have been described so far.I n this paper,w edemonstrate that the selective deuteration and tritiation of the oxazole, (benz)imidazole, triazole and carbazole pattern in complex molecules can be achieved using Ru nanoparticles (RuNps) as catalyst( see Figure 2). For the oxazole and imidazole substructures, this approach possesses ab roader scope of applications, ah ighers olventa pplicability,a nd allows, in most cases, higher isotopei ncorporations under mild reactions conditions comparedt op reviously described methods.
On top of that, this work describes also the first general methodf or the selectivedeuterium and tritium labelling of the 1,2,4-triazole and carbazole scaffold by HIE reactions. From an application point of view,t hese reactions can be used for the late stage radiolabelling of complex pharmaceuticals (applying al ow pressure of tritium gas) and the preparation of internal standards for LC-MS quantification.O namore fundamental aspect, adeeper understanding of the reactionmechanismsinvolved in CÀHa nd NÀHa ctivation processes at the surfaceo f the Ru nanocatalyst was providedb yD FT calculations.T hese studies notably revealed the formation of different types of key intermediates, explaining the isotope incorporation at a, b and g positions of the directing nitrogen atom.

Results and Discussion
Labelling of oxazole derivatives We decided to initiate our studies with the deuteration of oxazole derivatives because effective methodst ol abel this substructure are still scarce. Based on our previousr esults, we envisioned that Ru nanoparticles might be used as ac atalyst for the deuteration of such nitrogen containing heterocycles. [15] Different oxazole derivatives 1-4 were successfully deuterated at 50 8Ci nT HF or DMA as solventu sing ac atalytic amount of ruthenium nanoparticles (5 mol %) stabilized in ap olyvinylpyrrolidone matrix( RuNp@PVP) under 2bar of D 2 gas( see Figure 3). Under these reactionc onditions, diphenyloxazole 1 was labelledw ith at otal uptake of 2.6 deuterium atoms, incorporated at the oxazole core and in the ortho positions of the phenylg roup. The possibility to selectively activateC ÀHb onds at a and g positions to the nitrogen atom of 1 permitted to obtain higherd euterium incorporationt han the one achievable over homogeneous Ir I catalysis (where only two deuteriums (2D) can be theoretically introduced). [16] The substrates 2-4 were used to demonstrate the broader substrate scope of RuNp-catalyzed HIE, as they cannot be labeled by other existing approaches such as Ir I -catalysis due to their substitution pattern. Indeed, compounds 2 and 3 were successfully deuterated at the C 2 and C 4 positiono ft he oxazole ring (both a-positions relative to the nitrogen atom). Furthermore, regio-and chemoselective labelling at C 2 of carboxylic compound 4 was achieved by using DMA as solvent.
Theoretical calculations at the DFT-PBE level of theory were conducted in order to study the reaction pathway leading to the C(sp 2 )ÀHa ctivation at the a positions of the nitrogen  atom. To achieve this study, a0 .5 nm ruthenium cluster with 1.4 Ha toms per Ru surfacea tom (Ru 13 H 17 )w as used as RuNp model. [17] As we can see in Figure 4, the coordination of 3 to the RuNp model through the lone pair of the nitrogen atom is an exothermic process (3 N* :c a. À19 kcal mol À1 ;t he labellingi s explained in the SI). From this intermediate, as tabilizing agostic interaction can be established between the C 2 ÀH( 3 N*,C2H* , green pathway) or C 4 -H (3 N*,C4H* ,b lue pathway) group and one of the first-neighbored rutheniuma toms to the one that interacts with the nitrogen atom. The formation of this threecenter,t wo-electron bond between aC ÀHb onding orbital and an empty metal orbital is evidencedb yt he slight carbonp yramidalization (accompanied by the lifting of the hydrogen atom out of the plane of the oxazole ring). From both 4-membered dimetallacycle intermediates 3 N*,C4H* and 3 N*,C2H* ,t he CÀHb ond activation is ak inetically accessible process with an activation barriero f6.0 kcal mol À1 on C 4 position( 3 N*,C4H°* )a nd of 4.2 kcal mol À1 on C 2 position (3 N*,C2H°* ). However,from athermodynamic point of view,t he CÀHb ond breaking is an almost athermic process at the C 2 position (3 N*,C2* : + 1kcal mol À1 with respect to 3 N*,C2H* )w hereas it is clearly endothermic at the C 4 position (3 N*,C4* : + 4.3 kcal mol À1 w.r.t. 3 N*,C4H* ). The H* atom can then easily exchange its position with one of the numerous deuterides already presenta tt he surfaceo ft he RuNps (H*$D* step in Figure 4), as previously demonstrated by NMR experiments. [9a] One availabled euteride in the vicinity of the active site can then recombine with the C X atom, provided that kinetics and thermodynamics do not impede it (the full pathway is given in Figure S1). In this mechanism,t he first key parameter is the formation of a4 -membered dimetallacyle in both pathways (3 N*,CXH* , X = 2o r4 ). But owing to the small barrier heights, as econd key parameter is the competition between the (CÀH)*!(C)*(H)* reaction( i.e., 3 N*,CXH* !(3 N*,CX* )(H*)) and the (C)*(H)*!(CÀH)* back reaction. The lower deuterium incorporation at the C 4 position (15 %) versus the C 2 position( 98 %), experimentally observed forc ompound 3,c an therefore be explained by the small barrier( only 1.7 kcal mol À1 )f or the back reaction(from 3 N*,C4* to 3 N*,C4H* ), thereby reducing the efficiency of the global process (see also Figure S2 for af ully detailed pathway,t hat is, up to the final D-incorporation). As imilar explanation can probably be invoked for compound 2,w here the isotopic labellinga tt he C 2 position( 99 %) was found to be higher than that measured at the C 4 position (26 %).

Labelling of imidazole derivatives
Further,R uNp@PVPc atalyzed deuterationsw ere also conducted on various imidazole derivatives( see Figure 5). 2-Phenylimidazole 5 showed high deuterium incorporation in the ortho-   positions of the phenyla nd at both a-positions relative to the imidazole nitrogen atoms. This result represented another example where the use of RuNps as catalysty ieldedahigher deuterium incorporation (3.3D) than Ir I -based catalysts, which would lead to the incorporation of only two deuterium atoms at am aximum.
The deuterium incorporation on both sites of 5 has also been considered from at heoretical point of view in order to identifyn otably,t he key intermediate leading to the labelling at the g-positions of the coordinating nitrogen. In order to answer this question, two competitive pathways were investigated (g1 in red and g2 in blue, Figure 6a). Independent of the considered pathway,c ompound 5 is initially adsorbed at the RuNp surfacet hrough the lone pair of the N 3 nitrogen atom to give 5 N* ,f ollowed by the formation of as tabilizing CÀ Ha gostici nteraction. It is noteworthy that two different adducts can be formed, either with the same ruthenium atom (5 N*,g1H* )o rw ith two neighboring ruthenium atoms (5 N*,g2H* ). They lead, respectively to af ive-membered metallacyle (a key intermediate analogue to the one proposed in homogeneous catalysis) or as ix-membered dimetallacycle adduct. The pathway that involves as ix-membered dimetallacycle is both endothermica nd kinetically accessible (blue pathway in Figure 6a). This mechanism cannotb ee xcluded for the deuterium incorporation but it is probably inefficient due to the smallb arrier (1.9 kcal mol À1 )a nd the exothermicity (ca.-4 kcal mol À1 )o ft he (C)*(H)* $ (CÀH)* back reaction (i.e., 5 N*,g2* $5 N*,g2H* ). The CÀH activation involvingafive-membered metallacycle( red pathway in Figure 6a)i sa lso kinetically accessible, with an activation barriero f7 .4 kcal mol À1 ,b ut thermodynamically more favorable (À4.1 kcal mol À1 )o nthe contrary to the g 1 case.
Thus, the deuterium incorporation at the g-position of the nitrogen is most probably due to ap rocess that goes through af ive-memberedm etallacyle intermediate such as in homogeneous catalysis. The very efficient HIE in a to nitrogen atoms (96 %) corresponds to at hermodynamically favorable mechanism (À2.1 kcal mol À1 w.r.t. (CÀH)*, green pathway in Figure 6b)i nvolving a4 -membered dimetallacycle as keyi ntermediate and al ow activation barrier for the CÀHb ond breaking step (4.3 kcal mol À1 ). Thus, similart ot he C 2 Ha ctivation pathway in compound 3 (see Figure4,g reen pathway) the competition between the (C)*(H)*$(CÀH)* back reaction (i.e., (5 N*,a* )(H*) $5 N*,aH* )a nd the (C)*(D*)*$(CÀD)* (i.e., (5 N*,a* )(D*)$5 N*,aD* )i sotopice xchange is in favor of the latter. An explanation of the difference between the isotopic enrichmentso f6 2a nd 96 %i ss omewhat beyond the chemical accuracy of DFT,e ven though, interestingly, the barriert hat leads to the (C g -D)* turns out to be highert han its (C a ÀD)* counterpart (11.5 vs. 6.5 kcal mol À1 ), in agreementw ith the observed lower isotopic enrichmenta tt his position. Compound 6 was deuterated at the a-position of the unsubstituted nitrogen atom with 99 %o fi sotopice nrichmenta ccompanied by a slight deuterium incorporation on the hydroxymethyl group. The coordination of the nitrogen and the oxygen atom to the surfaceo ft he catalystp robably immobilizes the substrate in a certain conformation.T his constraint would increase the activation barrierf or CÀHa ctivation giving rise to al owered deuterium incorporation at the C(sp 3 )c enter.V ery high incorporations of deuterium wereo btained for benzimidazoles 7 and 8, which cannot be labelledb yo ther HIE procedures to the best of our knowledge.I ndeed, carbon centers in a and b positions of the nitrogen atoms of 7 were deuterated with 98 %a nd, re- spectively 81 %o fi sotopice nrichment. AD FT-based investigation was also achieved for compound 7 in order to identify the key intermediate involved in the labelling of the b-positions of the nitrogens (see Figure 7). Again, the reactions tarts with a favorable s-donation of the nitrogen lone pair (7 N* )c ombined with af urthers tabilization of the adducta saresult of at he C a ÀH( 7 N*,aH* )o rC b ÀH( 7 N*,bH* )a gostici nteraction on an eighboring Ru atom. The C b ÀHH IE reaction involves a5-membered dimetallacycle and is favored by an exothermic (CÀ H)*$(C)*(H)* reaction( 7 N*,b* is more stable than 7 N*,bH* by À2.3 kcal mol À1 )l eadingt ot he H/D exchange (see the discussion for compound 3)a nd ar elatively low CÀHa ctivation barrier (7 N*,bH°* :5 .6 kcal mol À1 ). As shown in Figure 7, the C a ÀHH IE reactioni sc haracterized by ap rofile (in green), very similar to the profiles calculated for the two other H/D exchanges in a, described above for oxazole and imidazole substructures.I ti s noteworthy that the most efficient labellingp rocess (98 %) goes first through an almostb arrierless (C)*(H)* pathway and is then followed by the formation of the new (C-D)* bond which also requires to overcome the lowest barrier (4.5 vs. 7.9 kcal mol À1 ,s ee Figure S3). Overall, the postulated CÀHa ctivation processes excelled in three key factorsw hich characterize an efficient HIE at C(sp 2 )c enters, that is, the formation of metallacycle intermediates, low barriers and their exothermicities.
Regarding 8,a ll CÀHb onds situated at the b-positions of nitrogen atoms (C(sp 3 )-H and C(sp 2 )-H) were activated, leading to the incorporationo f3 .7 deuterium atoms. Interestingly,t he reaction conducted in deuterated THF did not lead to an in-crease of the isotopic enrichment whereas running the reaction twice resulted in ah igheri sotopice nrichment on the methyl [ 80].

Labellingo ft riazole derivatives
To the best of our knowledge,n og eneral approach hasb een described so far for the labelling of the 1,2,4-triazoles caffold using direct HIE. To our delight, our Ru nanocatalyst also promoted effective HIE on theseh eterocyclesa sd emonstrated by the labelling of the differently functionalized compounds 9-12 (see Figure 8). Both, a and g positions of coordinating nitrogen atoms were labelled leading to high deuterium uptakes per substructure unit (up 3.8D in as ingle run of catalysis).S imilar to previous findings, it appeared very likely,t hat the underlying CÀHa ctivations for the labelling at the a positions of the triazolics caffold pass through 4-membered dimetallacyclic key intermediates (see Figure 4). In contrast to the targeted C 2 -H and C 4 -H of oxazoles, the C 3 ÀHa nd C 5 ÀHo ft he 1,2,4-triazole unit displaya lmostt he same reactivities in most cases, which was reflected in identical isotopic enrichments on C 3 and C 5 of compounds 9-12 (see Figure 8). Logically,C ÀHa ctivations in ortho of adjacent phenyl substituents musta lso proceed through 5-membered metallacyclic key intermediates as in the case of 2-phenylimidazole (see Figure 6). The efficient deuterium incorporationa nd the functional group tolerance for the methoxy unit in 10,t he amino unit in 11 and the acetamide unit in 12 supported the potentialo ft his methodt op repare stable isotopically labelled internal standards for LC/MSq uantification.I ndeed, in al ater section, the applicability of RuNp@PVP was also confirmed for the deuterium and tritium labellingo fm ore complex triazole based drugs and one agrochemical(see Figures 12 and1 3).

Labelling of carbazole derivatives
Up to now,only few methods for the deuteration of carbazoles have been reported using HIE. All of them employed harsh reaction conditions and led to unselective isotope incorporation. [18] Through Ru nanocatalysis, compounds 13-16,w ere deuterated with high isotopic enrichments at the b-positions relative to the nitrogen using mild reaction conditions (see Figure 9). Interestingly,w ef ound that the addition of ab ase enhanced the chemoselectivity and efficacy of the deuterium incorporation (see Supporting Information for results obtained without base). Indeed, without base, considerable amounts of reduced side-products were formed, reducing the overall yield of isolated products. In contrast, performing the reaction with 1equiv.o fC s 2 CO 3 ,l ed to the recovery of deuterated compounds 13-16 in nearly quantitative yields. The higher chemoselectivity (i.e.,t he non-formation of reduced side-products) and efficacy (higher isotopic enrichments), observed for this transformation in the presence of ab ase, might be explained by the fostering of reaction intermediates leadingt ot he isotope incorporationi nb positiono ft he nitrogen. To test this hypothesis, aD FT-based investigation was first performed withoutb ase using carbazole as model compound. Different coordination modes of the substrate were considered( see Figure S4). The most favored involved the coordination of Na nd the simultaneous agostici nteraction of C 1 -H to give 13 NH*,bH* (see Figure 10). In this case, 13 is weakly coordinated to the surface, just by À9kcal mol À1 (compared to the ca. À25 kcal mol À1 in the previousc ases). This is consistent with a p-coordination of the nitrogen atom instead of a s-coordination. Starting from 13 NH*,bH* two pathways could be stated:t he NÀHa ctivation (13 N*,bH* ,g reen line) followed by the C 1 ÀHa ctivation (13 N*,b* )a nd the C 1 ÀHa ctivation( 13 NH*,b* ,b lack line) followed by the NÀHa ctivation (13 N*,b* ). These both exothermic reactions involvem oderate barrier heights, but the CÀHa ctivation seems easier,w hatever the pathway is. Interestingly,i ft he NÀH activation takes place before (greenl ine), the barrier of the CÀ Ha ctivation is significantly loweredb y5 .3 kcal mol À1 (with an activation energy of 9.2 for TS 13 NH*,bH°* vs. 3.9 kcal mol À1 for TS 13 N*,bH°* ). In summary,t he optimal reaction consists in the coordination of a5 -membered dimetallacycle that partially breakst he conjugation.T his preludes ap ossible HIE both on N  and on C 1 through two pathways that require to overcome a 17.8 kcal mol À1 apparent barrier and going on to 13 ND*,bD* . Prompted by this data, we were eager to investigate the role of cesium carbonate (Cs 2 CO 3 )o nt hese activation processes. The exploration of these pathways in the presence of ab ase is not an easy task for transition-state search algorithms. It is, however, possible to give some energetic and structural clues regarding the role of Cs 2 CO 3 on the CÀHa nd NÀHa ctivation in compound 13.A ss hown in Figure 11 a, Cs 2 CO 3 can favorably interactw ith both the surfacea nd 13 NH*,bH* .I nterestingly,t his Cs 2 CO 3 /13 NH*,bH* complex is more stable by ca. 20 kcal mol À1 than the two speciesl ying far away from each other on the surface. However,t his is an energy minimum, that is,g eometry optimizations do not involveabarrierless Ht ransfer from C 1 or Nt owardt he base. Given that the species resultingf rom this transfer are thermodynamically less stable by % 4a nd % 6kcal mol À1 ,s uch pathways would not facilitate the deuterium incorporation. However,t he possible role of the base after af irst CÀ Ho rN ÀHa ctivation by the metal surfaceo ft he RuNp should be also considered to clarify the situation.A ss hown in Figure 11 b, aC s 2 CO 3 molecule in the vicinity of the 13 NH*,b* or 13 N*,bH* intermediates spontaneously-andh ence efficientlyabstracts the hydrogen of the NÀHa nd C 1 ÀHb onds, respectively.T he reaction is exothermic by % 19.5 kcal mol À1 .G iven the Ca nd Nc oordination on the surface, the H/D exchange can then occur from the resulting 13 N*,b* compound. To sum up, these mechanistic investigations have shown that after a prior CÀHo rN ÀHa ctivation on the surface, ab ase could potentially facilitate the second rate-determining steps (CÀHa nd NÀHa ctivation) of the reactions leadingt oh igheri sotope incorporation. [19] Labelling of pharmaceuticals and other bioactive molecules a) Deuterium labelling To illustrate the usefulness and the broad applicability of our RuNpsc atalyzed HIE, deuterium and tritium labelling of N-heterocyclec ontaining molecules of medicalr elevance and higher molecular complexity,w as then considered (see Figure 12). As af irst example, we have chosen the oxazole containing alkaloid, pimprinine 17 possessing anticonvulsant [20] and antiviral activities. [21] The use of RuNp@PVP (20 mol %) in CD 3 OD with 1equiv of Cs 2 CO 3 ,a llowed am uch higherd euteration of this sensitivem olecule on its indole moiety than without Cs 2 CO 3 , analogoust ot he findings madew ith carbazoles 13-16.V ery good isotopice nrichmenta lso occurred on the oxazole core (for the resultsw ithout Cs 2 CO 3 ,s ee Supporting Information). The benzimidazole containing drug astemizole 18 was used as ad rug in the treatment of allergies. [22] In the case of this complex molecule, deuteration occurs at the b-position of the unsubstituted nitrogen of the benzimidazole moiety with an isotopic enrichment of 87 %. Furthermore, the analysis of 2 HNMR data (@14T) also supported slightH /D exchange on one a Figure 12. Deuterium labelling of complex pharmaceuticals (at 55 8C, overnight). Figure 11. Abilityo fC s 2 CO 3 to coordinate to the catalystsurface and to adopt the role of aproton acceptor in the NÀHa nd CÀHa ctivationstep. a) Ht ransfer from 13 NH*,bH* to cesium carbonate, b) Htransfer to cesium carbonate after apreliminary CÀHo rN ÀHa ctivation. methylene of the tertiary amine (22 %). Imiquimod 19 has a convincing efficacy against malignant melanoma. [23] This poorly soluble molecule was successfully deuteratedu sing DMA as solvent, highlighting the broad solvent compatibility of our method. The human antifungal drugf luconazole 20,w as selectively labelled on the 1,2,4-triazoleu nits leading to av ery high isotope incorporation (almost 4D incorporated). Thisr esult repeatedlym anifests the potential of our method for the preparation of stable isotopically labelled internal standards of a commercial drug forL C-MS quantifications. This rapid deuterium labellingi si nsofar beneficiary as we consider that stable isotopically labelledi nternal standards of fluconazole were originally synthesized from deuterated precursors over four steps. [24] The efficient and selectived euteration of the triazole substructure of the complex agricultural fungicidefluquinconazole 21 is representative for the functional group tolerance of the described catalytic transformation. Despite the presence of differentc arbon-halogen bonds , the highly deuterated product waso btained in high yield after as imple filtration through aC18 cartridge. The 1,2,3-triazoledrug suvorexant 22,a dministered for insomnia treatment, is known to be labelledb yh ydrogen isotopes within other methods. Hence, H/D exchange occurs either on the CÀHb onds of the 1,2,3-triazole with ah omogeneous Fe 0 catalyst (leading to the incorporation of 0.6D) or in the ortho position of the adjacent phenylw ith Crabtree's catalyst (theoretically limited to the incorporation of 1D). [13] Here, the use of RuNpsa sc atalysta llowed the deuterium labelling of both, the 1,2,3-triazoleg roup (1.6D) andt he adjacent phenyl ring (0.3D), which gave ac onsiderably higherd euterium incorporation as aw hole, compared with the ones obtained using the previously described HIE procedures. Carvedilol 23 is an onselective beta/alpha-1 blockeru sed for treating congestive heart failure, left ventricular dysfunction and high blood pressure. [25] Using carvedilol 23 as as ubstrate led to the deuteration of the secondary amine with ah igh isotopic enrichment due to the highera ffinity of the alkylamine nitrogen compared to the one embedded in the carbazole moiety.B ys imply protecting the aliphatic amine with a Boc-group and adding one equivalent of Cs 2 CO 3 ,w e were able to exclusively label the carbazole moiety (molecule 24,s ee Figure12) with ah igh isotopic enrichment( 99 %). The possibility to modify the regioselectivity of the isotope incorporation on as uch complexs tructure using simple protecting group strategies, highlights the versatility of our RuNps catalyzed HIE reactions for the synthesis of labelled drug compounds for metabolic studies.

b) Tritium labelling of pharmaceuticals
As an ext step, we demonstrated that the late-stage tritiation of complex pharmaceuticals can succeed through our catalytic method( see Figure13). In this context,t he use of T 2 gas as isotopics ource is ag reat advantage because it is the easiest raw material to handlef or tritium labelling. Typically, reactions involving gaseous tritium were conducted using as ubatmospheric pressure of T 2 in order to minimize the risk of leakage and radioactive releases.
Up to date, the simplest way to label the N-heterocyclics ubstructures of astemizole and carvedilol with tritium was the transformationo fh alogenated precursors with Pd/C and T 2 ,a procedure that consisted of several other reactions teps. [26] With our method, radioactive analogueso fa stemizole 18*,f luconazole 20*,a nd N-Boc-protected carvedilol 24* were obtained with satisfying molar activities and in high yields using subatmospheric tritium gasp ressures. It is noteworthy that in every case (18*, 20*, 24*)t ritium labelling took place on positions which are not major metabolism sites. [27] The obtained molar activities of 18* and 20* (24 Ci mmol À1 ), under at ritium gas pressure of % 900 mbar,c learly outperformed the prerequisites for ADME studies (10-20 Ci mmol À1 ). Performing the radiolabellingu nder al ower tritium gas pressure of % 500 mbar,t ritiated 24* could be obtained with at ritium incorporation of % 9Cimmol À1 ,which was still within an acceptable range.

Conclusion
In this paper,w ed emonstrated that the applicationo fR u nanocatalysts can provide efficient deuteration and tritiation of different classes of heterocycles in complex molecules using D 2 or T 2 gas as isotopics ources. For the labelling of oxazole and imidazole substructures, this work represents as ignificant advance in practicalityc ompared to previously described methods, with notably higher isotope uptakes, ab roader substrate scope and ah ighers olventc ompatibility. This methodr epresents also the unique approach allowing the selective deuterium and tritium incorporation on 1,2,4-triazolea nd carbazole substructures using direct hydrogen isotope exchange. In terms of application, the high incorporation of deuterium atoms allows the synthesis of deuterated internal standards for LC/MSq uantification and the efficacy of the catalytic process permitst he preparation of complex radiolabelled drugs owning high specific activities by using asubatmospheric pressure of T 2 gas. From am ore fundamental point of view,t he detailed theoretical calculations have deciphered the processes Figure 13. Tritium labelling of complex pharmaceuticals. Chem. Eur.J.2020, 26,4988 -4996 www.chemeurj.org 2019 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim of CÀHbond activation at a, b-and g-positions of coordinating nitrogen atoms occurring att he surfaceo fR uc lusters. All the key intermediates involved in suchr eactions were disclosed: four-membered dimetallacycle for the CÀHa ctivation in a, five-membered dimetallacycle for the CÀHa ctivation in b and five-membered metallacycle for the CÀHa ctivation in g. Energy profiles qualitativelya ccount for the experimental isotopic enrichment rates. CÀHa ctivation equilibria or CÀDr ecombination equilibria exhibit characteristicp atterns that drive the overall reaction. In addition to the fact that this study paves the way for an easier access to valuablei sotopically labelled complex molecules, the theoretical aspects addressed will be useful for future developments of more efficient and selective nanocatalysts for CÀHa ctivations.