Steven Frippiat

Antoine Peresson

Thibaut Perse

Yvan Ramondenc

Cédric Schneider

Olivier Querolle

Patrick Angibaud

Virginie Poncelet

Lieven Meerpoel

Vincent Levacher

Laurent Bischoff

Christine Baudequin

Christophe Hoarau
email: christophe.hoarau@insa-rouen.fr

Pd(0)-Catalyzed Direct Inter-and Intramolecular C-H Functionalization of 4-Carboxyimidazoles

Keywords: catalysis, arylation, palladium, heterocycles, alkenation, C-H functionalization, palladium catalysis, imidazole, arylation, alkenylation C-H functionalization, palladium catalysis, imidazole, arylation, alkenylation

come

Imidazoles are abundantly present in various naturallyoccurring and biologically active compounds, 1 pharmaceutics 2 and materials. 3 This class of azaheterocycle is employed to produce N-heterocyclic carbenes (NHC) used as ligands in various transition-metal complexes in homogeneous catalysis 4 and organocatalysis. 5 Imidazolium-based ionic liquids are also often considered as green reaction media due to their unique chemical and physical properties. 6 In this context, concise synthetic access towards imidazole cores is of high importance. Since last decades, the synthesis of imidazoles over numerous standard condensation reactions has been intensively reported. 7 The main drawback arises from the introduction at an early stage of the adequate functional group which penalizes the subsequent cyclization sequence. One of modern synthetic alternative is focused on the late-stage palladium-catalyzed C-H functionalization reactions from C2, C4 & C5 unsubstituted imidazoles. 8,9,10,11,12 In particular, Pd(0)catalyzed C-H (hetero)arylation of N-substituted imidazoles with aryl halides at C2 and/or C5 positions has been widely studied by Bellina and Rossi group. 12 As some remarkable progresses, the orthogonal C2-H versus C5-H arylation of N-substituted imidazoles have been developed respectively under baseassisted Pd(0) / Cu(I) cooperative catalysis and Pd(0)-catalyzed concerted metalation-deprotonation (CMD), taking advantage of the acidic and the nucleophilic specific characters of respectively C2 and C5 sites. 12b,i Although being less reactive, the C4 position of the imidazole ring was also reached through rare C-H functionalization reactions methodology. 13 Inspired by the pioneering Suzuki work on intramolecular C-H arylation of Nbromophenyl imidazole-4-carboxamide 14 and, in line, with our ongoing interest for the regioselective Pd(0)-catalyzed C-H arylation and alkenylation with halides of (oxa)thiazole-4carboxylates, 15 as well as imidazole-5-carboxylate, 16 we turned our attention to the 4-carboxyimidazole series. Unlike the parent imidazole, the presence of the ester function at the C4 site offers several new synthetic and methodologic opportunities. Indeed, the carboxylate group may be gracelessly easily removed by catalytic extrusion of volatile CO 2 or converted into wealth of other moieties, and can be used as leaving group in various decarboxylative coupling with formation of C-C or C-heteroatom bonds. 17 Herein, we turned our attention to an exhaustive study of selective C2-H and C5-H arylation of different methyl Nsubstituted imidazole-4-carboxylate with aryl halides under Pd(0) catalysis through both inter-and intramolecular pathways. The later one opens access to innovative non-aromatic imidazoles-based tricycles offering potential for applications in pharmaceutical sciences.

We started our investigations by selecting the N-benzyl methyl imidazole-4-carboxylate 1a as substrate, which was readily prepared by N-benzylation of the commercially available methyl imidazole-4-carboxylate under S N 2-type nucleophilic substitution using NaH as base and benzylbromide as electrophilic reagent. 18,19 The first set of Pd(0)-catalyzed C-H arylation of 1a with of 4-bromotoluene (1.2 equivalent) was carried out by using Pd(OAc) 2 catalyst, PPh 3 ligand (Table 1) and Cu(I) as additive to reach selectively the C2 site in accordance to Bellina and Rossi reported work. 12i In fact, the reaction was found poorly efficient in the standardly-used high polar DMF solvent with both potassium and cesium carbonates leading mainly to degradation of starting material 1a (Table 1, entry 1). However in the less polar 1,4-dioxane solvent, the Pd(0)-catalyzed and CuIassisted C-H arylation of 1a with bromotoluene provided selectively the C2-H arylated imidazole-4-carboxylate 3a in 34% yield (Table 1,entry 2). a Yield of isolated compound. b Using 4-iodotoluene (1.2 eq) as electrophilic reagent instead of 4-bromotoluene. c Reaction conditions: 1a (1 eq), 4-iodotoluene (1 eq), Pd(OAc) 2 (5 mol %), PCy 3 .HBF 4 (10 mol %), DBU (2 eq), CuI (2 eq), in 1,4-dioxane ([1a] = 0.16M), 210 °C, 20 min under microwave irradiation.

Three additional bases, Cs 2 CO 3 , CsF and DBU were then evaluated. Cs 2 CO 3 proved to be deleterious to the CH arylation process leading mainly to degradation of the starting material 1a (Table 1, entries 3). On the other hand, CsF and DBU were found more operating than K 2 CO 3 base since the C2-arylated imidazole-4carboxylate 3a was produced in respectively 51 and 76% yields (Table 1, entries 4 and 5). Changing the electrophile for paraiodotoluene and keeping DBU as base led to the production of 3a in excellent 95% isolated yield (Table 1, entries 6). Then, the C-H arylation of the methyl imidazole-4-carboxylate 1a with paraiodotoluene was carried out under microwave irradiation using the optimal conditions. However, in this case, the use of PPh 3 as ligand which is prone to C-P cleavage reactions 20,21 led, as side product, to the introduction of a phenyl on the position 2 of the imidazole 1a. As a replacement, PCy 3 .HBF 4 ligand was then employed. In that case, the C2-arylated imidazole-4-carboxylate 3a was obtained as single product in 66% yield after 20 min irradiation ( With these optimal catalytic conditions in hands, the scope of the intermolecular arylation of the imidazole 1a with different bromo-and iodoarenes as electrophiles was investigated. 22,23 Interestingly, the methodology revealed to be operative over a wide range of aryl bromides incorporating electron-withdrawing groups and another electron-donating group. Notably, a broad series of C2-arylated imidazoles flanked with methyl (3a), methoxy (3b), cyano (3c-e), fluoro (3f), trifluoromethyl (3g), nitro (3h), methylketone (3i) and methyl ester (3j) groups were successfully isolated in moderate to good yields (Scheme 1). The yields could be significantly improved by using aryliodides as electrophile reagents as exemplified for methyl C2-arylated imidazole-4-carboxylates 3b, 3d, 3g and 3f. Importantly, the protocol remained remarkably highly efficient with bromoheterocycles as electrophile reagents. Indeed, several methyl C2-heteroarylated-imidazole-4-carboxylates have been synthetized in presence of standard heterocycles such as pyridine (3k), pyrimidine (3l), furan (3m) and 5-methylthiophene (3n).

[Br] = 37%, [I] = 57% (47%) a N N MeO 2 C Bn 3c CN [Br] = 86% ([I] = 56%) a N N MeO 2 C Bn 3d [Br] = 30%, [I] = 69% NC N N MeO 2 C Bn 3e [Br] = 86% CN N N MeO 2 C Bn 3f [Br] = 49%, [I] = 86% (28%) a N N MeO 2 C Bn 3g [Br] = 56%, [I] = 88% F CF 3 N N MeO 2 C Bn 3h [Br] = 77% ([I] = 56%) a N N MeO 2 C Bn 3i [Br] = 46%, [I] = 46% (49%) a NO 2 O MeO 2 C N N MeO 2 C Bn 3j [Br] = 80% ([I] = 37%) a N N MeO 2 C Bn 3k N [Br] = 90% ([I] = 26%) a CO 2 Me N N MeO 2 C Bn 3l N N [Br] = 86% (20%) a N N MeO 2 C Bn 3m, Y = O, R = H [Br] = 72% (11%) a Y (Het)Ar-X or vinyl-Br (1.2 equiv) N N MeO 2 C Bn 3o Ph [Br] = 67% b N N MeO 2 C Bn 3p [Br] = 81% b R 3n, Y = S, R = Me [Br] = 47% (10%)
As additional studies, some reactions were performed under microwave irradiation using iodoarenes. Although the time of the reaction was reduced to 20 min compared to conventional heating, in most of cases, lower performances were observed since methyl imidazole-4-carboxylates 3b-c,f,h-n were obtained in a range of poor to moderate yields (10-56%).

As already mentioned, the regioselective direct arylation to afford the C2-or C5-arylated imidazole compounds has attracted a lot of attention during the past decades. However, the C2-H alkenylation of naked-imidazole has been less successful in contrast to their benzo-fused azole derivatives such as benzimidazole under Pd (with or without copper co-catalysis) catalysis from the corresponding alkenyl bromides. 24,25 Nevertheless, a Pd-catalyzed cross-dehydrogenative coupling reaction was successfully developed by Ong in 2014 to afford the corresponding C2-alkenylated imidazole. 26 In this context, the C-H alkenylation of 1a with alkenyl halides was then examined. However, applying strictly the above optimized protocol, the direct alkenylation of 1a with the 2-bromostyrene failed.

Meanwhile, in accordance with our previous observations in C-H alkenylation of 4,4'-dialkylimidazolone series, 27 the direct CH alkenylation reaction of 1a with the 2-bromostyrene was found immediately conclusive when replacing only the PPh 3 by P(otolyl) 3 as ligand. In this case, the expected alkenylated imidazoles 3o-p were produced in 67% and 81% yields respectively from corresponding bromo-alkenes coupling partners (Scheme 1).

In continuation of this successful work on intermolecular selective C2-H (hetero)arylation of compound 1a, we then turned our attention to the development of an intramolecular CH arylation protocol starting from methyl 1-(2-bromobenzyl)-1Himidazole-4-carboxylate 4a. First, the optimized protocol used for intermolecular C2-H arylation of 1a revealed surprisingly ineffective. Other investigations under the same bis-catalytic mode but using carbonate bases were found also ineffective. 21 However, on the basis of our previous investigations in regioselective copper-free Pd(0)-catalyzed and carbonateassisted arylation of oxa(thia)zole-4-carboxylates, 15f-g intramolecular CH arylation of 4a was achieved under standard catalysis based on the use of Pd(OAc) 2 , carbonate bases and various phosphines in 1,4-dioxane. The results are summarized in table 2, and shows clearly that, under these conditions, a mixture of 4-carboxyimidazole-based tricyclic heterocycles possessing both imidazol-2-yl-aryl (5A-C2) and imidazole-5-yl-aryl (5A-C5) systems was identified with the isolated isomer 5A-C5 as the major one. Good performances of the direct C5-H arylation were observed using K 2 CO 3 as a base and PPh 3 as well as electron-rich PCy 3 .HBF 4 and electron-poor P(4-FC 6 H 4 ) 3 as ligand providing the 4-carboxyimidazole-based tricyclic heterocycles 5A-C5 in a range of 70 to 81% isolated yields (Table 2, entries 1-3). Only the bidendate ligand dppb proved to be less effective (Table 2, entry 4). The observed C5-H selectivity using K 2 CO 3 and electron-rich or electron-poor phosphines as optimal reagents are in favour a carbonate-assisted CMD-based process which, probably due to steric hindrance, was not effective for the intramolecular arylation of imidazole 1a reported above. In accordance to our observations in (oxa)thiazole-4-carboxylate series, the acetateassisted metalation-deprotonation process was found to be also ineffective with compound 4a (Table 2,entry 5). In addition, with stronger bases such as Cs 2 CO 3 , the intramolecular C-H arylation of 4a proceeded less efficiently as the 4-carboxyimidazole-based tricyclic heterocycle 5A-C5 was still isolated, as the main product but, in a poor 35% yield (Table 2, entry 6). Having previously highlighting the shift of selectivity from the most steric hindered C5 site to lower steric hindered C2 site through K 2 CO 3 -assited CMD-proceeding in oxazole and thiazole-4-carboxylates series by using bulky ligands, 15f-g the behavior of the intramolecular C-H arylation of 4a regarding the regioselectivity was examined when increasing phosphine bulkiness. First, P t Bu 2 Me gave predominantly the same 5A-C5 isomer in a good 79% yield (Table 2, entry 7). However, when further increasing the bulkiness of the phosphine and using the most steric hindered P t Bu 3 •HBF 4, the imidazole-based tricyclic heterocycle 5A-C2 was formed only in a slight excess compared to its regioisomer 5A-C5 (Table 2, entry 8).Therfore, full inversion of the regioselectivity was not observed as anticipated. The production of 4-carboxyimidazole-based tricyclic heterocycles 5-8 embedding with an 5-and up to 7-membered central ring was finally investigated starting from various methyl N-alkylated imidazole-4-carboxylates 4b-g and by selecting the optimized PCy 3 •HBF 4 ligand. Results are depicted in Scheme 2. The para-fluorinated imidazole-based tricyclic heterocycles 5B-C5 was produced in excellent 90% yield, the 5B-C2 isomer being isolated in very few amounts (8%). Similarly, to the preparation of 5-membered medium ring models, the imidazole-based 6membered tricyclic heterocyclic 6-C5 was also successfully obtained as the major product in excellent 81% yield. Again, the other isomer 6-C2 was produced as the only side product in 9% yield. Formation of compounds 6-C5 and 6-C2 has already been reported via a radical cyclisation although with lower yield and selectivity. 18a With these promising results in hands, the preparation of imidazole-based 7-membered tricyclic heterocycles 7 and 8 were next investigated. However, in this case, we found that even the ring-closing reaction occurred mainly at the C5 site of the imidazole ring leading to the 7-C5 and 8-C5 isomer in moderate 61 and 62% yields, a significant reduction of selectivity in favour of the less steric hindered C2-H site was observed and the 7-C2 and 8-C2 isomers were isolated in much more significant yields (39 and 37% respectively).

Unfortunately, this methodology failed to prepare the imidazolebased 8-membered tricyclic heterocycles. Finally, regarding the reactivity and the regioselectivity observed within this direct C-H arylation study in imidazole-4carboxylate series through both intramolecular and intermolecular and using both Pd(0)-or Pd(0) / CuI cooperative catalysis, assumptions of the modes of carbonate-assisted metalation-deprotonation might be done (Scheme 3). First, we assumed that the standard K 2 CO 3 -assisted CMD-based reactivity could not proceed at both highly sterically hindered C2-H and C5-H sites of 1a (model A and B) whilst, for the intramolecular pathway from compound 4a, the reactivity could be recovered due to a decrease of the steric constraint. In that case, the C5-H vs C2-H competition was observed showing that the energetic barriers might be very closed (mode C and D). Naturally, the intramolecular direct C2-H arylation under Pd(0) / CuI cooperative catalysis pathway is penalized with the difficulty to achieve within the same molecule the consecutive generation of imidazole-2-yl copper as well as the oxidative addition of Pd(0) to the aryl halide moiety prefiguring the intramolecular transmetalation key step. On the other hands, the CuI co-catalyst being only efficient for intermolecular C2-H arylation of 1a, the As summarized, this work reports the first full investigations of palladium-catalyzed direct C-H functionalization with halides in N-substituted imidazole-4-carboxyxlate series. In particular, the first selective direct C2-H (het)arylation and alkenylation of Nbenzyl imidazole-4-carboxylate with bromo-and iodo(het)arenes and bromoalkenes under Pd(0)/Cu(I) cooperative catalysis was described giving a neat access to a broad library of C2functionalized methyl imidazole-4-carboxylate compounds. By contrast to previous observations in 4-nitro N-substituted imidazole-4-carboxylate series, 29 in our case, the intermolecular CMD-based reactivity does not operate at C5 site when C4 site and nitrogen imidazole are both substituted. On almost the opposite, using N-(ortho-halogeno benzyl) imidazole-4-carboxylates as starting materials, the arylation took place successfully under K 2 CO 3 -assisted CMD-based conditions to produce new C5arylated fused imidazoles with a 5-to 7 membered central rings as major products. As major mechanistic highlighting in direct C-H functionalization of heterocycle, the standard cooperative Pd(0)/Cu(I) catalysis revealed inoperative when driving through intramolecular pathway due to probably the difficulty of getting in the same first the formation of the imidazole-2-yl copper intermediate followed by the oxidative addition of Pd(0) to the aryl halide moiety. Moreover, a loss of C5-H vs C2-H selectivity was clearly observed during the preparation of 7-membered tricyclic imidazole-based heterocycles probably due to the more important size of the linker. Additional studies are currently ongoing to further functionalize these tricyclic scaffolds in order to generate chemical diversity.

F

Experimental section 1. General procedure for alkylation

To a solution of methyl 4-imidazolecarboxylate (4 mmol, 1.0 eq) in anhydrous DMF (25 mL), sodium hydride (95% dry, 6 mmol, 1.5 eq) was introduced in small portions under argon atmosphere. The mixture was stirred at 80°C for 1 h. A solution of the alkylating agent (6 mmol, 1.5 eq) in 5 mL of DMF was introduced dropwise to the reaction mixture which was heated at 80°C for 12 h. The solution was cooled to room temperature and the salts were removed by filtration on a Celite® pad. The solution was evaporated under reduced pressure to afford two regioisomers which were separated by column chromatography using petroleum ether/ethyl acetate (1/4) as eluent.

All regioisomers methyl 1[(2-bromophenyl)alkyl)-1H-imidazole-5-carboxylate were previously prepared in our laboratory and reported. 1 In this paper, we do not used these regioisomers, also we have not reported the analysis of these compounds.

methyl 1-benzyl-1H-imidazole-4-carboxylate 1a:

Compound 1a was prepared according to the general procedure for alkylation. Compound 3a was prepared according to the method A. The crude product was purified by flash chromatography to afford 3a in 76% yield with aryl-bromide and 95% yield with aryl-iodide as (q, J = 3.7 Hz, CH), 123.8 (q, J = 270.8 Hz, CF3), 51.9 (OCH3), 51.2 (N-CH2). IR : 3126, 3079, 1695, 1547, 1456, 1325, 1230, 1166, 1109, 1071, 1007, 843, 730 7.66 (d, J = 8.3 Hz, 2Harom), 7.35-7.33 (m, 3Hbenzyl), 7.07-7.05 (m, 2Hbenzyl) Compound 3n was prepared according to the method A. The crude product was purified by flash chromatography to afford 3n in 47% as a yellow solid. mp : 127.1 -127.9 °C (DCM/Pentane).

1 H NMR (300 MHz, CDCl3) δ 7.58 (s, 1Him, H5), 7.36-7.34 (m, 3Hbenzyl)

Pd-catalyzed intermolecular direct C-H alkenylation of methyl N-benzyl-1Himidazole 4-carboxylate.

Methyl 1-benzyl-1H-imidazole-4-carboxylate 1a (0.5 mmol, 1 eq), the appropriate alkenylhalide (0.6 mmol, 1.2 eq), Pd(OAc)2 (25 µmol, 5 mol%), P(o-tolyl)3•HBF4 (50 µmol, 10 mol%), CuI (1 mmol, 2 eq) and anhydrous DBU (1 mmol, 2 eq) were placed in a dry sealed tube containing a magnetic stir bar. The tube was evacuated and filled back with N2 three rime before adding anhydrous 1,4-dioxane (3.0 mL). The tube was sealed and heated to 140 °C for 18 hours. The reaction was filtered over a Celite® pad (washed with DCM and EtOAc). The solvents were removed under reduced pressure and the crude product was then purified by column chromatography (DCM to DCM/EtOAc 9-1). The DCM layers were dried over anhydrous MgSO4 and concentrated in vacuo to afford the title product in quantitative yield. The crude sulfonates were used without purification.

2,4-dibromobenzyl methanesulfonate

White solid 1 H NMR (300 MHz, CDCl3) δ 7.79 (d, J = 1.8 Hz, 1H), 7.51 (dd, J = 8.2, 1.8 Hz, 1H), 7.37 (d, J = 8.3, 1H), 5.27 (s, 2H), 3.05 (s, 3H).

2-bromo-4-fluorobenzyl methanesulfonate

White solid 1 H NMR (300 MHz, CDCl3) δ 7.49 (dd, J = 8.6, 5.9 Hz, 1H), 7.36 (dd, J = 8.1, 2.5 Hz, 1H), 7.08 (td, J = 8.3, 2.5 Hz, 1H), 5.28 (s, 2H), 3.02 (s, 3H).

2-(2-bromophenyl)ethyl methanesulfonate. 2

Pale yellow oil 1 H NMR (300 MHz, CDCl3) δ 7.51 (d, J = 8.0 Hz, 1H), 7.26-7.24 (m, 2H), 7.13-7.07 (m, 1H), 4.39 (t, J = 6.9 Hz, 2H), 3.15 (t, J = 6.9 Hz, 2H), 2.85 (s, 3H

General procedure for alkylation

To a solution of sodium hydride (95% dry, 6 mmol, 1.5 eq) in anhydrous DMF (25 mL), methyl 4-imidazolecarboxylate (4 mmol, 1eq) was introduced in small portions under argon atmosphere. The mixture was stirred at 80°C for 1 h. A solution of the alkylating agent (6 mmol, 1.5 eq) in 5 mL of DMF was introduced dropwise to the reaction mixture which was heated at 80°C for 12 h. The solution was cooled to room temperature and the salts were removed by filtration on a Celite® pad. The solution was evaporated under reduced pressure to afford two regioisomers which were separated by column chromatography using petroleum ether/ethyl acetate (1/4) as eluent.

All regioisomers methyl 1[(2-bromophenyl)alkyl)-1H-imidazole-5-carboxylate were previously prepared in our laboratory and reported. 4 In this paper, we do not used these regioisomers, also we have not reported the analysis of these compounds.

methyl 1[(2-bromophenyl)methyl]-1H-imidazole-4-carboxylate 4a :

Compound 4a was prepared from 1-bromo-2-(bromomethyl)benzene (commercial source) following the general procedure for alkylation.

White solid (51% a) Conditions: 4a (1 eq), Pd(OAc)2 (5 mol%), ligand (10 mol%), base (2 eq), 1,4-dioxane (0.16 M), 140 °C, 18h. b) Yield of isolated compound. 9. General procedure for the Pd-catalyzed intramolecular direct C-H arylation of 2bromo N-substituted-imidazoles.

Method C: Bromo N-substituted-imidazole 4a-e (0.27 mmol, 1 equiv.) was placed in a dry sealed tube containing a magnetic stir bar with an appropriate Pd(OAc)2 (3 mg, 13 μmol, 5 mol %), PCy3•HBF4 (10 mg, 27 μmol, 10 mol%), K2CO3 (75 mg, 0.54 mmol, 2.0 eq). The tube was evacued and filled back with N2 three time before adding anhydrous 1,4-dioxane (final concentration of compound 5a-g in dioxane was 0.12M). The tube was sealed and heated to 140 °C for 18 hours. The reaction mixture was filtered over a Celite® pad (washed with DCM and then EtOAc) and the solvents were removed under reduced pressure. The crude product was then purified by flash column chromatography. Compound 5A-C5 was prepared according to the method C using 4a (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 5A-C5 in 81% as a white solid. mp : 150.9-151. Compound 6-C5 was prepared according to the method C using 4c (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 6-C5 in 81% as a white solid (81 %). mp = 187.1-188. Compound 6-C2 was prepared according to the method C using 4c (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 6-C2 in 9% yellow oil. 1 H NMR (300 MHz, CDCl3) δ 8.19-8.16 (m, 1Harom), 7.65 (s, 1H, C5, CHim), 7.38-7.29 (m, 2Harom), 7.26-7.23 (m, 1Harom), 4.23 (t, J = 6.9 Hz, 2H, N-CH2), 3.93 (s, 3H, OMe), 3.19 (t, J = 6.9 Hz, 2H, CH2-Ph). Compound 7-C2 was prepared according to the method C using 4d (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 7-C2 in 39%. 1 H NMR (300 MHz, CDCl3) δ 7.84-7.81 (m, 1Harom), 7.72 (s, 1H, C5, CHim), 7.36-7.33 (m, 2Harom), 7.25-7.23 (m, 1Harom), 3.94 (t, J = 6.9 Hz, 2H), 3.90 (s, 3H, OMe), 2.71 (t, J = 7.1 Hz, 2H), 2.33 (p, J = 6.9 Hz, 2H 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 3.90 Georg Thieme Publishers KG, Rüdigerstraße 14, 70469 Stuttgart, Germany Manuscript Submitted to Synlett

Scheme 2

 2 Scheme 2 Synthesis of 4-carboxyimidazole-based tricyclic heterocycles

Figure 1

 1 Figure 1 Plausible intermediates in catalytic C-H arylation

  the N-(CuI)-chelated imidazole-4-carboxylate complex, reported by Gorelsky group (model E and G), 28 might be here discarded in favour of the generation of imidazole-2-yl copper intermediate (model F), in accordance to the Bellina/Rossi suggestion. 11f

3 .

 3 Optimization of the Pd-catalyzed intermolecular direct C2-H arylation of the methyl 1-benzyl-1H-imidazole-4-carboxylate 1a under microwave irradiation conditions: of formation of compound 3a was evaluated by Liquid Chromatography coupled to Mass Spectroscopy b: mixture of compounds 3a and 3. Entry 1: 3a/3: 75/25 Entry 2: 3a/3: 70/30 4. Pd-catalyzed intermolecular direct C-H arylation of methyl N-benzyl-1Himidazole 4-carboxylate under thermic conditions or microwave irradiation conditions. Method A. General procedure for Pd-catalyzed intermolecular direct C2-H arylation of methyl N-benzyl-1H-imidazole 4-carboxylate under thermic conditions: Methyl 1-benzyl-1H-imidazole-4-carboxylate 1a (80 mg, 0.37 mmol, 1 eq), the appropriate halide 2 (1.2 eq), Pd(OAc)2 (18 µmol, 5 mol%), PPh3 (37 µmol, 10 mol%), CuI (0.74 mmol, 2 eq) and anhydrous DBU (0.74 mmol 2 eq) were placed in a dry sealed tube containing a magnetic stir bar. The tube was evacuated and filled back with N2 three rime before adding anhydrous 1,4-dioxane(2.5 mL). The tube was sealed and heated to 140 °C for 18 hours. The reaction was filtered over a Celite ® pad (washed with DCM and EtOAc). The solvents were removed under reduced pressure and the crude product was then purified by column chromatography (DCM to DCM/EtOAc 9-1).

  procedure for Pd-catalyzed intermolecular direct C2-H arylation of methyl N-benzyl-1H-imidazole 4-carboxylate under microwave irradiation conditions: To a solution of compound 1a in dioxane, in a septum sealed tube, were successfully added the appropriate halide (1.0 eq), ligand (10 mol%), CuI (2 eq) and DBU (2eq). Between each addition, the mixture was degassed and refilled with nitrogen. Then, Pd(OAc)2 (5 mol%) in solution in 1,4-dioxane (final concentration of compound 1a in 1,4-dioxane was 0.16M). Then, the tube was sealed and heated at 210°C using a single mode microwave (Biotage Initiator EXP60) with a power output ranging from 0 to 400W for 20mn. Then, the reaction mixture was diluted into dichloromethane and concentrated. The mixture was purified by reverse phase preparative chromatography (solid deposit with SiO2 63-200 µm) using a gradient of NH4CO3 2g/L and acetonitrile.

  procedure for the synthesis of 2-(2-bromophenyl)alkyl methanesulfonates 2-(2-bromophenyl)alkyl mesylates were prepared form the corresponding alcohols according to the procedure.2 Methanesulfonyl chloride (5.6 mmol, 1.5 eq) was added dropwise to a solution of 2-(2-bromophenyl)alkyl alcohol (3.7 mmol) and triethylamine (11.1 mmol) in toluene (30 mL) at 0°C. The reaction mixture was further stirred at room temperature for 18 h and then poured into water and DCM. The aqueous phase was extracted with DCM (2×15 mL).

  13 C NMR (75 MHz, CDCl3) δ 163.5 (C=O), 163.3 (d, J = 245.6 Hz, Cq, C-F), 153.4 (Cq), 137.9 (Cq), 137.1 (d, J = 2.7 Hz, Cq), 131.1 (d, J = 10.4 Hz, Cq), 125.0 (d, J = 9.1 Hz, CH), 122.5 (C5, CHim), 115.9 (d, J = 23.7 Hz, CHarom), 108.5 (d, J = 25.3 Hz, CHarom), 52.0 (OCH3), 48.9 (N-CH2). HRMS (TOF MS ESI+): calcd. For C12H10FN2O2 [M + H] + 233.0726; found 233.0724.

8 (Compound 7 - 4 (

 874 13 C NMR (75 MHz, CDCl3) δ 163.6 (C=O), 145.1 (C2, Cqim),133.3 (Cq), 132.Cq), 129.4 (CHarom), 127.9 (CHarom), 127.8 (CHarom), 126.3 (Cq), 125.5 (CH), 124.7 (CH), 51.9 (OCH3), 43.9 (N-CH2), 28.4 (CH2-Ph). This is a known compound. 2 methyl 6,7-dihydro-5H-benzo[c]imidazo[1,5-a]azepine-1-carboxylate 7-C5 : C5 was prepared according to the method C using 4d (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 7-C5 in 61%. mp : 185.7-186.2 °C (CH2Cl2/PE). 1 H NMR (300 MHz, CDCl3) δ 7.77-7.74 (m, 1Harom), 7.56 (s, 1H, C2, CHim),7.37-7.34 (m, 2Harom), 7.27-7.24 (m, 1Harom), 3.89-3.85 (m, 2H), 3.85 (s, 3H, OMe), 2.63 (t, J = 7.1 Hz, 2H), 2.25 (p, J = 6.9 Hz, 2H).13 C NMR (75 MHz, CDCl3) δ 163.5 (C=O), 138.Cq),138.2 (Cq), 136.3 (C2, CHim), 131.5 (CHarom), 129.7 (CHarom), 128.9 (CH), 128.6 (Cq), 126.6 (CHarom), 51.7 (OCH3), 43.2 (N-CH2), 31.3 (CH2), 30.2 (CH2). HRMS : Anal. Calcd for C14H15N2O2: [M+H] + 243.1134, found 243.1137.

7 (Compound 8 - 4 (

 784 4 (CH), 74.0 (O-CH2), 51.OCH3),43.5 (N-CH2). HRMS : Anal. Calcd for C13H13N2O3: [M+H] + 245.0926C2 was prepared according to the method C using 4e (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 8-C2 in 37% as a pale yellow solid. mp : 167.9-169.3 °C (CH2Cl2/PE). 1 H NMR (300 MHz, CDCl3) δ 8.59(d, J = 8.1 Hz, 1Harom), 7.66 (s, 1Him, H5), 7.28 (d, J = 8.0 Hz, 1Harom), 7.11 (t, J = 7.6 Hz, 1Harom), 7.02(d, J = 8.1 Hz, 1Harom), 4.48-4.41 (m, 4H), 3.92 (s, 3H, OMe).13 C NMR (75 MHz, CDCl3) δ 163.

F

Table 1

 1 Optimization of the palladium-catalysed intermolecular direct C-H arylation of 1a

				Pd(OAc) 2 (5 mol%)
				PPh 3 (10 mol%)
				CuI (X equiv)
	MeO 2 C	N	H	Base (2 equiv) Solvent, 140 °C, 18h	MeO 2 C	N	Me
	H	N		Br		H	N
		Bn					Bn
					Me
	1a			2a (1.2 equiv)		3a
	Entry		Base	Solvent b	CuI (n equiv)	Yield (%) a
	1		K 2 CO 3	DMF		2	< 5
	2		K 2 CO 3	1,4-dioxane	2	34
	3		Cs 2 CO 3	1,4-dioxane	2	4
	4		CsF	1,4-dioxane	2	51
	5		DBU	1,4-dioxane	2	76
	6		DBU	1,4-dioxane	2	95 b
	7		DBU	1,4-dioxane	2	66 c
	8		DBU	1,4-dioxane	1	65
	9		DBU	1,4-dioxane	-	<5
	10		Cs 2 CO 3	1,4-dioxane	--	< 5
	11		K 2 CO 3	1,4-dioxane	--	< 5

  Table 1, entry 7). As additional observations, lower amounts of CuI reduced the efficiency of the C-H arylation process with para-bromotoluene (Table 1, entry 8). Moreover, more importantly, without copper, very poor reactivity was observed (Table1, entry 9-11) using DBU as well as Cs 2 CO 3 and K 2 CO 3 as bases suggesting that the standard carbonate-assisted palladiumcatalyzed C-H arylation process is not operative.

			Pd(OAc) 2 (5 mol%)
			PPh 3 (10 mol%),
	MeO 2 C	N	DBU (2 equiv), CuI (2 equiv), 1,4-dioxane, 140 °C, 18 h	MeO 2 C	N	(Het)Ar
		N			N	or vinyl
		Bn			Bn
	1a				3b-p
		N
			OMe
		N
	3b		Bn

  Scope of (hetero)aryl and alkenyl halides. a Yields obtained under microwave irradiation conditions (Table1, entry 10) are given in brackets. b P(o-tolyl) 3 phosphine ligand was used instead of PPh 3 .

Scheme 1

Table 2

 2 Optimization of the reaction conditions of palladium-catalyzed intramolecular C-H arylation of 4a

					MeO 2 C	N
			Pd(OAc) 2 (5 mol%)		N
			ligand (10 mol%),
	MeO 2 C	N	1,4-dioxane, 140 °C, 18 h Base (2 equiv),		5A-C5
		H
	H	N			MeO 2 C	N
	4a					N
		Br
						5A-C2
	Entry		Ligand	Base	Yield (%) a,b 5A-C5	Yield (%) a,b 5A-C2
	1		PPh 3	K 2 CO 3	70	3
	2	PCy 3 •HBF 4	K 2 CO 3	81	9
	3	P(4-FC 6 H 4 ) 3	K 2 CO 3	81	17
	4		dppb	K 2 CO 3	42	nd
	5	PCy 3 •HBF 4	KOAc	nd	nd
	6	PCy 3 •HBF 4	Cs 2 CO 3	35	3
	7	P t Bu 2 Me•HBF 4	K 2 CO 3	79	7
	8	P t Bu 3 •HBF 4	K 2 CO 3	23	27
	a Conditions: 4a (1 eq.), Pd(OAc) 2 (5 mol%), ligand (10 mol%), base (2 eq.),
	1,4-dioxane (0.16 M), 140 °C, 18h.

b

Yield of isolated product.

General procedure for Pd-catalyzed intermolecular direct C2- H arylation of methyl N-benzyl-1H-imidazole 4-carboxylate

  Eur. J. Org. Chem. 2006, 693. (j) Bellina, F.; Cauteruccio, S.; Mannina, L.; Rossi, R.; Viel, S. J. Org. Chem. 2005, 70, 3997. (13) Joo, J. M.; Touré, B. B.; Sames, D. J. Org. Chem. 2010, 75, 4911. (14) Kuroda, T.; Suzuki, F. Tetrahedron Lett. 1991, 32, 6915. Organometallics 2015, 34, 3713. (f) Baba, K.; Tobisu, M.; Chatani, N. Angew. Chem. Int. Ed. 2013, 52, 11892. (g) Li, Z.; Zhou, H.; Xu, J.; Wu, X.; Yao, H. Tetrahedron 2013, 69, 3281. (h) Ma, M.-T.; Lu, J.-M. Tetrahedron 2013, 69, 2102. (21) See the supporting information for more details. (22)

	Synlett	Letter / Cluster / New Tools
	under thermic conditions: Methyl 1-benzyl-1H-imidazole-4-
	carboxylate 1a (80 mg, 0.37 mmol, 1 eq), the appropriate halide 2
	(1.2 eq), Pd(OAc) 2 (18 µmol, 5 mol%), PPh 3 (37 µmol, 10 mol%),
	CuI (0.74 mmol, 2 eq) and anhydrous DBU (0.74 mmol 2 eq) were
	placed in a dry sealed tube containing a magnetic stir bar. The tube
	was evacuated and filled back with N2 three rime before adding
	anhydrous 1,4-dioxane (2.5 mL). The tube was sealed and heated
	to 140 °C for 18 hours. The reaction was filtered over a Celite® pad
	(washed with DCM and EtOAc). The solvents were removed under
	reduced pressure and the crude product was then purified by
	column chromatography (DCM to DCM/EtOAc 9-1).
	(23) Compound 3a was prepared according to the general procedure.
	The crude product was purified by flash chromatography to afford
	3a in 76% yield with aryl-bromide and 95% yield with aryl-iodide
	as a pale yellow solid. mp : 89-90 °C. 1 H NMR (300 MHz, CDCl 3 ) δ 7.64 (s, 1H im , H5), 7.46 (d, J = 8.0 Hz, 2H arom ), 7.35-7.33 (m, 3H benzyl ), 7.22 (d, J = 8.0 Hz, 2H arom ), 7.09-7.07 (m, 2H benzyl ), 5.19 (s, 2H), 3.89 (s, 3H, OMe), 2.38 (s, 3H, CH 3 ). J. Org. Chem. 2016, 2421. (25) Sahnoun, S.; Messaoudi, S.; Brion, J.-D.; Alami, M. Eur. J. Org. Chem. R e r o P e r 2010, 6097. e (26) Lee, W.-C.; Wang, T.-H.; Ong, T.-G. Chem. Commun. 2014, 50, 3671. v (27) (a) Muselli, M.; Baudequin, C.; Perrio, C.; Hoarau, C.; Bischoff, L.
	i e w
	8111.
	(19) Two regioisomers were obtained, however concerning this study
	only methyl N-alkylated imidazole-4-carboxylates were evaluated.
	(20) For recent references, see: (a) Baba, K.; Masuya, Y.; Chatani, N.;

Template for SYNLETT © Thieme Stuttgart • New York 2020-02-27 page 6 of 7 60, 6079. (j) Kuroda, T.; Suzuki, F. Tetrahedron Lett. 1991, 32, 6915. (11) For palladium-catalyzed intermolecular direct arylation of imidazole derivatives, see: (a) Vellakkaran, M.; Lingayya, R.; Kumar, B. N.; Nagaiah, K.; Poornachandra, Y.; Kumar, C. G. RSC Adv. 2015, 5, 8057. (b) Grosse, S.; Pillard, C.; Massip, S.; Marchivie, M.; Jarry, C.; Bernard, P.; Guillaumet, G. J. Org. Chem. 2015, 80, 8539. (12) (a) Lessi, M.; Panzetta, G.; Marianetti, G.; Bellina, F. Synthesis 2017, 49, 4676. (b) Perego, L. A.; Grimaud, L.; Bellina, F. Adv. Synth. Catal. 2016, 358, 597. (c) Bellina, F. ; Lessi, M.; Manzini, C. Eur. J. Org. Chem. 2013, 5621. (d) Bellina, F.; Cauteruccio, S.; Di Fiore, A.; Marchetti, C.; Rossi, R. Tetrahedron 2008, 64, 6060. (e) Bellina, F.; Cauteruccio, S.; Di Fiore, A.; Rossi, R. Eur. J. Org. Chem. 2008, 5436. (f) Bellina, F.; Cauteruccio, S.; Rossi, R. Curr. Org. Chem. 2008, 12, 774. (g) Bellina, F.; Calandri, C.; Cauteruccio, S.; Rossi, R. Tetrahedron 2007, 63, 1970. (h) Bellina, F.; Cauteruccio, S.; Rossi, R. J. Org. Chem. 2007, 72, 8543. (i) Bellina, F.; Cauteruccio, S.; Mannina, L.; Rossi, R.; Viel, S. (15) (a) Martin, T.; Verrier, C.; Hoarau, C.; Marsais, F. Org. Lett. 2008, 10, 2909. (b) Verrier, C.; Martin, T.; Hoarau, C.; Marsais, F. J. Org. Chem. 2008, 73, 7383. (c) Roger, J.; Verrier, C.; Le Goff, R.; Hoarau, C.; Doucet, H. ChemSusChem 2009, 2, 951. (d) Verrier, C.; Hoarau, C.; Marsais, F. Org. Biomol. Chem. 2009, 7, 647. (e) Verrier, C.; Fiol-Petit, C.; Hoarau, C.; Marsais, F. Org. Biomol. Chem. 2011, 9, 6215. (f) Théveau, L.; Verrier, C.; Lassalas, P.; Martin, T.; Dupas, G.; Querolle, O.; Van Hijfte, L.; Marsais, F.; Hoarau, C. Chem. Eur. J. 2011, 17, 14451. (g) Théveau, L.; Querolle, O.; Dupas, G.; Hoarau, C. Tetrahedron 2013, 69, 4375. (16) Thireau, J.; Schneider, C.; Baudequin, C.; Gaurrand, S.; Angibaud, P.; Meerpoel, L.; Levacher, V.; Querolle, O.; Hoarau, C. Eur. J. Org. Chem. 2017, 2491. (17) For recent reviews, see: (a) Cai, X.-h.; Yang, M.; Xie, B. Curr. Org. Chem. 2018, 22, 1906. (b) Zhang, T.; Wang, N.-X.; Xing, Y. J. Org. Chem. 2018, 83, 7559. (c) Hosseinian, A.; Nasab, F. A. H.; Ahmadi, S.; Rahmani, Z.; Vessally, E. RSC Adv. 2018, 8, 26383. (d) Schwarz, J.; König, B., Green Chem. 2018, 20, 323. (e) Perry, G. J. P.; Larrosa, I. Eur. J. Org. Chem. 2017, 3517. (f) Wie, Y.; Hu, P.; Zhang, M.; Su, W. Chem. Rev. 2017, 117, 8864. (g) Jin, Y.; Fu, H. Asian J. Org. Chem. 2017, 6, 368. (h) Li, H.; Miao, T.; Wang, M.; Li, P.; Wang, L. Synlett 2016, 27, 1635. (18) (a) Allin, S. M.; Bowman, W. R.; Elsegood, M. R. J.; McKee, V.; Karim, R.; Rahman, S. S. Tetrahedron 2005, 61, 2689. (b) Aldabbagh, F.; Bowman, W. R.; Mann, E.; Slawin, A. M. Z. Tetrahedron 1999, 55, Tobisu, M. Chem. Lett. 2017, 46, 1296. (b) Zhou, H.; Li, J.; Yang, H.; Xia, C.; Jiang, G. Org. Lett. 2015, 17, 4628. (c) Zhou, Y.; Gan, Z.; Su, B.; Li, J.; Duan, Z.; Mathey, F. Org. Lett. 2015, 17, 5722. (d) Baba, K.; Tobisu, M.; Chatani, N. Org. Lett. 2015, 17, 70. (e) Karthik, V.; Gupta, V.; Anantharaman, G. 13 C NMR (75 MHz, CDCl 3 ) δ 163.5 (C=O), 149.5 (C2, Cq im ), 139.7 (C4', Cq arom ), 135.9 (C1', Cq benzyl ), 132.9 (C4 im, Cq), 129.3 (2CH), 129.2 (2CH), 129.1 (2CH), 128.4 (CH benzyl ), 127.1 (C5, CH im ), 126.9 (2CH benzyl ), 126.5 (Cq), 51.8 (OCH 3 ), 51.0 (N-CH 2 ), 21.4 (CH 3 ). IR (ATR ,cm -1 ): 3125, 3079, 2920, 2850, 1689, 1549, 1330, 1227, 1004, 725 cm -1 . HRMS : Anal. Calcd for C 19 H 19 N 2 O 2 : [M+H] + 307.1447, found 307.1455. (24) (a) Vabre, R.; Chevot, F.; Legraverend, M.; Piguel, S. J. Org. Chem. 2011, 76, 9542. (b) Vabre, R.; Legraverend, M.; Piguel, S. Dyes and Pigments 2014, 105, 145. (c) Baladi, T.; Granzhan, A.; Piguel, S. Eur. Chem. Eur. J. 2016, 22, 5520. (b) Muselli, M.; Baudequin, C.; Hoarau, C.; Bischoff, L. Chem. Commun. 2015, 51, 745. (28) Gorelsky, S. I.; Coordin. Chem. Rev. 2013, 257, 153. Gorelsky, S. I.; Lapointe, D.; Fagnou, K. J. Org. Chem. 2012, 77, 658. (29) Iaroshenko, V. O.; Gevorgyan, A.; Mkrtchyan, S.; Arakelyan, K.; Grigoryan, T.; Yedoyan, J.; Villinger, A.; Langer, P. J. Org. Chem. 2015, 80, 2103.

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catalyzed direct inter-and intramolecular C˗H functionalization of 4-carboxyimidazoles.

  Steven Frippiat, a Antoine Peresson, a Thibault Perse, b Yvan Ramondenc, a Cédric Schneider, a Olivier Querolle, b Patrick Angibaud, b Virginie Poncelet, b Lieven Meerpoel, c Vincent Levacher, b Laurent Bischoff, a Christine Baudequin a and Christophe Hoarau a Janssen Research & Development, Division of Janssen-Cilag S. A., Campus de Maigremont, BP 615 27106 Val de Reuil Cedex, France.

	Supporting Information
	a Normandie University, COBRA UMR 6014 et FR 3038, University Rouen; INSA Rouen; CNRS, IRCOF
	1 rue Tesnière 76821 Mont Saint Aignan, France.
	c Janssen Research & Development, Division of Janssen-Cilag a division of Janssen Pharmaceutica NV,
	Turnhoutseweg 30, B-2340, Beerse, Belgium. F
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	P
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b

Table of Contents

 of General information ................................................................................................................................ 2 Experimental section ............................................................................................................................... 3Coupling constants (J) are reported in Hertz (Hz). Signals were assigned as far as possible by means of two-dimensional NMR spectroscopy: 1 H-13 C-COSY (HSQC: Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple Bond Correlation).HRMS were recorder on a LC Waters Acquity coupled to a Waters LCT Premier XE instrument

	The infrared spectra of compounds were recorded on a Perkin Elmer Spectrum 100 FT IR
	spectrometers.
	Melting points (mp [°C]) were taken on open capillary tubes and are uncorrected, performed
	on a Stuart SMP3.
	F
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	S3

methyl 1-benzyl-2-(4-cyanophenyl)-1H-imidazole-4-carboxylate 3c: Compound

  The crude product was purified by flash chromatography to afford 3g in 88% as a white solid. mp : 123.1-123.8 °C (DCM/Pentane). 1 H

	129.2 (2CH), 129.1 (2CH), 128.4 (CHbenzyl), 127.1 (C5, CHim), 126.9 (2CHbenzyl), 126.5 7.07-7.04 (m, 2Hbenzyl), 5.24 (s, 2H), 3.90 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3) δ 163.1 1Harom, H6'), 7.70-7.68 (m, 2H, Harom 4'and H5), 7.52 (t, J = 7.8 Hz, 1Harom, H5'), 7.36-7.34
	(Cq), 51.8 (OCH3), 51.0 (N-CH2), 21.4 (CH3). IR (ATR ,cm -1 ): 3125, 3079, 2920, 2850, 1689, (C=O), 147.1 (C2, Cqim), 135.1 (C1', Cqbenzyl), 133.7 (Cq), 133.7 (Cq), 132.4 (2CHarom), 129.7 (m, 3Hbenzyl), 7.06-7.04 (m, 2Hbenzyl), 5.21 (s, 2H), 3.89 (s, 3H, OMe). 13 C NMR (75 MHz,
	1549, 1330, 1227, 1004, 725 cm -1 . HRMS : Anal. Calcd for C19H19N2O2: [M+H] + 307.1447, found (2CH), 129.5 (2CH), 128.8 (CHbenzyl), 128.3 (C5, CHim), 126.7 (2CHbenzyl), 118.3 (Cq), CDCl3) δ 163.0 (C=O), 146.7 (Cq), 135.0 (C1', Cqbenzyl), 133.4 (Cq), 133.2 (CHarom, C6'), 132.9 NMR (300 MHz, CDCl3) δ 7.71-7.64 (m, 4Harom), 7.69 (s, 1Him, H5), 7.36-7.34 (m, 3Hbenzyl),
	307.1455. 113.3 (Cq), 52.0 (OCH3), 51.3 (N-CH2). IR : 3120, 3077, 2945, 2226, 1695, 1611, 1548, 1454, (CHarom, C4'), 132.7 (CHarom, C2'), 130.8 (Cq), 129.6 (Cq), 129.5 (CHarom, C5'), 129.4 7.08-7.05 (m, 2Hbenzyl), 5.22 (s, 2H), 3.89 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3) δ 163.2
	methyl 1-benzyl-2-(4-methoxyphenyl)-1H-imidazole-4-carboxylate 3b: 1330, 1225, 1006, 723 cm -1 . HRMS : Anal. Calcd for C19H15N3O2: [M+H] + 318.1243, found (2CHbenzyl), 128.8 (CHbenzyl), 128.0 (C5, CHim), 126.8 (2CHbenzyl), 117.9 (Cq), 113.1 (Cq), (C=O), 147.7 (Cq, C2im), 135.3 (C1', Cqbenzyl), 135.5 (Cq), 132.9 (d, J = 1.2 Hz, Cq), 131.5 (q,
	318.1249. 52.0 (OCH3), 51.3 (N-CH2). IR : 3066, 2947, 2229, 1731, 1460, 1436, 1347, 1179, 1010, 898, J = 32.8 Hz), 129.5 (2 CH), 129.4 (2CH), 128.7 (CH), 127.9 (CH), 126.8 (2CHbenzyl), 125.68
	methyl 1-benzyl-2-(2-cyanophenyl)-1H-imidazole-4-carboxylate 3d: 720 cm -1 . HRMS : Anal. Calcd for C19H15N3O2: [M+H] + 318.1243, found 318.1244.
	methyl 1-benzyl-2-(4-fluorophenyl)-1H-imidazole-4-carboxylate 3f:
	F F F F
	o r o r o r o r
	P P P P
	e e e e
	e r e r e r e r	01-6.99
	R e v 135.8 (C1', Cqbenzyl), 132.8 (C4im, Cq), 130.7 (2CHarom, C2'), 129.2 (2CHbenzyl), 128.4 (CHbenzyl), 127.0 (C5, CHim), 126.9 (2CHbenzyl), 121.7 (Cq), 114.1 (2CHarom, C3'), 55.4 (OCH3), 51.8 (OCH3), 51.0 (N-CH2). IR (ATR ,cm -1 ): 2948, 2838, 1713, 1611, 1538, 1481, R e v (m, 2H), 5.10 (s, 2H), 3.87 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3) δ 163.0 (C=O), 145.1 (C2, Cqim), 134.8 (C1', Cqbenzyl), 133.5 (Cq), 133.3 (Cq), 133.2 (CHarom), 132.9 (CHarom), 131.8 (CHarom), 130.3 (CHarom), 129.2 (2CHbenzyl), 128.7 (CHbenzyl), 127.4 (2CHbenzyl), 127.2 (C5, R e v 1 H NMR (300 MHz, CDCl3) δ 7.64 (s, 1Him, H5), 7.53 (dd, J = 8.6, 5.4 Hz, 1H), 7.34-7.31 (m, R 3Hbenzyl), 7.10-7.02 (m, 4H), 5.16 (s, 2H), 3.87 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3) δ 163.3 (C=O), 163.5 (d, J = 248.6 Hz, Cq, C-F), 148.3 (Cq, C2im), 135.5 (C1', Cqbenzyl), 133.0 e v
	i e w 1347, 1328, 1251, 1175, 1004, 837 cm -1 . HRMS : Anal. Calcd for C19H18N2O3: [M+H] + 323.1396, found 323.1404. i e w CHim), 117.2 (Cq), 113.6 (Cq), 51.9 (OCH3), 51.4 (N-CH2). IR : 3137, 2947, 2226, 1717, 1545, 1437, 1345, 1324, 1006, 814, 767, 713 cm -1 . HRMS : Anal. Calcd for C19H15N3O2: [M+H] + 318.1243, found 318.1244. i e w i (Cq, C4im), 131.3 (d, J = 8.5 Hz, 2CHarom), 129.3 (2CHbenzyl), 128.5 (CHbenzyl), 127.3 (C5, e CHim), 126.8 (2CHbenzyl), 125.6 (d, J = 3.3 Hz, Cqarom), 115.8 (d, J = 21.9 Hz, 2CHarom) 51.9 (OCH3), 51.0 (N-CH2). IR : 3129, 3079, 1694, 1551, 1454, 1332, 1223, 1006, 840, 731 cm -1 . w
	HRMS : Anal. Calcd for C18H15F1N2O2: [M+H] + 311.1196, found 311.1204.
	methyl 1-benzyl-2-(4-(trifluoromethyl)phenyl)-1H-imidazole-4-carboxylate 3g:
	a pale yellow solid. mp : 89-90 °C. 1 H NMR (300 MHz, CDCl3) δ 7.64 (s, 1Him, H5), 7.46 (d,
		S7 S8 S9

J = 8.0 Hz, 2Harom),

7.35-7.33 (m, 3Hbenzyl), 7.22

(d, J = 8.0 Hz, 2Harom), 7.09-7.07 (m, 2Hbenzyl)

,

5.19 (s, 2H)

,

3.89 (s, 3H, OMe), 2.38 (s, 3H, CH3)

.

13

C NMR (75 MHz, CDCl3) δ 163.5 (C=O), 149.5 (C2, Cqim), 139.7 (C4', Cqarom), 135.9 (C1', Cqbenzyl), 132.9 (C4im, Cq), 129.3 (2CH), Compound 3b was prepared according to the method A. The crude product was purified by flash chromatography to afford 3b in 57% as a pale yellow solid. mp : 70-71 °C (Et2O). 1 H NMR (300 MHz, CDCl3) δ 7.63 (s, 1Him, H5), 7.49

(d, J = 8.6 Hz, 2 Harom), 7.35-7.33 (m, 3Hbenzyl), 7.09-7.06 (m, 2Hbenzyl), 6.92 (d, J = 8.6

Hz, 2Harom), 5.18 (s, 2H), 3.89 (s, 3H, OMe), 3.82 (s, 3H, OMe).

13

C NMR (75 MHz, CDCl3) δ 163.5 (C=O), 160.6 (Cqarom, C4'), 149.4 (C2, Cqim), 3c was prepared according to the method A. The crude product was purified by flash chromatography to afford 3c in 86% as a white solid. mp : 160-161 °C (DCM/Pentane). 1 H NMR (300 MHz, CDCl3) δ 7.71-7.69 (m, 5H, 4Harom and 1Him, H5),

7.37-7.35 (m, 3Hbenzyl)

, Compound 3d was prepared according to the method A. The crude product was purified by flash chromatography to afford 3d in 69% as a pale yellow gum. 1 H NMR (300 MHz, CDCl3) δ 7.76

(d, J = 7.2 Hz, 1Harom), 7.68 (s, 1Him, H5), 7.63 (d, J = 7.7 Hz, 1Harom), 7.56 (t, J = 8.1 Hz, 2Harom), 7.29-7.27 (m, 3Hbenzyl), 7.09-7.06 (m, 2Hbenzyl), 6.92 (d, J = 8.6 Hz, 2Harom)

,7

.

methyl 1-benzyl-2-(3-cyanophenyl)-1H-imidazole-4-carboxylate 3e:

Compound 3e was prepared according to the method A. The crude product was purified by flash chromatography to afford 3e in 86% as a pale yellow solid. mp : 160.1 -162.2 °C (DCM/Pentane). 1 H NMR (300 MHz, CDCl3) δ 7.87 (s, 1Harom, H2'), 7.79 (d, J = 7.9 Hz, Compound 3f was prepared according to the method A. The crude product was purified by flash chromatography to afford 3f in 86% as a yellow solid. mp : 116.3-118.1 °C (DCM/Pentane).

S10

Compound 3g was prepared according to the method A.

benzyl-2-(pyridin-2-yl)-1H-imidazole-4-carboxylate 3k:

  Compound 3k was prepared according to the method A. The crude product was purified by flash chromatography to afford 3k in 90% as a brown solid. mp : 128.2 -129.5 °C (DCM/Pentane).

	(OCH3), 51.2 (N-CH2). IR : 3130, 2949, 1715, 1612, 1437, 1350, 1278, 1202, 1103, 1007, 863,
	702 cm -1 . HRMS : Anal. Calcd for C20H18N2O4: [M+H] + 350.1345, found 335.1354.
	F methyl 1-1 H NMR (500 MHz, CDCl3) δ 8.56 (br d, J=4.73 Hz, 1Hpy,H15), 8.30 (d, J=8.20 Hz, 1 Hpy, H18), o r 7.73-7.79 (m, 1 Hpy, H17), 7.65 (s, 1Him, H5), 7.23-7.33 (m, 3Hbenzyl and 1Hpy), 7.20 (br d, J=7.57
	P Hz, 2Hbenzyl, H8 and H12), 5.93 (s, 2H, N-CH2), 3.90 (d, J=1.58 Hz, 3H, O-Me).
	e
	e r 128.86 (2CHbenzyl), 128.05 (CHbenzyl), 127.74 (2CHbenzyl), 123.79 (CHpy, C18), 123.37 (CHpy,
	C16), 52.34 (N-CH2), 51.74 (O-Me).	R
	IR : 3126, 2925, 1697, 1587, 1545, 1275, 1221, 1090, 1011, 777, 729, 706 cm -1 . HRMS : Anal. e v i Calcd for C17H15N3O2: [M+H] + 294.1243, found 294.1246.
		e
		w
		, 5.22 (s, 2H), 3.92
		S12

(s, 3H, OMe),

3.90 (s, 3H, OMe)

.

13

C NMR (75 MHz, CDCl3) δ 166.

5 (C=O), 163.3 (C=O), 148.1 (Cq), 135.4 (Cq), 133.7 (Cq), 133.4 (Cq)

, 131.0 (Cq), 129.9 (2 CHarom), 129.3 (2CHbenzyl), 129.2 (CH), 128.6 (CH), 127.9 (CH), 126.9 (2CHbenzyl), 52.4 (OCH3), 51.9 13 C NMR (126 MHz, CDCl3) δ ppm 163.31 (C=O), 149.89 (CHpy, C15), 148.24 (s, 1 Cqpy, C13), 145.71 (Cqim, C2), 136.7 (CHpy, C17), 136.7 (Cqbenzyl, C7), 132.62 (Cqim, C4), 129.07 (CHim, C5), methyl 1

-benzyl-2-(pyrimidin-5-yl)-1H-imidazole-4-carboxylate 3l:

	52.0 (OCH3), 51.4 (N-CH2). IR : 3140, 2945, 1724, 1553, 1437, 1345, 1209, 1126, 1007, 728,
	715 cm -1 . HRMS : Anal. Calcd for C16H14N4O2: [M+H] + 295.1195, found 295.1188.
	methyl 1-benzyl-2-(furan-2-yl)-1H-imidazole-4-carboxylate 3m:
	Compound 3m was prepared according to the method A. The crude product was purified by
	flash chromatography to afford 3m in 72% as a brown solid. mp : 121.6-123.4 °C F (DCM/Pentane). 1 H NMR (300 MHz, CDCl3) δ 7.54 (s, 1Him, H5), 7.47 (d, J = 8.8 Hz, 1H), o 7.32-7.28 (m, 3Hbenzyl), 7.15-7.12 (m, 2Hbenzyl), 6.98 (d, J = 3.4 Hz, 1H), 6.47 (dd, J = 3.3, 1.8 r Hz, 1H), 5.41 (s, 2H), 3.86 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3) δ 163.1 (C=O), 144.5 P (Cqfur), 143.2 (CHfur), 140.2 (C2, Cqim), 135.4 (C1', Cqbenzyl), 133.0 (C4im, Cq), 129.1 e (2CHbenzyl), 128.4 (CHbenzyl), 127.3 (2CHbenzyl), 127.2 (C5, CHim), 111.7 (CHfur), 111.5 e r (CHfur), 51.8 (OCH3), 51.5 (N-CH2). IR : 3126, 2945, 1719, 1602, 1521, 1344, 1178, 1008, 767,
	R 720 cm -1 . HRMS : Anal. Calcd for C16H14N2O3: [M+H] + 283.1083, found 283.1089.
	e v
	i
	e
	w
	Compound 3l was prepared according to the method A. The crude product was purified by flash
	chromatography to afford 3l in 86% as a yellow solid. mp : 166.9-167.8 °C (DCM/Pentane). 1 H
	NMR (300 MHz, CDCl3) δ 9.21 (s, 1Hpy), 8.91 (s, 2Hpy), 7.75 (s, 1Him, H5), 7.35-7.32 (m,
	S13

3Hbenzyl),

7.05-7.02 (m, 2Hbenzyl), 5.24 (s, 2H), 3.89 (s, 3H, OMe)

.

13

C NMR (75 MHz, CDCl3) δ 162.9 (C=O), 158.9 (CHpy), 156.4 (2CHpy), 143.0 C2, Cqim), 134.6 (C1', Cqbenzyl), 134.1 (C4im, Cq), 129.5 (2CHbenzyl), 128.9 (CH), 128.5 (C5, CHim), 126.6 (2CHbenzyl), 124.3 (Cqpy),

2-bromophenoxy)propyl methanesulfonate

	found 277.9612. This is a known compound and the spectral data are identical to those reported
	in literature. 2
	2-(2-bromophenyl)propyl methanesulfonate 3
	Yellow oil
	1 H NMR (300 MHz, CDCl3) δ 7.55 (d, J = 8.0 Hz, 1H), 7.27-7.24 (m, 2 H), 7.13-7.07 (m, 1H),
	4.27 (t, J = 6.3 Hz, 2H), 3.04 (s, 3H), 2.88 (t, J = 7.7 Hz), 2.13-2.03 (m, 2H). 13 C NMR (75
	MHz, CDCl3) δ 139.65 (Cq), 132.96 (CH), 130.56 (CH), 128.13 (CH), 127.65 (CH), 124.31
	(Cq), 69.15 (CH2), 37.36 (CH3, OMs), 32.03 (CH2), 29.07 (CH2). HRMS (TOF MS EI+): calcd.
	F For C10H13BrO3S 291.97602; found 291.97688. This is a known compound and the spectral F
	o r data are identical to those reported in literature. 3 o r
	P 1-Bromo-2-(2-methanesulfonyloxyethyloxy)benzene P
		e e
	Yellow oil.	e r e r
	R 1 H NMR (300 MHz, CDCl3) δ 7.54 (d, J = 8.2 Hz, 1H), 7.27 (t, J = 7.6 Hz, 1H), 6.91-6.85 (m, R
	e v i 2H), 4.59 (t, J = 4.3 Hz, 2H), 4.26 (t, J = 4.3 Hz, 2H), 3.14 (s, 3H). 13 C NMR (75 MHz, CDCl3) e v δ 154.3 (Cq), 133.4 (CH), 128.7 (CH), 122.8 (CH), 113.6 (CH), 111.9 (Cq), 68.3 (CH2),66.8 i
	(CH2), 37.7 (CH3, OMs).	e e
	3-(Yellow oil.	w w
	1 H NMR (300 MHz, CDCl3) δ 7.54 (dd, J = 7.9 and 1.4 Hz, 1H), 7.29-7.24 (m, 1 H), 6.92-6.83
	(m, 2H), 4.53 (t, J = 6.0 Hz, 2 H), 4.16 (t, J = 5.7 Hz, 2 H), 3.00 (s, 3H), 2.28 (q, J = 5.9 Hz, ). 13 C NMR (75 MHz, CDCl3) δ 2H). 13 C NMR (75 MHz, CDCl3) δ 154.7 (Cq), 133.4 (CHarom), 128.7 (CHarom), 122.3 (CHarom),
	135.64 (Cq), 133.09 (CH), 131.63 (CH), 129.02 (CH), 127.79 (CH), 124.49 (Cq), 68.58 (CH2), 113.3 (CHarom), 112.1 (Cq), 66.9 (CH2), 64.2 (CH2), 37.1 (CH3, OMs), 28.9 (CH2). HRMS (TOF
	37.30 (CH3, OMs), 35.96 (CH2). HRMS (TOF MS EI+): calcd. For C9H11BrO3S 277.9599; MS ESI+): calcd. For C10H17BrNO4S [M + NH4] + 326.0062; found 326.0067.
	2 S. M. Allin, W. R. Bowman, M. R. J. Elsegood, V. McKee, R. Karim, S. S. Rahman, Tetrahedron, 2005, 61,
	2689-2696. 3 L. Ripa, A. Hallberg, J. Org. Chem. 1998, 63, 84-91.
		S18 S19

  Hz, Cq, C-F), 143.30(d, J = 4.0 Hz), 136.81 (d, J = 2.7 Hz, Cqarom), 132.8 (C2, CHim), 131.28(d, J = 11.1 Hz), 124.69 (d, J = 9.1 Hz), 123.8 (Cq), 115.68 (d, J = 23.7 Hz), 111.48 (d,

	244.7 J = 25.9 Hz), 51.9 (OCH3), 48.8 (N-CH2). HRMS (TOF MS ESI+): calcd. For C12H10FN2O2 [M
	+ H] + 233.0726; found 233.0731.
	methyl 8-fluoro-5H-imidazo[2,1-a]isoindole-2-carboxylate 5B-C2 :
	2 °C. 1 H NMR (300 MHz, CDCl3) δ 8.34 (d, J = 7.5 Hz, 1Harom), 7.69 (s,
	1Him, H2), 7.49-7.37 (m, 3Harom), 5.04 (s, 2H), 3.97 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3)
	δ 163.7 (C=O), 144.3 (Cq), 141.5 (Cq), 132.5 (C2, CHim), 129.4 (Cq), 128.9 (CHarom), 128.5
	(CHarom), 124.1 (CHarom), 123.4 (CHarom), 123.2 (Cq), 51.7 (OCH3), 49.1 (N-CH2). HRMS (TOF Compound 5B-C2 was prepared according to the method C using 4b (0.27 mmol). The crude
	MS ESI+): calcd. For C12H11N2O2 [M + H] + 215.0821; found 215.0817. Compound methyl 5H-imidazo[2,1-a]isoindole-2-carboxylate 5A-C2: product was purified by flash chromatography (EtOAc) to afford 5B-C2 in 8% as a pale yellow F solid. mp : 177.0 °C (degradation). 1 H NMR (300 MHz, CDCl3) δ 7.91 (s, 1Him, H5), 7.61 (dd, o r J = 8.1, 2.0 Hz, 1Harom), 7.44 (dd, J = 8.3, 4.4 Hz, 1Harom), 7.11 (td, J = 8.8, 2.3 Hz, 1Harom),
	P 4.95 (s, 2H, N-CH2), 3.94 (s, 3H, OMe).
	e
	Compound 5A-C2 was prepared according to the method C using 4a (0.27 mmol). The crude e r
	product was purified by flash chromatography (EtOAc) to afford 5A-C2 in 9% as a pale yellow R solid. mp : 145.0-145.3 °C. 1 H NMR (300 MHz, CDCl3) δ 7.95 (d, J = 7.4 Hz, 1Harom), 7.91 (s, 1Him, H5), 7.49-7.38 (m, 3Harom), 4.97 (s, 2H), 3.94 (s, 3H, OMe). 13 C NMR (75 MHz, CDCl3) e v
	i δ 163.7 (C=O), 154.5 (Cq), 141.8 (Cq), 137.7 (Cq), 129.1 (Cq), 129.0 (CHarom), 128.7 (CHarom), e 123.6 (CHarom), 122.3 (CHarom), 121.2 (CH), 51.9 (OCH3), 49.1 (N-CH2). HRMS (TOF MS ESI+): calcd. For C12H11N2O2 [M + H] + 215.0821; found 215.0823. w
	methyl 8-fluoro-5H-imidazo[5,1-a]isoindole-1-carboxylate 5B-C5 :
	Compound 5B-C5 was prepared according to the method C using 4b (0.27 mmol). The crude
	product was purified by flash chromatography (EtOAc) to afford 5B-C5 in 90% as a pale yellow
	S24

solid. mp : 197.5 °C (degradation)

. 1 H NMR (300 MHz, CDCl3) δ 8.09 (dd, J = 8.7, 2.3 Hz, 1Harom), 7.74 (s, 1Him, H2), 7.41 (dd, J = 8.4, 4.7 Hz, 1H), 7.10 (td, J = 8.7, 2.6 Hz, 1H), 5.05 (s, 2H, N-CH2), 4.00 (s, 3H, OMe).

13

C NMR (75 MHz, CDCl3) δ 163.5 (C=O), 163.3

(d, J =

  2 °C (CH2Cl2/PE). 1 H NMR (300 MHz, CDCl3) δ 8.74 (d, J = 7.6 Hz, 1Harom), 7.53 (s, 1H, C2, CHim)7.42-7.37 (m, 1Harom) 7.34-7.26 (m, 2Harom), 4.16 (t, J = 6.5 Hz,

	2H, N-CH2), 3.95 (s, 3H, OMe), 3.09 (t, J = 6.6 Hz, 2H, CH2-Ph). 13 C NMR (75 MHz, CDCl3)
	δ 164.3 (C=O), 135.6 (C2, CHim), 133.9 (Cq), 133.1 (Cq), 129.2 (CHarom), 128.5 (CHarom), 128.3
	(Cq), 127.9 (CHarom), 127.7 (CHarom) , 126.0 (Cq), 52.0 (OCH3), 42.6 (N-CH2), 29.7 (CH2-Ph).

HRMS : Anal. Calcd for C13H13N2O2: [M+H] + 229.0977, found 229.0981. This is a known compound.

2

  ).13 C NMR (75 MHz, CDCl3) δ 163.6 (C=O), 149.4 (C2, Cqim), 138.0 (Cq), 132.5 Cq),130.2 (Cq), 129.8 (CHarom), 129.2 (CHarom), 129.2 (CHarom), 127.2 (CHarom), 126.8 (C5, CHim), 51.8 (OCH3), 45.0 (N-CH2), 31.0 (CH2), 30.4 (CH2).Compound 8-C5 was prepared according to the method C using 4e (0.27 mmol). The crude product was purified by flash chromatography (EtOAc) to afford 8-C5 in 62% as a white solid. mp : 190.2-191.4 °C (CH2Cl2/PE). 1 H NMR (300 MHz, CDCl3) δ 7.88 (dd, J = 7.7, 1.5 Hz, 1Harom),7.64 (s, 1Him, H2), 7.42 (td, J = 7.8, 1.6 Hz, 1Harom), 7.29 (t, J = 7.5 Hz, 1Harom), 7.18 (d, J = 8.0 Hz, 1Harom), 4.51 (t, J = 5.9 Hz, 2H, O-CH2), 4.11 (t, J = 5.9 Hz, 1H, N-CH2),3.88 (s, 3H, OMe).13 C NMR (75 MHz, CDCl3) δ 163.3 (C=O), 153.1 (Cq), 136.0 (Cq), 135.8 (CH), 132.3 (CH), 131.2 (CH), 129.4 (Cq), 124.6 (CH), 122.7 (Cq), 122.

	methyl 5,6-dihydrobenzo[f]imidazo[1,5-d][1,4]oxazepine-1-carboxylate 8-C5 :
	F
	o r
	P
	e
	e r
	R
	e v
	i
	e
	w

S26

(

Georg Thieme Publishers KG, Rüdigerstraße 14, 70469 Stuttgart, Germany

J. Thireau, C. Schneider, C. Baudequin, S. Gaurrand, P. Angibaud, L. Meerpoel, V. Levacher, O. Querolle, C. Hoarau, Eur. J.Org. Chem., 2017, 2491-2494.

Acknowledgment

This work has been partially supported by INSA Rouen, Rouen University, CNRS, EFRD, European INTERREG IV A France (Channel) and Labex SynOrg (ANR-11-LABX-0029). We also thank the CRIHAN for software optimization and technical support.

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Primary Data

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General information

All reagents were purchased from commercial suppliers and were used without further purification. Extra dry 1,4-Dioxane and DMF were purchased from Sigma-Aldrich® in sealed bottles over 3Å or 4Å molecular sieves and stored under N2.

The reactions were performed without any protection from the light and monitored by thinlayer chromatography (TLC) analysis using silica gel (60 F254) plates. Compounds were visualized by UV irradiation (λ = 254 nm) and/or spraying TLC stain such as a KMnO4 solution followed by heating at 200 °C. Flash chromatography columns were performed on silica gel 60 (230-400 mesh, 0.040-0.063 mm).

1 H and 13 C NMR spectra were recorded at room temperature on a Brucker Advance spectrometer operating at 300 or 500 MHz, and 75 or -125 MHz respectively. Chemical shifts are given in parts per million and 1 H and 13 C NMR spectra were referenced using the solvent signal as an internal standard. The following abbreviations are used for the proton spectra multiplicities: s: singulet, d: doublet, t: triplet, q: quartet, qt: quintuplet, m: multiplet, br: broad. 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.31 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.30 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.28 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.26 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.25 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.20 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 7.19 Page .75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 76.75 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.00 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26 77.26


Adeline Decambron

Nausikaa Devriendt

Nathanael Larochette

Mathieu Manaserro

Marianne Bourguignon

Hanane El-Hafci

Hervé Petite

Véronique Viateau

Delphine Logeart-Avramoglou
email: delphine.logeart@cnrs.fr

Mathieu Manassero

Effect of the Bone Morphogenetic Protein-2 Doses on the Osteogenic Potential of Human Multipotent Stromal Cells-Containing Tissue Engineered Constructs

Keywords: bone, tissue engineering, in vivo, BMP-2, MSC, dual delivery

come   L'archive ouverte pluridisciplinaire

INTRODUCTION:

Stem cell-based bone tissue engineering (TE) approaches are currently developed by combining osteoprogenitors (such as multipotent stromal cells [MSC]) with a supporting substrate for the repair of large bone defects. The efficiency of bone TE in both experimental and clinical studies, however, remains unsatisfactory. Indeed, in large animal models, in which full bone regeneration was demonstrated using MSC-containing TE constructs, inconsistent bone repair outcomes have limited large-scale clinical applications. [START_REF] Bensaïd | De Novo Reconstruction of Functional Bone by Tissue Engineering in the Metatarsal Sheep Model[END_REF][START_REF] Manassero | Bone Regeneration in Sheep Using Acropora Coral, a Natural Resorbable Scaffold, and Autologous Mesenchymal Stem Cells[END_REF][START_REF] Petite | Tissue-engineered bone regeneration[END_REF][START_REF] Viateau | Longbone critical-size defects treated with tissue-engineered grafts: A study on sheep[END_REF] This evidence emphasizes the great need for improving the efficacy of bone TE constructs. Among the causes responsible for failure to bone regeneration, an insufficient osteoinductive potential of these cell-containing TE constructs has been implicated. [START_REF] Manassero | Bone Regeneration in Sheep Using Acropora Coral, a Natural Resorbable Scaffold, and Autologous Mesenchymal Stem Cells[END_REF][START_REF] Amini | Bone tissue engineering: recent advances and challenges[END_REF][START_REF] Dumic-Cule | Biological aspects of segmental bone defects management[END_REF] To overcome this issue, the strategy of adding an osteoinductive cue to stem cells was addressed in the present study.

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive growth factor involved in the early stage of bone formation and repair. [START_REF] Tsuji | BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing[END_REF] The literature on the in vivo potential benefit of BMP-2 addition to stem cells (either bone marrow-or adipose-derived stem cells) on new bone formation or healing has been, however, controversial. In fact, some studies reported that such codelivery led to a significant increase in bone formation. [START_REF] Mehrkens | Intraoperative engineering of osteogenic grafts combining freshly harvested, human adipose-derived cells and physiological doses of bone morphogenetic protein-2[END_REF][START_REF] Gohil | Evaluation of rhBMP-2 and bone marrow derived stromal cell mediated bone regeneration using transgenic fluorescent protein reporter mice[END_REF][START_REF] Rui | Comparative study between coral-mesenchymal stem cells-rhBMP-2 composite and auto-bone-graft in rabbit critical-sized cranial defect model[END_REF][START_REF] Kim | Evaluation of bone regeneration with biphasic calcium phosphate substitute implanted with bone morphogenetic protein 2 and mesenchymal stem cells in a rabbit calvarial defect model[END_REF][START_REF] Decambron | Low-dose BMP-2 and MSC dual delivery onto coral scaffold for critical-size bone defect regeneration in sheep[END_REF] In particular, a recent study published by our group demonstrated that a bone TE construct containing autologous MSCs combined with BMP-2 used to treat critical-sized defect in sheep fully repaired 50% of the tested animals as only 20% and 30% of the animals repaired when treated with either BMP-2 alone or MSCs alone, respectively.12 In contrast, other studies showed no improvement in bone formation with codelivery of both BMP-2 and stem cells compared with results obtained with BMP-2 treatment alone. [START_REF] Boos | Directly auto-transplanted mesenchymal stem cells induce bone formation in a ceramic bone substitute in an ectopic sheep model[END_REF][START_REF] Kamal | Transplantation of mesenchymal stem cells, recombinant human BMP-2,and their combination in accelerating the union after osteotomy and increasing, the mechanical strength of extracorporeally irradiated femoral autograft in rat models[END_REF][START_REF] Herberg | Mesenchymal stem cell expression of SDF-1β synergizes with BMP-2 to augment cell-mediated healing of critical-sized mouse calvarial defects[END_REF][START_REF] Keibl | Human adipose derived stem cells reduce callus volume upon BMP-2 administration in bone regeneration[END_REF] Definite comparisons of these results, however, are not possible because the source of MSCs, the material scaffolds, the implantation model, and the doses of BMP-2 used were different. This latter point is critical as BMP-2 promotes bone formation in a dosedependent manner17 and most of the aforementioned studies assessed a single dose of BMP-2. Besides, BMP-2 has an optimal dose for therapeutic success, and delivery of supraphysiological doses generated deleterious side effects, including excessive bone formation and immune responses.18,19 For these reasons, substantial work needs to be done (i) to determine the effective dosage of BMP-2, which promotes MSCmediated bone formation and (ii) to provide detailed mechanistic insights on the relation among BMP-2, MSCs, and bone formation at this minimal dosage.

To this aim, we determined the effects of two BMP-2 dosages on the bone-forming potential of TE constructs containing human MSCs (hMSCs) and assessed the influence of BMP-2 on the host murine progenitor cells as well as on the direct and paracrine contribution of implanted hMSCs in a mice ectopic model.

MATERIALS AND METHODS:

Material scaffolds

Two hundred to 300 μm calcium carbonate particles (Coral from Porites sp.; Biocoral®, Inoteb, France) were used as the bioceramic scaffolds. These particles were sterilized by autoclaving at 120°C for 20 min. hMSC isolation and culture HMSCs were harvested from bone marrow obtained as discarded tissue during routine bone surgery from three patients (one woman and two men, 15, 22, and 31 years of age, respectively) at the Lariboisiere Hospital (Paris, France). The tissues were collected with the respective donor's consent in agreement with Lariboisiere Hospital regulations. hMSCs were isolated from each donor bone marrow using a procedure adapted from literature reports20 and were characterized for the expression of select CD markers (specifically, positive for CD90, CD73, CD105, and negative for CD45; data not shown). These cells were cultured in Alpha-Minimum Essential Medium (Sigma) containing 10% fetal bovine serum (PAA Laboratories) and antibiotics (PAA Laboratories) into tissue culture plates. Adherent cells from each donor were cultured separately until passage 3 and then pooled before seeding on coral scaffolds.

Bone morphogenetic protein-2

Recombinant human BMP-2 powder (InductOs® 12 mg; Medtronic, France) was reconstituted in sterile 1 mM HCl solution (pH ∼3) at a concentration higher than 100 μg/mL, aliquoted, and kept at -80°C un l use. The initial BMP-2 concentration was assessed by UV detection at 280 nm (using a NanoDropTM1000 UV-Vis Spectrophotometer) with the extinction coefficient fixed at 1.41 absorbance units per mg/mL.

Preparation of TE constructs

Bare coral particles

Twenty-five milligrams of sterile coral particles were washed with complete culture medium.

hMSC-containing coral particles

Twenty-five milligrams of sterile coral particles were immersed in complete culture medium at 37°C for at least 1 h. After the medium removal, 106 hMSCs were seeded onto the particles and allowed to adhere in static condition at 37°C overnight.

BMP-2-loaded coral particles

Twenty-five milligrams of sterile coral particles were washed in 1 mM HCl and loaded with 50 μL of BMP-2 diluted at either 10 or 100 μg/mL (defined as low dose [LD] and high dose [HD], respectively) in 1 mM HCl and maintained under mild shaking at 4°C overnight. The adsorption rates of BMP-2 onto coral particle were determined and found to be 81% and 84% for LD and HD, respectively (Table 1). The retention capacities of these bioceramic scaffolds for BMP-2 were determined by monitoring the kinetics of protein release during 48 h (Supplementary Fig. S1A; Supplementary Data are available online at www.liebertpub.com/tea). Both dose cases displayed similar release profiles with an initial burst release of BMP-2 occurring during the first hour of the experiment (due to desorption of unbound growth factor) followed by a minimal (up to 3%) BMP-2 released afterward. Maintenance of the biological activity of BMP-2 coated onto coral particles was confirmed using pluripotent C2C12 myoblasts as a cell model21 (Supplementary Fig. S1B). At similar doses of BMP-2, the bioactivity of BMP-2 adsorbed onto coral particles was slightly lower than the one of soluble BMP-2, but still significant.

Six different types of TE constructs were prepared by mixing at a 1:1 ratio the aforementioned types of particles in 8 mm wells (Table 1). Before mixing with other particles, BMP-2-loaded coral particles were quickly washed twice with culture medium (to neutralize the acidity). Before implantation, the mixed particles were embedded in 100 μL fibrin gel (prepared using 18 mg/mL fibrinogen and 50 U/mL Thrombin; Tissucol®, Baxter).

In vivo implantation

Eight-week-old, female, nude mice (RJ: NMRI-nu [nu/nu]) were obtained from JANVIER LABS and handled in accordance with the European Directive 2010/63/EU regarding the protection of animals used for scientific purposes. All animal experiments were performed following approval of the protocols by the Ethics Committee on Animal Research of Lariboisiere/Villemin (no. CEEA-LV/2014-10-20), Paris, France. Under general anesthesia of each animal, the TE constructs were randomly implanted subcutaneously into both the thoracic and abdominal dorsal regions on each side of the rachis. [START_REF] Monfoulet | The pH in the Microenvironment of Human Mesenchymal Stem Cells Is a Critical Factor for Optimal Osteogenesis in Tissue-Engineered Constructs[END_REF] The anesthesia protocol used included premedication with 0.05 mg/kg buprenorphine (Buprecare® 0.3 mg/mL; Axience, France) 20 min before anesthesia (intraperitoneal injection of 10 mg/kg xylazine, Rompun® 2%; Bayer HealthCare, France, and 100 mg/kg ketamine, Ketamine 1000®; Mérial, France).

Microcomputed tomography analysis, histology, histomorphometry, and immunohistochemistry

At 8 weeks postimplantation, the mice were sacrificed (using pentobarbital, Dolethal®; Vetoquinol) and the retrieved constructs (n = 12 per group) were immediately fixed in 10% neutral buffered formaldehyde solution for 24 h. The fixed specimens were imaged and analyzed using a high-resolution microcomputed tomography (μ-CT) machine (Skyscan 1172; Skyscan, Aartselaar, Belgium) to determine the volume of the explanted TE constructs. The data were collected without a filter at 80 kV and 100 μA for 150 ms. Visualization and reconstruction were obtained using NRecon software (Skyscan, Belgium). Because the explants displayed an oblate ellipsoid shape, the implant volumes were measured using the formula: Volume = [4 × π × length × width × height]/3. Quantification of bone and coral volumes using μ-CT were not possible because their respective gray-level thresholds did not provide conclusive discrimination. The constructs were, therefore, processed for undecalcified histology and histomorphometric analysis. The bone surface area (stained in red), coral surface area, and bone marrow-like tissue surface area were measured in each specimen section and expressed in μm2 according to established methods. [START_REF] Monfoulet | The pH in the Microenvironment of Human Mesenchymal Stem Cells Is a Critical Factor for Optimal Osteogenesis in Tissue-Engineered Constructs[END_REF] The atomic chemical composition analysis of TE construct bone (and compared with a mouse cortical bone) was performed from resin-embedded sections using scanning electron microscopy (SEM)-energy-dispersive X-ray (EDX) spectroscopy using an Analytical UHR Schottky Emission Scanning Electron Microscope SU-70 (HITACHI). A set of TE constructs (n = 4 per group) were implanted for 28 days, retrieved, fixed in 4% paraformaldehyde (pH 7.4), decalcified in ethylenediaminetetraacetic acid (14.5% w/v) at 4°C for 2 weeks, and embedded in paraffin. Some sequential sections were examined for the presence of human beta-2-microglobulin (a membrane protein that enables tracking human cells) immunodetection, using the Envision+ Kit (Dako) and polyclonal rabbit anti-beta-2-microglobulin (1/1000; Novocastra) as the primary antibody.22

Quantitative gene expression analysis by RT-PCR on explanted TE constructs

Another set of TE constructs (n = 6 per group) were prepared and either nonimplanted (day 0) or implanted for 7 and 28 days, retrieved, and stored at -80°C. The frozen explanted constructs were then finely chopped with a scalpel blade, and total RNA was extracted by adding 1 mL of TRIzol® (Thermo Scientific). The RNA concentration and purity were determined using a NanoDrop spectrophotometer (NanoDrop 1000; LabTech). cDNA was obtained after reverse transcription of 3 μg of purified RNA using the Superscript II enzyme (Life Technologies) and random primers. Quantitative PCR was then performed using 25 and 75 ng cDNA in 96-well plates (iCycler iQ PCR plates; Bio-Rad) and TaqMan gene expression assays (Life Technologies) for mouse genes and human genes, respectively, following the manufacturer's instructions and using the MyiQ™ Single-Color Real-Time PCR Detection System (Bio-Rad). The results were normalized to those of the respective GAPDH as internal standard. The full names of the genes monitored and the assay IDs are given in Supplementary Table S1.

Statistical analyses

Statistical analyses were performed using a commercially available software package (GraphPad Prism, version 6.0c; GraphPad Software, Inc., La Jolla). Quantitative data were expressed as mean ± SEM. The one-way analysis of variance (ANOVA) followed by Tukey's post hoc test was used to compare mean of more than two groups. The quantitative kinetics data were analyzed using two-ways ANOVA followed by Tukey's post hoc test. For all analyses, differences at p < 0.05 were considered as statistically significant.

Results

In vivo osteogenic performance of TE constructs

To determine the osteogenic potential of the constructs containing both the hMSCs and BMP-2, these specimens were implanted subcutaneously in the back of mice. The six conditions tested, including appropriate controls (specifically, constructs containing either bare coral or coral loaded with hMSCs or coral loaded with BMP-2 at either LD or HD) are listed in Table 1. Based on the determined adsorption rate, the total BMP-2 dose delivered in vivo was estimated to be 0.4 and 4.2 μg for the LD and HD, respectively. After 8 weeks, the hMSC-HD explanted constructs and their HD controls exhibited 1.6-2.4-fold larger volumes than the other constructs as determined by μ-CT analysis (p < 0.05) (Fig. 1A). Representative images from histology sections from each type of construct tested are shown in Figure 1B. Minimal new bone tissue was present both in the hMSC constructs and LD constructs, whereas no bone tissue was observed in the control coral constructs (Fig. 1Ba-c). More new bone tissue deposited around residual coral particles throughout the specimen was observed when hMSCs were combined with LD in constructs (Fig. 1Bd). In contrast, a bone tissue organized as a peripheral shell connected with thin internal trabeculae and enclosing bone marrow-like tissue, and few residual coral particles was found in hMSC-HD-containing constructs; the cell-free HD constructs exhibited same histological features (Fig. 1Be,f).

Examination under higher magnification of both hMSC-and hMSC-LDcontaining constructs revealed the presence of woven bone tissue with large nucleated cells and abundant osteoid encircled by bone-lining cells present all around the new bone tissue, illustrating its immature nature as well as high bone-forming activity (Fig. 2A,B). In contrast, newly formed bone tissue in the hMSCs-HD-containing constructs appeared more lamellar and mature (Fig. 2C). In the latter constructs, the peripheral bone tissue appeared still active with the presence of a layer of osteoid that is, however, much thinner than in the case of the hMSC-and hMSC-LDcontaining constructs (Fig. 2C); besides, the inner trabeculae bone tissue appeared either less active or quite inactive with total absence of osteoblasts laying down new bone tissue (Fig. 2D). Noteworthy, these HDcontaining constructs were filled with bone marrow containing numerous adipocytes.

SEM-EDX analyses revealed that the Ca/P ratios in bone tissue from all TE constructs were in the same range (from 1.92 to 1.98), with the exception of the hMSCs-HD-containing constructs, from which the Ca/P ratio was lower (1.67) (Supplementary Fig. S2A); it is worth to notice that the Ca/P ratio from mouse cortical bone was 1.5. While the P content was similar in all groups, the Ca content was lower in LD-and hMSCs-HD-containing constructs as well as in mouse cortical bone (Supplementary Fig. S2B). These data indicated that, with the exception of hMSCs-HD-containing constructs, the degrees of calcification of the ectopic induced bone tissue in TE constructs were similar and higher than the one of mature cortical bone.

Histomorphometric quantification confirmed the qualitative observations (Fig. 3):

-Bone quantification indicated that the addition of the LD to hMSC-containing constructs significantly (p < 0.05) enhanced the new bone tissue compared with constructs containing hMSCs only. The addition of the HD to hMSC-containing constructs, however, induced an amount of bone dramatically higher than those induced by the other groups, except the control HD construct. Thus, in this case, the presence of hMSCs in the HD constructs was not beneficial and did not change the osteogenic outcome (Fig. 3A).

-

The bone marrow tissue quantification confirmed that constructs containing both hMSCs and LD or either one of these components did not contain bone marrow, whereas the hMSCs-HDcontaining constructs were substantially (up to 50% of the surface area) filled with this tissue (Fig. 3B). It is worth to notice that the absence of hMSCs in the HD constructs significantly (p < 0.05) increased the volume of the bone marrow. However, independently of the bone-marrowcontaining explant analyzed, the adipocyte extent did not change and comprised 47% ± 21% of the bone marrow tissue (data not shown).

-

The extent of the scaffold material resorption in constructs was also quantified (Fig. 3C). All constructs containing BMP-2 exhibited levels of coral resorption significantly higher (p < 0.05) than that found with the control constructs of bare coral. Additionally, the absence of hMSCs in BMP-2-containing constructs enhanced resorption levels in comparison with the hMSC-containing constructs.

When comparing the efficacy of the two dosages, these results provided evidence that only low dose of BMP-2 enhances the osteogenic potential of hMSC-containing constructs compared with treatment with either hMSCs alone or BMP-2 alone. In contrast, at high dose, BMP-2 exhibited a strong bone-forming potential by itself, and its addition to hMSC constructs did not improve the osteogenic effect. For these reasons, the following studies, aiming at understanding the beneficial effect of BMP-2 on hMSC-containing constructs, were performed using BMP-2 at low dose only.

Role of BMP-2 on codelivered MSCs in TE constructs

Constructs containing both hMSCs and LD were specifically explanted at shorter postimplantation times and examined in comparison with control constructs containing either hMSCs or LD.

Immunostaining of human β2-microglobulin enabled visualization of the implanted hMSCs still present in sections of the explanted constructs 28 days postimplantation (Fig. 4). Positive human cells of both the hMSC and hMSCs-LD constructs were detected as bundles in the fibrous tissue surrounding the coral granules and bone tissue (Fig. 4A,B). In both types of constructs, while most of the osteocytes were not labeled (at a higher extent in the hMSCs-LD constructs), some human cells were observed along the periphery of the coral particles and embedded in osteocyte lacunae (arrows in Fig. 4A-C); these results provided evidence that the new bone tissue was partly formed by the murine host cells and partly by the human implanted cells.

To further characterize the relative implication of mouse and human osteoprogenitor cells in the newly formed bone tissue, kinetics analysis of the both expressed murine (Fig. 5) and human (Fig. 6) osteogenesisrelated genes in TE constructs explanted at 0, 7, and 28 days postimplantation were performed. The expressions of the mRUNX2, mIBSP, mbGLAP, and mSPP1 genes increased with the implantation time (Fig. 5). Interestingly, in comparison with coral-, hMSC-, and LD-containing constructs, expression of mRUNX2, mIBSP, and mbGLAP were significantly upregulated in the hMSC-LD-containing constructs (Fig. 5). Expression of mSPP1, a known regulator of mineral deposition in the bone extracellular matrix, was similar in all types of constructs (Fig. 5). Similarly, the expressions of human osteogenesis-related genes (i.e., hRUNX2, hIBSP, hbGLAP, and hSPP1) also increased with the implantation time (Fig. 6A), confirming the osteogenic differentiation of the surviving implanted human MSCs. No difference, however, was found in the expression of these genes in both hMSC-LD and hMSC-containing constructs at each postimplantation time tested.

To assess the paracrine effect of implanted human MSCs on host murine progenitors, gene expressions of hSDF1 and hPDGF-B (known for being involved in hMSC recruitment) as well as of hVEGF2 (known for its involvement in angiogenesis) were analyzed (Fig. 6B). Expressions of these three genes were upregulated at day 7 and downregulated at day 28 postimplantation. Only the expression of hPDGF-B was found significantly higher (5-fold) in hMSCs-LD constructs than in hMSC constructs at day 7 postimplantation.

The aforementioned data provided evidence that the higher bone formation observed with hMSCs-LD constructs was most probably due to higher murine osteogenic differentiation; moreover, the effect of BMP-2 at low dose to either induce the differentiation of the grafted human MSCs into osteoblasts or modulate their paracrine cues was null or minimal.

DISCUSSION:

In the present study, we assessed the advantage of combining BMP-2, a potent osteoinductive growth factor involved in the early stage of bone formation, with hMSCs to enhance the bone-forming potential of TE constructs in an ectopic mouse model; more specifically, we determined its effective dosage that improves the osteogenic potential of hMSCcontaining constructs. Our results provided evidence that the addition of high dose of BMP-2 induced a bone tissue in higher amount and more mature than those induced by low dose BMP-2; its codelivery with hMSCs, however, did not improve the osteogenic potential of constructs. In contrast, a significant benefit of BMP-2, combined with hMSCs on the bone-forming potential of these constructs, was found in the presence of a low dose of BMP-2. This effect seems to be due to BMP-mediated induction of the osteogenic differentiation of the host progenitors only and not of the grafted human cells.

Several previous studies have tested the potential cooperative effect of BMP-2 with MSCs on bone formation in both ectopic and orthotopic site with contradictory results. While some studies reported that such codelivery led to a significant increase in bone formation,8-11,23 other studies showed no improvement compared with results obtained with BMP-2 treatment alone.13-16 These inconsistent outcomes are likely due to the various and/or inappropriate doses of BMP-2 used in these studies. In fact, determining the accurate dosage of BMP-2 in association with MSCs is critical since BMP-2 induces bone formation in a dose-dependent manner17 and its delivery in high doses has been reported to be associated with adverse effects, including excessive bone formation, ectopic bone formation, inflammatory responses, osteoclast-mediated bone resorption, and inappropriate adipogenesis.18,24 In the present study, hMSCs were combined with BMP-2 at either low (10 μg/mL) or high (100 μg/mL) dose (corresponding to 0.4 and 4.2 μg per TE construct, respectively) to determine the effective BMP-2 dosage that enhances the intrinsic osteogenic potential of implanted hMSCs. It should be noted that the high dose used in the present study was lower than those that induce adverse side effects in rats (more than 300 μg/mL). [START_REF] Zara | High Doses of Bone Morphogenetic Protein 2 Induce Structurally Abnormal Bone and Inflammation In Vivo[END_REF] The osteogenic potential of the TE constructs was assessed in vivo in an ectopic mouse model, which has the advantage of evaluating the "true" osteogenic capacity of constructs since osteoconduction from the bone bed of the recipient animals does not occur at this location. Implantation of bone TE constructs in "unphysiological" ectopic site does not, however, enable the evaluation of high-dose BMP-2-mediated adverse effects. As expected, the amount of newly formed bone enhanced with the amount of implanted BMP-2 and this outcome was also observed in the presence of hMSCs.

When used at a high dose, the strong bone-forming potential of BMP-2 largely superseded the hMSC-mediated osteogenic outcome, and, therefore, addition of BMP-2 within hMSC-containing constructs had no additive effect on bone formation. Compared with the other constructs tested, the addition of high dose of BMP-2 along with or without hMSCs, led to larger-sized constructs. Histological analysis revealed that these constructs induced large bony ossicles, typical of BMP-2-induced ectopic bone induction with thin and mature bone trabeculae present mainly at the outer part of the implants forming a shell, and fatty bone marrow occupying the center of the ossicles. Additionally, weak or lacking boneforming activity was observed in these constructs.

Interestingly, codelivery of hMSCs with high dose of BMP-2, although not changing the amount of newly formed bone, significantly decreased the size of the explanted TE construct, limited coral resorption, reduced the amounts of fatty bone marrow, and promoted a bone tissue with a calcification degree comparable to those of murine cortical bone, suggesting that the presence of hMSCs modulates the excessive biological effects of high-dose BMP-2. Similarly, in a previous study, Keibl et al. noticed that the association of hMSCs to BMP-2 significantly reduces the periosteal callus volume in a femoral transcortical defect in rat, whereas observed in excess with BMP-2 alone.16 Further studies, however, are necessary to decipher the mechanism of action of hMSCs on the high-dose BMP-2-mediated excessive biological effects.

While BMP-2 delivered at high dose overcame the advantage of combining this growth factor with hMSCs, in contrast, when used at low dose, the BMP-2 combined with hMSCs significantly enhanced the boneforming potential of the TE constructs in comparison with constructs containing either hMSCs or BMP-2 only. This observation was in accordance with a recent study published by our group that provided evidence that the treatment of a critical-sized defect in sheep with codelivery of BMP-2 at low dose (10 μg/mL), autologous bone marrowderived MSCs and coral particles improved, but not significantly, the bone healing in comparison with that mediated by single treatment of either MSCs or low-dose BMP-2.12 In the present study, the ectopic newly formed bone (specifically, aspect and high activity with great extent of osteoid and bone-lining osteoblasts as well as degree of calcification) appeared similar to the one induced by hMSCs alone.

We next sought to determine the role of BMP-2 codelivered with hMSCs since it remains poorly understood so far. It is reported that the ectopic bone formation mediated by BMP-2 results from osteogenic differentiation of circulating mesenchymal precursor cells (including myoblast21 and adipocyte8,25 progenitors), which are locally exposed to the growth factor. In the present study, we wondered whether BMP-2 also enhanced the osteogenic potential of the implanted hMSCs resulting in a synergized effect on the bone formation. Because high dose of BMP-2 combined with hMSC did not exhibit beneficial effect on the bone-forming potential compared with BMP-2 alone, these analyzes were performed on BMP-2 codelivered at low dose only.

Immunostaining of the implanted human cells provided evidence that both murine and human cells were present in the osteocyte lacunae proving that the new bone tissue was hybrid, chiefly formed by the murine host cells, but also formed by the human implanted cells (the later ones being present at a low extent).

Expressions of the select osteogenesis-related genes of both murine and human origins in TE constructs increased with the implantation time, confirming the osteogenic differentiation of the host murine progenitors along with the surviving implanted hMSCs. However, expressions of the select murine osteogenesis-related genes in TE constructs tested were upregulated (except, SPP1, a regulator of mineral deposition) in the presence of hMSCs combined with low-dose BMP-2, compared with their respective expressions in the presence of either one of these components alone. In contrast, expressions of osteogenesis-related genes of human origin were similar in TE constructs containing hMSCs either with or without low-dose BMP-2. Additionally, because in stem cell-based TE, hMSCs play not only a direct role on new tissue formation, but also an indirect role by supporting resident progenitor cells through paracrine mechanisms,26 expression of key chemotactic and angiogenic factors in human MSCs was assessed. Only PDGF-B expression was found slightly higher in human MSCs in the presence of low-dose BMP-2, whereas those of SDF-1 and VEGF-A were similar in both types of constructs.

Taken altogether, these results suggested that whether the addition of low-dose BMP-2 to hMSC-containing construct promoted the host murine osteogenic differentiation, by contrast, its effect on implanted human MSCs toward either differentiation into osteoblasts or induction of paracrine cues was null or minimal. Therefore, the advantage of combining BMP-2 with hMSCs to enhance the bone-forming potential of hMSCs-containing TE constructs appeared to be an additive effect of both components rather than a synergistic one

CONCLUSION

The optimal bone repair and regeneration is a complex process that requires suitable scaffold, cells, and osteoinductive signals. Whether bone growth is promoted by the presence of either hMSCs or BMP-2, their combination is an attractive strategy to enhance the bone-forming potential of TE constructs. Results of the present study showed that the addition of low dose of BMP-2 was beneficial to improve the osteogenic potential of hMSCs containing TE constructs, whereas BMP-2 delivered at high dose overcame the advantage of combining this growth factor with hMSCs. Furthermore, this study demonstrated that this beneficial effect of low dose of BMP-2 with implanted hMSCs did not involve higher differentiation of these cells into osteoblasts or induction of paracrine cues. The knowledge gained from these evaluations will significantly help in designing improved combination strategies for tissue regeneration with better clinical outcomes. Kinetics of in vitro BMP-2 release from coral particles during 48 hours. Fifty mg of coral particles were coated with BMP-2 as described in the "Preparation of TE constructs" paragraph in the "Materials and Methods" section. Following removal of the non-adsorbed BMP-2 solution, the particles were rinsed, immersed in 1 mL of PBS (0.15M NaCl) at pH 7.4 containing 0.5% BSA and 0.02% NaN3 and kept at 37°C under gentle shaking. At 1, 3, 7, 24 and 48 h, the supernatants (containing the released BMP-2) were collected and replaced by 1 mL fresh buffer. The respective supernatants were aliquoted and stored at -80°C until BMP-2 quantification was performed using an ELISA assay kit (DuoSet ELISA Development System, R&D Systems, MN) according to the manufacturer's protocol. The amount of released BMP-2 in each supernatant was expressed as a percentage of the initial adsorbed amount of BMP-2. Values are means ± SEM from three separate experiments.

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(B)

Biological activity of the BMP-2 retained onto the coral particles. The bioactivity was determined by assaying the BMP-2induced alkaline phosphatase (ALP) activity (a marker of osteogenic differentiation) on murine C2C12 skeletal myoblasts (American Type Culture Collection) (Katagiri, J Cell Biol, 1994). C2C12 cells (100,000 in DMEM (Dulbecco's modified Eagle's medium) supplemented with 2 % FBS) were seeded on the particles (previously coated with BMP-2 and rinsed) and cultured under standard conditions. At day 6, the cells were rinsed with PBS and lysed using 200 µL of lysis-buffer (NaHCO3 0.1 M, Na2CO3 0.1 M, MgCl2 1 mM, Triton X-100 0.1 %, pH 10.2) and freeze/thawed three times. The ALP activity in each lysate was determined as previously described (Degat et al., J Biomed Mate Res, 2009) and was normalized to the DNA content of the respective cell population using the PicoGreen kit (Thermo scientific). The level of ALP activity induced by the coral-adsorbed BMP-2 was compared to that obtained from the soluble BMP-2 (at 0.1, 0.5 and 1 µg/ml) in the culture medium (positive controls). Values in parentheses are the estimated dose of BMP-2. Values are means ± SEM from three separate experiments.

Figure 1 :

 1 Figure 1: Ectopic bone formation in tissue-engineered constructs. (A) Box-and-whisker plot of volume of the TE constructs explanted after 8 weeks of implantation in nude mice and determined by μ-CT analysis. (n = 12) One-way ANOVA with Tukey's post hoc test: (a)p < 0.05 versus coral alone constructs; (b)p < 0.05 versus constructs containing hMSCs; (c)p < 0.05 versus constructs containing same dose of BMP-2 and no hMSCs. (B) Representative histology sections of each type of TE constructs tested (a-f) stained with Picrofuchsin and Stevenel's blue. Mineralized bone matrix is stained pink/red (scale bar = 500 μm). μ-CT, microcomputerized tomography; ANOVA, analysis of variance; BMP-2, bone morphogenetic protein-2; hMSC, human multipotent stromal cells; TE, tissue engineering.

Figure 2 :

 2 Figure 2: Histological analysis. Bone tissue cross-sections of tissue-engineered constructs prepared with either hMSCs alone (A), hMSCs and BMP-2 at low dose (hMSCs-LD) (B), or hMSCs and BMP-2 at high dose (hMSCs-HD) (C, D) stained with Picrofuchsin and Stevenel's blue. The presence of osteoblasts laying down the osteoid tissue is indicated by the black arrows. (c) coral particle; (o) osteoid; (b) mineralized bone; (m) bone marrow-like tissue; (a) adipocytes (scale bar = 100 μm).

Figure 3 :

 3 Figure 3: Box-and-whisker plots of the areas of bone tissue (A), bone marrow-like tissue (B), and residual coral scaffold (C) as determined by histomorphometric analysis. (n = 12) One-way ANOVA with Tukey's post hoc test: (a) p < 0.05 versus coral alone constructs; (b) p < 0.05 versus constructs containing hMSCs; (c) p < 0.05 versus constructs containing same dose of BMP-2 and no hMSCs

Figure 4 :

 4 Figure 4: Immunolabeling of implanted hMSCs in tissue-engineered constructs. Implanted hMSC still present in the constructs [prepared with either hMSCs alone (A) or hMSCs and BMP-2 at low dose (hMSCs-LD) (B-D)] explanted 28 days postimplantation was detected on paraffin-treated sections following immunostaining against human β2-microglobulin. The black arrows point at positive labeled osteocytes. (c) coral particle; (b) mineralized bone.

Figure 5 :

 5 Figure 5: Expression of murine osteogenesis-related genes in TE constructs. Results from RT-qPCR analysis of gene expression of mouse runt-related transcription factor 2 (mRUNX2), integrin-binding sialoprotein/bone sialoprotein (mIBSP), bone gamma-carboxyglutamate (Gla) protein/Osteocalcin (mbGLAP), secreted phosphoprotein 1/osteopontin (mSPP1) in TE constructs explanted at 7 and 28 days after implantation. The gene expression was normalized to that of the respective mGAPDH as internal standard. Values are mean ± SEM (n = 6). Two-way ANOVA with Tukey's post hoc test: (a) p < 0.05 versus coral alone constructs; (b) p < 0.05 versus constructs containing hMSCs; (c) p < 0.05 versus constructs containing low-dose BMP-2. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; SEM, standard error of the mean; RT-qPCR, quantitative reverse transcription polymerase chain reaction.

Figure 6 :

 6 Figure 6: Expression of either human osteogenesis (A) or human chemotactism and angiogenesis-related genes (B) in TE constructs. Results from RT-qPCR analysis of gene expression of Runt-related transcription factor 2 (hRUNX2), integrin-binding sialoprotein/bone sialoprotein (hIBSP), bone gammacarboxyglutamate (Gla) protein/osteocalcin (hbGLAP), secreted phosphoprotein 1/osteopontin (hSPP1), stromal cell-derived factor 1/C-X-C motif chemokine ligand 12 (hSDF-1), platelet-derived growth factor subunit B (hPDGF-B), and vascular endothelial growth factor A (hVEGF-A) in TE constructs after 0, 7, and 28 days of implantation. Gene expressions were normalized to that of the respective hGAPDH as internal standard. Values are mean ± SEM (n = 6). Two-way ANOVA with Tukey's post hoc test: (a) p < 0.05 versus constructs containing low dose BMP-2 alone.

Figure S1 :

 S1 Figure S1: (A)Kinetics of in vitro BMP-2 release from coral particles during 48 hours. Fifty mg of coral particles were coated with BMP-2 as described in the "Preparation of TE constructs" paragraph in the "Materials and Methods" section. Following removal of the non-adsorbed BMP-2 solution, the particles were rinsed, immersed in 1 mL of PBS (0.15M NaCl) at pH 7.4 containing 0.5% BSA and 0.02% NaN3 and kept at 37°C under gentle shaking. At 1, 3, 7, 24 and 48 h, the supernatants (containing the released BMP-2) were collected and replaced by 1 mL fresh buffer. The respective supernatants were aliquoted and stored at -80°C until BMP-2 quantification was performed using an ELISA assay kit (DuoSet ELISA Development System, R&D Systems, MN) according to the manufacturer's protocol. The amount of released BMP-2 in each supernatant was expressed as a percentage of the initial adsorbed amount of BMP-2. Values are means ± SEM from three separate experiments.

  Decambron A, Devriendt N, Larochette N, et al. Effect of the Bone Morphogenetic Protein-2 Doses on the Osteogenic Potential of Human Multipotent Stromal Cells-Containing Tissue Engineered Constructs. Tissue Eng. 2019;642-51. https://doi.org/10.1089/ten.tea.2018.0146

ACKNOWLEDGMENTS

The authors thank Medtronic, Inc. for kindly providing BMP-2, Prof R. Bizios for reading the article, Dr M. Bensidhoum for his help in μ-CT analysis, and F. Pillier (UPR15-CNRS-LISE) for SEM-EDX analyses. This study was supported by the Centre National de la Recherche Scientifique (CNRS) and the Fondation pour l'Avenir (project n° ET3 704, 2013)

Disclosure Statement

The authors declare no competing financial interests