Eduard Badarau

Frédéric Robert

Stéphane Massip

Florian Jakob

Simon Lucas

Sven Frormann

Léon Ghosez

Discovery of a subnanomolar and selective spirocyclic ligand of the glucocorticoid receptor

Pure diastereomeric spirocyclic analogs of fluorocortivazol were conveniently prepared by a short and efficient synthetic sequence recently developed in our laboratory. The structures and conformations of several key products were confirmed by single crystal X-ray diffraction analysis. Conformational assignments were also supported by DFT calculations. Preliminary biological testing led to the identification of a highly potent hGR ligand with an excellent activity in the subnanomolar range. All tested compounds from this series were also selective versus the progesterone receptors.

Introduction

Glucocorticoids (GCs) are potent therapeutic agents currently used for the treatment of allergic and inflammatory diseases. The currently accepted paradigm is that the therapeutic benefit of glucocorticoids is highly correlated with their transrepression versus transactivation effects. Transrepression occurs when the complex of GCs and their receptors (GR) inhibits post-transcriptional activity of other transcriptional factors, such as activator protein 1 (AP-1) and the nuclear factor-kappa B (NFκB) which are known to induce inflammatory effects. On the contrary, transactivation will trigger undesired side effects. Moreover GCs also interact with many other steroid receptors (MR -mineralocorticoid, PRprogesterone, ER -estrogen, AR -androgen receptors) thus leading to other side effects. Developing selective GR ligands with a better dissociating profile would certainly be of therapeutical benefit.

We recently designed a new class of potential ligands of glucocorticoid receptors [START_REF] Badarau | Design and Synthesis of Spirocyclic Ligands of Glucocorticoid Receptors[END_REF] which are spirocyclic analogs of the compounds MK disclosed by Ali et al. [START_REF] Ali | Novel N-Arylpyrazolo[3,2-c]-Based Ligands for the Glucocorticoid Receptor: Receptor Binding and in Vivo Activity[END_REF] (Scheme 1). The spirocyclic substructure was expected to increase the conformational pool while keeping the key hydroxyl pharmacophore in the same environment, as indicated by the good superposition of the reference MK molecule on the spirocyclic target (Figure 1). Scheme 1. Design of new potential spiro-corticoids Earlier examples of spirocyclic corticoids did not imply a modification of the core scaffold but consisted in the replacement of a carbonyl group by a spirolactone aiming at producing potent aldosterone antagonists devoid of undesired endocrinological side effects. These derivatives were highly potent binders for the MR, AR and PR 3 but they bound weakly to the GR. 4 We have recently reported a detailed study of a practical synthetic route to this new class of spirocyclic ligands. The observation of an IC50 of 27 nM for a representative example in this series prompted us to prepare more examples and study their biological activities in more details. [START_REF] Badarau | Design and Synthesis of Spirocyclic Ligands of Glucocorticoid Receptors[END_REF]

Results and discussion

Synthesis of the target alcohols

We had already optimized the synthetic route for the 5-and 6-membered analogues. [START_REF] Badarau | Design and Synthesis of Spirocyclic Ligands of Glucocorticoid Receptors[END_REF] We were also interested in testing the higher homologues which should show a higher conformational flexibility, possibly favouring a better interaction with the target protein. The synthetic route was similar to that reported for the lower homologues (Scheme 2). The commercially available cycloheptanone was formylated by treatment with sodium hydride and ethyl formate in dry diethyl ether. Base-catalyzed addition of 1 to methyl vinyl ketone followed by treatment of the Michael adduct 2 with a 1:1 mixture of morpholine and acetic acid delivered the desired spirocyclic intermediate 3. The selective protection of the saturated ketone was first conducted under the experimental conditions optimized for the 6-membered analogue: treatment of 3 with ethylene glycol in the presence of 1 eq. PTSA and of molecular sieves at room temperature. This gave a poor yield (≈ 10%) of the desired monoketal 4 while the lower homologue had led to a 72% yield in the same experimental conditions. A slightly better yield (25%) of 4 was obtained after treatment of 3 in a refluxing mixture of ethylene glycol/benzene (Dean-Stark) containing 0.05 eq. of PTSA.

Scheme 2. Synthesis of the spirodiketones 5. NaH, HCOOEt in Et2O, 48 h, rt ; (b) methyl vinyl ketone, DBU, TEA / THF, 12 h, rt, n = 3, 91%; (c) morpholine : AcOH (1:1) / DMSO, 24 h, rt, 63 %; (d) ethylene glycol, PTSA (0.05 eq.) / PhH / 12 h, reflux, 5c (25%);

The construction of the pyrazole ring was performed in two steps: formylation of the protected ketone 4 followed by treatement with p-fluorophenyl hydrazine to yield the tricyclic pyrazole 6 in 72% yield (Scheme 3). Hydrolysis of 6 delivered ketone 7c in a good 83% yield.

A comparative DFT conformational analysis (M06-2X/6-311+G(2d,p) level in chloroform) was performed on the homologous series of ketones 7 (n = 1, 2 and 3). Contrarily to the 6-membered derivative which has two low-energy conformations in a 1:1 ratio, [START_REF] Badarau | Design and Synthesis of Spirocyclic Ligands of Glucocorticoid Receptors[END_REF] for the more rigid five-membered ketone 7a (Figure 2) the major (73%) conformation resembles that of the six-membered ketone in the crystal. Surprisingly, the seven-membered ketone 7c (Figure 2) exhibited a preference (68%) for a twistboat conformation rather than the expected twist-chair. 5,6 The full details of the conformational analyses are described in the supplementary material. Ketone 7c and the lower homologs, ketones 7a and 7b synthesized as described earlier, [START_REF] Badarau | Design and Synthesis of Spirocyclic Ligands of Glucocorticoid Receptors[END_REF] were converted into the target alcohols by addition of phenylmagnesium bromide and benzyl magnesium bromide. The selection of a benzyl and a phenyl group resulted from our previous results, which showed that hydrophobic moieties led to favourable interactions with the GRs. For example, we have previously identified that a benzyl moiety triggered an IC50 of 27 nM in the case of the 6-membered analogue.

The addition reactions yielded mixtures of diastereoisomeric alcohols, which could be separated. (Scheme 4, Table 1). At this stage the formation of two diastereomerss was a bonus since they were easily separated. In our previous report [START_REF] Badarau | Design and Synthesis of Spirocyclic Ligands of Glucocorticoid Receptors[END_REF] we had assigned the preferred conformations of diastereoisomeric alcohols 8 and 9 by DFT calculations. The conformational assignment was also supported by an X-ray diffraction analysis conducted on only one diastereoisomer (R = 1-propynyl). Now we have been able to obtain suitable crystals for both diastereoisomers 8b and 9b (R = Ph). Our initial hypothesis concerning the conformation of all diastereoisomers was thus confirmed: an axial hydroxyl group and the bulkier alkyl or aryl substituent in equatorial position (Figure 3). Crystals of 7-membered analogues (8e and 9d) suitable for X-ray diffraction analyses could also be obtained. They also confirmed that both isomers adopt the conformation with an axial hydroxyl group as in the case of the related 5-and 6-membered alcohols (Figure 4). The spatial orientation of the hydroxyl group was expected to be an important factor for the interaction with the glucocorticoid receptor. It seemed thus important to confirm that the ligands conserve their conformations in physiological conditions. We therefore studied the conformational profile of the phenyl-substituted tertiary alcohols by DFT in water (M06-2X/6-311+G(2d,p) level). As expected, in both alcohols 8b and 9b, the favoured conformations were those with an equatorial phenyl group (higher A value: 3 vs 0.87 kcal/mol), This conformation was further stabilized as a result of a π-stacking interaction between the phenyl and pyrazole ring. For 8b in aqueous solution, 97% of the conformational population corresponded to conformers with an equatorial phenyl group but differing by the conformation around the N-Ar bond. One of these conformers was found in the crystal structure of 8b.

The situation is similar for the other diastereoisomer 9b, the conformers with an equatorial phenyl group representing 90% of the conformational population (Figure 5). The calculations also revealed some stabilization of these conformers by the π-stacking between the phenyl group and the pyrazole ring. The same factors were also responsible for the stabilization of the major (98%) conformation of the more rigid cyclopentanol 8a. Surprisingly, for the more flexible cycloheptanols 8d and 9d, the more stable twist-chair conformations with an equatorial phenyl group were also predominant (Figure 6).

Synthesis of the dehydrated analogues

We also synthesized a series of dehydrated analogs to investigate whether the hydroxyl group was essential for the interaction with the GRs. The mixture of diastereomeric alcohols 8b and 9b obtained from the reaction of ketone 7b with phenylmagnesium bromide was treated with triflic acid for 10 min in refluxing toluene to yield the expected elimination product 10a (Scheme 5). In order to offer a possible additional H-bonding in the binding site, we decided to introduce an OH group on the aromatic substituent. The reaction of the Grignard reagent derived from 4-bromo-anisole yielded the expected alcohol but the subsequent treatment with BBr3 aiming at cleaving the methyl group only yielded the dehydration product 10c. We thus decided to use a trialkylsilyl protecting group which could be easily cleaved under non acidic conditions. It was anticipated that a trimethylsilyl group would not stand the conditions of the first step. We thus selected the more stable triisopropylsilyl (TIPS) ethers. The halogen exchange step with sec-butyl lithium yielded the reactive lithiated nucleophiles. The corresponding diastereomeric mixture of adducts 8 and 9 was subsequently reacted with TBAF and subsequently with BBr3 to yield the elimination product 10b (Scheme 5).

Synthesis of α-substituted analogues

We expected the σ-position of the ketone 7b easy to modulate, in spite of the sterical hindrance imposed by the spirocyclic moiety. However, classic anionic conditions employing sodium hydride or LDA (1.2 to 3 eq.) and benzyl bromide as alkylating agents (1.2 to 3 eq.), at low temperature, conducted to full recovery of the starting material. De-aggregating the lithium enolates by coordinating the metal with N,N'-dimethylpropyleneurea (DMPU), a less toxic equivalent of the well-known coordinating reagent hexamethyl-phosphoramide (HMPA) delivered low yields of the alkylated products along with the recovery of the remaining starting material (Scheme 6). Interestingly, in the case of propargyl bromide, the best experimental conditions were LDA (1.2 eq) in THF at -78°C, while adding the DMPU additive showed only traces of the desired product. Surprisingly, dialklylated compounds 12 were also isolated. They were characterized by NMR, MS and also analysed by X-rays diffraction (Figure 7). The monovs poly-alkylation issue was discussed by several other groups. [7][8][9] It is generally accepted that the enolate reactivity relies on its stage of aggregation, less substituted enolate (thus more aggregated) should be less reactive. The π-π interaction between the enolate and the alkylating agent could also be an important factor in the polyalkylation process. 10 The structures of diastereoisomer 13a and 13b were confirmed by X-ray diffraction analyses. In both cases the preferred conformation was that with an axial hydroxyl group and an equatorial vicinal substituent (Figure 7).

Biological studies

A study of the binding affinity to the GR was performed using [ 3 H]dexamethasone as radioligand (see supporting information for details on binding measurements). It is important to underline that only pure diastereoisomeric alcohols were tested. In most cases their 3D structure was assigned by X-rays diffraction studies (vide supra). As shown in Table 2, a clear trend was observed: compounds having the hydroxyl group parallel to the axis of the central heterocyclic scaffold were all good GR binders with IC50 values in the nM range. Except for compound 8d, all other diastereoisomers, which have the hydroxyl moiety orthogonal to the axis of the main scaffold showed a low affinity for the glucocorticoid receptors. The best GR ligand identified in this study was compound 9b which showed a subnanomolar affinity (IC50 = 0.41 nM). This derivative was 30-fold more potent than prednisolone (IC50 = 13 nM), a classical reference GR ligand used in the same experiment. Increasing the size of the hydroxyl-carrying ring induced a 15-fold decrease of activity. However, the higher homologue 9d still remained a potent hGR ligand (IC50 = 6 nM). Unexpectedly, even if 50-fold less active than the corresponding tertiary alcohol 9b, the dehydrated compounds 10a,b still showed a binding affinity in the low nM range (10-20 nM), similar to prednisolone (IC50 = 13 nM) (Table 3). The corresponding methyl ether 10c showed a much weaker activity resulting perhaps from the replacement of H by a bulkier methyl group. An X-ray diffraction was performed on 10c. Superposition between the crystal structure of 10c and the crystal structures of the potent derivatives 9b and 9d shows that all these derivatives share the same aromatic/hydrophobic pharmacophore (Figure 8). In the case of compound 10c, the absence of a hydroxyl group, presumably responsible for a Hbonding within the binding site, decreases its potency 50-fold for the GRs. A subset of ligands was also evaluated for their binding to the progesterone receptors (PR). As shown in Table 4, these spirocycles were devoid of any affinity for this type of steroid receptors.

Conclusions

Spirocyclic glucocorticoids were designed, synthesized and evaluated for their activity towards hGR. Pure diastereoisomers were tested for their binding to the hGR. This led to the identification of novel highly active glucocorticoid ligands. The best ligand identified so far in this series, compound 9b, had an excellent IC50 of 0.4 nM, 30-fold more potent than prednisolone, the classical reference GR ligand used in the same experiment (IC50 = 13 nM). The corresponding diastereoisomer 8b was devoid of activity for hGR (IC50 = 1012 nM). Preliminary results also showed that this new class of hGR binders were selective over the PR. The dehydrated analogue (10a) of the most potent compound identified so far (9b) showed a 50-fold lower affinity for the GRs (10b, IC50 = 20 nM). This seems to indicate the importance of the H-bond generated by the hydroxyl moiety present on the ligand. However, as the affinity is still in the nM range, this could be an opportunity for the medicinal chemist to keep essential features for a nM potency, while removing the chemical complexity represented by an additional asymmetric center.

We believe that these preliminary synthetic and biological studies open a new window for the discovery of new potent and selective glucocorticoid ligands.

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Experimental

General experimental

The infrared (IR) spectra were recorded on a Bruker IRFT IFS 55 spectrometer on zinc selenide plates, unless stated otherwise.

1 H-NMR and 13 C-NMR were recorded with Bruker Avance II 300 (at 300.18 MHz and 75.48 MHz, respectively) in CDCl3-d1 and DMSO-d6, using tetramethylsilane (TMS) as an internal standard. Chemical shifts are reported in parts per million (ppm) and the coupling constants are reported in units of Hertz [Hz]. Multiplicities were abbreviated as follows: singlet (s), doublet (d), triplet (t), quartet (q). Low resolution mass spectra (MS) / High resolution mass spectra (HRMS) were recorded with a ThermoElectron LCQ Advantage and a ThermoElectron Exactive spectrometer, respectively.

Melting points were recorded using a Buchi B-540 apparatus and are uncorrected. Flash chromatography was performed on Merck 40-70 nM (230-400 mesh) silica gel under air pressure. Thin-layer chromatography (TLC) was carried out on Merck silica gel 60 F254 precoated plates. Visualization was made with ultraviolet light (λ = 254/365 nm) or using an aqueous potassium permanganate staining solution.

Experimental details and characterization data 4.2.1 2-oxo-1-(3-oxobutyl)cycloheptanecarbaldehyde (2)

Sodium hydride (60% dispersion in mineral oil, 10.5 g) was added under nitrogen to dry diethyl ether (400 ml). Subsequently, ethyl formate (11.68 g, 13 ml, 1.2 eq.) and then cyclohexanone (15 g, 12.6 ml, 1 eq.) were added and the mixture was stirred for 48 h at room temperature. The orange mixture was slowly poured on ice-cold water (200 ml), the aqueous layer separated and washed with diethyl ether (2 x 200 ml) before being acidified with hydrogen chloride (aq. soln., 6N). The organic materials were extracted with diethyl ether (3 x 150 ml), the combined organic layers were dried over magnesium sulfate and the solvent was evaporated to conduct to the desired α-formylated cyclohexanone 1 as a light-yellow liquid subsequently used without further purification. The crude compound 1 (18 g, 1 eq.) was dissolved in THF (130 ml), then DBU (2 ml, 0.1 eq.), TEA (2.69 ml, 0.1 eq.) and methyl acrylate (16.1 ml, 1.5 eq.) were consecutively added to the mixture and stirred for 24 h at room temperature under inert atmosphere. After the complete consumption of the starting material, the solvent was evaporated, the residue diluted with DCM (200 ml) and washed with ammonium chloride (sat. soln., 2 x 100 ml) and sodium chloride (sat. soln., 1 x 100 ml). The organic solvent was dried over magnesium sulfate and evaporated under vacuum. The crude product was purified by flash-chromatography (eluent: PE / EtOAc 7/3) to give the desired addition derivative 2 as a colorless oil (24.7 g

spiro[5.6]dodec-4-ene-3,12-dione (3)

Under inert atmosphere, morpholine (9.36 ml, 1 eq.) and acetic acid (6.2 ml, 1 eq.) were added to a solution of the previously obtained Michel adduct 2 (22.6 g, 1 eq.) in DMSO (150 ml). The mixture was vigorously stirred overnight at room temperature, then, after the complete consumption of the starting material, slowly added to sodium carbonate (satd. soln., 200 ml). The organics were extracted with ethyl acetate (4 x 100 ml), then washed with ammonium chloride (150 ml) before drying over magnesium sulfate. The solvent was evaporated under reduced pressure and the crude product was purified by flash chromatography (PE / EtOAc 7/3) to give the desired annulated compound 3 (13 g). C12H16O2; light-yellow oil; yield: 63 %; M = 192.25 g / mol; IR (ZnSe) ν = 2937, 1696, 1682, 1455, 1346, 1152, 1138 cm - 1 ; 1 H NMR (300 MHz, CDCl3) δ 1.40-1.91 (m, 9H), 2.16-2.50 (m, 4H), 2.59-2.68 (m, 1H), 5.91-5.99 (m, 1H), 6.67-6.74 (m, 1H); 13 C NMR (75 MHz, CDCl3) δ 24.3 (CH2), 26.5 (CH2), 30.1 (CH2), 31.6 (CH2), 33.9 (CH2), 36.4 (CH2), 40.1 (CH2), 53.3 (Cq), 129.3 (CH), 150.9 (CH), 198.4 (Cq), 212.3 (Cq); HRMS: calcd. for C12H17O2 193.12231, found 193.12250 (1.003 ppm).

1,4

-dioxadispiro[4.0.5-6.5-5]hexadec-7-en-9-one (4) Under nitrogen atmosphere, to a solution of the spiro-derivative 3 (1 g, 1 eq.) in benzene were successively added ethylene glycol (3 ml, 10 eq.) and PTSA (0.05 g, 0.05 eq.). The mixture was refluxed for 12 h with water distillation using a Dean-Stark trap. The solvent was removed under vacuum, the residue was dissolved in sodium bicarbonate (aq. satd. soln., 30 ml), then the organics extracted with DCM (3 x 30 ml). The combined organic layers were washed with brine (100 ml), dried over magnesium sulfate and the solvent was evaporated under vacuum. The desired ketal was isolated by flashchromatography (PE/EtOAc 15/5) in mixture with an unidentified product (1/2 ratio as showed by H-NMR).

9-oxo-1,4

-dioxadispiro[4.0.5 6 .5 5 ]hexadec-10-ene-8-carbaldehyde (5) Under inert atmosphere, the previously obtained ketal 4 (1.4 g, 1 eq.) was dissolved in dry toluene (50 ml) and the mixture cooled to -40°C. Ethyl formate (1.9 ml, 4 eq.) and then sodium hydride (60% dispersion in oil, 0.94 g, 4 eq.) were subsequently added before slowly adding methanol (0.1 ml, 0.5 eq.) at low temperature over 15 minutes. The mixture was stirred for 30 min at low temperature, then left to slowly come back to room temperature and stirred for two additional hours. After the complete consumption of the starting material, the mixture was slowly poured onto ice-cold water (100 ml), the organic layer was separated and washed two additional times with water (2 x 75 ml). The combined aqueous layers were subsequently acidified to with hydrogen chloride (aq. soln., 6N) before extracting the organic materials with diethyl ether (3 x 100 ml). The combined organic layers were dried over magnesium sulfate and the solvent evaporated to give a light yellow oil (turning brown on standing) and was subsequently used without further purification. C15H20O4; Light yellow oil; Yield: 89 %; M = 264.32 g/mol; IR (ZnSe) ν = 2933, 1641, 1576, 1444, 1415, 1192, 1043 cm - 1 ; 1 H NMR (300 MHz, CDCl3) δ 1.43-1.71 (m, 8H), 1.75-1.85 (m, 2H), 2.32 (dd, J = 14.7 Hz, J = 1.4 Hz, 1H), 2.68 (dd, J = 14.0 Hz, J = 0.8 Hz, 1H), 3.87-3.99 (m, 4H), 5.98 (d, J = 10.3 Hz, 1H), 6.79 (dd, J = 10.3 Hz, J = 1.7 Hz, 1H), 7.42 (s, 1H), 13.67 (ls, 1H); 13 C NMR (75 MHz, CDCl3) δ 21.2 (CH2), 22.3 (CH2), 27.5 (CH2), 29.0 (CH2), 32.6 (CH2), 32.7 (CH2), 48.3 (Cq), 64.5 (CH2), 64.9 (CH2), 106.7 (Cq), 113.6 (Cq), 126.3 (CH), 153.7 (CH), 166.7 (CH), 189.4 (Cq); HRMS: calcd. for C15H27O4 265.14344, found 265.14418 (2.807 ppm).

1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cycloheptane]-1'-one-ethylene-acetal (6)

At room temperature, sodium acetate (0.190 g, 1.2 eq.) and 4-F-phenylhydrazine (0.37 g, 1.2 eq.) were added to a solution of the previously-obtained α-formylation product 5 (0. 5 g, 1 eq.) in acetic acid (10 ml). The mixture was stirred until the complete consumption of the starting material (approx. 1.5 h), then the reaction was quenched by addition of sodium carbonate (sat. soln. 100 ml). The aqueous mixture was washed with ethyl acetate (3 x 100 ml), the combined organic layers were dried over magnesium sulfate and the solvent evaporated under reduced pressure. The crude product was subsequently purified by flash-chromatography (eluent: PE / EtOAc 8/2) to conduct to the desired indazole derivative 6. C21H23FN2O2; light brown viscous oil; yield: 72 %; M = 354.42 g / mol; IR (ZnSe) ν = 2922, 1517, 1225, 1076 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.45-1.72 (m, 8H), 1.82-1.87 (m, 2H), 2.74 (d, J = 15.9 Hz, 1H), 2.92 (d, J = 15.9 Hz, 1H), 3.95-4.02 (m, 4H), 6.01 (d, J = 10.2 Hz, 1H), 6.39 (d, J = 10.2, 1H), 7.11-7.16 (m, 2H), 7.42-7.46 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 21.1 (CH2), 21.6 (CH2), 25.7 (CH2), 27.4 (CH2), 32.0 (CH2), 32.8 (CH2), 49.0 (Cq), 64.6 (CH2), 64.9 (CH2), 113.6 (CH), 114.3 (Cq), 115.5 (Cq), 116.0 (CH), 116.3 (CH), 125.2 (CH), 125.3 (CH), 136.6 (CH), 136.7 (Cq), 138.7 (CH), 159.9 (Cq), 163.2 (Cq); HRMS: calcd. for C21H24FN2O2 355.18163, found 355.18277 (3.202 ppm).

1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cycloheptane]-1'-one (7c)

The ketal 6 was deprotected by adding hydrogen chloride (aq. soln. 6N, 6 ml) to a solution of indazole 6 (0.4 g, 1 eq.) in THF (12 ml). The reaction was conducted at 65°C for 1.5 h, and then quenched by pouring onto a solution of sodium bicarbonate (10% aq. soln. 100 ml). The aqueous solution was washed with ethyl acetate (3 x 100 ml), then the combined organic layers were dried over magnesium sulfate and the solvent evaporated under vacuum. The desired ketone 7c was isolated as a light yellow oil after purification on flash chromatography (eluent PE / EtOAc 8/2). C19H19FN2O; Light yellow visc. oil; Yield: 83 %; M = 310.37 g / mol; IR (ZnSe) ν = 2930, 1703, 1516, 1222, 841 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.40-1.71 (m, 6H), 1.80-1.84 (m, 2H), 2.52-2.68 (m, 3H), 3.23 (d, J = 16.1 Hz, 1H), 5.87 (d, J = 10.0 Hz, 1H), 6.45 (dd, J = 10.0 Hz, J = 0.5 Hz, 1H), 7.07-7.13 (m, 2H), 7.37-7.43 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 24.3 (CH2), 26.6 (CH2), 27.7 (CH2), 30.2 (CH2), 35.4 (CH2), 39.9 (CH2), 54.3 (Cq), 114.8 (Cq), 115.9 (CH), 115.9 (CH), 116.2 (CH), 125.0 (CH), 125.2 (CH), 132.8 (CH), 135.6 (Cq), 136.1 (Cq), 138.1 (CH), 159.9 (Cq), 163.1 (Cq), 213.2 (Cq); HRMS: calcd. for C13H20FN2O 311.15542, found 311.15486 (-1.793 ppm).

1-(4-fluorophenyl)-1'-phenyl-spiro[4H-indazole-5,2'-cyclohexane]-1'-ol

Under inert atmosphere and at 0°C, the previously obtained ketone 7b (0.3 g, 1 eq.) was dissolved in dry THF (10 ml). Phenylmagnesium bromide (1M soln. in THF, 1.02 ml, 3 eq.) was slowly added at low temperature and the mixture was stirred 2 additional hours at 0°C until the complete conversion of the starting material. The reaction was quenched by addition of ammonium chloride (satd. soln. 30 ml) and the organic-soluble materials extracted with ethyl acetate (3 x 50 ml). The organic phase was dried over magnesium sulfate and concentrated under vacuum. The solid residue was triturated with diethyl ether to give a white powder which was further crystallized in acetonitrile to conduct to the (5R, 8R)-diastereoisomer (0.180 g, 48%), as confirmed by X-rays. The recovered filtrate was subsequently evaporated to conduct to a solid residue, further purified by flash-chromatography. The (5R, 8S)-diastereoisomer (as showed by crystallization in ethanol) was eluted first (PE/EtOAc 9/1, 0.103 g, 27%), then the remaining (5R, 8R)-diastereoisomer (0.031 g, 8%).

1-(4-fluorophenyl)-1'-phenyl-spiro[4H-indazole-5,2'-cyclohexane]-1'-ol (8b)

(5R, 8R)-diastereoisomer: C24H23FN2O; colourless crystals; Mp = 234-236 °C; Yield: 56 %; M = 374.45 g / mol; IR (ZnSe) ν = 2922, 1513, 837 cm -1 ; 1 H NMR (300 MHz, DMSO) δ 1.49-1.66 (m, 5H), 1.79-1.88 (m, 2H), 2.46 (d, J = 16.4 Hz, 1H), 2.83 (d, J = 16.4 Hz, 1H), 4.85 (s, 1H), 6.28 (d, J = 10.3 Hz, 1H), 6.46 (d, J = 10.4 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 7.17

1-(4-fluorophenyl)-1'-phenyl-spiro[4H-indazole-5,2'-cyclohexane]-1'-ol (9b)

(5R, 8S)-diastereoisomer: C24H23FN2O; colourless crystals; Mp = 173-175 °C; Yield: 27 %; M = 374.45 g / mol; IR (ZnSe) ν = 3369, 2936, 1513, 1265, cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.56-1.77 (m, 5H), 1.86-1.97 (m, 2H), 2.56 (dd, J = 4.1 Hz, J = 9.5 Hz, 1H), 2.64 (d, J = 15.9 Hz, 1H), 2.90 (d, J = 15.9 Hz, 1H), 6.25 (d, J = 10.2 Hz, 1H), 6.41 (d, J = 10.3 Hz, 1H), 7.07 -7.18 (m, 3H), 7.23-7.33 (m, 5H), 7.46 -7.49 (m, 2H); 13

1-(4-fluorophenyl)-1'-phenyl-spiro[4H-indazole-5,2'-cycloheptane]-1'-ol

Phenylmagnesium bromide (1M in THF, 0.97 ml, 0.97 mmol, 3 eq.) was slowly added at 0°C to a solution of ketone 7b (0.1 g, 0.32 mmol, 1 eq.) in dry THF (10 ml). After 2 h at low temperature, the reaction was quenched by addition of water (20 ml), and the organics extracted with ethyl acetate (3 x 20 ml). The recovered organic layers were dried over magnesium sulfate, concentrated under vacuum and further purified by flash chromatography (PE / EtOAc 9/1). The (5R, 8S)diastereoisomer was eluted firstly (38%), then the (5R, 8R)-diastereoisomer (10%), followed by the recovered starting material (20%).

1-(4-fluorophenyl)-1'-phenyl-spiro[4H-indazole-5,2'-cycloheptane]-

1-(4-fluorophenyl)-1'-phenyl-spiro[4H-indazole-5,2'-cycloheptane]-1'-ol (9d)

(5R, 8S)-diastereoisomer: C25H25FN2O; White solid; Mp = 145-147 °C; Yield: 38 %; M = 388.48 g / mol; IR (ZnSe) ν = 3331, 2922, 1521, 1229, 846 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.50-2.04 (m, 9H), 2.12-2.22 (m, 1H), 2.55-2.65 (m, 1H), 2.82 (q, J = 15.9 Hz, 2H), 6.14 (dd, J = 10.3 Hz, J = 0.6 Hz, 1H), 6.31 (d, J = 10.3 Hz, 1H), 7.06-7.21 (m, 6H), 7.25-7.30 (m, 2H), 7.44-7.47 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 20.7 (CH2), 22.0 (CH2), 26.1 (CH2), 27.1 (CH2), 33.4 (CH2), 38.9 (CH2), 48.3 (Cq), 79.4 (Cq), 113.2 (CH), 115.4 (Cq), 115.9 (CH), 116.2 (CH), 125.1 (CH), 125.2 (CH), 126.9 (CH), 127.0 (CH), 127.2 (CH), 136.5 (Cq), 137.8 (CH), 138.9 (CH), 148.0 (Cq), 159.9 (Cq), 163.1 (Cq); HRMS: calcd. for C25H28FN2O 389.20237, found 389.20341 (2.677 ppm).

1'-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cycloheptane]-1'-ol

The title compound was obtained following the same experimental method employed for the synthesis of compound 8d-9d, after 2h of reaction at 0°C. Purification by flash-chromatography (eluent: 9/1 PE/EtOAc) delivered the (5R, 8R)-diastereoisomer followed by the (5R, 8S)-diastereoisomer. The secondly eluted diastereoisomer was crystallized in ACN and submitted to X-Rays diffractions in order to confirm the proposed stereochemistry.

1'-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cycloheptane

1'-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cycloheptane

1'-(4-fluorophenyl)-2-phenyl-1',4'-dihydrospiro-[cyclohex-2-ene-1,5'-indazole] (10a)

Dry toluene (10 ml) was added to a crude mixture of diasteroisomers 8b-9b (obtained from 0.5 g of ketone 7b as described above). Triflic anhydride (0.7 ml, 4.2 mmol, 2.5 eq.) was subsequently added to the solution and the mixture was heated at 100°C under stirring for 10 min. After cooling to room temperature, the mixture was diluted with ethyl acetate (25 ml) and poured onto sodium bicarbonate (satd. soln., 30 ml). Theo organic materials were extracted with ethyl acetate (3 x 50 ml), dried over magnesium sulfate and concentrated under vacuum. The crude product was purified by flash chromatography (PE / EtOAc 95 / 5) to conduct to the desired elimination product (15%) along with an unidentified product. C24H21FN2; light yellow oil; Yield: 15 %; M = 356.43 g / mol; IR (ZnSe) ν = 2932, 1515, 1224 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.60-1.70 (m, 3H), 1.80-1.88 (m, 1H), 2.12-2.22 (m, 2H), 2.49 (d, J = 16.3 Hz, 1H), 2.97 (d, J = 16.3 Hz, 1H), 5.85 (t, J = 3.9 Hz, 1H), 6.02 (d, J = 10.0 Hz, 1H), 6.32 (d, J = 10.0 Hz, 1H), 7.03-7.24 (m, 8H), 7.30-7.36 (m, 2H); 13

4-[1-(4-fluorophenyl)spiro[4H-indazole-5,6'-cyclohexene]-1'-yl]phenol (10b)

Under inert atmosphere, (4-bromophenoxy)-triisopropylsilane (0.56 g, 1.69 mmol, 2 eq.) was dissolved in dry THF (15 ml) and the mixture cooled at -78°C before slowly adding a solution of n-butyl lithium (1.5 M in THF, 1.25 ml, 1.85 mmol, 2.2 eq.). After 30 min of stirring at -78°C, a solution of the ketone 7b (0.25 g, 0.84 mmol, 1 eq.) in dry THF (5 ml) was added and the mixture was allowed to slowly come back to room temperature overnight. The reaction was quenched by addition of water (100 ml) and the organic materials were extracted with ethyl acetate (3 x 100 ml) before removing the solvent under vacuum. The crude product was purified by flash-chromatography (PE / EtOAc 9/1 to 8/2) to deliver the desired addition product. Starting material was also recovered during the purification (30%). C33H43FN2O2Si; Colorless vis. oil; Yield: 57 %; M = 546.79 g / mol; IR (ZnSe) ν = 3380, 2948, 1601, 1517, 1269, 835 cm - 1 ; 1 H NMR (300 MHz, CDCl3) δ 1.04 (d, J = 6.8 Hz, 18H), 1.12-1.29 (m, 3H), 1.6-1.83 (m, 8H), 2.41-2.52 (m, 1H), 2.51 (d, J = 16.3 Hz, 1H), 3.10 (d, J = 16.3 Hz, 1H), 6.24 (q, J = 10.3 Hz, 2H), 6.69 (d, J = 8.7 Hz, 2H), 7.09 (t, J = 8.6 Hz, 2H), 7.22 (s, 1H), 7.27-7.32 (m, 4H); 13 C NMR (75 MHz, CDCl3) δ 12.7 (3CH), 18.0 (6CH3), 21.5 (CH2), 21.8 (CH2), 27.6 (CH2), 34.9 (CH2), 35.8 (CH2), 44.9 (Cq), 76.6 (Cq), 115.5 (CH), 115.8 (CH), 116.1 (CH), 116.5 (Cq), 118.5 (2CH), 125.1 (CH), 125.2 (CH), 127.8 (2CH), 135.2 (CH), 135.9 (Cq), 138.0 (CH), 138.3 (Cq), 154.8 (Cq), 159.9 (Cq), 163.1 (Cq); HRMS: calcd. for C33H44FN2O2Si 547.31506, found 547.31572 (1.206 ppm).

The previously obtained alcohol (0.09 g, 0.0.164 mmol, 1 eq.) was dissolved in THF (3 ml) and the solution was cooled to 0°C in an ice-bath before slowly adding a solution of TBAF (1M in DCM, 0.5 ml, 0.5 mmol, 3 eq.). After 5 min the TLC indicated the complete consumption of the starting material. The reaction was quenched by addition of sodium bicarbonate (20% aq. soln. 20 ml) and the organics extracted by EtOAc (3 x 20 ml). The combined organic layers were dried over magnesium sulphate and further concentrated under vacuum to deliver a crude product. This was subsequently purified by flash-chromatography (DCM / MeOH 9/1), to conduct to the desired deprotected phenol. The isolated fraction was further triturated in diethyl ether to deliver a white powder, subsequently filtered on a funnel and dried under vacuum. The previously obtained phenol (0.1 g, 0.25 mmol, 1 eq.) was dissolved in dry DCM (2ml) then the mixture was cooled to -78°C before slowly adding boron tribromide (1M in DCM, 0.3 ml, 0.3 mmol, 1.2 eq.). The reaction was continued at low temperature for 1 h, and then quenched with sodium bicarbonate (20% aq. soln., 10 ml). The organic materials were extracted with DCM (3 x 10 ml), the combined organic layers were dried over magnesium sulfate and the crude product purified by flash-chromatography (PE / EtOAc 15/5) to deliver the dehydrated product 10b. C24H21FN2O; White solid; Mp = 77-79 °C; Yield: 83 %; M = 372.43 g / mol; IR (ZnSe) ν = 3320, 2933, 1609, 1517, 1265, 1229 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.68-1.91 (m, 4H), 2.20-2.24 (m, 2H), 2.57 (d, J = 16.3 Hz, 1H), 3.05 (d, J = 16.3 Hz, 1H), 5.85 (t, J = 3.9 Hz, 1H), 6.04 (d, J = 10.0 Hz, 1H), 6.35 (dd, J = 10.0 Hz, J = 0.5 Hz, 1H), 6.62-6.65 (m, 2H), 6.83 (s, 1H), 7.07-7.14 (m, 4H), 7.33 (s, 1H), 7.36-7.41 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 18.3 (CH2), 26.1 (CH2), 30.7 (CH2), 36.0 (CH2), 41.0 (Cq), 113.6 (CH), 114.7 (2CH), 115.5 (Cq), 116.1 (CH), 116.4 (CH), 125.5 (CH), 125.6 (CH), 127.7 (CH), 129.9 (2CH), 134.7 (Cq), 136.5 (Cq), 138.3 (CH), 141.3 (CH), 143.3 (Cq), 154.9 (Cq), 160.1 (Cq), 163.4 (Cq); HRMS: calcd. for C24H22FN2O 373.171068, found 373.16948 (-4.26 ppm).

1-(4-fluorophenyl)-1'-(4-methoxyphenyl)spiro[4H-indazole-5,6'-cyclohexene] (10c)

Under inert atmosphere, iPrMgCl (2M in THF, 0.84 ml, 3.55 mmol, 2.1 eq.) was added at -20°C to a solution of 4-iodoanisole (0.4 g, 1.69 mmol, 1 eq.) in dry THF (10 ml). After 1 h of stirring at -20°C, a solution of ketone precursor 7b (0.5 g, 1.69 mmol) in dry THF (3 ml) was slowly added. The temperature was allowed to come back to room temperature and the mixture stirred overnight. The reaction was quenched by addition of sodium bicarbonate (20% aq. soln., 50 ml), then extracted with ethyl acetate (3 x 50 ml). The combined organic layers were dried over magnesium sulfate, the solvent evaporated under reduced pressure. The crude product was purified by flash chromatography (Cy / EtOAc 8/2) to deliver the addition product (25%) along with recovery of the starting material (50%). The obtained alcohol (0.1 g, 0.25 mmol, 1 eq.) was subsequently dissolved in dry DCM (2ml), then the mixture was cooled to -78°C before slowly adding boron tribromide (1M in DCM, 0.3 ml, 0.3 mmol, 1.2 eq.). The reaction was continued at low temperature for 1 h, and then quenched with sodium bicarbonate (20% aq. soln., 10 ml). The organic materials were extracted with DCM (3 x 10 ml), the combined organic layers dried over magnesium sulfate and the crude product purified by flash-chromatography (PE / EtOAc 15/5) to deliver the dehydrated product 10c (85%). C25H23FN2O; White solid; Mp = 126-128 °C; Yield: 20 % (over two steps); M = 386.46 g / mol; IR (ZnSe) ν = 2933, 1609, 1517, 1284, 1247, 838 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.69-1.77 (m, 3H), 1.85-1.93 (m, 1H), 2.21-2.26 (m, 2H), 2.58 (d, J = 16.3 Hz, 1H), 3.07 (d, J = 16.3 Hz, 1H), 3.76 (s, 3H), 5.90 (t, J = 3.9 Hz, 1H), 6.06 (d, J = 10.0 Hz, 1H), 6.41 (d, J = 10.0 Hz, 1H), 6.77 (d, J = 8.8 Hz, 2H), 7.12-7.18 (m, 1H), 7.22 (d, J = 8.8 Hz, 2H), 7.33 (s, 1H), 7.41-7.46 (m, 2H); 13

6'-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cyclo-hexane]-1'-one (11a)

Under nitrogen atmosphere and at low temperature (-78°C), LHMDS (1M soln, in THF, 0.7 ml, 0.67 mmol, 1 eq.) and then DMPU (0.1 ml, 0.84 mmol, 1.2 eq.) were subsequently added to a solution of the starting ketone 7b (0.2 g, 0.67 mmol, 1 eq.) in dry THF (5 ml). The mixture was stirred at low temperature for 1 h, prior to addition of benzyl bromide (0.08 ml, 0.67 mmol, 1 eq.). The reaction was allowed to slowly come back to room temperature over 12 h under stirring, then quenched by addition of water (10 ml). The organic materials were extracted with ethyl acetate (3 x 10 ml) and subsequently concentrated under vacuum. The crude product was purified by flash-chromatography (PE / AcOEt 9 / 1). The dibenzylated product was eluted first followed by the mono-benzylated derivative as a mixture of 2/1 diastereoisomers. (

6',6'-dibenzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cyclohexane]-1'-one (12a)

The title compound was obtained during the synthesis of the compound 11a as indicated above.

6'-benzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cyclohexane]-1'-ol (13a)

Under inert atmosphere and at -78°C, DIBAL-H (1M soln. in cyclohexane, 0.23 ml, 0.23 mmol, 3 eq.) was slowly added to a solution of the starting ketone 11a (0.03 g, 0.08 mmol, 1 eq.) in dry THF (3 ml). The mixture was stirred at low temperature for 2 h, then, after the complete consumption of the starting material, the reaction was quenched with water (10 ml). The organics were extracted with ethyl acetate (3 x 10 ml) and the combined organic layers were concentrated under vacuum. The crude product was purified by flash-chromatography (PE / EtOAc 7/3) to give the desired alcohol.

6',6'-dibenzyl-1-(4-fluorophenyl)spiro[4H-indazole-5,2'-cyclohexane]-1'-ol (14a)

Under inert atmosphere and at 0°C, LiAlH4 (0.01 g, 0.23 mmol, 1.2 eq.) was added in one portion to a mixture of the starting ketone 12a (0.09 g, 0.19 mmol, 1 eq.) in dry THF (3 ml). The mixture was stirred for 2 h at 0°C, and then the reaction was quenched by addition of water (1 ml). After dilution with ethyl acetate (10 ml), the precipitated salts were filtrated on a celite pad. Water was subsequently added (10 ml) and the organics were extracted with ethyl acetate (3 x 10 ml). The combined organic layers were dried over magnesium sulfate and concentrated under vacuum. The crude product was purified by flashchromatography (PE / EtOAc 8/2) to conduct to the desired alcohol. C32H31FN2O; White solid; Mp = 124-126 °C; Yield: 56 %; M = 478.60 g / mol; IR (ZnSe) ν = 3390, 2933, 1517, 1225, 1072 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 0.88 (td, J = 13.3 Hz, J = 4.0 Hz, 1H), 1.14 (td, J = 13.7 Hz, J = 3.1 Hz, 1H), 1.36-1.43 (m, 1H), 1.48 (d, J = 3.5 Hz, 1H), 1.64 (dd, J = 14.0 Hz, J = 2.3 Hz, 1H), 1.81 (dd, J = 13.7 Hz, J = 2.4 Hz, 1H), 1.88-2.05 (m, 1H), 2.05 (d, J = 13.2 Hz, 1H), 2.98 -3.31 (m, 4H), 3.38 (d, J = 3.2 Hz, 1H), 5.34 (d, J = 10.0 Hz, 1H), 6.42 (d, J = 10.0 Hz, 1H), 7.10-7.32 (m, 12H), 7.40, 7.46 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 16.4 (CH2), 23.0 (CH2), 29.9 (CH2), 33.0 (CH2), 37.4 (CH2), 42.9 (Cq), 43.1 (CH2), 44.6 (Cq), 77.9 (CH), 115.1 (Cq), 116.1 (CH), 116.4 (CH), 116.4 (CH), 125.3 (CH), 125.4 (CH), 126.2 (CH), 126.2 (CH), 127.9 (2CH), 128.1 (2CH), 131.4 (2CH), 131.4 (2CH), 135.5 (Cq), 138.4 (Cq), 138.7 (Cq), 139.0 (CH), 141.3 (CH); HRMS: calcd. for C32H32FN2O 479.249318, found 479.24958 (0.55 ppm).

1-(4-fluorophenyl)-6'-prop-2-ynyl-spiro[4H-indazole-5,2'-cyclohexane]-1'-one (11b)

Under inert atmosphere, LDA (2M soln. in THF, 0.44 ml, 0.88 mmol, 1.3 eq.) was slowly added at -78°C to a solution of ketone 7b (0.2 g, 0.67 mmol, 1 eq.) in dry THF (6 ml). After 1h of reaction at low temperature, propargyl bromide (80% soln. in toluene, 0.11 ml, 1.01 mmol, 1.5 eq.) was added and the reaction allowed to slowly come back to room temperature over 12 h under stirring. The reaction was quenched by addition of water (10 ml) and the organic materials extracted with ethyl acetate (2 x 20 ml). The combined organic layers were dried over magnesium sulfate and concentrated under vacuum to give a crude product further purified by flash-chromatography (PE / EtOAc 9/1). C21H19FN2O; Light yellow visq oil; Yield: 18 %; M = 336.40 g / mol; IR (ZnSe) ν = 2937, 1711, 1517, 1225, 846 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.35-1.66 (m, 3H), 1.73-1.82 (m, 1H), 1.94-2.06 (m, 2H), 2.15-2.25 (m, 2H), 2.45 (d, J = 16.2 Hz, 1H), 2.45-2.62 (m, 2H), 2.91-3.02 (m, 1H), 3.42 (d, J = 16.2 Hz, 1H), 6.12 (d, J = 10.0 Hz, 1H), 6.52 (d, J = 10.0 Hz, 1H), 7.11-7.17 (m, 2H), 7.40-7.46 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 19.2 (CH2), 21.3 (CH2), 27.9 (CH2), 34.5 (CH2), 40.9 (CH2), 45.9 (CH), 51.9 (Cq), 69.7 (CH), 82.7 (Cq), 116.1 (CH), 116.4 (CH), 116.8 (Cq), 117.5 (CH), 125.1 (CH), 125.3 (CH), 130.0 (CH), 135.5 (Cq), 138.0 (CH), 160.0 (Cq), 163.3 (Cq); HRMS: calcd. for C21H19FN2NaO 357.13736, found 357.13660 (-2.14 ppm).

1-(4-fluorophenyl)-6'-prop-2-ynyl-spiro[4H-indazole-5,2'-cyclohexane]-1'-ol (13b)

The title compound was obtained following the same experimental protocol used for the synthesis of compound 13a starting from 0.05 g of the corresponding ketone.

Figure 1 .

 1 Figure 1. Example of superposition between the reference ligand MK (green) and a designed spirocyclic derivative (violet, n = 2, R = Bn)

Scheme 3 :

 3 Scheme 3 : Construction of the pyrazole ring. (a) : NaH, HCOOEt, MeOH / toluene / 48 h, rt; (b) 4-F-Ph-NHNH2, AcONa / AcOH / 24 h, rt, (over two steps, 6: 64%); (c) HCl / THF / 1.5 h, 65°C, (7c: 83%).

Figure 2 :

 2 Figure 2 : Conformational analysis of the 5-and 7-membered ketones 10a and 10c

Scheme 4 .

 4 Scheme 4. Reaction of organometallics with ketones 7a-c. (a) RMgBr / THF, 12h, 0°C to r.t. (seeTable 1 for details)

Figure 3 .Figure 4 .

 34 Figure 3. Crystal structures of tertiary alcohols 8b and 9b

Figure 5 .Figure 6 .

 56 Figure 5. Representative conformers for the alcohols 8b (97% population) and 9b (90 % population)

Scheme 6 .

 6 Scheme 6. Synthesis of α-substituted analogues 13 and 14. (a) (i) LHMDS (1 eq.), DMPU (1.2 eq.), BnBr (1.2 eq.) / THF, 12 h, -78°C to r.t. (R = Bn, 11a (12%, isolated as 2/1 mix of diastereoisomers), 12a (14%); (ii) LDA (1.2eq.), propargyl bromide (1.5 eq.) / THF, 12 h, -78°C to r.t. (R = propargyle, 11b (18%), 12b (10%); (b) DIBAL-H (3 eq.) / THF, 3 h then recrystallization in ACN (R = Bn, 13a (55%); R = propargyl, 13b (18%)); (c) LAH (1.2 eq.) / THF, 2 h, 0°C (R = Bn, 56%).

Figure 7 .

 7 Figure 7. Crystal structures of dibenzylated product 12a and of the isolated reduction products 13a and 13b. H-atoms were omitted for clarity.

Table 3 .Figure 8

 38 Figure 8 Crystal structure of compound 10c and superposition of crystal structures of 9b (green), 9d (blue) and 10c (yellow)

  C14H20O3; light-yellow visc. oil; yield: 25%; M = 236,14g / mol; IR (ZnSe) ν = 2933, 1682, 1455, 1225, 1050 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.42-1.79 (m, 10H), 1.84-1.98 (m, 1H), 2.05-2.19 (m, 1H), 2.26-2.52 (m, 2H), 3.76-3.91 (m, 4H), 5.84 (d, J = 10.4 Hz, 1H), 6.83 (dd, J = 10.4 Hz, J = 1.4 Hz, 1H); 13 C NMR (75 MHz, CDCl3) δ 21.1 (CH2), 22.5 (CH2), 27.1 (CH2), 27.6 (CH2), 32.6 (CH2), 33.3 (CH2), 34.0 (CH2), 46.8 (Cq), 64.5 (CH2), 64.7 (CH2), 114.0 (Cq), 127.5 (CH), 155.5 (CH), 199.7 (Cq); HRMS: calcd. for C14H21O3 237.14852, found 237.14902 (2.104 ppm).

  (t, J = 7.5 Hz, 2H), 7.27-7.37 (m, 5H), 7.50 (d, J = 7.4 Hz, 2H) (1H masked by the DMSO signal); 13 C NMR (75 MHz, DMSO) δ 21.0 (CH2), 21.4 (CH2), 27.0 (CH2), 34.0 (CH2), 34.2 (CH2), 44.4 (Cq), 75.2 (Cq), 114.5 (CH), 115.7 (Cq), 115.9 (CH), 116.2 (CH), 124.8 (CH), 124.9 (CH), 125.9 (CH), 126.6 (2CH), 127.0 (2CH), 135.3 (Cq), 136.3 (CH), 137.8 (CH), 147.0 (Cq), 158.9 (Cq), 162.2 (Cq); MS: m/z = 375.2 [M + H] + ; HRMS: calcd. for C24H24FN2O 375.1867, found 375.1863 (-1.2371 ppm).

  C NMR (75 MHz, CDCl3) δ 20.1 (CH2), 21.0 (CH2), 25.1 (CH2), 30.3 (CH2), 33.7 (CH2), 45.5 (Cq), 76.7 (Cq), 114.3 (CH), 115.0 (Cq), 115.9 (CH), 116.2 (CH), 125.1 (CH), 125.2 (CH), 127.1 (CH), 127.1 (2CH), 127.5 (2CH), 136.4 (Cq), 137.9 (CH), 146.3 (Cq), 159.9 (Cq), 163.2 (Cq); MS: m/z = 375.2 [M + H] + ; HRMS: calcd. for C24H24FN2O 375.18672, found 375.18586 (-2.287 ppm).

  ]-1'-ol (8e) (5R, 8S)-diastereoisomer: C25H25FN2O; White solid; Mp = 144-146 °C; Yield: 20 %; M = 402.50 g / mol; IR (ZnSe) ν = 3574, 2926, 1515, 1222, 1079, 839 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.41-1.87 (m, 10H), 2.52 (d, J = 13.5 Hz, 1H), 2.75 (d, J = 16.6 Hz, 1H), 3.12 (d, J = 13.5 Hz, 1H), 3.28 (d, J = 16.6 Hz, 1H), 5.93 (d, J = 10.3 Hz, 1H), 6.47 (d, J = 10.3 Hz, 1H), 7.14 -7.35 (m, 7H), 7.45-7.50 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 20.0 (CH2), 21.1 (CH2), 27.0 (CH2), 27.7 (CH2), 31.4 (CH2), 35.7 (CH2), 45.6 (CH2), 48.6 (Cq), 63.5 (CH2) 77.0 (Cq), 114.6 (CH), 116.1 (CH), 116.4 (CH), 125.3 (CH), 125.4 (CH), 126.7 (CH), 128.5 (2CH), 131.4 (2CH), 135.6 (CH), 137.4 (Cq), 139.0 (CH); MS m/z = 403.2 [M+H] + ; HRMS: calcd. for C26H28FN2O 403.21855, found 403.21711 (-2.252 ppm).

  ]-1'-ol (9e) (5R, 8R)-diastereoisomer: C25H25FN2O; White solid; Mp = 127-129 °C; Yield: 18 %; M = 402.50 g / mol; IR (ZnSe) ν = 3390, 2926, 1514, 1225, 1079, 838 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.48-1.71 (m, 9H), 2.02-2.11 (m, 1H), 2.73-3.02 (m, 4H), 6.36 (d, J = 10.3 Hz, 1H), 6.47 (d, J = 10.3 Hz, 1H), 7.13-7.35 (m, 7H), 7.45-7.50 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 21.0 (CH2), 22.5 (CH2), 25.5 (CH2), 26.5 (CH2), 31.1 (CH2), 35.9 (CH2), 44.7 (CH2), 48.8 (Cq), 77.3 (Cq), 113.7 (CH), 115.2 (Cq), 116.1 (CH), 116.4 (CH), 125.2 (CH), 125.3 (CH), 126.8 (CH), 128.5 (2CH), 131.5 (2CH), 136.9 (Cq), 137.5 (Cq), 138.4 (CH), 138.7 (CH), 160.0 (Cq), 163.3 (Cq); HRMS: calcd. for C26H28FN2O 403.2180, found 403.21873 (1.765 ppm).

  C NMR (75 MHz, CDCl3) δ 18.3 (CH2), 26.2 (CH2), 30.8 (CH2), 36.1 (CH2), 41.0 (Cq), 113.8 (CH), 115.4 (Cq), 116.0 (CH), 116.3 (CH), 125.3 (CH), 125.4 (CH), 126.4 (CH), 127.8 (2CH), 128.8 (CH), 128.8 (2CH), 136.1 (Cq), 138.3 (CH), 140.8 (CH), 142.7 (Cq), 144.0 (Cq), 160.0 (Cq), 163.2 (Cq); HRMS: calcd. for C24H22FN2 357.17615, found 357.17532 (-2.333 ppm).

  C32H29FN2O; white solid; m.p. 188-190C Yield: 14 %; M = 476.58 g / mol; IR (ZnSe) ν = 2933, 1689, 1517, 1222, 1079, 838 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.48-1.55 (m, 1H), 1.67-1.89 (m, 5H), 2.52 (d, J = 13.2 Hz, 1H), 2.64 (d, J = 6.3 Hz, 1H), 2.69 (d, J = 8.8 Hz, 1H), 2.96 (d, J = 15.7 Hz, 1H), 3.23 (d, J = 13.2 Hz, 1H), 3.35 (d, J = 13.2 Hz, 1H), 5.54 (d, J = 10.0 Hz, 1H), 6.36 (d, J = 10.0 Hz, 1H), 7.08-7.16 (m, 6H), 7.20-7.33 (m, 6H), 7.39-7.44 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 16.4 (CH2), 29.1 (CH2), 29.4 (CH2), 32.2 (CH2), 43.9 (CH2), 44.4 (CH2), 51.0 (Cq), 54.5 (Cq), 113.8 (Cq), 115.2 (CH), 116.1 (CH), 116.4 (CH), 125.1 (CH), 125.2 (CH), 126.6 (CH), 126.8 (CH), 128.2 (2CH), 128.2 (2CH), 131.3 (2CH), 131.3 (2CH), 134.2 (CH), 135.9 (Cq), 137.1 (Cq), 137.8 (Cq), 138.3 (CH), 160.0 (Cq), 163.3 (Cq), 217.4 (Cq); HRMS: calcd. for C32H30FN2O 477.23367, found 477.23231 (-2.846 ppm).

  C25H25FN2O; White solid; Mp = 203-205 °C; Yield: 77 %; M = 388.48 g / mol; IR (ZnSe) ν = 3342, 2922, 1517, 1218, 992 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.36-1.70 (m, 7H), 1.83-1.94 (m, 1H), 2.43 (d, J = 16.0 Hz, 1H), 2.55-2.70 (m, 2H), 2.92 (d, J = 16.0 Hz, 1H), 3.41 (ls, 1H), 6.06 (d, J = 10.1 Hz, 1H), 6.38 (d, J = 10.1 Hz, 1H), 7.12-7.24 (m, 7H), 7.32-7.39 (m, 3H); 13 C NMR (75 MHz, CDCl3) δ 21.5 (CH2), 25.4 (CH2), 29.7 (CH2), 30.5 (CH2), 39.8 (CH2), 40.5 (CH), 41.2 (Cq), 71.9 (CH), 115.8 (CH), 115.8 (Cq), 116.1 (CH), 116.4 (CH), 125.1 (CH), 125.2 (CH), 126.0 (CH), 128.4 (2CH), 129.1 (2CH), 135.7 (CH), 137.2 (Cq), 138.3 (CH), 140.8 (Cq), 163.3 (Cq); HRMS: calcd. for C25H26FN2O 389.20237, found 389.20147 (-2.31 ppm).

  C21H21FN2O; White solid; Mp = 173-175 °C; Yield: 18 %; M = 336.40g / mol; IR (ZnSe) ν = 3302, 2930, 1517, 1225 cm - 1 ; 1 H NMR (300 MHz, CDCl3) δ 1.39-1.68 (m, 5H), 1.93 (t, J = 2.7 Hz, 1H), 2.11 (d, J = 5.8 Hz, 1H), 2.19 (t, J = 2.6 Hz, 1H), 2.22 (t, J = 3.0 Hz, 1H), 2.42 (d, J = 16.0 Hz, 1H), 2.96 (d, J = 16.0 Hz, 1H), 3.64 (d, J = 5.2 Hz, 1H), 6.09 (d, J = 10.1 Hz, 1H), 6.45 (d, J = 10.1 Hz, 1H), 7.13-7.19 (m, 2H), 7.38 (s, 1H), 7.42-7.47 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 21.3 (CH2), 22.8 (CH2), 25.3 (CH2), 29.6 (CH2), 30.2 (CH2), 38.2 (CH), 41.2 (Cq), 69.6 (CH), 71.5 (CH), 83.4 (Cq), 115.9 (Cq), 115.9 (CH), 116.1 (CH), 116.4 (CH), 125.0 (CH), 125.1 (CH), 135.5 (CH), 137.2 (Cq), 138.3 (CH), 160.0 (Cq), 163.3 (Cq); HRMS: calcd. for C21H22FN2O 337.171068, found 337.17086 (-0.62 ppm).

Table 1

 1 for details)

Table 1 .

 1 Reaction of organometallics with ketones 10

	n	R	Diastereoisomers c
	1	Ph	8a (12%)	not isolated
	2	Ph	8b (56%)	9b (27%)
	2	Bn 1	8c (81%)	9c (13%)
	3	Ph	8d (10%)	9d (38%)
	3	Bn	8e (20%)	9e (18%)

Scheme 5. Synthesis of dehydrated analogues 10a-c. For X = H: (

	a) PhMgBr / THF; (b) Tf2O / PhCH3, 10 min, reflux (15% over two
	steps); for X = OH: (a) 4-OTIPS-bromobenzene, n-BuLi; (b) i: TBAF / THF, 5 min, 0°C; ii: BBr3 / DCM, 1 h, 0°C (36% over three
	steps); for X = OMe: (a) 4-OMe-phenylmagnesium chloride / THF, 1h, 0°C; (b) BBr3 / DCM, 1 h, 0°C (20% over two steps).

Table 2 .

 2 Binding affinities of the new compounds to the GRs

	R1	R2	n
				Cpd	IC50 (nM)	Cpd	IC50 (nM)
	Ph	H	1	8a	> 1000	-	-
	Ph	H	2	8b	1012	9b	0.41
	Bn *	H	2	8c	5729	9c	27
	Ph	H	3	8d	9.5	9d	6
	Bn	H	3	8e	130	9e	89
	H	Bn	2	13a	> 1000	-	-
	H	Propargyl	2	13b	> 1000	-	-
	H	Bn	2	14a	> 1000	-	-

prednisolone 13 nM * previously reported, Ar = 4-F-Ph

Table 4 .

 4 Affinities for the glucocorticoid and the progesterone receptors

	Cpd	IC50 (nM) GR	IC50 (nM) PR
	9b	0.41	780
	9c	27	> 1000
	9d	6	> 1000
	10a	20	> 1000
	prednisolone	13	> 1000

  3) Nickisch, K.; Bittler, D.; Laurent, H.; Losert, W.; Casals-Stenzel, J.; Nishino, Y.; Schillinger, E.; Wiechert, R. Aldosterone Antagonists. 2. New 7.Alpha.-(Acetylthio)-15,16-Methylene Spirolactones. J. Med. Chem. 1987, 30 (8), 1403-1409. (4) Claire, M.; Faraj, H.; Grassy, G.; Aumelas, A.; Rondot, A.; Auzou, G. Synthesis of New 11.Beta.-Substituted Spirolactone Derivatives. Relationship with Affinity for Mineralocorticoid and Glucocorticoid Receptors. J. Med. Chem. 1993, 36 (16), 2404-2407. (5) Bocian, D. F.; Strauss, H. L. Conformational Structure and Energy of Cycloheptane and Some Related Oxepanes. J. Am. Chem. Soc. 1977, 99 (9), 2876-2882. (6) Dorofeeva, O. V.; Gurvich, L. V.; Mastryukov, V. S. On Conformations of Cycloheptane and Cyclooctane in the Gas Phase. Journal of Molecular Structure 1985, 129 (1), 165-168. (7) Nicolaou, K. C.; Liu, J.-J.; Yang, Z.; Ueno, H.; Sorensen, E. J.; Claiborne, C. F.; Guy, R. K.; Hwang, C.-K.; Nakada, M.; Nantermet, P. G. Total Synthesis of Taxol. 2. Construction of A and C Ring Intermediates and Initial Attempts to Construct the ABC Ring System. J. Am. Chem. Soc. 1995, 117 (2), 634-644. (8) Moffat, D.; Nichols, C. J.; Riley, D. A.; Simpkins, N. S. The Synthesis of Bioactive Indolocarbazoles Related to K-252a. Org. Biomol. Chem. 2005, 3 (16), 2953-2975. (9) Streitwieser, A.; Kim, Y.-J.; Ze-Rong Wang, D. Why Is Alkylation of an Enolate Accompanied by So Much Polyalkylation? Org. Lett. 2001, 3 (16), 2599-2601. (10) Díez-Barra, E.; Merino, S.; Sánchez-Verdú, P.; Torres, J. On the π-π Interaction in the Benzylation of Ketones.

Tetrahedron 1997, 53 (33), 11437-11448.

  MS m/z = 389.2 [M+H] + ; HRMS: calcd. for C25H28FN2O 389.20285, found 389.20136 (-2.590 ppm).

	(CH2), 27.0 (CH2), 28.8 (CH2), 30.8 (CH2), 41.6 (CH2), 49.2 (Cq), 79.8 (Cq), 115.0 (CH), 116.0 (CH), 116.3 (CH), 116.5
	(Cq), 125.1 (CH), 125.2 (CH), 126.6 (CH), 126.7 (2CH), 127.7 (2CH), 135.4 (CH), 136.1 (Cq), 138.6 (CH), 148.4 (Cq), 163.2
	(Cq);
	1'-ol (8d)
	(5R, 8R)-diastereoisomer: C25H25FN2O; White solid; Mp = 130-131 °C; Yield: 10 %; M = 388.48 g / mol; IR (ZnSe) ν =
	3333, 2923, 1515, 1222, 839 cm -1 ; 1 H NMR (300 MHz, CDCl3) δ 1.52-1.73 (m, 6H), 1.85-2.11 (m, 4H), 2.49-2.59 (m, 1H),
	2.64 (d, J = 16.1 Hz, 1H), 2.81 (d, J = 16.1 Hz, 1H), 6.15 (dd, J = 10.4 Hz, 1H), 6.34 (dd, J = 10.3 Hz, J = 0.5 Hz, 1H),
	7.08-7.17 (m, 3H), 7.23-7.28 (m, 3H), 7.34-7.38 (m, 2H), 7.46-7.49 (m, 2H); 13 C NMR (75 MHz, CDCl3) δ 20.8 (CH2), 21.9


Audrey Beillard

Xavier Bantreil
email: xavier.bantreil@umontpellier.fr

Thomas-Xavier Me
email: thomas-xavier.metro@umontpellier.fr

Jean Martinez

Lamaty

Mechanochemistry for facilitated access to N,N-diaryl NHC metal complexes †

A user-friendly and solvent-free mechanosynthetic strategy allowed light-sensitive silver(I) complexes featuring N,N-diaryl N-heterocyclic carbenes (NHC), including challenging and sterically hindered ligands, to be yielded efficiently. The first transmetalations using ball-milling to obtain palladium and copper NHC complexes were performed. A convenient one-pot two-step metalation/transmetalation procedure was also realized.

Introduction

Mechanochemistry and, more specifically, ball-milling is becoming an increasingly valuable technique for solvent-free and environmentally friendly synthesis of organometallic complexes. 1 Among the advantages of ball-milling, the lowering of solvent use 2 along with excellent agitation generally induced enhanced reaction rates while working without external heating. Additionally, ball-milling provides user-friendly procedures with a simplified set-up of reactions and recovery of products, and applications in various domains can be found in the literature. 3 Organometallic synthesis has been demonstrated to be efficient using solid-state protocols (by grinding reagents using a mortar and pestle) to give generally better and faster reactions than those obtained by solution chemistry. 4 With ball-milling technology, along with more controlled and homogeneous agitation, the same advantages have been found for the synthesis of organometallic complexes featuring a wide variety of metals and ligands. 5 For example, solvent-free ball-milling could supersede solution chemistry in the multicomponent synthesis of rhenium species. 5d Similarly, a one-pot two-step mechanosynthesis of analytically pure salen-zinc complexes is possible within only 1 h of milling. 5c As a demonstration that mechanochemistry can lead to enhanced reactivity, a relevant example is the reaction of asymmetrically substituted azobenzene with palladium(II) acetate. Just as in solution, initially a cyclopalladated compound can be obtained, but further milling with additional Pd(OAc) 2 yields quantitatively the double-palladated complex, which cannot be obtained under classical solution conditions. 5f In this context, recently we built on our expertise in mechanochemistry 6 to demonstrate that the synthesis of N,N-dialkylimidazolium salts and corresponding N-heterocyclic carbene (NHC) silver complexes in a ball-mill are extremely efficient. 7 Moreover, the first and only mechano-transmetalation to date yielded a NHC gold complex, which could also be obtained through a one-pot three-step procedure without isolating intermediates. This report was, nevertheless, limited to N,N-dialkyl ligands, whereas organometallic complexes featuring N,N-diaryl-NHC are widely described in the literature. 8 In addition, reports involving modification or synthesis of NHC-metal complexes using grinding or ball-milling are limited. Nolan et al. described the modification of [AuCl(NHC)] complexes into the corresponding [Au(OR)(NHC)] complexes using a mortar and pestle. 4h In 2015, Adams et al. demonstrated that the synthesis of PEPPSI-type compounds could be performed efficiently in a ball-mill from the imidazolium salt, pyridinium precursor, potassium hydroxide and the appropriate metal source. 9 More challenging due to their generally lower nucleophilicity and higher steric hindrance, 10 the metalation in solution of N,N-diaryl imidazolium salts in the presence of silver(I) oxide often require longer reaction times (24 h) than their N,N-dialkyl analogues. 11 In addition, working in refluxing solvent was sometimes necessary. Thus, we wanted to assess if ball-milling could facilitate this transformation and yield N,N-diaryl-NHC silver complexes (Scheme 1), thereby opening a synthetic route to other N,N-diaryl-NHC metal complexes. It is noteworthy that, in addition to their importance as NHC transfer reagents, several N,N-diaryl-NHCs silver complexes have shown high cytotoxicity against cancer cell lines. 12

Results and discussion

Classical conditions for solvent-free milling, using a stainlesssteel ball and jar agitated with a vibratory ball-mill (vbm) at a frequency of 25 Hz, were first applied. To our delight, the reaction of IMesÁHCl (1,3-bis(2,4,6-trimethylphenyl)imidazolium chloride, Fig. 1) with Ag 2 O yielded 82% of the corresponding [AgCl(IMes)] complex in only 1 h 40 min (Table 1, entry 1). As a comparison, the use of a planetary ball-mill (pbm) at 450 rpm allowed [AgCl(IMes)] to be obtained in a similar manner, 13 thus allowing envisioning of a facilitated scale-up. An important feature is that silver(I) complexes are sensitive to light, and reactions in solution must be protected from light. Stainless-steel milling jars are not transparent to light, and decomposition of the complexes was not observed which, in addition to a short reaction time, renders the grinding method even more user-friendly. To support the robustness of the method, a prolonged synthesis of [AgCl(IMes)] for 10 h in the vbm gave the pure complex in 79% yield, and degradation was not observed.

In all cases, before the work-up on the whole reaction mixture, a sample was deposited on cotton and CDCl 3 was added to quickly solubilize and filter the mixture. Conversions were then calculated by 1 H NMR. To ensure that the conversion measured was obtained by mixing solid reagents together, and that the reaction was not occurring during this work-up, a control experiment was performed. IMesÁHCl and Ag 2 O were gently mixed together for o1 min to obtain a homogeneous mixture. A sample was directly taken and the work-up described above was applied. 1 H NMR spectra showed 0% conversion, indicating that no reaction had occurred during the work-up. In addition, the same reaction was performed again and stopped after 15 min of milling. 1 H NMR spectra were recorded before and after work-up of the whole reaction mixture. In both cases, E30% conversion was measured, indicating that no reaction had occurred during treatment of the reaction using solvent.

When SIMesÁHCl (1,3-bis(2,4,6-trimethylphenyl)imidazolidinium chloride) was used, milling at 25 Hz did not give reasonable conversions. However, increasing the frequency to 30 Hz, thus transferring more energy to the reaction mixture, allowed Scheme 1 Synthetic strategy for NHC-transition metal mechanosynthesis. [AgCl(SIMes)] to be obtained in 89% yield after 1 h 30 min (Table 1, entry 2). IPrÁHCl andSIPrÁHCl (1,3-bis(2,6-diisopropylphenyl)imidazolium and imidazolidinium chlorides), which are known to be more sterically hindered, as witnessed by the %V bur 21 of the ligand from literature data, 20 induced a lowered reactivity.

Nevertheless, the milling method again proved to be highly efficient when a slightly prolonged reaction time of 3 h was applied. Under these conditions, [AgCl(IPr)] and [AgCl(SIPr)] were obtained in 89% and 73% yield, respectively (Table 1, entries 3 and 4). Having these results in hand with the most classical NHC ligands used for organometallic synthesis, we decided to extend this methodology to less common and even more sterically hindered NHC bearing methyl groups on the imidazole ring backbone. To ensure full conversion in each case, reaction conditions were kept at 30 Hz for 3 h. Satisfyingly, [AgCl(IMes Me )] and its saturated congeners, [AgCl(SIMes Me-cis )] and [AgCl(SIMes Me-trans )], could be isolated in 81-86% yield (Table 1, entries 5-7). It is important to note that the two isomers, SIMes Me-cis ÁHCl and SIMes Me-trans ÁHCl, were separated during their synthesis, characterized and assigned thanks to X-ray diffraction of the trans isomer (Fig. 2). To the best of our knowledge, it is the first time that they were reacted separately to furnish the two diastereomeric silver(I) complexes.

Similarly, [AgCl(IPr Me )] and unprecedented [AgCl(SIPr Me )], which was obtained as a mixture of isomers, could be produced in 82% and 77% yield, respectively (Table 1, entries 8 and9). Finally, the methodology was also extended to the triazolium salt TPTÁHCl (1,3,4-triphenyl-1,2,4-triazolium chloride, Fig. 1), which was converted to the corresponding silver complex in 89% yield (Table 1, entry 10). The mechanochemical method appeared to be quite general and allowed to access previously described as well as unprecedented NHC-silver(I) complexes in good-to-excellent yields and in short reaction times. For comparison, conditions from the literature to obtain the complexes have been added to Table 1. The mechanochemical method was, in general, found to be more efficient than classical solution conditions, either with dichloromethane or water, yielding the desired complexes in similar yields than those described in the literature, but in reduced reaction times and without the need to protect the reaction mixture from light. Conditions using microwave activation were found to be faster because the metalation occurred in 30 min, but heating at 110 1C in toxic toluene or THF was necessary. The mechanochemical method is, therefore, one of the most efficient ways to produce NHC silver complexes.

We were pleased to find that the mechanosynthesis of [AgCl(NHC)] complexes was highly practical. We questioned if transmetalation involving N,N-diaryl NHC could be feasible in the ball-mill. In particular, transmetalation leading to gold, copper and palladium complexes was envisioned. Indeed, such complexes are widely used in catalysis and [AuCl(IMes)] and [CuCl(IMes)] have also been reported to display higher cytotoxicity than cisplatin in various cancer cell lines. 12b,22 Gratifyingly, mechanical agitation allowed an almost quantitative [AuCl(IMes)], [CuCl(IMes)] and [PdCl(Z 3 -allyl)(IMes)] yield starting from [AgCl(IMes)] in only 1 h to 1.5 h of reaction, using a stainlesssteel ball and jar agitated with a vbm at a frequency of 25 Hz (Scheme 2). Importantly, the last two examples represent the first transmetalations in a ball-mill leading to copper and palladium complexes. Conditions for the synthesis of the gold complex were directly applied from our previous report involving N,N-dialkyl NHC ligands. 7 A rapid optimization of the stoichiometry and reaction time allowed a full conversion to be reached, as confirmed by 1 H NMR, when copper and palladium reagents were used in slight excess.

In order to increase the usefulness of this methodology, we attempted to perform both metalation and transmetalation in a one-pot two-step process, starting directly from IMesÁHCl without isolating the [AgCl(IMes)] intermediate (Scheme 3). Using this procedure, both the use of a solvent for isolating the intermediate and the light-exposure issue of the silver complexes could be avoided. Satisfyingly, this one-pot two-step strategy allowed the isolation of [AuCl(IMes)] in 76% yield, in only 2 h 40 min of reaction time in the ball-mill. Similarly, [CuCl(IMes)] and [PdCl(Z 3 -allyl)(IMes)] could be synthesized in higher yield than with the procedure involving isolation of the intermediate silver complex (90% and 92% yield vs. 81% and 80% yield, respectively).

Conclusion

Silver complexes featuring N,N-diaryl N-heterocyclic carbenes could be obtained in o3 h at room temperature under solventfree ball-milling. This user-friendly and highly efficient method allowed transmetalation with copper, gold and palladium without observable decomposition of the silver complex. Ultimately, a highly practical one-pot two-step metalation/transmetalation procedure allowed copper, gold and palladium complexes to be yielded directly in short reaction times and without isolation of the silver intermediates.

Experimental

General remarks

All reagents were purchased from Sigma-Aldrich, Fluka and Alfa Aesar and used without further purification. The milling treatments were carried out either in a vibrating Retsch Mixer Mill 200 (vbm) or in a Retsch PM100 Planetary Mill (pbm). ''Milling load'' is defined as the sum of the mass of the reactants per free volume in the jar. 1 H NMR spectra were recorded on Bruker Avance DPX 300 MHz, 400 MHz or Bruker Avance III 600 MHz spectrometers. They are reported in ppm using deuterated solvent used for calibration (CDCl 3 at 7.26 ppm or DMSO-d 6 at 2.50 ppm). Data are reported as s = singlet, d = doublet, t = triplet, q = quadruplet, qt = quintuplet, m = multiplet, sept = septuplet; coupling constant in Hz; integration. 13 C NMR spectra were recorded on Bruker Avance AM 75 MHz or 101 MHz spectrometers and are reported in ppm using deuterated solvent used for calibration (CDCl 3 at 77.2 ppm or DMSO-d 6 at 39.5 ppm). HRMS analysis was performed on a Q-Tof (Waters, ESI, † 2001) spectrometer.

All of reaction mixtures were recovered with a solvent and filtrated over celite to remove metallic particles lost by the reactor during ball-milling.

All of the reactions using a vbm were made at 25 Hz or 30 Hz under air with no interruption of the milling.

Syntheses

General procedure for the synthesis of [AgCl(IMes)] in a vbm. 1,3-Bis- (2,4,6-trimethylphenyl)imidazolium chloride (132.4 mg, 0.389 mmol, 1.00 eq.) and silver oxide (49.5 mg, 0.214 mmol, 0.55 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h and 40 min in a vbm operated at 25 Hz. The grey powder was recovered with dichloromethane and the suspension was filtrated over celite. The filtrate was concentrated under vacuum to afford [1,3-bismesitylimidazol-2-ylidene]silver chloride (142.3 mg, 0.318 mmol, 82%) as a pale-brown solid.

Synthesis of [AgCl(IMes)] in a pbm. 1,3-Bis-(2,4,6-trimethylphenyl)imidazolium chloride (122.0 mg, 0.358 mmol, 1.00 eq.) and silver oxide (45.6 mg, 0.197 mmol, 0.55 eq.) were introduced in a 12 mL stainless-steel grinding bowl with 50 stainless-steel balls (diameter, 5 mm). Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 2 h in a pbm operated at 450 rpm. The grey powder was recovered with dichloromethane and the suspension was filtrated over celite. The filtrate was concentrated under vacuum to afford [1,3-bismesitylimidazol-2-ylidene]silver chloride (140.3 mg, 0.313 mmol, 88%) as a pale-brown solid.

[AgCl(IMes)]. 1 H NMR (300 MHz, CDCl 3 ) d 7.14 (d, J = 1.5 Hz, 2H), 7.00 (s, 4H), 2.36 (s, 6H), 2.08 (s, 12H); 13 C NMR (101 MHz, CDCl 3 ) d 183. 1 (dd, J = 256.0, 18.3 Hz), 139.9, 135.3, 134.7, 129.7, 122.9, 122.8, 21.2, 17.8. Data in agreement with the literature. 23 [AgCl(SIMes)]. White solid, 69% yield. 1 H NMR (400 MHz, CDCl 3 ) d 6.96 (s, 4H), 4.01 (s, 4H), 2.31 (s, 6H), 2.30 (s, 12H); 13 C NMR (101 MHz, CDCl 3 ) d 164. 2, 138.9, 135.4, 135.1, 129.9, 51.2, 51.1, 21.0, 17.9. Data in agreement with the literature. 23 [AgCl(IPr)]. White powder, 89% yield. A mixture of [AgCl(IPr)] and [Ag(IPr) 2 ]Cl were obtained in 95 : 5 ratio. Peaks for [AgCl(IPr)] (major product) are reported: 1 H NMR (400 MHz, CDCl 3 ) d 7.50 (t, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz, 4H), 7.22 (d, J = 1.8 Hz, 2H), 2.62-2.47 (m, 4H), 1.28 (d, J = 6.9 Hz, 12H), 1.22 (d, J = 6.9 Hz, 12H); 13 C NMR (101 MHz, CDCl 3 ) d 184.6 (dd, J = 258.5, 21.4 Hz), 145. 7, 134.6, 130.9, 124.5, 123.8, 123.7, 28.8, 24.8, 24.1. Data in agreement with the literature. 23 [AgCl(SIPr)]. White solid, 73% yield. 1 H NMR (400 MHz, CDCl 3 ) d 7.42 (t, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 4H), 4.06 (s, 4H), 3.05 (hept, J = 6.9 Hz, 4H), 1.342 (d, J = 6.9 Hz, 12H), 1.337 (d, J = 6.9 Hz, 12H); 13 C NMR (101 MHz, CDCl 3 ) d 13 C of carbene was not observed, 146. 7, 134.6, 130.2, 124.9, 54.1, 54.0, 29.0, 25.6, 24.2. Data in agreement with the literature. 23 [AgCl(IMes Me )]. Pale-brown solid, 81% yield. A mixture of [AgCl(IMes Me )] and [Ag(IMes Me ) 2 ]Cl was obtained in 90 : 10 ratio. Peaks for [AgCl(IMes Me )] (major product) are reported: 1 H NMR (300 MHz, CDCl 3 ) d 6.99 (s, 4H), 2.35 (s, 6H), 2.01 (s, 12H), 1.91 (s, 6H); 13 C NMR (101 MHz, CDCl 3 ) d 178. 8 (dd, J = 258.4, 18.4 Hz), 139.5, 135.0, 134.0, 129.7, 126.13, 126.07, 21.2, 17.8, 9.3. HRMS calcd for C 23 H 28 N 2 Ag [M À Cl À ] + : 439.1303; found: 439.1308.

[AgCl(SIMes Me-cis )]. White solid, 86% yield. 1 H NMR (300 MHz, CDCl 3 ) d 6.96 (s, 2H), 6.94 (s, 2H), 4.47 (s, 2H), 2.36 (s, 6H), 2.30 (s, 6H), 2.27 (s, 6H), 1.19 (d, J = 5.5 Hz, 6H); 13 C NMR (101 MHz, CDCl 3 ) d 206. 1 (dd, J = 242.4, 17.4 Hz), 138.5, 136.3, 135.5, 134.1, 130.1, 130.0, 61.4, 61.3, 21.0, 19.1, 18.1, 13.0; HRMS calcd for C 46 H 60 N 4 Ag [M À Cl] + : 775.3869; found: 775.3879.

[AgCl(SIMes Me-trans )]. White solid, 86% yield. 1 H NMR (400 MHz, CDCl 3 ) d 6.94 (s, 2H), 6.91 (s, 34H), 4.07-3.91 (m, 2H), 2.32 (s, 6H), 2.28 (s, 6H), 2.26 (s, 6H), 1.28 (d, J = 5.8 Hz, 6H); 13 C NMR (101 MHz, CDCl 3 ) d 207.64 (dd, J = 242.0, 17.4 Hz), 138.6, 136.6, 135.3, 134.0, 130.1, 130.0, 66.64, 66.54, 21.0, 19.1, 18.4, 18.0; HRMS calcd for C 46 H 60 N 4 Ag [M À Cl] + : 775.3869; found: 775.3870.

[AgCl(IPr Me )]. Pale-brown solid, 82% yield. After purification, a mixture of [AgCl(IPr Me )] and [Ag(IPr Me ) 2 ]Cl was obtained in 97 : 3 ratio. Peaks for [AgCl(IPr Me )] (major product) are reported: 1 H NMR (400 MHz, DMSO-d 6 ) d 7.60-7.53 (m, 2H), 7.44 (d, J = 7.7 Hz, 4H), 2.39 (sept, J = 6.8 Hz, 4H), 1.95 (s, 6H), 1.22 (d, J = 6.8 Hz, 12H), 1.18 (d, J = 6.8 Hz, 12H); 13 C NMR (101 MHz, DMSO-d 6 ) d 178.9 (dd, J = 253. 1, 18.1 Hz), 146.0, 133.2, 131.1, 127.3, 127.2, 124.9, 28.6, 25.5, 23.4, 9.6. Data in agreement with the literature. 20 [AgCl(SIPr Me )]. White solid, 77% yield. Mixture of diastereoisomers (57 : 43) 1 H NMR (400 MHz, DMSO-d 6 ) d 7.46 (t, J = 7.7 Hz, 2H), 7.36 (t, J = 6.7 Hz, 4H), 4.57 (s, 2H, 43%), 4.01 (s, 2H, 57%), 3.26 (sept, J = 6.8 Hz, 2H, 57%), 3.15 (sept, J = 6.9 Hz, 2H, 43%), 3.06 (sept, J = 6.9 Hz, 2H, 43%), 2.95 (sept, J = 6.8 Hz, 2H, 57%), 1.35-1.26 (m, 18H), 1.23 (d, J = 6.7 Hz, 12H, 57%), 1.19 (d, J = 6.7 Hz, 6H, 43%), 1.14 (d, J = 4.8 Hz, 6H, 43%); major diastereoisomer: 13 C NMR (101 MHz, DMSO-d 6 ) d 206.3 (dd, J = 201.0, 17.2 Hz), 147.5, 146.8, 132.9, 129.8, 124.7, 124.4, 68.3, 68.2, 28.2, 28.1, 25.9, 25.1, 23.6, 22.8, 17.4; minor diastereoisomer: 13 C NMR (101 MHz, DMSO-d 6 ) d 203.9 (dd, J = 201. 2, 17.8 Hz), 147.4, 146.8, 133.2, 129.6, 124.5, 63.0, 62.9, 28.3, 28.0, 25.9, 25.2, 23.4, 23.1, 12.8; HRMS calcd 5, 154.1, 139.5, 137.0, 131.6, 130.6, 130.3, 130.0, 129.6, 129.2, 127.5, 124.7, 123.3; HRMS calcd for C 20 H 15 N 3 Ag [M À Cl] + : 404.0317; found: 404.0317.

Preparation of [CuCl(IMes)] from [AgCl(IMes)]. [1,3-Bismesitylimidazol-2-ylidene]silver chloride (136.6 mg, 0.305 mmol, 1.00 eq.) and copper(I) chloride (45.3 mg, 0.458 mmol, 1.50 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h in a vbm operated at 25 Hz. The white powder was recovered with dichloromethane and the suspension was filtrated over celite. The filtrate was concentrated under vacuum to afford [1,3-bismesitylimidazol-2ylidene]copper chloride (122.7 mg, 0.304 mmol, 100%) as a white powder.

One-pot two-step preparation of [CuCl(IMes)] from IMesÁ HCl. 1,3-Bis- (2,4,6-trimethylphenyl)imidazolium chloride (100.6 mg, 0.295 mmol, 1.00 eq.) and silver oxide (37.6 mg, 0.162 mmol, 0.55 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). The bowl was closed and subjected to grinding for 1 h and 40 min in a vbm operated at 25 Hz. The bowls were opened and copper(I) chloride (43.8 mg, 0.442 mmol, 1.50 eq.) added. Total mass of the reagents was calculated so that milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h in a vbm operated at 25 Hz. The grey powder was recovered with dichloromethane and the suspension was filtrated over celite. The filtrate was concentrated under vacuum to afford [1,3-bismesitylimidazol-2-ylidene] 9, 139.6, 135.5, 134.5, 129.6, 129.2, 122.6, 121.9, 21.2, 17.8. Data in agreement with the literature. 24 Preparation of [AuCl(IMes)] from [AgCl(IMes)]. [1,3-Bismesitylimidazol-2-ylidene]silver chloride (101.7 mg, 0.227 mmol, 1.00 eq.) and chloro(dimethylsulfide)gold (80.3 mg, 0.273 mmol, 1.20 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h in a vbm operated at 25 Hz. The yellow powder was recovered with dichloromethane and the suspension was filtrated over silica. The filtrate was concentrated under vacuum to afford [1,3bismesitylimidazol-2-ylidene]gold chloride (117.5 mg, 0.219 mmol, 96%) as a pale-yellow powder.

One-pot two-step preparation of [AuCl(IMes)] from IMesÁHCl. 1,3-Bis- (2,4,6-trimethylphenyl)imidazolium chloride (75.5 mg, 0.221 mmol, 1.00) and silver oxide (28.2 mg, 0.122 mmol, 0.55 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). The bowl was closed and subjected to grinding for 1 h and 40 min in a vbm operated at 25 Hz. Afterwards, chloro(dimethylsulfide)gold (78.3 mg, 0.266 mmol, 1.20 eq.) were added. Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h and 30 min in a vbm operated at 25 Hz. The grey powder was recovered with dichloromethane and the suspension was filtrated over silica. The filtrate was concentrated under vacuum to afford [1,3bismesitylimidazol-2-ylidene]gold chloride (90.2 mg, 0.168 mmol, 76%) as a pale-yellow solid. 1 H NMR (600 MHz, CDCl 3 ) d 7.10 (s, 1H), 6.99 (s, 2H), 2.35 (s, 3H), 2.11 (s, 6H); 13 C NMR (151 MHz, CDCl 3 ) d 173. 5, 139.8, 134.71, 134.68, 129.5, 122.4, 21.2, 17.8. Data in agreement with the literature. 25 Preparation of [PdCl(g 3 -allyl)(IMes)] from [AgCl(IMes)]. [1,3-Bismesitylimidazol-2-ylidene]silver chloride (118.8 mg, 0.265 mmol, 1.00 eq.) and allylpalladium chloride dimer (63.1 mg, 0.173 mmol, 0.65 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h and 30 min in a vbm operated at 25 Hz. The black powder was recovered with dichloromethane and the suspension was filtrated over celite. The filtrate was concentrated under vacuum and the resulting solid was put on silica, filtrated with cyclohexane to remove excesses of allylpalladium chloride dimer and ethyl acetate to collect the desired product. Ethyl acetate was evaporated to afford [1,3bismesitylimidazol-2-ylidene](Z 3 -2-propen-1-yl)palladium chloride (122.0 mg, 0.250 mmol, 94%) as a pale-yellow powder.

One-pot two-step preparation of [PdCl(g 3 -allyl)(IMes)] from IMesÁHCl. 1,3-Bis- (2,4,6-trimethylphenyl)imidazolium chloride (87.8 mg, 0.258 mmol, 1.00) and silver oxide (32.8 mg, 0.142 mmol, 0.55 eq.) were introduced in a 10 mL stainless-steel grinding bowl with one stainless-steel ball (diameter, 10 mm). The bowl was closed and subjected to grinding for 1 h and 40 min in a vbm operated at 25 Hz. Afterwards, allylpalladium chloride dimer (61.3 mg, 0.167 mmol, 0.65 eq.) was added. Total mass of the reagents was calculated so that the milling load was 19.2 mg mL À1 . The bowl was closed and subjected to grinding for 1 h and 30 min in a vbm operated at 25 Hz. The black powder was recovered with dichloromethane and the suspension was filtrated over celite. The filtrate was concentrated under vacuum and the resulting solid was put on silica, filtrated with cyclohexane to remove excesses of allylpalladium chloride dimer and ethyl acetate to collect the desired product. Ethyl acetate was evaporated to afford [1,3bismesitylimidazol-2-ylidene](Z 3 -2-propen-1-yl)palladium chloride (110.8 mg, 0.227 mmol, 88%) as a pale-yellow solid. 8, 139.0, 136.0, 135.6, 129.2, 129.1, 123.1, 114.4, 72.5, 49.3, 21.3, 18.43, 18.37; minor diastereoisomer: 13 C NMR (101 MHz, CDCl 3 ) 183.3, 139.0, 136.0, 135.5, 129.3, 129.2, 123.2, 113.9, 71.7, 52.5, 21.3, 18.71, 18.66. Data in agreement with the literature. 26 Notes and references

d

Fig. 1

 1 Fig. 1 Structure of the NHC used in this study.

Fig. 2 Scheme 2

 22 Fig.2ORTEPs (probability at 50% level) of SIMesMe-trans . H and Cl atoms have been omitted for clarity.

  for C 29 H 42 N 2 Ag [M À Cl] + : 525.2399; found: 525.2404. [AgCl(TPT)]. Brown crystallized powder, 89% yield. 1 H NMR (400 MHz, DMSO-d 6 ) d 8.08-8.00 (m, 2H), 7.64-7.48 (m, 9H), 7.46-7.41 (m, 4H); 13 C NMR (151 MHz, DMSO-d 6 ) d 183.

Table 1

 1 Mechanosynthesis of silver complexes a

	Entry	Product	%V bur b	t (h)	Yield (%)	Conditions from lit.
	1 2 3	[AgCl(IMes)] [AgCl(SIMes)] [AgCl(IPr)]	35.1 34.5 42.9	1.7 1.5 3	82 c 89 89	Ag 2 O (1.15 eq.), CH 2 Cl 2 , 7 h, reflux, 73% 14 Ag 2 O (0.65 eq.), H 2 O, 24 h, reflux, 92% 15 Ag 2 O (0.65 eq.), toluene, 30 min, 110 1C, MW, 83% 16 Ag 2 O (1.5 eq.), CH 2 Cl 2 , 24 h, r.t., 95% 17 Ag 2 O (0.65 eq.), H 2 O, 24 h, reflux, 88% 15 Ag 2 O (0.65 eq.), THF, 30 min, 110 1C, MW, 90% 16 Ag 2 O (0.5 eq.), CH 2 Cl 2 , 6 h, reflux, 94% 18 Ag 2 O (0.65 eq.), H 2 O, 24 h, reflux, 87% 15 Ag 2 O (0.65 eq.), toluene, 30 min, 110 1C, MW, 85% 16
	4	[AgCl(SIPr)]	44.0	3	73	Ag

2 O (0.5 eq.), CH 2 Cl 2 , 24 h, r.t., 92%

17

Ag 2 O (0.65 eq.), H 2 O, 24 h, reflux, 74% 15

Ag 2 O (0.65 eq.), THF, 30 min, 110 1C, MW, 92% 16 5 [AgCl(IMes Me )] n.a. 3 81 Unprecedented 6 [AgCl(SIMes Me-cis )] n.a. 3 86 Ag 2 O (1.0 eq.), CH 2 Cl 2 , 24 h, 82% 19 7 [AgCl(SIMes Me-trans )] n.a. 3 86 8 [AgCl(IPr Me )] 44.9 3 82 Ag 2 O (0.65 eq.), CH 2 Cl 2 , 3 h, r.t., 82% 20 9 [AgCl(SIPr Me )] n.a. 3 77 Unprecedented 10 [AgCl(TPT)] n.a. 3 89 Unprecedented a Total mass of reactants: 182 mg, vbm = vibratory ball-mill. b Data from lit. 20 n.a. = not available. c Reaction performed at 25 Hz.

  copper chloride (107.2 mg, 0.266 mmol, 90%) as a white powder. 1 H NMR (300 MHz, CDCl 3 ) d 7.07 (s, 2H), 7.01 (s, 4H), 2.36 (s, 6H), 2.11 (s, 12H); 13 C NMR (75 MHz, CDCl 3 ) d 179.

  1 H NMR (400 MHz, CDCl 3 ) d 7.09 (s, 2H), 6.97 (s, 4H), 4.93- 4.77 (m, 1H), 3.94-3.83 (m, 1H), 3.39 (d, J = 6.1 Hz, 1H, 25%), 3.20 (d, J = 6.1 Hz, 1H, 75%), 2.81 (d, J = 13.5 Hz, 1H, 75%), 2.76 (d, J = 13.4 Hz, 1H, 25%), 2.33 (s, 6H), 2.23 (s, 6H), 2.20 (s, 6H), 1.87 (d, J = 12.1 Hz, 1H, 25%), 1.80(d, J = 11.8 Hz, 1H, 75%); major diastereoisomer: 13 C NMR (101 MHz, CDCl 3 ) d 183.

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