RhIII‐Catalyzed C−H Activation of Aryl Hydroxamates for the Synthesis of Isoindolinones

Abstract RhIII‐catalyzed C−H functionalization reaction yielding isoindolinones from aryl hydroxamates and ortho‐substituted styrenes is reported. The reaction proceeds smoothly under mild conditions at room temperature, and tolerates a range of functional groups. Experimental and computational investigations support that the high regioselectivity observed for these substrates results from the presence of an ortho‐substituent embedded in the styrene. The resulting isoindolinones are valuable building blocks for the synthesis of bioactive compounds. They provide easy access to the natural‐product‐like compounds, isoindolobenzazepines, in a one‐pot two‐step reaction. Selected isoindolinones inhibited Hedgehog (Hh)‐dependent differentiation of multipotent murine mesenchymal progenitor stem cells into osteoblasts.


General Information
Unless otherwise noted, all commercially available compounds were used as provided without further purification. Solvents for chromatography were technical grade.
High resolution mass spectra (HR-MS) were recorded on an LTQ Orbitrap mass spectrometer coupled to an Accela HPLC-System (HPLC column: Hypersyl GOLD, 50 mm x 1 mm, particle size 1.9 μm, ionization method: electron spray ionization). Fourier transform infrared spectroscopy (FT-IR) spectra were obtained with a Bruker Tensor 27 spectrometer (ATR, neat) and are reported in terms of frequency of absorption (cm -1 ).
Acid chlorides were either commercially available or made from their corresponding carboxylic acid following the reported procedure. [1] 2-Bromostyrenes were also commercially available or made from their corresponding 2-Bromobenzadehyde following a modified version of the reported procedure. [2]

General Procedure for the synthesis of OBoc-aryl-hydroxamates 1
Following a modified procedure by Fagnou et al. [3] , a flask was charged with hydroxylamine hydrochloride (1.5 equiv.), potassium carbonate (2.0 equivalent) and solvent (EtOAc/water; 2:1). The reaction was cooled to 0 °C with vigorous stirring before dropwise addition of acid chloride (1 equiv.) dissolved in EtOAc (2 ml). The reaction was stirred at RT for 14 h. The reaction mixture was diluted with water and extracted with EtOAc (2 x 20 ml). The combined organic layers were washed with brine (10 ml), dried over anhydrous sodium sulfate and concentrated in vacuo. The crude hydroxamic acid was used in the next step without further purification.
All the aryl-hydroxamates are reported and their characterization matches the literature. [4]

General Procedure for the synthesis of styrenes 2
According to the reported procedure [5] , a 100 mL round bottom flask was charged with 2-bromostyrene (1.0 eq.) and THF [0.2M]. The solution was then cooled to -78 °C and n-BuLi (1.2 eq., 1.6 M solution in hexanes) was added dropwise. The resulting mixture was then stirred at -78 °C for 1 h. Ethylene oxide (3.0 eq., 2.5 M solution in THF) was then added to the reaction mixture and stirred for an additional 0.5 h at -78 °C. The reaction was then warmed to RT and stirred for 14 h. A saturated solution of NH4Cl (10 mL) was added, and the aqueous layer washed with Et2O (3 x 25 mL). The combined organic layers were dried over Na2SO4, filtered and concentrated in vacuo to afford the corresponding alcohol which was used in the next step without further purification.
The solvent was removed under reduced pressure and the crude material was purified by silica gel chromatography (Pent/EtOAc, 4:1) to afford the title styrenes 2.
Some of the styrenes are reported and our characterization matches the literature. [6]

General Procedure for the synthesis of Isoindolobenzazepine scaffolds 4
In a dram vial equipped with a stirring bar was added [RhCp*Cl2]2 (1.0 mol%), KOAc (0.0025 mmol, 0.25 eq.), N-((tert-butoxycarbonyl)oxy)aryl-hydroxamate substrate (1) (0.1 mmol, 1.0 eq.) and dry MeOH [0.1 M]. The mixture was stirred for 30 seconds before the addition of the alkene substrate (2), (0.15 mmol, 1.5 eq.). The reaction was left to stir at room temperature for 16 h. The solvent was then evaporated under reduced pressure. Dry DCM [0.05 M] was added to the crude mixture followed by tBuOK (0.30 mmol, 3.0 eq.). The reaction mixture was stirred for 30 minutes at room temperature. The reaction was then quenched with H2O and extracted with DCM (2 x 5 mL). The organic layer was washed with brine, dried over MgSO4, filtered, and the solvent was evaporated. The crude material was purified by silica gel chromatography (Pent/EtOAc, 4:1) to afford the title compounds 4.

X-ray Structure Analyses
Data collection was conducted on a Bruker D8 Venture four-circle diffractometer by Bruker AXS GmbH using a PHOTON II CPAD detector by Bruker AXS GmbH. X-ray radiation was generated by microfocus sources IµS and IµS 3.0 Cu or Mo by Incoatec GmbH with HELIOS mirror optics and a single-hole collimator by Bruker AXS GmbH.
For the data collection, the programs APEX 3 Suite (v.2018.7-2) with the integrated programs SAINT (integration) and SADABS (adsorption correction) by Bruker AXS GmbH were used. Using Olex2, [7] the structures were solved with the ShelXT [8] structure solution program using Intrinsic Phasing and refined with the XL [9] refinement package using Least Squares minimization.

Reagents
Purmorphamine was purchased from Cayman Chemical (#10009634) and DMSO from (Sigma Aldrich, #67685). The compound collection was synthesized as described in the paper. All other reagents used are mentioned in the respective protocols below, with the corresponding sources.

Osteoblast differentiation and viability assay
The osteoblast differentiation assay and the viability assay were performed using C3H10T1/2 cells. [10] For the screening and IC50 determinations 800 C3H10T1/2 cells per well were seeded in white 384-well plates. After incubation overnight, cells were treated with 1.5 µM purmorphamine and different concentrations of the compounds or DMSO as a control. After 96 h the cell culture medium was aspirated and the commercial luminogenic ALP substrate CDP-Star (Roche, #11685627001) was added. The cells were incubated for one hour at room temperature and in absence of light. Afterwards the luminescence signal was read. To identify and exclude toxic compounds, which would also lead to a reduced luminescent signal, cell viability measurements were conducted in parallel. For this purpose, C3H10T1/2 cells were seeded and treated as described above. The cellular ATP content was determined as a measure of cell viability using the Cell Titer Glo reagent (Promega). Compounds were considered as hit compounds if they caused at least 50% reduction in the luminescent signal in the osteoblast differentiation assay while retaining cell viability ≥ 80% at a concentration of 10 µM. To determine IC50 curves for hit compounds, three-fold dilution curves starting from 10 μM, were used. Calculations of the IC50 values were conducted, using GraphPad Prism 7. (GraphPad Software, USA). After 96 h, the activity of alkaline phosphatase was detected using a chemiluminescent substrate. The purmorphamine control was set to 100%. Data are mean values ± SD and representative of three biological replicates, each performed in three technical replicates. C: Chemical structure of the known Hh inhibitor Vismodegib.

DFT Calculations
Geometry optimizations and frequency analysis were carried out using Gaussian 16 Rev. B [11] employing tight convergence criteria using the M06-2X functional. [12] The 6-31G(2d,p) basis set was used for all elements except for Rh, for which LabL2DZ including pseudo potentials was used. The solvent environment was simulated by the IEFPCM of methanol as implemented in the software package.

Cartesian coordinates
Structure II*