Supporting Information for Design, Synthesis and Biological Evaluation of Novel Biphenylsulfonamide Derivatives as Selective AT2 Receptor Antagonists

for Design, Synthesis and Biological Evaluation of Novel Biphenylsulfonamide Derivatives as Selective AT2 Receptor Antagonists Danhui Wang a, Wenjie Zhaoa, Zuzhi Zhanga, Yanchun Zhang* a,b , Jiaming Lia,b, Weijun Huanga aCollege of Pharmacy, Anhui University of Chinese Medicine, Hefei, 230012, China bAnhui Province Key Laboratory of Chinese Medicinal Formula, Hefei, Anhui, 230012, China * Corresponding author: Yanchun Zhang , E-mail address: yczhang2017@163.com ☨These authors contributed equally to this work.


Synthesis of intermediate 4a-4c 1.1 General procedure A: Synthesis of intermediates (2a-2c)
Isobutylbenzene, butylbenzene or butoxybenzene (74.50 mmol) was dissolved in the CH2Cl2 (200 mL). Subsequently, chlorosulfonic acid (298.01 mmol) was added slowly to the solution in an ice bath and the mixture was stirred at rt for 1 h. The reaction was quenched by dropped ice-cold water (50 mL) into the mixture. The reaction mixture was extracted twice with CH2Cl2 (20 mL) and washed with water (30 mL×2) and brine (30 mL×2). The organic layer was dried with anhydrous Na2SO4, filtered, and evaporated to achieve 2a-2c.

General procedure B: Synthesis of intermediates (3a-3c)
To a 250 mL closed pressure vessel was added 2a, 2b, or 2c (70.90 mmol) dissolved in DCM (200 mL). Tert-butylamine (11.18 mL, 106.35 mmol) was added to the mixture in an ice bath. The reaction was stirred at r.t for 8 h. The reaction was extracted with DCM (50 mL), the organic layers were combined and washed with NaCl, dried over Na2SO4, and concentrated to give 3a-3c.

General procedure C: Synthesis of intermediates (4a-4c)
To a 100 mL round-bottom flask were added 3a, 3b, 3c (7.42 mmol) followed by dry THF (20 mL). N-BuLi (1.6 M in hexane, 9.28 mL, 14.85 mmol) was added to the mixture at -78 ℃.The reagent was added under nitrogen and the reaction was stirred for 1 h. After the flask was warmed to -20 ℃, kept for 3 h and subsequently decreased to -78 ℃. Triisopropyl borate (2.57 mL, 11.14 mmol) was then added. The reaction mixture was stirred over night at room temperature. The reaction mixture was treated with an excess of 2 M HCl solution in an ice bath. The mixture was extracted with ethyl acetate (50 mL ⅹ 2 ). The combined organic phase was washed with water and brine, dried with Na2SO4, filtered and evaporated. Using silica gel column chromatography (PE/EA as eluent), the residue was purified to obtain 4a-4c.
2. NMR and HRMS spectra of compounds 8a-8l, 9a-9h. 1 H and 13 C NMR spectra were recorded at room temperature at 400 MHz and 100 MHz respectively using a QNP probe. NMR spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d6) at room temperature unless otherwise stated. Chemical shifts (δ values) are reported in parts per million, and are referenced to the deuterated residual Solvent peak. NMR data was reported as: δ value (chemical shift, J-value(Hz), integration, where s = singlet, d = doublet, t = triplet, q = quartet, brs = broad singlet). High-resolution mass spectra (HRMS) were recorded with a Bruker microTOF ESI-TOF mass spectrometer in positive ion mode unless otherwise specified. For all experiments, cells were plated at the same initial density of 3.6 × 10 4 cells/35 mm Petri dish. To determine a good test concentration, all compounds were tested at various concentrations ranging from 1 pM to 1 uM. It was only at the highest concentration of compounds 8d and 8h that any evidence of cell death was observed, and that was most probably due to a higher concentration of DMSO (due to low solubility). Cells were treated without (control cells), or with Angiotensin II (100 nM) or with compound 8d (100 nM), 8h (100 nM), 9h (10 and 100 nM), 8i (10 and 100 nM), 8j (100 nM), 8k (100 nM), or 8l (10 and 100 nM) in the absence or in the presence of PD-123,319 (10 uM), an AT2 receptor antagonist. The antagonist was introduced daily 30 min prior to Ang II, compound 8d, 8i, 8k, 8l, 8h or 9h, to evaluate antagonistic properties.
Cells were examined under a phase contrast microscope, and micrographs were taken after 3 days under the various experimental conditions. Cells with at least one neurite longer than a cell body were counted as positive for neurite outgrowth. The number of cells with neurites represents the percentage of the total amount of cells in the micrographs. At least three different experiments were conducted for each condition, each in duplicate [33] . At least five images were taken per petri dish. Hence, a total of 250−400 cells from each of the duplicate dishes were examined.