Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy

Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We show that the clinically used ebselen and related analogues promote thermal stability of A4V SOD1 when binding to Cys111 only. We have developed a A4V SOD1 differential scanning fluorescence-based assay on a C6S mutation background that is effective in assessing suitability of compounds. Crystallographic data show that the selenium atom of these compounds binds covalently to A4V SOD1 at Cys111 at the dimer interface, resulting in stabilisation. This together with chemical amenability for hit expansion of ebselen and its on-target SOD1 pharmacological chaperone activity holds remarkable promise for structure-based therapeutics for MND using ebselen as a template.

The figures are mean ± standard deviation of symmetrical bond distances in all dimer interfaces in the asymmetric unit.

Supplementary table 2.
Distance between Cα of the residues at the dimer interface.
The figures are mean ± standard deviation of the distances of all dimer interfaces in the asymmetric unit.
Highlights are the distances of the residues in loopVI between two monomers of the C2 A4V SOD1 structures. The starting boronic acids for compounds 9-10 were not available and therefore were synthesised in by S N 2 chemistry to afford the desired boronic acids in good to excellent yields. These were subjected to the Suzuki conditions to afford the biaryl aniline intermediates as shown in Scheme S3.

General procedure 2: Benzisoselenazolone formation via CuI ring closure
A solution of copper iodide (0.2-1.0 eq) and 1,10-phenanthroline (0.2-1.0 eq) in DMF (30 mL/g) was stirred under nitrogen for 15 minutes (dark orange/brown solution). The required amide (1 eq), selenium (1.2 eq) and potassium carbonate (1.5 eq) were then added before heating to 110°C for 36 hours under a nitrogen atmosphere. The reaction mixture was then stirred in brine for 3 hours before extracting with ethyl acetate (3 x 30mL). The combined organic extracts were then dried over anhydrous MgSO 4 , filtered and concentrated under reduced pressure. The crude products were purified by flash chromatography.

General Procedure 3: Suzuki couplings
To a mixture of THF (1.5M) and water (1.5M) was added 3-bromoaniline (3 eq) and potassium carbonate (3 eq) the flask was evacuated and backfilled with N 2 . The selected boronic acid (1 eq) was added, the flask evacuated and backfilled with N 2 . Finally palladium tetrakis (2.5 mol%) was added and the reaction vessel was evacuated and backfilled with N 2 before refluxing at 80 °C overnight. The reaction mixture was diluted with brine and product extracted into ethyl acetate. The Organic phases were dried with MgSO 4 and product purified by FCC.

General Procedure 4: Preparation of amine coupled aryl side chains
To (4-(bromomethyl)phenyl)boronic acid (1 eq) was added THF (0.5M), DIPEA (1.2 eq) and selected amine (1.2-1.5 eq). The reaction was stirred at room temperature under a N 2 atmosphere overnight. The reaction was diluted with brine and extracted into ethyl acetate (x 3), dried with MgSO 4 and solvents removed in vacuo to yield the desired product. No further purification.

General Procedure 5-Amide coupling conditions for final products
To a flask at room temperature was added 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetic acid (1.0 eq), DMF (5 ml) EDC.HCl (1.2 eq) and pyridine (2.0 eq). The reaction mixture was left to stir for 15 minutes followed by the addition of the desired amine (1.0 eq) and further pyridine (2.0 eq.) and the reaction stirred overnight at room temperature. The reaction was then diluted with ethyl acetate, acidified with 1 M HCl, basified with 1 M NaOH, washed with water, brine, dried over MgSO 4 and concentrated to yield the crude product.

General Procedure 7 -Alkylation of the BIZT core using benzyl bromides
To a flask at room temperature was added benzo[d]isothiazol-3(2H)-one (1eq), THF, potassium carbonate (2.5 eq) and the desired benzyl bromide (2.0 eq), and the reaction stirred at room temperature overnight. The reaction was then concentrated, diluted with ethyl acetate, washed with a saturated NaHCO 3 solution, water, brine, dried over MgSO 4 , concentrated to yield the crude product.
The crude product was purified by FCC (100 % EtOAc) and the residue was recrystallised from ethyl acetate to give the target as a white solid (0.088 g, 15 %
Compound used in the next step without further purification.