Carboxylic Acid Bioisosteres Boost Nurr1 Agonist Selectivity

Abstract

Nuclear receptor related 1 (Nurr1) is a neuronal ligand-activated transcription factor implicated in neurodegenerative diseases including Alzheimer's disease, Parkinson's disease and multiple sclerosis, which has fueled the development of Nurr1 modulators. Among them, the clinically studied dihydroorotate dehydrogenase (DHODH) inhibitor vidofludimus was found to exhibit strong Nurr1 agonism. Here, we aimed to establish a vidofludimus-derived Nurr1 agonist lacking DHODH inhibitor potency as a tool. We explored bioisosteric replacement of the drug's carboxylate motif and succeeded in boosting selectivity for Nurr1 over DHODH to >100-fold. Dopaminergic neural cells treated with the optimized tetrazole-based Nurr1 agonist revealed induction of genes involved in neuroprotection and neuronal health, supporting the potential of Nurr1 activation in neurodegenerative diseases.

Figure 1. In vitro profiling of 32.

(a) Activity of 32 on NR4A receptors. Data are the mean±S.E.M. fold activation from Gal4 hybrid reporter gene assays; n ≥ 3. (b) Binding of 32 to the Nurr1 LBD in isothermal titration calorimetry (ITC) with a K_d value of 0.2 µM. The upper panel shows the isotherm of the 32-protein titration; the lower panel shows the fitting of the heat of binding. (c) 32 revealed no relevant activity on lipid-sensing and promiscuous nuclear receptors at 1 µM. The heatmap shows the mean relative activation [%] compared to reference agonists.

Figure 2

Impact of 32 on Nurr1 dimerization in a homogenous time-resolved fluorescence resonance energy transfer (HTRF) based assay monitoring the interaction of Tb³⁺-cryptate labeled Nurr1 LBD (FRET donor) and sGFP labeled Nurr1 LBD (FRET acceptor). Data are the mean±S.E.M. ΔHTRF; n = 3.

Figure 3. Effects of 32 on mRNA expression in rat dopaminergic neuronal cells (N27).

TH−tyrosine hydroxylase, SOD2−superoxide dismutase 2, Sesn3−sestrin 3, BIRC5−baculoviral inhibitor of apoptosis repeat-containing 5 (also termed survivin), XIAP−X-linked inhibitor of apoptosis, BDNF−brain-derived neurotrophic factor, CCND2−cyclin D2, FLRT2−fibronectin leucine rich transmembrane protein 2, CRMP4−collapsin response mediator protein 4. mRNA levels were referenced to GAPDH and analyzed by the 2^-ΔCt method. Boxplots are min.-max.; n = 6; ^* p <0.05, ^** p <0.001, ^*** p <0.0001 (ANOVA with Dunnett’s multiple comparisons test).

Scheme 1. Synthesis of 13–16.^a

^a Reagents&Conditions: (a) 12c, 15b with oxalyl chloride, 0°C to rt, 2 h, evap.; NaH, THF, 10 min, 0°C, then acid chloride, 0°C to rt, 6 h, 49%; (b) n-BuLi (2.5 M in THF), THF, –78°C, 30 min, then 1,4-diazabicyclo[2.2.2]octane sulfur dioxide complex, –78°C, 30 min, 23%; (c) hydroxylamine-O-sulfonic acid, NaOAc, MeCN/H₂O (1:1), rt, 16 h, 36%; (d) cat. ZnCl₂, Ac₂O, rt, 1 h, 50 %; (e) m-CPBA, CH₂Cl₂, rt, 1 h, 8%; (f) (NH₄)₂CO₃, (diacetoxyiodo)benzol, MeOH, rt, overnight, 24%.

Scheme 2. Synthesis of 17

^a Reagents&Conditions: (a) n-BuLi (2.5 M in THF), THF, –78°C, 30 min, then 3,4-diisopropoxycyclobut-3-ene-1,2-dione, –78°C, 30 min, 40%; (b) AcOH/1N aq. HCl (6:1), rt, overnight, 32%.

Scheme 3. Synthesis of 18 and 21.^a

^a Reagents&Conditions: (a) NH₄Cl, EDC, DMAP, CH₂Cl₂ rt to 60°C, 3 h, 80% (18c), NH₄Cl, EDC, DMAP, DMF, rt to 60°C, overnight, 50–52% (21c, 30c); (b) cyanuric chloride, DMF, 0°C, 3 h, 70–78% (18b, 30b), Burgess reagent, THF, 0°C to rt, 94% (21b); (c) H₂NOH•HCl, Hünig's base, MeOH, 70°C, 4 h, 46% (18a), H₂NOH, EtOH/H₂O, 50°C, 5 h, 88% (21a); (d) CDI, 1,8-diaza-bicyclo[5.4.0]undec-7-en, 1,4-dioxane, 100°C, 3 h, 36% (18), CDI, DMF, 100°C, 15 h, 21% (21).

Scheme 4. Synthesis of Building Block 31a.^a

^a Reagents&Conditions: (a) 31b, SOCl₂, CH₂Cl₂, 0°C, 2 h, evap.; 12b, NaH, THF, 0°C, 30 min, then acid chloride, 0°C, 2h, 18%.

Scheme 5. Synthesis of 19, 22, 30 and 31.^a

^a Reagents&Conditions: (a) NaN₃, NH₄Cl, DMF, 125°C, overnight, 25–46% (19, 22, 30), NaN₃, Et₃N•HCl, DMF, 120°C, 4 h, 11% (31).

Scheme 6. Synthesis of 23 and 28.^a

^a Reagents&Conditions: (a) 1,1'-thiocarbonyldiimidazole, Hünig's base, DMF, 100°C, overnight, 33%; (b) SOCl₂, pyridine, CH₂Cl₂, −50°C to rt, 2 h, 29%.

Scheme 7. Synthesis of 24 and 25.^a

^a Reagents&Conditions: (a) MeHNOH•HCl, Hünig's base, EtOH, 80°C, 5 h, 54%; (b) 1,1'-carbonyldiimidazole, Hünig's base, DMF, 80°C, overnight, 62%; (c)1,1′-thiocarbonyldiimidazole, Hünig's base, DMF, 80°C, 15 h, 60%.

Scheme 8. Synthesis of 26 and 27.^a

^a Reagents&Conditions: (a) oxalyl chloride, CH₂Cl₂, cat. DMF, 0°C to 50°C, 1 h, evap., then NH₂NH₂•H₂O, Hünig's base, THF, rt, overnight, 49%; (b) 1,1'-carbonyldiimidazole, Hünig's base, DMF, rt, 4 h, 35%; (c) 1,1′-thiocarbonyldiimidazole, Hünig's base, DMF, 80°C, 15 h, 60%.

Scheme 9. Synthesis of 11.^a

^a Reagents&Conditions: (a) isobenzofuran-1,3-dione 11a, AlCl₃, CHCl₃, 70°C, 12 h, 50%.

Scheme 10. Synthesis of 32.^a

^a Reagents&Conditions: (a) oxalyl chloride, CH₂Cl₂, cat. DMF, 0°C to 50°C, 1 h, evap., then NH₃•H₂O, THF, 5°C to rt, overnight, 81%; (b) Burgess reagent, THF, 0°C to rt, 1 h, 81%; (c) NaN₃, NH₄Cl, DMF, 120°C, 15 h, 9%.

Chart 1. Nurr1 modulators

Chart 2. Design of the lead 12