Optimization of tricyclic Nec-3 necroptosis inhibitors for in vitro liver microsomal stability

Abstract

Necroptosis is a regulated caspase-independent cell death pathway with morphological features resembling passive non-regulated necrosis. Several diverse structure classes of necroptosis inhibitors have been reported to date, including a series of 3,3a,4,5-tetrahydro-2H-benz[g]indazoles (referred to as the Nec-3 series) displaying potent activity in cellular assays. However, evaluation of the tricyclic necroptosis inhibitor's stability in mouse liver microsomes indicated that they were rapidly degraded. A structure-activity relationship (SAR) study of this compound series revealed that increased liver microsomal stability could be accomplished by modification of the pendent phenyl ring and by introduction of a hydrophilic substituent (i.e., α-hydroxyl) to the acetamide at the 2-position of the tricyclic ring without significantly compromising necroptosis inhibitory activity. Further increases in microsomal stability could be achieved by utilizing the 5,5-dioxo-3-phenyl-2,3,3a,4-tetrahydro-[1]benzothiopyrano[4,3-c]pyrazoles. However, in this case necroptosis inhibitory activity was not maintained. Overall, these results provide a strategy for generating potent and metabolically stable tricyclic necrostatin analogs (e.g., 33, LDN-193191) potentially suitable for in vivo studies.

Figure 1

Necrostatins. The numbering convention of 2 is also shown.

Figure 2

Thermal ellipsoidal drawing of 20b as determined by single crystal x-ray analysis.

Scheme 1

(a) Ar(CH₂)_nCHO, 8N NaOH, EtOH, rt, 2 h or Ar(CH₂)_nCHO, conc HCl, MeOH, Δ, 4 h (60 – 75%); (b) R³CO₂H, NH₂NH₂·xH₂O, 120 °C, 15 h (70 – 80% when n = 0; 20% when n = 1); (c) H₂ (1 atm), 10% Pd/C, EtOH (47–75%); (d) MCPBA, DCM, rt, 16 h (90–96%).

Scheme 2

(a) NaN(TMS)₂, THF, 0 °C, then 4-MeO-PhC(O)Cl, 0 °C to rt (80%); (b) NaN(TMS)₂, THF, 0 °C, then MeI, 0 °C to rt (91%); (c) NH₂NH₂·xH₂O, DCM, 4Å molecular sieves (52%); (d) MeC(O)Cl, DCM, −78 °C, then NaBH(OAc)₃, −78 °C to rt (95%).