Synthesis and Biochemical Evaluation of Warhead-Decorated Psoralens as (Immuno)Proteasome Inhibitors

Abstract

The immunoproteasome is a multicatalytic protease that is predominantly expressed in cells of hematopoietic origin. Its elevated expression has been associated with autoimmune diseases, various types of cancer, and inflammatory diseases. Selective inhibition of its catalytic activities is therefore a viable approach for the treatment of these diseases. However, the development of immunoproteasome-selective inhibitors with non-peptidic scaffolds remains a challenging task. We previously reported 7H-furo[3,2-g]chromen-7-one (psoralen)-based compounds with an oxathiazolone warhead as selective inhibitors of the chymotrypsin-like (β5i) subunit of immunoproteasome. Here, we describe the influence of the electrophilic warhead variations at position 3 of the psoralen core on the inhibitory potencies. Despite mapping the chemical space with different warheads, all compounds showed decreased inhibition of the β5i subunit of immunoproteasome in comparison to the parent oxathiazolone-based compound. Although suboptimal, these results provide crucial information about structure-activity relationships that will serve as guidance for the further design of (immuno)proteasome inhibitors.

Keywords: electrophilic compounds; immunoproteasome; non-peptidic; psoralen core; warhead scan.

Figure 1

Structures of the most studied iCP-selective peptidic inhibitors. For a more thorough overview on subunit-selective iCP inhibitors, the reader is referred to recent reviews [32,35].

Figure 2

A selection of non-peptidic iCP inhibitors. ‘Compound 42’ [39] was the most selective irreversible β5i subunit inhibitor from the initial series of psoralen-based inhibitors. It represents the parent compound for studies in this manuscript.

Figure 3

Schematic representation of the work described in this study. The numbering system for the psoralen ring is shown for clarity, as well as general nomenclature for the warhead moieties used.

Scheme 1

Synthesis of compounds with allyl (4) and vinyl (7) warheads attached at position 3 of the psoralen ring. Reagents and conditions: (a) allyl bromide, NaH (60%), THF, 0 °C to rt, overnight; (b) resorcinol, 98% H₂SO₄, dioxane, 0 °C to rt, overnight; (c) 2-bromoacetophenone, K₂CO₃, KI, dioxane, 100 °C, 24 h; (d) 1 M NaOH, propan-2-ol, 80 °C, 40 min; (e) crotonyl chloride, K₂CO₃, acetone, 60 °C, 24 h; (f) 1 M KOH, EtOH, 85 °C, 2 h.

Scheme 2

Synthesis of compounds 8, 9, 10, and 11 with ketone, vinyl sulfone, 3-bromo-4,5-dihydroisoxazole, and pinacolate ester, respectively, as warheads. Reagents and conditions: (a) Dess–Martin periodinane, Pd(OAc)₂, CH₃CN, H₂O, 50 °C, overnight; (b) DMSO, H₂O, NH₄I, 130 °C, 36 h; (c) 1,1-dibromoformaldoxime, DMF, NaHCO₃, −15 °C to rt, 5 h; (d) bis(pinacolato)diboron, CsF, 1,4-dioxane, MeOH, 100 °C, 12 h.

Scheme 3

Synthesis of compounds with aldehyde- (15) and α,β-unsaturated aldehyde-based (16) warheads attached at position 3 of the psoralen ring. Reagents and conditions: (a) 1 M HCl, dioxane, reflux, 2 h; (b) i. SOCl₂, DMF, toluene, rt, 17 h; ii. H₂, Pd/BaSO₄, toluene, 100 °C, 2 h; (c) 1 M NaOH, propan-2-ol, 60 °C, 15 min; (d) SeO₂, dioxane, H₂O, MW, 150 °C, 1 h. Synthesis of compound 12 was described previously [39].

Figure 4

COSY experiment for 16. Circled cross-peaks indicate coupling between aldehyde proton CHO and the adjacent C2′-H, and between C2′-H and C3′-H.

Figure 5

NOESY experiment for 16. Only cross-peaks that indicate coupling between CH₃ protons and C4-H and C3′-H are shown.

Scheme 4

Synthesis of epoxyketone-based compounds 20 and 21. Reagents and conditions: (a) i. TFA, CH₂Cl₂, 0 °C, 30 min; ii. HATU, HOBt×H₂O, DIPEA, DMF (20) or CH₂Cl₂ (for 21), rt, 24 h. Syntheses of compounds 17, 18, and 19 were based on previously described procedures [39], compound 17; [47], compound 18; [48], compound 19. All spectral data (¹H-NMR, HRMS) corresponded well to the original reports.

Scheme 5

Synthesis of alkyl bromide-based psoralen 23 and psoralen 24 with azetidin-2-one as a warhead. Reagents and conditions: (a) 3-bromopropanoyl chloride, K₂CO₃, CH₂Cl₂, 0 °C to rt, 3 h; (b) NaOtBu, DMF, 0 °C to rt, 24 h. Synthesis of compound 22 was described previously [39].

Figure 6

Inhibition results represented as bar charts of inhibition percentage. carf.: carfilzomib.

Figure 7

Molecular modelling. Binding site residues are presented as green sticks with labels for some of the key residues. (A) Covalent docking of ‘42′ (magenta) into the β5i subunit (PDB: 5M2B). Please note that only the initial intermediate formed after the nucleophilic attack of OH group of Thr1 onto the carbonyl group of the oxathiazolone is represented. Co-crystalized ligand Ro19 is presented with blue sticks and dashed yellow lines for hydrogen bonds. (B) Noncovalent docking of 10 (cyan) and 16 (yellow) reveals good alignment of the psoralen core with the proposed pose of ‘42′ (magenta). However, the distance from the electrophilic carbons of 10 and 16 to the catalytic Thr1O^γ is too large to form a covalent bond.