LP99: Discovery and Synthesis of the First Selective BRD7/9 Bromodomain Inhibitor

Abstract

The bromodomain-containing proteins BRD9 and BRD7 are part of the human SWI/SNF chromatin-remodeling complexes BAF and PBAF. To date, no selective inhibitor for BRD7/9 has been reported despite its potential value as a biological tool or as a lead for future therapeutics. The quinolone-fused lactam LP99 is now reported as the first potent and selective inhibitor of the BRD7 and BRD9 bromodomains. Development of LP99 from a fragment hit was expedited through balancing structure-based inhibitor design and biophysical characterization against tractable chemical synthesis: Complexity-building nitro-Mannich/lactamization cascade processes allowed for early structure-activity relationship studies whereas an enantioselective organocatalytic nitro-Mannich reaction enabled the synthesis of the lead scaffold in enantioenriched form and on scale. This epigenetic probe was shown to inhibit the association of BRD7 and BRD9 to acetylated histones in vitro and in cells. Moreover, LP99 was used to demonstrate that BRD7/9 plays a role in regulating pro-inflammatory cytokine secretion.

Keywords: bromodomain; cascade reactions; enantioselective catalysis; epigenetics; organocatalysis.

Figure 1

Fragment hit for BRD9. A) 1 (pale sticks) binds ATAD2 BRD via H bonds (dotted lines) to N1064 and conserved water molecules (red spheres; PDB 4QST). B) Electrostatic surface representation of BRD9 overlaid with 1 and conserved water molecules from ATAD2 for reference. The black arrow in (B) indicates the attachment point targeted for the design of selective inhibitors.

Figure 2

A) Co-crystal structure of compound 6 (yellow sticks) and BRD9 BRD (grey sticks and red ribbon) with the ligand electron density (2FoFc, blue mesh). B) Compound 6 binds to BRD9 with a K_D value of 612 nm according to ITC analysis.

Scheme 1

Synthesis of heterocycle-substituted quinolones. a) ethyl acetoacetate, xylenes, reflux, 24 %; b) H₂SO₄, 88 %; c) NaH, MeI, DMF, 76 %; d) heterocycle, [Pd₂(dba)₃], Xantphos, Cs₂CO₃, 1,4-dioxane, 100 °C, 5–96 %. dba=dibenzylideneacetone, Xantphos=4,5-bis(diphenylphosphanyl)-9,9-dimethylxanthene.

Scheme 2

Synthesis of analogues for SAR studies around the lead scaffold. Reagents and conditions: a) R³CHO, NH₄OAc, EtOH, 90 °C, 26–86 %, d.r. 2:1–>20:1; b) NiCl₂⋅6 H₂O, NaBH₄, MeOH, 0 °C; c) Boc₂O, 39–91 % (2 steps); d) 4, [Pd₂(dba)₃], Xantphos, Cs₂CO₃, 1,4-dioxane, 100 °C, 2–75 %; e) 4, K₃PO₄, CuI, (±)-trans-1,2-diaminocyclohexane, 1,4-dioxane, 97 °C, 7–65 %; f) HCl/dioxane, 96 %; g) R²Cl, TEA, CH₂Cl₂ or RNCO, CH₂Cl₂, 12–60 %. Boc=tert-butyloxycarbonyl, TFA=trifluoroacetic acid.

Scheme 3

Organocatalytic enantioselective synthesis of BRD9 inhibitors. Reagents and conditions: a) 11, K₂CO₃, 68 (10 mol %), TBME, −20 °C, 70 %, d.r. 7:1, ee_major 90 %/ee_minor 90 %; b) TFA, CH₂Cl₂; c) DBU, CH₂Cl₂, 73 % (2 steps); d) NiCl₂⋅6 H₂O, NaBH₄, MeOH, 0 °C; e) Boc₂O, 74 % (2 steps); f) 4, K₃PO₄, CuI, (±)-trans-1,2-diaminocyclohexane, 1,4-dioxane, 97 °C, 65 %; g) HCl/dioxane, 96 %; h) RCl, TEA, CH₂Cl₂ or RNCO, CH₂Cl₂, 25–40 %. DBU=1,8-diazabicyclo[5.4.0]undec-7-ene, TBE=tert-butyl methyl ether, TEA=triethylamine.

Figure 3

LP99 is a potent and selective BRD7/9 inhibitor. Selectivity panel of LP99 against 48 BRDs (bold type) at 10 μm in terms of the ΔT_m values determined by DSF.

Figure 4

LP99 is active in cellular assays. A) BRET assay of LP99 on BRD7 and BRD9 fusion proteins. B) LP99 inhibits the expression of IL6 in LPS-stimulated THP-1 cells.