[1,2,4]triazolo[4,3-a]phthalazines: inhibitors of diverse bromodomains

Abstract

Bromodomains are gaining increasing interest as drug targets. Commercially sourced and de novo synthesized substituted [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of both the BET bromodomains such as BRD4 as well as bromodomains outside the BET family such as BRD9, CECR2, and CREBBP. This new series of compounds is the first example of submicromolar inhibitors of bromodomains outside the BET subfamily. Representative compounds are active in cells exhibiting potent cellular inhibition activity in a FRAP model of CREBBP and chromatin association. The compounds described are valuable starting points for discovery of selective bromodomain inhibitors and inhibitors with mixed bromodomain pharmacology.

Figure 1

(A) Triazole-containing BET inhibitors. (B) The bromodomain family is made of eight subfamilies (large italic). Family members screened in this work are shown in larger typeface.

Figure 2

Commercial [1,2,4]triazolo[4,3-a]phthalazines are potent inhibitors of multiple bromodomains by DSF screening.

Scheme 1. The Synthesis of Compounds 13 and 24–33

Reagents and conditions: (a) NH₂NHAc, nBuOH, reflux (41%); (b) NH₂NH₂, THF; (c) R₅CO₂H, p-dioxane, reflux (32–42%); (d) Boc-glycine, THF, reflux (39%); (e) 35, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O (29–80%); (f) HCl, EtOAc (100%); (g) 36, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O (25%); (h) (i) 37, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O, (ii) SnCl₂, EtOH, reflux, (iii) PhSO₂Cl, pyridine, THF (16% over 3 steps); (i) 39, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O (35%); (j) (i) 38 or 40, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O, (ii) KOH, MeOH (39–79% over 2 steps).

Figure 3

Docking of compound 17 (orange ball and stick) in bromodomain of CREBBP (PDB ID, 3SVH; protein, green ribbon and sticks; water molecules, red and blue spheres; top loop removed for clarity in first image). The triazole moiety forms two H-bonds (dashed line) to a conserved water (red sphere) and asparagine 1168 (N1168). The sulfonamide accepts two H-bonds from arginine 1173 (R1173).

Figure 4

Synthetic inhibitors of multiple bromodomains.

Scheme 2. The Synthesis of Compounds 49–57

Reagents and conditions: (a) morpholine, iPrOH (100%); (b) 4-methylpiperazine, Et₃N, iPrOH (100%); (c) (BOpin)₂, Pd(dppf)Cl₂, KOAc, p-dioxane/DMSO (47–72%); (d) 19, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O (32–50%); (e) SnCl₂, EtOH, reflux (26–100%); (f) RSO₂Cl, Et₃N, p-dioxane or RSO₂Cl, pyridine, DCM (47–81%); (g) 22, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O (62%); (h) (i) 23, Pd(PPh₃)₄, K₂CO₃, p-dioxane/H₂O, (ii) HCl, EtOAc (60%).

Figure 5

Triazolophthalazines with para-aminophenyl substituents are potent bromodomain inhibitors.

Figure 6

(A) Compound 51 (green stick) in complex with BRD4(1) (PDB ID: 4NQM, blue ribbon and stick, top loop removed for clarity in first image) shows H-bonds (dashed lines) between the triazole moiety, the conserved asparagine (N140), and a pocket water (red sphere). The anionic sulfonamide forms an additional hydrogen bond to tryptophan (W81). (B) In complex with BRD9 (PDB ID: 4NQN, yellow ribbon and stick (the ZA loop was removed for clarity in first image)), compound 51 forms H-bonds to N100 and water but acts as an H-bond acceptor via a sulfonamide oxygen to Y106. The electron density map from the X-ray refinement is shown as a dark-blue mesh around the ligand.

Figure 7

(A) Cells transfected with a trimerized CREBBP-BRD-GFP construct show rapid recovery of fluorescent intensity after photobleaching (FRAP) (black). Recovery time is increased by pretreating cells with 2.5 μM SAHA* (green) and restored by transfecting with incompetent mutant protein (N1168F, red). (B) Cells treated with SAHA^a (2.5 μM) and compounds 50 (blue), 51 (yellow), 53 (purple), and 55 (red) (1 μM) show increased recovery rates. (C) Recovery half-lives of transfected (black), SAHA treated (green), and SAHA plus compound treated cells. (D) Fluorescent images of cells show rapid recovery of photobleached area (red circle) after compound treatment. ^aSAHA treated cells; ^bN1168F, mutation of N1168 to Phe.