BAY-069, a Novel (Trifluoromethyl)pyrimidinedione-Based BCAT1/2 Inhibitor and Chemical Probe

Abstract

The branched-chain amino acid transaminases (BCATs) are enzymes that catalyze the first reaction of catabolism of the essential branched-chain amino acids to branched-chain keto acids to form glutamate. They are known to play a key role in different cancer types. Here, we report a new structural class of BCAT1/2 inhibitors, (trifluoromethyl)pyrimidinediones, identified by a high-throughput screening campaign and subsequent optimization guided by a series of X-ray crystal structures. Our potent dual BCAT1/2 inhibitor BAY-069 displays high cellular activity and very good selectivity. Along with a negative control (BAY-771), BAY-069 was donated as a chemical probe to the Structural Genomics Consortium.

Figure 1

Previously reported BCAT inhibitors. The activities listed are taken from the literature.^,,

Figure 2

Biochemical BCAT1 HTS assay and hit selection.

Figure 3

Co-crystal structures of human BCAT1 in complex with 1, 2, and substrate-mimic 3PP. (A) HTS hits 1 (left) and 2 (right), with IC₅₀ values from the biochemical assays. (B) Superimposition of the crystal structures of BCAT1 in complex with 1 (PDB 7NWB chain A, ligand carbon atoms in green, protein carbon atoms in gray, and cofactor PLP in magenta) and in complex with 3PP (PDB 7NTR, for clarity, only the ligand is shown, with carbon atoms in yellow). (C) Same superimposition as B, now rotated and with the molecular surface to show the view from the solvent into the deep active site cavity. (D) Same view as B into the active site of BCAT1 in complex with 2 (chain A, ligand carbon atoms in cyan, protein carbon atoms in gray, and cofactor PLP in magenta (PDB 7NWC). (E) Superimposition of the co-crystal structures of BCAT1 with 1 and 2 (surface only shown for 1).

Figure 4

Co-crystal structures of BCAT1 in complex with the 5′-fluoro analogue of 21 (carbon atoms in green, PDB accession code 7NXN) and in complex with the 5′-fluoro analogue of 24 (carbon atoms of protein in gray and of inhibitor in orange, PDB accession code 7NXO), shown from two different orientations (A,B). The carbon atoms of the cofactor PLP are depicted in magenta. Shown are the active sites in the B chains. For clarity, the molecular surface is shown only in chain A and only for 24.

Figure 5

Hybrid design of compound 38. (A) Structure of compound 38, combining substructural elements of compound B¹³ with the trifluoromethyl-pyrimidinedione core, exemplified by compound 12. (B) Superposition of two X-ray co-crystal structures that inspired the design: Co-crystal structure of BCAT2 with the compound B¹³ (PDB accession code 5HNE, carbon atoms in wheat) and BCAT1 structure in complex with 12 (chain A, ligand carbon atoms in cyan, PDB 7NWM). (C) Co-crystal structure of BCAT1 with hybrid compound 38 (PDB accession code 7NY9), protein carbon atoms in gray and inhibitor carbon atoms in yellow).

Figure 6

(A): X-ray co-crystal structures of BCAT1 in complex with 35a (PDB accession code 7NY2, inhibitor carbon atoms in blue). (B) X-ray co-crystal structure of 36a (PDB accession code 7NYA, inhibitor carbon atoms in green).

Scheme 1. Synthetic Route for Compound 36

Reagents and conditions: (a) o-cresol, CuCl, Cs₂CO₃, 2,2,6,6-tetramethylheptane-3,5-dione, 1-methyl-2-pyrrolidinone (NMP), 120 °C, overnight, and 23%; (b) CuCl₂, ^tBuONO, MeCN, 70 °C, 2 h, and 45%; (c) Fe, AcOH, rt, 2 h, and 74%; (d) methyl chloroformate, pyridine, rt, 4 h, and 56%; (e) (i) NaH, ethyl 3-amino-4,4,4-trifluorobut-2-enoate DMF, 0 °C, and 10 min; and (ii) addition of 42, 100 °C, 24 h, and 38%.