Dihydroquinazolines as a novel class of Trypanosoma brucei trypanothione reductase inhibitors: discovery, synthesis, and characterization of their binding mode by protein crystallography

Abstract

Trypanothione reductase (TryR) is a genetically validated drug target in the parasite Trypanosoma brucei , the causative agent of human African trypanosomiasis. Here we report the discovery, synthesis, and development of a novel series of TryR inhibitors based on a 3,4-dihydroquinazoline scaffold. In addition, a high resolution crystal structure of TryR, alone and in complex with substrates and inhibitors from this series, is presented. This represents the first report of a high resolution complex between a noncovalent ligand and this enzyme. Structural studies revealed that upon ligand binding the enzyme undergoes a conformational change to create a new subpocket which is occupied by an aryl group on the ligand. Therefore, the inhibitor, in effect, creates its own small binding pocket within the otherwise large, solvent exposed active site. The TryR-ligand structure was subsequently used to guide the synthesis of inhibitors, including analogues that challenged the induced subpocket. This resulted in the development of inhibitors with improved potency against both TryR and T. brucei parasites in a whole cell assay.

Figure 1

The TryR-catalyzed reduction of trypanothione disulfide (T[S]₂) to dihydrotrypanothione (T[SH]₂, N¹,N⁸-bis(glutathionyl)spermidine).

Figure 2

3,4-Dihydroquinazolines 1a and 1b, small molecule inhibitors of T. cruzi and T. brucei TryR (see Table 1).

Scheme 1. General Synthetic Route to 3,4-Dihydroquinazoline Analogues (Also See Schemes S1, S3, and S4 of the Supporting Information; For Details of Individual Substituents, See Tables 2–5; Note That Products of This Route Are Racemates)

Reagents and conditions: (i) R²COCl, DMAP, pyridine, CH₂Cl₂, 25 °C, 16 h; (ii) H₂N(CH₂)_nR³, EtOH, 160 °C (MW), 2 h; (iii) NaBH₄, DMF, or EtOH, 50 °C, 16 h; (iv) POCl₃, CH₂Cl₂, 0–25 °C, 16 h; (v) EtOH, 160 °C (MW) 2 h; (vi) NaBH₄, EtOH, 78 °C, 16 h.

Scheme 2. Synthetic Routes for the Preparation of Two 3,4-Dihydroquinazoline Amide Arrays (For Additional Details, See Scheme S3 of the Supporting Information; For Details of Individual Substituents, See Table 3)

Reagents and conditions: (i) (1) HCl, dioxane/THF, 25 °C, 2 h; (2) RCOCl, pyridine, CH₂Cl₂, 40 °C, 16 h.

Scheme 3. Synthetic Routes for the Preparation of Bi-aryl 3,4-Dihydroquinazoline Analogues (See Schemes S4 and S5 of the Supporting Information for the Synthesis of 15c and 18, Respectively; For Details of Individual Substituents, See Table 4)

Reagents and conditions: (i) (HO)₂B-R^6/8, K₃PO₄, Pd(PPh₃)₄, dioxane/H₂O, 120 °C (MW), 0.5 h.

Scheme 4. Synthetic Routes for the Preparation of 3,4-Dihydroquinazoline Analogues Containing Different Substitutions at R⁴ (29a–f) (Also See Scheme S7 of the Supporting Information; For Details of Individual Substituents, See Table 5)

Reagents and conditions: (i) Ac₂O, 138 °C, 2 h; (ii) (1) R⁴Li, THF, 0 °C, 2 h; (2) TMSCl, 0–25 °C, 15 min; (iii) R⁴ MgX, Et₂O, 0–25 °C, 16 h; (iv) AcCl, DMAP, pyridine, CH₂Cl₂, 25 °C, 16 h; (v) R⁴ MgX, THF, 25 °C, 30 min, 67 °C, 2 h; (vi) H₂N(CH₂)_nCH₂R³, EtOH, 160 °C (MW), 2 h; (vii) NaBH₄, EtOH, 78 °C, 16 h.

Scheme 5. Shortened Synthetic Strategy for the Preparation of 3,4-Dihydroquinazoline Analogues Substituted at R⁴ (For Details of Individual Substituents, See Table 5)

Reagents and conditions: (i) (1) 1-(2-aminoethyl)piperidine, CH₂Cl₂, 25 °C, 1 h; (2) formamide, 130 °C (MW), 10 min; (ii) R⁴ MgCl, THF, 0–25 °C, 2–4 h; (iii) BF₃·OEt, HSiEt₃, CH₂Cl₂, 25 °C, 16 h.

Figure 3

SAR derived from screening analogues 1a–n (Table 1).

Figure 4

The X-ray protein crystal structure of 1a in complex with T. brucei TryR. (A) and (B) show two orientations, rotated about 90°, of the TryR–compound 1a complex active site showing experimental density (magenta) for the inhibitor contoured at 2.5 σ. Compound 1a is shown in blue and FAD in turquoise. Carbon atoms are shown in brown, oxygens in red, nitrogens in dark blue, and sulfurs in yellow. Important residues have been highlighted in black including Trp21 and Met113, forming the hydrophobic wall and electron donor Glu18. Those residues involved in binding the halogen component are labeled in green, and those involved in the novel hydrophobic pocket are in blue. (C) Shows the same complex with the solvent accessible surface shown in beige and the ligand in green to demonstrate where the inhibitor is occupying subpockets within the active site cleft. (D) Surface representation of TryR active site cleft with bound compound 1a. The structure of T[SH]₂ (yellow) bound to TryR from PDB entry 2wow has been overlaid.

Figure 5

The crystallographically determined binding orientations of compounds 6a, 11e, 13e, and 29a overlaid in the active site of the TryR–1a complex structure. 1a is displayed in cyan, 6a in orange, 11e in magenta, 13e in green, and 29a in yellow. The 3,4-dihydroquinazoline and phenyl ring systems overlay well, while the more variable and flexible N3-substituents point out into the active site opening in a variety of orientations.

Figure 6

Summary of the development of hit compound 1a by an initial chemistry-driven approach and subsequent structure-based inhibitor design strategy. The units for ligand efficiencies (Lig. eff.) are kcal mol^–1 per non-hydrogen atom.