Discovery of Potent Orally Bioavailable WD Repeat Domain 5 (WDR5) Inhibitors Using a Pharmacophore-Based Optimization

Abstract

WD repeat domain 5 (WDR5) is a nuclear scaffolding protein that forms many biologically important multiprotein complexes. The WIN site of WDR5 represents a promising pharmacological target in a variety of human cancers. Here, we describe the optimization of our initial WDR5 WIN-site inhibitor using a structure-guided pharmacophore-based convergent strategy to improve its druglike properties and pharmacokinetic profile. The core of the previous lead remained constant while a focused SAR effort on the three pharmacophore units was combined to generate a new in vivo lead series. Importantly, this new series of compounds has picomolar binding affinity, improved cellular antiproliferative activity and selectivity, and increased kinetic aqueous solubility. They also exhibit a desirable oral pharmacokinetic profile with manageable intravenous clearance and high oral bioavailability. Thus, these new leads are useful probes toward studying the effects of WDR5 inhibition.

Figure 1.

Chemical structures and in vitro profiles of representative WDR5 WIN-site inhibitors and WDR5 PROTAC. Compound 1 is also known as 16 in our previous report.

Figure 2.

X-ray co-crystal structure of 1 bound to the WDR5 WIN site (PDB ID: 6UCS). (A) Compound 1 (yellow carbon-capped sticks) bound to WDR5 represented as semitransparent electrostatic potential surface with labeled S₂, S₄, and S₇ binding regions. (B) Key H-bond and π–π stacking binding interactions (red dashed lines) of the core unit and denoted WDR5 residues (green sticks). (C) Key H-bond and π–π stacking binding interactions (red dashed lines) of the P₂ unit in the S₂ subsite and denoted WDR5 residues (green sticks). (D) Binding interactions of the P₄ unit represented as space-filling dots in the S₄ subsite (gray surface). (E) Binding interactions of the P₇ unit represented as space-filling dots in the S₇ subsite (gray surface).

Figure 3.

Profile overview of 1 and considerations for the pharmacophore-based optimization strategy.

Figure 4.

(A) Surface representation of the WDR5 WIN site (PDB ID: 3EG6) when bound to MLL1 peptide (magenta carbon-capped lines and sticks (P₂ residue)) with labeled S₂, S₄, and S₇ binding regions. (B) Surface representation of the WDR5 WIN site (PDB ID: 4QL1) when bound to OICR-9429 (cyan carbon-capped lines and sticks (S₂ binding moiety)) with labeled S₂, S₄, and S₇ binding regions. (C) Overlay of MLL1 peptide (magenta) and OICR-9429 (cyan) bound WDR5 protein structures in cartoon representation. The side chains of WDR5 residues F133, F149, and F263 of both structures are represented as sticks to demonstrate conformational changes. (D) Overlay of MLL1 peptide (magenta) and compound 1 (green) bound WDR5 protein structures in cartoon representation. The side chains of WDR5 residues F133, F149, and F263 for both structures are represented as sticks to demonstrate retention of binding poses.

Figure 5.

X-ray co-crystal structure of 20 bound to WDR5 (PDB ID: 7U9Y). (A) Compound 20 (orange carbon-capped sticks) bound to WDR5 represented as a semitransparent electrostatic potential surface with labeled S₂, S₄, and S₇ binding regions. (B) Overlay of 20 (orange sticks) and 1 (yellow sticks; PDB ID: 6UCS). (C) Key π–π stacking binding interactions (red dashed lines) of the 2-methyl imidazole P₂ unit of 20 in the S₂ subsite with denoted WDR5 residues. (D) Overlay of MLL1 peptide (magenta sticks, PDB ID: 3EG6), 1 (green sticks, PDB ID: 6UCS), and 20 (yellow sticks) bound WDR5 protein structures in cartoon representation. The side chains of WDR5 residues F133, F149, and F263 for the structures are represented as sticks to demonstrate retention of binding poses.

Figure 6.

X-ray co-crystal structure of 37 bound to WDR5 (PDB ID: 7UAS). (A) Compound 37 (white carbon-capped sticks) bound to WDR5 represented as semitransparent electrostatic potential surface with labeled S₂, S₄, and S₇ binding regions. (B) Binding interactions of the (S)-1-cyclopropyl-1-(4-methylpyridine-2-yl)methyl P₇ unit in the S₇ subsite, pro(R)-H represented as a light orange ball. (C) Overlay of 20 (orange sticks) and 37 (white sticks).

Scheme 1.

Synthesis of Imidazole-Imine Series Compounds 2–15^{a a}R¹ is defined in Table 1. Conditions: (a) 3,5-dimethoxybenzylamine, NaBH(OAc)₃, CH₂Cl₂, rt, overnight, then 1,4-dioxane, 110 °C, 99%; (b) phenyl triflimide, i-Pr₂NEt, THF/CH₂Cl₂, rt, overnight, 79%; (c) B₂Pin₂, KOAc, PdCl₂(dppf)·CH₂Cl₂, 1,4-dioxane, 100 °C overnight, 99%; (d) CuBr₂, MeOH/H₂O, 80 °C, 90%; (e) LiBHEt₃ (1 M THF), THF, 0 °C, 1 h, 41%; (f) PBr₃, CH₂Cl₂, 0 °C, 42%; (g) 1-methyl-1H-imidazol-2-amine hydrochloride, i-Pr₂NEt, MeCN, 60 °C, overnight, quant.; (h) R¹–B(OH)₂ or R¹–B(Pin), PdCl₂(dppf)·CH₂Cl₂, K₂CO₃, 1,4-dioxane/H₂O, 80 °C, overnight, 20–70%.

Scheme 2.

Synthesis of Nonbasic Warhead Series Compounds 16–24^{a a}R² is defined in Table 2. Conditions: (a) (1-methyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)boronic acid, PdCl₂(dppf)·CH₂Cl₂, K₂CO₃, 1,4-dioxane/H₂O, 90 °C, overnight, 85%; (b) LiBHEt₃ (1 M THF), THF, 0 °C, 1 h, 95%; (c) PBr₃, CH₂Cl₂, 0 °C, 90%; (d) R²–azole, MeCN, 50 °C, overnight, 45–60%.

Scheme 3.

Synthesis of Pyridyl Series Compounds 25–33^{a a}R³ and R⁴ are defined in Table 3. Synthesis of 58 was reported in Scheme S1. Conditions: (a) R³-boronic acid, PdCl₂(dppf)·CH₂Cl₂, K₂CO₃, 1,4-dioxane/H₂O, 90 °C, overnight, 90%; (b) TFA, anisole, CH₂Cl₂, rt, overnight, 87%; (c) LiBHEt₃ (1 M THF), THF, 0 °C, 1 h, quant.; (d) PBr₃, CH₂Cl₂, 0 °C, quant.; (e) 2-methyl-1H-imidazole, MeCN, 50 °C, overnight, 70%; (f) R⁴-pyridylmethyl halide, NaH, DMF, 0 °C, 2 h, 25–71%.

Scheme 4.

Synthesis of Compounds 34, 35, 38, and 39^{a a}R⁶ is defined in Table 4. Conditions: (a) (S)- or (R)-2-methylpropane-2-sulfinamide, Cs₂CO₃, CH₂Cl₂, rt, overnight, 65%; (b) MeMgBr (3.4 M 2-MeTHF), THF, −78 °C, 84–92%; (c) HCl (4 M 1,4-dioxane), THF, 94%—quant.; (d) chiral amine, i-Pr₂NEt, NaBH(OAc)₃, CH₂Cl₂, rt, overnight, then 1,4-dioxane, 110 °C, 84%; (e) phenyl triflimide, i-Pr₂NEt, THF/CH₂Cl₂, rt, overnight, 93%; (f) (1-ethyl-3-(trifluoromethyl)-1H-pyrazol-4-yl)boronic acid, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane/H₂O, 80 °C, 96%; (g) LiBH₄ (2 M THF) or LiBHEt₃ (1 M THF), THF, 0 °C, 1 h, 72–85%; (h) CH₃SO₂Cl, i-Pr₂NEt, CH₂Cl₂, 0 °C, then LiBr, THF, reflux, 53% or PBr₃, CH₂Cl₂, 0 °C, 98%; (i) R⁶-imidazole, MeCN, 50 °C, overnight, 73%.