Preparation of von Hippel-Lindau (VHL) E3 ubiquitin ligase ligands exploiting constitutive hydroxyproline for benzylic amine protection

Abstract

The von Hippel-Lindau (VHL) protein serves as the substrate recognition subunit of the multi-subunit Cullin-2 RING E3 ubiquitin ligase (CRL2^VHL), which regulates intracellular concentrations of hypoxia inducible factors (HIFs) through a ubiquitin proteasome system (UPS) cascade. Strategic recruitment of CRL2^VHL by bi- or trifunctional targeted protein degraders (e.g., PROTACs®) offers the prospect of promoting aberrant polyubiquitination and ensuing proteasomal degradation of disease-related proteins. Non-peptidic, l-hydroxyproline-bearing VHL ligands such as VH032 (1) and its chiral benzylic amine analog Me-VH032 (2), are functional components of targeted protein degraders commonly employed for this purpose. Herein, we compare two approaches for the preparation of 1 and 2 primarily highlighting performance differences between Pd(OAc)₂ and Pd-PEPPSI-IPr for the key C-H arylation of 4-methylthiazole. Results from this comparison prompted the development of a unified, five-step route for the preparation of either VH032 (1) or Me-VH032 (2) in multigram quantities, resulting in yields of 56% and 61% for 1 and 2, respectively. Application of N-Boc-l-4-hydroxyproline rather than N-tert-butoxycarbonyl to shield the benzylic amine during the coupling step enhances step economy. Additionally, we identified previously undisclosed minor byproducts generated during arylation steps along with observations from amine deprotection and amidation reaction steps that may prove helpful not only for the preparation of 1 and 2, but for other VHL recruiting ligands, as well.

Scheme 1. Synthetic routes (A) converging to key intermediate 13 en route to VH032 (1) and (B) diverging from 19 en route to Me-VH032 (2).

Fig. 1. ¹H NMR comparison of crude 9 formed using (A) 0.1 mol% Pd(OAc)₂ and (B) 0.5 mol% Pd-PEPPSI-IPr. (C) ¹H NMR spectrum of purified 9.

Scheme 2. Initial route used to prepare VH032 (1). Reagent and conditions: (i) 0.5 mol% Pd-PEPPSI-IPr, K₂CO₃ (2 equiv.), PivOH (0.3 equiv.), DMA, 125 °C, 2 h; (ii) (a) (ⁱBu)₂AlBH₄ (1.1 equiv.), THF, 0 °C to r.t., 2 h; (b) 6 M HCl, reflux, 3 h; (iii) HATU (1.3 equiv.), DIPEA (3.5 equiv.), DMF, r.t., 19 h; (iv) (a) CH₂Cl₂ : TFA (1 : 1), 0 °C, 1 h; (b) NaOH solution until pH = 12.5–13; (v) HATU (1.3 equiv.), DIPEA (3.5 equiv.), DMF, r.t., 18 h; (vi) (a) CH₂Cl₂ : TFA (1 : 1), 0 °C, 1 h; (b) NaOH solution until pH = 12.5–13.

Fig. 2. ¹H NMR and photographic comparison of crude 10 formed using: (A) 6 mol% Pd(OAc)₂; (B) 1 mol% Pd(OAc)₂; (C) 0.5 mol% Pd-PEPPSI-IPr. Residual reaction solvent, DMA, and azeotroping solvent, toluene, are indicated.

Scheme 3. Optimized syntheses of VH032 (1) and Me-VH032 (2). Reagent and conditions: (i) EDC·HCl (1.3 equiv.), HOBt (1.3 equiv.), DIPEA (2.3 equiv.), CH₂Cl₂ : DMF (5 : 1), −10 °C to 0 °C, 1 h then 0 °C to r.t., 17 h; (ii) 0.5 mol% Pd-PEPPSI-IPr, K₂CO₃ (2 equiv.), PivOH (0.3 equiv.), DMA, 125 °C, 2.5 h; (iii) (a) CH₂Cl₂ : TFA (1 : 1), 4 °C, 1.5 h; (b) NaOH solution until pH = 12.5–13; (iii′) (a) 4 M HCl in MeOH, 4 °C, 2 h; (b) NaOH solution until pH = 12.5–13; (iv) EDC·HCl (1.3 equiv.), HOBt (1.3 equiv.), DIPEA (2.3 equiv.), CH₂Cl₂ : DMF (5 : 1), −10 °C to 0 °C, 1 h then 0 °C to r.t., 18 h; (v) (a) CH₂Cl₂ : TFA (1 : 1), 4 °C, 0.5–1 h; (b) NaOH solution until pH = 12.5–13; (v′) (a) 4 M HCl in MeOH, 4 °C, 2 h; (b) NaOH solution until pH = 12.5–13.