SAR studies of 4-pyridyl heterocyclic anilines that selectively induce autophagic cell death in von Hippel-Lindau-deficient renal cell carcinoma cells

Abstract

We recently identified a class of pyridyl aniline thiazoles (PAT) that displayed selective cytotoxicity for von Hippel-Lindau (VHL) deficient renal cell carcinoma (RCC) cells in vitro and in vivo. Structure-activity relationship (SAR) studies were used to develop a comparative molecular field analysis (CoMFA) model that related VHL-selective potency to the three-dimensional arrangement of chemical features of the chemotype. We now report the further molecular alignment-guided exploration of the chemotype to discover potent and selective PAT analogues. The contribution of the central thiazole ring was explored using a series of five- and six-membered ring heterocyclic replacements to vary the electronic and steric interactions in the central unit. We also explored a positive steric CoMFA contour adjacent to the pyridyl ring using Pd-catalysed cross-coupling Suzuki-Miyaura, Sonogashira and nucleophilic displacement reactions to prepare of a series of aryl-, alkynyl-, alkoxy- and alkylamino-substituted pyridines, respectively. In vitro potency and selectivity were determined using paired RCC cell lines: the VHL-null cell line RCC4 and the VHL-positive cell line RCC4-VHL. Active analogues selectively induced autophagy in RCC4 cells. We have used the new SAR data to further develop the CoMFA model, and compared this to a 2D-QSAR method. Our progress towards realising the therapeutic potential of this chemotype as a targeted cytotoxic therapy for the treatment of RCC by exploiting the absence of the VHL tumour suppressor gene is reported.

Figure 1. PAT chemotype and biologically relevant alignment

Figure 2. New biologically relevant alignment

Figure 3

Induction of Autophagy by PATs in RCC4 Cells ^{a a} RCC4 cells, 20×, after 5 μM drug exposure for 24 h.

Scheme 1

Reagents and conditions: (a) KOH, NEt₃, EtOH, 80 °C; (b) NaOMe, MeOH, then NH₄Cl; (c) 3-methylphenyl isothiocyanate, iPr₂NEt, DMF, then DIAD; (d) MeI, EtOH; (e) 4-pyridinecarbohydrazide, pyridine; (f) MeNH₂, EtOH; (g) NH₂-NH₂·H₂O, EtOH; (h) isonicotinic acid, EDCI, DCM. $ = commercially available.

Scheme 2

Reagents and conditions: (a) DMF-DMA, reflux; (b) 56, NaOH, iPrOH, reflux; (c) Pd(PPh₃)₄, 4-pyridineboronic acid, K₂CO₃, 1,4-dioxane, 80 °C; (d) m-toluidine, p-TsOH, 1,4-dioxane, reflux; (e) m-toluidine, Pd(OAc)₂, BINAP, NaOtBu, toluene, DMF, 90 °C; (f) m-bromotoluene, Pd(OAc)₂, BINAP, NaOtBu, toluene, DMF, 90 °C. $ = commercially available.

Scheme 3

Reagents and conditions: (a) (COCl)₂, DMF, DCM; (b) Me(OMe)NH·HCl, NEt₃, DCM; (c) MeMgBr, THF; (d) Br₂, HOAc/HBr; (e) arylthiourea 58, EtOH; (f) TMSCH₂N₂, THF; then HBr. $ = commercially available.

Scheme 4

Reagents and conditions: (a) CuI, NaI, H₂N(CH₂)₂NH₂, 1,4-dioxane, 110 °C; (b) PdCl₂(dppf), Ar-boronic acid or ester, K₂CO₃, Tol/EtOH/H₂O/DMF, 90 °C; (c) PdCl₂(PPh₃)₂, CuI, HC≡C-R, NEt₃/DMF, 50–70 °C; (d) Boc₂O, DMAP, DCM; (e) i) MsCl, NEt₃, DCM, −20 °C, ii) morpholine or N-methylpiperazine, K₂CO₃, DMF, iii) TFA, DCM; (f) CuSO₄, Na ascorbate, BnN₃, DCM/EtOH/H₂O, 20 °C; (g) ROH or RNH₂, neat, Cs₂CO₃, 160–180 °C.