Discovery of NCT-501, a Potent and Selective Theophylline-Based Inhibitor of Aldehyde Dehydrogenase 1A1 (ALDH1A1)

Abstract

Aldehyde dehydrogenases (ALDHs) metabolize reactive aldehydes and possess important physiological and toxicological functions in areas such as CNS, metabolic disorders, and cancers. Increased ALDH (e.g., ALDH1A1) gene expression and catalytic activity are vital biomarkers in a number of malignancies and cancer stem cells, highlighting the need for the identification and development of small molecule ALDH inhibitors. A new series of theophylline-based analogs as potent ALDH1A1 inhibitors is described. The optimization of hits identified from a quantitative high throughput screening (qHTS) campaign led to analogs with improved potency and early ADME properties. This chemotype exhibits highly selective inhibition against ALDH1A1 over ALDH3A1, ALDH1B1, and ALDH2 isozymes as well as other dehydrogenases such as HPGD and HSD17β4. Moreover, the pharmacokinetic evaluation of selected analog 64 (NCT-501) is also highlighted.

Figure 1

Representative small molecule ALDH1A1 inhibitors and theophylline-based qHTS hits.

Figure 2

Rapid dilution assay. The experiment was conducted using 64 as the inhibitor in the presence of ALDH1A1 (20 nM), substrate (propionaldehyde, 80 µM), and cofactor (NAD⁺, 1 mM) in 1536-well format.

Scheme 1. Synthetic Routes Assessing Theophylline-Based ALDH1A1 Inhibitorsa

^aReagents and conditions: (a) R¹-Br, K₂CO₃, DMF, rt to 60 °C, 1–24 h, 8 (98%), 9 (95%), 10 (99%); (b) 1-ethoxycarbonylpiperazine, DMSO, microwave, 160 °C, 30 min, 11 (58%); (c) methyl 4-hydroxybenzoate, K₂CO₃, DMF, microwave, 160 °C, 1 h, 66–83%; (d) 1.5 N LiOH_(aq), THF/MeOH, 50 °C, 2–3 h, 48–70%; (e) MeNH₂ (2 M in THF), HATU, DMF, (i-Pr)₂NEt, rt, 2 h, 46–59%; (f) N-Boc-4-hydroxypiperidine or N-Boc-4-hydroxyazetidine (for 14–16 and 18), NaH, DMF, rt, 0.5–2 h, 81–96%; or N-Boc-4-aminopiperidine (for 17), DMSO, microwave, 160 °C, 45 min, 66%; (g) 4 M HCl in dioxane, rt, 3–24 h, 90–99%; (h) 2-chloro-4-methylpyrimidine or 2-bromo-5-methylthiazole, K₂CO₃, DMF, microwave, 160 °C, 1 h; (i) corresponding substituted carbonyl chloride, Et₃N, CH₂Cl₂, rt, 0.5–1 h, for 26–27, 29–34, and 36–38; or ethyl isocyanate, Et₃N, CH₂Cl₂, rt, 0.5 h, for 28; or cyclopropanesulfonyl chloride, Et₃N, CH₂Cl₂, rt, 0.5 h, for 35; (j) 16, cyclopropanecarbonyl chloride, Et₃N, CH₂Cl₂, rt, 0.5 h, 21 (96%); (k) Pd(OH)₂ (20 mol %), EtOAc/EtOH, 70 °C, 24 h, 22 (75%); (l) corresponding alkyl or benzyl bromide or chloride, K₂CO₃, DMF, 50–−60 °C, 3–24 h, for 39–52; or corresponding hydroxyl compounds, ^tBuO₂CN═NCO₂ ^tBu, PPh₃, THF, rt or 60 °C, 1–3 h, for 53–56.

Scheme 2. Synthesis of Piperazine-Substituted, Theophylline-Based ALDH1A1 Inhibitorsa

^aReagents and conditions: (a) 2-hydroxyacetic acid, neat, 100 °C, 3 h, 49%; (b) NaOH_(aq), 100 °C, 2 h, 24 (82%); (c) R¹-Br, K₂CO₃, DMF, rt to 60 °C, 3–24 h; (d) Dess–Martin periodinane, CH₂Cl₂, 0 °C to rt, 1–2 h; (e) piperazine or homopiperazine derivatives (or its HCl salt), NaBH(OAc)₃, Et₃N, rt, 4 h.