Studies Towards Hypoxia-Activated Prodrugs of PARP Inhibitors

Abstract

Poly(ADP-ribose)polymerase (PARP) inhibitors (PARPi) have recently been approved for the treatment of breast and ovarian tumors with defects in homologous recombination repair (HRR). Although it has been demonstrated that PARPi also sensitize HRR competent tumors to cytotoxic chemotherapies or radiotherapy, normal cell toxicity has remained an obstacle to their use in this context. Hypoxia-activated prodrugs (HAPs) provide a means to limit exposure of normal cells to active drug, thus adding a layer of tumor selectivity. We have investigated potential HAPs of model PARPi in which we attach a bioreducible "trigger" to the amide nitrogen, thereby blocking key binding interactions. A representative example showed promise in abrogating PARPi enzymatic activity in a biochemical assay, with a ca. 160-fold higher potency of benzyl phthalazinone 4 than the corresponding model HAP 5, but these N-alkylated compounds did not release the PARPi upon one-electron reduction by radiolysis. Therefore, we extended our investigation to include NU1025, a PARPi that contains a phenol distal to the core binding motif. The resulting 2-nitroimidazolyl ether provided modest abrogation of PARPi activity with a ca. seven-fold decrease in potency, but released the PARPi efficiently upon reduction. This investigation of potential prodrug approaches for PARPi has identified a useful prodrug strategy for future exploration.

Keywords: NU1025; PARP inhibitor; anti-cancer agents; hypoxia; hypoxia-activated prodrugs; prodrug; radiolytic reduction; tumor targeting.

Figure 1

Olaparib 1 bound in the PARP-2 binding site (4tvj) [41].

Figure 2

Reduction of nitroheterocyclic isoquinolinones 3a–c [42,43].

Figure 3

Poly(ADP-ribose)polymerase inhibitor (PARPi) (2a, 2c, 4, 6, 9 and 12) and corresponding model hypoxia-activated prodrugs (HAPs) (3c, 5, 7, 8, 10, 11 and 13).

Scheme 1

Synthesis of PARPi 4, 6, and 12. i) NaOH, H₂NNH₂·H₂O, 70 °C 18 h, 75%; ii) K₂CO₃, KI, BnCl, Δ 18 h, 21%; iii) 5% Pd/C, EtOH, H₂ 60 psi, 16 h, 99%; iv) CDI, DMF, 70 °C, NH₃(aq), 16 h, 74%; v) AcCl, pyridine, THF 16 h, then 0.5 M NaOH, 1 h, 48%; and vi) BBr₃, DCM, 30 °C 72 h, 63%.

Scheme 2

Synthesis of prodrug triggers. i) DIPEA, MsCl, 0.5 h, 91%; ii) MnO₂, Δ 18h, 76%; iii) NH₄OAc, 45 °C 2h, then NaCNBH₄, 48h, then used without purification, see Scheme 4; iv) MeMgBr, TiCl₄, −78 °C to −30 °C, 20, −30 °C to −20 °C 3 h, 53%; v) MnO₂, Δ 18h, 81%; vi) NH₄OAc, 40 °C 1 h, then NaCNBH₄, 18 h, then used without purification, see Scheme 4.

Scheme 3

Synthesis of model HAPs 3c, 5, 10, 11, and 13. i) NaH, 0.5 h, 50%; ii) NaH, 0.5 h, 10%; iii) LiHMDS, 2 h, NaI, 18 h, 49%; iv) LiHMDS, 18 h, 55%; v) K₂CO₃, 72 h, 47%.

Scheme 4

Synthesis of primary amide PARPi prodrugs 7 and 8. i) K₂CO₃, BnBr, Δ 18 h, then NaOH, Δ 2 h, 82%; ii) (COCl)₂, 18 h, then 21, pyridine, 0 °C–rt, 4 h, 70%; iii) (COCl)₂, 18 h, then 24, pyridine, 0 °C–rt, 18 h, 29%.

Figure 4

LC/MS analysis of the radiolytic reduction of model HAP 5. (A) Diode array chromatograms at 276 nm (bandwidth 16 nm), with 0 and 40 Gy. (B) Total ion chromatograms (positive mode, scanning m/z 100–600), with 0 and 40 Gy. (C) Mass spectrum of peak at retention time 4.701 min (identified as hydroxylamine). (D) Mass spectrum of peak at 7.932 min (identified as prodrug by comparison with authentic 5). ▽ Indicates the retention time of the parent PARPi 4.

Figure 5

LC/MS analysis of the radiolytic reduction of model HAP 3c. (A) Diode array chromatograms at 276 nm (bandwidth 16 nm), with 0 and 40 Gy. (B) Total ion chromatograms (positive mode, scanning m/z 100–600), with 0 and 40 Gy. (C) Mass spectrum of peak at 3.891 min (identified as hydroxylamine). (D) Mass spectrum of peak at 7.159 min (identified as prodrug by comparison with authentic 3c) ▽ Indicates the retention time of the parent PARPi 2c.

Figure 6

LC/MS analysis of the chemical reduction of model HAP 3c. (A) Extracted ion chromatogram at m/z 333 corresponding to the Br-79 isotopologue of the amino derivative of 3c. (B) Mass spectrum of peak at 5.580 min. (C) Extracted ion chromatogram at m/z 224 corresponding to the ⁷⁹Br isotopologue of 2c from injection of a 1.8 µM solution. (D) Mass spectrum of peak at 6.839 min.

Figure 7

LC/MS analysis of the radiolytic reduction of model HAP 13. (A) Diode array chromatograms at 276 nm (bandwidth 16 nm), with 0 and 40 Gy. (B) Total ion chromatograms (negative mode, scanning m/z 100–600), with 0 and 40 Gy. (C) Mass spectrum of peak at 8.849 min (identified as effector by comparison with authentic 12). (D) Mass spectrum of peak at 10.529 min (identified as prodrug by comparison with authentic compound 13).