Design, Synthesis, Biological Evaluation, and Crystallographic Study of Novel Purine Nucleoside Phosphorylase Inhibitors

Abstract

Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC₅₀ values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC₅₀ values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 μM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.

Figure 1

Example structures of published PNP inhibitors.

Figure 2

General structure of designed PNP inhibitors.

Scheme 1. Two Strategies Show How to Perform the Key Transformation Using the Ullmann Coupling

Scheme 2. Optimized Multigram Synthesis of Compound 4

Reagents and conditions: (a) BnOH, Na, rt, 5 h, SiO₂ filtration, crystallization; (b) NIS, THF, rt, 5 min, crystallization; (c) SEM-Cl, NaH, DMF, rt, 15 min, crystallization.

Scheme 3. Synthesis of Compounds 7a–f via Ullmann Coupling of Compound 4 with Substituted Thiophenols

Reagents and conditions: (a) compound 4, CuI, 1,10-phenanthroline, Et₃N, toluene, MW, 120 °C, 1–2 h; (b) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min.

Scheme 4. Synthesis of Compound 9

Reagents and conditions: (a) CH₃COSK, CuI, 1,10-phenanthroline, Et₃N, toluene, 125 °C, 4 h; (b) MeOH, K₂CO₃, 50 °C, 1 h; (c) air, MeOH, rt, 1 h.

Scheme 5. Synthesis of Aliphatic Acyclic Nucleoside Phosphonates (ANPs) 13a–b from compound 9

Reagents and conditions: (a) PME-Cl or PEE-Cl, NaH, DMF, 80 °C, overnight; (b) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min; (c) (1) TMSBr, pyridine, rt, overnight; (2) Dowex 50-Na⁺.

Scheme 6. Synthesis of Aryl Iodides 15a–c

Reagents and conditions: (a) P(Oi-Pr)₃, toluene, MW, 160 °C, 1 h; (b) CH₂[P(O)(OEt)₂]₂, 5 M NaOH (aq), DCM, rt, 48 h; (c) TfOCH₂P(O)(Oi-Pr)₂, NaH, THF, 0 °C, 1 h.

Scheme 7. Synthesis of PNP Inhibitors Containing the Phosphonate Moiety

Reagents and conditions: (a) compound 9, CuI, 2-isobutyrylcyclohexanone, Cs₂CO₃, toluene, MW, 120 °C, 1–3 h; (b) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min; (c) (1) TMSBr, pyridine, rt, overnight; (2) Dowex 50-Na⁺.

Scheme 8. Synthesis of meta-Phenylphosphonate 21

Reagents and conditions: (a) HP(O)(OEt)₂, Pd₂(dba)₃, xantphos, Et₃N, dioxane, 100 °C, on; (b) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min; (c) (1) TMSBr, pyridine, rt, overnight; (2) Dowex 50-Na⁺.

Scheme 9. Synthesis of Phosphonate 23

Reagents and conditions: (a) TfOCH₂P(O)(Oi-Pr)₂, NaH, THF, 0 °C, 1 h; (b) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min; (3) TMSBr, pyridine, rt, overnight; (4) Dowex 50-Na⁺.

Figure 3

Target compounds with the extra-substituted central phenyl scaffold.

Scheme 10. Synthesis of Brominated Analogs 27 and 31

Reagents and conditions: (a) DIBAH, DCM, 0 °C–rt, overnight; (b) PDC, DCM, rt, 4 h; (c) CH₂[P(O)(OEt)₂]₂, t-BuO^–K⁺, THF, rt, 1 h; (d) NBS, (BzO)₂, DCE, reflux, 4 h; (e) NMMO, 4 Å mol sieves, ACN, 0 °C, 2 h.

Scheme 11. Attempted Synthesis of Hydroxybenzaldehydes 34a–c via ortho-Lithiation

Reagents and conditions: (a) (CH₃NHCH₂)₂, toluene, reflux, 15 min; (b) (1) t-BuLi, Et₂O, −78 °C–rt, overnight; (2) (CH₂I)₂, Et₂O, −78 °C–rt, 30 min.

Scheme 12. Unintended Preparation of Hydroxybenzaldehydes 35 and 36

Reagents and conditions: (a) t-BuLi, Et₂O, −78 °C–rt, overnight; (2) (CH₂I)₂, Et₂O, −78 °C–rt, 30 min. (b) I₂, KI, NH₄OH, H₂O, rt, 3 h.

Scheme 13. Synthesis of Benzaldehyde 34b

Reagents and conditions: (a) I₂, AgNO₃, MeOH, rt, 1 h; (b) (1) BBr₃, DCM, −78 °C–rt, overnight; (2) aq NaHCO₃.

Scheme 14. Synthesis of Benzaldehyde 34c

Reagents and conditions: (a) (CH₃NHCH₂)₂, toluene, reflux, 15 min; (b) (1) t-BuLi, Et₂O, −78 °C–rt, overnight; (2) (CH₂I)₂, Et₂O, −78 °C–rt, 30 min; (c) (1) BBr₃, DCM, −78 °C–rt, overnight; (2) aq. NaHCO₃.

Scheme 15. Synthesis of Vinyl Phosphonates 42a–c

Reagents and conditions: (a) [P(O)(OEt)₂]CH₂COOH, piperidine, acetic acid, toluene, reflux, overnight, 87%; (b) [P(O)(OEt)₂]₂CH₂, K₂CO₃, EtOH, reflux, 1 h, 88%; (c) [P(O)(OEt)₂]₂CH₂, NaH, DMF, 40 °C, overnight.

Scheme 16. Synthesis of Vinyl Phosphonates 43a–l

Reagents and conditions: (a) MeI, NaH, DMF, 0 °C–rt, 1 h; (b) i-PrI, NaH, DMF, 0 °C–rt, 1 h; (c) C₆F₅, NaH, DMF, 70 °C, 1 h; (d) PFBnBr, NaH, DMF, 0 °C–rt, 1 h.

Scheme 17. Synthesis of Target Phosphonates 45a–q

Reagents and conditions: (a) compound 9, CuI, 2-isobutyrylcyclohexanone, Et₃N, DMF, MW, 120 °C, 1–3 h; (b) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min; (3) TMSBr, pyridine, rt, overnight; (4) Dowex 50-Na⁺.

Scheme 18. Synthesis of Oxamethylphosphonates 49a–b

Reagents and conditions: (a) TfOCH₂P(O)(Oi-Pr)₂, NaH, THF, 0 °C, 1 h; (b) compound 9, CuI, 2-isobutyrylcyclohexanone, Et₃N, DMF, MW, 120 °C, 1–3 h; (c) (1) TFA, rt, 15 min; (2) NH₃/EtOH, rt, 1 min; (3) TMSBr, pyridine, rt, overnight; (4) Dowex 50-Na⁺.

Scheme 19. Synthesis of Phosphonate Prodrugs

Reagents and conditions: (a) (1) TMSBr, pyridine, rt, overnight; (2) TEAB, rt, 5 min; (3) l-amino acid ethyl ester hydrochloride, phenol, Et₃N, 2-Aldrithiol, PPh₃, pyridine, 60 °C, overnight.

Scheme 20. Optimized Synthesis of TAF-like Prodrug 50a

Reagents and conditions: (a) (1) TMSBr, pyridine, rt, overnight; (2) TEAB, TBAH, rt, 5 min; (b) l-alanine ethyl ester hydrochloride, phenol, Et₃N, 2-Aldrithiol, PPh₃, pyridine, 60 °C, overnight.

Scheme 21. Optimized Multigram-Scale Synthesis of Compound 52 and Prodrug 50a

Reagents and conditions: (a) (1) 2-mercaptophenol, CuI, 1,10-phenanthroline, Et₃N, toluene, 120 °C, 2 h; (2) TsOCH₂P(Oi-Pr)₂, t-BuO^–K⁺, DMF, 60 °C, 3 h; (3) TFA, rt, 15 min; (4) NH₃/EtOH, rt, 1 min; (b) (1) TMSBr, pyridine, rt, overnight; (2) TEAB, TBAH, rt, 5 min; (c) l-alanine ethyl ester hydrochloride, phenol, Et₃N, 2-Aldrithiol, PPh₃, pyridine, 60 °C, overnight.

Figure 4

Inhibition kinetics of compound 18c against hPNP (competitive model).

Figure 5

PK profile of compound 18c in mice and rats.

Figure 6

hPNP in complex with compounds 18c, PDB 7ZSL (A), 45b, PDB 7ZSM (B), 45q, PDB 7ZSN, (C) 45i, PDB 7ZSO (D), and 45n, PDB 7ZSP (E). Hydrogen-bond forming residues are shown as sticks and highlighted in bold; other interacting residues are shown as lines and labeled. Protein carbon atoms are shown with gray color; inhibitor carbons are shown in green (A), magenta (B), orange(C), gray (D), and red (E) color, while nitrogen atoms are blue, phosphorus atoms are orange, sulfur is yellow, and oxygen is red. Direct hydrogen bonds are shown as blue-dashed lines, water-mediated—cyan-dashed lines, and halogen bonds as red-dashed lines. In panels F and G, protein carbons are shown in the same color as inhibitors, and highlighted residues are shown as sticks and labeled. Panel F shows overlay of hPNP in complex with 18c (green), 45b (magenta), and 45q (orange). Panel G shows an overlay of hPNP in the complex with 18c (green), 45i (gray), and 45n (red).

Figure 7

MtPNP in complex with 18c, PDB 7ZSQ (A), 45b, PDB 8C25 (B), and 45q, PDB 7ZSR (C). Carbon atoms are shown in salmon red (A), cyan (B), and black (C). Oxygen atoms are red, nitrogen—blue, sulfur—yellow, and phosphorus—orange. Residues that form direct hydrogen bonds are shown as sticks and labeled with bold fonts; other residues are shown as lines and labeled. Hydrogen bonds are represented by blue, and turquois dashed lines are for direct and water-mediated hydrogen bonds. The halogen bond is shown as a yellow dashed line. Panel D shows the overlay of all three ligands and the part of alpha helix 7 with His243 shown as sticks.