Antitubercular 2-Pyrazolylpyrimidinones: Structure-Activity Relationship and Mode-of-Action Studies

Abstract

Phenotypic screening of a Medicines for Malaria Venture compound library against Mycobacterium tuberculosis (Mtb) identified a cluster of pan-active 2-pyrazolylpyrimidinones. The biology triage of these actives using various tool strains of Mtb suggested a novel mechanism of action. The compounds were bactericidal against replicating Mtb and retained potency against clinical isolates of Mtb. Although selected MmpL3 mutant strains of Mtb showed resistance to these compounds, there was no shift in the minimum inhibitory concentration (MIC) against a mmpL3 hypomorph, suggesting mutations in MmpL3 as a possible resistance mechanism for the compounds but not necessarily as the target. RNA transcriptional profiling and the checkerboard board 2D-MIC assay in the presence of varying concentrations of ferrous salt indicated perturbation of the Fe-homeostasis by the compounds. Structure-activity relationship studies identified potent compounds with good physicochemical properties and in vitro microsomal metabolic stability with moderate selectivity over cytotoxicity against mammalian cell lines.

Figure 1

Generic structure of pyrazolylpyrimidones.

Figure 2

(a) Structure of PZP; and (b) depiction of metal chelation by pyrazolylpyrimidinone 1.

Scheme 1. Synthetic Route to Explore R1 and R2 on the Pyrimidinone and Polar Modifications at R5 on the Pyrazole

Reagents and conditions: (i) (a) thiourea, KOH, EtOH, reflux (b) iodomethane, NaOH, H₂O/EtOH; (ii) NH₂NH₂·H₂O, EtOH, reflux; (iii) Br₂, AcOH, 60 °C; (iv) phenylboronic acid, Pd(PPh₃)₂Cl₂, 1,4-dioxane, K₂CO₃; (v) acetylacetone, EtOH, AcOH, reflux; (vi) 3-aminocrotonitrile, EtOH, reflux; (vii) acetyl chloride, pyridine, CH₂Cl₂; and (viii) ethyl acetoacetate, EtOH, AcOH, reflux.

Scheme 2. Synthetic Routes to Access Core Modifications

Reagents and conditions: (i) MeOH, PPh₃, DIAD, THF, 25 °C; (ii) MeI, K₂CO₃, DMF, 100 °C; (iii) NaOCH₃, MeOH, 80 °C; (iv) phenylboronic acid, Pd(PPh₃)₂Cl₂, K₂CO₃, 1,4-dioxane, reflux; (v) 3,5-dimethylpyrazole, xanphos, Pd(OAc)₂, Cs₂CO₃, 1,4-dioxane, 130 °C; (vi) BBr₃, MeOH; (vii) BBr₃, CH₂Cl₂; (viii) urea, 1,4-dioxane, 140 °C, microwave; (ix) POCl_3, DMF; (x) 3,5-dimethylpyrazole, Cs₂CO₃, 1,4-dioxane, 80 °C, microwave; and (xi) 20% aq NaOH, 80 °C.

Scheme 3. Synthetic Route for Pyrazole Replacements

Reagents and conditions: (i) urea, 1,4-dioxane, 140 °C, microwave; (ii) POCl₃, DMF; (iii) 20% aq NaOH, 80 °C (iv) 5-membered heterocycle, Cs₂CO₃, 1,4-dioxane, 80 °C, microwave; and (v) EtOH, reflux, 1 h.

Scheme 4. Synthetic Route to Polar Substitutions on R1 of the Pyrimidinone

Reagents and conditions: (i) aqueous NaOH, dioxane; (ii) boronic acid, Cs₂CO₃, Pd(OAc)₂, dppf, 1,4-dioxane, 70 °C or K₂CO₃, PdCl₂(dppf)₂, 1,4-dioxane, 70 °C; and (iii) 3,5-dimethylpyrazole, Cs₂CO₃, 1,4-dioxane, 130 °C, microwave.

Scheme 5. Synthetic Route to Amide 26

Reagents and conditions: (i) CH₃ONa, THF, 0 °C; (ii) 3,5-dimethylpyrazole, Cs₂CO₃, 1,4-dioxane, 110 °C, microwave; (iii) 4-fluoroaniline, HATU, Et₃N, DMF; and (iv) BBr₃, CH₂Cl₂.

Scheme 6. Synthetic Route to Polar Substitutions on R2 of the Pyrimidinone

Reagents and conditions: (i) ethyl 2-cyanoacetate, KOH, ethanol, reflux; (ii) (a) MeI, NaOH, H₂O (b) NH₂NH₂·H₂O, EtOH, reflux; (iii) acetylacetone, AcOH, EtOH, 85 °C; (iv) H₂SO₄, 80 °C; and (v) tert-butyl nitrite, AcOH, 75 °C.

Scheme 7. Synthetic Routes Used to Explore the Pyrazole R5 Position

Reagents and conditions: (i) diketone, EtOH, AcOH, reflux; (ii) 3-aminocrotonitrile, EtOH, reflux; (iii) acetaldehyde, DMF, AcOH, NaCNBH₄, MeOH, 25 °C; (iv) N-(2-methoxyethyl)-3-oxobutanamide, Lawesson’s reagent, THF, 25 °C; and (v) Cs₂CO₃, Pd₂(dba)₃, XantPhos, 1,4-dioxane, aryl halide, 120 °C

Scheme 8. Synthetic Route to Compounds 36–39

Reagents and conditions: (i) NH₂-R, EtOH, reflux; (ii) 33, AcOH, EtOH, 100 °C.

Scheme 9. Synthesis of Compound 35

Reagents and conditions: (i) (a) thiourea, KOH, EtOH and (b) MeI, NaOH, H₂O; (ii) mCPBA, CH₂Cl₂, 25 °C; (iii) N₂H₂. H₂O, EtOH; (iv) EtI, K₂CO₃, DMF, 70 °C; and (v) Cs₂CO₃, 1,4-Dioxane, 130 °C.

Scheme 10. Synthesis of Compounds with Pyrazole C4 Substitutions

Reagents and conditions: (i) Br₂, AcOH, 100 °C, 2h; (ii) pyridine-3-boronic acid, K₂CO₃, Pd(dppf)Cl₂, THF/water (10:1), 70 °C, 16h; (iii) N-bromosuccinamide, p-toluene sulphonic acid, CH₂Cl₂; 0 °C; (iv) morpholine, Et₃N, CH₂Cl₂; and (v) EtOH/AcOH reflux, 16 h.

Scheme 11. Synthesis of Compounds with Pyrazole C3 Modifications

Reagents and conditions. (i) DMF, 130 °C, 2 h; (ii) Cs₂CO₃, 1,4-dioxane, 130 °C, microwave; (iii) N,N-dimethylamine, HATU, Et₃N, DMF; (iv) (a) DIBALH, CH₂Cl₂, −78 °C; (b) N,N-dimethylamine, NaCNBH₃, THF, RT, 24 h; and (v) Cs₂CO₃, 1,4-dioxane, 130 °C, microwave.

Figure 3

Essential pharmacophoric features and scope for further SAR studies.

Figure 4

Fe²⁺ plays a critical role in the MoA of 45. (A) Fe²⁺ rescue of Mtb from growth inhibition mediated by 45. (B) Spectroscopic titration of 45 with Fe²⁺. The data are representative of two biological replicates performed in duplicate.