Structure-Activity Relationships of Pyrazolo[1,5- a]pyrimidin-7(4 H)-ones as Antitubercular Agents

Abstract

Pyrazolo[1,5-a]pyrimidin-7(4H)-one was identified through high-throughput whole-cell screening as a potential antituberculosis lead. The core of this scaffold has been identified several times previously and has been associated with various modes of action against Mycobacterium tuberculosis (Mtb). We explored this scaffold through the synthesis of a focused library of analogues and identified key features of the pharmacophore while achieving substantial improvements in antitubercular activity. Our best hits had low cytotoxicity and showed promising activity against Mtb within macrophages. The mechanism of action of these compounds was not related to cell-wall biosynthesis, isoprene biosynthesis, or iron uptake as has been found for other compounds sharing this core structure. Resistance to these compounds was conferred by mutation of a flavin adenine dinucleotide (FAD)-dependent hydroxylase (Rv1751) that promoted compound catabolism by hydroxylation from molecular oxygen. Our results highlight the risks of chemical clustering without establishing mechanistic similarity of chemically related growth inhibitors.

Keywords: antitubercular activity; pyrazolo[1,5-a]pyrimidin-7(4H)-one; structure−activity relationship; tuberculosis.

Figure 1

Chemical structure of the hit compound, 1, and analogues 2–4 that have been previously reported.⁻

Figure 2

(A) Three probable tautomeric structures (4a–c) of the pyrazolo[1,5-a]pyrimidin-7(4H)-one scaffold in 4; (B) X-ray crystal structure of 4.

Scheme 1. General Synthetic Scheme of Pyrazolo[1,5-a]pyrimidin-7(4H)-one Derivatives 1, 2, 4, and P6, P8–P27

Reagents and conditions: (a) toluene, reflux, overnight; (b) AcOH, reflux, overnight.

Scheme 2. Synthesis of P1 and P2

Reagents and conditions: (a) POCl₃ (2.5 equiv), DIPEA (0.2 equiv), 90 °C, overnight; (b) NaOMe in MeOH (2.0 equiv), rt, overnight; (c) MeI (5.0 equiv), Cs₂CO₃ (3.0 equiv), rt, overnight.

Scheme 3. Synthesis of Analogues P3–P5

Reagents and conditions: (a) 7 (1.0 equiv), 8 (10 equiv), NaOMe (2.0 equiv), 90 °C, overnight; (b) N₂H₄·H₂O (2.0 equiv), EtOH, reflux, overnight; (c) 5 (1.0 equiv), EtOH, reflux, overnight; (d) 11 (1.0 equiv), 12 (2.0 equiv), K₂CO₃ (4.0 equiv), CuBr₂ (0.1 equiv), DMSO, 120 °C, overnight; (e) methanolic HCl, rt, overnight; (f) ethyl chloroformate (1.2 equiv), K₂CO₃ (1.0 equiv), CH₂Cl₂, rt, 36 h; (g) EtOH, AcOH, reflux, 5 h.

Scheme 4. Synthesis of Analogue P7

Reagents and conditions: (a) 1 (1.0 equiv), Cs₂CO₃ (3 equiv), CF₃I in DMSO, 365 nm UV irradiation during overnight rt stirring.

Figure 3

(A) Murine macrophage cytotoxicity was measured using a standard luminescence-based assay. Curves represent the average of three trials done in duplicates. (B) J774 cells were infected with Mtb H37Rv (MOI 1:5) and treated with the corresponding compounds for 7 days. Survival of intracellular bacteria was measured by plating serial dilutions following J774 lysis. Bars: mean ± SEM obtained from two trials done in duplicates.

Figure 4

(A) Effect of iron levels on the potency of test compounds. MICs were measured in the presence of 100 μM desferrioxamine (DFO) or 250 μM FeCl₃. The increase in potency of the compounds is expressed as MIC ratios equal to MIC in pure 7H9 media divided by MIC in 7H9 supplemented with either DFO or FeCl₃. Bars represent mean ± SEM (n = 2). (B) Effect of 1 in the iron uptake of Mtb in vitro. Cells treated with the specified compound were fed ⁵⁵Fe, and the radioactivity inside H37Rv was measured over a 72 h time course. Data represent mean ± SD (n = 3). CPM, counts per minute.

Figure 5

(A) Calculated structure of Rv1751 (red ribbons) superimposed on crystal structure of rifampicin monooxygenase (Rox, green ribbons) from S. venezuelae. Rifampicin (RIF) is shown in magenta, and FAD is shown in cyan. (B) Co-crystal structures of RIF and FAD are superimposed onto the calculated structure of Rv1751 (red ribbons). Residues mutated in P19^R are highlighted in yellow (conferring high-level resistance) and cyan (conferring low-level resistance).

Figure 6

(A) Degree of compound degradation upon exposure to WT H37Rv and the indicated P19^R strains was measured via LC/MS. Bars represent mean ± SEM (n = 3). (B) The correlation between compound degradation by Mtb and in vitro MIC shows the dependence of antimycobacterial activity to compound metabolism.