Identification of Phenylpyrazolone Dimers as a New Class of Anti‐Trypanosoma cruzi Agents

Abstract Chagas disease is becoming a worldwide problem; it is currently estimated that over six million people are infected. The two drugs in current use, benznidazole and nifurtimox, require long treatment regimens, show limited efficacy in the chronic phase of infection, and are known to cause adverse effects. Phenotypic screening of an in‐house library led to the identification of 2,2′‐methylenebis(5‐(4‐bromophenyl)‐4,4‐dimethyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one), a phenyldihydropyrazolone dimer, which shows an in vitro pIC50 value of 5.4 against Trypanosoma cruzi. Initial optimization was done by varying substituents of the phenyl ring, after which attempts were made to replace the phenyl ring. Finally, the linker between the dimer units was varied, ultimately leading to 2,2′‐methylenebis(5‐(3‐bromo‐4‐methoxyphenyl)‐4,4‐dimethyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (NPD‐0228) as the most potent analogue. NPD‐0228 has an in vitro pIC50 value of 6.4 against intracellular amastigotes of T. cruzi and no apparent toxicity against the human MRC‐5 cell line and murine cardiac cells.


General methods parasitology
The phenotypic assays against T. brucei, T. cruzi, L. infantum and cytotoxicity against MRC-5 cells were done as previously described in Blaazer et al. 1
Subsequently oxalyl dichloride (13.7 mL, 162 mmol) and DMF (0.08 mL, 1.08 mmol) were added and the mixture was allowed to warm up to room temperature. The mixture was stirred for 2 h after which volatiles were evaporated. The remaining solids were redissolved in 50 ml of THF. In a separate flask methyl isobutyrate (18.6 mL, 162 mmol) was stirred in THF (50 mL) at -78°C and a 2M LDA (65 mL, 130 mmol) was added dropwise while maintaining -78°C. Upon full addition, the mixture was stirred for 45m after which the acid chloride of 6 in THF was added dropwise, again maintaining the temperate at -78°C. The reaction was allowed to warm up to room temperature after which crude was quenched with water and extracted with diethyl ether. The organic phase was washed twice with water and once with brine. The organic layer was then dried with MgSO4, filtered and evaporated to dryness. The crude was used in the next step without further purification.

General method II: ring closure of β-keto-estes
Crude keto-ester 5s (34 g, 79 mmol) was dissolved in ethanol (75 mL) and hydrazine hydrate (64%) (38.6 mL, 793 mmol) was added. The reaction was stirred at room temperature for 48h after which white precipitation was visible. To the stirred solution 20 mL of water was added to allow further precipitation, after which solids were filtered off. Collected solids were dried in vacuo yielding the desired product.
The vial was sealed and refluxed in a sand bath at 60°C overnight after which the reaction mixture was a clear solution. The solution was diluted with DCM (30 mL), and the organic layer was extracted with water (3 x 30 mL). The organic layer was dried over MgSO4 and the solvent evaporated under vacuo to give a white/yellow solid. The obtained solids were recrystallized from MeOH.

General method IV: Installing aliphatic linkers
Pyrazolone 6s (1.0 g, 3.4 mmol) was added to a flask and DMF (14 mL) was added after which the mixture was cooled to 0°C. Subsequently, NaH (60% in mineral oil)(0.14 g, 3.4 mmol) was added. The reaction mixture was stirred at room temperature for 30 min, after which 1,2-dibromoethane (0.145 mL, 1.68 mmol) was added dropwise at 0°C. The reaction was stirred overnight at room temperature. Upon completion, the reaction was quenched with sat.
aqueous NH4Cl and extracted with EtOAc (50 mL). The organic layer was washed with water (50 mL) and brine (40 mL). After this, the organic layer was dried over MgSO4 and volatiles were evaporated under vacuo. The crude product was purified over SiO2 using a gradient from 20% EtOAc in n-heptane towards 65% EtOAc to give the title compound as an off-white solid.

Experimental data
The experimental data of 5s, 6s, 7s, 12b, 12e, 13, 14 and 15 can be found in the main text of this publication. Prepared according to general method I and II using 2.0 g (9.9 mmol) of 3bromobenzoic acid. Formed little precipitate, the crude was purified over SiO2 using a gradient from 20% EtOAc in n-heptane towards 60% EtOAc to give 1.5 g (5.6 mmol, 57%) of the title compound as a yellow solid. 1   Prepared according to general method I and II using 2.0 g (9.9 mmol) of 2bromobenzoic acid. Formed little precipitate, the crude was purified over SiO2 using a gradient from 20% EtOAc in n-heptane towards 60% EtOAc to give 1.8 g (6.7 mmol, 68%) of the title compound as a white solid. 1

3-(3-fluorophenyl)-4,4-dimethyl-1H-pyrazol-5(4H)-one (6h)
N N O H F Prepared according to general method I and II using 2.0 g (14.3 mmol) of 3fluorobenzoic acid. Formed little precipitate, the crude was purified over SiO2 using a gradient from 20% EtOAc in n-heptane towards 60% EtOAc to give 1.32 g (6.4 mmol, 45%) of the title compound as a white solid. 1   Prepared according to general method I and II using 0.5 g (3.5 mmol) of 3methoxybenzoic acid. Formed little precipitate, the crude was purified over SiO2 using a gradient from 20% EtOAc in n-heptane towards 60% EtOAc to give 0.45 g (2.1 mmol, 60%) of the title compound as a white solid. 1