Structure‐Activity Relationship of Phenylpyrazolones against Trypanosoma cruzi

Abstract Chagas disease is a neglected parasitic disease caused by the parasitic protozoan Trypanosoma cruzi and currently affects around 8 million people. Previously, 2‐isopropyl‐5‐(4‐methoxy‐3‐(pyridin‐3‐yl)phenyl)‐4,4‐dimethyl‐2,4‐dihydro‐3H‐pyrazol‐3‐one (NPD‐0227) was discovered to be a sub‐micromolar inhibitor (pIC50=6.4) of T. cruzi. So far, SAR investigations of this scaffold have focused on the alkoxy substituent, the pyrazolone nitrogen substituent and the aromatic substituent of the core phenylpyrazolone. In this study, modifications of the phenyldihydropyrazolone scaffold are described. Variations were introduced by installing different substituents on the phenyl core, modifying the geminal dimethyl and installing various bio‐isosteres of the dihydropyrazolone group. The anti T. cruzi activity of NPD‐0227 could not be surpassed as the most potent compounds show pIC50 values of around 6.3. However, valuable additional SAR data for this interesting scaffold was obtained, and the data suggest that a scaffold hop is feasible as the pyrazolone moiety can be replaced by a oxazole or oxadiazole with minimal loss of activity.


Introduction
The protozoan parasite Trypanosoma cruzi is the causative agent of Chagas disease. This parasite is transmitted by the triatomine bug vector that used to be endemic only in Latin America, but is now slowly moving towards North America as well. [1] It is estimated that currently around 8 million people are infected and many more are at risk of being infected. [2] Upon infection, the disease first enters into an acute phase in which symptoms are generally mild, fever-like, uncharacteristic or even absent. [3] As a result, Chagas disease is often not diagnosed in this stage and proceeds untreated towards a chronic phase. [4] Although the initial symptoms will disappear after a few weeks, the parasite will persist and evolve to an indeterminate symptomatic chronic phase. [5] This ultimately develops into progressive chronic cardiomyopathy in 30 % of the patients while another 10 % develop neurological, digestive or mixed clinical symptoms. Although the remaining 60 % do not develop any symptoms, they remain infective if untreated and therefor remain an infection risk. [6] While currently only two drugs are on the market, these are far from optimal as they have long treatment regimes, cause adverse drug effects and have limited efficacy during the chronic phase. [7] Benznidazole (1, Figure 1) and nifurtimox (2) are nitro-heteroaromatic drugs that were developed in the late 1960s. Their efficacy in the acute phase is widely accepted, however, their effectiveness during the chronic phase is still under debate. [8] In addition, they are known to cause adverse drug effects such as weight loss, depression and amnesia. [7c,9] With these limitations of the current drugs, it is clear that the need for novel chemotherapies is high. The drug discovery pipeline of Chagas disease has a few potential clinical candidates, mostly from private-public partnerships originating from the last decade. Meanwhile, drug resistance has been reported for both benznidazole (1, Figure 1) and nifurtimox (2) in in vitro strains. [11] Within the PDE4NPD (phosphodiesterase inhibitors for neglected parasitic disease) consortium, an European Union-funded public-private partnership to target several neglected tropical diseases, Chagas disease was one of the focus points. We previously reported the discovery of NPD-0227 (3, Figure 1), a sub-micromolar inhibitor of T. cruzi and modifications of this hit have been described with focus on substituents of the aromatic substituent (R 1 ), pyrazolone nitrogen (R 2 ) and the alkoxy substituent (R 3 ). [10] In this previous work, modifications on R 2 and R 3 did not result in increased activities. To further investigate the structure-activity-relationships of this scaffold, the present work focused on the effect of modifications of the core phenyl ring replacing the gem-dimethyl moiety of the dihydropyrazolone and replacement of the dihydropyrazolone ring with various heterocyclic and aromatic moieties.
N-Alkylation of the diydropyrazolones (33-44, Scheme 1) was done using sodium hydride and isopropylbromide, installing the desired isopropyl moiety (45-56). The final step was a Suzuki cross-coupling to install a 3-pyridinyl ring on the 3position yielding twelve analogues (57-68) of NPD-0227 (3, Figure 1) with variations on the central phenyl ring. The 4cyanophenyl (74) was prepared via a similar route: starting from 4-bromo-3-chlorobenzoic acid (69, scheme 2). This benzoic acid was transofrmed to the corresponding isopropyl-pyrazolone (72) in three steps. The key step was the conversion of the bromine towards the cyano moiety by using CuCN in DMF, after which this intermediate (73) was used in a Suzuki cross-couping to install the final 3-pyridinyl moiety (74).
Modifications of the gem-dimethyl moiety present in NPD-0227 (3) were installed in an early stage of the synthesis route (Scheme 3). Similar conditions were used as in Schemes 1 and 2; benzoic acid 75 was transformed to the corresponding acid chloride, after which the desired lithium enolates were added to yield the cyclopentene (77), methylpiperidine (79) and tetrahydropyran analogues (80). Exception was the cyclopentyl substituted keto-ester (78), which was prepared by a one-pot reaction in which carboxylic acid (75) was converted to the corresponding imidazolide, followed by addition of ethyl potassium malonate and subsequent decarboxylation. This unsubstituted keto-ester (76) was then dialkylated with 1,4dibromobutane to yield the desired cyclopentyl ring. These four keto-esters (77-80) were then exposed to the same sequence as in Scheme 1 to yield spiro analogues 89-92.
Subsequent ring closure with hydrazinecarbothioamide yielded the final thiadiazole 98.
The dihydropyridazinone moiety (Scheme 7) has some similarity to the dihydropyrazolone moiety of NPD-0227 (3), although this ring contains one additional carbon. Installation of this moiety started with a Friedel-Craft acylation of 2bromoanisole 100, resulting in β-keto-acid 101. Ring closure of this molecule with hydrazine gave the dihydropyridazinone ring (102) which was subsequently alkylated using sodium hydride to give the N-isopropyl derivative (103). Similar to previously described synthetic routes, last step of installing the 3-pyridinyl moiety was done using Suzuki conditions as described earlier, resulting in dihydropyridazinone 104.
Pyrolotriazole 109 (Scheme 8) is a bicyclic heterocycle with similarity to the original dihydropyrazolone. Preparation starts from the previously reported dihydropyrazolone 105 which was refluxed with Lawessons reagent to yield pyrazol-3-thione 106. [10] Addition of hydrazine to this building block yielded hydrazineylidine 107, from which the second heterocyclic ring was formed by adding cyclopropanecarbonyl chloride, resulting to the pyrolotriazole moiety (108). A final Suzuki cross-coupling yielded pyrolotriazole 109. Attempts to install an isopropyl, cyclopentyl or n-propyl did not succeed as the ring-closure step proved problematic.
Oxazoles 120-124 (Scheme 9) were prepared from benzoic acid 75; first step of this route was a sodium hydride promoted nucleophilic attack of the benzoic acid on selected bromoketones, yielding keto-esters 110-114. Subsequent ring closure with ammonium acetate yielded mixtures of respective imidazoles and oxazoles (115-119), which were relatively easily separated by column chromatography. Although attempts were made to isolate the imidazoles, these could not be obtained in sufficient purity. The subsequent Suzuki cross coupling yielded the desired 3-pyridine substituted phenyloxazoles 120-124.
Oxadiazoles 132-136 (Scheme 10) were prepared from benzoic acid 75, which was transformed to the ethyl ester by refluxing in EtOH in the presence of H 2 SO 4 . Addition of hydrazine to ester 125, yielded hydrazide 126, which was ring closed with the desired acid chlorides in the presence of POCl 3 to yield oxadiazoles 127-131. The final step was Suzuki crosscoupling to yield 3-pyridinyl substituted phenyloxadiazoles 132-136.
The final heterocyclic replacement investigated the thiazole ring (Scheme 11). The starting material was benzoic acid 75, which was chlorinated using oxalyl chloride, followed by a quench of ammonium hydroxide to yield benzamide 137. The benzamide was converted to the corresponding thioamide 138 using Lawessons reagent. This intermediate (138) could be used to form the desired thiazoles upon addition of bromoketones, yielding analogues 139 and 140. To finalize the molecules a Suzuki cross-coupling was used to install the 3-pyridinyl moiety, resulting in compounds 141 and 142.

Results and Discussion
In this work, the SAR around NPD-0227 (3) has been further investigated with modifications on the central phenyl ring, the gem-dimethyl moiety and the dihydropyrazolone headgroup. In Table 1, the screening results are shown of various substituents on the core phenyl group. Attempts to move the 4-methoxy of NPD-0227 to the 5-position (57) leads to a decrease in activity with a pIC 50 value of 5.1, while introducing a methoxy substituent on both the 4-and 5-position (58), shows an even larger decrease in activity (pIC 50 = 4.3).
Constraining the methoxy-substituent in a heterocycle resulting in dihydrobenzofuran 59 only resulted in a small decrease (pIC 50 = 5.8) in activity compared to NPD-0227. Installing a bromine on the phenyl ring next to the 4-methoxy substituent (60) resulted in a decrease in activity with a pIC 50 value of 5.1. Removal of the 4-methoxy led to analogue 61, which resulted in a tenfold drop in potency (pIC 50 = 5.5), showing that a methoxy substituent on this position is beneficial. Installation of a fluorine (62), chlorine (63), or methyl Scheme 7. Preparation of the dihydropyridazinone analogue 104 ( (64) group instead of the original 4-methoxy substituent resulted in compounds with similar pIC 50 values around 5.5 as the unsubstituted analogue 61. Introduction of a cyano moiety (74) instead of a methoxy resulted in an even further drop in activity with a pIC 50 value of 4.3.
As the 4-methoxy substituted analogue (3) performed the best thus far, additional substituents along with the methoxy were investigated. Both the 2-fluoro-4-methoxy (65) and the 6fluoro-4-methoxy (66) showed an approximately tenfold decrease compared to NPD-0227 (3); the 4-methoxy-5-methyl (67) and 4-methoxy-6-methyl (68) showed an even larger decrease with pIC 50 values of 4.9 and 4.5, respectively. Selectivity index between T. cruzi activity and MRC-5 cytotoxicity are lower then seen with NPD-0227, with the best compounds showing a SI of > 25-fold. Exact SIs are not known as the lowest concentration measured for cytotoxicity is 62 μM Four compounds were prepared with variations of the gemdimethyl moiety of NPD-0227 (3); installing a cyclopentyl (89, Table 2) and a cyclopentene (90) both resulted in a more than tenfold loss in activity compared to NPD-0227 (3). Introduction of the more polar methylpiperidine (91) and a tetrahydropyran (92) resulted in an even further decrease in potency with both compounds having a pIC 50 value of 4.5. Selectivity index between T. cruzi activity and MRC-5 cytotoxicity are poor for these series, with the best compounds (89, 90) showing fivefold selectivity.
Diydropyrazolo-oxazole 95 (Table 3), in which the dihydropyrazolone oxygen is constrained in a second five-membered ring shows a loss in activity with a pIC 50 value of 4.5. An even larger loss in activity is observed with aminothiodiazole 98, which is inactive (pIC 50 < 4.2). Replacing the oxygen of the dihydropyrazolone with a sulfur atom, leads to dihydropyrazolethione 99, which had similar activity (pIC 50 = 6.3) as NPD-0227 (3, pIC 50 = 6.4), although a small increase in toxicity against human MRC-5 cells (pIC 50 = 4.8) is observed, which is a 32-fold selectivity. Adding an extra carbon to the ring, resulting in pyridazinone 104 leads to a pIC 50 value of 5.0. Also pyrazolotriazole 109, which has a bicyclic system with quite some To investigate if the dihydropyrazolone moiety could be replaced, three different aromatic heterocycles were installed on this position while still being able to address the same region as the isopropyl moiety of NPD-0227 (3). This resulted in a series of five membered heterocycles: oxazoles (120-124, Table 4), oxadiazoles (132-136) and thiazoles (141-142). The synthesized oxazoles (120-124) all showed fairly similar activities with pIC 50 values around 6.0, although large differences can be seen in toxicity towards MRC-5 cells. While the tert-butyl (120), phenyl (121) and 3-fluorophenyl (122) substituted oxazole and phenyl substituted methyloxazole (124) all   Addition of an extra nitrogen to the heteroaromatic ring lead to oxadiazoles 132-136 which showed no toxicity towards MRC-5 cells at the lowest concentration screened, as all compounds reported pIC 50 values below 4.2 (62 μM). Aliphatic substituents (132-134) on the oxadiazole gave some activity against T. cruzi with pIC 50 values around 4.9. However, aromatic substituents (135-136) are preferred as both phenyloxazole (135) and 3-fluorophenyl (136) showed activities around pIC 50 5.8. Finally, the thiazoles showed decent activities (pIC 50 around 5.8) but toxicity of these compounds against MRC-5 cells is equally high, making these compounds less favorable for future studies.

Conclusion
Multiple approaches were explored to optimize the activity of NPD-0227 (3). Modification of the core phenyl moiety delivered interesting SAR data, but activity was generally quite low, with dihydrobenzofuran (59) performing best with a pIC 50 value of 5.8. Replacing the gem-dimethyl moiety with several spiroanalogues resulted in four analogues with a maximum activity of 5.2 (pIC 50 ) hence showing substantially lower activities than NPD-0227 (pIC 50 = 6.4). The sulfur analogue of NPD-0227, dihydropyrazolethione 99 showed a similar activity as NPD-0227 (3) with a pIC 50 of 6.3, however this was accompanied with an increase in cytotoxicity. As these compounds could not match the activity of NPD-0227, no further screenings were done on other strains or life stages, focusing on SAR of these series. Introduction of heterocycles instead of the pyrazolone moiety also yielded several compounds with promising activities, amongst which oxazole 123 showed a pIC 50 value of 6.2 (SI > 100-fold over MRC-5 cytotoxicity) and oxadiazole 135 which had a pIC 50 value of 5.8. Although these compounds do not show higher activities then optimized lead NPD-0227 (3), these scaffold hops could be new starting points for future hitto-lead optimization, especially with the promising selectivity index seen for oxadiazole 123.

Experimental Section Biology
Trypanosoma cruzi in vitro assay: Bloodstream trypomastigotes (BT) of the Y strain of T. cruzi were obtained by cardiac puncture of infected Swiss Webster mice on the parasitaemia peak. [12] For the standard in vitro susceptibility assay on intracellular amastigotes, T. cruzi Tulahuen CL2, β-galactosidase strain (DTU VI, nifurtimoxsensitive) was used. The strain is maintained on MRC-5 SV2 (human lung fibroblast) cells in MEM medium, supplemented with 200 mM L-glutamine, 16.5 mM NaHCO 3 , and 5 % heat inactivated fetal calf serum (FCSi). After incubation at 37°C for 7 d, parasite growth was assessed by adding the α-galactosidase substrate chlorophenol red-α-d-galactopyranoside. The color reaction was read at 540 nm after 4 h, and absorbance values were expressed as a percentage of the blank controls. All cultures and assays are conducted at 37°C under 5 % CO 2 . [13] Benznidazole was used as a reference compound.
MRC-5 cytotoxicity in vitro assay: MRC-5-SV2 cells, originally from a human diploid lung cell line, were cultivated in MEM, supplemented with L-glutamine (20 mM), 16.5 mM sodium hydrogen carbonate and 5 % FCSi. For the assay, 10 4 cells/well were seeded onto the test plates containing the pre-diluted sample and incubated at 37°C and 5 % CO 2 for 72 h. Cell viability was assessed fluorometrically 4 h after addition of resazurin (excitation 550 nm, emission 590 nm). The results are expressed as percentage reduction in cell viability compared to untreated controls. Tamoxifen was used as a reference compound.

Chemistry
Chemicals and reagents were obtained from commercial suppliers and were used without further purification. Anhydrous DMF, THF and CH 2 Cl 2 were obtained by passing them through an activated alumina column prior to use. Microwave reactions were executed