Lead Optimization of the 5-Phenylpyrazolopyrimidinone NPD-2975 toward Compounds with Improved Antitrypanosomal Efficacy

Human African trypanosomiasis (HAT) still faces few therapeutic options and emerging drug resistance, stressing an urgency for novel antitrypanosomal drug discovery. Here, we describe lead optimization efforts aiming at improving antitrypanosomal efficacy and better physicochemical properties based on our previously reported optimized hit NPD-2975 (pIC50 7.2). Systematic modification of the 5-phenylpyrazolopyrimidinone NPD-2975 led to the discovery of a R4-substituted analogue 31c (NPD-3519), showing higher in vitro potency (pIC50 7.8) against Trypanosoma brucei and significantly better metabolic stability. Further, in vivo pharmacokinetic evaluation of 31c and experiments in an acute T. brucei mouse model confirmed improved oral bioavailability and antitrypanosomal efficacy at 50 mg/kg with no apparent toxicity. With good physicochemical properties, low toxicity, improved pharmacokinetic features, and in vivo efficacy, 31c may serve as a promising candidate for future drug development for HAT.


■ INTRODUCTION
Human African trypanosomiasis (HAT), also known as sleeping sickness, is an infectious disease that mainly occurs in remote and rural areas of sub-Saharan Africa.In 2020, 663 new HAT cases were reported worldwide, most of which were reported in the Democratic Republic of the Congo. 1 As a member of neglected parasitic diseases (NPDs), there are undoubtedly more patients without diagnosis and proper treatment.To date, people in more than 30 countries are still at risk of contracting HAT. 2 Two Trypanosoma brucei subspecies cause HAT.−5 During the first hemolymphatic stage, parasites proliferate in the lymphatic system and cause acute febrile illness. 6In the second meningo-encephalitic stage, parasites invade the central nervous system, causing neurological disorders, coma, and eventually death without proper treatment. 7reatment of HAT depends on the parasite subspecies and stages of the disease. 8,9Current treatments include pentamidine, melarsoprol, a nifurtimox−eflornithine combination, and fexinidazole. 10,11−18 Taking the side effects, the population at risk, underreporting, inconvenient administration, and drug resistance into account, there clearly is an urgent need for innovative drug discovery efforts to achieve the goal of HAT eradiation by 2030, as set by the WHO. 19ne of the goals of the PDE4NPD consortium 20 aimed to explore novel treatments for four NPDs, e.g., HAT, Chagas disease, leishmaniasis, and schistosomiasis.A phenotypic screening strategy was employed for hit discovery next to a structure-based strategy focusing on parasite cyclic nucleotide phosphodiesterases (PDEs).A promising "hit" series has already been reported as a result of the phenotypic screening approach, where the most potent compound NPD-2975 (2)  showed an IC 50 of 70 nM against T. b. brucei with confirmed in vivo efficacy. 21Despite high potency both in vitro and in vivo, one of the drawbacks was its moderate metabolic stability.Taking NPD-2975 (2) as a starting point, the present study reports our lead optimization efforts to further improve the antitrypanosomal efficacy and pharmacological/pharmacokinetic profile in the series of 5-phenylpyrazolopyrimidinones.
■ RESULTS Design Strategy.As a modification of the phenyl ring of the 5-phenylpyrazolopyrimidinone scaffold was previously reported, optimization efforts here focused on the other positions of the NPD-2975 scaffold (Figure 1).As an initial step to further understand the structure−activity relationship (SAR) of this scaffold, we introduced methyl groups at R 1 −R 3 to explore the chemical space around the three nitrogen atoms.Several substituents with various physiochemical properties were introduced to the position where methylation resulted in the highest antitrypanosomal activity (R 2 , Table 1).Meanwhile, to improve solubility and structural diversity, an analogue with an amino group instead of the carbonyl group was prepared.Finally, substituents with different sizes and physicochemical properties were introduced to explore the R 4 position.
Chemistry.−28 The synthesis of the R 1 -methyl analogue 7 originally started from a direct N-methylation of 2. However, only mixtures of two N-methyl regioisomers (11 and 15) were obtained after several trials (synthetic conditions not shown).Ultimately, analogue 7 was prepared with the route depicted in Scheme 1. Starting from intermediate 3 (reported in the synthesis of 2), a methyl amine group was introduced to form the amide 4. A subsequent reduction with palladium on carbon and hydrogen gas yielded the amino intermediate 5.The last ring-closure step was initially performed with 4-fluorobenzoic acid, as previously  reported. 22,29However, reactions with 6 under basic conditions did not work out probably due to the steric hindrance of the extra methyl group.−32 For the R 2 -methyl analogues, synthesis of an R 2 -N-methylated BIPPO (1) analogue directly with dimethyl sulfate (DMS) was reported previously, but the synthesis of the corresponding R 3 analogue was not reported and the regiochemistry of the reaction was not investigated. 29Here, we report the synthesis of R 2 and R 3 analogues of 2 using a different synthetic route (Scheme 2) with confirmed regiochemistry using 1D-NOESY experiments (Figures S14 and S18).Starting from 3, introduction of a methyl group with iodomethane yielded two separable regioisomers 8 and 12. Next, the amidation and reduction reactions afforded the key intermediates 10 and 14 without purification of 9 and 13.The last ring-closure steps were completed with 4-fluorobenzoic acid as reported for 2. 21 To improve solubility and structural diversity, an amino group was introduced with the route shown in Scheme 3. Starting with 2, a chlorination reaction in POCl 3 yielded 16, which was further converted to 17 with an ammonia solution.After this initial modification, we focused on the R 2 position with the highest activity (7, 11, 15, and 17) whereby the synthetic route for 11 was applied for the synthesis of R 2 analogues 21a-e but slightly modified for 21f (Scheme 4).In the first step of the synthesis of 21f, introduction of the decarboxylation side-product 24'.Thus, an extra step to convert 3 to ester 23 was performed before the pyrazole alkylation reaction.Lastly, our synthetic efforts focused on the R 4 position.Analogues (31a−f) with aliphatic substituents of various sizes were prepared (Scheme 5).Due to physiochemical properties, some intermediates in Schemes 4 and 5 were difficult to isolate and were used in the next step without further purification.Phenyl analogue 35 was prepared by a different route (Scheme 6).The first step formed a phenylpyrazole scaffold with benzyl cyanide and ethyl 2diazoacetate under basic conditions. 33After the amide coupling and amidation reactions, 34 was obtained and used for the next step without further purification.The ring-closure reaction of 34 was completed under the same basic conditions as described for the synthesis of 2. 21 In Vitro Evaluation against T. brucei.In our initial optimization of the 5-phenylpyrazolopyrimidinone 2 (NPD-2975) (Table 1), we investigated the effect of methylation on the three nitrogen atoms and replacement of the carbonyl group with an amino group.The synthesized analogues were tested against T. b. brucei, and inhibition of MRC-5 (human lung fibroblasts MRC-5 SV40 ) cell proliferation was included as

Journal of Medicinal Chemistry
cytotoxicity control.Only the R 2 methyl analogue 11 showed an equal potency (pIC 50 7.1) to 2. The other two (R 1 and R 3 ) N-methyl analogues 7 and 15 exhibited significantly decreased activity (>100-fold and 20-fold, respectively).Analogue 17 with an amino group instead of the carbonyl group was prepared to improve solubility for further modification.However, a >100-fold activity decrease and slight cytotoxicity increase (pIC 50 4.6) were observed, discouraging the amino substituents at this position.The R 2 analogue 11 which displayed the highest antitrypanosomal activity was selected as the basis for further SAR investigations.
The second round of modifications further focused on the R 2 position, introducing several aliphatic substituents and one aromatic group on this position using the same synthetic route as used for 11 (Scheme 4).To increase diversity and solubility, analogues 21a−f and 22 were synthesized and evaluated in vitro (Table 2).A clear trend of decreasing activity (from pIC 50 7.1 for 11 to pIC 50 < 4.2 for 21d and 21f) can be correlated with the increasing size of the R 2 substituent.Analogues 21a and 21b with an ethyl and n-propyl substituent showed a marginally lower activity (pIC 50 6.6 and 6.8) compared to 2. However, introduction of an isopropyl group at the R 2 position decreased the antitrypanosomal activity of 21c (pIC 50 5.2) almost 100-fold compared with 2 (Table 2).Introducing an extra oxygen atom in the linker of 21b to increase flexibility was also detrimental to the activity, and 21d showed no antitrypanosomal activity at all.All these results indicate limited chemical space around this R 2 position.Based on these SAR results, 21e and 22 were prepared to increase the solubility.They exhibited micromolar IC 50 values (3.2 and 0.8 μM, respectively) against T. brucei with lower cLogP values (1.9 and 2.3, respectively) compared with 21a and 21b (cLogP 3.4 and 3.9, respectively).Analogue 21f with a bulky aromatic substituent showed no antitrypanosomal activity as can be expected from the above-described SAR.
Although a few analogues with lower cLogP and antitrypanosomal activity were identified in the R 2 analogue series, none of them exhibited improved activity combined with better drug-like physiochemical properties.Thus, our last modification focused on the R 4 position.Based on structure 2, analogues with a phenyl group and different aliphatic substituents were synthesized and evaluated in vitro (Table 3).For analogues with aliphatic substituents at the R 4 position, a difference in IC 50 values against T. brucei of more than three log units was observed, with the most potent being 31a (pIC 50 8.0) with a cyclopentyl substituent, and the least active being 31e (pIC 50 4.6) with a methyl substitution at R 4 (Table 3).An increase in antitrypanosomal activity is nicely correlated with the increasing size of R 4 substituents with a maximum activity for the cyclopentyl group.Analogues (31d−f) with substituents smaller than an isopropyl group are less potent than 2. Analogues (31a and 31c) with larger R 4 substituents exhibit improved potency (6 and 4-fold) compared to 2. Introduction of a cyclohexyl (31b) or phenyl group (35) at R 4 decreased the pIC 50 values to 7.4 and 7.3, which is 4-fold lower compared to 31a (Table 3).Due to the very low synthesis yield toward the phenyl analogue (Scheme 6), no more aryl analogues were prepared.It can be concluded that R 4 is a key position in the 5phenylpyrazolopyrimidinone structure, and proper substitution can significantly affect the antitrypanosomal activity potential.It also should be noted that none of the R 4 substituted analogues showed noticeable cytotoxicity.
Parasite Selectivity Panel and Metabolic Stability.Based on their promising antitrypanosomal activity, analogues 31a and 31c were selected for further antiparasitic profiling.First, they were tested in vitro against the protozoan species Trypanosoma cruzi and Leishmania infantum and the clinically

Journal of Medicinal Chemistry
relevant T. b. rhodesiense.Their potency against T. b. rhodesiense was similar to T. b. brucei while no activities were observed against T. cruzi and L. infantum as well as no cytotoxicity against MRC-5 cells and peritoneal mouse macrophages (PMM) (Table 4).
Next, the metabolic stability of 31a and 31c was tested in human and mouse liver microsomes and compared with the previously published hit compound 2. As shown in Figure 2, a significant difference in metabolic stability in the Phase-I metabolism was shown as a result of R 4 substitution.Analogue 31a with a cyclopentyl group at R 4 was metabolized within 15 min by mouse liver microsomal Phase-I metabolism; 31c with a tert-butyl group at R 4 exhibited improved metabolic stability compared with 2. No significant metabolism was observed for 31a and 31c by human liver microsomal Phase-I metabolism, as was also observed for 2. For Phase-II metabolism, both R 4substituted compounds showed good stability with at least 69% of parent compound remaining after a 1 h incubation in both mouse and human liver microsomes, indicating that the Phase-I metabolism is indeed the main route of metabolism.For the other analogues (11, 31b, and 35) with pIC 50 > 7.0, metabolic stability results are summarized in Table S1.
In Vivo Pharmacokinetics.Due to its acceptable in vitro metabolic stability, the in vivo pharmacokinetic properties of 31c were measured after either oral (PO) or intraperitoneal (IP) administration (Figure 3), and pharmacokinetic parameters were derived based on the measured blood concentrations (Table 5).Both routes of administration quickly led to micromolar blood concentrations that exceed the in vitro IC 50 value against T. brucei by more than 300-fold (Table 3, Figure 3).With regard to metabolic stability, 31c showed a slightly higher T 1/2 than 2 after IP administration.Whereas the T 1/2 of 31c and 2 after PO administration were comparable, a more than 7-fold higher AUC 0−6 h was observed after PO administration of 31c compared with 2 (Table 5).Since the oral bioavailability of 31c was significantly higher, this route of administration was used for subsequent evaluation of antiparasitic efficacy in a mouse model of acute T. b. brucei infection.Remarkably, an average concentration of 296 nM 31c was observed in the mice brain after a 24 h treatment (Table S2), which is more than 18 times of its IC 50 value against T. brucei.This result shows its promising application for the treatment of second-stage HAT in the future.
In Vivo Evaluation of 31c.In our previous in vivo results with 2, 21 all animals survived until the end of the experiment at 50 mg/kg dose.However, in the group of 25 mg/kg, all animals died at 11 days postinfection (dpi), which could be a result of its moderate metabolic stability.With promising pharmacokinetic parameters, 31c was evaluated in a mouse model of acute T. b. brucei infection and compared to suramin at 10 mg/kg IP once a day (s.i.d.) for 5 days as positive control.Treatment with 31c at 25 mg/kg and 50 mg/kg twice a day (b.i.d.) PO for 5 consecutive days led to apparent full clearance of parasitemia (Figure 4), with the exception of an accidental death in the 25 mg/kg group.All other animals in the experiment survived throughout the 60 days postinfection follow-up period without relapse, similar to the positive control suramin.The difficulty to detect trypanosome Spliced Leader (SL) RNA by qPCR in blood, spleen, fat, and brain tissue further corroborates the effective clearance of the acute T. b. brucei infection by exposure to 31c (Figure S1).These data indicate a markedly improved in vivo potential compared with 2. 21

■ DISCUSSION
Starting from our previously reported antitrypanosomal hit compound 2 (NPD-2975), lead optimization efforts toward substituted 5-phenylpyrazolopyrimidinones with higher potency and improved physiochemical properties are presented.Systematic modification of the pyrazolopyrimidinone scaffold led to a library of 18 new compounds, with slightly higher molecular weight (average of 298 compared with 272 Da of 2) Figure 2. Metabolic stability of 31a and 31c in comparison with 2. (A) Phase-I metabolic stability of 31a and 31c in the presence of mouse and human liver microsomes.(B) Phase-II metabolic stability of 31a and 31c against mouse and human liver microsomes.Source data are provided in Table S1.and diverse physiochemical properties (cLogP and tPSA).These compounds were tested phenotypically against T. b. brucei in vitro.Our first modification focused on the three nitrogen atoms in the core scaffold of 2. Substitution reactions on both nitrogen atoms of the imidazole ring yielded two regioisomers, which allowed us to study the influence on the antiparasitic potency of a methyl group at different positions of 2. The developed synthetic routes can be utilized for lead optimization of this scaffold in the future.Drastic potency differences between analogues with a methyl group at R 1 , R 2 , and R 3 positions (7, 11, and 15) guided us to focus on the R 2 position, which maintained activity with small substituents.Although no potency improvement was observed for compounds with R 2 substituents after further modification, its tolerance for polar groups can be explored to improve solubility in the future.Modification at the R 4 position was not synthetically convenient based on the synthetic route for 2, since for every analogue the R 4 substituent had to be introduced at the beginning of the synthetic route.However, R 4 turned out to be a key position for potency.A clear potency improvement is correlated with the increasing size of R 4 groups up to a cyclopentyl group, with three analogues (31a-c) showing low nanomolar IC 50 values (<100 nM) against T. brucei.Further replacement of the phenyl group in 35 exhibited comparable antitrypanosomal potency compared with 2. As follow-up, analogues with pIC 50 > 7 were tested against human and mouse liver microsomes for their metabolic stability, which was suboptimal for 2. Remarkable metabolic stability was observed for 31c (NPD-3519, pIC 50 7.8) next to its low toxicity for a number of other protozoan parasites and human cell lines.
As suggested by its metabolic stability, 31c showed improved pharmacokinetic features, such as longer half-life (1.56 h after IP administration) and more than 7-fold increase in AUC 0−6 h after PO dosing compared with 2. Also, 31c was detected after 24 h in the brain tissues at significantly higher concentrations than its antitrypanosomal IC 50 .In an acute mouse model of HAT, 31c yielded full clearance of parasitemia at 25 and 50 mg/kg b.i.d. for 5 days.These results warrant further exploration as drug candidate for HAT.The mode of action of 31c is still unknown and is currently being investigated next to 2 with a metabolomics approach 34,35 and an RNAi method, as previously reported. 36CONCLUSIONS To conclude, our lead optimization study starting from the previously reported 2 (NPD-2975) yielded a series of compounds with improved antitrypanosomal potency.Among them, 31c with a tert-butyl group at R 4 exhibits an IC 50 of 17 nM against T. b. brucei and acceptable metabolic stability.Pharmacokinetic evaluation revealed improved druglike properties (T 1/2 and AUC).Most importantly, the absence of detectable parasitemia in peripheral blood following an oral dose of 50 mg/kg or 25 mg/kg b.i.d. for 5 days in mice unveiled its promising in vivo potential; hence, 31c could serve as an antitrypanosomal candidate for future drug development against HAT.

■ EXPERIMENTAL SECTION
In Vitro and In Vivo Evaluation.All compounds tested are confirmed to pass a publicly available pan-assay interference compounds filter. 37,38The antiparasitic assays were carried out exactly as described in Blaazer et al. 39 Metabolic stability, pharmacokinetics, acute mouse infection model results were collected as described previously. 21All animal experiments were conducted in compliance with institutional guidelines and following approval by the Ethical Committee of the University of Antwerp, Belgium [UA-ECD 2014−96].Female Swiss mice (15−20 g), were purchased from Janvier (Le Genest Saint Isle, France).The PK properties of two compounds (2 and 31c) were compared after a single 10 mg/kg intraperitoneal (IP) or 50 mg/kg oral (PO) dose in uninfected mice (n = 3/group, 12 mice in total).A total of 12 mice were used to evaluate the in vivo potency of 31c at two doses, including a vehicle and reference (Suramin) control group.Animals were treated PO b.i.d. for 5 days at 25 and 50 mg/kg 31c.For the SL-RNA qPCR experiments blood was collected sublingually and subjected to erythrocyte lysis before extracting RNA with the QIAamp RNA Blood Mini kit (Qiagen).The mice were sedated after blood sampling  with a mixture of ketamine and xylazine, allowing extensive perfusion to eliminate blood contamination in other tissues.Small pieces of fat, brain, and spleen tissue were excised and immediately transferred to RNA later (Qiagen) and incubated overnight at 4 °C.RNA extraction was performed as instructed in the RNeasy Mini Plus Kit manual (Qiagen).The one-step SensiFAST SYBR Hi-Rox PCR kit (Bioline USA Inc., via Gentaur Belgium BVBA, Kampenhout, Belgium) was used for the PCR with the following forward and reverse primers, respectively 5′-AACTAACGCTATTATTAGAA-3′ and 5′-CAATA-TAGTACAGAAACTG-3′.An initial activation step of 10 min at 45 °C and 10 min at 95 °C was used, followed by the amplification step for 40 cycles (15 s at 95 °C, 15 s 50 °C and 15 s at 60 °C).At last, the melting curves were generated with an increment of 0.3 °C (15 s at 95 °C 1 min at 45 °C and 15 s at 95 °C).The PCR was run on the Step One Plus real-time PCR system (Applied Biosystems, California, USA).An additional qPCR was performed with the mouse housekeeping gene Eef2 to confirm successful RNA extraction.
Parasite and Cell Cultures.In vitro experiments were carried out with the bloodstream form of the T. b. brucei Squib strain (suraminsensitive).Parasites were routinely cultured in T25 culture flasks containing 10 mL of HMI-9 medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies) and 2.5 μg/mL Geneticin (Life Technologies).MRC-5 SV2 cells were cultured in MEM + Earl's salts-medium, supplemented with 2 mM L- glutamine, 16.5 mM NaHCO 3 , and 5% inactivated fetal calf serum.All cultures and assays were conducted at 37 °C under an atmosphere of 5% CO 2 .
Trypanosoma Susceptibility Assay.The compound stock solutions in 100% DMSO at 20 mM were first 4-fold serially diluted in DMSO and next in water to obtain a highest in-test compound concentration of 64 μM and of DMSO not exceeding 1%.Parasites were counted in a KOVA counting chamber and diluted to 1.5 × 10 4 parasites/well of a 96-well plate (200 μL total volume), upon which the prediluted test compounds were added.Drug exposure covered a 72 h period without renewal of the culture medium.After 3 days of incubation, parasite growth was assessed fluorimetrically after addition of 50 μL of resazurin per well.After 6 h (T.b. rhodesiense) or 24 h (T.b. brucei) at 37 °C, fluorescence is measured (λ ex 550 nm, λ em 590 nm).Parasite viability was assessed using the resazurin viability assay, and drug activity was calculated as percentage viability reduction compared to nontreated controls.Results were used to determine the 50% inhibitory concentration (IC 50 ).
MRC-5 SV2 Cytotoxicity Assays.Assays are performed in sterile 96-well microtiter plates, each well containing 10 μL of the watery compound dilutions together with 190 μL of MRC-5 SV2 inoculum (1.5 × 10 5 cells/mL).Cell growth is compared to untreated-control wells (100% cell growth) and medium-control wells (0% cell growth).After 3 days of incubation, cell viability is assessed fluorimetrically after addition of 50 μL of resazurin per well.After 4 h at 37 °C, fluorescence is measured (λ ex 550 nm, λ em 590 nm).The results are expressed as % reduction in cell growth/viability compared to control wells and an IC 50 and an IC 90 (50 and 90% inhibitory concentrations) are determined.
Pharmacokinetics.Compounds 2 and 31c were evaluated for their pharmacokinetic properties after a single 10 mg/kg intraperitoneal (IP) or 50 mg/kg oral (PO) dose in uninfected mice.Blood drops were sampled before treatment and at 0.5, 1, 2, 4, 6, and 24 h after PO dosage; samples after IP dosage were identical with an additional time point of 0.25 h.The blood drops were analyzed adopting the dry blood spot technique and analysis by LC-MS 2 .Briefly, blood was collected from the retro-orbital complex using capillary tubes and dropped (15 μL) on WhatmanFTADMPK cards (B).The spots were left to air-dry at room temperature for at least 2 h.For analysis, a 6 mm disk was punched out and extracted in 75:25 MeCN/water containing the internal standard tolbutamide.The amount of parent compound was determined using liquid chromatography (UPLC) (Waters Aquity) coupled with tandem quadrupole mass spectrometry (MS 2 ) (Waters Xevo) equipped with an electrospray ionization (ESI) interface and operated in multiple reaction monitoring mode.Standard curves in whole blood were made for calibration and validation.Standard PK parameters were determined using Topfit software.
Brain tissue of the animals was collected on ice at autopsy 24 h post-treatment (50 mg/kg oral dose) after perfusion.For perfusion, mice were sedated with ketamine/xylazine allowing transcardial perfusion with 10 mL of KREBS Henseleit solution (Sigma-Aldrich) to eliminate blood contamination in the tissues.The tissues were immediately homogenized using a GentleMacs tissue homogenizer.The tissue samples were subjected to protein precipitation by adding MeCN, followed by a centrifugation step at 4 °C for 5 min at 21, 130 g.The supernatant was further diluted in 75:25 MeCN/water for LC-MS 2 analysis as described above.
Acute Mouse Model.Mice were allocated to groups of three and were infected by IP injection of 10 4 T. brucei Squib 427 trypomastigotes.Compounds 2 and 31c were formulated in PEG 400 at 12.5 and 6.25 mg/mL envisaging a maximal dosing volume of 100 μL/25 g live body weight.Suramin was included as a reference for T. brucei and injected IP s.i.d. for 5 days at 10 mg/kg.A PEG 400 vehicle control group was also included.Compounds 2 and 31c were administered PO b.i.d. for 5 days at 25 and 50 mg/kg.The first treatment was given 30 min prior to the artificial infection.Drug efficacy was evaluated by microscopic determination of the parasitemia in a blood drop collected from the tail vein at several time points until 63 days postinfection (dpi).Animals were observed for the occurrence/presence of clinical or adverse effects during the experiment.An SL-RNA qPCR assay was performed in all surviving animals to confirm parasitological cure.For the SL-RNA qPCR, peripheral blood was subjected to erythrocyte lysis before extracting RNA with the QIAamp RNA Blood Mini kit (Qiagen).The mice were sedated after blood sampling with a mixture of ketamine and xylazine, allowing extensive perfusion to eliminate blood contamination in other tissues.Small pieces of fat, brain, and spleen tissues were excised and immediately transferred to RNA later (Qiagen) and incubated overnight at 4 °C.RNA extraction was performed as instructed in the RNeasy Mini Plus Kit manual (Qiagen).The onestep SensiFAST SYBR Hi-Rox PCR kit (Bioline USA Inc., via Gentaur Belgium BVBA, Kampenhout, Belgium) was used for the PCR with the following forward and reverse primers, respectively, 5′-AACTAACGCTATTATTAGAA-3′ and 5′-CAATATAGTACA-GAAACTG-3′.An initial activation step of 10 min at 45 °C and 10 min at 95 °C was used, followed by the amplification step for 40 cycles (15 s at 95 °C, 15 s 50 °C, and 15 s at 60 °C).At last, the melting curves were generated with an increment of 0.3 °C (15 s at 95 °C, 1 min at 45 °C, and 15 s at 95 °C).The PCR was run on the Step One Plus real-time PCR system (Applied Biosystems, California, USA).An additional qPCR was performed with the mouse housekeeping gene Eef2 to confirm successful RNA extraction.
Chemistry.General Information.All starting materials were obtained from commercial suppliers and used without purification.Preparation of 2 and 3 has been reported previously. 21Anhydrous THF, DCM, and DMF were obtained by passing through an activated alumina column prior to use.All reactions were carried out under a nitrogen atmosphere unless mentioned otherwise.TLC analyses were performed using Merck F 254 aluminum-backed silica plates and visualized with 254 nm UV light.Flash column chromatography was executed using Biotage Isolera equipment.All HRMS spectra were recorded on a Bruker microTOF mass spectrometer using ESI in positive-ion mode.Nuclear magnetic resonance (NMR) spectra were determined with a Bruker Avance II 300 MHz, a Bruker Avance II 500 MHz or a Bruker Avance III HD 600 MHz spectrometer.Chemical shifts are reported in parts per million (ppm) against the reference compound using the signal of the residual nondeuterated solvent (CDCl 3 δ = 7.26 ppm ( 1 H), δ = 77.16ppm ( 13 C); DMSO-d 6 δ = 2.50 ppm ( 1 H), δ = 39.52 ppm ( 13 C)).NMR spectra were processed using MestReNova 14.0 software.The peak multiplicities are defined as follows: s, singlet; d, doublet; t, triplet; q, quartet; dd, doublet of doublets; ddd, doublet of doublets of doublets; dt, doublet of triplets; dq, doublet of quartets; td, triplet of doublets; tt, triplet of triplets; qd, quartet of doublets; p, pentet; dp, doublet of pentets; br, broad signal; m, multiplet.For NMR listings, in addition to specific instructions that are given by the journal in the guidelines for authors, the following additional procedures were used: (1) multiplicity is not solely reported based on peak shapes, but it also distinguishes the coupling to all nonequivalent protons that have similar J values; (2) if additional smaller couplings are observed but are too small for accurate quantitation because the precision is smaller than the digital resolution, a symbol Δ will be used; (3) the notation "m" is used in case of obscured accurate interpretation as a result of (i) overlapping signals for different protons or (ii) a result of overlapping signal lines within the same proton signal; (4) for any rotamers or diastereomers, signals will be listed separately; (5) NMR signals that could only be detected with HSQC analysis are denoted with a # symbol; (6) NMR signals that could only be detected with HMBC analysis are denoted with a * symbol; (7)   13 C signals are visible in the spectra due to the tautomerism of non-N-substituted pyrazoles.HSQC and HMBC were measured to assign 13 C signals if applicable.IUPAC names were adapted from ChemBioDraw Ultra 19.0.Purities were measured using analytical LC-MS using a Shimadzu LC-20AD liquid chromatography pump system with a Shimadzu SPDM20A diode array detector with the MS detection performed with a Shimadzu LCMS-2010EV mass spectrometer operating in positive ionization mode.The column used was an Xbridge (C18) 5 μm column (100 mm × 4.6 mm).The following solutions are used for the eluents.Solvent A: H 2 O/ HCOOH 999:1 and solvent B: MeCN/HCOOH 999:1.The eluent program used is as follows: flow rate: 1.0 mL/min, start with 95% A in a linear gradient to 10% A over 4.5 min, hold 1.5 min at 10% A, in 0.5 min in a linear gradient to 95% A, hold 1.5 min at 95% A, total run time: 8.0 min.Compound purities were calculated as the percentage peak area of the analyzed compound by UV detection at 254 nm.All final compounds are >95% pure by HPLC analysis.

4-Amino-3-cyclohexyl-1H-pyrazole-5-carboxamide (30b).
Oxalyl chloride (15.0 mL, 11.1 mmol) was added dropwise to a solution of 28b (2.42 g) in DCM (80 mL) containing two drops of DMF at 0 °C, which was then warmed to RT and stirred for 2 h.The reaction mixture was evaporated under reduced pressure, and coevaporated three times with toluene.The residue was then dissolved in toluene, added dropwise to a solution of NH 3 in MeOH (7 M, 7.2 mL, 50 mmol) at 0 °C, and stirred at RT for 18 h.The resulting suspension was concentrated under reduced pressure and used for the next step without further purification.The crude intermediate 29b (2.5 g) was combined with 10% palladium on carbon (0.24 g) in EtOH (50 mL) and stirred under H 2 gas insert at 60 °C for 16 h.The reaction mixture was filtered through Celite and the solid was washed with MeOH (50 mL).The filtrate was concentrated under reduced pressure and used in the next step without further purification.
4-Amino-3-(tert-butyl)-1H-pyrazole-5-carboxamide (30c).Oxalyl chloride (6.16 mL, 70.4 mmol) was added dropwise to a suspension of 28c (5.00 g, 23.5 mmol) in DCM (240 mL) containing DMF (0.082 mL, 1.1 mmol) under nitrogen at 0 °C.The reaction mixture was stirred at 0 °C for 1 h, allowed to warm to RT, and stirred for a further 2 h.The reaction mixture was concentrated in vacuo and coevaporated with toluene three times.The residue was dissolved in DCM (100 mL) and added dropwise to 7 M NH 3 in MeOH (10.1 mL, 70.4 mmol) at 0 °C.After stirring for 3 h, the reaction mixture was concentrated in vacuo and used in the next step without further purification.The crude intermediate 29c was combined with 10% palladium on carbon (0.85 g, 0.80 mmol) in EtOH (90 mL) and stirred under a H 2 gas insert at 60 °C for 6 h.The reaction mixture was filtered through Celite and the solid was washed with MeOH (50 mL).The filtrate was concentrated under reduced pressure and the residue was used in the next step without further purification.
4-Amino-3-ethyl-1H-pyrazole-5-carboxamide (30d).Amide 29d (830 mg, 4.51 mmol) and 10% palladium on carbon (200 mg) in EtOH (90 mL) were stirred under a H 2 insert at 60 °C for 6 h.The reaction mixture was filtered and the residue was washed with MeOH (50 mL).The filtrate was concentrated in vacuo under reduced pressure and the residue was used for the next step without further purification.
In vitro metabolism: intrinsic clearance of 11, 31a−c and 35 in comparison with 2; measured brain concentrations of 31c in the pharmacokinetic experiments at 24 h after treatment; detection of parasite infection by SL-RNA detection by qPCR; LCMS, and 1 H NMR, 13

Figure 4 .
Figure 4.In vivo evaluation of 31c in a stage-I mouse model of HAT.Parasitemia (A) and survival rate (B) of stage-I T. b. brucei-infected mice treated with vehicle (n = 3), suramin (n = 3) at 10 mg/kg or 31c (n = 3) at 50 or 25 mg/kg.Results in figure A are expressed as mean number of bloodstream forms (BSF)/mL ± standard error of mean (SEM).VIC, vehicle.

Table 2 .
In Vitro Antitrypanosomal Activity and Cytotoxicity of R 2 Analogues a cLogP and tPSA are adopted from CDD Vault.b Mean values ± standard deviations, n ≥ 2.

Table 3 .
In Vitro Antitrypanosomal Activity and Cytotoxicity of R 4 Analogues