Discovery of 5-Phenylpyrazolopyrimidinone Analogs as Potent Antitrypanosomal Agents with In Vivo Efficacy

Human African Trypanosomiasis (HAT), caused by Trypanosoma brucei, is one of the neglected tropical diseases with a continuing need for new medication. We here describe the discovery of 5-phenylpyrazolopyrimidinone analogs as a novel series of phenotypic antitrypanosomal agents. The most potent compound, 30 (NPD-2975), has an in vitro IC50 of 70 nM against T. b. brucei with no apparent toxicity against human MRC-5 lung fibroblasts. Showing good physicochemical properties, low toxicity potential, acceptable metabolic stability, and other pharmacokinetic features, 30 was further evaluated in an acute mouse model of T. b. brucei infection. After oral dosing at 50 mg/kg twice per day for five consecutive days, all infected mice were cured. Given its good drug-like properties and high in vivo antitrypanosomal potential, the 5-phenylpyrazolopyrimidinone analog 30 represents a promising lead for future drug development to treat HAT.


■ INTRODUCTION
Human African Trypanosomiasis (HAT), also known as African sleeping sickness, is a neglected parasitic disease (NPD). 1 Although the detected cases have dropped from 25,000 in 1995 to less than 1000 in 2019, the parasite still represents a risk for 65 million people living in 36 African countries. 2 The kinetoplastid parasite Trypanosoma brucei is the causative agent of HAT 3 and is transmitted by tsetse flies (Glossina sp.). Two subspecies are responsible for the infection in human beings. One of the subspecies, T. b. gambiense, causes most human infections (95%) and occurs in Central and West Africa, which is also known as West African trypanosomiasis. While T. b. rhodesiense is responsible for infections (5%) in East and Southern Africa, known as East African trypanosomiasis. 4 HAT develops in two clinical stages. 3 In the first stage, the parasites multiply in the bloodstream and lymphatic system, causing non-specific symptoms such as headache, fever, and joint pain. Once the parasites penetrate the central nervous system, the disease reaches the second stage, leading to the most common symptom, namely a disturbed sleep pattern. If left untreated, the second stage of HAT will eventually result in a coma or death. 2 There are only a few treatment options available to control both stages of trypanosomiasis. 5−7 Thus far, all of them, except fexinidazole ( Figure S1), suffer from difficult administration and moderate to severe side effects. 8−10 As for many infectious diseases, drug resistance often develops, and the most recently approved fexinidazole shares cross-resistance with nifurti-mox. 11−13 Although the clinical trial of a promising new drug, acoziborole ( Figure S1), is in progress, it is not easy to reach the goal of HAT elimination by 2030. 14 Hence, a high urgency to explore novel antitrypanosomal agents remains.
Despite this urgency, only a limited number of programs are currently involved in research toward a new HAT treatment. 15 −18 Among them, phosphodiesterases (PDEs) are an important class of targets for HAT drug discovery. PDEs are enzymes that hydrolyze the second messengers cAMP and cGMP to their non-cyclic analogs, AMP and GMP. As these enzymes are found in both humans and parasites, some of them have been proven to be important targets for treating human diseases and potential targets for parasitic diseases. 19−22 For instance, sildenafil was developed for the treatment of erectile dysfunction by targeting human PDE5. 23 With abundant knowledge regarding human PDEs, we aimed to develop new antiparasitic treatments via a PDE target-based drug discovery strategy and a phenotypic screening strategy within the EU-funded consortium PDE4NPD (Phosphodiesterase Inhibitors for Neglected Parasitic Diseases). 24 In 2007, T. brucei PDE B1 (TbrPDEB1) 19 was validated as a target, and in 2018, Blaazer et al. 25 reported optimization efforts toward TbrPDEB1-selective compounds targeting a parasitic-specific pocket (P-pocket 26 ). Besides these target-based efforts, phenotypic screening/evaluation also played an important role. In 2015, BIPPO (1, Figure 1) analogs were reported as a novel class of potent anti-Plasmodium falciparum agents, potentially acting via their effect on cyclic nucleotide levels. 27 Considering their antimalarial potency, low molecular weights, good drug-like properties (Table S1), and the fact that these molecules might act as parasite PDE inhibitors, a small additional series of BIPPO analogs (Table 1) was synthesized and phenotypically screened against a panel of protozoan parasites containing T. b. brucei, Trypanosoma cruzi, and Leishmania infantum. This study presents our efforts to characterize novel 5-phenylpyrazolopyrimidinone analogs as potent antitrypanosomal agents with in vivo efficacy.

■ RESULTS
Chemistry. The designed pyrazolopyrimidinone analogs were synthesized via the route shown in Scheme 1. The synthesis of 1, 4−8, 11, 12, and 15 was reported previously. 27−30 This route starts with condensation and ring closure reactions to form the pyrazole ester intermediate 4.
These two steps can be combined into a one-pot reaction. Following hydrolysis and nitration, intermediate 6 was obtained. The nitration reaction is a key step, and the rate of adding the reagent and the reaction temperature need to be carefully controlled. 31 The first four (a−d) steps can be performed at a four hundred-gram scale without column purification. Following primary amide formation and reduction of the nitro group, the key intermediate 8 was formed. The final compounds can be obtained with two different methods from 8. Analogs 9−37 and 40−42 were prepared by amide coupling followed by ring closure reactions under basic conditions (Scheme 1A), whereas analogs 38 and 43−45 were obtained by ring closure reactions with the corresponding aldehydes and iodine (Scheme 1B) due to starting material availability and reactivity. Analog 39 was obtained following hydrolysis of 38, and [2 + 3] cycloaddition of 37 with NaN 3 yielded tetrazole 46 with a decent yield. Due to tautomerism of the pyrazole ring in the structures, some carbon signals are too broad or invisible in the 1D NMR spectra, and earlier publications 27,29 did not report complete chemical characterizations (especially 13 C NMR signals) for the published analogs. 25,27 Here, we report the results of 13 C NMR combined with 2D NMR (HSQC and HMBC) or high-temperature NMR needed to obtain full characterization. For analog 30, additional efforts with salt formation to prevent tautomerism ended up with sharp 13 C NMR signals; the further "1,n-ADEQUATE" experiment confirmed its structure ( Figures  S90−94).
In Vitro Evaluation of Anti-T. brucei Activity. In our PDE4NPD program, we initially synthesized a small library of 5-benzyl-3-isopropyl-1H-pyrazolo[4,3-d]pyrimidin-7(6H)-one (BIPPO, 1, Figure 1) analogs as this scaffold was reported to inhibit PDEs from P. falciparum. 27 Consequently, the pyrazolopyrimidinone scaffold was considered an interesting start to identifying antitrypanosomal compounds. The first small series focused on variations in the 5-position of the BIPPO scaffold (Table 1). The benzyl moiety was replaced with a number of aromatic and aliphatic substituents, and the linker length and flexibility between the pyrazolopyrimidinone moiety and the terminal aromatic substituent were varied to explore structure−activity relationships (SARs). The analogs were tested for activity against T. b. brucei, T. cruzi, L. infantum, and toxicity against the human MRC-5 cell line was determined to measure general toxicity and selectivity. Based on the results shown in Table S2, only T. b. brucei inhibition was observed; thereafter, we focused on modifications for improved antitrypanosomal activity.  As shown in Table 1, BIPPO (1) is only weakly active against T. b. brucei. A pyridine instead of a phenyl was introduced in 9 to increase solubility, but no potency improvement was observed. The same results were obtained with 10 and 11, in which, respectively, an oxygen atom or a methylene group was introduced into the linker. Pyrazolopyrimidinones with aliphatic substituents (12, 13, and 14) at R 1 also exhibited low potency. The potency of analogs with different aromatic substituents varied considerably. When the aromatic rings were directly attached to the pyrazolopyrimidinone moiety, a drastic improvement in antiparasitic activity was observed. Analogs 15 and 16 were 125 and 16 times more potent than 1, respectively. However, 17 with a thiazole exhibited comparable activity to 1.
The phenyl-analog 15 had a pIC 50 of 6.6 against T. b. brucei without noticeable toxicity against MRC-5 cells and was the most active compound in our first round of modifications.
Our second round of modifications (Table 2) logically were based on compound 15. Analogs with different substituents at the ortho, meta, and para positions were synthesized, and activities against T. b. brucei ( Figure S2) and MRC-5 fibroblasts were determined. Analogs with an ortho substituent   (18−21) showed comparable activity to 15, with the orthosubstituted methyl analog 21 being the most potent (pIC 50 6.8). The analogs with an ortho-halogen substituent (18−20) were slightly less potent than 15. The ortho-methoxysubstitued 22 was clearly less effective, with a 25-fold reduction in potency. The meta-halogen analogs 23 and 24 proved to be significantly more potent than their ortho-analogs 18 and 19, with the meta-fluoro analog 23 showing a pIC 50 of 7.0. Analog 25 with a meta-methyl group was equipotent to 15, while 26 with a meta-methoxy group was slightly less potent. In contrast, the introduction of meta-OH, −N(CH 3 ) 2 , and −SO 2 CH 3 (27−29) led to a more than 10-fold reduction in potency compared with 15.
For analogs with para-substituents, 30 with a para-F substituent was the most potent in this series with a pIC 50 of 7.2 (Table 2). A comparable potency was observed for the para-Cl analog 31, but a four-fold reduction in potency was observed for the bromide 32. The para-methoxy group in 33 decreased the pIC 50 to 6.1, which is three times less active than 15.
Based on the SAR results from Table 2, our third round of modifications (Table 3) focused on the para-position of the phenyl ring in 15 to further improve chemical diversity and physicochemical properties, such as polarity and solubility. Analogs with relatively small substituents 35−37 exhibited comparable potency with 15. Once bigger or polar substituents (38−46) were introduced, pIC 50 values decreased dramatically (<5.0), which indicates that only a restricted set of substituents is accepted at the para-position of this phenyl ring.
As 30 (NPD-2975) turned out to be the most interesting compound in this series in terms of in vitro potency and physicochemical properties (cLogP), this compound was selected for further antiparasitic profiling. Table 4 shows the activity of 30 against a panel of protozoan parasites, revealing nanomolar potency against T. b. brucei and no activity against T. cruzi, L. infantum, or P. falciparum. Moreover, no toxicity was observed against the human MRC-5 cell line or peritoneal mouse macrophages (PMM), endorsing its high selectivity.
In Vitro Profiling of 30. Given its good potency and lack of toxicity, 30 was evaluated in detail in several in vitro assays. The target-based approach in the PDE4NPD consortium had shown that targeting both TbrPDEB1 and TbrPDEB2 with specific inhibitors 25 will kill trypanosomes, thereby confirming earlier target validation work by Seebeck et al. 19,32 As the scaffold of 30 was earlier identified to target PDE enzymes, 27 30 was tested against TbrPDEB1 but proved to be inactive (Table 5, Figure S3). Since both TbrPDEB1 and TbrPDEB2 need to be inhibited to kill trypanosomes, 19 this observation immediately dismisses the PDE-hypothesis for the observed antiparasitic activity of 30.
To identify potential safety issues, 30 was subsequently tested at 10 μM in the Eurof ins Safety-47 panel, which includes 24 GPCRs, two nuclear hormone receptors, three transporters, eight ion channels, six non-kinase enzymes, and four kinase enzymes (Table S3). The activity of these targets was modulated <50% at 10 μM, with the exception of human    (Table 5). Metabolic Stability. Metabolic stability was assessed by incubating 30 and the control diclofenac with mouse, rat, and human liver microsomes in the absence or presence of uridine diphosphate glucuronic acid (UDPGA) to stimulate phase-II metabolism. The results indicated that 30 was metabolized the slowest by human microsomes and to a moderate extent in rodent microsomes (Figure 2). Phase-I metabolism in mouse microsomes resulted in 55% of the parent drug remaining after 30 min of incubation, which can be defined as acceptable metabolic stability. 33 No significant difference was observed when metabolism by UGT enzymes was induced by the addition of UDPGA (phase-II metabolism), suggesting that phase-I metabolism is the main route of metabolism in 30.
Based on its low cytotoxicity, potent in vitro activity (Table  2), good selectivity (Table 4), and acceptable metabolic stability in mouse microsomes (Figure 2), 30 was progressed to in vivo evaluation in mouse.
In Vivo Pharmacokinetics. The pharmacokinetic profiles of 30 were determined after either oral (PO) or intraperitoneal (IP) administration (Figure 3), and their blood concentrations were used to derive various pharmacokinetic parameters ( Table 6). Both PO and IP administration quickly led to micromolar blood concentrations that exceeded the IC 50 more than 50-fold (Table 6, Figure 3). For the subsequent evaluation of 30 in a mouse model of acute T. b. brucei infection, 50 mg/kg PO administration was used.
In Vivo Evaluation of 30. Next to suramin (10 mg/kg IP s.i.d. for 5 days) as a positive reference in a mouse model of acute T. b. brucei infection, treatment with 30 at 50 mg/kg twice daily PO for 5 consecutive days resulted in apparent full clearance of parasitemia (Figure 4). In contrast to the high parasitemia and early mortality in the vehicle-treated mice, all 30-treated animals in the highest dosing group were devoid of peripheral blood parasites throughout the 60 days postinfection (dpi) follow-up period. An additional SL RNA qPCR confirmed the absence or undetectable levels of parasites in peripheral blood. A clear dose-dependent in vivo efficacy was recorded, as all animals treated at 25 mg/kg b.i.d. for 5 days relapsed and succumbed within 11 dpi.

■ DISCUSSION
We present a series of 5-phenylpyrazolopyrimidinone analogs with low nanomolar IC 50 values and promising in vivo efficacy against T. b. brucei. Based on the published anti-P. falciparum inhibitor 1 (BIPPO), a library of 38 analogs was designed and phenotypically screened against T. b. brucei. The shortening of the linker between the phenyl group and the pyrazolopyrimidinone moiety drastically increased its potency, resulting in 15 with a pIC 50 of 6.6. Further modification based on the structure of 15 with various substituents on its phenyl ring led to the discovery of 30 with an IC 50 of 70 nM against T. b. brucei and no noticeable toxicity for a number of other protozoan parasites or human cell lines. Additional modifications based on the structure of 30 to improve solubility did not yield analogs with improved activity. Potentially, this might be a result of limited space or a lipophilic environment in the binding pocket of its target, which is currently unknown.
Follow-up analysis revealed that 30 did not inhibit the TbrPDEB1 enzyme, a validated target for HAT, and showed good selectivity over a range of targets (24 GPCRs, 2 nuclear hormone receptors, 3 transporters, 8 ion channels, 6 nonkinase enzymes, and 4 kinase enzymes). Also, 30 tested negative in a mini-Ames test and showed no hERG liability, two crucial criteria in drug discovery for a 'lead' compound.  With respect to metabolism, 30 did not inhibit CYP2C9, CYP2D6, or CYP3A4 but did have moderate CYP1A2 and 2C19 liability and was metabolically stable in human and rodent liver microsomes, resulting in micromolar levels of 30 in mouse serum after PO and IP administration. In an acute mouse model of HAT, 30 yielded a 100% survival rate at 50 mg/kg b.i.d. for 5 days, making it an interesting lead for HAT treatment. Unfortunately, its mode of action is still unknown and is currently being investigated with a metabolomics approach 34,35 and an RNAi method, as previously reported. 36 Although the number of HAT patients is decreasing every year, many people living in sub-Saharan Africa are still at risk of infection. In the past few years, a number of publications 25,33,37−44 indeed specifically focused on antitrypanosomal drug discovery. Compared to these published scaffolds, the pyrazolopyrimidinones (including 30) have a relatively low molecular weight, which is for most of the analogs lower than 300 Dalton. This is a very nice feature of this compound series and can be further exploited during lead optimization. Next to that, other physicochemical properties (such as cLogP, number of hydrogen bond donors/acceptors) of 30 fit with Lipinski's rule of five, 45 indicating good drug-like properties. Also, the selectivity and metabolic stability of 30 are remarkable. Last but not least, its in vivo pharmacokinetic features and in vivo efficacy are outstanding since a complete cure was obtained at 50 mg/kg b.i.d. PO for 5 days without relapse at 60 dpi.

■ CONCLUSIONS
To conclude, our pyrazolopyrimidinone analogs with phenyl substituents are novel, potent, and selective antitrypanosomal agents. Compound 30 with a para-fluorophenyl group exhibits an IC 50 of 70 nM against T. b. brucei. Follow-up physiochemical feature analysis, metabolic stability, and pharmacokinetic testing revealed its excellent drug-like properties. Importantly, the absence of detectable parasite levels in peripheral blood following an oral dose of 50 mg/kg b.i.d. for 5 days in mice disclosed its promising in vivo potential and deserves further exploration for future drug development.

■ EXPERIMENTAL SECTION
In Vitro Evaluation. All compounds tested (1, 9−46) pass a publicly available pan-assay interference compound filter. 46,47 The antiparasitic assays and the TbrPDEB1 enzyme assay were carried out as described in Blaazer et al. 25 Briefly, phosphodiesterase activity assays were performed using the PDELight HTS cAMP phosphodiesterase Kit (Lonza, Walkersville, USA) at 25°C in non-binding, low volume 384 wells plates (Corning, Kennebunk, ME, USA). PDE activity was measured in "stimulation buffer" (50 mM Hepes, 100 mM NaCl, 10 mM MgCl 2 , 0.5 mM EDTA, 0.05 mg/mL BSA, pH 7.5). Dose response curves were measured in triplo. The compounds were diluted in DMSO. Inhibitor dilutions (2.5 μL) were transferred to the 384 wells plate, 2.5 μL PDE in stimulation buffer was added and mixed, 5 μL cAMP (at 2 × km up to 20 μM) was added, and the assay was incubated for 20 min at 300 rpm. The reaction was terminated with 5 μL Lonza Stop Buffer supplemented with 10 μM NPD-0001. Luminescence was determined in a Victor3 luminometer. Antitrypanosomal assays were carried out with the T. brucei Squib 427 strain cultured in Hirumi's modified Iscove's medium 9 (HMI-9) supplemented with 10% fetal bovine serum at 37°C in a 5% CO 2 atmosphere. Parasites were seeded at a concentration of 1.5 × 10 4 parasites/well. Four-fold dilutions of the test compounds were added, with the highest in-test concentration of 64 μM. After 72 h of drug exposure, viability was determined by incubation with 10 μg/mL resazurin (Sigma-Aldrich, St. Louis, MO, USA) and fluorescence reading after 24 h. The percentage growth inhibition compared to untreated control wells was used to calculate the 50% inhibitory concentration (IC 50 ). The CYP450 assays, hERG cardiotoxicity assay, mitochondrial toxicity assay, and data analysis were carried out as described in Moraes et al. 48 The mini-Ames test (Wuxi 49 ) and the Eurofins Safety47 panel (Eurofins 50 ) screens were outsourced to CROs.
Metabolic Stability. The microsomal stability assay was carried out based on the BD Biosciences Guidelines for Use (TF000017 Rev1.0) with minor adaptations. Male mouse and pooled human liver microsomes (Corning) were purchased and stored at −80°C until use. Both CYP450 and other NADPH-dependent enzymes (phase-I metabolism) and UGT enzymes (phase-II metabolism) were evaluated for NPD-2975 (30) with a working concentration of 5 μM. Diclofenac was used as a reference drug. Compound 30 was incubated with 0.5 mg/mL liver microsomes in potassium phosphate buffer, and reactions were initiated by the addition of 1 mM NADPH and 2 mM UDPGA cofactors for phase-I and phase-II metabolism, respectively. Samples were collected after 0, 15, 30, and 60 min. At these time points, 20 μL was withdrawn from the reaction mixture,  and 80 μL cold acetonitrile (MeCN), containing the internal standard tolbutamide, was added to inactivate the enzymes and precipitate the proteins. The mixture was vortexed for 30 s and centrifuged at 4°C for 5 min at 15,000 rpm. The supernatant was stored at −80°C until analysis. The loss 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 the multiple reaction monitoring (MRM) mode. Pharmacokinetics. Analog 30 (NPD-2975) was evaluated for its pharmacokinetic properties after a single dose, either IP at 10 mg/kg or PO at 50 mg/kg. Blood drops from the animals were sampled before treatment and at 0.5, 1, 2, 4, 6, and 24 h after PO dosage; sampling after IP dosage was identical with an additional time point of 0.25 h. The blood drops were analyzed adopting the dry blood spot collection and analysis by LC−MS 2 . Briefly, blood samples were collected from the tail vein using capillary tubes and dropped (15 μL) on Whatman FTA DMPK 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 brain tissue of the animals was collected on ice at autopsy 24 h post-treatment after perfusion. The tissue was immediately homogenized using a GentleMacs tissue homogenizer. The homogenates were then either immediately processed for analysis or stored at −80°C. The tissue samples were subjected to protein precipitation by adding MeCN, followed by a centrifugation step at 4°C for 5 min at 15,000 rpm. The supernatant was further diluted in 75:25% MeCN/water. The bio-analysis used liquid chromatography (UPLC) (Waters Aquity) coupled with tandem quadrupole mass spectrometry (MS 2 ) (Waters Xevo), equipped with an ESI interface, and operated in the MRM mode. Standard curves in whole blood were made for calibration and validation. Standard pharmacokinetic parameters were determined using Topfit software.
Acute Mouse Model. Mice were allocated to groups of three and were infected by an IP injection with 10 4 trypomastigotes of T. b. brucei (suramin-sensitive Squib 427 strain). Compound 30 (NPD-2975) was 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. Next to including a vehicle control group with only PEG 400 , suramin was included as the reference drug and was injected IP s.i.d. for 5 consecutive days at 10 mg/kg. 30 was administered PO b.i.d. for 5 consecutive days at 25 or 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 tail vein blood samples at several time points until 63 dpi. An additional SL RNA qPCR assay was performed for all surviving animals to confirm parasitological cure. Animals were observed for the occurrence/ presence of clinical or adverse effects during the course of the experiment. In cases of very severe clinical signs, either due to toxicity or clinical disease, animals were euthanized for animal welfare reasons. All 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].
Chemistry. General Information. All starting materials were obtained from commercial suppliers and used without purification. Anhydrous 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. All NMR spectra were recorded on either a Bruker Avance 300, 400, 500, or 600 spectrometer at 25°C, unless mentioned otherwise. The peak multiplicities are defined as follows: s, singlet; d, doublet; t, triplet; q, quartet; p, pentet; dd, doublet of doublets; dt, doublet of triplets; td, triplet of doublets; br, broad; m, multiplet, app, and apparent. The spectra were referenced to the internal solvent peak as follows: CDCl 3 (δ = 7.26 ppm in 1 H NMR, δ = 77.16 ppm in 13 C NMR), DMSO-d 6 (δ = 2.50 ppm in 1 H NMR, δ = 39.52 ppm in 13 C NMR). IUPAC names were adapted from ChemBioDraw Ultra 19.0. Purities were measured with the aid of analytical LC−MS using a Shimadzu LC-20AD liquid chromatography pump system with a Shimadzu SPDM20A diode array detector, with MS detection performed with a Shimadzu LCMS-2010EV mass spectrometer operating in the positive (or negative) 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, and 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 (1,  are >95% pure by HPLC analysis (Figures S4−S142). Note: not all 13 C signals are visible in the spectrum due to the rapid tautomerism of non-N-substituted pyrazoles. HSQC and HMBC were measured to assign 13 C signals if needed.
The reaction mixture was concentrated in vacuo and purified by flash column chromatography on silica gel with a gradient elution of MeOH in DCM (0−10%) to yield the title compound 7 as a white solid (8. 29 4-Amino-3-isopropyl-1H-pyrazole-5-carboxamide (8). Carboxamide 7 (5.70 g, 28.8 mmol) and 10% palladium on carbon (1.00 g, 0.940 mmol) in EtOH (90 mL) were stirred under H 2 atmosphere at 60°C for 18 h. The reaction mixture was filtered, and the solid was washed with MeOH (50 mL). The filtrate was concentrated in vacuo and purified by flash column chromatography on silica gel eluting with DCM/MeOH 9:1 to give the title product 8 as an off-white solid Spectral data agree with a previous report. 29 General Procedure for the Synthesis of Analogs 1, 9−46. Method A: 4-Amino-3-isopropyl-1H-pyrazole-5-carboxamide 8 (1.0 equiv) and the corresponding acid (1.0 equiv), PyBrop (1.1 equiv), and TEA (2.0 equiv) were combined in DCE and heated using microwave irradiation at 120°C for 20 min. The reaction mixture was purified by column chromatography with an eluent of DCM and MeOH to get amide intermediates as off-white solids. Then, the amide intermediate was combined with KO t Bu (2.0 equiv) in i PrOH (10 mL) and heated using microwave irradiation at 130°C for 30 min. The reaction mixture was concentrated in vacuo and purified by column chromatography to get the final products.