Isoxazolopyrimidine-Based Inhibitors of Plasmodium falciparum Dihydroorotate Dehydrogenase with Antimalarial Activity

Abstract

Malaria kills nearly 0.5 million people yearly and impacts the lives of those living in over 90 countries where it is endemic. The current treatment programs are threatened by increasing drug resistance. Dihydroorotate dehydrogenase (DHODH) is now clinically validated as a target for antimalarial drug discovery as a triazolopyrimidine class inhibitor (DSM265) is currently undergoing clinical development. We discovered a related isoxazolopyrimidine series in a phenotypic screen, later determining that it targeted DHODH. To determine if the isoxazolopyrimidines could yield a drug candidate, we initiated hit-to-lead medicinal chemistry. Several potent analogues were identified, including a compound that showed in vivo antimalarial activity. The isoxazolopyrimidines were more rapidly metabolized than their triazolopyrimidine counterparts, and the pharmacokinetic data were not consistent with the goal of a single-dose treatment for malaria.

Introduction

Malaria remains one of the most significant infectious diseases worldwide, with people in 91 countries at risk for infection by the Plasmodium parasite that is the causative agent of the disease.¹⁻⁴ The WHO reported 216 million cases in 2016 with nearly 0.5 million deaths.⁵ Five Plasmodium species cause malaria in humans, but most deaths are caused by Plasmodium falciparum, in African children under 5 years old. With the implementation of artemisinin combination therapies (ACT) for treatment, combined with expanded insect control measures, the number of malaria deaths has been declining in the past decade.⁵ However, the progress is threatened by the emergence of ACT treatment failures in Southeast Asia (first reported in Cambodia).^6,7 Delayed parasite clearance times have been linked to mutations in the propeller domain of a scaffolding protein called Kelch 13 that lead to an increased survival of the ring-stage parasites upon artemisinin treatment.⁸⁻¹⁰ A recent evidence suggests that additional resistance mechanisms may also be emerging,¹¹ and of particular concern is the increased association between ACT treatment failures and resistance toward the partner compound.^3,12 The emergence of ACT resistance combined with treatment failures with other antimalarial agents (e.g., chloroquine and sulfadoxine/pyrimethamine) highlights the need for new drug development for the treatment of malaria.

A robust global effort to identify new drugs for the treatment of malaria is being fostered by collaborative efforts among not-for-profit organizations, academia, and industry.^3,13,14 This effort has led to the identification of four new compounds that are currently in phase II clinical development, including a fully synthetic peroxide OZ439,^15,16 a novel imidazolopiperazine KAF156(17) of an unknown mechanism of action, a spiroindolone KAE609(18) that inhibits P. falciparum ATP4, and DSM265 (1)¹⁹ (Figure 1), an inhibitor of Plasmodium dihydroorotate dehydrogenase (DHODH). A number of additional compounds are either in phase I clinical development or undergoing preclinical testing.^3,13,14 The need to introduce all new agents as combination therapies to protect against resistance, as well as the requirement to provide compounds efficacious against several Plasmodium species, and for use in both treatment and prevention, requires a robust pipeline to meet clinical needs.²⁰ Additionally, the emphasis on identifying compounds with pharmacokinetic properties that can support a single-dose cure to improve patient compliance increases the challenge of identifying compounds with the required properties.

Figure 1.

Structures of PfDHODH inhibitors.

The identification of 1 has led to the clinical validation of DHODH as a new target for the treatment of malaria.²¹⁻²⁴ DHODH catalyzes the fourth step in de novo pyrimidine biosynthesis, a pathway that is essential to the malaria parasite, because it lacks the salvage enzymes typically found in other eukaryotic cells (including in humans).²⁵Plasmodium DHODH is a mitochondrial enzyme that utilizes flavin mononucleotide (FMN) and coenzyme Q (CoQ) to catalyze the oxidation of dihydroorotate to orotic acid. We used a target-based high-throughput screening approach to identify the inhibitors of P. falciparum DHODH (PfDHODH), leading to the identification of a triazolopyrimidine-based series with a potent and selective activity against both Plasmodium DHODH and P. falciparum.²⁵ Through the use of structure-based iterative medicinal chemistry, we identified 1 from this series,^19,26,27 and this compound is currently progressing through clinical development where it has shown good safety and excellent efficacy for the treatment of patients infected with P. falciparum as a single dose.²¹⁻²⁴ Subsequently, we also identified additional compounds from the triazolopyrimidine series with improved physicochemical properties (e.g., 2)²² (Figure 1).

In our search to identify additional chemical scaffolds with the DHODH inhibitory activity, which could be progressed for the treatment of malaria, we uncovered an isoxazolopyrimidine scaffold that is closely related to the triazolopyrimidine series (e.g., 1 and 2). The initial isoxazolopyrimidine hit 3 (Figure 1 and Table 1) was identified in a phenotypic screen of the GSK compound library²⁸ and was later found to inhibit DHODH. To determine if the isoxazolopyrimidine series had unique properties relative to the triazolopyrimidine series, we undertook a hit-to-lead expansion, identifying several potent and selective analogues.

Table 1. Structure–Activity Relationship^a.

^aThe IC₅₀ and EC₅₀ data of DHODH and P. falciparum were collected in triplicate across the full dose range; the 95% confidence interval of the fit is provided in parenthesis. For compound 3, error represents the standard deviation for four replicates.

^bAlbumax-based media were used to collect the indicated data. Data for 1 and 2 were previously reported.^31,32

Results and Discussion

Identification of the Isoxazolopyrimidine Series

The lead isoxazolopyrimidine (3; Table 1) was identified in a large-scale phenotypic screen for antimalarial compounds; the dataset for this full screen has previously been made public.²⁸ The compound 3 was subsequently identified as a DHODH inhibitor with moderate potency against both the enzyme and the P. falciparum parasite in whole-cell assays (Table 1). The identification of the target was based on structural similarity to the triazolopyrimidine series (Figure 1), and on secondary screening assays, including the screening of a transgenic parasite line that expresses cytoplasmic yeast DHODH²⁹ and is therefore resistant to inhibitors that target mitochondrial-type DHODHs such as Plasmodium DHODH.

Chemistry

To improve the potency of 3 and to build in the characteristics required for good in vivo activity, we undertook a hit-to-lead expansion of the series. Compound 3 showed some disconnect between the enzyme and parasite data, suggesting potential permeability issues, as potency on the parasite was less than that on the enzyme (Table 1). It was also rapidly metabolized in mouse liver microsomes (Table 2) and showed poor exposure when dosed orally in mice (C_max = 0.014 μM; AUC_inf = 0.0069 μM·h at 10 mg/kg based on blood levels). Given the structural similarities to the triazolopyrimidine series, we reasoned that the approaches used to improve the potency and metabolic stability in the triazolopyrimidine series might translate to the isoxazolopyrimidines, allowing us to use both the prior data from the triazolopyrimidines and the X-ray structure of 1 bound to PfDHODH as guides for medicinal chemistry (Figure 2). In the triazolopyrimidine series, the aniline substituents that had para-substituted −CF₃ or −SF₅ functional groups (e.g., Figure 1; 1) showed both the best potency and metabolic stability of those that were tested,^26,27,30 whereas the addition of the pyridinyl CF₃-aniline reduced log D and improved the aqueous solubility (e.g., Figure 1; 2).³¹ Thus, we systematically incorporated each of these substitutions into the isoxazolopyrimidine series. In the triazolopyrimidines, substantial increases in potency were achieved by substituting the C2 carbon with linear fluorohydrocarbons (e.g., CF₂CH₃ of 1);²⁶ however, as this position was not available for substitution in the isoxazolopyrimidines, we also tested the effect of replacing the isoxazolopyrimidine ring methyls with CF₃ on the potency and metabolic stability. Because of the proximity of the C2 carbon to the hydroxyl group of Y528 in the PfDHODH structure bound to 1 (Figure 2), we also tried the substitution of the methyl group with CH₂OH. Finally, in the triazolopyrimidines, we found that the replacement of the aniline with tetrahydro-2-naphthyl derivatives could boost the potency, whereas that with 2-indanyls provided good metabolic stability;³² hence, the derivatives with these substitutions were also prepared.

Table 2. Physicochemical Properties and In Vitro Metabolism in Liver Microsomes.

compd	log DpH7.4a	kinetic solubility pH 6.5 (μg/mL)b	CLint (H/M μL/min/mg protein)c	predicted Eh (H/M)d
1e	3.6	12.5–25	4.3/2.8	<0.2/<0.2
2e	2.5	>100	2.2/<7	0.003/<0.01
3	nd	Nd	6.2/28	nd
9	4.3	1.6–3.1	36/183	0.59/0.89
10	4.3	1.6–3.1	23/179	0.47/0.89
11	4.4	<1.6	32/233	0.56/0.91
12	4.4	<1.6	19/138	0.42/0.86
13	3.7	3.1–6.3	17/143	0.39/0.86
14	4	6.3–12.5	20/38	0.44/0.63
15	3.6	6.3–12.5	24/28	0.48/0.55
16	2.9	25–50	8/13	0.25/0.36
30	4.7	1.6–3.1	26/328	0.5/0.93
31	4.8	1.6–3.1	13/76	0.33/0.77
32	5	<1.6	13/73	0.34/0.76
35	4.5	1.6–3.1	18/36	0.41/0.61
36	4.7	1.6–3.1	10/26	0.28/0.53

^aDetermined chromatographically.

^bKinetic solubility range following a 30 min incubation at room temperature.

^cIn vitro intrinsic clearance in human (H) and mouse (M) liver microsomes.

^dPredicted hepatic extraction ratio. E_h < 0.3 represents low, E_h 0.4–0.7 moderate, and E_h > 0.8 high predicted clearance.

^eValues for compounds 1 and 2 have been previously reported;^19,26,31 nd, not determined.

Figure 2.

X-ray structure comparison of the inhibitor-binding site for PfDHODH bound to 1 versus 15. Select residues within 4 Å of the inhibitor in the binding site are shown for the comparison of 1 (4rx0) (purple, highlighted with transparent spheres) and 15 (green, highlighted with transparent spheres). The X-ray structure of 1 bound to PfDHODH has been previously reported.¹⁹ The FMN cofactor and the product orotic acid (l-Oro) are also shown bound in the pocket. The coordinates for the structure of PfDHODH bound to 15 have been submitted to the Protein Data Bank (PDB, 6GJG).

The target compounds were synthesized as outlined in Schemes 1–4. The preparation of the 3-substituted-5-amino-4-carbamoyl isoxazoles (6, 19, 27) required different approaches depending on the final target. The generation of these intermediates was followed by cyclization with triethyl orthoacetate or trifluoroacetic anhydride, which afforded the corresponding isoxazolopyrimidone intermediates (7, 20, 28). Subsequently, these were converted to the desired isoxazolopyrimidine derivatives (9–16, 23, 30–32, 35–37) by chlorination and amination with appropriate amines.

Scheme 1.

Scheme 4.

The synthesis of the isoxazolopyrimidines (9–16) is illustrated in Scheme 1(33) The compound 5-amino-3-methylisoxazole-4-carbonitrile (5) was prepared by the reaction of methylethoxymethylenemalononitrile (4) with hydroxylamine under basic conditions. This amino nitrile (5) was converted to 5-amino-3-methylisoxazole-4-carboxamide (6) by treatment with concentrated sulfuric acid. Further, cyclization with a mixture of triethyl orthoacetate and acetic anhydride under microwave conditions gave 3,6-dimethylisoxazolo[5,4-d]-pyrimidin-4(5H)-one (7). Pyrimidinone (7) was converted to 4-chloro-3,6-dimethylisoxazolo[5,4-d]-pyrimidine (8), with the excess phosphorous oxychloride refluxing at 80 °C for 10 h. This upon treatment with the substituted amines in EtOH/diisopropylethylamine (DIPEA)–isopropyl alcohol (IPA) MW/tetrahydrofuran (THF)-Seal tube/NaOtBu-tert-butylxphos–THF resulted in the desired 3,6-dimethyl-substituted isoxazolopyrimidine derivatives (9–16). The displacement of 4-chloropyrimidines by amines generally proceeded smoothly by heating with organic bases like DIPEA in ethanol or simple heating in alcoholic solvents like ethanol or IPA. However, these standard conditions failed to work in the following two isoxazolopyrimidines. Mostly, unreacted starting materials were recovered, and hence subjecting to harsher conditions like refluxing in sealed tube for 3 days and Buchwald conditions was adopted to obtain 15 and 16, respectively.

An alternate route was attempted to achieve the target 6-methyl isoxazolo[5,4-d]pyrimidin-3-yl)methanol derivative 23 (Scheme 2).^34,35 The reaction of cyanoacetamide (17) with ethyl oximinochloroacetate (18) in the presence of sodium ethoxide yielded ethyl 5-amino-4-carbomoylisoxazole-3-carboxamide (19). Followed by cyclization, chlorination and amination reactions were carried out under similar conditions as shown in Scheme 1 to obtain the intermediate 22. Finally, the reduction of ester (22) with sodium borohydride in THF–MeOH afforded ((6-methyl-4-((4-(pentafluorosulfanyl)phenyl)amino)isoxazolo[5,4-d]pyrimidin-3-yl)methanol (23).

Scheme 2.

The synthesis of 30–32 was performed as outlined in Scheme 3. To prepare the starting precursor 5-amino-3-(trifluoromethyl)isoxazole-4-carboxamide (27), we utilized the route previously described.³⁶ First, trifluoroacetaldehyde hydrate (24) treated with hydroxylamine hydrochloride in methanol at ambient temperature for 16 h afforded trifluoroacetaldehyde oxime (25). Bromination of the resulting oxime with n-bromosuccinamide in dry dimethylformamide (DMF) afforded trifluoroacetohydroximoyl bromide (26). Compound 26 was cyclized with cyanoacetamide (17) to give the corresponding 5-amino-3-(trifluoromethyl)isoxazole-4-carboxamide (27). Finally, the 6-methyl-3-trifluoromethyl isoxazolo[5,4-d]pyrimidine derivatives (30–32) were prepared from 27 by using similar conditions, as illustrated in Scheme 1.

Scheme 3.

Finally, we adopted the synthetic route in Scheme 4 to prepare 3-methyl-6-trifluoromethyl isoxazolopyrimidines (35–37) from the corresponding isoxozolo carboxamide (6) by cyclization with trifluoroacetic anhydride, followed by chlorination and amination with the appropriate amines.

DHODH Inhibition and Antiplasmodial Efficacy in Vitro

The compounds were tested against DHODH from P. falciparum and evaluated for activity against the P. falciparum 3D7 parasites in whole-cell assays (Table 1). Similar to the triazolopyrimidine series,^26,32 the best potency on both PfDHODH and P. falciparum 3D7 parasites was observed for compounds containing SF₅-aniline (14 and 32) and those with tetrahydro-2-naphthyl amines (9 and 11). The SF₅-aniline group was 2–5-fold more potent within this series than the CF₃-aniline group (14 vs 15; 32 vs 31; 36 vs 35). In contrast, these two groups were fairly equivalent with respect to in vitro potency within the triazolopyrimidines,²⁶ though clearly in that case SF₅-aniline provided better plasma exposure and more potent in vivo activity. Within the tetrahydro-2-naphthyl amines, compounds with the halogens substituted at the 7-position (9 and 11) were significantly more potent than those with the halogens substituted at the 6-position (10 and 12). However, the most potent compounds from the series (9, 11, 14) were 2–4-fold less potent than 1 on P. falciparum parasites, although they were close to meeting the development criteria in this assay (EC₅₀ < 10 nM). Of note, the compounds containing tetrahydro-2-naphthyl amines were assayed as racemic mixtures; so, the potency of the pure active enantiomer would be expected to be ∼2-fold better, based on the data for similarly substituted triazolopyrimidines.³² The substitution of either the R₁ or R₂ methyl group with CF₃ led to a reduction in potency, with the R₂ substitution leading to a greater loss than the R₁ substitution. Similarly, substitution of the R₁ methyl group with CH₂OH also led to reduced potency, suggesting that this group was unable to form an H-bond with Y528 in the pocket, or was too sterically hindered to bind well. The compounds with 2-indanyl amines (13, 30, 37) were less potent than those with tetrahydro-2-naphthyl amine or SF₅-aniline, similar to what we observed previously for the triazolopyrimidines.³² Select compounds were also tested against DHODH from Plasmodium vivax, and to assess species selectivity, the human enzyme was also assayed (Table 1). The activity on P. vivax DHODH ranged from 2–3-fold more potent to 2-fold less potent, depending on the compound. The four compounds that were tested also retained good selectivity versus the human enzyme, although 31 showed modest activity on the human enzyme, suggesting that replacing the R₁ methyl group with CF₃ could lead to reduced species selectivity depending on the context.

X-ray Crystallography

To assess the binding mode, a co-crystal structure was obtained between PfDHODH and 15. The structure was solved by molecular replacement to 1.99 Å resolution, with an R_work/R_free of 0.163/0.206 (Table S1). The compound 15 bound to the same site as observed previously for the triazolopyrimidines,^{19,26,32,37,38} and the overall structure was similar to these previous examples (Figure 2). The compound 15 was shifted in the pocket, so that the isoxazolopyrimidine ring was closer to the FMN cofactor than the triazolopyrimdine ring. In response to this shift, several other residues in the binding pocket also moved slightly, including F188, to accommodate the slightly different binding mode. However, like 1, 15 makes H-bonds between the bridging NH (N1) and His185, and between the pyrimidine nitrogen (N3) and Arg265, that are likely to contribute significantly to the binding energy.

Physicochemical Properties and in Vitro Metabolism

To provide a preliminary indication if the compounds were likely to show good plasma exposure after oral dosing, selected compounds were tested to measure their physicochemical properties and to assess the in vitro metabolism in the presence of human and mouse liver microsomes (Table 2). Overall, the isoxazolopyrimidines showed poor solubility at pH 6.5, and when comparing matched compounds between the isoxazolopyrimidines and the triazolopyrimidines, solubility was 2–4-fold lower for the isoxazolopyrimidines [e.g., 1 vs 14; 2 vs 16; 38(32) vs 13 (see Table 3)]. For all compounds tested, metabolic stability was significantly worse for the isoxazolopyrimidines compared to the triazolopyrimidines, and no compounds were identified that showed similar metabolic stability compared to 1 or 2.^19,31 These data suggested that the isoxazolopyrimidines were unlikely to provide the type of sustained plasma exposure necessary to meet the product profile of a single-dose cure.

Table 3. Mouse Pharmacokinetics (PK) Comparison between Isoxazolopyrimidines (13 and 14) and Triazolopyrimidines (1 and 38) Following Oral Administration.

	1a,b	14c	38a,d	13a
dose (mg/kg)	10	10	20	20
apparent t1/2 (h)	2.5	1.9	2.5	0.9
Cmax (μM)	3.1	1.3	29	2.1
Tmax (h)	1.5	0.75	1	1.0
AUCinf (μM·h)	23	3.8	170	4.2

^aBased on plasma concentrations.

^bData taken from ref (19).

^cBased on blood concentrations.

^dData taken from ref (32).

In Vivo Pharmacokinetic Studies in Mice

To directly assess the exposure after oral dosing, two compounds in the series were dosed in mice. The compound 14 was chosen as one of the most potent compounds because it was the isoxazolopyrimidine equivalent to the clinical candidate 1, allowing the assessment of the performance of SF₅-aniline within the isoxazolopyrimidine series. The compound 13 was dosed as the comparator to 38,³² which also contains difluoro 2-indanyl amine and showed good plasma exposure within the context of the triazolopyrimidine series (Table 3). In both cases, the triazolopyrimidines (1 and 38) showed a much higher exposure than the matched isoxazolopyrimidine (14 and 13), as measured by both C_max and AUC (Figure 3).

Figure 3.

Exposure profiles following a single oral administration of 10 (1 and 14) or 20 (13 and 38) mg/kg to mice, respectively. The data represent plasma concentrations for all compounds except 14, which represents blood concentration. To aid comparison between the compounds, data for 13 and 38 have been scaled to a dose of 10 mg/kg, assuming dose-proportional kinetics. The symbols represent individual data points (1, 38, 13) or mean ± SD (n = 5) (14). Note that the error bars for 14 are smaller than the symbols at most time points.

In Vivo Efficacy in the SCID Mouse Model of P. falciparum

To assess the in vivo efficacy of 14, it was dosed in the standard severe combined immunodeficient (SCID) mouse model of P. falciparum infection. The compound was dosed orally for 4 days (dose levels of 10, 20, 30, 40, 50, and 60 mg/kg). Parasitemia was monitored throughout the 7-day study, and blood levels were measured for 24 h after the first dose (Figure 4). The dose required to reach ED₉₀ (90% reduction in parasitemia) was determined from the data collected 24 h after the last dose. The ED₉₀ for 14 of ∼63 mg/kg was minimally 8-fold higher than that for 1 when dosed in the same model using once daily (QD) dosing.²⁶ The poor efficacy was likely a reflection of the much lower plasma exposure of 14 relative to 1 (Table 3). Additionally, as 60 mg/kg was the highest dose tested, the minimal dose to provide the maximum parasite clearance was not reached.

Figure 4.

P. falciparum efficacy study in SCID mice treated with 14. The mice were infected with P. falciparum on day 0 and were dosed with 14 orally once per day for 4 days (i.e., days 3–7). Parasitemia and blood levels of 14 were monitored daily to assess the efficacy. (A) Parasitemia levels. (B) Blood levels of 14 measured after the first dose on day 3. The dose levels are indicated in the figure legend.

Conclusions

Plasmodium DHODH is a clinically validated new target for the treatment of malaria. Triazolopyrimidine 1 is currently in phase II clinical development for this disease.²² To assess the potential for a related isoxazolopyrimidine series to yield a potential backup candidate that could be progressed to preclinical development, we undertook a hit-to-lead optimization of a series based on a hit from a previously published whole-cell screen (Figure 5). Although we identified analogues with good in vitro potency that met the development criteria, the isoxazolopyrimidines were less attractive at all levels than their matched triazolopyrimidine partners. Most importantly, the isoxazolopyrimidines were less soluble and were much less metabolically stable. As a consequence, they showed significantly lower plasma exposure and substantially reduced in vivo efficacy. The best analogue 14, which, like 1, contains an SF₅-aniline substituent, required too high a dose for efficacy in vivo to meet the development criteria,^14,20 and the relatively short half-life in mice suggests as well that it would not have the pharmacokinetic profile needed to reach the goal of a single-dose cure.

Figure 5.

Schematic depicting the hit-to-lead chemistry pathway and key results.

Experimental Procedures

DHODH Enzyme Purification and Assay

P. falciparum, P. vivax, and human DHODH were expressed as N-terminal truncations lacking the mitochondrial transmembrane domain as His₆-tag fusions to facilitate purification from the Escherichia coli BL21 phage-resistant cells by Ni²⁺–agarose column chromatography/gel-filtration column chromatography, as previously described.^26,30,37PfDHODH for crystallography was additionally truncated to remove a surface loop to facilitate crystallization, also as previously described.^37,39 A dye-based spectrophotometric assay that follows the reduction of 2,5-dichloroindophenol (DCIP) at 600 nm catalyzed by DHODH was used to determine the inhibitor potency as previously described^26,32 The enzyme and substrate concentrations were as follows: DHODH (5–10 nM), l-dihydroorotate (0.2 mM), CoQ_d (0.02 mM), and DCIP (0.12 mM). The assay buffer contained 100 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid (HEPES), pH 8.0, 150 mM NaCl, 10% glycerol, and 0.05% Triton X-100. The stock solutions of the isoxazolopyrimidine-based compounds and any control DHODH inhibitors were prepared in dimethyl sulfoxide (DMSO) and protected from light in dark amber vials. The IC₅₀ values (concentration that gave 50% enzyme inhibition) were determined by collecting the dose response data over a range of inhibitor concentrations (0.01–100 μM) and fitting the data to the log[I] versus response (three parameters) equation in GraphPad Prism. The reported error represents the 95% confidence interval of the fit. All data were collected in triplicate.

In Vitro Parasite Assays

The P. falciparum 3D7 parasites were grown in RPMI-1640 containing 0.5% Albumax-II as previously described.²⁶ The final DMSO concentration in the media was held constant at 0.2%. The P. falciparum growth assays were performed using the SYBR-green method after 72 h of growth in the presence of drug.³⁸ The log[I] versus response variable slope (four parameters) equation (GraphPad prism) was used to fit the data and to define the effective concentration (EC₅₀) that reduced parasitemia by 50%. The reported errors were calculated from the data collected minimally in triplicate and represent the 95% confidence interval of the fit.

X-ray Crystallography

PfDHODH (amino acids 158–569, Δ384–413) was expressed and purified as described.³⁷ The protein/ligand complex was made by incubating 50 μL of protein at 12.8 mg/mL (in 10 mM HEPES pH 7.8, 100 mM NaCl, 5% glycerol, 10 mM dl-dithiothreitol, and 1 mM N,N-dimethyldodecylamine N-oxide) with 1 μL N,N-dimethyldodecylamine N-oxide (500 mM stock), 0.5 μL l-dihydroorotate (200 mM stock), and 0.5 μL compound (200 mM stock in DMSO) at room temperature for 30 min. The crystals were grown by hanging drop vapor diffusion at 20 °C using drops consisting of 1 μL protein solution and 1 μL crystallization solution (24% PEG 3350, 100 mM potassium fluoride, and 100 mM MES pH 6.5) suspended over a reservoir of the crystallization solution. The crystals were harvested using the reservoir solution, containing 20% glycerol, and flash frozen in liquid nitrogen. The X-ray diffraction data were collected at 100 K at the European Synchrotron Radiation Facility. The data were processed and scaled using autoPROC,⁴⁰ utilizing XDS,⁴¹ POINTLESS,⁴² AIMLESS,⁴² STARANISO,⁴³ and the CCP4 suite of programs.⁴⁴ The crystal space group is P2₁, with the unit cell dimensions a = 58.7 Å, b = 163.0 Å, c = 64.7 Å, α = 90°, β = 112.9°, γ = 90°, and two protein molecules in the asymmetric unit. The data collection statistics are given in the Supporting Information, Table S1. The structures were determined using the coordinates of an isomorphous unliganded protein model (unpublished), with the preliminary refinement carried out using autoBUSTER.⁴⁵ The ligands were clearly visible in the resulting F_o–F_c electron density maps (Supporting Information, Figure S1). The initial structure refinement was carried out at 2.65 Å, which was subsequently extended to 1.99 Å to include additional reflections, following the analysis of merged intensity data for anisotropy of diffraction using STARANISO. Coot⁴⁶ was used for model building, with the refinement completed using autoBUSTER. The statistics for the final models are given in the Supporting Information, Table S1. The coordinates and structure factors have been deposited in PDB under the accession code 6GJG.

Physicochemical Properties

log D_7.4 was estimated using a chromatographic method as described previously.²⁴ The aqueous solubility was assessed using a plate-based method, whereby the compound in DMSO was spiked into a pH 6.5 phosphate buffer (final DMSO content 1 v/v %) and the samples analyzed by nephelometry to determine the solubility range based on a method described previously.²⁴

In Vitro Metabolism

The microsome in vitro intrinsic clearance (in vitro CL_int) and predicted hepatic extraction (E_h) ratio of the selected compounds were determined using human and mouse liver microsomes (BD Gentest, BD Biosciences, Bedford, MA or Sekisui XenTech, LLC, Kansas City, KS) following incubation (37 °C) at a compound concentration of 0.5 or 1 μM and microsomal protein concentration of 0.4 or 0.5 mg/mL. The metabolic reactions were initiated by the addition of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-regenerating system (1 mg/mL NADPH, 1 mg/mL glucose-6-phosphate, and 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl₂ (0.67 mg/mL) and were quenched by the addition of ice-cold acetonitrile. The amount of compound remaining in the sample supernatant at selected time points over 0–60 min was quantified by liquid chromatography–mass spectrometry (LC–MS). The methods used for the determination of in vitro CL_int and E_h values have been described previously.²⁶

Animal Studies

All animal studies were ethically reviewed and carried out in accordance with either the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (mouse PK studies for 13) or European Directive 2010/63/EEC and the GSK Policy on the Care, Welfare, and Treatment of Animals (mouse PK studies for 3 and 14 and SCID mouse studies for 14).

Mouse PK

In vivo PK studies of 13 were conducted in male Swiss outbred mice. The compound was administered by oral gavage (10 mL/kg) of a suspension formulation containing 0.5 w/v % carboxymethylcellulose, 0.5 v/v % benzyl alcohol, and 0.4 v/v % Tween 80. Blood was collected over 24 h by either submandibular bleed or terminal cardiac puncture (two samples per mouse) into heparinized tubes, and plasma was separated by centrifugation and stored at −80 °C until analysis. The compound concentrations were quantified by LC–MS following protein precipitation with acetonitrile containing an internal standard. A Waters Acquity UPLC with a Supelco Ascentis Express RP Amide column (50 × 2.1 mm, 2.7 μm) was used for chromatography with the mobile phase buffer 0.05% formic acid in water and 0.05% formic acid in methanol mixed by a linear gradient (flow rate 0.4 mL/min; injection volume 3 μL; and cycle time 4 min). Positive electrospray ionization mass spectrometry with multiple reaction monitoring was used for detection on a Waters Xevo TQ mass spectrometer (cone voltage 35 V and collision energy 20 V). The concentrations were quantified by comparison to calibration standards prepared in blank mouse plasma, and the pharmacokinetic parameters were determined by noncompartmental analysis.

For 3 and 14, mouse PK was performed by oral gavage of suspension formulations prepared in 0.1 w/v % methylcellulose. Peripheral blood samples (25 μL) were taken serially from 0.5 to 24 h, mixed with water (25 μL) to lyse the erythrocytes, and stored at −80 °C until use. A 96-well plate filter system (MultiScreen Solvinert 0.45 μm Hydrophobic PTFE; Millipore) was used to perform protein precipitation by liquid–liquid extraction. Per well, acetonitrile/methanol (80:20, v/v) (0.12 mL) and internal standard was mixed with blood lysate (10 μL). The plates were vortexed (10 min), filtered, and analyzed by LC–MS/MS (Sciex API 4000 Triple Quadrupole Mass Spectrometer, Sciex, Division of MDS Inc., Toronto, Canada) in positive ion mode with electrospray ionization. Phoenix, version 6.3 (Phoenix WinNonlin, Certara L.P.) was used to perform noncompartmental analysis to determine the pharmacokinetic parameters.

SCID Mouse Efficacy Studies

The in vivo efficacy of 14 was measured against P. falciparumPf3D7^0087/N9 growing in the peripheral blood of NOD-scidIL2Rγ^null mice (Jackson Laboratory, USA) (23–36 g) engrafted with human erythrocytes as described.⁴⁷ Human biological samples were sourced and used in accordance with the terms defined in the informed consent of the IRB/EC-approved protocol. The parasites (20 × 10⁶) were inoculated by intravenous injection, and antimalarial efficacy was assessed using a standard “4-day test”. Blood parasitemia was measured by FACS analysis. Compound 14 was administered in vehicle (1% methylcellulose) by oral gavage QD for four consecutive days starting on the third day after infection. The actual administered doses were determined by the measurement of the formulation concentrations. The compound concentrations in either the formulations or the blood from the infected mice were measured by LC–MS–MS (described above). The efficacy markers (ED₉₀, AUC_ED90) were determined on the seventh day after infection, one day after the last dose.

Chemistry

General Methods

Commercial suppliers were used to source reagents and starting materials, which were then used without further purification unless indicated. Flash chromatography was performed with the Biotage (prepacked) silica-gel columns forming the stationary phase and the analytical grade solvents as the eluent. Preloaded silica gel 60 F₂₅₄ plates were employed to monitor the reaction progress by thin layer chromatography, and the results were visualized by UV light and iodine vapor. The reported yields were for purified product and were not optimized. The proton nuclear magnetic resonance (¹H NMR) spectra were determined at room temperature using Bruker AVANCE 300 and 400 MHz spectrometers with chemical shifts provided in parts per million (δ) and coupling constants in Hertz. The residual solvent peaks from the ¹H NMR spectra were used as internal standards (7.26 ppm for CDCl₃, 2.50 ppm for DMSO-d₆, and 3.34 ppm for MeOD). Spin multiplicities are defined with the following abbreviations: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), and m (multiplet). An Agilent Liquid Chromatograph—6320 Ion trap or 6130 mass spectrometer was used to record the total ion current traces by electrospray positive and negative ionization (ES+/ES−). The LC–MS conditions for analytical chromatography were as follows: Column, Zorbax XDB C18 (Agilent Technologies, 50 × 4.6 mm) or Atlantis dC18 (Waters, 50 × 4.6 mm), both with a stationary phase particle size of 5 μM. The aqueous solvent (A) was 0.1% HCOOH or TFA in water and the organic solvent (B) was acetonitrile. The samples were separated at a column temperature of 25 °C using a run time of 7 min and the following gradient steps: 0–4.0 min 10–95% of B, 4.0–5.0 min 95% of B, 5.0–5.5 min 95–10% of B, and 5.5–7.0 min 10% of B, with a flow rate of 1.0 mL/min (injection volume 2 μL). Detection was at the UV wavelength range (210–400 nm). An Atlantis dC18 column from Waters (5 μm, 250 × 4.6 mm) with a linear elution gradient ranging from 0 to 100% ACN over 30 min at a flow rate of 1 mL/min was used for analytical high-performance liquid chromatography analyses. A purity of >95% LC–MS has been established for all the reported compounds.

3-Amino-4-cyano-5-methyl isoxazole (5)

To a solution of hydroxylamine hydrochloride (11.72 g, 0.1688 mol) in water (36.8 mL), sodium hydroxide (6.75 g, 0.01688 mol) and ethanol (82.8 mL) were added and stirred for 30 min. To this stirred solution, (1-ethoxy ethylidene)malonitrile (4) (23 g, 0.1688 mol) was added carefully, and the reaction mixture was heated to 50 °C for 30 min and then stirred at 25 °C for 16 h. After the completion of the reaction, ethanol was removed under reduced pressure. The residue was triturated with diethylether; the solid precipitated out was filtered, and the filtered product was washed with water and dried under reduced pressure to get 18.35 g (90% yield) of 3-amino-4-cyano-5-methyl isoxazole (5). ¹H NMR (400 MHz, DMSO-d₆): δ 8.34 (s, 2H), 2.13 (s, 3H).

5-Amino-3-methylisoxazole-4-carboxamide (6)

Concentrated sulfuric acid (128.45 mL) was added slowly to 5-amino-4-cyano-3-methylisoxazole (5) (18.3 g, 0.1490 mol), and the solution was stirred at 50 °C for 1 h and stirred for an additional hour at 25 °C. After the completion of the reaction, the mixture was carefully added to crushed ice, yielding a white solid, which slowly dissolved as the stirred mixture was allowed to warm to room temperature. To this mixture, concentrated ammonium hydroxide was carefully added, with the ice cooling to pH 9, and the resulting suspension was refrigerated overnight to give a crystalline white solid; the solid was filtered and dried under reduced pressure to get 14 g (66.5% yield) of 5-amino-3-methylisoxazole-4-carboxamide (6). ¹H NMR (400 MHz, DMSO-d₆): δ 7.46 (s, 2H), 6.69 (s, 2H), 2.24 (s, 3H).

3,6-Dimethylisoxazolo[5,4]pyrimidin-4-ol (7)

A mixture of 5-amino-3-methyl isoxazole-4-carboxamate (6) (1 g, 0.07 mol), triethyl ortho acetate (6 mL), and acetic anhydride (6 mL) was microwaved at 170 °C for 30 min. After the completion of the reaction, the reaction mixture was cooled to 25 °C; the precipitated product was collected by filtration, washed with cold ether, and dried under reduced pressure to get 0.74 g (62.16% yield) of 3,6-dimethylisoxazolo[5,4]pyrimidin-4-ol (7). ¹H NMR (400 MHz, DMSO-d₆): δ 12.77 (s, 1H), 2.42 (s, 3H), 2.39 (s, 3H).

4-Chloro-3,6-dimethyl-isoxazolo[5,4]pyrimidine (8)

A mixture of 3,6-dimethylisoxazolo[5,4]pyrimidin-4-ol (7) (10.18 g, 0.061 mol) and phosphorus oxychloride (39.61 g, 0.258 mol) was refluxed for 6 h at 105 °C. After the completion of the reaction, the resultant dark red solution was concentrated to remove excess POCl₃ and then slowly added to a mixture of ice and water. The solution was neutralized with 10% aq Na₂CO₃, and the product was extracted with dichloromethane (DCM). Further extractions of the aqueous layer with DCM were performed; the organic layers were combined and washed with water, followed by brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was then purified by column chromatography, yielding 8.5 g (75.5% yield) of 4-chloro-3,6-dimethyl-isoxazolo[5,4]pyrimidine (8).

N-(7-Chloro-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (9)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (5) (0.15 g, 0.000819 mol) in IPA (5 mL), 7-chloro-1,2,3,4-tetrahydronaphthalen-2-ylamine HCl (0.17 g, 0.000819 mol) and diisopropylethylamine (0.15 g, 0.0012 mol) were added, and the reaction was microwaved at 135 °C for 30 min. After the completion of the reaction, the solvent was removed under reduced pressure, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.067 g (25% yield) of pure N-(7-chloro-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (9). LC–MS APCI: calculated for C₁₇H₁₇ClN₄O, 328.80; observed m/z: [M + H]⁺ 329.4 (purity: 99.69%), ¹H NMR (400 MHz, MeOD): δ 7.43 (d, J = 8.04 Hz, 1H), 7.26 (d, J = 8.44 Hz, 1H), 7.06 (d, J = 8.04 Hz, 1H), 4.73–4.78 (m, 1H), 3.17–3.20 (m, 1H), 2.90–3.00 (m, 3H), 2.61 (s, 3H), 2.53 (s, 3H), 2.21–2.23 (m, 1H), 1.93–1.98 (m, 1H).

N-(6-Chloro-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (10)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.1 g, 0.00054 mol) in IPA (4 mL), 6-chloro-1,2,3,4-tetrahydronaphthalen-2-amine HCl (0.12 g, 0.00054 mol) and diisopropylethylamine (0.104 g, 0.00081 mol) were added, and the reaction was microwaved at 135 °C for 30 min. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.075 g (28% yield) of pure N-(6-chloro-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (10). LC–MS APCI: calculated for C₁₇H₁₇ClN₄O, 328.80; observed m/z: [M + H]⁺ 329.2 (purity: 99.73%), ¹H NMR (400 MHz, MeOD): δ 7.17 (s, 1H), 7.08–7.14 (m, 2H), 4.71–4.77 (m, 1H), 3.17–3.22 (m, 1H), 2.88–3.01 (m, 3H), 2.62 (s, 3H), 2.53 (s, 3H), 2.22–2.25 (m, 1H), 1.94–2.01 (m, 1H).

N-(7-Bromo-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (11)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.17 g, 0.00092 mol) in IPA (4 mL), 7-bromo-1,2,3,4-tetrahydronaphthalen-2-amine HCl (0.24 g, 0.00092 mol) and diisopropylethylamine (0.18 g, 0.00139 mol) were added, and the reaction was microwaved at 135 °C for 30 min. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.055 g (16% yield) of pure N-(7-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (11). LC–MS APCI: calculated for C₁₇H₁₇BrN₄O, 373.25; observed m/z: [M + H]⁺ 373.4 (purity: 99.89%), ¹H NMR (400 MHz, MeOD): δ 7.43 (d, J = 8.04 Hz, 1H), 7.26 (d, J = 8.44 Hz, 1H), 7.06 (d, J = 8.04 Hz, 1H), 4.73–4.78 (m, 1H), 3.17–3.20 (m, 1H), 2.90–3.00 (m, 3H), 2.61 (s, 3H), 2.53 (s, 3H), 2.21–2.23 (m, 1H), 1.93–1.98 (m, 1H).

N-(6-Bromo-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (12)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.1 g, 0.00043 mol) in IPA (4 mL), 6-bromo-1,2,3,4-tetrahydronaphthalen-2-amine HCl (0.11 g, 0.00043 mol) and diisopropylethylamine (0.18 g, 0.00064 mol) were added, and the reaction was microwaved at 135 °C for 30 min. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.077 g (22.5% yield) of pure N-(6-bromo-1,2,3,4-tetrahydronaphthalen-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (12). LC–MS APCI: calculated for C₁₇H₁₇BrN₄O, 373.25; observed m/z: [M + 2]⁺ 375.2, (purity: 99.69%), ¹H NMR (400 MHz, MeOD): δ 7.32 (s, 1H), 7.26 (d, J = 1.80 Hz, 1H), 7.03 (d, J = 8.28 Hz, 1H), 4.71–4.75 (m, 1H), 3.14–3.19 (m, 1H), 2.97–2.99 (m, 2H), 2.85–2.92 (m, 1H), 2.62 (s, 3H), 2.53 (s, 3H), 2.20–2.24 (m, 1H), 1.96–2.00 (m, 1H).

N-(5,6-Difluoro-2,3-dihydro-1H-inden-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (13)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.1 g, 0.00054 mol) in IPA (4 mL), 5,6-difluoro-2,3-dihydro-1H-inden-2-amine(0.09 g, 0.00054 mol) and diisopropylethylamine (0.139 g, 0.00108 mol) were added, and the reaction was microwaved at 150 °C for 30 min. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.089 g (51.7% yield) pure N-(5,6-difluoro-2,3-dihydro-1H-inden-2-yl)-3,6-dimethylisoxazolo[5,4-d]pyrimidin-4-amine (13). LC–MS APCI: calculated for C₁₆H₁₄F₂N₄O, 316.31; observed m/z: [M + H]⁺ 317.2, (purity: 95.65%), ¹H NMR (400 MHz, MeOD): δ 7.13 (t, J = 9.08 Hz, 2H), 5.21–5.29 (m, 1H), 3.36–3.42 (m, 2H), 3.03–3.09 (m, 2H), 2.60 (s, 3H), 2.54 (s, 3H).

3,6-Dimethyl-N-(4-(pentafluoro-l6-sulfanyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (14)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.1 g, 0.00054 mol) in EtOH(5 mL), 4-(pentafluoro-l6-sulfanyl)aniline (0.18 g, 0.00054 mol) was added, and the reaction was heated at 60 °C for 3 h. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for 8, to yield 0.049 g (25% yield) 3,6-dimethyl-N-(4-(pentafluoro-l6-sulfanyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (14). LC–MS APCI: calculated for C₁₃H₁₁F₅N₄OS, 366.31; observed m/z: [M + H]⁺: 367.0, (purity: 99.80%), ¹H NMR (400 MHz, MeOD): δ 7.98 (d, J = 9.04 Hz, 2H), 7.85 (d, J = 2.08 Hz, 2H), 2.75 (s, 3H), 2.63 (s, 3H).

3,6-Dimethyl-N-(4-(trifluoromethyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (15)

To a solution of 4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.15 g, 0.00081 mol) in THF (10 mL), 4-trifluoromethylaniline (0.13 g, 0.00081 mol) was added, and the reaction was heated at 140 °C for 72 h. After the completion of the reaction, the solvent was removed under reduced pressure. The reaction mixture was cooled and diluted with water and extracted with EtOAc. The organic layer was washed with brine solution and dried over anhydrous Na₂SO₄, filtered, concentrated under reduced pressure, and purified by column chromatography, yielding 0.095 g (38% yield) of 3,6-dimethyl-N-(4-(trifluoromethyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (15). LC–MS APCI: calculated for C₁₄H₁₁F₃N₄O, 308.26; observed m/z: [M + H]⁺ 309.2, (purity: 99.63%), ¹H NMR (400 MHz, MeOD): δ 7.97 (d, J = 8.44 Hz, 2H), 7.70 (d, J = 8.52 Hz, 2H), 2.74 (s, 3H), 2.60 (s, 3H).

3,6-Dimethyl-N-(6-(trifluoromethyl)pyridin-3-yl)isoxazolo[5,4-d]pyrimidin-4-amine (16)

4-chloro-3,6-dimethylisoxazolo[5,4-d]pyrimidine (8) (0.15 g, 0.000816 mol) and 6-(trifluoromethyl)pyridine-3-amine (0.105 g, 0.000653 mol) were dissolved in THF (15mL) under nitrogen. Sodium tert-butoxide (0.23 g, 0.00244 mol) was added and degassed for 10 min. tert-Butylxphos (0.067 g, 0.0000979 mol) was added and heated to reflux for 3 h. After the completion of the reaction, the reaction mixture was cooled to 25 °C and diluted with water and extracted with EtOAc (2 × 10 mL). The combined organic layers were washed with brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography, yielding 0.5 g (20% yield) of 3,6-dimethyl-N-(6-(trifluoromethyl)pyridin-3-yl)isoxazolo[5,4-d]pyrimidin-4-amine (16). LC–MS APCI: calculated for C₁₃H₁₀F₃N₅O, 309.25; observed m/z: [M + H]⁺ 310.2, (purity: 99.83%), ¹H NMR (400 MHz, MeOD): δ 9.10 (d, J = 2.40 Hz, 1H), 8.52–8.55 (m, 1H), 7.87 (d, J = 8.60 Hz, 1H), 2.78 (s, 3H), 2.63 (s, 3H).

Ethyl 5-Amino-4-carbamoylisoxazole-3-carboxylate (19)

2-Cyanoacetamide (17) was added to the freshly prepared NaOEt from sodium (0.13 g, 0.0058 mol) and ethanol (3 mL), and the reaction mixture was stirred at 25 °C for 1 h. The reaction mixture was cooled to 0 °C, and a solution of ethoxy-2-chloro-2-ethyl-hydroxy oximido acetate (18) (0.45 g, 0.0029 mol) was added in EtOH (2 mL) and the reaction mixture was heated for 4 h at 60 °C. The reaction mixture was cooled and diluted with water and extracted twice with EtOAc; the organic layer was washed with a brine solution and dried over anhydrous Na₂SO₄, filtered, concentrated, and purified by column chromatography, yielding 0.04 g (7% yield) pure ethyl 5-amino-4-carbamoylisoxazole-3-carboxylate (19).

Ethyl 6-Methyl-4-oxo-4,5-dihydroisoxazolo[5,4-d]pyrimidine-3-carboxylate (20)

A mixture of ethyl 5-amino-4-carbamoylisoxazole-3-carboxylate (19) (0.25 g, 0.0012 mol), triethyl ortho acetate (3.1 g, 0.0188 mol), and acetic anhydride (1.9 g, 0.0188 mol) was microwaved for 20 min at 130 °C. After the completion of the reaction, DCM was added, and the solid precipitated out was filtered and dried to get 0.14 g (51% yield) of ethyl 6-methyl-4-oxo-4,5-dihydroisoxazolo[5,4-d]pyrimidine-3-carboxylate (20). ¹H NMR (400 MHz, DMSO-d₆): δ 12.98 (s, 1H), 4.40 (q, J = 9.44 Hz, 2H), 2.42 (s, 3H), 1.32 (t, J = 9.44 Hz, 3H).

Ethyl 4-Chloro-6-methylisoxazolo[5,4-d]pyrimidine-3-carboxylate (21)

A mixture of ethyl 6-methyl-4-oxo-4,5-dihydroisoxazolo[5,4-d]pyrimidine-3-carboxylate (20) (0.1 g, 0.000448 mol) and POCl₃ (3 mL) was refluxed for 4 h at 100 °C. After the completion of the reaction, the resultant dark red solution was concentrated to remove excess POCl₃ and then slowly added to a mixture of ice and water. The solution was neutralized with 10% aq NaCO₃ and the product was extracted with DCM as described for compound 8. The solvent was removed under reduced pressure, and the crude product was purified by column chromatography to yield 0.07 g (70% yield) of ethyl 4-chloro-6-methylisoxazolo[5,4-d]pyrimidine-3-carboxylate (21).

Ethyl 6-Methyl-4-((4-(pentafluorosulfanyl)phenyl)amino)isoxazolo[5,4-d]pyrimidine-3-carboxylate (22)

A mixture of ethyl 4-chloro-6-methylisoxazolo[5,4-d]pyrimidine-3-carboxylate (21) (0.11 g, 0.00045 mol) and 4-(pentafluorosulfanyl)aniline (0.099 g) in EtOH (5 mL) was heated to 90 °C for 4 h. After the completion of the reaction, the solvent was removed under reduced pressure. The crude product was dissolved in DCM and washed with water as described for compound 8, and the product was purified by column chromatography, yielding 0.105 g (54.4% yield) pure ethyl 6-methyl-4-((4-(pentafluorosulfanyl)phenyl)amino)isoxazolo[5,4-d]pyrimidine-3-carboxylate (22). ¹H NMR (400 MHz, CDCl₃): δ 10.52 (s, 1H), 8.03 (d, J = 11.48 Hz, 2H), 7.80 (d, J = 12.20 Hz, 2H), 4.65 (q, J = 9.56 Hz, 2H), 2.77 (s, 1H), 1.55 (t, J = 9.56 Hz, 3H).

(6-Methyl-4-((4-(pentafluorosulfanyl)phenyl)amino)isoxazolo[5,4-d]pyrimidin-3-yl)methanol (23)

A solution of ethyl 6-methyl-4-((4-(pentafluorosulfanyl)phenyl)amino)isoxazolo[5,4-d]pyrimidine-3-carboxylate (22) (0.07 g, 0.000165 mol) in THF (3 mL): MeOH (2 mL) was cooled to 0 °C, and NaBH₄ (0.024 g, 0.00066 mol) was added and allowed to stir at 25 °C for 4 h. After the completion of the reaction, the solvents were removed under reduced pressure. The crude product was dissolved in DCM, washed, and purified as described for compound 8 by column chromatography to get 0.049 g (77.7% yield) (6-methyl-4-((4-(pentafluorosulfanyl)phenyl)amino)isoxazolo[5,4-d]pyrimidin-3-yl)methanol (23). LC–MS APCI: calculated for C₁₃H₁₁F₅N₄O₂S, 382.31; observed m/z: [M + H]⁺ 383.2, (purity: 97.69%), ¹H NMR (400 MHz, DMSO-d₆): δ 10.49 (s, 1H), 8.03 (d, J = 9.08 Hz, 2H), 7.97 (t, J = 2.20 Hz, 2H), 7.35–7.37 (m, 1H), 5.00 (s, 2H), 2.65 (s, 3H).

2,2,2-Trifluoroacetaldehyde oxime (25)

A solution of trifluoroacetaldehyde methyl hemiacetal (24) (20 g, 0153 mol) and hydroxylamine hydrochloride (10.9 g, 0.1568 mol) in MeOH (30 mL) was cooled to 0 °C; to this cooled reaction mixture, 50% NaOH aq solution (36 mL) was added and stirred for 16 h at 25 °C. Heptane and water were added to the reaction, and the layers were separated. The aqueous layer was acidified with 6 N HCl and extracted with diethylether. The organic layer was dried over Na₂SO₄, filtered, and concentrated to get 22.5 g of 2,2,2-trifluoroacetaldehyde oxime (25) as yellow oil.

2,2,2-Trifluoro-N-hydroxyacetimidoyl Bromide (26)

A solution of 2,2,2-trifluoroacetaldehyde oxime (25) (22.5 g, 0.199 mol) in DMF (15 mL) was cooled to 0 °C. To this cooled solution, NBS (35.3 g, 0.0199 mol) in DMF (15 mL) was added dropwise at 0 °C and stirred at 25 °C for 4 h. After the completion of the reaction, the reaction mixture was diluted with water and diethylether; the layers were separated and the organic layer dried over Na₂SO₄, filtered, and concentrated under reduced pressure to get 22.7 g (59% yield) of 2,2,2-trifluoro-N-hydroxyacetimidoyl bromide (26) as yellow oil.

5-Amino-3-(trifluoromethyl)isoxazole-4-Carboxamide (27)

A mixture of 2-cyano acetamide (17) (17.7 g, 0.211 mol) and sodium methoxide (10.3 g, 0.192 mol) in MeOH (50 mL) was stirred at 25 °C. To this, 2,2,2-trifluoro-N-hydroxyacetimidoyl bromide (26) (17.7 g, 0.211 mol) in MeOH (50 mL) was added dropwise and stirred at 25 °C for 1 h. After the completion of the reaction, methanol was removed under reduced pressure, and the residue was washed with cold dil. aq ammonia; the solid separated out was filtered and dried to get 1.5 g (10% yield) of 5-amino-3-(trifluoromethyl)isoxazole-4-carboxamide (27). ¹H NMR (400 MHz, DMSO-d₆): δ 8.01 (s, 2H), 6.97 (s, 2H).

6-Methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one (28)

To a solution of 5-amino-3-(trifluoromethyl)isoxazole-4-carboxamide (27) (0.5 g, 0.0025 mol) in acetic anhydride (3 mL), trimethyl orthoacetate (3 mL) was added and microwaved at 120 °C for 1 h. After the completion of the reaction, the solvent was removed and triturated with ether to get 0.3 g (53% yield) pure 6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one (28). ¹H NMR (400 MHz, DMSO-d₆): δ 13.27 (s, 1H), 2.83 (s, 3H).

4-Chloro-6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (29)

To a slurry of 6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one (28) (0.25 g, 0.00114 mol) in toluene (10 mL), catalytic amounts of dimethyl aniline and POCl₃ (1.77 g, 0.0114 mol) were added and heated to 120 °C for 3 h. After the completion of the reaction, the volatiles were removed, the residue was dissolved in EtOAc, and washed with water and brine solution. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure, and the crude product was purified by column chromatography yielding 0.155 g (55% yield) of 4-chloro-6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (29).

N-(5,6-Difluoro-2,3-dihydro-1H-inden-2-yl)-6-methyl-3-(Trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine (30)

To a solution of 4-chloro-6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (29) (0.07 g, 0.00031 mol) in IPA (5 mL), 5,6-difluoro-2,3-dihydro-1H-inden-2-amine (0.054 g, 0.00031 mol) and diisopropylethylamine (0.083 g, 0.00063 mol) were added, and the reaction was microwaved at 150 °C for 30 min. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM and processed, as described for compound 8, yielding 0.08 g (68.4% yield) pure N-(5,6-difluoro-2,3-dihydro-1H-inden-2-yl)-6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine, after column chromatography (30). LC–MS APCI: calculated for C₁₆H₁₁F₅N₄O, 370.28; observed m/z: [M + H]⁺ 371.2, (purity: 97.72%), ¹H NMR (400 MHz, MeOD): δ 7.15 (t, J = 9.04 Hz, 2H), 5.23–5.27 (m, 1H), 3.39–3.44 (m, 2H), 3.02–3.07 (m, 2H), 2.61 (s, 3H).

6-Methyl-3-(trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (31)

To a solution of 4-chloro-6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (29) (0.1 g, 0.00045 mol) in EtOH, 4-(trifluoromethyl)aniline (0.074 g, 0.00045 mol) was added and the reaction was heated at 60 °C for 16 h. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM and processed, as described for compound 8, to yield 0.073 g (45% yield) pure 6-methyl-3-(trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (31). LC–MS APCI: calculated for C₁₄H₈F₆N₄O, 362.23; observed m/z [M + H]⁺ 363.2, (purity: 99.85%), ¹H NMR (400 MHz, CDCl₃): δ 7.90 (d, J = 8.52 Hz, 2H), 7.72 (d, J = 8.64 Hz, 2H), 7.36 (s, 1H), 2.78 (s, 3H).

6-Methyl-N-(4-(pentafluoro-l6-sulfanyl)phenyl)-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine (32)

To a solution of 4-chloro-6-methyl-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (29) (0.1 g, 0.00045 mol) in EtOH, 4-(pentafluorosulfanyl)aniline (0.1 g, 0.00045 mol) was added, and the reaction was heated at 60 °C for 16 h. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, processed, as described for 8, and purified by column chromatography to yield 0.055 g (28% yield) 6-methyl-N-(4-(pentafluoro-l6-sulfanyl)phenyl)-3-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine (32). LC–MS APCI: calculated for C₁₃H₈F₈N₄OS, 420.28; observed m/z: [M – H] 419.0, (purity: 99.42%), ¹H NMR (400 MHz, CDCl₃): δ 7.83–7.90 (m, 4H), 7.36 (s, 1H), 2.79 (s, 3H).

3-Methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one (33)

5-Amino-3-methylisoxazole-4-carboxamide (6) (0.2 g, 0.0014 mol) and trifluoroacetic anhydride (0.59 g,0.0028 mol) were added to freshly prepared NaOEt and stirred at 60 °C for 16 h. After the completion of the reaction, the volatiles were removed under reduced pressure. The product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, to yield 0.15 g (48% yield) 3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one (33).

4-Chloro-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (34)

To a slurry of 3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4(5H)-one (33) (0.15 g, 0.00114 mol) in toluene (10 mL), catalytic amounts of dimethyl aniline and POCl₃ (5 mL) were added and heated to 120 °C for 3 h. After the completion of the reaction, the volatiles were removed, and the residue was dissolved in EtOAc and washed with water and brine solution. The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure; the crude product was purified by column chromatography to yield 0.10 g (60% yield) of 4-chloro-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (34).

3-Methyl-6-(trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine (35)

To a solution of 4-chloro-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (34) (0.07 g, 0.00031 mol) in EtOH, 4-(trifluoromethyl)aniline (0.051 g, 0.00031 mol) was added, and the reaction was heated at 60 °C for 16 h. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, to yield 0.085 g (73% yield) pure 3-methyl-6-(trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)isoxazolo[5,4-d]pyrimidin-4-amine(35). LC–MS APCI: calculated for C₁₄H₈F₆N₄O, 362.23; observed m/z: [M – H] 361.2, (purity: 99.79%), ¹H NMR (400 MHz, CDCl₃): δ 7.89 (d, J = 8.60 Hz, 2H), 7.74 (d, J = 8.60 Hz, 2H), 7.21 (s, 1H), 2.82 (s, 3H).

3-Methyl-N-(4-(pentafluoro-l6-sulfanyl)phenyl)-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine (36)

To a solution of 4-chloro-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (34) (0.08 g, 0.000365 mol) in EtOH, 4-(pentafluoro-l6-sulfanyl)aniline (0.08 g, 0.000365 mol) was added, and the reaction was heated at 60 °C for 16 h. After the completion of the reaction, the solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.066 g (42% yield) 3-methyl-N-(4-(pentafluoro-l6-sulfanyl)phenyl)-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine (36). LC–MS APCI: calculated for C₁₃H₈F₈N₄OS, 420.28; observed m/z: [M – H] 419.0, (purity: 99.54%), ¹H NMR (400 MHz, CDCl₃): δ 7.87–7.81 (m, 4H), 7.20 (s, 1H), 2.83 (s, 3H).

N-(5,6-Difluoro-2,3-dihydro-1H-inden-2-yl)-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine (37)

To a solution of 4-chloro-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidine (34) (0.15 g, 0.00062 mol) in IPA (5 mL), 5,6-difluoro-2,3-dihydro-1H-inden-2-amine (0.105 g, 0.00062 mol) and diisopropylethylamine (0.161 g, 0.0012 mol) were added and the reaction microwaved at 150 °C for 30 min. The solvent was removed, and the product was dissolved in DCM, washed, and purified by column chromatography, as described for compound 8, yielding 0.095 g (41% yield) pure N-(5,6-difluoro-2,3-dihydro-1H-inden-2-yl)-3-methyl-6-(trifluoromethyl)isoxazolo[5,4-d]pyrimidin-4-amine, (37). LC–MS APCI: calculated for C₁₆H₁₁F₅N₄O, 370.28; observed m/z: [M – H] 369.0, (purity: 99.08%), ¹H NMR (400 MHz, DMSO-d₆): δ 8.22 (d, J = 6.92 Hz, 1H), 7.34 (t, J = 9.24 Hz, 2H), 5.10–5.12 (m, 1H), 3.32–3.38 (m, 2H), 3.08–3.14 (m, 2H), 2.66 (s, 3H).

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b01573.

• X-ray data collection and refinement statistics for PfDHODH bound to 15 and electron density difference maps for the 15 binding site on PfDHODH (PDF)

• Molecular weights and formulas of the compounds(XLSX)

Accession Codes

The coordinates and structure factors have been deposited in the Protein Data Bank under the accession code 6GJG.

The authors declare no competing financial interest.