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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 August 11.
Published in final edited form as:
PMCID: PMC3156099
NIHMSID: NIHMS310702

Structure-guided lead optimization of triazolopyrimidine-ring substituents identifies potent Plasmodium falciparum dihydroorotate dehydrogenase inhibitors with clinical candidate potential

Abstract

Drug therapy is the mainstay of antimalarial therapy, yet current drugs are threatened by the development of resistance. In an effort to identify new potential anti-malarials we have undertaken a lead optimization program around our previously identified triazolopyrimidine-based series of Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of PfDHODH was used to inform the medicinal chemistry program allowing the identification of a potent and selective inhibitor (DSM265) that acts through DHODH inhibition to kill both sensitive and drug resistant strains of the parasite. This compound has similar potency to chloroquine in the humanized SCID mouse P. falciparum model, can be synthesized by a simple route, and rodent pharmacokinetic studies demonstrated it has excellent oral bioavailability, a long half-life and low clearance. These studies have identified the first candidate in the triazolopyrimidine series to meet previously established progression criteria for efficacy and ADME properties, justifying further development of this compound towards clinical candidate status.

Introduction

Malaria is an ancient human enemy with descriptions of malarial-like febrile illness documented over two thousand years ago in the writings of the Greek physician Hippocrates.1 The identification of the parasite and the link to mosquito-based transmission in 1880 led to our modern understanding of the disease. Drugs to treat malaria predate our knowledge of its etiology (e.g. quinine was isolated from the cinchona bark in 1820), yet today malaria still leads to 220 million cases and approximately 1 million deaths per year, with over 2 billion people at risk for the disease.2, 3 The world community is currently involved in its second attempt at global malaria eradication.4 While some progress has been made in developing a vaccine, the best candidate RTSS provides only partial immunity.5 Thus the mainstay of anti-malarial treatment, and the key to successful eradication continues to be chemotherapy. Against a backdrop of widespread drug resistance to traditional therapies (e.g. chloroquine and pyrimethamine/sulfadoxine), the introduction of artemisinin-based combination therapies (ACTs) has become the most powerful tool to combat the disease.6, 7 ACTs are highly effective for the treatment of Plasmodium falciparum malaria leading to significant reduction in the mortality and morbidity of the disease. Recent emergence of potential artemisinin resistance along the Thai-Cambodia border threatens to derail these successes, as well as any hope at global eradication.8 Few clinically approved treatment options will remain if artemisinin resistance becomes widespread.

A continual pipeline to identify and develop anti-malarial agents is required to combat the ability of the parasite to rapidly acquire resistance to chemotherapy in the field.9 The global effort to identify new drugs is being fueled by not-for-profit organizations and the development of substantial portfolios of candidate molecules ranging from identified hits to compounds in clinical development.1012 The completion of whole organism screens of large chemical libraries has identified thousands of new chemical species with anti-malarial activity 13, 14, while target-based approaches are also being successfully used to find novel chemotypes. The translation of these findings to clinical successes still presents a formidable challenge to the development of new drugs.

Dihydroorotate dehydrogenase (DHODH) has emerged as the best validated new target for the development of novel anti-malarials since the identification of the bc1 complex as the target of atovaquone, based on the discovery of several classes of inhibitors with anti-malarial activity.15 DHODH catalyses the fourth step in the de novo pyrimidine biosynthetic pathway, the flavin mononucleotide (FMN)-dependent oxidation of dihydrorotate to orotic acid. P. falciparum DHODH (PfDHODH) is localized to the inner mitochondrial-membrane and utilizes ubiquinone (CoQ) as the final electron acceptor to regenerate oxidized FMN. The malaria parasite is uniquely vulnerable to inhibition of this pathway because it lacks the salvage enzymes that serve as an additional source of pyrimidine nucleosides in other organisms, including the human host. Several different chemical series that are potent inhibitors of PfDHODH have been identified by enzyme activity-based HTS and were subsequently found to have anti-malarial activity both in vitro and in vivo. These include the triazolopyrimidines (1) (Fig. 1) identified by our group1619 and N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides identified by Genzyme and colleagues.20, 21 Structural analysis of the bound inhibitor enzyme complexes has revealed that these different chemical classes have overlapping but distinctly different binding modes owing to conformational flexibility in the enzyme inhibitor binding-site.21, 22 Most recently, metabolically stable triazolopyrimidines (24)(Fig. 1) have been identified that are able to suppress parasites in a mouse model of infection, however these compounds lacked the potency required in a clinical development candidate.16, 19 In order to improve the potency of the series, we have utilized the X-ray structural information to guide additional medicinal chemistry. The enzyme bound structure of 1 and 2 showed that the triazolopyrimidines filled most of the available binding pocket, but that a channel between the C2 position of the triazolopyrimidine ring and the FMN cofactor was available that could potentially accommodate additional functionality.22 In a major advance forward, herein we describe the lead optimization program to modify the C2 position of the triazolopyrimidine scaffold that has yielded several potent compounds with pharmacokinetic profiles and in vivo efficacy in mouse models that meet previously established progression criteria.

Fig. 1
Structures of select triazolopyrimidines

Chemistry

The focus of this study was to examine the SAR associated with modifications of the substituent at C2 of the triazolopyrimidine ring system. The development of a synthetic strategy for these triazolopyrimidine derivatives was dictated by the nature of the atom used as linker at the C2- position of the ring. The synthesis of the 2-substituted analogs 11–55 shown in Table 1 proceeded through the intermediate 7-hydroxy-triazolopyrimidines 9 shown in Scheme 1, which was accessed by two routes. The most general approach involved the condensation of ethyl 3-oxobutanoate with aminoguanidine hydrochloride to afford diaminopyrimidone 7 in moderate yield (11–42 and 45–55). This route had the advantage that 7 served as a common intermediate from which a diverse set of C2 alkyl analogs could be prepared. Reaction of 7 with a variety of acid chlorides (R defined in Table 1) gave the C2 substituted triazolopyrimidoness 9 in good to excellent yields, even with sterically hindered R-groups. This reaction apparently proceeded by initial acylation of 7 followed by a slower cyclization step yielding 9 and, in some cases, required more forcing conditions to effect complete conversion to 9.23 Alternatively, to generate 43, 44 and 100, we used our previously described chemistry16, 17, 19 in which the 5-aminotriazoles 8 were either commercially available or prepared initially (see experimental). In these cases, the reaction of 8 with ethyl 3-oxobutanoate proceeded through the acetoacetate ketone with formation of an aminocrotonate intermediate that cyclizes at the N-2 of the triazole ring yielding the desired isomer 9.24, 25 The limitation of this approach was the availability of starting 3-substituted-5-amino-1,2,4-triazole derivatives due to lengthy reaction times, inefficiency and variable results during their preparation. Treatment of 9 with phosphoryl chloride then gave the chloro triazolopyrimidnes 10, which could be converted to the desired 2-substituted 7-aminoaryl products 11–55, 100 by reaction with the requisite aniline under a variety of conditions.

Scheme 1
Synthesis of the triazolopyrimidine compounds 11–55, 100
Table 1
Structure activity relationships of the triazolopyrimidine compounds

As shown in Scheme 2, the preparation of analogs with either S- or O-linked substituents at C2 (58–84) started with the commercially available triazolopyrimidone 56. Conversion of 56 to the chloride (57, R = SMe) under the standard conditions followed by displacement of the chloro group with the requisite aniline afforded compounds 58–61 in good yields. Oxidation with hydrogen peroxide in the presence of catalytic Na2WO4 in hot aqueous acetic acid proved to be very straight forward, although competitive nitrogen oxidation lowered the yield of this step. Nevertheless, compounds 62–65 were easily prepared by this procedure. These compounds then served as intermediates for the preparation of compounds 66–84 by taking advantage of the methylsulfonyl group to act as a leaving group. Thus, treatment of these C2 methylsulfonyl analogs with a variety of alkyl and functionalized alkyl alkoxides under microwave conditions afforded the desired compounds (66–84) in good yields.

Scheme 2
Synthesis of the triazolopyrimidine compounds 66–84

Analogs with amino (89–97) or hydroxyl (98, 99, 101) functionality at the R position were obtained from specific intermediates 4547 (prepared as in Scheme 1) and further derivatized as shown in Schemes 3 and and4.4. Compounds 89–97 were generated in moderate to good yields by treating the fully elaborated chloromethyl (45a, 45b) or chloroethyl (46a, 46b) analogs with the appropriate amine in methanol or THF in the microwave at 120°C (Scheme 3). The hydoxy alkyl analogs 98–99, 101 were prepared by hydrogenolysis (Scheme 4) of the requisite benzyl ether (47a, 47b) using flow chemistry with an H-cube instrument.

Scheme 3
Synthesis of triazolopyrimidines 89 – 97
Scheme 4
Synthesis of triazolopyrimidines 98, 99 and 101

Results

Optimization of inhibitor potency

The original triazolopyrimidine lead compound (1) was identified by HTS in a PfDHODH-based enzyme activity based screen.17 This compound was a potent and selective inhibitor of PfDHODH, however it was rapidly metabolized and showed no activity against the parasite in vivo. Replacement of the naphthyl amine with para-substituted anilines led to the identification of metabolically stable analogs (e.g. 2 – 4) that were able to suppress parasite growth in the P. berghei mouse model 16, 19 (Figure 1). Additionally these studies identified the key anilines (4-CF3 and 4-SF5) that provided the optimal balance between parasite activity and metabolic stability. These compounds (2 – 4) however were less potent than expected of a drug candidate and required large doses for in vivo efficacy. The X-ray structures of PfDHODH bound to 1 and 2 were determined and this analysis identified a narrow channel leading from the C2 carbon of the triazolopyrimidine ring towards the bound FMN cofactor.22 This channel is lined by several hydrophobic side chains, the aliphatic side chain portion of Arg265 and by a single polar group – the side chain hydroxyl of Tyr528. This hydroxyl donates a hydrogen bond to the carbonyl oxygen of Gly226. In the co-crystal structure of PfDHODH with 1, but not with 2, a single ordered water molecule is trapped between bound inhibitor and protein on one side of the channel where it donates hydrogen bonds to the hydroxyl of Tyr528 and to N1 of 1. The crystal structures indicate that the triazolopyrimidine in 2 penetrates about 0.3 Å more deeply into the narrow channel than is the case with 1 which may force a slight movement of the water molecule destabilizing its hydrogen bonding interactions and disordering its binding. In order to improve the potency of the compound series, we sought to exploit potential additional binding energy by building functionality from the C2 position of the ring. We reasoned that an unbranched alkyl substituent at C2 would fit well into the allowed space in the pocket. But we also recognized that protein conformational changes seen previously in PfDHODH as a response to binding different inhibitor chemotypes22 could possibly alter the shape and electronic characteristics of this narrow channel if probed by suitable C2 substituents.

In order to identify the most active and promising compound, we undertook a systematic evaluation of modification of the triazlopyrimidine ring at the C2 position including some larger and more polar substituents that were designed to probe the sensitivity of the channel to possible ligand induced conformational changes. The chemistry was designed to allow study of different structural effects including chain length effects, branch-group effects at different distances in C2, electronic effects on the 5-member ring of the triazolopyrimidine, heteroatom effects at different distances in C2, polarity effects at different distances in C2 or donor- and acceptor- hydrogen-bonding effects in C2 looking for an extra interaction with the FMN co-factor. In order to evaluate whether the previously optimized anilines (4-CF3 and 4-SF5) remained appropriate for the various functional groups that were coupled to C2, four different anilines (4-Cl, 3-Cl, 4-CF3 and 4-SF5) were tested within each series.

Compounds were initially evaluated for potency against PfDHODH and against the P. falciparum parasite in whole cell assays (Tables 1). Generally we observed a good correspondence between activity against PfDHODH and inhibition of P. falciparum 3D7 cell growth for the compounds in the series. Alkyl R-groups at C2 were tested to explore the size of the pocket, including ethyl (1114), iso-propyl (1518), iso-butyl (19–22), tert-butyl (23 and 24), sec-butyl (25 and 26), and cyclopropyl (27 – 30). All but the iso-butyl substituents were tolerated and had similar activity to the parent compounds (26), with a small (2 – 3-fold) improvement in potency against PfDHODH observed for 13, 17 and 29, or a larger 5 – 10 fold improvement observed for the 3-Cl aniline (12 and 16) or the 4-Cl aniline (11 and 15) derivatives. These data suggest the geometry of the channel is wide enough to accept steric hindrance on the atom adjacent to the C2 carbon of the triazolopyrimidine ring but that it narrows beyond the second atom from C2. Compounds with a direct ether linkage to the triazolopyrimidine ring, OMe (66 and 67) and OEt (68 – 71), also showed similar to modestly improved potency when compared to the parent compounds (26). In the context of 4-CF3 or 4-SF5 –aniline, the addition of OEt at C2 (70 and 71) led to 4–8-fold improved potency against PfDHODH, however both were less active than those containing a haloalkyl (37 and 38). Addition of a linker carbon between C2 and cylclopropyl (3134) led to reduced inhibitory activity as did increasing the chain length of the ethyl ether group at C2 (-OCH2CH2OCH3) (72 and 73), demonstrating that the pocket is not able to accommodate these larger groups at the C2 position. Compounds that contained an ether linkage in the R group (4855) that were not directly linked to the triazolopyrimidine ring showed in general lower activity than the parent compounds (26), as did those containing an alcohol (-CH2OH (98, 99) or - CH2CH2OH (101)). Addition of an amine functionality into the R at C2 (7484, 89 – 97) led to complete loss in inhibitory activity independent of the substitution pattern on the N atom or its position in the chain, thus confirming the lipophilic character of the channel. Transformation of C2 into a carbonyl moiety (85 and 86) also led to complete loss in inhibitory activity.

The most potent inhibitory activity against PfDHODH was from compounds that contained an unbranched haloalkyl R group at C2, including -CF2CH3 (35 – 38), -CF2CH2CH3 (39 – 42) or -CF3 (43 and 44), an unbranched alkyl sulfur or oxygen containing group like -SCH3 (58 – 61) or -SO2CH3 (62 – 65). Of these compounds, 37 (4-CF3-aniline), 60 (4-CF3-aniline) and 38 (4-SF5-aniline) demonstrated the best potency against the parasite in whole cell assays (EC50’s below 20 nM against P. falciparum 3D7). For 35 – 38, the addition of CF2CH3 to the C2 position improved activity by 4–40-fold against PfDHODH and of 25–240-fold against the parasite compared to the matched analogs (2 – 6) containing a hydrogen at C2 (Figure 2). A similar improvement in potency was observed for addition of SCH3 at C2 (60). Compounds containing 4-Cl (35, 39) or 3-Cl (36, 40) aniline showed greater fold improvement in potency against PfDHODH than the 4-CF3 (37) and 4-SF5 (38) anilines, but the larger fold improvement has its origin in the poorer activity of the parent chloro compounds (R = H, 5 and 6), and the absolute potency of these compounds against PfDHODH is similar to those with 4-CF3 and 4-SF5 aniline.

Fig. 2
P. falciparum in vitro growth curves and genetic rescue with yeast DHODH for 38. Data were collected in human serum and were fitted to the 4 parameter dose response equation in Graphpad Prism to obtain EC50 values. 38 (P. falciparum 3D7; EC50 = 0.019 ...

Compounds 37 and 38 were also tested for whole cell activity in media supplemented with Albumax instead of human serum because we previously observed these two media gave different results for compounds containing the 4-SF5 aniline.19 As observed for compound 3, compound 38 also showed 6–7–fold better activity against parasites cultured in media supplemented with Albumax in comparison to human serum, whereas for 37 results were similar in the two media (Table 1).

Physicochemical properties and plasma protein binding

A selection of compounds were chosen for analysis of their physicochemical properties including LogD, aqueous solubility and plasma protein binding (Table 2). The compounds in the series all have physicochemical properties that are consistent with good oral absorption (MW <430, H bond donors <2 and H bond acceptors <6, polar surface area < 90 Å2). The LogD for the series of compounds was found to range from 2.8 – 3.9 and was typically highest for compounds containing a 4-SF5 aniline. Aqueous solubility varied from poor to good, and compounds with 4-SF5 aniline were the least soluble while those with 3- or 4-Cl aniline were among the best. The most potent compounds (37 and 38) showed mid range solubility and again, the 4-SF5 aniline (30 – 60 μM) derivative was less soluble than the 4-CF3 aniline (70 – 140 μM). Human plasma protein binding was evaluated by two methods: 1) a chromatographic method that allowed rapid evaluation of a larger number of compounds and was useful for comparative purposes, and 2) a more rigorous ultracentrifugation method that provided an accurate absolute value for assessing total versus free drug concentrations in the in vivo models. Using the chromatographic method, compounds in the series typically ranged from 85 – 98% protein bound, with those containing the 4-SF5 aniline having both the highest LogD and the highest plasma protein binding. For those compounds (37 and 38) that were evaluated with both methods, the ultracentrifugation method yielded a higher protein binding value than the chromatographic method. Protein binding for 38 was found to be very high in both human and mouse plasma (99.9 and 99.6%, respectively), whereas the binding of 37 was somewhat lower (98.8 and 97.4%, respectively). For both 37 and 38, protein binding in rat plasma (94.2 and 97.3%, respectively) was lower than that in either mouse or human plasma, but the binding of 38 was still higher than that of 37. Protein binding in the Albumax media used for in vitro activity assessment was not measured but it is likely that the intrinsic “free” in vitro potency of compound 38 is extremely high.

Table 2
Physicochemical properties and in vitro metabolism

In vitro metabolic stability

As an initial screen for good in vivo pharmacokinetic properties, metabolic stability was evaluated in vitro using human and mouse hepatic microsomes. We have previously observed a good correlation between stability in microsomal assays and exposure in rodents for the triazolopyrimidine series 16, 19. In this assay, in vitro intrinsic clearance (CLint) values were measured and used to predict an in vivo intrinsic clearance and subsequently, an in vivo hepatic extraction ratio (Eh) (fraction of compound predicted to be metabolized on the first pass through the liver) with the assumption that hepatic metabolism would represent the primary clearance mechanism. Compounds that contained - CF2CH3 (37 – 38), -CF2CH2CH3 (41 – 42), or -CF3 (43 – 44) at the C2 position showed low intrinsic clearance (CLint <6 μL/min/mg microsomal protein in human microsomes and <10 μL/min/mg in mouse microsomes) in these assays, provided that these C2 R groups were coupled with the 4-CF3 (37, 41, 43) or 4-SF5 (38, 42, 44) aniline (Table 2). The low intrinsic clearance values suggested that these compounds would exhibit good metabolic stability in both mice and humans. Compounds containing 4- Cl (11, 35) or 3-Cl (12, 36) aniline had much higher intrinsic clearance values suggesting that they would be more rapidly metabolized in vivo. Compounds containing an -CH2CH3 (13 and 14), an - OCH2CH3 (70 and 71), -cyclopropyl (29 and 30), -CH2OCH3 (50 and 51), -CH2CH2OCH3 (54 and 55) or -CH2CH2OH (101) at C2 also showed good metabolic stability, again provided that these C2 R groups were coupled to either 4-CF3 or 4-SF5 rather than to 3- or 4-Cl aniline. However, these compounds were all less potent than those containing a haloalkyl (37 and 38) group. Compounds that contained sulfur (60 and 61) were among the most potent PfDHODH inhibitors in the series, however they showed poor metabolic stability and were therefore not likely to show sufficient exposure in vivo to provide efficacy. Thus, the compounds that provided the best balance between potency and metabolic stability were identified to be 37 and 38, and these were selected as the front-runners for further characterization.

Species selectivity and evaluation of cytotoxicity

Select compounds were evaluated for activity against P. berghei, P. vivax and human DHODH (Table 1). The addition of groups larger than hydrogen at C2 systematically reduced binding affinity to the P. berghei enzyme; for example, 37 and 38 were 100-fold less potent on P. berghei DHODH than on the P. falciparum enzyme. In contrast to the results on PbDHODH, P. vivax DHODH was inhibited with similar potency to PfDHODH by both 37 and 38 (P. vivax DHODH IC50 = 0.084 and 0.073 μM, respectively), suggesting that both compounds will have good activity against P. vivax parasites. Compounds in the series continued to show excellent selectivity against the human enzyme, and the IC50 for hDHODH was >100 μM (Table 1) for all of the evaluated compounds with the exception of 44 which showed weak activity against the human enzyme (IC50 = 41 μM).

The two front-runner compounds 37 and 38 were tested against an expanded panel of P. falciparum strains (Table 3). Both compounds showed similar potency against drug sensitive and drug resistant (chloroquine and atovaquone) strains. Cytotoxicity was assessed against both a mouse (L1210) and human (HepG2) cell line and no activity within the tested range (up to 50 μM) was observed for either compound against these cell lines.

Table 3
Comparison of P. falciparum cell lines and mammalian cytotoxicity data

Evaluation of the antimalarial mode of action

In order to confirm that cell killing by the DHODH inhibitors results from inhibition of PfDHODH, we tested the inhibitors against a P. falciparum D10 cell line transfected with cytoplasmic yeast DHODH (D10_yDHODH) that uses fumarate instead of mitochondrial CoQ as the final electron acceptor.26 This cell line has previously been demonstrated to rescue parasites from the toxicity of both, mitochondrial bc1 complex inhibitors such as atovoquone26 and PfDHODH inhibitors such as our triazlopyrimidines.13, 27 Addition of proguanil has been shown to restore sensitivity of D10_yDHODH to bc1 complex inhibitors but not to specific PfDHODH inhibitors. The D10_yDHODH cell line is resistant to both 37 and 38 and this result was independent of the presence of proguanil in the culture media, demonstrating that the antimalarial effect of these compounds is due to inhibition of PfDHODH. Representative data for 38 are shown in Fig. 2.

X-ray structure determination of PfDHODH in complex with 37

The X-ray structure of PfDHODH in complex with 37 was solved to 2.95 Å resolution (Table S1; Fig. S1 supplemental). Both the triazolopyrimidine ring and the aniline ring superimpose closely onto the position of 2 (pdb 3I6R)22 though there is a slight shift of both rings away from FMN and towards Phe188 (RMSD = 0.4Å)(Fig. 3A). The key interactions are preserved, including an H-bond between His185 and the pendant nitrogen that bridges between the triazolopyrimidine ring and the phenyl ring, and an H-bond between Arg265 and the pyrimidine nitrogen N4. The CF2CH3 R-substituent of 37 binds into the channel that extends from the triazolopyrimidine ring towards the FMN and makes good van der Waals interactions throughout the pocket, which is composed of Ile263, Ile272, the hydrophobic portion of the Arg265 side chain, and of the hydroxyl and ring of Tyr528 (Fig. 3B). Thus the addition of the -CF2CH3 R-substituent to the scaffold provides key hydrophobic interactions with the enzyme in the one significant space that was left unoccupied by the previous analogs from the series. Furthermore the electron withdrawing effect of the fluorine atoms will reduce electron density on the nitrogens within the 5-membered triazole ring. This may, therefore, contribute to the potency increase as a result of decreased desolvation. These observations provide a structural explanation for the improved potency of these analogs. The structure also provides insight as to why R groups that are larger than propyl are inactive (e.g. 19 – 22) as the pocket has insufficient room to accommodate a branched chain functionality as large as iso-butyl. The complete inactivity of R groups containing an amine functionality is explained by the hydrophobicity of the R-pocket and the lack of H-bonding partners in the R-pocket except for lone pair electrons of the Tyr528 side chain hydroxyl. Finally, the X-ray structure provides insight into the poor binding of 37 to PbDHODH. The replacement of Gly181 with Ser in PbDHODH may lead to a restriction in the size of the R-group that can be accommodated by this enzyme.

Fig. 3Fig. 3
X-ray structure determination of PfDHODH in complex with 37. A. Structural alignment of PfDHODHΔ384-413-37 (green) in comparison to PfDHODH bound with 2 (pdb 3I6R) (pink). Residues within 4Å of the bound inhibitor are displayed. L-orotate ...

Pharmacokinetic properties in mice and rats

To further assess the potential for 37 and 38 to be selected as clinical development candidates, their pharmacokinetic properties were assessed in mice and rats. In non-infected mice, 37 and 38 showed good exposure following single dose oral administration (Fig. 4 and Table 4B). Maximum plasma concentrations (Cmax) and AUC0-inf values of 38 increased approximately in proportion to the increase in dose over the range 0.4 – 10 mg/kg range, however the relative increase was somewhat reduced at 50 mg/kg dose. Compound 37 exhibited dose proportional increases in Cmax and AUC0-inf across the full dose range (0.4 – 50 mg/kg). Four other compounds (13, 41, 70 and 71) with good potency (EC50 < 0.1 μM) against the parasite and good metabolic stability were also tested to evaluate plasma exposure in mice (Table 4B). Compounds 13 and 70 showed similar exposure to 37 and 38 as evaluated by both Cmax and AUC0-inf, while compounds 41 and 71 had somewhat lower exposure.

Fig. 4
Pharmacokinetic analysis of A) 38 and B) 37 after oral dosing in mice (n=1 mouse per time point). Plots represent plasma concentration versus time after a single dose. Dose levels and route are shown in the graph legend.
Table 4
In vivo pharmacokinetic data for key triazolopyrimidines in mice

The pharmacokinetic properties of 37 and 38 in rats were examined after both intravenous (i.v.) and oral (p.o.) administration. After i.v. dosing, 38 had low clearance (4–6 mL/min/kg), moderately high volume of distribution (3–6 L/kg), and a long half-life (10–13 h) whereas 37 exhibited higher clearance (13–23 mL/min/kg), similar volume of distribution (3 L/kg), and a shorter half-life (4–6 h). Neither compound was excreted unchanged in urine. After oral dosing, both 37 and 38 demonstrated good oral bioavailability and long half-lives (Table 5 and Fig. 5), however the increase in both the time to reach the maximum concentration (Tmax) and the half-life with increasing dose suggested a slower rate of absorption with increasing dose. Compound 38 showed dose-proportional kinetics over the oral dose range of 2–50 mg/kg while for 37, there was evidence for dose-dependency over the range of 2–20 mg/kg.

Fig. 5
Pharmacokinetic analysis of A) 38 and B) 37 after oral (PO) or IV dosing in rats (n=2 rats per dose route). Plots represent plasma concentration versus time after a single dose. Dose levels and route are shown in the graph legend.
Table 5
In vivo pharmacokinetic properties for 37 and 38 in rats (average of n=2 per dose level)

In vivo efficacy of 37 and 38 in the P. falciparum SCID mouse model

The observation of good bioavailability of 37 and 38 in mice and rats led us to test these compounds in an in vivo efficacy model. The poor activity of 37 and 38 against PbDHODH prohibited the use of the P. berghei mouse model to evaluate their in vivo efficacy. To overcome this issue, 37 and 38 were tested against the human malaria parasite P. falciparum in the humanized SCID mouse model.28 To provide a direct comparator to our prior studies that used the P. berghei mouse model, compound 4 (in vivo mouse P. berghei ED50 = 10 mg/kg for 4)19, was included in the SCID mouse study. Unlike 37 and 38, 4 has similar activity against PfDHODH and PbDHODH, thus was able to be evaluated in both the SCID mouse and the P. berghei mouse models. Chloroquine was also included as a control to represent a standard anti-malarial agent with known behavior in the SCID mouse model.

Compounds (4, 37 and 38) were dosed orally 1× daily for 4 days beginning 3 days after mice were infected with parasites. The ED50’s were determined 24 h after the final dose as described. 28 Both 37 and 38 markedly inhibited parasitemia in peripheral blood of mice (ED90 = 25.9 mg/kg and 8.1 mg/kg, respectively), with 38 showing efficacy at doses in the order of chloroquine (ED90 = 4.9 ± 0.5 mg/kg) and artesunate (12.7 ± 1.3 mg/kg)28 (Table 6 and Fig. 6). Compound 38 has a clear potency advantage over 37, however both were more potent than 4 (by 7-fold and 2-fold respectively) and both meet our development criteria for a preclinical candidate (defined as an ED90 < 30 mg/kg in this model). Interestingly, aberrant late trophozoites/early schizonts were detected in peripheral blood of mice treated effectively with compounds 37 and 38, which is consistent with a mechanism of action affecting parasite metabolism. The comparator compound 4, showed similar efficacy in the SCID model (ED50 = 19 mg/kg) to what was observed previously in the P. berghei model (ED50 = 10 mg/kg)19.

Fig. 6
Efficacy of 38 in SCID mice infected with P. falciparum after oral dosing. Mice were infected with parasites on day zero and a single oral dose/day was given on days 3, 4, 5 and 6. The limit of detection of the assay is 0.01% parasitemia. Dose levels ...
Table 6
In vivo Efficacy and pharmacokinetic properties in the SCID mouse P. falciparum model after oral dosing (average of n=3 mice per compound)

A pharmacokinetic study in uninfected and infected SCID mice engrafted with human erythrocytes was performed in order to interpret the efficacy results. The oral exposure of 4, 37, and 38 upon p.o. administration at 10 mg/kg indicated that infection did not significantly change compound disposition of compounds (Table 6). Interestingly, the overall exposure in whole blood for the three compounds studied was similar, which suggest that differences in intrinsic potency rather than changes in pharmacokinetic parameters are most likely responsible for the differences in efficacy detected in vivo.

Discussion

The burden of malaria falls disproportionately on the world’s poorest countries and while effective treatments are in place, the continual challenge is to stay ahead of the parasite’s unyielding ability to select for drug resistance. The identification of new chemical classes with different modes of action than existing drugs, and which also show good drug-like properties and efficacy against the parasite is the key to treatment of the disease. Through a structure-guided lead optimization program that integrated pharmacokinetic and metabolism assessment into the medicinal chemistry plan, we have identified two potent PfDHODH inhibitors (37 and 38) from the triazolopyrimidine series that meet previously established progression criteria. The antiparasitic activity of these compounds in the humanized SCID mouse P. falciparum model (ED90 < 30 mg/kg) suggests that both compounds show good in vivo efficacy. Parallel pharmacokinetic studies in mice and rats demonstrated sustained plasma concentrations after oral dosing consistent with once daily dosing, and both compounds are active against drug sensitive and drug resistant strains of the parasite, all of which are necessary to meet the target product profile.11 These compounds represent a significant breakthrough in potency compared to previously identified compounds from this series, and they represent the first that are potent enough to be progressed for clinical development.

The triazolopyrimidines were identified early in our program to develop PfDHODH inhibitors for the treatment of malaria, however previous compounds from the series have either lacked metabolic stability (1) or have shown insufficient potency against the parasite (2 – 4).16, 17, 19 The X-ray structure of 1 and 2 bound to PfDHODH showed that a narrow channel leading from the C2 position of the triazolopyrimidine ring towards the FMN cofactor afforded the best chance to build additional functionality into the molecule to improve potency.22 We have systematically evaluated a series of alkyl, haloalkyl, ether and amine substituents in this position leading to the identification of several functional groups that provided the desired boost in potency. Most of the best functional groups at C2 are electron withdrawing and hydrophobic, and the rank order of potency against both the enzyme and the parasite in whole cell assays was -CF2CH3, -SCH3 > -CF3, -CF2CH2CH3 > -OCH2CH2 > -CH2CH3. Larger substituents and branched chain or cyclic groups were less active. These results match well with the observation from the X-ray structure of 37 bound to PfDHODH, which shows that the C2 binding pocket is narrow and dominated by hydrophobic residues. As was observed in prior compounds that contain hydrogen at the C2 position 19, anilines substituted with 4-CF3 or 4-SF5 provided the best balance between potency and good metabolic stability. Compounds with 4-Cl or 3-Cl showed somewhat lower potency and were notably less metabolically stable.

In contrast to the good activity of the haloalkyl substituents, all compounds containing amines at C2 were inactive. The X-ray structure analysis shows that except for the hydroxyl oxygen of Tyr528, there are no H bond acceptors in the pocket that would bind a C2 substituent. In the absence of a major ligand induced reorganization of the pocket, the Tyr528 side chain oxygen is geometrically inaccessible to any potential hydrogen bonding group such as an amine directly linked through a tetrahedral carbon attached to the triazolopyrimidine at C2. The addition of alcohols at C2 also led to reduced activity. These compounds (particularly –CH2CH2OH at C2) were designed to displace the bound water molecule (W15) observed to bind between the Tyr528 hydroxyl and N3 on the triazolopyrimidine in the X-ray structure of PfDHODH bound to 122, and to test the hypothesis that the resulting gain in entropy would improve binding affinity. However, compounds with -CH2OH (98 and 99) and –CH2CH2OH (101) at C2 were less active then the parent compounds 2, 5 and 6, suggesting that even if the bound water was displaced, this mechanism did not provide additional binding energy possibly owing to the energetic cost of desolvating the hydroxyl of 101 upon binding to PfDHODH.

The most potent of the analogs with -CF2CH3 at C2 (37 and 38) were identified as the front-runners and evaluated in a series of additional assays to determine their development potential. These compounds showed 25–50-fold better activity against P. falciparum 3D7 cells in whole cell assays than compounds containing hydrogen at C2. This improvement in potency was achieved while remaining on target, as parasites transfected with yeast DHODH were resistant to both inhibitors as would be expected if the cell killing occurred via DHODH inhibition. Both compounds also showed good activity against PvDHODH, suggesting that they will also be useful to treat P. vivax infections, and both were fully active against drug resistant strains of the parasite, including atovoquone and chloroquine resistant strains.

Compounds 37 and 38 exhibited good metabolic stability when evaluated in hepatic microsomal assays, and pharmacokinetc profiles confirmed low clearance and good oral bioavailability in both mice and rats leading to high and extended plasma exposure profiles. In rats, compound 37 showed significant dose dependency in the kinetic profile even over a relatively low dose range. The greater than proportional increase in AUC0-inf for 37, and the corresponding apparent bioavailability of >100% at the higher dose, suggested saturation of the clearance pathway(s) with increasing dose for this compound. In comparison, 38 exhibited reasonably dose proportional kinetics in rats over a broad dose range. The delay in Tmax and prolongation of the half-life with increasing dose for both 37 and 38 suggests a dose-dependent reduction in the rate of absorption, possibly due to dissolution rate-limited absorption resulting from the relatively low aqueous solubility (Table 2). Importantly, the low solubility did not appear to limit the extent of absorption based on the high bioavailability for both compounds.

Compounds 37 and 38 required a significantly lower dose to clear parasites in a SCID mouse model than prior compounds from the triazolopyrimidine series, and this finding can be attributed to the combination of the significantly improved potency and the good plasma exposure profile. For 38, the dose required (ED50/ED90 2.8/8.1 mg/kg) was similar to that for chloroquine examined in the same study, and these values were 7-fold lower than for the previous most efficacious compound from the series (4) when tested in the same SCID mouse model. The matched analog of 38 containing a hydrogen at C2 (3) has not been tested in this model. However because 3 and 4 have similar activity against PbDHODH and PfDHODH they were both previously tested for efficacy in the P. berghei mouse model.19 In this experiment the ED50 for 3 was 2-fold higher than for 4, suggesting that the addition of the CF2CH3 group to C2 may have improved in vivo efficacy by up to 14-fold (e.g. 38 was 7-fold more potent than 4 in the SCID model, and 4 is 2-fold more potent than 3 in the P. berghei model). These data also differentiate these compounds from the reported N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides where the ED50 in the P. berghei in vivo model for the most potent of these compounds (Genz-667348) was 2–6-fold higher (depending on the parasite strain) and required BID dosing 21, in comparison to the results obtained for 38 where QD dosing was used.

Results for the plasma protein binding studies with 37 and 38 indicate that the binding of 38 was higher than that of 37 in each of the species tested (e.g. human, rat and mouse), meaning that the free fraction of 38 would be lower than that of 37. While binding in the Albumax media used for the in vitro cell-based assay has not been assessed, the plasma protein binding trends would suggest that 38 would also exhibit higher binding, and lower free concentrations, in the Albumax media. Since 37 and 38 had similar activity in the P. falciparum cell-based assay, a lower free fraction for 38 may indicate that it is actually more potent than 37 if the free concentrations are considered. This hypothesis, combined with the longer half-life and more extended exposure profile for 38 measured in normal mice, may explain the difference in potency for the two compounds in the SCID mouse model despite their similar measured whole blood concentrations.

Based on the better in vivo efficacy and the observation of more linear pharmacokinetics in rats, 38 has been chosen as the lead compound for further workup towards clinical candidate status. While none of the other compounds with potency in the same range as 37 and 38 (EC50 < 20 nM in the whole cell P. falciparum assay) were metabolically stable, compounds 13 and 70 were the next best compounds with respect to the combination of potency and metabolic stability. Both compounds also showed good exposure after oral dosing in non-infected mice, and thus might serve as backup compounds to the identified front-runner (38).

Conclusion

We have identified a compound (38) from the triazolopyrimidine series of PfDHODH inhibitors that meets development criteria, showing efficacy against P. falciparum in vivo in a murine model, activity against drug resistant parasite strains, and activity against PvDHODH. Importantly the excellent in vivo efficacy was obtained with QD dosing, attributable to the long half-life and excellent oral exposure, and suggesting that 38 will be able to meet the target product profile of cure in three consecutive daily doses in patients. Final validation of the development potential of 38 will require toxicological studies in animals and the successful clinical proof of concept of a DHODH inhibitor for the treatment of malaria in man.

Materials and Methods

Determination of enzyme inhibition constants

The plasmids used for the E. coli expression of His-tagged PfDHODH, PbDHODH, and hDHODH have been previously described.1618 The P. vivax DHODH expression construct was generated as follows. A 1266-bp DNA fragment of PvDHODH was amplified by PCR from genomic P. vivax strain Belem DNA provided by John Barnwell (CDC) using primers 5′-GTA GTA GCT CTA TAC ATG TAT TTC GAG TCC TAC GAC CCC G -3′ and 5′-CTC GAG GGC GGC CCG CCG GTG GGC CCG CCC GAC GGC GT -3′ and ligated into Zero Blunt TOPO vector (Invitrogen). QuickChange site-directed mutagenesis kit (Stratagene) was used to remove an internal NdeI site from PvDHODH using primers 5′-TGT CTA CTC ACA TGA TTT CTC AAA TG-3′ and 5′-CAT TTG AGA AAT CAT GTG AGT AGA CA-3′. The resultant clone was used to PCR amplify the PvDHODH region introducing XhoI and NdeI restriction sites, respectively with primers 5′-TGG AAT TCG CCC TCG AGG GCG GCC CGC CGG-3′ and 5′-GTA GTA GCT CTA CAT ATG TAT TTC GAG TCC TAC GAC-3′. This PCR product was ligated into Zero Blunt TOPO vector (Invitrogen) and digested with XhoI and NdeI to excise the DHODH coding sequence, which was then ligated to pET22b expression vector (Novagen) to generate the C-terminal 6×His-tagged truncated PvDHODH construct used for recombinant protein expression. All four proteins were expressed as soluble truncated proteins in the absence of the N-terminal domain that includes the membrane-spanning region of the protein. Proteins were purified as previously described by Ni+2-agarose chromatography and gel filtration.17, 18

Steady-state kinetic assays to determine the IC50’s for inhibitors were performed as previously described 17, 18. Briefly, a dye-based assay that couples the final oxidation of CoQ to the reduction of 2,6- dichloroindophenol (DCIP) was followed at 600 nm (ε=18.8 mM−1cm−1). Enzyme stocks were diluted into assay buffer containing 0.1 mM BSA to make a 100× working stock solution, which was kept on ice. Assays were initiated by adding 5 μl of this stock solution to 500 μl assay buffer containing substrates and inhibitors. Conditions were as follows: DHODH (ET = 5 – 10 nM), substrates (0.2 mM L-dihydroorotate and 0.02 mM CoQD), DCIP (0.12 mM) and assay buffer (100 mM HEPES, pH 8.0, 150 mM NaCl, 10% Glycerol, 0.1%Triton) at 20 °C. Inhibitor concentration was varied in a 3-fold dilution series (0.01 – 100 uM). The percent inhibition relative to the no inhibitor control was determined (vi/vo ×100) and data were fitted to the log[I] vs response (three parameters) equation in Graph Pad Prism to determine the IC50.

X-ray structure determination of PfDHODHΔ384-413-37

Protein purification. Protein for crystallization was expressed from a different construct than that used for the inhibition studies. Deletion of a surface loop (Δ384-413) was necessary to generate a protein that could be crystallized, and both the construct and the purification procedure have been previously described.21, 22 These prior studies also showed that deletion of this loop had no effect on enzyme activity. Preliminary crystallization conditions were found using the random crystallization screen AmSO4 suite (Nextal). The initial conditions were refined by variation of pH, precipitant, and protein concentrations to find optimal conditions. Crystallizations were setup using the hanging drop vapor diffusion method at 20°C. Crystals of PfDHODHΔ384-413-37 complex were obtained by mixing Reservoir solution (1.64 – 1.74 M Ammonium sulfate, 0.1 M Sodium Acetate, pH 4.1, and 10 mM DTT) with an equal volume of PfDHODHΔ384-413 (20 mg/ml) pre-equilibrated with 0.5 mM 37 and 2 mM dihydroorotate. Crystals typically grew in 4–7 days.

Diffraction data were collected at 100K on beamline 19ID at Advanced Photon Source (APS) using an ADSC Q315 detector. The crystal of PfDHODHΔ384-413-37 diffracted to 2.95 Å and has a space group of P64 with the cell dimension of a=b=86.6, c=138.2. The structure contains only one molecule of PfDHODH in the asymmetric unit. Diffraction data were integrated and intensities were scaled with HKL2000 package 29.

Crystallographic phases for PfDHODH inhibitors were solved by molecular replacement with Phaser 30 using the previously reported structure of PfDHODHΔ384-413 (PDB ID 3I6522) with ligands removed as the search model. Structures were rebuilt with COOT31, refined with REFMAC32 to R and Rfree of 0.236 and 0.285 respectively (Table S2 and Fig. S1, supplemental materials). The final structure contains residues 161–347, 355–383, and 414–565 and 5 water molecules. Electron density was not observed for loop 348–354 is missing. All residues in the final structure were within the allowed section of the Ramachandran plot (Table S2, supplemental materials). Water molecules were added if the density was stronger than 3.4 σ and removed if the density was weaker than 1 σ in the density map with ARP/warp.33

Molecular Modeling

Structures were displayed using PyMol (DeLano Scientific LLC, San Carlos, CA). The RMSD between the structures of PfDHODH bound to 37 versus 2 was calculated using DaliLite.34

In vitro assay of P. falciparum

Parasites were grown in Gibco-Invitrogen RPMI-1640 supplemented with 20% human type A+ plasma and 2% (w/v) red blood cells 35 or with Gibco-Invitrogen 0.5% Albumax I. Before transferring cultures to media with in Albumax, cells were washed three times in RPMI-1640 to remove plasma-supplemented medium. The D10 cell line containing yeast DHODH was cultured in the absence and in the presence of proguanil (5μM). Low-passage L1210 mouse leukemia cells (American Type Culture Collection) were also grown in plasma-supplemented RPMI-1640, while HepG2 human hepatocarcinoma cells (American Type Culture Collection) were grown in F12/DMEM (1:1). Blood products were obtained from Biochemed Services, Virginia. [3H]-hypoxanthine uptake was used to measure cell growth and incorporation of no-drug samples was compared to drug-treated cells as described previously. 36 Data were fitted to the log[I] vs response – variable slope (4 parameter) model in Graph Pad Prism to determine the ED50’s for inhibition of parasite growth.

Physicochemical Properties

Partition coefficients (LogDpH 7.4) were estimated by comparing their chromatographic retention properties to a set of standard compounds with known partition coefficients. Data were collected using a Waters 2795 HPLC instrument with a Waters 2487 dual channel UV detector with a Phenomenex Synergi Hydro-RP 4 μm (30×2 mm) column. The mobile phase was aqueous buffer (50 mM ammonium acetate, pH 7.4) and acetonitrile with an acetonitrile gradient of 0 to 100% over 10 min. Compound elution was monitored at 220 and 254 nm.

Aqueous solubility was estimated by nephelometry. Compound in DMSO was spiked into either pH 6.5 phosphate buffer or 0.01M HCl (approx. pH 2.0) with the final DMSO concentration being 1%. Samples were then analyzed by Nephelometry to determine the solubility range as described previously.37

Plasma protein binding was assessed using two different methods: 1) chromatographic retention on a human albumin column, or 2) by ultracentrifugation. The chromatographic method utilized a human albumin column (ChromTech Chiral-HSA 50 × 3.0mm, 5 μm, Sigma-Aldrich) and compared retention characteristics to a series of standard compounds with known human plasma protein binding values using a modification of a published method.38 A Waters 2795 HPLC system equipped with a Waters 2487 dual channel UV detector (monitored at 220 and 254 nm) was used with a mobile phase comprised of aqueous buffer (25 mM ammonium acetate buffer pH 7.4) and 30% isopropanol in the same buffer. The isopropanol concentration was varied over a 10 min gradient and the column was reconditioned prior to the next injection.

Measurement of plasma protein binding by ultracentrifugation was performed using human, rat and mouse plasma. Human blood was obtained from the Australian Red Cross Blood Service), rat blood from male Sprague Dawley rats, and mouse blood from male Swiss outbred mice. Plasma was separated from whole blood by centrifugation and stored frozen at −80°C (human) or −20°C (rat and mouse). Compound stock solutions were prepared in DMSO (1 mg/mL) and then further diluted in 50% (v/v) acetonitrile (20 and 200 μg/mL). Plasma at 37°C was spiked with compound stock solutions to give concentrations of 200 and 2000 ng/mL (maximum final DMSO and acetonitrile concentrations were 0.2% and 0.5%, respectively), followed by equilibration at 37°C for 1 h and ultracentrifugation (Beckman Rotor type 42.2 Ti; 223,000 × g and 37°C) for 4.2 h to separate plasma proteins from plasma-water. An aliquot of supernatant (i.e. plasma-water) was taken from each tube to determine free concentration. The total concentration was determined in non-centrifuged plasma samples similarly incubated. The concentration of test compound in samples was determined by LC-MS using calibration standards prepared in the respective matrices. The percentage of compound bound to plasma proteins (% bound) was calculated according to the following equation: %bound = 100 × (Cplasma – C plasma-water)/Cplasma, where Cplasma was the concentration observed in the non-centrifuged plasma 37°C sample and Cplasma-water was the plasma-water phase following centrifugation.

In vitro human and mouse metabolism

Human and mouse liver microsomes (BD Gentest, Discovery Labware Inc., Woburn, Massachusetts) were suspended in 0.1 M phosphate buffer (pH 7.4) at a final protein concentration of 0.4 mg/mL and incubated with compounds (1 μM) at 37°C. An NADPH-regenerating system (1 mg/mL NADP, 1 mg/mL glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase) and MgCl2 (0.67 mg/mL) was added to initiate the metabolic reactions, which were subsequently quenched with ice-cold acetonitrile at time points ranging from 0 – 60 min. Samples were also incubated in the absence of the NADPH-regenerating system to monitor for non-cytochrome P450-mediated metabolism in the microsomal matrix. Samples were then subjected to centrifugation and the amount of parent compound remaining in the supernatant was monitored by LC-MS. The first order rate constant for substrate depletion was determined by fitting the data to an exponential decay function and these values were used to calculate the in vitro intrinsic clearance value (Clint) and the predicted in vivo intrinsic clearance value (CLint vivo) as described.39 The predicted in vivo hepatic extraction ratio (Eh) was calculated using the following relationship: Eh = CLint vivo /(Q + CLint vivo) where Q is liver blood flow (20.7 mL/min/kg, 55.2 mL/min/kg and 90 mL/min/kg in for humans, rats and mice, respectively).40

Pharmacokinetic analysis

Animal studies were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and the study protocol was approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee.

Systemic exposure was studied following oral administration of compounds to non-fasted male Swiss outbred mice after oral administration and to overnight fasted male Sprague Dawley rats after IV and oral administration. Mice were given unrestricted access to food and water throughout the pre- and post-dose phases of the study. Rats were given unrestricted access to water throughout the pre- and post-dose sampling period, and access to food was re-instated 4 h post-dose. For oral dosing, compounds were administered to mice and rats in an aqueous suspension vehicle (0.5% w/v sodium caboxymethylcellulose, 0.5% v/v benzyl alcohol and 0.4% v/v Tween 80; 0.1 mL dose volume per mouse, n=1 mouse per time point; 1.0 mL per rat, n=2 rats per compound) by gavage. After the oral dose was administered to rats, the dosing tube was rinsed with 1 mL of Milli-Q water to collect any residual formulation, and the rinse was also administered. For intravenous dosing to rats, compounds were administered in an aqueous solution vehicle as a 10 min constant rate infusion into the jugular vein at two dose levels (1.0 mL per rat, n=2 rats per dose level for each compound).

At specific time points following oral dosing to mice, animials were anaesthetized with gaseous isofluorane and a single blood sample was collected via cardiac puncture. For sample collection from rats, samples of arterial blood were collected via an in-dwelling carotid cannula. Blood samples collected from mice and rats were immediately transferred to heparinized tubes that contained a stabilization cocktail (Complete® inhibitor cocktail, potassium fluoride and EDTA) to minimizes the potential for ex vivo degradation of the test compound. Plasma for analysis was then collected by centrifugation.

Compound concentrations were quantitated in plasma samples by LC-MS (on either a Micromass Quattro Premier or Micromass Xevo TQ) using calibration standards that were prepared in blank plasma. Plasma samples and standards were prepared by precipitation with acetonitrile, followed by centrifugation and analysis of the supernatant. The analytical lower limit of quantitation (LLQ) value for each compound in plasma was typically 1 ng/mL or below.

Pharmacokinetic parameters were calculated using non-compartmental methods. The terminal elimination half-life (t1/2), the area under the plasma concentration versus time profile from time zero to either the last sample time point (AUC0-24h) or extrapolated to infinity (AUC0-inf), plasma clearance, and volume of distribution at steady-state were determined using WinNonlin software (version 5.2.1, Pharsight Corporation, Mountain View, CA). The maximum plasma concentration (Cmax), the time to reach the maximum concentration (Tmax) and the time plasma concentrations remained above 1 μM (TC >1 μM) were taken directly from the concentration versus time profiles. Oral bioavailability (%F) was estimated by comparing the average dose-normalized AUC0-inf after oral administration to the average dose-normalized AUC0-inf after IV administration.

P. falciparum in vivo efficacy model and pharmacokinetic analysis

All the experiments were approved by the DDW Ethical Committee on Animal Research, performed at the DDW Laboratory Animal Science facilities accredited by AAALAC, and conducted according to European Union legislation and GlaxoSmithKline policy on the care and use of animals.

Compound efficacy was evaluated against P. falciparum Pf3D70087/N9 propagated in NOD-scid IL-2Rγnull mice that were engrafted with human erythrocytes as previously described. 41 Mice (n = 3) were infected on Day 0 with 2 × 107 parasites and compound was administered QD on days 3–6 post infection by oral gavage in an aqueous suspension vehicle (0.5% w/v sodium caboxymethylcellulose, 0.5% v/v benzyl alcohol and 0.4% v/v Tween 80) at 20 ml/kg. Control animals received vehicle alone. ED50 and ED90 were estimated by fitting a 4-decade logistic equation for the log10 of parasitemia at day 7 versus log10 of the dose administered using GraphPad Prism 5.0 (GrahPad Software). Parasitemia was assessed by FACS as previously described.28

In a parallel study, pharmacokinetic properties were also evaluated in the SCID mouse model both in the presence and absence of infection. Compound was administered in a single oral dose of 10 mg/kg and whole blood levels were followed for 24 h. Analytical methods for the detection of compounds 37 and 38 were as described above. Groups of 3 animals received a single 10 mg/kg oral dose of compound formulated in water/0.5 % carboxymethylcellulose/0.4% Tween 80/0.5 % benzyl alcohol. Blood samples were collected over 48 h post-dosing by puncture of the lateral tail vein, and mixed 1:1 with de-ionized water 0.1% saponin and stored frozen at −70°C until analysis. After protein precipitation and liquid/liquid extraction, the samples were assayed by LC/MS using ESI conditions by selected ion monitoring in an API 4000 mass spectrometer (Applied Biosystems Sciex, Foster City, CA) coupled to a HPLC chromatograph (Agilent HP1100 Series, Agilent Technologies, Spain). Quantification was conducted by comparison to calibration curves prepared in blood. Blood concentration versus time data were analyzed by non-compartmental analysis (NCA) methods using WinNonlin® Professional Version 5.2.

Chemistry – Synthetic methods and experimental

General methods

Starting materials were obtained from commercial suppliers and used without further purification unless otherwise stated. Flash chromatography was carried out using pre-packed Isolute Flash or Biotage silica-gel columns as the stationary phase and analytical grade solvents as the eluent unless otherwise stated.

NMR spectra were determined on a Varian Unity spectrometer. Chemical shifts are reported as δ values (ppm) downfield from tetramethylsilane, used as an internal standard in the solvent indicated. All coupling constants are reported in hertz (Hz), and multiplicities are labelled br (broad), s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), td (triplet of doublets), ddd (double-doubledoublet) and m (multiplet).

Total ion current traces were obtained for electrospray positive and negative ionisation (ES+ / ES−) on a Waters ZMD 2000. Analytical chromatographic conditions used for the LC/MS analysis: Column: ACE, 4.6mm × 30mm. The stationary phase particle size is 3 μm and the flow rate was 1ml/min. Solvents were A: Aqueous solvent = Water + 0.1% Formic Acid; B: Organic solvent = Acetonitrile + 0.1% Formic Acid; Methods: 5 minute run time (0 – 0.2 min 10%B, 0.2 – 3.5 min 10 – 90% B, 3.5 – 4.01 min 90% B; 4.01 – 5 min 90 – 10% B. The following additional parameters were used: injection volume (5 μl), column temperature (30 °C), UV wavelength range (220–330 nm)

The purity of all tested compounds was ≥95% using the analytical method described above unless stated otherwise.

A table matching the synthetic intermediates to the final compounds is provided as Table S1, supplemental materials.

Synthesis of compounds 11–55, 100

These compounds were synthesized following the route described in Scheme 1 through intermediates 7, 8a–b, 9a–p and 10a–p.

2,3-diamino-6-methyl-4(3H)-pyrimidinone (7)

To a solution of EtONa prepared from sodium (9.19 g, 400 mmol) and Ethanol (350 mL), aminoguanidine hydrochloride (44.2 g, 400 mmol) was added and the reaction was heated at 50°C for 30 min. Then, the reaction was filtered to remove NaCl and ethyl 3- oxobutanoate (25.3 mL, 200 mmol) was added to the filtrate, the reaction mixture was heated at reflux for 5 h and, then, stirred at RT overnight. The precipitate obtained was filtered and dried under vacuum to afford intermediate 7, a pale pink solid (15.2 g, 54% yield) 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.02 (br s, 2H), 5.51 (br, 1H), 5.29 (s, 2H), 1.98 (s, 3H); ESIMS m/z 141 (MH)+.

General procedure for the synthesis of compounds 9a–9g and 9j–9k, 9m–9o

A solution of 7 (2.5 mmol) and the appropriate alkyl chloride (2.5 mmol) in a mixture of 1,4-dioxane (4 mL) and N,N-dimethylformamide (DMF) (1 mL) was heated at reflux overnight. In cases where the open, uncyclized intermediate was obtained, the reaction mixture was concentrated under vacuum, and the crude was dissolved in acetic acid (5 mL) and heated at reflux overnight. The reaction was then concentrated under vacuum and the residue was purified by flash chromatography on a silica gel column.

2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9a)

Eluting with DCM:MeOH mixtures from 100:0 to 90:10% to yield the title compound as a white solid (75% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 5.89 (s, 1H), 2.87 (q, J= 7.4 Hz, 2H), 2.50 (s, 3H), 1.41 (t, J= 7.5 Hz, 3H); ESIMS m/z 179 (MH)+.

5-methyl-2-(1-methylethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9b)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (45% yield).1H NMR (300 MHz, DMSO-d6) δ ppm: 13.21 - 12.82 (br, 1H), 5.76 (s, 1H), 3.06-2.96 (m, 1H), 2.28 (s, 3H), 1.28 (d, J= 6.9 Hz, 6H); ESIMS m/z 193 (MH)+.

5-methyl-2-(2-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9c)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (46% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.01 (br s, 1H), 5.76 (s, 1H), 2.54 (d, J= 7.1 Hz, 2H), 2.49 (s, 3H), 2.09 - 2.04 (m, 1H), 0.92 (d, J= 6.6 Hz, 6H); ESIMS m/z 207 (MH)+.

2-(1,1-dimethylethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9d)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 85:15% to yield the title compound as a white solid (71% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.26 - 12.85 (br, 1H), 5.76 (s, 1H), 2.28 (s, 3H), 1.33 (s, 9H); ESIMS m/z 207 (MH)+.

5-methyl-2-(1-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9e)

Eluting with DCM:MeOH mixtures, gradient from 99:1 to 85:15% to yield the title compound as a white solid (55% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.33 - 12.78 (br, 1H), 5.76 (s, 1H), 2.85 - 2.76 (m, 1H), 2.28 (s, 3H), 1.79 - 1.68 (m, 1H), 1.65 - 1.55 (m, 1H), 1.25 (d, J= 6.8 Hz, 3H), 0.83 (t, J= 7.3 Hz, 3H); ESIMS m/z 207 (MH)+.

2-cyclopropyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9f)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 85:15% to yield the title compound as a white solid (22% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.26 - 12.77 (br, 1H), 5.75 (s, 1H), 2.27 (s, 3H), 2.07 (m, 1H), 1.02 - 0.97 (m, 2H), 0.9 - 0.86 (m, 2H); ESIMS m/z 191 (MH)

2-(cyclopropylmethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9g)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a pale yellow solid (24% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.29 - 12.92 (br, 1H), 5.76 (s, 1H), 2.59 (d, J= 7.1 Hz, 2H), 2.29 (s, 3H), 1.14 - 1.03 (m, 1H), 0.50 - 0.46 (m, 2H), 0.24 - 0.21 (m, 2H); ESIMS m/z 205 (MH)+.

5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9j)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (87% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.01 (br s, 1H), 5.81 (s, 1H), 4.45 (s, 2H), 3.32 (s, 3H), 2.30 (s, 3H); ESIMS m/z 195 (MH)+.

5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9k)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (58% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.04 (br s, 1H), 5.77 (s, 1H), 3.70 (t, J= 6.6 Hz, 2H), 3.23 (s, 3H), 2.90 (t, J= 6.6 Hz, 2H), 2.28 (s, 3H); ESIMS m/z 209 (MH)+.

2-(2-chloroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9m)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (31% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.12 (br s, 1H), 5.79 (s, 1H), 3.98 (t, J= 6.6 Hz, 2H), 3.16 (t, J= 6.6 Hz, 2H), 2.29 (s, 3H); ESIMS m/z 213 (MH)+.

2-(chloromethyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9n)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (58% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.52 - 12.72 (br, 1H), 5.85 (s, 1H), 4.78 (s, 2H), 2.32 (s, 3H); ESIMS m/z 199 (MH)+.

5-methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]triazolo[1,5-a] pyrimidin-7-ol (9o)

Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to yield the title compound as a white solid (72% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.27 (m, 5H), 5.84 (s, 1H), 4.76 (s, 2H), 4.55 (s, 2H), 2.45 (s, 3H); ESIMS m/z 271 (MH)+.

General procedure for the synthesis of compounds 9h and 9i

To a solution of NaEtO prepared from sodium (143 mmol) and ethanol (150 mL), Intermediate 7 (71.4 mmol) was added and the reaction mixture was heated at 80 °C for 30 min. Then, the reaction mixture was cooled down to RT and ethyl 2,2-difluoropropanoate (1.2 equiv.) or ethyl 2,2-difluorobutanoate (1.2 equiv.) were added to synthesise 9h or 9i respectively. The mixture was stirred at RT for 30 min before being heated to 80 °C for 1.5 – 3 h. The reaction mixture was concentrated to dryness and water (200 mL) was added. The reaction mixture pH was adjusted to 4 by addition of 2N aq. HCl solution whilst a white solid precipitated. The solid was filtered off, washed with water and dried under vacuum to afford compounds 9h or 9i. The mother liquors were further extracted with DCM (5 × 35 mL) and the combined organic layers were dried over Na2SO4, filtered, and concentrated under vacuum to yield additional desired product.

2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9h)

White solid (73% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 13.39 (br s, 1H), 5.91 (s, 1H), 2.33 (s, 3H), 2.06 (t, J= 19 Hz, 3H); ESIMS m/z 215 (MH)+.

2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9i)

White solid (53% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 13.36 (br s, 1H), 5.90 (s, 1H), 2.40-2.32 (m, 2H), 2.32 (s, 3H), 0.99 (t, J= 7.6 Hz, 3H); ESIMS m/z 229 (MH)+.

General procedures for compounds 9l, 9p (Scheme 1)

2-(trifluoromethyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9l)

A mixture of ethyl 3- oxobutanoate (20 mmol) and 5-(trifluoromethyl)-4H-1,2,4-triazol-3-ylamine 8a (20 mmol) (commercially available) was heated under reflux in acetic acid (10 mL) for 4–6 h and then concentrated. The product was then cooled to RT, filtered, washed with water, and dried under vacuum to give a pale pink solid with 50–65% yield. Mp: 172–174 °C, 1H NMR (300 MHz, CDCl3): δ ppm: 6.5 (br s, 1H), 6.0 (s, 1H), 2.40 (s, 3H). MS m/z 219.2 [M + H]+.

3-{2-[(phenylmethyl)oxy]ethyl}-1H-1,2,4-triazol-5-amine (8b)

To a solution of NaEtO prepared from sodium (0.237 g, 10.30 mmol) and Ethanol (10 mL), aminoguanidine hydrochloride (1.138 g, 10.30 mmol) was added and the reaction was heated at 50°C for 30 min. The reaction mixture was filtered to remove NaCl and methyl-3-benzyloxypropanoate (1 g, 5.15 mmol) was added. The reaction mixture was then heated at reflux for 5h, the formation of product being observed by TLC (90:10 DCM/MeOH) staining with KMnO4. The reaction mixture was concentrated under vacuum and product was purified by flash chromatography (silica gel column, eluting with DCM/MeOH mixtures from 100:0 to 80:20%). Upon collection of the appropriate fractions, the title compound was obtained as a white solid (0.320 g, 29% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.36-7.28 (m, 5H), 4.57 (s, 2H), 3.79 (t, J= 6.6 Hz, 2H), 2.95 (t, J= 6.6 Hz, 2H); ESIMS m/z 219 (MH)+.

5-methyl-2-{2-[(phenylmethyl)oxy]ethyl}[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9p)

A solution of Intermediate 8b (0.31 g, 1.42 mmol) and ethyl 3-oxobutanoate (0.216 mL, 1.704 mmol) in acetic acid (5 mL) was heated at 80°C overnight. The reaction mixture was concentrated under vacuum to obtain a pale yellow solid that was triturated in hexane to remove the excess of ethyl 3-oxobutanoate. After drying the solid under vacuum, the title compound was obtained as a pale yellow solid (0.39 g, 97% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.05 (br s, 1H), 7.33-7.24 (m, 5H), 5.77 (s, 1H), 4.49 (s, 2H), 3.81 (t, J= 6.6 Hz, 2H), 2.96 (t, J= 6.6 Hz, 2H), 2.28 (s, 3H); ESIMS m/z 285 (MH)+.

General procedure for the synthesis of compounds 10a–10p

A suspension of Intermediate 9a–9p (1.17 mmol) in phosphorous oxychloride (3.5 mmol) was heated at reflux for 1 h. The reaction mixture was added dropwise into iced water, neutralized with solid Na2CO3 and product was extracted with DCM. The combined organic layers were washed with brine and dried over anh. Na2SO4. A brown oil was obtained upon solvent removal in vacuo which was purified by flash chromatography on silica gel, eluting with Hexanes:EtOAc mixtures from 100:0 to 40:60%.

7-chloro-2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10a)

White solid (76% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 7.01 (s, 1H), 2.98 (q, J= 7.6 Hz, 2H), 2.68 (s, 3H), 1.43 (t, J= 7.6 Hz, 3H); ESIMS m/z 197 (MH)+.

7-chloro-5-methyl-2-(1-methylethyl)[1,2,4]triazolo[1,5-a] pyrimidine (10b)

White solid (65% yield).1H NMR (300 MHz, CDCl3) δ ppm: 7.01 (s, 1H), 3.35-3.25 (m, 1H), 2.68 (s, 3H), 1.45 (d, J=7.1 Hz, 6H); ESIMS m/z 211 (MH)+.

7-chloro-5-methyl-2-(2-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidine (10c)

White solid (85% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.01 (s, 1H), 2.82 (d, J= 7.1 Hz, 2H), 2.68 (s, 3H), 2.34-2.29 (m, 1H), 1.45 (d, J= 6.6 Hz, 6H); ESIMS m/z 225 (MH)+.

7-chloro-2-(1,1-dimethylethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10d)

Pale yellow solid (58% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 6.99 (s, 1H), 2.67 (s, 3H), 1.50 (s, 9H); ESIMS m/z 225 (MH)+.

7-chloro-5-methyl-2-(1-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidine (10e)

Colourless needles (56% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.01 (s, 1H), 3.12-3.03 (m, 1H), 2.68 (s, 3H), 2.02-1.91 (m, 1H), 1.81-1.7 (m, 1H), 1.42 (d, J= 7.1 Hz, 3H), 0.93 (t, J=7.3 Hz, 3H); ESIMS m/z 225 (MH)+.

7-chloro-2-cyclopropyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10f)

White solid (13% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 6.97 (s, 1H), 2.66 (s, 3H), 2.28-2.22 (m, 1H), 1.26-1.22 (m, 2H), 1.15-1.10 (m, 2H); ESIMS m/z 209 (MH)+

7-chloro-2-(cyclopropylmethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10g)

Yellow solid (36% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 7.57 (s, 1H), 2.76 (d, J= 7.1 Hz, 2H), 2.660 (s, 3H), 1.24-1.12 (m, 1H), 0.52-0.48 (m, 2H), 0.28-0.24 (m, 2H) ESIMS m/z 223 (MH)+

7-chloro-2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo[1,5-a] pyrimidine (10h)

White solid (86% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 7.17 (s, 1H), 2.75 (s, 3H), 2.18 (t, J= 18.7 Hz, 3H); ESIMS m/z 233 (MH)+.

7-chloro-2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo[1,5-a] pyrimidine (10i)

White solid (68% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 7.17 (s, 1H), 2.75 (s, 3H), 2.53-2.41 (m, 2H), 1.13 (t, J= 7.6 Hz, 3H); ESIMS m/z 247 (MH)+.

7-chloro-5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo[1,5-a]pyrimidine (10j)

White solid (75% yield) 1H NMR (300 MHz, CDCl3) δ ppm: 7.07 (s, 1H), 4.78 (s, 2H), 3.56 (s, 3H), 2.71 (s, 3H); ESIMS m/z 213 (MH)+.

7-chloro-5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo[1,5-a]pyrimidine (10k)

White solid (85% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.03 (s, 1H), 3.93 (t, J= 6.6 Hz, 2H), 3.38 (s, 3H), 3.24 (t, J= 6.6 Hz, 2H), 2.69 (s, 3H); ESIMS m/z 227 (MH)+.

7-chloro-5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyrimidine (10l)

Mp: 102–105 °C, 1H NMR (300 MHz, CDCl3): δ ppm: 7.29 (s, 1H), 2.80 (s, 3H). MS m/z 237.0 [M + H]+.

7-chloro-2-(2-chloroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10m)

White solid (67% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.06 (s, 1H), 4.04 (t, J= 6.6 Hz, 2H), 3.43 (t, J= 6.6 Hz, 2H), 2.70 (s, 3H); ESIMS m/z 231 (MH)+.

7-chloro-2-(chloromethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10n)

White solid (65% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.11 (s, 1H), 4.82 (s, 2H), 2.73 (s, 3H); ESIMS m/z 217 (MH)+.

7-chloro-5-methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]triazolo[1,5-a]pyrimidine (10o)

White solid (87% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 7.44-7.35 (m, 5H), 7.07 (s, 1H), 4.84 (s, 2H), 4.77 (s, 2H), 2.71 (s, 3H); ESIMS m/z 289 (MH)+.

7-chloro-5-methyl-2-{2-[(phenylmethyl)oxy]ethyl}[1,2,4]triazolo[1,5-a]pyrimidine (10p)

White solid (0.31 g, 75% yield).1H NMR (300 MHz, CDCl3) δ ppm: 7.33-7.31 (m, 5H), 7.02 (s, 1H), 4.58 (s, 2H), 4.01 (t, J= 6.6 Hz, 2H), 3.28 (t, J= 6.6 Hz, 2H), 2.69 (s, 3H); ESIMS m/z 303 (MH)+.

General procedure for the synthesis of compounds 11–55, 100

To a suspension of Intermediate 10a10p (1 mmol) in Ethanol (5 mL), the corresponding aniline (1 mmol) was added and the mixture was stirred at RT or at 50°C until the reaction reached completion. Ammonia in methanol (1 mmol) was added and solvent was removed in vacuo and the crude mixture was purified by flash chromatography (silica gel, eluting with Hexane:EtOAc mixtures from 75:25 to 25:75%) to yield the title compound.

N-(4-chlorophenyl)-2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (11)

Pale yellow solid (87% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.55 (d, J= 8.3 Hz, 2H), 7.45 (d, J= 8.3 Hz, 2H), 6.48 (s, 1H), 2.86 (q, J= 7.6 Hz, 2H), 2.43 (s, 3H), 1.34 (t, J= 7.6 Hz, 3H); ESIMS m/z 288 (MH)+.

N-(3-chlorophenyl)-2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (12)

Pale yellow solid (82% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.54-7.51 (m, 1H), 7.48-7.36 (m, 2H), 6.51 (s, 1H), 2.85 (q, J= 7.6 Hz, 2H), 2.44 (s, 3H), 1.34 (t, J= 7.6 Hz, 3H); ESIMS m/z 288 (MH)+.

2-ethyl-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (13)42

White solid (90% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.37 (s, 1H), 7.80 (d, J= 8.5 Hz, 2H), 7.66 (d, J= 8.5 Hz, 2H), 6.60 (s, 1H), 2.84 (q, J= 7.6 Hz, 2H), 2.42 (s, 3H), 1.33 (t, J= 7.6 Hz, 3H); ESIMS m/z 322 (MH)+.

2-ethyl-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo [1,5-a]pyrimidin-7-amine (14)42

White solid (87% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.44 (br s, 1H), 7.95 (d, J= 8.3 Hz, 2H), 7.66 (d, J= 8.3 Hz, 2H), 6.68 (s, 1H), 2.83 (q, J= 7.3 Hz, 2H), 2.43 (s, 3H), 1.32 (t, J= 7.3 Hz, 3H); ESIMS m/z 380 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-(1-methylethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (15)

Colourless solid (35% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.77-7.63 (br, 1H), 7.49-7.45 (m, 2H), 7.36-7.31 (m, 2H), 6.27 (s, 1H), 3.29-3.19 (m, 1H), 2.58 (s, 3H), 1.46 (d, J= 7.1 Hz, 6H); ESIMS m/z 302 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-(1-methylethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (16)

White solid (14 % yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.79-7.68 (br, 1H), 7.45-7.39 (m, 2H), 7.33-7.27 (m, 2H), 6.36 (s, 1H), 3.30-3.19 (m, 1H), 2.56 (s, 3H), 1.45 (d, J=6.9 Hz, 6H); ESIMS m/z 302 (MH)+.

5-methyl-2-(1-methylethyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (17)42

White solid (29% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 8.00-7.86 (br, 1H), 7.77 (d, J= 8.3 Hz, 2H), 7.50 (d, J= 8.3 Hz, 2H), 6.47 (s, 1H), 3.32-3.22 (m, 1H), 2.58 (s, 3H), 1.46 (d, J= 6.9 Hz, 6H); ESIMS m/z 336 (MH)+.

5-methyl-2-(1-methylethyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (18)42

White solid (35% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.96-7.83 (br, 3H), 7.47 (d, J= 8.8 Hz, 2H), 6.50 (s, 1H), 3.30-3.20 (m, 1H), 2.58 (s, 3H), 1.45 (d, J=6.9 Hz, 6H); ESIMS m/z 394 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-(2-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (19)

White solid (87% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.15 (s, 1H), 7.50 (d, J= 8.3 Hz, 2H), 7.46 (d, J= 8.3 Hz, 2H), 6.36 (s, 1H), 2.66 (d, J= 7.1 Hz, 2H), 2.38 (s, 3H), 2.26-2.22 (m, 1H), 0.96 (d, J= 6.6 Hz, 6H); ESIMS m/z 316 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-(2-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (20)

White solid (88% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.39 (s, 1H), 7.79 (d, J= 7.9 Hz, 2H), 7.67 (d, J= 7.3 Hz, 2H), 6.60 (s, 1H), 2.67 (d, J= 7.1 Hz, 2H), 2.42 (s, 3H), 2.21-2.17 (m, 1H), 0.96 (d, J= 6.6 Hz, 6H); ESIMS m/z 316 (MH)+.

5-methyl-2-(2-methylpropyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21)42

White solid (96% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.19 (s, 1H), 7.55-7.41 (m, 3H), 7.33-7.31 (m, 1H), 6.41 (s, 1H), 2.67 (d, J= 7.1 Hz, 2H), 2.40 (s, 3H), 2.20-2.17 (m, 1H), 0.96 (d, J= 6.6 Hz, 6H); ESIMS m/z 350 (MH)+.

5-methyl-2-(2-methylpropyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (22)42

White solid (92% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.45 (s, 1H), 7.95 (d, J= 8.7 Hz, 2H), 7.66 (d, J= 8.7 Hz, 2H), 6.67 (s, 1H), 2.68 (d, J= 7.1 Hz, 2H), 2.43 (s, 3H), 2.22-2.17 (m, 1H), 0.96 (d, J= 6.6 Hz, 6H); ESIMS m/z 408 (MH)+.

2-(1,1-dimethylethyl)-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (23)

White foam (66% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.15-10.04 (br, 1H), 7.81 (d, J= 8.6 Hz, 2H), 7.69-7.67 (m, 2H), 6.54 (s, 1H), 2.41 (s, 3H). 1.43 (s, 9H); ESIMS m/z 350 (MH)+.

2-(1,1-dimethylethyl)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24)

White solid (49% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.2-10.08 (br, 1H), 7.96 (d, J= 9.1 Hz, 2H), 7.75-7.62 (m, 2H), 6.53 (s, 1H), 2.42 (s, 3H). 1.43 (s, 9H); ESIMS m/z 408 (MH)+.

5-methyl-2-(1-methylpropyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (25)

White solid (35% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.41-10.3 (br, 1H), 7.8 (d, J= 8.3 Hz, 2H), 7.69-7.67 (m, 2H), 6.59 (s, 1H), 2.98-2.89 (m, 1H), 2.42 (s, 3H), 1.9-1.79 (m, 1H), 1.73-1.62 (m, 1H), 1.33 (d, J= 7.07 Hz, 3H), 0.86 (t, J= 7.3 Hz, 3H); ESIMS m/z 350 (MH)+.

5-methyl-2-(1-methylpropyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (26)

White solid (39% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.49-10.31 (br, 1H), 7.97-7.95 (m, 2H), 7.68-7.66 (m, 2H), 6.67 (s, 1H), 2.98-2.89 (m, 1H), 2.43 (s, 3H), 1.9-1.79 (m, 1H), 1.73-1.62 (m, 1H), 1.32 (d, J= 7.07 Hz, 3H), 0.88-0.84 (m, 3H); ESIMS m/z 408 (MH)+.

N-(4-chlorophenyl)-2-cyclopropyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (27)

White solid (69% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.66-10.25 (br, 1H), 7.55-7.52 (m, 2H), 7.48-7.45 (m, 2H), 6.40 (s, 1H), 2.39 (s, 3H), 2.20-2.13 (m, 1H), 1.11-1.01 (m, 4H); ESIMS m/z 300 (MH)+.

N-(3-chlorophenyl)-2-cyclopropyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (28)

Beige solid (82% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.53-10.26 (br, 1H), 7.52-7.48 (m, 2H), 7.45-7.42 (m, 1H), 7.38-7.35 (m, 1H), 6.45 (s, 1H), 2.41 (s, 3H), 2.20-2.13 (m, 1H), 1.10-1.01 (m, 4H); ESIMS m/z 300 (MH)+.

2-cyclopropyl-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (29)42

White solid (26% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.79-10.36 (br, 1H), 7.82 (d, J= 8.34 Hz, 2H), 7.67 (d, J= 8.34 Hz, 2H), 6.62 (s, 1H), 2.42 (s, 3H), 2.21-2.14 (m, 1H), 1.11-1.01 (m, 4H); ESIMS m/z 334 (MH)+.

2-cyclopropyl-5-methyl-N-[4-(pentafluoro-l6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (30)42

Beige solid (60% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.89-10.35 (br, 1H), 8.01-7.95 (m, 2H), 7.66 (d, J= 8.8 Hz, 2H), 6.69 (s, 1H), 2.43 (s, 3H), 2.20-2.14 (m, 1H), 1.10-1.01 (m, 4H); ESIMS m/z 392 (MH)+.

N-(4-chlorophenyl)-2-(cyclopropylmethyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (31)

White solid (60% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.65-10.29 (br, 1H), 7.56-7.53 (m, 2H), 7.49-7.45 (m, 2H), 6.44 (s, 1H), 2.76 (d, J= 6.8 Hz, 2H), 2.42 (s, 3H), 1.24-1.14 (m, 1H), 0.54-0.50 (m, 2H), 0.31-0.27 (m, 2H); ESIMS m/z 314 (MH)+.

N-(3-chlorophenyl)-2-(cyclopropylmethyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (32)

White solid (75% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.65-10.28 (br, 1H), 7.53-7.49 (m, 2H), 7.46-7.43 (m, 1H), 7.38-7.36 (m, 1H), 6.48 (s, 1H), 2.75 (d, J= 71 Hz, 2H), 2.43 (s, 3H), 1.24-1.15 (m, 1H), 0.54-0.49 (m, 2H), 0.31-0.27 (m, 2H); ESIMS m/z 314 (MH)+.

2-(cyclopropylmethyl)-5-methyl-N-(4-(trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (33)

White solid (11% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.77-10.36 (br, 1H), 7.82 (d, J= 8.5 Hz, 2H), 7.68 (d, J= 8.5 Hz, 2H), 6.64 (s, 1H), 2.76 (d, J= 7.1 Hz, 2H), 2.44 (s, 3H), 1.25-1.14 (m, 1H), 0.54-0.49 (m, 2H), 0.30-0.27 (m, 2H); ESIMS m/z 348 (MH)+.

2-(cyclopropylmethyl)-5-methyl-N-(4-(pentafluoro-λ6-sulfanyl)phenyl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (34)

White solid (14% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.67-10.38 (br, 1H), 7.99-7.95 (m, 2H), 7.67 (d, J= 8.8 Hz, 2H), 6.70 (s, 1H), 2.75 (d, J=7.1 Hz, 2H), 2.45 (s, 3H), 1.25-1.14 (m, 1H), 0.53-0.49 (m, 2H), 0.30-0.26 (m, 2H); ESIMS m/z 406 (MH)+.

N-(4-chlorophenyl)-2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (35)

Beige solid (91% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.35 (s, 1H), 7.53 (d, J= 8.3 Hz, 2H), 7.46 (d, J= 8.3 Hz, 2H), 6.45 (s, 1H), 2.42 (s,3H), 2.12 (t, J= 18.7 Hz, 3H); ESIMS m/z 324 (MH)+.

N-(3-chlorophenyl)-2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (36)42

Beige solid (91% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.39 (s, 1H), 7.53-7.47 (m, 3H), 7.45-6.36 (m, 1H), 6.52 (s, 1H), 2.44 (s, 3H), 2.13 (t, J= 18.8 Hz, 3H); ESIMS m/z 324 (MH)+.

2-(1,1-difluoroethyl)-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4] triazolo[1,5-a]pyrimidin-7-amine (37)42

White solid (81% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.58 (s, 1H), 7.83 (d, J= 8.5 Hz, 2H), 7.70 (d, J= 8.5 Hz, 2H), 6.73 (s, 1H), 2.47 (s, 3H), 2.14 (t, J= 18.8 Hz, 3H); ESIMS m/z 358 (MH)+.

2-(1,1-difluoroethyl)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl] [1,2,4]triazolo[1,5-a]pyrimidin-7-amine (38)42

White solid (81% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.60 (br s, 1H), 7.97 (d, J= 9.3 Hz, 2H), 7.67 (d, J= 9.1 Hz, 2H), 6.79 (s, 1H), 2.47 (s, 3H), 2.13 (t, J= 18.9 Hz, 3H); ESIMS m/z 416 (MH)+.

N-(4-chlorophenyl)-2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (39)

White solid (88% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.34 (s, 1H), 7.53 (d, J= 8.3 Hz, 2H), 7.48 (d, J= 8.3 Hz, 2H), 6.47 (s, 1H), 2.42-2.39 (m, 5H), 1.02 (t, J= 7.3 Hz, 3H); ESIMS m/z 338 (MH)+.

N-(3-chlorophenyl)-2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (40)

White solid (77% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.38 (s, 1H), 7.54-7.48 (m, 3H), 7.38-7.36 (m, 1H), 6.52 (s, 1H), 2.44-2.39 (m, 5H), 1.02 (t, J= 7.3 Hz, 3H); ESIMS m/z 338 (MH)+.

2-(1,1-difluoropropyl)-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (41)42

White solid (83% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.58 (br s, 1H), 7.82 (d, J= 8.5 Hz, 2H), 7.67 (d, J= 8.4 Hz, 2H), 6.69 (s, 1H), 2.45 (s, 3H), 2.43-2.37 (m, 2H), 1.02 (t, J= 7.6 Hz, 3H); ESIMS m/z 372 (MH)+.

2-(1,1-difluoropropyl)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (42)42

1H NMR (400 MHz, DMSO-d6) δ ppm: 10.63 (br s, 1H), 7.97 (d, J= 9.0 Hz, 2H), 7.67 (d, J= 8.7 Hz, 2H), 6.78 (s, 1H), 2.47 (s, 3H), 2.45-2.37 (m, 2H), 1.02 (t, J= 7.3 Hz, 3H); ESIMS m/z 430 (MH)+. Mp 132 – 134°C.

2-(trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (43)42

Products were purified by column chromatography with 15–20% EtOAc/Hexane. Yields ranged from 20–30%. Mp: 124–126 °C; 1H NMR (300 MHz, CDCl3): δ 7.93 (brs, NH, exchangeable), 7.82 (d, J = 8.1 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 6.61 (s, 1H), 2.65 (s, 3H). MS m/z 362.3 [MH]+.

2-(trifluoromethyl)-N-(4-(sulfurpentafluoro)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (44)42

Products were purified by column chromatography with 15–20% EtOAc/Hexane. Yields ranged from 20–30%. Mp: 178–180 °C. 1H NMR (300 MHz, CDCl3): δ 10.79 (brs, NH, exchangeable), 8.01 (d, J = 8.9 Hz, 2H), 7.70 (d, J = 9.2 Hz, 2H), 6.90 (s, 1H), 2.51 (s, 3H). MS m/z 420.3 [MH]+.

2-(chloromethyl)-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (45a)

Brown foam (82% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.97-7.63 (br, 1H), 7.51-7.46 (m, 2H), 7.35-7.30 (m, 2H), 6.33 (s, 1H), 4.80 (s, 2H), 2.56 (s, 3H); ESIMS m/z 308 (MH)+.

2-(chloromethyl)-N-(3-chlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (45b)

Orange oil (91% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.96-7.67 (br, 1H), 7.48-7.39 (m, 2H), 7.36-7.27 (m, 2H), 6.41 (s, 1H), 4.80 (s, 2H), 2.59 (s, 3H). m/z 308 (MH)+.

2-(2-chloroethyl)-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (46a)

White solid (81% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.69 (s, 1H), 7.46 (d, J= 8.3 Hz, 2H), 7.29 (d, J= 8.3 Hz, 2H), 6.29 (s, 1H), 4.04 (t, J= 6.6 Hz, 2H), 3.37 (t, J= 6.6 Hz, 2H), 2.53 (s, 3H); ESIMS m/z 322 (MH)+.

2-(2-chloroethyl)-N-(3-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (46b)

White solid (96% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.76 (s, 1H), 7.43-7.28 (m 4 H), 6.38 (s, 1H), 4.04 (t, J= 6.6 Hz, 2H), 3.38 (t, J= 6.6 Hz, 2H), 2.56 (s, 3H); ESIMS m/z 322 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (47a)

Pale yellow solid (96% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.56 (d, J= 8.3 Hz, 2H), 7.47 (d, J= 8.3 Hz, 2H), 7.36-7.20 (m, 5H), 6.50 (s, 1H), 4.75 (s, 2H), 4.65 (s, 2H), 2.45 (s, 3H); ESIMS m/z 380 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (47b)

Pale yellow solid (97% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.52-7.30 (m, 9H), 6.54 (s, 1H), 4.75 (s, 2H), 4.65 (s, 2H), 2.46 (s, 3H); ESIMS m/z 380 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (48)

White solid (70% yield).1H NMR (400 MHz, DMSO-d6) δ ppm: 10.28 (s, 1H), 7.52 (d, J= 8.3 Hz, 2H), 7.46 (d, J= 8.3 Hz, 2H), 6.41 (s, 1H), 4.59 (s, 2H), 3.37 (s, 3H), 2.40 (s, 3H); ESIMS m/z 304 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (49)

White solid (79% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.32 (s, 1H), 7.51-7.45 (m, 3H), 7.35-7.32 (m, 1H), 6.46 (s, 1H), 4.60 (s, 2H), 3.37 (s, 3H), 2.42 (s, 3H); ESIMS m/z 304 (MH)+.

5-methyl-2-[(methyloxy)methyl]-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (50)

Pale yellow solid (90% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.52 (s, 1H), 7.81 (d, J= 8.5 Hz, 2H), 7.68 (d, J= 8.5 Hz, 2H, 6.66 (s, 1H), 4.61 (s, 1H), 3.38 (s, 3H), 2.44 (s, 3H); ESIMS m/z 338 (MH)+.

5-methyl-2-[(methyloxy)methyl]-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (51)

White solid (92% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.59 (s, 1H), 7.98 (d, J= 8.7 Hz, 2H), 7.69 (d, J= 8.7 Hz, 2H), 6.75 (s, 1H), 4.63 (s, 2H), 3.39 (s, 3H), 2.47 (s, 3H); ESIMS m/z 396 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo[1,5-a]pyrimidine-7-amine (52)

White solid (93% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.17 (s, 1H), 7.51 (d, J= 7.3 Hz, 2H), 7.45 (d, J= 7.3 Hz, 2H), 6.37 (s, 1H), 3.78 (t, J= 6.6 Hz, 2H), 3.25 (s, 3H), 3.03 (t, J= 6.6 Hz, 2H), 2.38 (s, 3H); ESIMS m/z 318 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (53)

White solid (55% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.19 (s, 1H), 7.51-7.46 (m, 2H), 7.37-7.34 (m, 1H), 6.42 (s, 1H), 3.79 (t, J= 6.6 Hz, 2H), 3.25 (s, 3H), 3.03 (t, J= 6.6 Hz, 2H), 2.40 (s, 3H); ESIMS m/z 318 (MH)+.

5-methyl-2-[2-(methyloxy)ethyl]-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (54)

White solid (86% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.42 (s, 1H), 7.80 (d, J= 8.5 Hz, 2H), 7.66 (d, J= 8.5 Hz, 2H), 6.61 (s, 1H), 3.79 (t, J= 6.6 Hz, 2H), 3.25 (s, 3H), 3.04 (t, J= 6.6 Hz, 2H), 2.42 (s, 3H); ESIMS m/z 352 (MH)+.

5-methyl-2-[2-(methyloxy)ethyl]-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (55)

White solid (72% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.48 (s, 1H), 7.96 (d, J= 8.7 Hz, 2H), 7.66 (d, J= 8.7 Hz, 2H), 6.69 (s, 1H), 3.79 (t, J= 6.6 Hz, 2H), 3.25 (s, 3H), 3.04 (t, J= 6.6 Hz, 2H), 2.43 (s, 3H); ESIMS m/z 410 (MH)+

Synthesis of compounds 66–84

These compounds were synthesized following the route described in Scheme 2 through intermediates 56–65.

7-chloro-5-methyl-2-(methylthio)[1,2,4]triazolo[1,5-a]pyrimidine (57)

A suspension of commercially available 7-hydroxy-5-methyl-2-methylthio-S-triazolo[1,5-a]pyrimidine (56) (2 g, 10.19 mmol) in phosphorus oxychloride (5 mL, 53.6 mmol) was heated at reflux for 10h, the starting material solubilizing and the mixture turning bright orange. TLC analysis (Hexane/EtOAC 1:1) showed a very messy reaction that had not reached completion, but it was decided to stop it to prevent further product degradation. Hence, the reaction mixture was added dropwise to iced water. The solution was neutralized with aq. 1N Na2CO3 and product was extracted with DCM. The aqueous layer was further extracted with DCM and the combined organic layers were washed with brine and dried over anh. Na2SO4. Solvent was removed under reduced pressure yielding a reddish solid which was purified by flash chromatography (silica gel, eluting with Hexane/EtOAc mixtures from 95:5 to 40:60%). Upon collection of the appropriate fractions, the title compound was obtained as a white solid (1.09 g, 47% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 6.99 (s, 1H), 2.74 (s, 3H), 2.68 (s, 3H); ESIMS m/z 215 (MH)+.

General Procedures for the synthesis of compounds 58–61

To a suspension of Intermediate 57 (1 mmol) in Ethanol (10 mL), the appropriate aniline (1 mmol) was added and the mixture was stirred at RT until reaching completion. Anhydrous ammonia (7M solution in MeOH, 1 mmol) was then added to the mixture and solvent was removed in vacuo. The crude mixture was purified by flash chromatography (silica gel, eluting with Hexane/EtOAc mixtures form 95:5 to 40:60%)

N-(4-chlorophenyl)-5-methyl-2-(methylthio)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (58)

Beige solid (86% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.81-10.15 (br, 1H), 7.57-7.54 (m, 2H), 7.48-7.45 (m, 2H), 6.42 (s, 1H), 2.69 (s, 3H), 2.42 (s, 3H); ESIMS m/z 306 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-(methylthio)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (59)

Pale yellow solid (61% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 11.26-10.10 (br, 1H), 7.55-7.5 (m, 2H), 7.45-7.39 (m, 2H), 6.48 (s, 1H), 2.69 (s, 3H), 2.44 (s, 3H); ESIMS m/z 306 (MH)+.

5-methyl-2-(methylthio)-N-[4-(trifluoromethyl)phenyl][1,2,4]-triazolo[1,5-a]pyrimidin-7-amine (60)42

White solid (86% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.33 (s, 1H), 7.81 (d, J= 8.3 Hz, 2H), 7.67 (d, J= 8.3 Hz, 2H), 6.62 (s, 1H), 2.68 (s, 3H), 2.42 (s, 3H); ESIMS m/z 340 (MH)+.

5-methyl-2-(methylthio)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (61)42

Light yellow solid (72% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.39 (s, 1H), 7.98-7.96 (m, 2H), 7.66 (d, J= 8.8 Hz, 2H), 6.70 (s, 1H), 2.68 (s, 3H), 2.44 (s, 3H); ESIMS m/z 398 (MH)+.

General Procedures for the synthesis of compounds 62–65 (Scheme 2)

To a mixture of the corresponding derivative 58–61 (1 mmol) and acetic acid (7 mL), sodium tungstate dihydrate (0.034 mmol) was added at RT. The reaction mixture was vigorously stirred and hydrogen peroxide (2 mmol) was added slowly at 40 °C. The resulting mixture was then heated at 50 °C until reaching completion. The excess hydrogen peroxide was destroyed by the addition of an aqueous solution of sodium sulfite, product being extracted with DCM several times. The combined organic layers were washed with brine and dried over anh. Na2SO4. A white solid was obtained upon solvent removal in vacuo which was purified by flash chromatography (silica gel, eluting with Hexane/EtOAc mixtures from 95:5% to 0:100%).

N-(4-chlorophenyl)-5-methyl-2-(methylsulfonyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (62)

White solid (36% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.58 (s, 1H), 7.57-7.54 (m, 2H), 7.50-7.47 (m, 2H), 6.57 (s, 1H), 3.48 (s, 3H), 2.46 (s, 3H); ESIMS m/z 338 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-(methylsulfonyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (63)

White solid (35% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.62 (br s, 1H), 7.55-7.37 (m, 4H), 6.62 (s, 1H), 3.48 (s, 3H), 2.48 (s, 3H); ESIMS m/z 338 (MH)+.

5-methyl-2-(methylsulfonyl)-N-[4-(trifluoromethyl)phenyl][1,2,4] triazolo[1,5-a]pyrimidin-7-amine (64)

White solid (56% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 8.18 (br s, 1H), 7.81-7.79 (m, 2H), 7.53 (d, J= 8.3 Hz, 2H), 6.63 (s, 1H), 3.44 (s, 3H), 2.64 (s, 3H); ESIMS m/z 372 (MH)+.

5-methyl-2-(methylsulfonyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl] [1,2,4]triazolo[1,5-a]pyrimidin-7-amine (65)

White solid (51% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 8.23-8.10 (br s, 1H), 7.93-7.91 (m, 2H), 7.50 (d, J= 8.6 Hz, 2H), 6.67 (s, 1H), 3.44 (s, 3H), 2.66 (s, 3H); ESIMS m/z 430 (MH)+.

General Procedures for the synthesis of compounds 66–86 (Scheme 2)

The appropriate derivative 62–65 (0.15 g, 0.349 mmol) was added to sodium methoxide (3 mmol) in Methanol (9 mL) (66, 67), or sodium ethoxide (3 mmol) in Ethanol (9 mL) (68–71) or to the appropriate alcohol (1.2 mmol) with sodium hydride (2 mmol) in THF (7 mL) (72–84). The mixture was heated under microwave irradiation at 120 °C for 0.5 – 1 h. Solvent was removed under vacuum and the crude mixture was purified by flash chromatography (silica gel, eluting with DCM: MeOH mixtures from 100:0 to 90:10%) or preparative HPLC (SunFire 30× 250 mm, H2O 0.1% TFA-ACN 0.1% TFA gradient from 20 to 80%).

N-(3-chlorophenyl)-5-methyl-2-(methyloxy)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (66)

White solid (33% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.44-9.95 (br, 1H), 7.52-7.41 (m, 3H), 7.37-7.34 (m, 1H), 6.43 (s, 1H), 4.06 (s, 3H), 2.39 (s, 3H); ESIMS m/z 290 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-(methyloxy)[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (67)

White solid (57% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.25-9.96 (br, 1H), 7.55-7.49 (m, 2H), 7.49-7.42 (m, 2H), 6.38 (s, 1H), 4.05 (s, 3H), 2.37 (s, 3H); ESIMS m/z 290 (MH)+.

N-(3-chlorophenyl)-2-(ethyloxy)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (68)

White solid (74% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.42-9.97 (br, 1H), 7.52-7.40 (m, 3H), 7.37-7.34 (m, 1H), 6.43 (s, 1H), 4.46 (q, J= 7.1 Hz, 2H), 2.39 (s, 3H), 1.39 (t, J= 7.1 Hz, 3H); ESIMS m/z 304 (MH)+.

N-(4-chlorophenyl)-2-(ethyloxy)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (69)

White solid (74% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.24-9.99 (br, 1H), 7.54-7.52 (m, 2H), 7.47-7.43 (m, 2H), 6.38 (s, 1H), 4.46 (q, J= 7.0 Hz, 2H), 2.37 (s, 3H), 1.39 (t, J= 7.0 Hz, 3H); ESIMS m/z 304 (MH)+.

2-(ethyloxy)-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (70)42

Beige solid (66% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.79-7.71 (br, 3H), 7.46 (d, J= 8.3 Hz, 2H), 6.47 (s, 1H), 4.56 (q, J= 7.1 Hz, 2H), 2.55 (s, 3H), 1.49 (t, J= 7.1 Hz, 3H); ESIMS m/z 338 (MH)+.

2-(ethyloxy)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (71)42

White solid (41% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.91-7-85 (m, 2H), 7.82-7.76 (br, 1H), 7.43 (d, J= 8.8 Hz, 2H), 6.51 (s, 1H), 4.56 (q, J= 7.1 Hz, 2H), 2.56 (s, 3H), 1.49 (t, J= 7.1 Hz, 3H); ESIMS m/z 396 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-{[2-(methyloxy)ethyl]oxy}[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (72)

White solid (46% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.51-7.49 (m, 2H), 7.33-7.31 (m, 2H), 6.26 (s, 1H), 4.63 (t, J= 4.6 Hz, 2H), 3.71 (d, J= 84.6 Hz, 2H), 3.45 (s, 3H), 2.5 (s, 3H); ESIMS m/z 334 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-{[2-(methyloxy)ethyl]oxy}[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (73)

White solid (58% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.26-9.98 (br, 1H), 7.51-7.47 (m, 2H), 7.43-7.40 (m, 1H), 7.36-7.33 (m, 1H), 6.43 (s, 1H), 4.53-4.51 (m, 2H), 3.71-3.69 (m, 2H), 3.3 (s, 3H), 2.38 (s, 3H); ESIMS m/z 334 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-{[2-(methylamino)ethyl]oxy}[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (74)

White solid (61% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.05-9.84 (br, 1H), 8.82-8.59 (br, 1H), 7.57-7.5 (m, 2H), 7.47-7.43 (m, 2H), 6.39 (s, 1H), 4.67-4.63 (m, 2H), 3.49-3.37 (br, 2H), 2.68-2.64 (m, 3H), 2.38 (s, 3H); ESIMS m/z 333 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-{[2-(methylamino)ethyl]oxy}[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (75)

White solid (38% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.09-9.90 (br, 1H), 8.84-8.6 (br, 1H), 7.53-7.48 (m, 2H), 7.44-7.35 (m, 2H), 6.45 (s, 1H), 4.65 (t, J= 4.8 Hz, 2H), 3.56-3.33 (br, 2H), 2.68-2.64 (m, 3H), 2.40 (s, 3H); 1H NMR (300 MHz, CDCl3) δ ppm: 10.36-9.91 (br, 2H), 7.44-7.27 (m, 4H), 6.18 (s, 1H), 4.99-4.78 (br, 2H), 3.7-3.49 (br, 2H), 2.91 (s, 3H), 2.32 (s, 3H); ESIMS m/z 333 (MH)+.

5-methyl-2-{[2-(methylamino)ethyl]oxy}-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (76)

White solid (48% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.25-10.15 (br, 1H), 8.82-8.6 (br, 1H), 7.84-7.81 (m, 2H), 7.65 (d, J= 8.5 Hz, 2H), 6.64 (s, 1H), 4.68-4.64 (m, 2H), 3.64-3.26 (br, 2H), 2.68-2.64 (m, 3H), 2.42 (s, 3H); ESIMS m/z 367 (MH)+.

5-methyl-2-{[2-(methylamino)ethyl]oxy}-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (77)

White solid (25% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 9.92-9.67 (br, 2H), 7.88-7.86 (m, 2H), 7.55 (d, J= 8.6 Hz, 2H), 6.34 (s, 1H), 4.93-4.82 (m, 2H), 3.70-3.51 (br, 2H), 2.96-2.84 (br, 3H), 2.43 (s, 3H); ESIMS m/z 425 (MH)+.

N-(4-chlorophenyl)-2-{[2-(dimethylamino)ethyl]oxy}-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (78)

White solid (20% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 13.26-11.23 (br, 1H), 7.52-7.46 (m, 2H), 7.36-7.34 (m, 2H), 6.26 (s, 1H), 4.9-4.88 (m, 2H), 3.66-3.64 (m, 2H), 3.00 (s, 6H), 2.51 (s, 3H); ESIMS m/z 347 (MH)+.

N-(3-chlorophenyl)-2-{[2-(dimethylamino)ethyl]oxy}-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (79)

White solid (38% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.46-7.4 (m, 2H), 7.36-7.27 (m, 2H), 6.34 (s, 1H), 4.9-4.88 (m, 2H), 3.66-3.64 (m, 2H), 2.97 (s, 6H), 2.53 (s, 3H), ESIMS m/z 347 (MH)+.

2-{[2-(dimethylamino)ethyl]oxy}-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (80)

White solid (7% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.76 (d, J= 8.6 Hz, 2H), 7.53 (d, J= 8.6 Hz, 2H), 6.47 (s, 1H), 4.93-4.91 (m, 2H), 3.63 (t, J= 4.9 Hz, 2H), 2.99 (s, 6H), 2.55 (s, 3H); ESIMS m/z 381 (MH)+.

2-{[2-(dimethylamino)ethyl]oxy}-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (81)

White solid (13% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.88 (d, J= 8.8 Hz, 2H), 7.51 (d, J= 8.6 Hz, 2H), 6.51 (s, 1H), 4.92 (t, J= 5.1 Hz, 2H), 3.61 (t, J= 5.1 Hz, 2H), 2.97 (s, 6H), 2.56 (s, 3H); ESIMS m/z 439 (MH)+.

2-[(2-aminoethyl)oxy]-N-(3-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (82)

White solid (54% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10 (s, 1H), 8.05 (br, 2H), 7.53-7.49 (m, 2H), 7.44-7.42 (m, 1H), 7.38-7.36 (m, 1H), 6.44 (s, 1H), 4.59 (t, J= 4.9 Hz, 2H), 3.35-3.31 (m, 2H), 2.39 (s, 3H); ESIMS m/z 319 (MH)+.

2-[(2-aminoethyl)oxy]-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (83)

White solid (46% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.35-10.08 (br, 1H), 8.06 (br, 2H), 7.83 (d, J= 8.6 Hz, 2H), 7.67-7.65 (m, 2H), 6.63 (s, 1H), 4.61-4.56 (m, 2H), 3.35-3.31 (m, 2H), 2.41 (s, 3H); ESIMS m/z 353 (MH)+.

2-[(2-aminoethyl)oxy]-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (84)

White solid (18% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 9.97 (s, 1H), 8.04 (br, 2H), 7.54 (d, J= 8.6 Hz, 2H), 7.45 (d, J= 8.6 Hz, 2H), 6.39 (s, 1H), 4.59 (t, J= 4.9 Hz, 2H), 3.34-3.31 (m, 2H), 2.37 (s, 3H); ESIMS m/z 319 (MH)+.

General Procedures for the synthesis of compounds 85–86

These compounds were obtained as a by-product in the reaction yielding 78–79, probably due to the presence of NaOH in the reaction medium.

7-[(4-chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-2(1H)-one (85)

White solid (21% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.09 (br s, 1H), 7.51 (d, J= 8.8 Hz, 2H), 7.42 (d, J=8.8 Hz, 2H), 6.44 (br s, 1H), 5.75 (s, 1H), 2.32 (s, 3H); 1H NMR (300 MHz, DMSO-d6+D2O) δ ppm: 7.51-7.49 (m, 2H), 7.42 (d, J=8.8 Hz, 2H), 6.45 (s, 1H), 5.73 (s, 1H), 2.32 (s, 3H); ESIMS m/z 276 (MH)+.

7-[(3-chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-2(1H)-one (86)

White solid (38% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.16 (br s, 1H), 7.50-7.46 (m, 2H), 7.41-7.39 (m, 1H), 7.35-7.33 (m, 1H), 6.49 (s, 1H), 5.76 (s, 1H), 2.34 (s, 3H); 1H NMR (300 MHz, DMSO-d6+D2O) δ ppm: 7.50-7.46 (m, 2H), 7.39-7.33 (m, 2H), 6.48 (s, 1H), 5.69 (s, 1H), 2.33 (s, 3H); ESIMS m/z 276 (MH)+.

General Procedures for the synthesis of 89–97 (Scheme 3)

The corresponding amine (10 equiv.) in MeOH or THF was added to a solution of the corresponding 2-(chloromethyl) triazolopyrimidine derivative (Intermediates 45a,b or 46a,b) (1 mmol) in Tetrahydrofuran (THF) (6 mL). The mixture was heated under microwave irradiation at 120 °C for 30–45 minutes. Whenever reaction completion had not been reached, more amine in MeOH or THF (10 mmol) was added. The mixture was microwave irradiated at 120 °C until reaction completion had been reached. The mixture was then filtered and solvent was removed under reduced pressure. The crude mixture was purified by preparative HPLC (SunFire 19× 150 mm, H2O 0.1% TFA-ACN 0.1% TFA gradient from 10 to 100%). Upon freeze-drying of the corresponding fractions, desired product was obtained.

N-(4-chlorophenyl)-5-methyl-2-[2-(methylamino)ethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (89)

White solid (68% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.57 (br s, 1H), 7.53 (d, J= 7.3 Hz, 2H), 7.47 (d, J= 7.3 Hz, 2H), 6.42 (s, 1H), 3.33 (s, 3H), 3.32-3.27 (m, 2H), 2.61-2.33 (m, 4H); ESIMS m/z 317 (MH)+.

2-(aminomethyl)-N-(4-chlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (90)

White solid (16% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 9.52-8.38 (br, 2H), 7.35 (d, J= 8.6 Hz, 2H), 7.30-7.27 (m, 2H), 6.20 (s, 1H), 4.53 (br s, 2H), 2.46 (s, 3H); ESIMS m/z 289 (MH)+.

N-(4-chlorophenyl)-5-methyl-2-((methylamino)methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (91)

White solid (71% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 10.61-9.65 (br, 1H), 7.53-7.44 (m, 4H), 6.24 (s, 1H), 4.40 (s, 2H), 2.97 (s, 3H), 2.49 (s, 3H); ESIMS m/z 303 (MH)+.

N-(4-chlorophenyl)-2-((dimethylamino)methyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (92)

White solid (31% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.50-7.47 (m, 2H), 7.41-7.37 (m, 2H), 6.42 (s, 1H), 4.53 (s, 2H), 3.05 (s, 6H), 2.58 (s 3H); ESIMS m/z 317 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-[2-(methylamino)ethyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (93)

White solid (53% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 9.57 (br s, 1H), 7.53-7.33 (m, 4H), 7.47 (d, J= 7.3 Hz, 2H), 6.48 (s, 1H), 3.33 (s, 3H), 3.23-3.20 (m, 2H), 2.64-2.34 (m, 4H); ESIMS m/z 317 (MH)+.

N-(3-chlorophenyl)-2-((dimethylamino)methyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (94)

Pale yellow solid (56% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.46-7.42 (m, 2H), 7.35-7.32 (m, 2H), 6.48 (s, 1H), 4.49 (s, 2H), 3.03 (s, 6H), 2.59 (s 3H); ESIMS m/z 317 (MH)+.

2-(aminomethyl)-N-(3-chlorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (95)

Pale yellow solid (11% yield). 1H NMR (300 MHz, MeOH-d4) δ ppm: 7.53-7.50 (m, 2H), 7.43-7.40 (m, 2H), 6.50 (s, 1H), 4.42 (s, 2H), 2.52 (s, 3H); ESIMS m/z 289 (MH)+.

N-(3-chlorophenyl)-5-methyl-2-((methylamino)methyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (96)

White solid (21% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 10.7-10.08 (br, 1H), 7.39-7.32 (m, 2H), 7.28-7.27 (m, 2H), 6.31 (s, 1H), 4.39 (s, 2H), 2.97 (s, 3H), 2.50 (s, 3H); ESIMS m/z 303 (MH)+.

2-(2-aminoethyl)-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (97)

White solid (40% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.77 (br s, 2H), 7.53 (d, J= 7.3 Hz, 2H), 7.47 (d, J= 7.3 Hz, 2H), 6.42 (s, 1H), 3.27 (m, 2H), 3.25 (m, 2H), 2.40 (s, 3H); ESIMS m/z 303 (MH)+.

General procedures for the synthesis of 98 and 99 (Scheme 4)

10% Palladium on activated charcoal (330.06 mmol) was placed in a hydrogenation flask under N2 atmosphere; intermediate 47a or 47b (1 mmol) dissolved in Acetic Acid (10 mL) was added. The reaction mixture was hydrogenated at 35 psi and checked at 16 h, 30 h and 40 h. After this time, LCMS showed a mixture of starting material, de-halogenated starting material, desired product and dehalogenated desired product in ratio (35/5/50/10). The reaction mixture was concentrated under vacuum and purified by preparative HPLC (SunFire, H2O 0.1% TFA-ACN 0.1% TFA gradient from 10 to 100%). After collection of the appropriate fractions, solvent was evaporated under reduced pressure and the product was dried under vacuum in presence of phosphorous pentoxide overnight to give the desired product.

{7-[(4-chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}methanol (98)

White solid (11% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.52 (br s, 1H), 7.53 (d, J= 8.3 Hz, 2H), 7.46 (d, J= 8.3 Hz, 2H), 6.45 (s, 1H), 4.66 (s, 2H), 2.42 (s, 3H); ESIMS m/z 290 (MH)+.

{7-[(3-chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-2-yl}methanol (99)

White solid (12% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.36 (br s, 1H), 7.52-7.45 (m, 3H), 7.37-7.34 (m, 1H), 6.50 (s, 1H), 4.66 (s, 2H), 2.42 (s, 3H); ESIMS m/z 290 (MH)+.

Synthesis of compound 101 (Scheme 4)

5-methyl-2-{2-[(phenylmethyl)oxy]ethyl}-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (100)

A suspension of Intermediate 10p (0.302 g, 0.997 mmol) and 4-(trifluoromethyl)aniline (0.125 mL, 0.997 mmol) in Ethanol (5 mL) was heated at 50°C for 1h. The reaction mixture was concentrated under vacuum, taken up with DCM (20 mL) and washed with Na2CO3 (2 × 15 mL). The organic layer was dried over anh. Na2SO4 filtered and concentrated under vacuum to afford a pale yellow solid (0.407 g, 95% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.02 (s, 1H), 7.79 (d, J= 8.6 Hz, 2H), 7.67 (d, J= 8.6 Hz, 2H), 7.32-7.25 (m, 5H), 6.61 (s, 1H), 4.51 (s, 2H), 3.90 (t, J= 6.6 Hz, 2H), 3.10 (t, J= 6.6 Hz, 2H), 2.42 (s, 3H); ESIMS m/z 428 (MH)+.

2-(5-methyl-7-{[4-(trifluoromethyl)phenyl]amino}[1,2,4]triazolo[1,5-a]pyrimidin-2-yl)ethanol (101)

A solution of Intermediate 100 (0.25 g, 0.585 mmol) in methanol (3 mL) was hydrogenated using 10% Pd/C as catalyst. The reaction mixture was concentrated and the residue was purified by flash chromatography (silica gel column, eluting with DCM:MeOH mixtures from 100:0 to 90:10%). Upon collection of the appropriate fractions, the title compound was obtained as a white solid (0.055 g, 30% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.40 (s, 1H), 7.80 (d, J= 8.6 Hz, 2H), 7.67 (d, J= 8.6 Hz, 2H), 6.60 (s, 1H), 4.72 (t, J= 5.5 Hz, 1H), 3.85 (q, J= 6.8 Hz, 2H), 2.96 (t, J= 6.8 Hz, 2H), 2.43 (s, 3H); ESIMS m/z 338 (MH)+.

Supplementary Material

1_si_001

Acknowledgments

The team acknowledges Dr. Dennis Kyle for providing the atovaquone resistant lines TM90C2B and TM90C2A, Dr. Akhil Vaidya for providing the D10 cell line harboring yeast DHODH, and Dr. Leonard D. Shultz and The Jackson Laboratory for providing access to NOD-scid IL-2Rγnull for the in vivo evaluation of compounds in the P. falciparum murine model through collaboration with GlaxoSmithKline. The team also acknowledges Emma Castro and Angel Pajares (Red Cross Blood Bank, Madrid, Spain) for the supply of human erythrocytes to perform the in vivo experiments in the P. falciparum murine model.

This work was supported by the United States National Institutes of Health grants, U01AI075594 (to MAP, PKR, SAC and IB) and R01AI53680 (to MAP and PKR). MAP acknowledges support of the Welch Foundation (I-1257) and PKR also acknowledges a Grand Challenge Explorations Award from the Bill and Melinda Gates Foundation. MAP holds the Carolyn R. Bacon Professorship in Medical Science and Education.

Abbreviations

PfDHODH
P. falciparum dihydroorotate dehydrogenase
PbDHODH
P. berghei DHODH
PvDHODH
P. vivax DHODH
hDHODH
human DHODH
CoQ
ubiquinone
FMN
flavin mononucleotide
HTS
high throughput screen
ACTs
artemesinin-based combination therapies
ADME
adsorption, distribution, metabolism, excretion
SAR
structure activity relationships

Footnotes

The X-ray structure coordinates for PfDHODH in complex with 37 have been deposited in the Protein data bank as PDB ID code 3SFK.

Supplemental Materials. A table of synthetic intermediates (Table S1), a table of the crystallographic refinement statistics (Table S2) and figure of the Fo-Fc difference map (Figure S1) are included as supplemental material.

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