Lead-optimization of aryl and aralkyl amine based triazolopyrimidine inhibitors of Plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity in mice
Malaria is one of the leading causes of severe infectious disease worldwide, yet our ability to maintain effective therapy to combat the illness is continually challenged by the emergence of drug resistance. We previously reported identification of a new class of triazolopyrimidine based P. falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitors with antimalarial activity, leading to the discovery of a new lead series and novel target for drug development. Active compounds from the series contained a triazolopyrimidine ring attached to an aromatic group through a bridging nitrogen atom. Herein we describe systematic efforts to optimize the aromatic functionality with the goal of improving potency and in vivo properties of compounds from the series. These studies led to the identification of two new substituted aniline moieties (4-SF5-Ph and 3,5-Di-F-4-CF3-Ph) which, when coupled to the triazolopyrimidine ring showed good plasma exposure and better efficacy in the P. berghei mouse model of the disease, than previously reported compounds from the series.
Introduction<!>Chemistry<!>Systematic substitution of the aryl amine moiety<!>Non-aromatic substitutions and addition of linkers between the aniline and the triazolopyrimidine<!>Triazolopyrimidine ring modifications<!>Species selectivity of inhibitor binding<!>In vitro ADME Properties<!>Mouse exposure and pharmacokinetic analysis in rats<!>Triazolopyrimidines 21 and 32 show good suppression of P. berghei growth in a mouse model<!>Discussion<!>Materials<!>Enzyme purification and assay<!>P. falciparum whole cell assays<!>In vitro ADME<!>Pharmacokinetic analysis<!>In vivo P. berghei efficacy in mice<!>Chemistry general methods<!>General procedure for compounds 5a-c<!>5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (5a)<!>5-Ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (5b)<!>5-Trifluoromethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (5c)<!>[1,2,4]triazolo[1,5-a]pyrimidin-5,7-diol (5d)<!>General procedure for compounds 6a-c<!>7-Chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (6a)<!>7-Chloro-5-ethyl-[1,2,4]triazolo[1,5-a]pyrimidine (6b)<!>5,7-Di-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (6d)<!>General procedure for compounds 7-44 and 52-61<!>N-(4-Ethylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (7).29<!>N-(4-Propylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (8).29<!>N-(4-Butylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (9).29<!>N-(4-Pentylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (10).29<!>N-(4-Hexylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (11).29<!>N-(4-Heptylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (12).29<!>N-(4-Octylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (13).29<!>N-(4-Isopropylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (14).29<!>N-(4-tert-Butylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (15).29<!>N-(4-Cyclopropylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (16)<!>N-(4-Vinylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (17).29<!>N-(4-Ethynylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (18)<!>N-(4-(2,2,2-trifuoroethyl)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (19)<!>5-Methyl-N-(4-(trifuoromethylthio)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (20)<!>N-(4-Pentafluorosulfenylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (21).29<!>N-(4-(benzyloxy)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (22).29<!>N1,N1-Dimethyl-N4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)benzene-1,4-diamine(23)29<!>5-Methyl-N-(4-(pyrrolidin-1-yl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24).29<!>N-(4-Morpholinophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (25).29<!>N1-tert-Butyl-N4(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)benzene-1,4-diamine (26)<!>N-(4-tert-Butoxyphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (27).29<!>2-Methyl-2-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamino)phenyl)propanenitrile (28)<!>N-(4-tert-Butyl-3-fluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (29)<!>N-(4-Chloro-3-fluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (30)<!>N-(3,4-Bis(trifluoromethyl)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (31)<!>N-(3,5-difluoro-4-(trifluoromethyl)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (32)<!>5-methyl-N-(5,6,7,8-tetrahydronaphthalen-2yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (33)29<!>N-(2,3-dihydro1H-inden-5-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (34).29<!>N-(Benzo[d][1,3]dioxol-5yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (35).29<!>N-(Benzo[b]thiophen-5yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (36)<!>5-Methyl-N-(2-(trifluoromethyl)-1H-benzo[d]imidazol-5yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (37)<!>N-Cyclopropyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (38)<!>N-Cyclobutyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (39)<!>N-Cyclopentyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (40)<!>N-Cyclohexyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (41)<!>N-Cycloheptyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (42)<!>N-Cyclooctyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (43)<!>N-Cyclododecyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (44)<!>N-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-O-phenylhydroxylamine (52)<!>5-Methyl-N-(4-trifluoromethyl)benzyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (55)<!>5-Methyl-N-phenethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (56)<!>5-Methyl-N-(3-phenyl)propyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (57)<!>N-Heptyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (58). mp 75 \xc2\xb0C<!>5-Ethyl-N-(4-trifluoromethyl)phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (59)<!>5-(Trifluoromethyl)-N-(4-trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (60)<!>5-Chloro-N-(4-trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (61).29<!>General procedure for compounds 45-51<!>N-(2,3-Dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (45)<!>N-(5-Fluoro-2,3-dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (46)<!>N-(5-Bromo-2,3-dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (47)<!>N-(5,6-Dimethoxy-2,3-dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (48)<!>5-Methyl-N-(1,2,3,4-tetrahydronaphthalen-2-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (49)<!>N-(6-Fluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (50)<!>N-(6-Choro-1,2,3,4-tetrahydronaphthalen-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (51)<!>Procedure for N-tert-butylbenzene-1,4-diamine (69)<!>Procedure for N-tert-butoxyaniline (70)
<p>Malaria remains one of the most significant infectious diseases world-wide. It is endemic in over 90 countries and leads to the death of 1-2 million people yearly, with a high prevalence of severe disease in children and pregnant woman.1, 2 The goal of eventual world-wide malaria eradication is now widely recognized. The approval of artemisinin-based combination therapies (ACTs) has led to substantial reductions in the burden of the disease and these drugs are a key component of the eradication ambitions.3 Past efforts to eliminate malaria have ended in the face of wide spread drug resistance to the available agents.4, 5 In a worrying development, decreased artemesinin effectiveness has recently been found along the Cambodia-Thailand border raising the concern that the value of ACTs will be increasingly compromised.6, 7 The challenge for the future is to find successful strategies to overcome the parasites unwavering ability to circumvent drug therapies through resistance. This goal depends on a continuing pipeline of new antimalarials as effective vaccines have not yet been identified.8, 9</p><p>Pyrimidine metabolism has proven to be a vulnerable component of the malaria parasite's biology and is the target of a significant number of the clinically effective therapies, including pyrimethamine and other dihydrofolate reductase inhibitors, and atovaquone, an inhibitor of the bc1 complex.1, 10 The malaria parasite relies on the de novo pyrimidine biosynthetic pathway to obtain pyrimidine nucleotides, and unlike the host it cannot salvage preformed pyrimidine bases or nucleosides.11 This vulnerability has led to a significant focus on identifying new targets within the pyrimidine biosynthetic pathway. Efforts to target the fourth enzyme in the de novo biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) are the most advanced.</p><p>DHODH catalyzes the flavin-mononucleotide (FMN)-dependent oxidation of dihydroorotate to generate orotic acid.11 The enzyme from different species can be divided into two classes, those that are cytoplasmic and utilize fumarate or NADH to oxidize the FMN cofactor (class 1), or those that are localized in the mitochondria and utilize ubiquinone (Coenzyme Q; CoQ) as the final oxidant (class 2). Both malarial and human DHODHs are class 2 mitochondrial enzymes. Potent and species selective inhibitors of Plasmodium falciparum DHODH (PfDHODH), which are inactive against the human enzyme, and which showed good activity against the parasites in whole cell assays have been identified by high-throughput screening (HTS).12-14 From these efforts, two series have emerged as viable leads for the development of a new anti-malarial agent targeting DHODH; the triazolopyrimidines identified by our group (Figure 1)13, 15 and the thiophenecarboxamides identified by Genzyme and collaborators.14, 16 Both series are currently undergoing lead optimization programs and these efforts have led to the identification of compounds with some activity against the malaria parasite in mouse models.15, 16 The structural basis for the species selectivity of these lead inhibitors has been demonstrated by X-ray structure determination of compounds from both series in complex with PfDHODH.16, 17 These studies have identified a number of differences in amino acid composition between the human and malarial enzyme in the inhibitor binding pockets that account for the species selectivity of the series.</p><p>The initial lead compound 1 (5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-naphthalen-2-yl-amine (DSM1)) in the triazolopyrimidine series showed good activity against both the enzyme and the parasite (IC50's in the range of 50 – 100 nM), but showed poor plasma exposure after repeat dosing.15 Subsequent medicinal chemistry efforts identified a metabolically stable compound 2 (5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-(4-trifluoromethyl-phenyl)-amine (DSM74)) that was able to suppress parasite growth in the P. berghei mouse model of the disease, providing the first proof of concept that PfDHODH inhibitors could have antimalarial activity in vivo.15 However, while compound 2 showed improved plasma exposure it was less potent than the original lead compound 1 as assessed in assays against PfDHODH and P. falciparum in culture. Our goal here was to identify a novel set of triazolopyrimidines with improved potency and prolonged plasma exposure, with the assumption that this would lead to improved in vivo efficacy against the malaria parasite in mouse models of malaria.</p><!><p>Compounds 7-52 and 55-61 were synthesized as shown in scheme 1 using previously described methods.13, 15, 18 Briefly, condensation of 3-amino-[1,2,4]triazole 4 with substituted ethyl acetoacetate 3a-c in acetic acid or with diethyl malonate 3d in NaOEt in EtOH gave the substituted 7-hydroxy-[1,2,4]triazolo[1,5-a]pyrimidine 5a-c/ 5,7-dihydroxy-[1,2,4]triazolo[1,5-a]pyrimidine 5d. Chlorination with POCl3 gave the corresponding 7-chloro-[1,2,4]triazolo[1,5-a]pyrimidine 6a-c/ 5,7-dichloro-[1,2,4]-triazolo[1,5-a]pyrimidine 6d, which upon treatment with substituted amines in ethanol/DMF-K2CO3 resulted in the desired products 7-52 and 55-61.</p><p>For compounds 26 and 27 it was necessary to synthesize the corresponding aryl amine precursors 69 and 70 (scheme 2). The aryl amines 69 and 70 were prepared using 1-fluoro-4-nitrobenzene 66 by aromatic nucleophilic substitution with tert-butyl amine/potassium tert-butoxide to provide nitro compounds 67 and 68. The nitro groups were then reduced with hydrogen in the presence of Pd/C catalyst to their corresponding amino compounds 69 and 70.</p><!><p>Previously identified triazolopyrimidines in the series showed either poor metabolic stability leading to poor plasma exposure, or lacked potency, so we undertook an extensive search for aryl amine replacements that would improve potency while maintaining good plasma exposure after oral dosing. The X-ray structure of 1 and 2 bound to PfDHODH demonstrated that the binding pocket for the aryl amine is completely hydrophobic and unable to form any H-bonding interactions (Figure 2). 17 The structures also showed that the pocket between the aryl amine (N1) (Figure 1) and the aromatic ring binding site is narrow, seemingly consistent with the observation that ortho substituents on the aniline ring are not tolerated.15 Therefore no additional compounds of this type were synthesized. However, structural data from several analogs with differing aromatic ring systems and substitutions indicated significant conformational flexibility in the hydrophobic pocket accommodating the aromatic ring, which supported further investigation of the effects of modifications in this region of the inhibitor molecule. To this end, we identified a series of amines (mostly commercially available) and coupled these to the triazolopyrimidine scaffold using the procedures established for the synthesis of 1 and 2 (Experimental methods) (Scheme 1). These analogs were designed to further explore binding requirements in this region of the enzyme.</p><p>All the new compounds were analyzed for their ability to inhibit PfDHODH and select compounds were evaluated against the human and P. berghei enzymes to explore species selectivity (Table 1). Compounds were also evaluated for efficacy against P. falciparum 3D7 cells in vitro. A good correlation between PfDHODH inhibitor activity and inhibition of P. falciparum 3D7 cell growth was observed throughout the series (Figure 3). The most potent analogs in the series were highly hydrophobic and similar in size to the naphthyl moiety of 1. These compounds include 15 (para-tert butyl-Ph), 29 (para-tert-butyl-Ph, meta-fluorine), 33 (5,6,7,8-tetrahydro-2-naphthyl) and 36 (5-benzothiophenyl). All four compounds showed similar potency to 1 against PfDHODH (IC50 < 100 nM) and P. falciparum 3D7 cells (EC50 50 –150 nM), while retaining selectivity against the human enzyme. Notably two of these compounds, 33 and 36, are naphthyl-analogs. The replacement of 5,6,7,8-tetrahydro-2-naphthyl 33 with the similar but smaller 5-indanyl 34 reduced activity (IC50 = 0.035 vs 0.22 μM respectively), suggesting that subtle differences in size or shape appear to have a significant impact on binding and, hence, potency in this series. Unlike 36 where the addition of sulfur into the aromatic ring system was favorable, the addition of nitrogen (37) or oxygen (35) was highly unfavorable, consistent with the hydrophobic nature in this area of the binding site (Figure 2).</p><p>Aliphatic substitution at the para position on the aniline moiety was explored with respect to potency against the PfDHODH enzyme (7 – 18). Consistent with the hydrophobic nature this binding interaction, the tert-butyl analog 15 was the most potent. The branched chain iso-propyl analog 1419, the straight-chain analogs (7, 8, 9 and 10) and the unsaturated analogs 17 and 18 were somewhat less potent. Also consistent with our structural data, as chain size increased beyond 5 carbons, potency decreased apparently associated with the fact that the binding site could no longer accommodate the increase in size. In fact, the apparent penalty in binding affinity increased with increasing chain length from n-pentyl to n-octyl (10-13). The activity of cyclopropyl analog 16 was similar to n-propyl analog 8 and the isopropyl analog 14. The substitution of larger 5- and 6-membered rings containing heteroatoms at the para position was strongly disfavored (24 and 25) as was the addition of an N or O linker to the tert-butyl group (26 and 27) further suggesting significant steric constraints in this area of the enzyme binding pocket. An additional demonstration that hydrophilic/polar functionality is not tolerated is evidenced by the lower potency of the 4-N(CH3)2 analog 23, which is similar in size to iso-propyl 14 but 4-fold less potent on PfDHODH. The substitution of CN for a methyl in the tert-butyl structure (28 vs 15) lead to a 13-fold reduction in inhibitory activity, again, possibly due to the introduction of polar functionality.</p><p>Next we evaluated a set of fluorinated groups at the para and meta positions of the aniline group. The substitution of 4-SF5 for 4-CF3 resulted in a compound 21 with 2-3 fold better activity against PfDHODH than 2 (Table 1), possibly due to the increase in hydrophobicity with SF5 compared to CF3. However, the addition of a second CF3 group onto the ring at the meta position in 31 (3,4-CF3-Ph) led to a decrease in potency relative to 2. The addition of a sulfur linker between the phenyl and the CF3 (20) reduced activity by several fold versus 2, while the addition of a methyl linker (19) reduced activity by nearly 10-fold. Both of these results are consistent with the observed size constraints in this area of the molecule. The addition of a meta-fluorine to the aniline ring was tested in combination with some of the better para-substituents (CF3 or tert-butyl) and provided somewhat improved potency in each case (2-fold decreases in PfDHODH IC50 were observed for 2 vs 32 and 15 vs 29). We previously reported the activity of the 4-Cl-Ph aryl substituent (DSM8915). In comparison, the addition of the meta-fluorine 30 to 4-Cl-Ph decreases the PfDHODH IC50 by 3-fold over the 4-Cl alone.</p><p>The EC50 for 21 and 32 against the parasite in our standard whole cell assay that utilizes serum was 10-fold higher than the activity observed on the enzyme (Table 1). However when these compounds were retested using Albumax as a replacement for serum, the EC50 for inhibition of 3D7 growth decreased and became similar to the enzyme inhibition data (EC50 = 0.18 and 0.22 μM respectively). In contrast the activity of 1 was similar in the two media. Previous studies have suggested that protein binding can influence how compounds behave under these different assay conditions20, providing a possible explanation as to why compounds 21 and 32 perform differently in the two media.</p><!><p>A series of non-aromatic cyclic and linear alkyl substitutions were also explored as aryl amine replacements (Table 2). The non-aromatic cyclic compounds 38 - 44 ranged in ring size from 3-10 carbons. These compounds were all inactive against both PfDHODH and P. falciparum 3D7 cells showing that an aromatic ring in this position is important for activity. The addition of a carbon linker between the pendant nitrogen (N1) and the phenyl ring was also explored. The single methyl linker compounds (54 – 55) were both inactive, as was 52 containing an oxygen linker. The addition of an ethyl (56) or propyl (57) linker yielded compounds with mid-micromolar range activity against PfDHODH, but these compounds were substantially less active than compounds that do not contain a linker. Compound 58, containing a simple n-heptyl group, showed similar activity to 56 and 57, consistent with the hydrophobic nature of the binding pocket. This was the only non-aromatic functionality to show activity in this position. In contrast to the inactivity of the straight chain carbon linkers, 2-indanyl (45) and derivatives (46 - 48) and 1,2,3,4-tetrahydro-2-naphthyl (49) and derivatives (50 - 51) all showed substantial activity, with most compounds in this series having an IC50 against PfDHODH in the 0.5 - 1.5 μM range. These data suggest that by conformationally restricting the aliphatic linker a favorable interaction with the pocket can be generated even in the absence of a direct conjugation between the pendant nitrogen (N1) and the aromatic ring. The exception is 48, where the addition of OCH3 groups onto the 2-indanyl led to reduced activity.</p><!><p>The X-ray structure of 1 and 2 bound to PfDHODH suggested that there is limited room to increase the size of the triazolopyrimidine ring substituent at positions R and R1 (Figure 2). An exploration of SAR was undertaken to probe the ability of the protein to accommodate additional bulk in these positions, potentially by inducing a conformational change (Table 3). Replacement of the methyl group at R with ethyl (59) or CF3 (60) lead to a 2- and 4-fold reduction in PfDHODH inhibitory activity, while in contrast replacement with chloro (61) reduced the IC50 by 2-fold. Addition of an ethyl to R1 (62 and 63) increased the IC50 by 3-6-fold over 14, while addition of a propyl (64) in this position led to complete loss in activity. Finally cyclyzing the carbon chains of positions R and R1 (65) was moderately well tolerated as this compound had an IC50 on PfDHODH that was only 2-fold higher than the previously reported analog with the same aryl group where R=methyl, R1 = H.15</p><!><p>All compounds tested in the series that showed activity against PfDHODH retained good selectivity over the human enzyme, and in no case did we see any activity against hDHODH (Tables 1-3). However compounds with good activity against PfDHODH did not always show good activity against P. berghei DHODH (PbDHODH), showing that significant species variability can occur within the compound series even between the closely related Plasmodium species. We previously observed that 1 showed a 5-fold higher IC50 for PbDHODH than PfDHODH, while 2 was similarly potent against both enzymes.15 A similar trend was observed in the extended inhibitor set. Compounds with the most bulky aryl substituents showed the greatest difference in potency, which we interpret as differential binding, between PfDHODH and PbDHODH, with the largest difference (∼30-fold) observed for 10 (4-n-pentyl-Ph) and 35 (5-benzodioxolyl). Compounds with similar size to 1 showed similar differential binding (∼5-fold) between the two enzymes, including 33, 34 and 48-51. In contrast compounds with smaller aryl groups more similar in size to 2 were similarly active against the two enzymes, including 21 and 32.</p><!><p>The goal of identifying triazolopyrimidines with improved potency over 2 was realized for a number of the compounds in Table 1. However compound potency is only one of the factors important for efficacy in vivo, and thus select compounds were evaluated to assess their physicochemical properties, protein binding and in vitro metabolic stability in human and mouse liver microsomes (Table 4). All of the compounds examined had molecular weight (<400), polar surface area (<85 Å2), number of hydrogen bond donors (<2) and acceptors (≤7), and number of freely rotating bonds (≤5), consistent with the ranges typically targeted for good oral absorption. Log DpH 7.4 varied from 1.6 - 3.6 and the aqueous solubility at pH 6.5 varied from poorly soluble (9, 15, 20, 29, 32) to highly soluble (8, 16, 35, 45, 49). Plasma protein binding, estimated using a rapid chromatographic method calibrated with known protein binding values for reference compounds, was in the range of 87-95% for the majority of compounds.</p><p>The most potent compounds, 15, 29, 33 and 36, all showed CLint values above 30 μL/min/mg microsomal protein corresponding to predicted hepatic extraction ratios of greater than 0.7 for both mouse and human and suggesting that they would be rapidly metabolized by the liver. The next most potent tier of compounds (21 and 32) with IC50's below 0.3 μM and 2-3-fold lower than for 2, showed more promising results. The CLint values for both 21 and 32 were <6 μL/min/mg protein (corresponding to predicted hepatic extraction ratios of <0.2) in both mouse and human microsomes suggesting that these compounds will undergo much slower rates of hepatic metabolism in vivo. Compounds 18, 20, 45 and 49 were also predicted to have low in vivo metabolism (CLint values of < 10 μL/min/mg protein) in humans, however they were more rapidly metabolized in mouse liver microsomes (CLint > 25 μL/min/mg protein) suggesting that hepatic clearance could potentially limit systemic exposure of these compounds in the mouse efficacy model. The remaining compounds that were tested all showed high intrinsic clearance values and are therefore likely to undergo significant in vivo hepatic metabolism in both humans and mice.</p><!><p>The systemic exposure of a selection of the more potent compounds was evaluated in mice after a single oral dose of 20 mg/kg, with plasma concentrations measured by LC/MS and followed for 24 h after dosing (Table 5 and Figure 4a). No adverse reactions were observed for any of the compounds after oral administration. Plasma exposure appeared to correlate closely with the in vitro hepatic clearance for most compounds. Compounds 21 and 32, both of which were stable in mouse liver microsomes, showed significant in vivo exposure with high Cmax values and prolonged time periods (18 and 19 h respectively) over which plasma concentrations remained above 1 μM. Interestingly, 32 displayed a high Cmax and prolonged exposure despite its poor aqueous solubility suggesting that the permeability of this compound was sufficiently high to overcome the potential solubility limitation. Both compounds displayed a higher Cmax and more prolonged exposure profiles than compound 2, based on evaluation of total plasma levels. Compound 49, which had an intermediate CLint in mouse liver microsomes, had a plasma exposure profile intermediate between the two other groups although the lower CLint value observed for human liver microsomes suggests this compound might show relatively better exposure in humans than observed in mice. Compounds with high CLint values (29, 14, 36, 33, 45) in mouse liver microsomes had poor in vivo exposure, exhibiting a low Cmax and rapid clearance from the plasma. Compound 29 also had very low aqueous solubility, which may also have contributed to its poor exposure profile. While compounds 29, 36 and 33 are significantly more potent than 2, based on their poor systemic exposure they would not be expected to show good efficacy in vivo and were therefore not evaluated further.</p><p>A more in depth pharmacokinetic analysis of 21 and 32 was performed in rats (Table 6 and Figure 4b) following single IV or oral administration. No adverse reactions were observed for either compound following administration by either dose route. Consistent with the results in mice, both compounds 21 and 32 showed prolonged exposure in rats after oral dosing (20 mg/kg) with a high Cmax (31 and 34 μM respectively) and long T1/2 (33 and 18 h, respectively). These compounds also had displayed similar binding to rat plasma proteins (95.2% and 97.3% for 21 and 32, respectively) suggesting that their free concentrations would also be comparable. Oral bioavailability was very high for both compounds, however an estimated bioavailability of >100% suggests that the pharmacokinetics were dose-dependent over the range of plasma concentrations observed. The IV kinetics of both compounds were investigated at two different dose levels. While 21 showed apparently good dose linearity over the 5-fold IV dose range, 32 exhibited clear dose-dependent kinetics over a similar dose range. This dose dependence was not the result of concentration dependent protein binding, but rather suggests that the in vivo clearance of 32 is saturable at high dose. It is likely that saturable clearance of 21 is also responsible for the apparent dose dependency with oral administration.</p><!><p>The combined P. falciparum whole cell activity data and pharmacokinetic profiles of 21 and 32 in mice suggested that these compounds had the best potential to show good anti-malarial activity of the synthesized compounds (Tables 1-3). Both compounds showed a slightly improved potency against P. falciparum 3D7 cells when compared to 2 (based on the data collected in albumax), and like 2, both compounds had similar activity against PfDHODH and PbDHODH, suggesting that they would show activity against P. berghei. Both compounds also showed more prolonged exposure profiles when compared with 2, suggesting they might show superior efficacy.</p><p>Compounds 21 and 32 were assessed using the standard Peter's test 20 in Swiss Webster mice infected with P. berghei NK65 ANKA strain. Compounds were dosed orally QD at four dose levels (3, 10, 30 and 100 mg/kg) and parasitemia was assessed 24 h after the last dose to determine the ED50 (Figure 5). As a comparator, mice were also dosed with chloroquine (2 mg/kg QD) using the same 4-day dosing protocol. After QD dosing the ED50 was determined to be 17 and 10 mg/kg respectively for 21 and 32. Chloroquine (2 mg/kg) suppressed parasitemia on average by ∼50% (24 h after the last dose) in the two studies, consistent with previous reports that the ED50 for chloroquine in this model is ∼2 mg/kg.20 The effect of BID dosing was also assessed for both 21 and 32. For 21, mice treated with either 100 mg/kg QD or 50 mg/kg BID showed low parasitemia levels on day 5 (8 and 2% of controls respectively, 24 h after the last dose), however none of the mice were parasite free on any day. In contrast, mice treated with 32 at 100 mg/kg QD or 50 mg/kg BID were parasite free on day 5, with parasitemia reemerging on day 7. Of the mice treated with 100 mg/kg BID 4/5 remained parasite free through day 7 before parasites reemerged on day 9. Thus while neither compound was capable of a complete cure, 32 demonstrated superior efficacy to 21, and both compounds showed better efficacy than 2, where 50 mg/kg BID dosing was only able to suppress parasitemia by 90% 24 h after the last dose.15 No adverse reactions were observed that were attributed to drug at any of the dose levels for either compound.</p><!><p>The development of new anti-malarial agents is necessary to keep pace with the ongoing evolution of drug resistance and is vital to arm public health efforts against the malaria parasite. The identification of novel chemical agents with anti-malarial activity is facilitated by the identification of new targets that provide the opportunity to develop anti-malarial drugs from new chemical space.13, 15 Our HTS-based discovery of the triazolopyrimidine-centered PfDHODH inhibitors with anti-malarial activity has provided both a new target and a novel lead series for future development. However in order to advance the triazolopyrimidine series to clinical development it was necessary to identify an optimized compound that shows both potency and good pharmacokinetic properties. In pursuit of this objective we undertook a systematic investigation of replacements/modifications of the aromatic ring systems of compounds 1 and 2. This effort led to the identification of two compounds, 21 and 32, with improved in vivo properties over prior analogs in the series, including better efficacy in the mouse P. berghei model.</p><p>We explored a variety of aryl, alkyl and alicyclic/aralkyl groups linked to the triazolopyrimidine scaffold through the pendant nitrogen (N1) (Figure 1). As with previous analogs in the series13, 15 we observed a close correlation between the potency of the compounds as inhibitors of PfDHODH and the activity against P. falciparum 3D7 cells. These data provide strong evidence that PfDHODH is the target of cell killing throughout the series. The most potent analogs in the series were naphthyl (1, 33, 36) and the 4-tert-butyl-phenyl analogs (15 and 29). The best of these compounds (29 and 33) had IC50's against PfDHODH in the 30 nM range. The next most potent analogs showing mid range IC50s (200 – 400 nM) contained a hydrocarbon of less than 5 carbons, or a fluorinated species (CF3 or SF5) at the para position of the aniline ring, with addition of a meta-fluorine providing an incremental increase in compound potency. Larger para substituents led to reduced activity and most of the other characterized compounds were substantially less potent. Taken together the data show that the preferred functionality is an aromatic ring system containing 10-12 carbons with 2 fused rings, or an aniline with a bulky hydrophobic group at the para position. This observed SAR is consistent with our structural studies, which demonstrate the pocket to be entirely hydrophobic and, based on the X-ray structures of 1 and 2, little free space remains to accommodate additional groups in the absence of conformational changes in the binding-site (Figure 2).</p><p>Comparison of the more potent analogs with those that showed poor activity provides additional insight into the nature of the inhibitor binding-site. We analyzed an expanded set of heteroatoms in the aryl ring and these results extend our prior findings that N and O heteroatoms are not tolerated in the aryl group. In contrast, the naphthyl analog possessing a lipophilic sulfur atom 36 showed good potency, consistent with the highly hydrophobic nature of the aryl binding pocket (Figure 2). We explored the effects of adding either linear alkyl groups or aliphatic ring systems as linkers between the triazolopyrimidine ring and the aryl group. Linear hydrocarbon or heteroalkyl linkers were inactive. However, by utilizing cycloalkyl ring as the link between the aromatic ring (49 and 45) and the pendant nitrogen (N1), activity in the same range as 2 was obtained. These data illustrate that improved potency is obtained by constraining the linker. However, compounds for which the aryl ring was conjugated directly to the pendant nitrogen (N1) (33 and 34) were more potent (15- and 2-fold, respectively) than the equivalent analog where the cycloalkyl ring is instead bound to N1 (49 and 45). We previously noted that the pyridine nitrogen (N5) is likely to be electron-rich in character, which facilitates the formation of a H-bond with R265 (Figure 2). 17 These data suggest that the optimal electronics to promote this interaction occur when N1 is coupled directly to an aryl group. Additionally better π-stacking interactions maybe formed with F227 and F188 when the aryl ring is closer to the opening of the hydrophobic pocket (Figure 2). The importance of these interactions is also suggested by the relative inactivity of the alicyclic and alkyl amines and by the inactivity of compounds containing a carbon or oxygen bridge between N1 and the aryl group. Compound 2 (IC50 = 0.28 μM) is for example significantly more potent than 55 (IC50 > 100 μM, benzyl), 56 (IC50 = 15 μM, phenethyl) or 57 (IC50 = 26 μM, phenpropyl).</p><p>All of the tested compounds retain excellent species selectivity showing activity on PfDHODH but not against the human enzyme. Amino acid sequence differences in the aryl pocket likely to account for this selectivity.17 Activity of the compounds in the series differs between the P. falciparum and P. berghei DHODH enzymes. The difference is greatest for large aryl or aralkyl groups, while compounds that contain smaller para substituents on the aniline show similar binding affinity for both enzymes. Two amino acid differences can be identified in the inhibitor binding-pocket between PfDHODH and PbDHODH (G181S and M536V). The residue at position 536 is in the aryl binding-pocket suggesting that this change is responsible for the observed species differences in binding between the two Plasmodium enzymes.</p><p>In vitro physicochemical and metabolism studies followed by pharmacokinetic analysis in mice and rats was used to evaluate the likelihood that the compounds would have the in vivo properties required of a drug candidate. The most potent compounds including 29 and 33 were rapidly metabolized in mouse and human microsomes which is likely the basis for poor plasma exposure after oral dosing in mice; 29 also has very low aqueous solubility at a pH representative of the small intestine which may have further contributed to its poor oral absorption. While the rapid metabolism prevents 29, 33 and 36 from being useful in vivo, all three compounds have similar in vitro activity to 1 and thus should be useful as alternatives to 1 for genetic selection in P. falciparum using the yeast DHODH marker that we recently described.21</p><p>The optimal aryl groups that have the best balance between potency, metabolic stability and plasma exposure were identified as 4-SF5-Ph (21) and 3,5-F-4-CF3-Ph (32). While these compounds were less potent than 29 and 33 against the enzyme, both showed prolonged plasma exposure in the mouse (Table 5) and better efficacy in the P. berghei mouse model of malaria when compared with 2. Compound 32 is the first triazolopyrimidine to be identified that is able to fully clear parasites from the infected mouse, though parasites remerged several days later. The EC50's against P. falciparum in whole cell assays were ∼200 nM for both 21 and 32, 10-200-fold above the levels for the comparator anti-malarial drugs (Table 1). Furthermore, the ED50's against P. berghei were 17 and 10 mg/kg, respectively for 21 and 32. These values are higher than reported for standard antimalarials (e.g. ED50's for chloroquine 1.8 mg/kg; mefloquine 3.8 mg/kg and artesunate 5.9 mg/kg)20, 22 possibly explaining why 21 and 32 were not able to fully clear the infection. Regardless, 21 and 32 compare favorably in the Peter's test of P. berghei infection to the most potent of the N-alkyl-5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides PfDHODH inhibitors described by Booker et al., which also were reported to have an ED50 of 10 - 15 mg/kg.16</p><p>In conclusion the studies described herein have identified the optimized aryl groups for coupling to the triazolopyrimdine ring that provide the best balance between potency and metabolic stability. Despite the improved properties of 21 and 32, these compounds likely would benefit from additional optimization to improve potency prior to selection of a drug candidate.</p><!><p>Compounds 53, 54, and 62-65 were purchased from ChemDiv. The amine precursors for 31 and 29, 3,4-bis(trifluoromethyl)aniline and 4-tert-butyl-3-fluoroaniline, respectively were purchased from the Boston University Chemistry Core (Michael P. Pollastri supervisor). Reagents for enzyme assay and purification were purchased from Sigma unless otherwise noted.</p><!><p>PfDHODH, PbDHODH and hDHODH were expressed and purified from E. coli as His6-tagged recombinant enzymes as previously described 13, 15, 23. All three enzymes were expressed as truncated soluble proteins without the N-terminal membrane spanning-domain. DHODH activity was assessed using the 2,6-dichloroindophenol (DCIP)-dye based assay as described 13, 15 using final assay conditions as follows: assay buffer (100 mM HEPES, pH 8.0, 150 mM NaCl, 10% Glycerol, 0.1%Triton), substrates (0.2 mM L-dihydroorotate and 0.02 mM CoQD), and DCIP (0.12 mM). Reactions were started by the addition of DHODH (ET = 10 nM) and monitored at 600 nm (ε=18.8 mM-1cm-1) for 5 – 10 min at 20 °C. Initial rates were used to determine the reaction velocity in the absence (vo) and presence (vi) of inhibitor. Inhibitors were added over a range of 0.01 – 100 μM using a 3-fold dilution series. Data for each inhibitor concentration were collected in triplicate and the measured vi/vo values were fitted to the log[I] vs response (three parameters) equation in Graph Pad Prism to determine the IC50.</p><!><p>P. falciparum 3D7 cells were grown in Gibco-Invitrogen RPMI-1640 supplemented with 2% (w/v) red blood cells and either 20% human type A+ plasma 24 or with Gibco-Invitrogen 0.5% Albumax I. Cultures used in Albumax testing were washed three times in RPMI-1640 to remove plasma-supplemented medium. Cell growth was monitored by [3H]-hypoxanthine uptake as described previously.25 Data at each concentration point were collected in quadruplicate and were fitted to the log[I] vs response - variable slope (4 parameter) model in Graph Pad Prism to determine the concentration of inhibitor that resulted in 50% growth inhibition (ED50).</p><!><p>Partition coefficients (LogDpH 7.4) were estimated by comparison of their chromatographic retention properties to a series of standard compounds with known values. The method utilized a Waters 2795 HPLC instrument with a Waters 2487 dual channel UV detector and employed a Phenomenex Synergi Hydro-RP 4 μm (30×2 mm) column and a mobile phase consisting of an aqueous buffer (50 mM ammonium acetate, pH 7.4) and acetonitrile. The acetonitrile content was varied from 0 to 100% over 10 min and the column re-conditioned prior to the next injection. Compound elution was monitored at 220 and 254 nm.</p><p>Protein binding values were estimated by comparison of their chromatographic retention properties on a human albumin column (ChromTech Chiral-HSA 50 × 3.0mm, 5 μm, Sigma-Aldrich) to those of a series of standard compounds with known values. The HPLC system was as above. The mobile phase consisted of an aqueous buffer (25 mM ammonium acetate buffer pH 7.4) and 30% isopropanol in the same aqueous buffer. Isopropanol composition was varied using a 10 min gradient after which the column was reconditioned prior to the next injection. Compound elution was monitored at 220 and 254 nm.</p><p>For 21 and 32, protein binding in rat plasma was assessed by ultracentrifugation to separate protein bound and free compound. Plasma samples were spiked with compound (200 and 2000 ng/mL) and centrifuged at 37°C for 4.2 h using a Beckman Optima XL 100K ultracentrifuge (Beckman Rotor type 42.2 Ti; 223,000 × g). Supernatant plasma water was assayed for the free compound concentration and this value was compared to the total concentration in non-centrifuged plasma controls incubated for the same time period at 37°C to determine the fraction bound.</p><p>Non-equilibrium solubility measurements were conducted by dilution of concentrated DMSO stock solutions into pH 6.5 phosphate buffer (final DMSO concentration of 1% v/v) and monitored for precipitate formation using a NEPHOLOstar Galaxy Nephelometer (BMG LABTECH GmbH, Offenburg, Germany). The solubility (expressed as a range) was taken to lie within the sample concentration range just prior to detection of a turbidimetric signal above the background reading.</p><p>To assess the in vitro metabolic properties compounds (1 μM) were incubated at 37°C with human and mouse liver microsomes (BD Gentest, Discovery Labware Inc., Woburn, Massachusetts) suspended in 0.1 M phosphate buffer (pH 7.4) at a final protein concentration of 0.4 mg/mL. Metabolic reactions were initiated by the addition of 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) and were quenched at various time points up to 60 min by the addition of ice-cold acetonitrile. Control samples lacking the NADPH-regenerating system were included to monitor for non-cytochrome P450-mediated metabolism in the microsomal matrix. Quenched samples were centrifuged and the relative loss of parent compound in the supernatant over the course of the incubation was monitored by LC-MS. Concentration versus time data for each compound were fitted to an exponential decay function to determine the first order rate constant for substrate depletion and the in vitro intrinsic clearance (CLint, μL/min/mg microsomal protein). These values were then used to calculate a predicted in vivo intrinsic clearance value (CLint vivo) according to the methods of Obach.26 For simplicity, CLint vivo was converted to a predicted in vivo hepatic extraction ratio (Eh) using the following equation: Eh = CLint vivo / Q + CLint vivo where Q is liver blood flow (20.7 mL/min/kg and 90 mL/min/kg in for humans and mice, respectively).27</p><!><p>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.</p><p>The systemic exposure of compounds listed in Table 5 was studied following oral administration to non-fasted male Swiss outbred mice. Mice had free access to food and water continuously throughout the pre- and post-dose phases of the study. Compounds were administered 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, 0.1 mL dose volume per mouse). At predefined time points, mice (n=1 mouse per time point for each compound) were anaesthetized with gaseous isofluorane and a single blood sample was collected via cardiac puncture. Blood was transferred to heparinized tubes containing a stabilization cocktail (Complete® inhibitor cocktail, potassium fluoride and EDTA) to minimize the potential for ex vivo degradation of test compound. Samples were immediately centrifuged to collect plasma for analysis.</p><p>The pharmacokinetics properties of 21 and 32 were also assessed in overnight fasted male Sprague Dawley rats after IV and oral administration. Rats had access to water ad libitum throughout the pre- and post-dose sampling period, and access to food was re-instated 4 h post-dose. Compounds were administered intravenously in an aqueous solution vehicle (21: 10% DMSO, 1% 1 M HCl in 0.1 M Captisol in water (CyDex Pharmaceuticals Inc., Lenexa, KS); 32: 10% DMSO, 1% 1 M HCl, 5% Tween 80 in 5% glucose/0.8% Tween 80 in water) 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) and orally by gavage (1.0 mL per rat, n=2 rats per compound) as an aqueous suspension (21: 5% DMSO, 0.5% hydroxypropylmethyl cellulose, 0.5% benzyl alcohol, 0.4% Tween 80 in water; 32: 1% DMSO, 0.5% carboxymethyl cellulose, 0.5% benzyl alcohol, 0.4% Tween 80 in water). After the oral dose was administered the tube was rinsed with 1 mL of Milli-Q water to collect any residual formulation, and this volume was also administered to the animal. Samples of arterial blood were collected up to 24 h post-dose via an in-dwelling carotid cannula. Blood was collected directly into heparinized tubes containing stabilization cocktail as described for the mouse studies, and maintained at 4°C. At the end of the collection period samples were centrifuged to collect plasma for analysis.</p><p>Quantitative analysis of each compound in plasma samples from both the mouse and rat studies was conducted by LC-MS (on either a Micromass Quattro Premier or Micromass Xevo TQ) against calibration standards prepared in blank plasma. Both 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 <0.002 μM for all compounds.</p><p>Intravenous and oral pharmacokinetic parameter estimation for 21 and 32 in rats was conducted using non-compartmental methods. WinNonlin software (version 5.2.1, Pharsight Corporation, Mountain View, CA) was utilized for estimation of 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. The maximum plasma concentration (Cmax), the time to reach the maximum concentration (Tmax) and the time plasma concentrations remained above 1 μM (T>1 μM) were taken directly from the concentration versus time profiles. The oral bioavailability (%F) for 21 and 32 in rats was estimated by comparing the average dose-normalized AUC0-24 h (n=2 rats) after oral administration to the average dose-normalized AUC0-24 h (n=2 rats) after IV administration at the higher of the two IV dose levels (i.e. 5 mg/kg for 21 and 3 mg/kg for 32). Due to the apparent dose dependency in the kinetics, the calculated bioavailability value is an estimate only.</p><!><p>Efficacy was assessed using the standard Peter's test as previously described.20, 28 The P. berghei NK65 ANKA strain was obtained from MR4 (Malaria Research and Reference Resource Center, Manassas, VA). Eight to ten week old female Swiss Webster mice (20-22 grams) were obtained from Charles River (Wilmington, MA). Mice were infected with 2 × 107 infected erythrocytes in 200 μL by intraperitoneal injection. Once a day oral dosing was performed at 3, 24, 48 and 72 h after infection. Test compounds or chloroquine (Sigma-Aldrich, St. Louis, MO) were suspended in the CMC vehicle described above for PK analysis and administered in volumes of 200 μL. Parasitemia was monitored daily starting 24 h post-infection. A minimum of 10 fields (1000× oil immersion) of >100 erythrocytes per field were counted by light microscopy. Animals demonstrating ≥20% loss in pre-infected body weight were euthanized in compliance with institutional guidelines. For ED50 determination parasitemia was counted 24 h after the final dose and data were fitted to the log (inhibitor) versus dose response variable slope (four parameter) equation in GraphPad Prism to determine the ED50.</p><!><p>All commercial chemicals and solvents are reagent grade and were used without further purification, unless otherwise specified. Reaction progress was monitored by thin layer chromatography (TLC) using silica gel 60 F-254 (0.25 mm) plates. Visualization was achieved with UV light and iodine vapor. Flash chromatography was carried out with silica gel (32-63 μm). 1H NMR spectra were recorded on dilute solutions in CDCl3 or DMSO-d6 at 300 MHz. Chemical shifts are reported in parts per million (δ) downfield from tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin multiplicities are described as s (singlet), brs (broad singlet), d (doublet), t (triplet), q (quartet) and m (multiplet). Electrospray ionization mass spectra were acquired on a Bruker Esquire Liquid Chromatograph-Ion Trap Mass Spectrometer. FABHRMS data were obtained on JEOL HX-110 mass spectrometer. Melting points (Pyrex capillary) were determined on a Mel-Temp apparatus and are uncorrected. As a test of purity, compounds were subjected to HPLC analysis on Beckman system gold using a gradient of 20% MeOH to 100% MeOH over 30 min, using a Beckman (ultrasphere) ODS column (5 μ, 4.6 mm × 15 cm). Compounds eluted as a single peak and were judged to be >95% pure.</p><!><p>A mixture of 3-amino-1,2,4-triazole 4 (20 mmol) and appropriate substituted ethyl acetoacetate 3a-c (20 mmol) was heated under reflux in 10 mL of acetic acid for 3.5-8 h. After the reaction mixture cooled to RT, the precipitated solid was filtered, washed with acetic acid followed by ethanol, and dried under vacuum, provided the desired products 5a-c with 45-55% yield.</p><!><p>mp 287 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.15 (s, 1H), 5.82 (s, 1H), 2.30 (s, 3H). MS m/z 151.1 (M + H)+.</p><!><p>mp 215 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.18 (s, 1H), 5.82 (s, 1H), 2.60 (q, 2H), 1.21 (t, 3H). MS m/z 162.9 (M - H)+.</p><!><p>mp 263 °C. 1H NMR (300 MHz, DMSO-d6): 8.40 (s, 1H), 8.04 (s, 1H, OH), 6.14 (s, 1H). MS m/z 202.9 (M - H)+.</p><!><p>To a stirred solution of 4.05 g (59.5 mmol) of NaOEt in 50 mL of absolute EtOH, 9.52 g (59.5 mmol) of ethyl malonate and 9.52 g (59.5 mmol) of 5-amino-[1,2,4]triazole were added and the mixture was refluxed for 8 h. After cool to room temperature, the precipitated white sodium salt was collected, dissolved in H2O, filtered with charcoal, and the filtrate was acidified with conc. HCl. The resulting precipitate was collected, washed with water and dried gave 4.5 g (50% yield) of white solid. mp 240 °C (lit 238 °C).18 1H NMR (300 MHz, DMSO-d6): 8.36 (s, 1H), 5.11 (s, 1H).</p><!><p>The proper [1,2,4]triazolo[1,5-a]pyrimidin-7-ol 5a-c (10 mmol) was added to 2.75 mL (30 mmol) of phosphorus oxychloride and heated under reflux for 45 min in a round bottom flask. Excess phosphorus oxychloride was removed by distillation at reduced pressure on a steam-bath and the residue triturated with ice water. Product was extracted from the aqueous mixture with methylene chloride, evaporated and purified by column chromatography using 60% EtOAc/Hexane. This afforded the products 6a-b with 50-65% yield. Compound 6c was used for next step without purification.</p><!><p>mp. 150 °C. 1H NMR (300 MHz, CDCl3): δ 8.50 (s, 1H), 7.15 (s, 1H), 2.75 (s, 3H). MS m/z 169.1 (M + H)+.</p><!><p>mp. 184 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.52 (s, 1H), 7.13 (s, 1H), 3.04 (q, 2H), 1.40 (t, 3H). MS m/z 183.1 (M + H)+.</p><!><p>A mixture of 304 mg (2 mmol) [1,2,4]triazolo[1,5-a]pyrimidin-5,7-diol 5d, 5 mL of POCl3 and 0.25 mL (2 mmol) of N,N dimethyl aniline (DMA) was refluxed for 1.5 h, forming a clear solution. The solution was concentrated in a reduced pressure to syrup, which was poured with stirring ice. The aqueous solution was extracted with CHCl3, the extract was washed with H2O, dried, evaporated and purified by column chromatography using 20% EtOAc/Hexane afforded 257 mg (68% yield) product 6d. 1H NMR (300 MHz, CDCl3): δ 8.58 (s, 1H), 7.3 (s, 1H).</p><!><p>The appropriate amine (1.1 mmol) was added to proper-7-chloro-[1,2,4]triazolo[1,5-a]pyrimidine 6a-d (1 mmol) in absolute ethanol (10 mL). The stirring was continued for 2-10 h at room temperature by monitoring with TLC until the starting material 6a-d was disappeared. Products were purified by column chromatography with CH2Cl2/MeOH/NH4OH (23:1:1) or EtOAc/Hexane. Yields ranged from 50-92%.</p><!><p>mp 174 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.10 (brs, NH, exchangeable), 8.48 (s, 1H), 7.37-7.29 (m, 4H), 6.32 (s, 1H), 2.68-2.61 (m, 2H), 2.40 (s, 3H), 1.24-1.19 (m, 3H). MS m/z 254.1 [M + H]+.</p><!><p>mp 150 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.10 (brs, NH, exchangeable), 8.48 (s, 1H), 7.37-7.27 (m, 4H), 6.32 (s, 1H), 2.61-2.56 (m, 2H), 2.40 (s, 3H), 1.68-1.56 (m, 2H), 0.95-0.90 (t, 3H). MS m/z 268.1[M + H]+.</p><!><p>mp 162 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.07 (brs, NH, exchangeable), 8.46 (s, 1H), 7.33-7.28 (m, 4H), 6.29 (s, 1H), 2.61-2.58 (m, 2H), 2.42 (s, 3H), 1.58-1.56 (m, 2H), 1.34-1.32 (m, 2H), 0.91-0.89 (t, 3H). MS m/z 282.2[M + H]+.</p><!><p>mp 163 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.17 (brs, NH, exchangeable), 8.46 (s, 1H), 7.33-7.28 (m, 4H), 6.29 (s, 1H), 2.60-2.58 (m, 2H), 2.43 (s, 3H), 1.78-1.69 (m, 2H), 1.54-1.42 (m, 4H), 1.12-0.89 (m, 3H). MS m/z 225.3 [M + H]+.</p><!><p>mp 160 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.1 (brs, NH exchangeable), 8.48 (s, 1H), 7.35-7.30 (m, 4H), 6.32 (s, 1H), 2.62 (m, 2H), 2.41 (s, 3H), 1.67-1. 53 (m, 2H), 1.38-1.26 (m, 6H), 0.89-0.86 (m, 3H). MS m/z 310.3 [M + H]+.</p><!><p>mp 153 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.2 (brs, NH exchangeable), 8.34 (s, 1H), 7.33-7.36 (m, 4H), 6.28 (s, 1H), 2.7 (m, 2H), 2.39 (s, 3H), 1.6-1.52 (m, 2H), 1.40-1.22 (m, 8H), 0.85 (t, J = 6.2 Hz, 3H). MS m/z 324.4 [M + H]+.</p><!><p>mp 144 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.1 (brs, NH exchangeable), 8.44 (s, 1H), 7.39-7.36 (m, 4H), 6.29 (s, 1H), 2.70-2.55 (m, 2H), 2.45 (s, 3H), 1.63-1.20 (m, 12H), 0.82 (t, J = 6.6 Hz, 3H). MS m/z 338.3 [M + H]+.</p><!><p>mp 185 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.12 (brs, NH, exchangeable), 8.48 (s, 1H), 7.37-7.27 (m, 4H), 6.32 (s, 1H), 3.20-2.95 (m, 1H), 2.41 (s, 3H), 1.28-1.15 (m, 6H). MS m/z 268.1[M + H]+.</p><!><p>mp 237 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.20 (brs, NH, exchangeable), 8.47 (s, 1H), 7.47 (d, J = 8.4 Hz, 2H), 7.35 (d, J = 8.1 Hz, 2H), 6.35 (s, 1H), 2.40 (s, 3H), 1.30 (s, 9H). MS m/z 282.2 [M + H]+.</p><!><p>mp 159 °C. 1H NMR (300 MHz, CDCl3): δ 10.1 (Brs, 1H, NH-exchangable), 8.47 (S, 1H), 7.29 (m, 4H), 6.28 (S, 1H), 2.48 (s, 3H), 1.94 (m, 1H), 0.97-0.67 (m, 4H) MS m/z 266.3 [M + H]+.</p><!><p>mp 180 °C. 1H NMR (300 MHz, CDCl3): δ 8.34 (s, 1H), 8.0 (brs, NH, exchangeable), 7.55-7.52 (m, 2H), 7.36-7.33 (m, 2H), 6.80-6.71 (m, 1H), 6.47 (s, 1H), 5.835-5.776 (d, 1H), 5.362-5.326 (d, 1H), 2.45 (s, 3H). MS m/z 252.2 [M + H]+.</p><!><p>mp 219 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.25 (brs, NH, exchangeable), 8.01 (s, 1H), 7.55-7.45 (m, 4H), 6.52 (s, 1H), 4.20 (s, 1H), 2.45 (s, 3H), 1.78-1.69 (m, 2H), 1.54-1.42 (m, 4H), 1.12-0.89 (m, 3H). MS m/z 250.1 [M + H]+.</p><!><p>mp 204 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.24 (brs, NH, exchangeable), 8.51 (s, 1H), 7.50-7.43 (m, 4H), 6.44 (s, 1H), 3.76-3.64 (m, 1H), 2.43 (s, 3H). MS m/z 308.2 [M + H]+.</p><!><p>mp 236 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.45 (brs, NH, exchangeable), 8.52 (s, 1H), 7.85-7.79 (m, 2H), 7.65-7.57 (m, 2H), 6.64 (s, 1H), 2.48 (s, 3H). MS m/z 326.2 [M + H]+.</p><!><p>mp 250 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.51 (brs, NH exchagable), 8.51 (s, 1H), 7.95 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 7.9 Hz, 2H), 6.74 (s, 1H), 2.45 (s, 3H). MS m/z 352.2 [M + H]+. FABHRMS calcd for [C12H10F5N5S + H]+ 352.06552, determined 352.06586.</p><!><p>mp 137 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.05 (brs, NH, exchangeable), 8.55 (s, 1H), 7.55-7.35 (m, 7H), 7.21-7.01 (m, 1H), 6.22 (s, 1H), 5.71 (s, 2H), 2.41 (s, 3H). MS m/z 332.3 [M + H]+.</p><!><p>mp 269 °C. 1H NMR (300 MHz, CDCl3): δ 9.89 (brs, NH, exchangeable), 8.45 (s, 1H), 7.22 (d, J = 9 Hz, 2H), 6.79 (d, J = 7.8 Hz, 2H), 6.08 (s, 1H), 2.93 (s, 6H), 2.36 (s, 3H). MS m/z 269.1 [M + H]+.</p><!><p>mp 271 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.86 (brs, NH, exchangeable), 8.45 (s, 1H), 7.20-7.18 (d, 2H), 6.62-6.59 (m, 2H), 6.04 (s, 1H), 3.05-3.25 (m, 4H), 2.36 (s, 3H), 2.01-1.95 (m, 4H). MS m/z 295.2 [M + H]+.</p><!><p>mp 230 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.95 (brs, NH, exchangeable), 8.48 (s, 1H), 7.35 (d, 2H), 7.05 (d, 2H), 6.22 (s, 1H), 3.76 (m, 4H), 3.15 (m, 4H), 2.40 (s, 3H). MS m/z 311.2 [M + H]+.</p><!><p>1H NMR (300 MHz, CDCl3): δ 8.38 (s, 1H), 7.72 (brs, NH, exchangeable), 7.15-7.05 (m, 2H), 6.85-6.70 (m, 2H), 6.18 (s, 1H), 2.55 (s, 3H), 1.34 (s, 9H). MS m/z 297.3 [M + H]+.</p><!><p>mp 176 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.15 (brs, NH, exchangeable), 8.50 (s, 1H), 7.34 (m, 2H), 7.06 (m, 2H), 6.28 (s, 1H), 2.38 (s, 3H), 1.35 (m, 9H). MS m/z 298.2 [M + Na]+.</p><!><p>mp 239 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.3 (brs, NH, exchangeable), 8.5 (s, 1H), 7.7-7.4 (m, 4H), 6.4 (s, 1H), 2.4 (s, 3H), 1.7 (m, 6H). MS m/z 293.3 [M + H]+.</p><!><p>mp 276 °C. 1H NMR (300 MHz, CDCl3): δ 10.22 (brs, NH, exchangeable), 8.5 (s, 1H), 7.5-7.2 (m, 3H), 6.5 (s, 1H), 2.5 (s, 3H), 1.35 (s, 9H). MS m/z 322.4 [M + Na]+.</p><!><p>mp 285 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.38 (brs, NH, exchangeable), 8.52 (s, 1H), 7.70-7.65 (m, 1H), 7.58-7.55 (m, 1H), 7.38-7.35 (m, 1H), 6.62 (s, 1H), 2.45 (s, 3H). MS m/z 278.2 [M + H]+.</p><!><p>1H NMR (300 MHz, DMSO-d6): δ 10.75 (brs, NH, exchangeable), 8.55 (s, 1H), 8.13-7.92 (m, 3H), 6.88 (s, 1H).</p><!><p>mp 238 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.76 (brs, NH exchangeable), 8.54 (s, 1H), 7.45 (m, 2H), 6.99 (s, 1H), 2.53 (s, 3H). MS m/z 330.2 [M + H]+. FABHRMS calcd for [C13H8F5N5 + H]+ 330.07789, determined 330.07626.</p><!><p>mp 187 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.01 (brs, NH, exchangeable), 8.50 (s, 1H), 7.30-7.05 (m, 3H), 6.25 (s, 1H), 2.76-2.65 (m, 2H), 2.48 (s, 3H), 1.96-1.65 (m, 4H). MS m/z 280.2 [M + H]+.</p><!><p>mp 228 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.07 (brs, NH exchangeable), 8.47 (s, 1H), 7.35-7.14 (m, 3H), 6.27 (s, 1H), 2.97-2.80 (m, 4H), 2.39 (s, 3H), 2.13-1.98 (m, 2H). MS m/z 266.3 [M + H]+.</p><!><p>mp 241 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.1 (brs, NH exchangeable), 8.42 (s, 1H), 7.4-7.26 (m, 3H), 6.29 (s, 1H), 5.46 (s, 2H), 2.38 (s, 3H). MS m/z 270.2 [M + H]+.</p><!><p>mp 244 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.28 (brs, NH exchangeable), 8.50 (s, 1H), 8.11 (d, J = 8.2, 1H), 7.95 (s, 1H), 7.87 (d, J = 4.9 Hz, 1H), 7.51 (d, J = 5.4, 1H), 7.45 (d, J = 8.2, 1H), 6.35 (s, 1H), 2.39 (s, 3H). MS m/z 282.3 [M + H]+.</p><!><p>mp 274 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.3 (brs, NH exchangeable), 8.51 (s, 1H), 7.84-7.74 (m, 2H), 7.47 (d, J = 9 Hz, 1H), 6.35 (s, 1H), 2.38 (s, 3H). MS m/z 334.2 [M + H]+.</p><!><p>mp 133 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.52 (brs, 1H, NH-exchangable), 8.36 (s, 1H), 6.44 (s, 1H), 2.78-2.62 (m, 1H), 2.48 (s, 3H) 0.97-0.67 (m, 4H).</p><!><p>mp 103 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.43 (d, J = 7.16 Hz, 1H, NH-exchangable), 8.38 (s, 1H), 6.26 (s, 1H), 4.27-4.10 (m, 1H), 2.42 (s, 3H), 2.39-2.14 (m, 4H), 1.81-1.63 (m, 2H).</p><!><p>1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 8.04 (d, J = 7.45, 1H, NH exchangalbe), 6.37 (s, 1H), 4.12-3.98 (m, 1H), 2.44 (s, 3H), 2.09-1.52 (m, 8H). MS m/z 302.1 [M + H]+.</p><!><p>1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 7.90 (brs, NH, exchangeable), 6.41 (s, 1H), 3.65-3.49 (m, 1H), 2.43 (s, 3H), 1.97-1.0 (m, 10H).</p><!><p>mp 92 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 7.89 (d, J = 7.68 Hz, NH exchangable), 6.32 (s, 1H), 3.87-3.66 (m, 1H), 2.44 (s, 3H), 2.0-1.40 (m, 12H).</p><!><p>mp 114 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 7.9 (d, J = 8.50 Hz), NH, exchangeable), 6.31 (s, 1H), 3.86-3.71 (m, 1H), 2.44 (s, 3H), 1.95-1.44 (m, 14H).</p><!><p>mp. 163 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.36(s, 1H), 7.80 (d, J = 8.5 Hz, NH, exchangeable), 6.27 (s, 1H), 3.83-3.67 (m, 1H), 2.45 (s, 3H), 1.86-1.22 (m, 22H).</p><!><p>mp 143 °C. 1H NMR (300 MHz, DMSO-d6): δ 9.38 (brs, NH, exchangeable), 8.35 (s, 1H), 7.31-7.15 (m, 3H), 7.05-7.15 (m, 1H), 7.95-6.85 (m, 2H), 2.13 (s, 3H). MS m/z 242.3 [M + H]+.</p><!><p>mp 190 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.1 (brs, NH exchagable), 8.51 (s, 1H), 7.92 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 7.6 Hz, 2H), 6.79 (s, 1H), 4.41-4.32 (m, 2H), 2.39 (s, 3H). MS m/z 308.2 [M + H]+.</p><!><p>mp 118 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.36 (s, 1H), 7.27-7.15 (m, 5H), 6.33 (s, 1H), 3.63-3.58 (m, 2H), 2.97-2.92 (m, 2H), 2.41 (s, 3H). MS m/z 254.1 [M + H]+.</p><!><p>mp 271 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.38 (s, 1H), 7.32-7.12 (m, 5H), 6.24 (s, 1H), 2.73-2.62 (m, 2H), 2.42 (s, 3H), 2.01-1.90 (m, 2H), 1.69-1.58 (m, 2H). MS m/z 268.1 [M + H]+.</p><!><p>1H NMR (300 MHz, DMSO-d6): δ 8.36 (s, 1H), 6.31 (s, 1H), 2.44 (s, 3H), 1.73-0.66 (m, 15H). MS m/z 248.3 [M + H]+.</p><!><p>mp 222 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.70 (brs, NH, exchangeable), 8.65 (s, 1H), 7.81-7.69 (m, 4H), 6.75 (s, 1H), 2.74-2.71 (m, 2H), 1.23-1.20 (m, 3H). MS m/z 308.3 [M + H]+.</p><!><p>mp 211 °C. 1H NMR (300 MHz, CDCl3): δ 8.62 (s, 1H), 8.46 (brs, NH, exchangeable), 7.95-7.85 (m, 2H), 7.58-7.47 (m, 2H), 6.95 (s, 1H). MS m/z 370.4 [M + Na]+.</p><!><p>mp 224 °C. 1H NMR (300 MHz, CDCl3): δ 8.7 (s, 1H), 7.9-7.7 (m, 4H), 6.6 (s, 1H). MS m/z 314.3 [M + H]+.</p><!><p>The appropriate amine (1.1 mmol) was added to 7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine 6a (1 mmol) in 5 mL of DMF, K2CO3 (1.2 eq) stirred under N2 atmosphere at room temperature for 5-20 h until the compound 6a disappeared. The crude product was purified by column chromatography using CH2Cl2/MeOH/NH4OH or EtOAc/Hexane resulted in the desired products 45-51. Yields ranged from 70-78%.</p><!><p>mp 192 °C. 1H NMR (300 MHz, CDCl3): δ 8.25 (s, 1H), 7.30-7.24 (m, 4H), 6.14 (brs, NH, exchangeable), 6.13 (s, 1H), 4.38 (m, 1H), 3.56-3.47 (m, 2H), 3.14-3.07 (m, 2H), 2.62 (s, 3H). MS m/z 266.1 [M + H]+.</p><!><p>mp 216 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.40 (brs, NH, exchangeable), 8.38 (s, 1H), 7.25-7.20 (m, 1H), 7.12-7.05 (m, 1H), 7.04-6.97 (m, 1H), 6.52 (s, 1H), 4.62-4.55 (m, 1H), 3.34-3.32 (m, 2H), 3.12-3.05 (m, 2H), 2.46 (s, 3H). MS m/z 284.2 [M + H]+.</p><!><p>mp 164 °C. 1H NMR (300 MHz, CDCl3): δ 8.25 (s, 1H), 7.43 (m, 1H), 7.39-7.36 (m, 1H), 7.17-7.14 (m, 1H), 6.28 (brs, NH, exchangeable), 6.12 (s, 1H), 4.59-4.49 (m, 1H), 3.54-3.42 (m, 2H), 3.22-3.01 (m, 2H), 2.62 (s, 3H). MS m/z 346 [M +2]+.</p><!><p>mp 191 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.38 (s, 1H), 8.36 (brs, NH, exchangeable), 6.88-6.85 (m, 2H), 6.52 (s, 1H), 4.62-4.52 (m, 1H), 3.72 (s, 6H), 3.30-3.20 (m, 2H), 3.12-3.02 (m, 2H), 2.45 (s, 3H). MS m/z 326.3 [M + H]+.</p><!><p>mp 57 °C. 1H NMR (300 MHz, CDCl3): δ 8.28 (s, 1H), 7.24-7.08 (m, 4H), 6.17 (brs, NH, exchangeable), 6.12 (s, 1H), 4.04 (m, 1H), 3.39-3.25 (m, 1H), 3.08-2.87 (m, 3H), 2.62 (s, 3H), 2.38-2.24 (m, 1H), 2.10-1.92 (m, 1H). MS m/z 280.2 [M + H]+.</p><!><p>mp 178 °C. 1H NMR (300 MHz, CDCl3): δ 8.25 (s, 1H), 7.18-7.08 (m, 1H), 6.97-6.78 (m, 2H), 6.17-6.02 (m, 1H and NH, exchangeable), 4.04 (m, 1H), 3.35-3.20 (m, 1H), 3.08-2.78 (m, 3H), 2.55 (s, 3H), 2.26-2.14 (m, 1H), 2.10-1.92 (m, 1H). MS m/z 320.3 [M + Na]+.</p><!><p>1H NMR (300 MHz, CDCl3): δ 8.28 (s, 1H), 7.25-7.18 (m, 2H), 7.12-7.01 (m, 1H), 6.17-6.12 (m, 1H and NH, exchangeable), 4.08 (m, 1H), 3.30-3.20 (m, 1H), 3.05-2.82 (m, 3H), 2.62 (s, 3H), 2.40-2.30 (m, 1H), 2.15-2.05 (m, 1H). MS m/z 314.3 [M + H]+.</p><!><p>A mixture of 1-fluoro-4-nitrobenzene (66) 1.41 g (10 mmol) and the tert-butyl amine 3.15 mL (30 mmol) in DMSO (20 mL) was placed in a RB flask and heated at 50 °C for 96 h. After the completion of the reaction, the mixture was poured on to crushed ice and extracted with EtOAc. The organic layer was dried, evaporated and purified by column chromatography (2% EtOAc/Hex) gave the 1.5 g (77% yield) of N-tert-butyl-4-nitroaniline (67).30 1H NMR (300 MHz, CDCl3): δ 8.03 (d, J = 9 Hz, 2H), 6.55 (d, J = 8.9 Hz, 2H), 4.64 (brs, NH, exchangeable), 1.43 (s, 9H).</p><p>N-tert-Butyl-4-nitroaniline (67) (194 mg, 1 mmol) was dissolved in the 10 mL of MeOH and added to 20 mg of 10% Pd-C and hydrogenated at room temperature for 3 h. Reaction completion was monitored by TLC and filtered through celite. The filtrate (69) was evaporated and used for the next step without further purification. 1H NMR (300 MHz, CDCl3): δ 6.95 (d, J = 8.9 Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 3.72 (brs, NH, exchangeable), 1.41 (s, 9H).</p><!><p>To the stirred solution of potassium tert-butoxide 795 mg (7.1 mmol) in 10 mL of DMF under N2 was added 1-fluoro-4-nitrobenzene (66) 0.75 mL (7.1 mmol) and continued the stirring for 2 h at room temperature. Quenched the reaction with ice cold water, extracted with EtOAc, dried, evaporated and purified (2% EtOAc/Hex) gave the 656 mg (56% yield) of 1-tert-butoxy-4-nitroaniline (68).31 1H NMR (300 MHz, CDCl3): δ 8.20 (m, 2H), 7.02 (m, 2H), 1.50 (s, 9H).</p><p>1-tert-Butoxy-4-nitroaniline (68) (150 mg, 0.769 mmol) was dissolved in the 10 mL of MeOH and added 20 mg of 10% Pd-C and hydrogenated at room temperature for overnight. Reaction completion was monitored by TLC and filtered through celite. The filtrate (70) was evaporated and used for the next step without further purification. 1H NMR (300 MHz, CDCl3): δ 6.90 (m, 2H), 6.70 (m, 2H), 3.50 (brs, NH, exchangeable), 1.21 (s, 9H).</p>