Lead optimization of aryl and aralkyl amine-based triazolopyrimidine inhibitors of plasmodium falciparum dihydroorotate dehydrogenase with antimalarial activity in mice

Article abstract of DOI:10.1021/jm200265b

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 Plasmodium 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- CF ₃-Ph), which, when coupled to the triazolopyrimidine ring, showed good plasma exposure and better efficacy in the Plasmodium berghei mouse model of the disease than previously reported compounds from the series.

Full text of DOI:10.1021/jm200265b

ARTICLE
pubs.acs.org/jmc
Lead Optimization of Aryl and Aralkyl Amine-Based
Triazolopyrimidine Inhibitors of Plasmodium falciparum
Dihydroorotate Dehydrogenase with Antimalarial Activity in Mice
Ramesh Gujjar,† Farah El Mazouni,§ Karen L. White,|| John White,† Sharon Creason,‡ David M. Shackleford,||
Xiaoyi Deng,§ William N. Charman,|| Ian Bathurst,^ Jeremy Burrows,^ David M. Floyd,^ David Matthews,^
Frederick S. Buckner,‡ Susan A. Charman,|| Margaret A. Phillips,*,§ and Pradipsinh K. Rathod*,†
Departments of †Chemistry and Global Health and ‡Medicine, University of Washington, Seattle, Washington 98195, United States
§Department of Pharmacology, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Boulevard, Dallas,
Texas 75390-9041, United States
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus),
Parkville, VIC 3052, Australia
^Medicines for Malaria Venture, Geneva, Switzerland
ABSTRACT: 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 Plasmodium 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 Plasmodium berghei mouse model of the disease
than previously reported compounds from the series.
’ INTRODUCTION
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 salv-
age preformed pyrimidine bases or nucleosides.11 This vulner-
ability 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, dihydroor-
otate dehydrogenase (DHODH), are the most advanced.
DHODH catalyzes the flavinmononucleotide(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 nicotinamide
adenine dinucleotide (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 dihydroorotate dehydrogenase (PfDHODH), which
Malaria remains one of the most significant infectious diseases
worldwide. It is endemic in over 90 countries and leads to the
death of 1ꢀ2 million people yearly, with a high prevalence of
severe disease in children and pregnant woman.1,2 The goal of
eventual worldwide malaria eradication is now widely recog-
nized. 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 widespread drug resistance to the available agents.4,5 In a
worrying development, decreased artemesinin effectiveness has
recently been found along the CambodiaꢀThailand 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 vac-
cines have not yet been identified.8,9
Pyrimidine metabolism has proven to be a vulnerable com-
ponent of the malaria parasite's biology and is the target of a
significant number of the clinically effective therapies, including
Received: March 7, 2011
Published: April 25, 2011
r
2011 American Chemical Society
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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.
Figure 1. Structures of triazolopyrimidine PfDHODH inhibitors 1
and 2.
’ CHEMISTRY
Scheme 1. Synthesis of the Triazolopyrimidine Compounds
7ꢀ52, 55ꢀ61a
Compounds 7ꢀ52 and 55ꢀ61 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 3aꢀc 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 5aꢀc/5,7-dihydroxy-[1,2,4]triazolo[1,5-a]pyr-
imidine 5d. Chlorination with POCl3 gave the corresponding
7-chloro-[1,2,4]triazolo[1,5-a]pyrimidine
6aꢀc/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 de-
sired products 7ꢀ52 and 55ꢀ61.
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.
’ RESULTS
a Reagents and conditions: (i) AcOH, 3.5ꢀ8 h, reflux, 45ꢀ55% (5aꢀc)/
NaOEt, EtOH, 8 h, 50% (5d); (ii) POCl3, 30ꢀ45 min, reflux, 50ꢀ65%
(6aꢀc)/POCl3, DMA, 1.5 hr, reflux, 68% (6d); (iii) R1ꢀNH2, EtOH/
DMF-K2CO3, 2ꢀ20 h, rt, 50ꢀ92%.
Systematic Substitution of the Aryl Amine Moiety. Pre-
viously 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 main-
taining 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 analogues 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 Section)
(Scheme 1). These analogues were designed to further explore
binding requirements in this region of the enzyme.
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).12ꢀ14 From these
efforts, two series have emerged as viable leads for the development
of a new antimalarial agent targeting DHODH: the triazolopyrimi-
dines identified by our group (Figure 1)13,15 and the thiophene-
carboxamides 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 com-
pounds from both series in complex with PfDHODH.16,17 These
studies have identified a number of differences in amino acid com-
position between the human and malarial enzyme in the inhibitor
binding pockets that account for the species selectivity of the series.
The initial lead compound 1 {5-methyl-[1,2,4]triazolo[1,5-a]-
pyrimidin-7-yl)-naphthalen-2-yl-amine (DSM1)} in the tria-
zolopyrimidine series showed good activity against both the
enzyme and the parasite (IC50 values in the range of 50ꢀ100
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
All of 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
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Scheme 2. Synthesis of Aryl Amine Precursors 69 and 70a
a Reagents and conditions: (i) t-Bu-NH2, DMSO, 96 h, 50 ꢀC, 77% (67)/t-BuOK, DMF, 2 h, 56% (68). (ii) Pd-C, H2, 3ꢀ10 h, room temperature,
80ꢀ85%.
against P. falciparum 3D7 cells in vitro. A good correlation between
PfDHODH inhibitor activity and inhibition of P. falciparum 3D7
cell growth were 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-benzo-
thiophenyl). All four compounds showed similar potency to 1
against PfDHODH (IC50 < 100 nM) and P. falciparum 3D7 cells
(EC50 = 50ꢀ150 nM), while retaining selectivity against the
human enzyme. Notably, two of these compounds, 33 and 36, are
naphthyl analogues. 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
Figure 2. Inhibitor binding site of 2 bound to PfDHODH. The pdb file
(37) or oxygen (35) was highly unfavorable, consistent with the
3I6R was used to generate the figure.17 A limited set of residues within the
4 Å shell of 2 are displayed (residues behind 2 were removed for clarity).
hydrophobic nature in this area of the binding site (Figure 2).
Aliphatic substitution at the para position on the aniline moiety
Residues and ligands are shown as space-filling balls, with the exception of
was explored with respect to potency against the PfDHODH
residues in the front of 2, where they are shown as sticks for clarity. Amino
enzyme (7ꢀ18). Consistent with the hydrophobic nature of this
acid residues and orotate (Oro) are colored purple, FMN in yellow, and 2
binding interaction, the tert-butyl analogue 15 was the most potent.
The branched chain iso-propyl analogue 14,19 the straight chain
analogues (7ꢀ10), and the unsaturated analogues 17 and 18 were
somewhat less potent. Also consistent with our structural data, as the
chain size increased beyond five carbons, the 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 (10ꢀ13). The activity of cyclopropyl
analogue 16 was similar to n-propyl analogue 8 and the isopropyl
analogue 14. The substitution of larger five- and six-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 demonstra-
tion that hydrophilic/polar functionality is not tolerated is evidenced
by the lower potency of the 4-N(CH3)2 analogue 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) leads to a 13-fold reduction in inhibitory activity, again possibly
due to the introduction of polar functionality.
in tan. Nitrogen, blue; oxygen, red; sulfur, yellow; and fluorine, light blue.
The figure is displayed using PyMol (DeLano, W. L. The PyMOL
Molecular Graphics System, 2002; http://www.pymol.org).
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.
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 com-
pounds 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 con-
ditions,20 providing a possible explanation as to why compounds 21
and 32 perform differently in the two media.
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 2ꢀ3-fold better activity
against PfDHODH than 2 (Table 1), possibly due to the increase
in hydrophobicity with SF5 as compared to CF3. However, the
addition of a second CF3 group onto the ring at the meta position
in 31 (3,4-di-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
Nonaromatic Substitutions and Addition of Linkers be-
tween the Aniline and the Triazolopyrimidine. A series of
nonaromatic cyclic and linear alkyl substitutions were also explored
as aryl amine replacements (Table 2). The nonaromatic cyclic
compounds 38ꢀ44 ranged in ring size from 3 to 12 carbons.
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Table 1. SAR of Substituted Aryl Amine-Based Triazolopyrimidine Derivatives
IC50(μM)
EC50 (μM)
Pf 3D7 cells
compd
name
Ar
PfDHODH
PbDHODH
hDHODH
1a
2a
DSM1
2-naphthyl
4-CF3-Ph
4-ethyl-Ph
0.047
0.28
0.61
0.99
0.28
0.82
4.2
0.23
0.38
3.9
>200
>100
>100
>100
>100
>100
ND
0.076, 0.066b
0.34
3.3
DSM74
7
DSM101
DSM102
DSM104
DSM116
DSM117
DSM118
DSM119
DSM103
DSM129
DSM316
DSM115
DSM157
DSM214
DSM217
DSM161
DSM156
DSM130
DSM216
DSM158
DSM229
DSM248
DSM251
DSM228
DSM215
DSM238
DSM190
DSM159
DSM160
DSM127
DSM191
DSM86
8
4- n-propyl-Ph
4- n-butyl-Ph
9.0
6.8
9
5.25
22
4.8
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
4- n-pentyl-Ph
4- n-hexyl-Ph
>5
20
>5
4- n-heptyl-Ph
4- n-octyl-Ph
24
>50
>100
0.88
0.4
ND
>5
>50
0.22
0.078
0.42
0.80
1.1
ND
>5
4-iso-propyl-Ph
4-tert-butyl-Ph
4-cyclo-propyl-Ph
4-venyl-Ph
>100
>100
>100
>100
>100
ND
0.78
0.32
1.4
2.9
4.5
0.34
5.1
4-ethynyl-Ph
4.1
4-CH2-CF3-Ph
4-S-CF3-Ph
2.5
8.8
11
0.61
0.13
5.4
1.2
>50
0.27
1.3, 0.18b
>10
2.7
4-SF5-Ph
0.28
>100
12
>100
ND
4- OCH2C6H5Ph
4-N(CH3)2-Ph
4-pyrrolenyl-Ph
4-morpholinyl-Ph
4-NHC(CH3)3-Ph
4-OC(CH3)3-Ph
4-C(CH3)2CN-Ph
3-F-4-C(CH3)3-Ph
3-F-4-Cl-Ph
0.82
3.5
>100
ND
>50
>100
>100
ND
ND
0.11
1.3
>20
>10
>5
>100
>50
44
ND
ND
ND
>10
3.7
1.0
ND
0.035
0.55
0.54
0.19
0.064
0.22
1.6
ND
0.097
3.5
>50
3,4-di-CF3-Ph
0.55
0.28
0.19
1
>100
>30
2.3
3,5-di-F-4-CF3-Ph
5,6,7,8-tetrahydro-2-naphthyl
5-indanyl
1.1,0.22b
0.16
0.8
>100
>100
>100
ND
5-benzodioxolyl
5-benzothiophenyl
2-CF3-5-benzo- imidazolyl
55
21
0.09
21
0.6
0.066
25
>50
ND
a Reported previously.13,15 ND, not determined. b Data were collected in media supplemented with albumax instead of serum. As comparators, data were
collected against Pf 3D7 cells for chloroquine, pyrimethamine, and atovaquone, EC50 = 7.1, 23, and 1.5 nM, respectively, which are similar to reported
values.32ꢀ34
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 and 55) were both inactive, as
was 52 containing an oxygen linker. The addition of an ethyl (56) or
propyl (57) linker yielded compounds with midmicromolar 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 nonaromatic functionality to show activity
in this position. In contrast to the inactivity of the straight chain
carbon linkers, 2-indanyl (45) and derivatives (46ꢀ48) and 1,2,3,4-
tetrahydro-2-naphthyl (49) and derivatives (50 and 51) all showed
substantial activity, with most compounds in this series having an
IC50 against PfDHODH in the 0.5ꢀ1.5 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.
Triazolopyrimidine Ring Modifications. The X-ray structure
of 1 and 2 bound to PfDHODH suggested that there is limited
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ARTICLE
room to increase the size of the triazolopyrimidine ring sub-
stituent at positions R and R1 (Figure 2). An exploration of SAR
was undertaken to probe the ability of the protein to accom-
modate 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) leads 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
3ꢀ6-fold over 14, while the 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 analogue with the
same aryl group where R = methyl, and R1 = H.15
Species Selectivity of Inhibitor Binding. 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 human dihydroorotate dehy-
drogenase (hDHODH) (Tables 1ꢀ3). However, compounds
with good activity against PfDHODH did not always show good
activity against Plasmodium berghei dihydroorotate dehydrogen-
ase (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-benzo-
dioxolyl). Compounds with similar size to 1showed similar differential
binding (∼5-fold) between the two enzymes, including 33, 34, and
48ꢀ51. In contrast, compounds with smaller aryl groups more similar
Figure 3. Structureꢀactivity correlation between PfDHODH and
P. falciparum 3D7 cells. Compounds from Tables 1ꢀ3 (using the human
serum media) were included in the analysis for those where both enzyme
and cell data yielded a determined IC50 value. Nonlogarithmic data were
fitted by linear regression to determine the parameters of the fit (slope =
0.99; R2 = 0.67). Data are plotted in log format for clarity of presentation.
Table 2. SAR of Alicyclic/Aralkyl and Alkyl Amine-Based Triazolopyrimidine Derivatives
IC50 (μM)
EC50 (μM)
Pf 3D7 cells
compd
name
R
PfDHODH
PbDHODH
hDHODH
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53a
54a
55
56
57
58
DSM139
DSM136
DSM134
DSM135
DSM137
DSM140
DSM138
DSM162
DSM218
DSM220
DSM219
DSM192
DSM236
DSM237
DSM194
DSM178
DSM179
DSM193
DSM213
DSM77
cyclo-propyl
cyclo-butyl
>100
>100
>100
>100
>100
>100
58
ND
ND
ND
ND
ND
ND
ND
2.4
ND
ND
ND
ND
ND
ND
ND
ND
>50
>50
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.4
cyclo-pentyl
cyclo-hexyl
cyclo-heptyl
cyclo-octyl
cyclo-dodecyl
2-indanyl
0.50
0.61
1.2
5-F-2-indanyl
5-Br-2-indanyl
1.4
1.3
3.3
6.6
5,6-di-OCH3-2-indanyl
1,2,3,4-tetrahydro-2-naphthyl
6-F-1,2,3,4-tetra-hydro-2-naphthyl
6-Cl-1,2,3,4-tetra-hydro-2-naphthyl
phenyloxy
4.6
41
7.7
0.54
0.63
0.67
>100
>100
>100
>100
15
2.3
1.8
1.9
2.5
10
10
>100
ND
ND
>100
27
ND
>10
>10
ND
>20
25
benzyl
4-F-benzyl
4-CF3-benzyl
2-phenylethyl
3-phenylpropyl
26
>100
55
DSM76
n-heptyl
8.6
13
a Compounds commercially obtained; ND, not determined.
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Table 3. SAR of Pyrimidine Ring-Modified Triazolopyrimidine Derivatives
IC50(μM)
EC50 (μM)
Pf 3D7 cells
compd
R/R1
Ar
PfDHODH
PbDHODH
hDHODH
59 (DSM247)
60 (DSM235)
61 (DSM252)
62 (DSM171)a
63 (DSM172)a
64 (DSM166)a
65 (DSM174)a
C2H5/H
CF3/H
4-CF3-Ph
0.78
1.7
2.0
2.3
>100
>100
>100
>100
>100
>100
>100
2.3
4-CF3-Ph
>10
3.4
Cl/H
4-CF3-Ph
0.17
1.7
0.13
4.5
CH3/C2H5
CH3/C2H5
CH3/C3H7
ꢀC3H6ꢀ
4-C(CH3)2-Ph
4-OCF3-Ph
4-CH3-Ph
>10
>10
>10
8.3
3.2
5.7
>100
1.4
>100
1.3
3-F-4-CH3-Ph
a Compounds commercially obtained; ND, not determined.
in size to 2 were similarly active against the two enzymes, including 21
and 32.
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 com-
pounds 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 humanlivermicrosomes suggests that this compound
might show relatively better exposure in humans than observed in
mice. Compounds with high CLint values (29, 14, 36, 33, and 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 toshowgoodefficacyinvivo
and were therefore not evaluated further.
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
In Vitro Absorption, Distribution, Metabolism, and Excre-
tion (ADME) Properties. The goal of identifying triazolopyr-
imidines 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 hu-
man and mouse liver microsomes (Table 4). All of the com-
pounds examined had molecular weight (<400), polar surface area
(<85 Å2), number of hydrogen bond donors (<2) and acceptors
(e7), and number of freely rotating bonds (e5), consistent with
the ranges typically targeted for good oral absorption. Log DpH 7.4
varied from 1.6 to 3.6, and the aqueous solubility at pH 6.5 varied
frompoorly soluble (9, 15, 20, 29, and 32) tohighlysoluble (8, 16,
35, 45, and 49). Plasma protein binding, estimated using a rapid
chromatographic method calibrated with known protein binding
values for reference compounds, was in the range of 87ꢀ95% for
the majority of compounds.
The most potent compounds, 15, 29, 33, and 36, all showed
CLint values above 30 μL/min/mg microsomal protein, corre-
sponding 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 values below 0.3 μM and
2ꢀ3-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 com-
pounds 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 metabo-
lized 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.
Mouse Exposure and Pharmacokinetic Analysis in Rats.
The systemic exposure of a selection of the more potent compounds
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Table 4. Physicochemical Properties, Plasma Protein Binding, and in Vitro Intrinsic Clearance (CLint) in Liver Microsomes for
Selected Triazolopyrimidine Derivatives
compd
Log DpH 7.4
aqueous solubility pH 6.5 (μM)
human plasma binding (%)b
CLint human/mouse (μL/min/mg protein)
Eh human/mouse
1
3.2
2.5
2.6
3.1
3.6
3.0
3.4
2.7
2.4
2.2
3.0
3.0
2.2
3.5
2.9
3.4
2.9
1.6
2.6
2.5
2.9
45ꢀ90
21ꢀ43
25ꢀ50
187ꢀ374
11ꢀ22
47ꢀ94
11ꢀ22
>377
93.8
86.9
88.0
93.5
96.7
91.3
93.2
86.8
84.4
81.8
96.1
95.4
ND
96.4
96.6
95.5
92.5
ND
89.8
76.1
ND
96/229a
8/NDa
61/ND
36/ND
36/ND
30/241
43/ND
53/287
32/168
8/27
0.84/0.91a
0.29/NDa
0.77/ND
0.66/ND
0.67/ND
0.64/0.91
0.71/ND
0.75/0.93
0.64/0.88
0.31/0.54
<0.2/0.84
<0.2/<0.2
0.4/0.91
2
7
8
9
14
15
16
17
18
20
21
23
29
32
33
34
35
36
45
49
25ꢀ50
100ꢀ200
5ꢀ10
71ꢀ142
93ꢀ186
<2.7
<5/119
<5/<6
9/226
32/68
0.64/0.75
<0.2/<0.2
0.80/0.94
0.81/0.94
0.37/ND
0.78/0.88
<0.2/0.59
<0.2/0.61
9ꢀ18
<5/<6
72/361
77/341
11/ND
64/162
<5/32
89ꢀ179
47ꢀ94
186ꢀ372
22ꢀ44
188ꢀ376
>358
<5/35
a Reported previously.13,15 ND, not determined. For reference, Eh < 0.3 is indicative of low predicted metabolism, Eh = 0.3ꢀ0.6 is medium predicted
metabolism, and Eh > 0.7 is high predicted metabolism. b Data calculated using the human albumin chromatographic method.
Table 5. Summary of Mouse Exposure Data Following a
Single Oral Dose (20 mg/kg)
PbDHODH, suggesting that they would show activity against
P. berghei. Both compounds also showed more prolonged exposure
profiles when compared with 2, suggesting that they might show
superior efficacy.
compd
Cmax (μM)
time above 1 μM (h)
Compounds 21 and 32 were assessed using the standard Peter's
test20 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 com-
pound 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.
21
32
29
14
36
33
45
49
12
19
18
<1
<1
<1
1
33
0.25
0.8
0.4
2.2
0.5
7.8
<1
6
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.
Triazolopyrimidines 21 and 32 Show Good Suppression
of P. berghei Growth in a Mouse Model. 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 antimalarial activity of the synthe-
sized compounds (Tables 1ꢀ3). 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
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Figure 4. Rodent pharmacokinetic data. (a) Plasma concentration vs time profiles in mice after a single oral dose of 20 mg/kg. Data for 2 were taken
from ref 15. (b) Plasma concentration vs time profiles in rats after a single oral or IV dose. Dose and route of dosing are indicated in the figure legend.
of two compounds, 21 and 32, with improved in vivo properties
over prior analogues in the series, including better efficacy in the
mouse P. berghei model.
Table 6. Rat Pharmacokinetic Parameters for 21 and 32
(Average n = 2 Rats)
parameter
21
1, 5.1
32
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 series,13,15
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 analogues in the series were naphthyl (1, 33, and 36)
and the 4-tert-butyl-phenyl analogues (15 and 29). Thebestofthese
compounds (29 and 33) had IC50 values against PfDHODH in the
30 nM range. The next most potent analogues showing midrange
IC50 values (200ꢀ400 nM) contained a hydrocarbon of less than
five 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 10ꢀ12 carbons with two 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 on the basis of 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).
IV dose (mg/kg)
0.9, 2.9
2.3, 1.5
0.4, 0.5
1.6, 4.5
18.6
clearance (mL/min/kg)a
volume of distribution at steady state (L/kg)a
t1/2 (h)a
1.5, 1.2
1.4, 1.5
11.4, 14.5
20.1
oral dose (mg/kg)
Tmax (min)
270
600
Cmax (μM)
31
34.3
t1/2 (h)a
33
17.6
AUC0-inf (μM min)a
bioavailability (F %)b
101 693
75 007
>100
>100
a Values are approximations as the terminal elimination phase was not
well-defined. b Data suggest dose-dependent kinetics.
’ DISCUSSION
The development of new antimalarial 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 antimalarial activity
is facilitated by the identification of new targets that provide the
opportunity to develop antimalarial drugs from new chemical
space.13,15 Our HTS-based discovery of the triazolopyrimidine-
centered PfDHODH inhibitors with antimalarial activity has
provided both a new target and a novel lead series for future
development. However, 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
Comparison of the more potent analogues 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 analogue possessing a lipophilic
sulfur atom 36 showed good potency, consistent with the highly
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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 The 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 M536 V). 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 Plas-
modium enzymes.
In vitro physicochemical and metabolism studies followed by
pharmacokinetic analysis in mice and rats were 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
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-di-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, although parasites re-emerged several days later. The
EC50 values against P. falciparum in whole cell assays were ∼200
nM for both 21 and 32, 10ꢀ200-fold above the levels for the
comparator antimalarial drugs (Table 1). Furthermore, the ED50
values 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 values 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
Figure 5. In vivo efficacy of 21 and 32 in the P. berghei mouse malarial
model. The zero time point represents the day of infection, and once
daily oral dosing was performed at 3, 24, 48, and 72 h after infection.
Dose levels are indicated on the graphs. Five mice were dosed per group,
and error bars represent the standard error of the mean. Percent
parasitemia vs time from initial innoculum (days) for 21 (A) and 32
(B). Not all dose levels are shown for clarity of presentation. (C and D)
Parasite levels were determined 24 h after the final dose and are plotted
as the normalized values (relative to the no drug vehicle control) vs log
of drug dose. Data were fitted to the log(inhibitor) vs doseꢀresponse
variable slope (four parameter) equation in GraphPad Prism to deter-
mine the ED50. The ED50 = 17 (10ꢀ29 mg/kg) and 10 mg/kg (7.6ꢀ13
mg/kg), respectively, for 21 and 32, where the 95% confidence interval is
indicated in parentheses.
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 the 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 analogue 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 may be formed with
F227 and F188 when the aryl ring is closer to the opening of the
hydrophobic pocket (Figure 2). The importance of these inter-
actions 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. Com-
pound 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).
PfDHODH inhibitors described by Booker et al., which also were
reported to have an ED50 of 10ꢀ15 mg/kg.16
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.
’ EXPERIMENTAL SECTION
Materials. Compounds 53, 54, and 62ꢀ65 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
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Journal of Medicinal Chemistry
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Boston University Chemistry Core (Michael P. Pollastri supervisor). Re-
agents for enzyme assay and purification were purchased from Sigma unless
otherwise noted.
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, and 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
noncytochrome 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 com-
pound 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 þ CLint vivo, where Q is liver
blood flow (20.7 and 90 mL/min/kg in for humans and mice,
respectively).27
Methods. Enzyme Purification and Assay. PfDHODH, PbDHO-
DH, and hDHODH were expressed and purified from Escherichia 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 asses-
sed using the 2,6-dichloroindophenol (DCIP) dye-based assay as descri-
bed13,15 using final assay conditions as follows: assay buffer (100 mM
HEPES, pH 8.0, 150 mM NaCl, 10% glycerol, and 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 mMꢀ1 cmꢀ1) for 5ꢀ10 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 0.01ꢀ100 μM using a 3-fold dilution series. Data for each in-
hibitor 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. falciparum Whole Cell Assays. P. falciparum 3D7 cells were grown
in Gibco-Invitrogen RPMI-1640 supplemented with 2% (w/v) red
blood cells and either 20% human type Aþ plasma24 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 responseꢀvariable slope
(four-parameter) model in Graph Pad Prism to determine the concen-
tration of inhibitor that resulted in 50% growth inhibition (ED50).
In Vitro ADME. 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 mm ꢁ 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 was reconditioned prior
to the next injection. Compound elution was monitored at 220 and 254 nm.
Protein binding values were estimated by comparison of their chrom-
atographic retention properties on a human albumin column (Chrom-
Tech Chiral-HSA 50 mm ꢁ 3.0 mm, 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.
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; 223000g). Supernatant
plasma water was assayed for the free compound concentration, and this
value was compared to the total concentration in noncentrifuged plasma
controls incubated for the same time period at 37 ꢀC to determine the
fraction bound.
Pharmacokinetic Analysis. Animal studies were performed in accor-
dance 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.
The systemic exposure of compounds listed in Table 5 was studied
following oral administration to nonfasted male Swiss outbred mice.
Mice had free access to food and water continuously throughout the pre-
and postdose 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.
The pharmacokinetics properties of 21 and 32 were also assessed in
overnight fasted male SpragueꢀDawley rats after IV and oral administra-
tion. Rats had access to water ad libitum throughout the pre- and postdose
sampling period, and access to food was reinstated 4 h postdose.
Compounds were administered intravenously in an aqueous solution
vehicle [21: 10% DMSO and 1% 1 M HCl in 0.1 M Captisol in water
(CyDex Pharmaceuticals Inc., Lenexa, KS); 32: 10% DMSO, 1% 1 M
HCl, and 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,
and 0.4% Tween 80 in water; 32: 1% DMSO, 0.5% carboxymethyl
cellulose, 0.5% benzyl alcohol, and 0.4% Tween 80 in water). After the
oraldosewas administered, thetubewasrinsedwith1 mLofMilli-Qwater
to collect any residual formulation, and this volume was also administered
to the animal. Samples of arterial blood were collected up to 24 h postdose
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.
Nonequilibrium 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 LAB-
TECH 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.
Quantitative analysis of each compound in plasma samples from both
the mouse and the rat studies was conducted by LC-MS (on either a
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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.
precipitated solid was filtered, washed with acetic acid followed by
ethanol, and dried under vacuum and provided the desired products
5aꢀc with 45ꢀ55% yield.
5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (5a). 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 þ H)þ.
1
Intravenous and oral pharmacokinetic parameter estimation for 21
and 32 in rats was conducted using noncompartmental 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 (AUC0ꢀ24 h) or the
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 bioavail-
ability (%F) for 21 and 32 in rats was estimated by comparing the
average dose-normalized AUC0ꢀ24 h (n = 2 rats) after oral administra-
tion to the average dose-normalized AUC0ꢀ24 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). Because of the apparent dose dependency in the
kinetics, the calculated bioavailability value is an estimate only.
In Vivo P. berghei Efficacy in Mice. The 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 (20ꢀ22 g) were obtained from Charles River
(Wilmington, MA). Mice were infected with 2 ꢁ 107 infected erythro-
cytes 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
postinfection. A minimum of 10 fields (1000ꢁ oil immersion) of >100
erythrocytes per field were counted by light microscopy. Animals
demonstrating g20% loss in preinfected 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 doseꢀresponse variable slope (four para-
meter) equation in GraphPad Prism to determine the ED50.
Chemistry General Methods. All commercial chemicals and solvents
were 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 (32ꢀ63 μm). 1H NMR
spectra were recorded on dilute solutions in CDCl3 or DMSO-d6 at 300
MHz. Chemical shifts are reportedin parts per million(δ) downfield from
tetramethylsilane (TMS). Coupling constants (J) are reported in Hz. Spin
multiplicitiesare described ass(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 20ꢀ100% MeOH over 30 min, using a Beckman
(ultrasphere) ODS column (5 μ, 4.6mmꢁ 15cm). Compoundselutedas
a single peak and were judged to be >95% pure.
5-Ethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (5b). mp 215 ꢀC. H
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)þ.
5-Trifluoromethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (5c). mp
1
263 ꢀC. H 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)þ.
[1,2,4]Triazolo[1,5-a]pyrimidin-5,7-diol (5d). 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 it was cooled to room
temperature, the precipitated white sodium salt was collected, dissolved in
H2O, and filtered with charcoal, and the filtrate was acidified with
concentrated 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).
General Procedure for Compounds 6aꢀc. The proper
[1,2,4]triazolo[1,5-a]pyrimidin-7-ol 5aꢀc (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 was triturated with ice water. The 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 50ꢀ65% yield. Compound 6c was used for the next
step without purification.
7-Chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (6a). 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 þ H)þ.
7-Chloro-5-ethyl-[1,2,4]triazolo[1,5-a]pyrimidine (6b). 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 þ H)þ.
5,7-Dichloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (6d). A mix-
ture 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) were
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) of product 6d. 1H NMR (300
MHz, CDCl3): δ 8.58 (s, 1H), 7.3 (s, 1H).
General Procedure for Compounds 7ꢀ44 and 52ꢀ61. The
appropriate amine (1.1 mmol) was added to proper-7-chloro-[1,2,4]
triazolo[1,5-a]pyrimidine 6aꢀd (1 mmol) in absolute ethanol (10 mL).
The stirring was continued for 2ꢀ10 h at room temperature by
monitoring with TLC until the starting material 6aꢀd disappeared.
Products were purified by column chromatography with CH2Cl2/
MeOH/NH4OH (23:1:1) or EtOAc/hexane. Yields ranged from 50
to 92%.
N-(4-Ethylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (7)29. mp 174 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.10 (brs,
NH, exchangeable), 8.48 (s, 1H), 7.37ꢀ7.29 (m, 4H), 6.32 (s, 1H),
2.68ꢀ2.61 (m, 2H), 2.40 (s, 3H), 1.24ꢀ1.19 (m, 3H). MS m/z 254.1
[M þ H]þ.
N-(4-Propylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (8)29. mp 150 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.10 (brs,
NH, exchangeable), 8.48 (s, 1H), 7.37ꢀ7.27 (m, 4H), 6.32 (s, 1H),
2.61ꢀ2.56 (m, 2H), 2.40 (s, 3H), 1.68ꢀ1.56 (m, 2H), 0.95ꢀ0.90
(t, 3H). MS m/z 268.1 [M þ H]þ.
General Procedure for Compounds 5aꢀc. A mixture of 3-amino-
1,2,4-triazole 4 (20 mmol) and appropriate substituted ethyl acetoace-
tate 3aꢀc (20 mmol) was heated under reflux in 10 mL of acetic acid for
3.5ꢀ8 h. After the reaction mixture cooled to room temperature, the
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N-(4-Butylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-a-
mine (9)29. mp 162 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.07 (brs,
NH, exchangeable), 8.46 (s, 1H), 7.33ꢀ7.28 (m, 4H), 6.29 (s, 1H),
2.61ꢀ2.58 (m, 2H), 2.42 (s, 3H), 1.58ꢀ1.56 (m, 2H), 1.34ꢀ1.32 (m,
2H), 0.91ꢀ0.89 (t, 3H). MS m/z 282.2 [M þ H]þ.
δ 10.05 (brs, NH, exchangeable), 8.55 (s, 1H), 7.55ꢀ7.35 (m, 7H),
7.21ꢀ7.01 (m, 1H), 6.22 (s, 1H), 5.71 (s, 2H), 2.41 (s, 3H). MS m/z
332.3 [M þ H]þ.
N1,N1-Dimethyl-N4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-
benzene-1,4-diamine (23)29. 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 þ H]þ.
N-(4-Pentylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (10)29. mp 163 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.17
(brs, NH, exchangeable), 8.46 (s, 1H), 7.33ꢀ7.28 (m, 4H), 6.29 (s,
1H), 2.60ꢀ2.58 (m, 2H), 2.43 (s, 3H), 1.78ꢀ1.69 (m, 2H), 1.54ꢀ1.42
(m, 4H), 1.12ꢀ0.89 (m, 3H). MS m/z 225.3 [M þ H]þ.
5-Methyl-N-(4-(pyrrolidin-1-yl)phenyl)-[1,2,4]triazolo[1,5-a]pyr-
imidin-7-amine (24)29. mp 271 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ
9.86 (brs, NH, exchangeable), 8.45 (s, 1H), 7.20ꢀ7.18 (d, 2H),
6.62ꢀ6.59 (m, 2H), 6.04 (s, 1H), 3.05ꢀ3.25 (m, 4H), 2.36 (s, 3H),
2.01ꢀ1.95 (m, 4H). MS m/z 295.2 [M þ H]þ.
N-(4-Hexylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (11)29. mp 160 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.1
(brs, NH exchangeable), 8.48 (s, 1H), 7.35ꢀ7.30 (m, 4H), 6.32 (s,
1H), 2.62 (m, 2H), 2.41 (s, 3H), 1.67ꢀ1. 53 (m, 2H), 1.38ꢀ1.26 (m,
6H), 0.89ꢀ0.86 (m, 3H). MS m/z 310.3 [M þ H]þ.
N-(4-Morpholinophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-
7-amine (25)29. 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 þ H]þ.
N-(4-Heptylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
1
amine (12)29. mp 153 ꢀC. H NMR (300 MHz, DMSO-d6): δ 10.2
(brs, NH exchangeable), 8.34 (s, 1H), 7.33ꢀ7.36 (m, 4H), 6.28 (s,
1H), 2.7 (m, 2H), 2.39 (s, 3H), 1.6ꢀ1.52 (m, 2H), 1.40ꢀ1.22 (m, 8H),
0.85 (t, J = 6.2 Hz, 3H). MS m/z 324.4 [M þ H]þ.
N1-tert-Butyl-N4(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)b-
enzene-1,4-diamine (26). 1H NMR (300 MHz, CDCl3): δ 8.38
(s, 1H), 7.72 (brs, NH, exchangeable), 7.15ꢀ7.05 (m, 2H),
6.85ꢀ6.70 (m, 2H), 6.18 (s, 1H), 2.55 (s, 3H), 1.34 (s, 9H). MS m/z
297.3 [M þ H]þ.
N-(4-Octylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (13)29. mp 144 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.1
(brs, NH exchangeable), 8.44 (s, 1H), 7.39ꢀ7.36 (m, 4H), 6.29 (s,
1H), 2.70ꢀ2.55 (m, 2H), 2.45 (s, 3H), 1.63ꢀ1.20 (m, 12H), 0.82 (t,
J = 6.6 Hz, 3H). MS m/z 338.3 [M þ H]þ.
N-(4-tert-Butoxyphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-
7-amine (27)29. 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 þ Na]þ.
2-Methyl-2-(4-(5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ylami-
no)phenyl)propanenitrile (28). mp 239 ꢀC. 1H NMR (300 MHz,
DMSO-d6): δ 10.3 (brs, NH, exchangeable), 8.5 (s, 1H), 7.7ꢀ7.4 (m,
4H), 6.4 (s, 1H), 2.4 (s, 3H), 1.7 (m, 6H). MS m/z 293.3 [M þ H]þ.
N-(4-tert-Butyl-3-fluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]p-
yrimidin-7-amine (29). mp 276 ꢀC. 1H NMR (300 MHz, CDCl3): δ
10.22 (brs, NH, exchangeable), 8.5 (s, 1H), 7.5ꢀ7.2 (m, 3H), 6.5 (s,
1H), 2.5 (s, 3H), 1.35 (s, 9H). MS m/z 322.4 [M þ Na]þ.
N-(4-Chloro-3-fluorophenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyr-
N-(4-Isopropylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (14)29. mp 185 ꢀC. 1H NMR (300 MHz, DMSO-
d6): δ 10.12 (brs, NH, exchangeable), 8.48 (s, 1H), 7.37ꢀ7.27
(m, 4H), 6.32 (s, 1H), 3.20ꢀ2.95 (m, 1H), 2.41 (s, 3H), 1.28ꢀ
1.15 (m, 6H). MS m/z 268.1 [M þ H]þ.
N-(4-tert-Butylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-
7-amine (15)29. 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 þ H]þ.
N-(4-Cyclopropylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-
7-amine (16). 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), 0.97ꢀ0.67 (m, 4H) MS m/z 266.3 [M þ H]þ.
N-(4-Vinylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
1
imidin-7-amine (30). mp 285 ꢀC. H NMR (300 MHz, DMSO-d6):
δ 10.38 (brs, NH, exchangeable), 8.52 (s, 1H), 7.70ꢀ7.65 (m, 1H),
7.58ꢀ7.55 (m, 1H), 7.38ꢀ7.35 (m, 1H), 6.62 (s, 1H), 2.45 (s, 3H). MS
m/z 278.2 [M þ H]þ.
1
amine (17)29. mp 180 ꢀC. H NMR (300 MHz, CDCl3): δ 8.34
(s, 1H), 8.0 (brs, NH, exchangeable), 7.55ꢀ7.52 (m, 2H), 7.36ꢀ7.33
(m, 2H), 6.80ꢀ6.71 (m, 1H), 6.47 (s, 1H), 5.835ꢀ5.776 (d, 1H),
5.362ꢀ5.326 (d, 1H), 2.45 (s, 3H). MS m/z 252.2 [M þ H]þ.
N-(4-Ethynylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (18). mp 219 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.25 (brs,
NH, exchangeable), 8.01 (s, 1H), 7.55ꢀ7.45 (m, 4H), 6.52 (s, 1H), 4.20
(s, 1H), 2.45 (s, 3H), 1.78ꢀ1.69 (m, 2H), 1.54ꢀ1.42 (m, 4H),
1.12ꢀ0.89 (m, 3H). MS m/z 250.1 [M þ H]þ.
N-(3,4-Bis(trifluoromethyl)phenyl)-5-methyl-[1,2,4]triazolo[1,5-
a]pyrimidin-7-amine (31). 1H NMR (300 MHz, DMSO-d6): δ 10.75
(brs, NH, exchangeable), 8.55 (s, 1H), 8.13ꢀ7.92 (m, 3H), 6.88 (s, 1H).
N-(3,5-Difluoro-4-(trifluoromethyl)phenyl)-5-methyl-[1,2,4]triaz-
olo[1,5-a]pyrimidin-7-amine (32). 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 þ H]þ. FABHRMS
calcd for [C13H8F5N5 þ H]þ, 330.07789; determined, 330.07626.
5-Methyl-N-(5,6,7,8-tetrahydronaphthalen-2yl)-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (33)29. mp 187 ꢀC. 1H NMR (300 MHz,
DMSO-d6): δ 10.01 (brs, NH, exchangeable), 8.50 (s, 1H), 7.30ꢀ7.05
(m, 3H), 6.25 (s, 1H), 2.76ꢀ2.65 (m, 2H), 2.48 (s, 3H), 1.96ꢀ1.65
(m, 4H). MS m/z 280.2 [M þ H]þ.
N-(4-(2,2,2-trifuoroethyl)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]
pyrimidin-7-amine (19). mp 204 ꢀC. 1H NMR (300 MHz, DMSO-d6):
δ 10.24 (brs, NH, exchangeable), 8.51 (s, 1H), 7.50ꢀ7.43 (m, 4H), 6.44
(s, 1H), 3.76ꢀ3.64 (m, 1H), 2.43 (s, 3H). MS m/z 308.2 [M þ H]þ.
5-Methyl-N-(4-(trifuoromethylthio)phenyl)-[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (20). mp 236 ꢀC. 1H NMR (300 MHz, DMSO-d6):
δ 10.45 (brs, NH, exchangeable), 8.52 (s, 1H), 7.85ꢀ7.79 (m, 2H),
7.65ꢀ7.57 (m, 2H), 6.64 (s, 1H), 2.48 (s, 3H). MS m/z 326.2 [M þ H]þ.
N-(4-Pentafluorosulfenylphenyl)-5-methyl-[1,2,4]triazolo[1,5-a]
pyrimidin-7-amine (21)29. 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 þ H]þ.
FABHRMS calcd for [C12H10F5N5S þ H]þ, 352.06552; determined,
352.06586.
N-(2,3-Dihydro1H-inden-5-yl)-5-methyl-[1,2,4]triazolo[1,5-a]py-
rimidin-7-amine (34)29. mp 228 ꢀC. 1H NMR (300 MHz, DMSO-d6):
δ 10.07 (brs, NH exchangeable), 8.47 (s, 1H), 7.35ꢀ7.14 (m, 3H), 6.27
(s, 1H), 2.97ꢀ2.80 (m, 4H), 2.39 (s, 3H), 2.13ꢀ1.98 (m, 2H). MS m/z
266.3 [M þ H]þ.
N-(Benzo[d][1,3]dioxol-5yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (35)29. mp 241 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ
10.1 (brs, NH exchangeable), 8.42 (s, 1H), 7.4ꢀ7.26 (m, 3H), 6.29 (s,
1H), 5.46 (s, 2H), 2.38 (s, 3H). MS m/z 270.2 [M þ H]þ.
N-(Benzo[b]thiophen-5yl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-
7-amine (36). mp 244 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.28 (brs,
N-(4-(Benzyloxy)phenyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimid-
in-7-amine (22)29. mp 137 ꢀC. 1H NMR (300 MHz, DMSO-d6):
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ARTICLE
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 þ H]þ.
5-Chloro-N-(4-trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyri-
midin-7-amine (61)29. mp 224 ꢀC. 1H NMR (300 MHz, CDCl3): δ 8.7
(s, 1H), 7.9ꢀ7.7 (m, 4H), 6.6 (s, 1H). MS m/z 314.3 [M þ H]þ.
General Procedure for Compounds 45ꢀ51. The appropriate amine
(1.1 mmol) was added to 7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyri-
midine 6a (1 mmol) in 5 mL of DMF, K2CO3 (1.2 equiv) stirred under
N2 atmosphere at room temperature for 5ꢀ20 h until the compound 6a
disappeared. The crude product was purified by column chromatography
using CH2Cl2/MeOH/NH4OH or EtOAc/hexane and resulted in the
desired products 45ꢀ51. Yields ranged from 70 to 78%.
5-Methyl-N-(2-(trifluoromethyl)-1H-benzo[d]imidazol-5yl)-[1,2,4]
triazolo[1,5-a]pyrimidin-7-amine (37). mp 274 ꢀC. 1H NMR (300 MHz,
DMSO-d6): δ 10.3 (brs, NH exchangeable), 8.51 (s, 1H), 7.84ꢀ7.74 (m,
2H), 7.47 (d, J=9 Hz, 1H), 6.35 (s, 1H), 2.38 (s, 3H). MSm/z 334.2 [M þ
H]þ.
N-Cyclopropyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
1
(38). mp 133 ꢀC. H NMR (300 MHz, DMSO-d6): δ 8.52 (brs, 1H,
N-(2,3-Dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo[1,5-a]p-
yrimidin-7-amine (45). mp 192 ꢀC. 1H NMR (300 MHz, CDCl3): δ
8.25 (s, 1H), 7.30ꢀ7.24 (m, 4H), 6.14 (brs, NH, exchangeable), 6.13 (s,
1H), 4.38 (m, 1H), 3.56ꢀ3.47 (m, 2H), 3.14ꢀ3.07 (m, 2H), 2.62 (s,
3H). MS m/z 266.1 [M þ H]þ.
NH-exchangable), 8.36 (s, 1H), 6.44 (s, 1H), 2.78ꢀ2.62 (m, 1H), 2.48
(s, 3H) 0.97ꢀ0.67 (m, 4H).
N-Cyclobutyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (39).
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), 4.27ꢀ4.10 (m, 1H), 2.42 (s,
3H), 2.39ꢀ2.14 (m, 4H), 1.81ꢀ1.63 (m, 2H).
N-(5-Fluoro-2,3-dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (46). mp 216 ꢀC. 1H NMR (300 MHz,
DMSO-d6): δ 8.40 (brs, NH, exchangeable), 8.38 (s, 1H), 7.25ꢀ7.20
(m, 1H), 7.12ꢀ7.05 (m, 1H), 7.04ꢀ6.97 (m, 1H), 6.52 (s, 1H),
4.62ꢀ4.55 (m, 1H), 3.34ꢀ3.32 (m, 2H), 3.12ꢀ3.05 (m, 2H), 2.46 (s,
3H). MS m/z 284.2 [M þ H]þ.
N-Cyclopentyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
(40). 1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 8.04 (d, J = 7.45,
1H, NH exchangalbe), 6.37 (s, 1H), 4.12ꢀ3.98 (m, 1H), 2.44 (s, 3H),
2.09ꢀ1.52 (m, 8H). MS m/z 302.1 [M þ H]þ.
N-Cyclohexyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
(41). 1H NMR (300 MHz, DMSO-d6): δ 8.37 (s, 1H), 7.90 (brs, NH,
exchangeable), 6.41 (s, 1H), 3.65ꢀ3.49 (m, 1H), 2.43 (s, 3H), 1.97ꢀ1.0
(m, 10H).
N-(5-Bromo-2,3-dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (47). mp 164 ꢀC. 1H NMR (300 MHz, CD-
Cl3):δ8.25 (s, 1H), 7.43 (m, 1H), 7.39ꢀ7.36(m, 1H), 7.17ꢀ7.14 (m, 1H),
6.28 (brs, NH, exchangeable), 6.12 (s, 1H), 4.59ꢀ4.49 (m, 1H), 3.54ꢀ3.42
(m, 2H), 3.22ꢀ3.01 (m, 2H), 2.62 (s, 3H). MS m/z 346 [M þ 2]þ.
N-(5,6-Dimethoxy-2,3-dihydro-1H-inden-2-yl)-5-methyl-[1,2,4]-
N-Cycloheptyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
(42). 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), 3.87ꢀ3.66 (m, 1H), 2.44
(s, 3H), 2.0ꢀ1.40 (m, 12H).
1
triazolo[1,5-a]pyrimidin-7-amine (48). mp 191 ꢀC. H NMR (300
MHz, DMSO-d6): δ 8.38 (s, 1H), 8.36 (brs, NH, exchangeable),
6.88ꢀ6.85 (m, 2H), 6.52 (s, 1H), 4.62ꢀ4.52 (m, 1H), 3.72 (s, 6H),
3.30ꢀ3.20 (m, 2H), 3.12ꢀ3.02 (m, 2H), 2.45 (s, 3H). MS m/z 326.3
[M þ H]þ.
N-Cyclooctyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (43).
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), 3.86ꢀ3.71 (m, 1H), 2.44 (s, 3H),
1.95ꢀ1.44 (m, 14H).
5-Methyl-N-(1,2,3,4-tetrahydronaphthalen-2-yl)-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (49). mp 57 ꢀC. 1H NMR (300 MHz,
CDCl3): δ 8.28 (s, 1H), 7.24ꢀ7.08 (m, 4H), 6.17 (brs, NH, ex-
changeable), 6.12 (s, 1H), 4.04 (m, 1H), 3.39ꢀ3.25 (m, 1H),
3.08ꢀ2.87 (m, 3H), 2.62 (s, 3H), 2.38ꢀ2.24 (m, 1H), 2.10ꢀ1.92 (m,
1H). MS m/z 280.2 [M þ H]þ.
N-Cyclododecyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine
(44). 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), 3.83ꢀ3.67 (m, 1H),
2.45 (s, 3H), 1.86ꢀ1.22 (m, 22H).
N-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-O-phenylhydr-
oxylamine (52). mp 143 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 9.38
(brs, NH, exchangeable), 8.35 (s, 1H), 7.31ꢀ7.15 (m, 3H), 7.05ꢀ7.15
(m, 1H), 7.95ꢀ6.85 (m, 2H), 2.13 (s, 3H). MS m/z 242.3 [M þ H]þ.
5-Methyl-N-(4-trifluoromethyl)benzyl)-[1,2,4]triazolo[1,5-
a]pyrimidin-7-amine (55). 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), 4.41ꢀ4.32 (m, 2H), 2.39 (s, 3H).
MS m/z 308.2 [M þ H]þ.
N-(6-Fluoro-1,2,3,4-tetrahydronaphthalen-2-yl)-5-methyl-[1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (50). mp 178 ꢀC. H NMR (300
MHz, CDCl3): δ 8.25 (s, 1H), 7.18ꢀ7.08 (m, 1H), 6.97ꢀ6.78 (m, 2H),
6.17ꢀ6.02 (m, 1H and NH, exchangeable), 4.04 (m, 1H), 3.35ꢀ3.20
(m, 1H), 3.08ꢀ2.78 (m, 3H), 2.55 (s, 3H), 2.26ꢀ2.14 (m, 1H),
2.10ꢀ1.92 (m, 1H). MS m/z 320.3 [M þ Na]þ.
N-(6-Choro-1,2,3,4-tetrahydronaphthalen-2-yl)-5-methyl-[1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (51). 1H NMR (300 MHz, CDCl3):
δ 8.28 (s, 1H), 7.25ꢀ7.18 (m, 2H), 7.12ꢀ7.01 (m, 1H), 6.17ꢀ6.12 (m,
1H and NH, exchangeable), 4.08 (m, 1H), 3.30ꢀ3.20 (m, 1H), 3.05ꢀ
2.82 (m, 3H), 2.62 (s, 3H), 2.40ꢀ2.30 (m, 1H), 2.15ꢀ2.05 (m, 1H). MS
m/z 314.3 [M þ H]þ.
5-Methyl-N-phenethyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (56).
mp 118 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 8.36 (s, 1H), 7.27ꢀ7.15
(m, 5H), 6.33 (s, 1H), 3.63ꢀ3.58 (m, 2H), 2.97ꢀ2.92 (m, 2H), 2.41
(s, 3H). MS m/z 254.1 [M þ H]þ.
5-Methyl-N-(3-phenyl)propyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine (57). mp 271 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 8.38 (s,
1H), 7.32ꢀ7.12 (m, 5H), 6.24 (s, 1H), 2.73ꢀ2.62 (m, 2H), 2.42 (s, 3H),
2.01ꢀ1.90 (m, 2H), 1.69ꢀ1.58 (m, 2H). MS m/z 268.1 [M þ H]þ.
N-Heptyl-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (58).
Procedure for N-tert-Butylbenzene-1,4-diamine (69). A mixture of
1.41 g of 1-fluoro-4-nitrobenzene (66) (10 mmol) and 3.15 mL of the tert-
butyl amine (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) and gave 1.5 g (77% yield) of N-tert-butyl-4-nitroaniline (67).30 1H
NMR (300 MHz, CDCl3):δ8.03 (d, J= 9 Hz, 2H), 6.55 (d, J= 8.9 Hz, 2H),
4.64 (brs, NH, exchangeable), 1.43 (s, 9H).
1
mp 75 ꢀC. H NMR (300 MHz, DMSO-d6): δ 8.36 (s, 1H), 6.31
(s, 1H), 2.44 (s, 3H), 1.73ꢀ0.66 (m, 15H). MS m/z 248.3 [M þ H]þ.
5-Ethyl-N-(4-trifluoromethyl)phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-
7-amine (59). mp 222 ꢀC. 1H NMR (300 MHz, DMSO-d6): δ 10.70
(brs, NH, exchangeable), 8.65 (s, 1H), 7.81ꢀ7.69 (m, 4H), 6.75 (s, 1H),
2.74ꢀ2.71 (m, 2H), 1.23ꢀ1.20 (m, 3H). MS m/z 308.3 [M þ H]þ.
5-(Trifluoromethyl)-N-(4-trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-
a]pyrimidin-7-amine (60). mp 211 ꢀC. 1H NMR (300 MHz, CDCl3): δ
8.62 (s, 1H), 8.46 (brs, NH, exchangeable), 7.95ꢀ7.85 (m, 2H), 7.58ꢀ7.47
(m, 2H), 6.95 (s, 1H). MS m/z 370.4 [M þ Na]þ.
N-tert-Butyl-4-nitroaniline (67) (194 mg, 1 mmol) was dissolved in
10 mL of MeOH, added to 20 mg of 10% PdꢀC, and hydrogenated at
room temperature for 3 h. The 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,
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ARTICLE
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).
Silamut, K.; Imwong, M.; Chotivanich, K.; Lim, P.; Herdman, T.; An,
S. S.; Yeung, S.; Singhasivanon, P.; Day, N. P.; Lindegardh, N.; Socheat,
D.; White, N. J. Artemisinin resistance in Plasmodium falciparum malaria.
N. Engl. J. Med. 2009, 361, 455–467.
(8) Wells, T. N.; Alonso, P. L.; Gutteridge, W. E. New medicines to
improve control and contribute to the eradication of malaria. Nat. Rev.
Drug Discovery 2009, 8, 879–891.
(9) Olliaro, P.; Wells, T. N. The global portfolio of new antimalarial
medicines under development. Clin. Pharmacol. Ther. 2009, 85, 584–
595.
(10) Vaidya, A. B.; Mather, M. W. Mitochondrial Evolution and
Functions in Malaria Parasites. Annu. Rev. Microbiol. 2009, 63, 249–267.
(11) Phillips, M. A.; Rathod, P. K. Plasmodium dihydroorotate
dehydrogenase: a promising target for novel anti-malarial chemother-
apy. Infect. Disord.: Drug Targets 2010, 10, 226–239.
Procedure for N-tert-Butoxyaniline (70). To the stirred solution of
potassium tert-butoxide 795 mg (7.1 mmol) in 10 mL of DMF under N2
was added 0.75 mL of 1-fluoro-4-nitrobenzene (66) (7.1 mmol), and
stirring was continued for 2 h at room temperature. The reaction was
quenched with ice cold water, extracted with EtOAc, dried, evaporated,
and purified (2% EtOAc/Hex) and gave 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).
1-tert-Butoxy-4-nitroaniline (68) (150 mg, 0.769 mmol) was dis-
solved in the 10 mL of MeOH, 20 mg of 10% PdꢀC was added, and it was
hydrogenated at room temperature overnight. The reaction completion
was monitored by TLC and filtered through Celite. The filtrate (70) was
1
evaporated and used for the next step without further purification. H
(12) Baldwin, J.; Michnoff, C. H.; Malmquist, N. A.; White, J.; Roth,
M. G.; Rathod, P. K.; Phillips, M. A. High-throughput screening for
potent and selective inhibitors of Plasmodium falciparum dihydroorotate
dehydrogenase. J. Biol. Chem. 2005, 280, 21847–21853.
NMR (300 MHz, CDCl3): δ 6.90 (m, 2H), 6.70 (m, 2H), 3.50 (brs, NH,
exchangeable), 1.21 (s, 9H).
(13) Phillips, M. A.; Gujjar, R.; Malmquist, N. A.; White, J.; El
Mazouni, F.; Baldwin, J.; Rathod, P. K. Triazolopyrimidine-based
dihydroorotate dehydrogenase inhibitors with potent and selective
activity against the malaria parasite, Plasmodium falciparum. J. Med.
Chem. 2008, 51, 3649–3653.
(14) Patel, V.; Booker, M.; Kramer, M.; Ross, L.; Celatka, C. A.;
Kennedy, L. M.; Dvorin, J. D.; Duraisingh, M. T.; Sliz, P.; Wirth, D. F.;
Clardy, J. Identification and characterization of small molecule inhibitors
of Plasmodium falciparum dihydroorotate dehydrogenase. J. Biol. Chem.
2008, 283, 35078–35085.
(15) Gujjar, R.; Marwaha, A.; El Mazouni, F.; White, J.; White, K. L.;
Creason, S.; Shackleford, D. M.; Baldwin, J.; Charman, W. N.; Buckner,
F. S.; Charman, S.; Rathod, P. K.; Phillips, M. A. Identification of a
metabolically stable triazolopyrimidine-based dihydroorotate dehydro-
genase inhibitor with antimalarial activity in mice. J. Med. Chem. 2009,
52, 1864–1872.
(16) Booker, M. L.; Bastos, C. M.; Kramer, M. L.; Barker, R. H., Jr.;
Skerlj, R.; Bir Sdhu, A.; Deng, X.; Celatka, C.; Cortese, J. F.; Guerrero
Bravo, J. E.; Krespo Llado, K. N.; Serrano, A. E.; Angulo-Barturen, I.;
Belꢀen Jimꢀenez-Díaz, M.; Viera, S.; Garuti, H.; Wittlin, S.; Papastogian-
nidis, P.; Lin, J.; Janse, C. J.; Khan, S. M.; Duraisingh, M.; Coleman, B.;
Goldsmith, E. J.; Phillips, M. A.; Munoz, B.; Wirth, D. F.; Klinger, J. D.;
Wiegand, R.; Sybertz, E. Novel inhibitors of Plasmodium falciparum
dihydroorotate dehydrogenase with anti-malarial activity in the mouse
model. J. Biol. Chem. 2010, 285, 33054–33064.
(17) Deng, X.; Gujjar, R.; El Mazouni, F.; Kaminsky, W.; Malmquist,
N. A.; Goldsmith, E. J.; Rathod, P. K.; Phillips, M. A. Structural plasticity
of malaria dihydroorotate dehydrogenase allows selective binding of
diverse chemical scaffolds. J. Biol. Chem. 2009, 284, 26999–27009.
(18) Makisumi, Y. Synthesis of potential anticancer agents. 5,7-Disub-
stituted s-triazolo[2,3-a]pyrimidines. Chem. Pharm. Bull. 1961, 9, 801.
(19) Guiguemde, W. A.; Shelat, A. A.; Bouck, D.; Duffy, S.;
Crowther, G. J.; Davis, P. H.; Smithson, D. C.; Connelly, M.; Clark, J.;
Zhu, F.; Jimenez-Diaz, M. B.; Martinez, M. S.; Wilson, E. B.; Tripathi,
A. K.; Gut, J.; Sharlow, E. R.; Bathurst, I.; El Mazouni, F.; Fowble, J. W.;
Forquer, I.; McGinley, P. L.; Castro, S.; Angulo-Barturen, I.; Ferrer, S.;
Rosenthal, P. J.; Derisi, J. L.; Sullivan, D. J.; Lazo, J. S.; Roos, D. S.;
Riscoe, M. K.; Phillips, M. A.; Rathod, P. K.; Van Voorhis, W. C.; Avery,
V. M.; Guy, R. K. Chemical genetics of Plasmodium falciparum. Nature
2010, 465, 311–315.
’ AUTHOR INFORMATION
Corresponding Author
*Tel: 214-645-6164. E-mail: margaret.phillips@UTSouthwestern.
edu (M.A.P.). Tel: 206-221-6069. E-mail: rathod@chem.washington.
edu (P.K.R.).
Funding Sources
This work was supported by the United States National Institutes
of Health Grants, U01AI075594 (to M.A.P., P.K.R., S.A.C., and
I.B.; Malaria Medicines Venture). M.A.P. also acknowledges the
support of the Welch Foundation (I-1257), and P.K.R. also
acknowledges support from NIH Grants AI089688, AI082617,
and the Grand Challenge Explorations Award from the Bill and
Melinda Gates Foundation. M.A.P. holds the Carolyn R. Bacon
Professorship in Medical Science and Education.
’ ABBREVIATIONS USED
PfDHODH, Plasmodium falciparum dihydroorotate dehydrogen-
ase; PbDHODH, Plasmodium berghei dihydroorotate dehydro-
genase; hDHODH, human dihydroorotate dehydrogenase; -
CoQ, ubiquinone/coenzyme Q; FMN, flavin mononucleotide;
HTS, high-throughput screen; ACTs, artemisinin-based combi-
nation therapies; NADH, nicotinamide adenine dinucleotide;
ADME, absorption, distribution, metabolism and excretion
’ REFERENCES
(1) Greenwood, B. M.; Fidock, D. A.; Kyle, D. E.; Kappe, S. H.;
Alonso, P. L.; Collins, F. H.; Duffy, P. E. Malaria: Progress, perils, and
prospects for eradication. J. Clin. Invest. 2008, 118, 1266–1276.
(2) Feachem, R. G.; Phillips, A. A.; Hwang, J.; Cotter, C.; Wielgosz,
B.; Greenwood, B. M.; Sabot, O.; Rodriguez, M. H.; Abeyasinghe, R. R.;
Ghebreyesus, T. A.; Snow, R. W. Shrinking the malaria map: Progress
and prospects. Lancet 2010, 376, 1566–1578.
(3) Eastman, R. T.; Fidock, D. A. Artemisinin-based combination
therapies: A vital tool in efforts to eliminate malaria. Nat. Rev. Microbiol.
2009, 7, 864–874.
(4) White, N. J. Antimalarial drug resistance. J. Clin. Invest. 2004,
113, 1084–1092.
(5) Mackinnon, M. J.; Marsh, K. The selection landscape of malaria
parasites. Science 2010, 328, 866–871.
(6) Dondorp, A. M.; Yeung, S.; White, L.; Nguon, C.; Day, N. P.;
Socheat, D.; von Seidlein, L. Artemisinin resistance: Current status and
scenarios for containment. Nat. Rev. Microbiol. 2010, 8, 272–280.
(7) Dondorp, A. M.; Nosten, F.; Yi, P.; Das, D.; Phyo, A. P.; Tarning,
J.; Lwin, K. M.; Ariey, F.; Hanpithakpong, W.; Lee, S. J.; Ringwald, P.;
(20) Fidock, D. A.; Rosenthal, P. J.; Croft, S. L.; Brun, R.; Nwaka, S.
Antimalarial drug discovery: Efficacy models for compound screening.
Nat. Rev. Drug Discovery 2004, 3, 509–520.
(21) Ganesan, S. M.; Morrisey, J. M.; Ke, H.; Painter, H. J.; Laroiya,
K.; Phillips, M. A.; Rathod, P. K.; Mather, M. W.; Vaidya, A. B. Yeast
dihydroorotate dehydrogenase as a new selectable marker for Plasmo-
dium falciparum transfection. Mol. Biochem. Parasitol. 2011, 177, 29–34.
(22) Nzila, A.; Rottmann, M.; Chitnumsub, P.; Kiara, S. M.; Kam-
chonwongpaisan, S.; Maneeruttanarungroj, C.; Taweechai, S.; Yeung, B. K.;
3948
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Journal of Medicinal Chemistry
ARTICLE
Goh, A.; Lakshminarayana, S. B.; Zou, B.; Wong, J.; Ma, N. L.;
Weaver, M.; Keller, T. H.; Dartois, V.; Wittlin, S.; Brun, R.; Yuthavong,
Y.; Diagana, T. T. Preclinical evaluation of the antifolate QN254,
5-chloro- N060-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine, as
an antimalarial drug candidate. Antimicrob. Agents Chemother. 2010,
54, 2603–2610.
(23) Baldwin, J.; Farajallah, A. M.; Malmquist, N. A.; Rathod, P. K.;
Phillips, M. A. Malarial dihydroorotate dehydrogenase: Substrate and
inhibitor specificity. J. Biol. Chem. 2002, 277, 41827–41834.
(24) Desjardins, R. E.; Canfield, C. J.; Haynes, J. D.; Chulay, J. D.
Quantitative assessment of antimalarial activity in vitro by a semiauto-
mated microdilution technique. Antimicrob. Agents Chemother. 1979,
16, 710–718.
(25) Jiang, L.; Lee, P.; White, J.; Rathod, P. Potent and selective
activity of a combination of thymidine and 1843U89, a folate-based
thymidylate synthase inhibitor, against Plasmodium falciparum. Antimi-
crob. Agents Chemother. 2000, 44, 1047–1050.
(26) Obach, R. S. Prediction of human clearance of twenty-nine
drugs from hepatic microsomal intrinsic clearance data: An examination
of in vitro half-life approach and nonspecific binding to microsomes.
Drug Metab. Dispos. 1999, 27, 1350–1359.
(27) Davies, B.; Morris, T. Physiological parameters in laboratory
animals and humans. Pharm. Res. 1993, 10, 1093–1095.
(28) Dominguez, J. N.; Leon, C.; Rodrigues, J.; Gamboa de Dom-
inguez, N.; Gut, J.; Rosenthal, P. J. Synthesis of chlorovinyl sulfones as
structural analogs of chalcones and their antiplasmodial activities. Eur.
J. Med. Chem. 2009, 44, 1457–1462.
(29) Phillips, M. A.; Rathod, P. K.; Gujjar, R.; Marwaha, A. S.;
Charman, S. A. Dihydroorotate dehydrogenase inhibitors with selective
anti-malarial activity. WO Patent 2009/082691; U.S. Patent
20090209557, 2009.
(30) Kotsuki, H.; Kobayashi, S.; Matsumoto, K.; Suenaga, H.;
Nishizawa, H. A convenient synthesis of aromatic amines from activated
aromatic fluorides. Synthesis. Synthesis 1990, 1147–1148.
(31) Woiwode, T. F.; Rose, C.; Wandless, T. J. A simple and efficient
method for the preparation of hindered alkyl-aryl ethers. J. Org. Chem.
1998, 63, 9594–9596.
(32) Sidhu, A. B.; Verdier-Pinard, D.; Fidock, D. A. Chloroquine
resistance in Plasmodium falciparum malaria parasites conferred by pfcrt
mutations. Science 2002, 298, 210–213.
(33) Yuvaniyama, J.; Chitnumsub, P.; Kamchonwongpaisan, S.;
Vanichtanankul, J.; Sirawaraporn, W.; Taylor, P.; Walkinshaw, M. D.;
Yuthavong, Y. Insights into antifolate resistance from malarial DHFR-TS
structures. Nat. Struct. Biol. 2003, 10, 357–365.
(34) Khositnithikul, R.; Tan-Ariya, P.; Mungthin, M. In vitro atova-
quone/proguanil susceptibility and characterization of the cytochrome b
gene of Plasmodium falciparum from different endemic regions of
Thailand. Malar. J. 2008, 7, 23.
3949
dx.doi.org/10.1021/jm200265b |J. Med. Chem. 2011, 54, 3935–3949