Triazolopyrimidine-based dihydroorotate dehydrogenase inhibitors with potent and selective activity against the malaria parasite Plasmodium falciparum

Article abstract of DOI:10.1021/jm8001026

A Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitor that is potent (K_I = 15 nM) and species-selective (>5000-fold over the human enzyme) was identified by high-throughput screening. The substituted triazolopyrimidine and its structural analogues were produced by an inexpensive three-step synthesis, and the series showed good association between PfDHODH inhibition and parasite toxicity. This study has identified the first nanomolar PfDHODH inhibitor with potent antimalarial activity in whole cells (EC ₅₀ = 79 nM).

Full text of DOI:10.1021/jm8001026

J. Med. Chem. 2008, 51, 3649–3653
3649
Triazolopyrimidine-Based Dihydroorotate Dehydrogenase Inhibitors with Potent and Selective
Activity against the Malaria Parasite Plasmodium falciparum
Margaret A. Phillips,*,§ Ramesh Gujjar,# Nicholas A. Malmquist,§ John White,# Farah El Mazouni,§ Jeffrey Baldwin,§ and
Pradipsinh K. Rathod*,#
Department of Pharmacology, UniVersity of Texas Southwestern Medical Center at Dallas, 6001 Forest Park BouleVard,
Dallas, Texas 75390-9041, and Department of Chemistry and Global Health, UniVersity of Washington, Seattle, Washington 98195
ReceiVed January 31, 2008
A Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) inhibitor that is potent (KI ) 15
nM) and species-selective (>5000-fold over the human enzyme) was identified by high-throughput screening.
The substituted triazolopyrimidine and its structural analogues were produced by an inexpensive three-step
synthesis, and the series showed good association between PfDHODH inhibition and parasite toxicity. This
study has identified the first nanomolar PfDHODH inhibitor with potent antimalarial activity in whole cells
(EC50 ) 79 nM).
Introduction
Malaria infects up to 900 million people and causes as many
as 2.7 million deaths worldwide each year.1,2 Nearly 40% of
the world’s population is at risk for contracting the disease,
which has been a major cause of mortality throughout history.
Antimalarial drugs have been the mainstay for managing new
infections and established disease. In recent decades widespread
drug resistance has been encountered for chloroquine and for
almost every other available antimalarial agent.3 Further, there
are indications that drug resistance may be appearing at faster
rates in some parts of the world.4 Multidrug combinations offer
temporary relief,5,6 but given current trends, it is clear that the
disease will continue to have an unacceptable impact on global
health unless novel drugs are developed. A significant portion
of the current arsenal of malaria drug therapies is rooted in
natural remedies, starting with the discovery of quinine nearly
350 years ago.7,8 The current challenge is to couple our
knowledge of malaria genomics and biochemistry with modern
platforms for drug discovery.
Pyrimidines are essential metabolites, required for DNA and
RNA biosynthesis and the biosynthesis of phospholipids and
glycoproteins. Unlike mammalian cells, the malaria parasite
cannot salvage preformed pyrimidine bases or nucleosides, and
pyrimidines must be acquired through the de novo biosynthetic
pathway.9–12 These biochemical results have been confirmed
by the genome sequence showing that pyrimidine salvage
enzymes are missing from the parasite.13 Dihydrofolate reduc-
tase is a validated target for malaria treatment14 and inhibitors
of thymidylate synthase have potent antimalarial activity,15–17
illustrating the importance of the pyrimidine biosynthetic
pathway to parasite survival.
Figure 1. Structure of PfDHODH active site bound to inhibitor 1.
Residues conserved between P. falciparum and human DHODH are
displayed in gray, and variable residues are displayed in purple. Orotic
acid (Oro), FMN, and 1 are displayed in yellow. The figure was
generated using PyMol from the file 1TV5.pdb.
inner mitochondrial membrane and utilize ubiquinone (CoQ)
as the physiological oxidant in the reaction.18,19 Recent studies
have suggested that the sole function of the parasite mitochon-
drial electron transport chain is to provide oxidized CoQ to
DHODH for the synthesis of pyrimidines, confirming the
essential role that DHODH plays in the biology of the parasite.20
Inhibitors of human DHODH have proven efficacy for the
treatment of rheumatoid arthritis,21,22 with an approved com-
pound on the market for this application (2-cyano-3-hydroxy-
N-[4-(trifluoromethyl)phenyl]-2-butenamide 1 (A77 1726)23
(Figure 1), the active metabolite of 5-methyl-N-[4-(trifluorom-
ethyl)phenyl]isoxazole-4-carboxamide), thus demonstrating that
DHODH is a “druggable” target. The X-ray structures of human
and malarial DHODH have been determined.24,25 Orotate and
FMN stack against each other in the center of the ꢀ/R barrel,
and the inhibitor-binding site is formed adjacent to this site by
two R-helices, which lie between the predicted N-terminal
transmembrane domain and the canonical ꢀ/R barrel domain
(Figure 1). The inhibitor binding-pocket has extensive variation
in amino acid sequence between the human and malarial
enzymes, providing the structural basis for the identification of
species-specific inhibitors.
The fourth and rate-limiting step of pyrimidine biosynthesis
is catalyzed by DHODH,a a flavin mononucleotide-dependent
enzyme. The human and malarial enzymes are localized to the
* To whom correspondence should be addressed. For M.A.P.: phone,
(214) 645-6164; fax, (214) 645-6166; e-mail, margaret.phillips@
UTSouthwestern.edu. For P.K.R.: phone, (206) 221-6069; fax, (206) 685-
8665; e-mail, rathod@chem.washington.edu.
§
University of Texas Southwestern Medical Center at Dallas.
University of Washington.
We previously utilized a high-throughput screen (HTS) to
identify a series of halogenated phenyl benzamide/naphthamides
that are potent and species-selective inhibitors of PfDHODH.26
Tricyclic aromatic amines have also been reported to be parasite
#
a Abbreviations: DHODH, dihydroorotate dehydrogenase; PfDHODH,
Plasmodium falciparum dihydroorotate dehydrogenase; CoQ, ubiquinone;
FMN, flavin mononucleotide; HTS, high-throughput screen.
10.1021/jm8001026 CCC: $40.75
2008 American Chemical Society
Published on Web 06/04/2008

3650 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Brief Articles
Figure 3. Selective and potent inhibition of PfDHODH and P.
falciparum cells by 7: (A) inhibition profile for PfDHODH (orange
circles, IC50 ) 0.047 ( 0.022 µM, n ) 6) compared to human DHODH
(black diamonds, IC50 > 200 µM); [E]T ) 10 nM; (B) activity in whole
cell assays against P. falciparum 3D7 (orange circles) or mouse L1210
(black diamonds) cells (EC50 ) 0.079 ( 0.045, n ) 9); (C) relationship
between IC50 and substrate concentration ([E]T ) 5 nM). KI was
determined by fitting the data to eq 2 (KI ) 0.015 ( 0.0011 µM). (D)
Rapid kinetic analysis showing 7 inhibits the CoQD-dependent oxidative
half-reaction (bottom) but not the DHO-dependent reductive half-
reaction (top): blue trace (1), no 7; orange trace (2), 7 (50 µM).
Figure 2. Compound 7: (A) chemical structure; (B) X-ray crystal
structure; (C) H NMR in DMSO-d6; (D) mass spectra.
1
specific inhibitors of PfDHODH.27 However, the inhibitors
described in these previous studies had poor antimalarial activity
in whole cell assays. Thus, while DHODH has received
significant attention as a promising new target for the develop-
ment of antimalarials, chemical validation of DHODH as a target
in malaria had not been fully established.
Results and Discussion
drug resistant strains of P. falciparum, including the multiple-
drug-resistant Dd2 (EC50 ) 0.14 ( 0.05 µM).
HTS-Based Discovery of an Antimalarial. Here, we de-
scribe the identification of a PfDHODH inhibitor, (5-methyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-2-ylamine 7 (DSM
1)28 (Figure 2A), that shows potent and species-selective
antiproliferative effects against the P. falciparum malaria
parasite. Compound 7 was discovered by HTS of a 220 000
compound library of “druglike” molecules using a colorometric
enzyme assay.26 P. falciparum DHODH was inhibited by 7 with
IC50 ) 0.047 ( 0.022 µM, and it is >5000-fold selective when
compared to the human enzyme (Figure 3A, Table 1). It inhibits
the proliferation of P. falciparum parasites in whole cell assays
with similar potency (EC50 ) 0.079 ( 0.048 µM for clone 3D7,
Figure 3B) and it does not inhibit the growth of a mouse cell
line (L1210) (EC50 > 10 µM). It is also highly active against
The initial structural annotation of 7 in the HTS chemical
library database was incorrect as judged by high-resolution mass
spectroscopy. The chemical identity of 7 was elucidated using
NMR analysis of the original material, and these data were
consistent with the structure depicted in Figure 2A. Compound
7 was resynthesized using a simple three-step synthetic method
(Scheme 1). The absolute structure of resynthesized 7 was
determined by X-ray crystallography (Figure 2B) and further
confirmed by mass spectrometry and NMR (Figure 2C,D).
Structure–Activity Relationships (SARs). A series of tri-
azolopyrimidine analogues of 7 were synthesized and tested
against the enzyme and against parasites in whole cell assays
(Table 1). These compounds show a wide range of IC50 values

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3651
Scheme 2
Table 1. Structure and Activity of the Triazolopyrimidine-Based Series
against PfDHODH and P. falciparum in Whole Cell Assaysa
an emerging and preliminary SAR for inhibitory activity on
PfDHODH and parasites. From these data we now know that
(i) R and R1 alkyl substituients can be modified with only a
modest decrease in activity (8-10), (ii) substitutions at R2
resulted in a loss of potency (11, 12), (iii) the introduction of
heteroatoms on, or in, the napthyl ring reduced activity (13-17),
(iv) the naphthalene attached at the 2-position is optimal,
whereas naphthalene attached at the 1-position showed reduced
activity (18); (v) replacement of naphthalene with the smaller
aniline group significantly reduced activity (19), while the larger
anthracene moiety was well tolerated (20).
Binding Mode and Species Selectivity. The biochemical
mechanism of inhibition by 7 was studied in detail to gain
insight into the structural basis for the selective and potent
binding of this compound to PfDHODH. Steady-state kinetic
analysis shows that the IC50 for 7 increases linearly with
increasing CoQD concentration (Figure 3C) as expected for a
competitive tight binding inhibitor.29 These data were fitted to
eq 2 yielding a KI in the low nanomolar range (KI ) 0.015 (
0.001 µM). Pre-steady-state stopped flow spectroscopy was
performed to characterize the effect of 7 on the individual
oxidative and reductive half-reactions catalyzed by PfDHODH.
Compound 7 inhibited the oxidative half-reaction (kox), prevent-
ing the transfer of electrons from FMNH2 to CoQ, while it did
not affect the DHO dependent reductive half-reaction (kred
)
(Scheme 2, Figure 3D). We previously observed a similar pattern
of inhibition for 1 and the biphenylamide inhibitors from our
HTS screen.30 Thus, these data suggest that all three inhibitor
classes utilize the same mechanism to inhibit DHODH. Finally,
site-directed mutagenesis of residues in the inhibitor binding-
site (F227A and R265A) of PfDHODH increased the IC50 of 7
by 940-fold and 130-fold, respectively (IC50 ) 44 ( 10 µM
for F227A; IC50 ) 6.1 ( 1.1 µM for R265A), providing strong
evidence that 7 is also bound in this site. As the inhibitor binding
site is not conserved in amino acid composition between P.
falciparum and human DHODH (Figure 1), this binding mode
explains the profound species-selective binding of 7 and its
derivatives.
a Errors represent the standard error of the mean. The IC50 for inhibition
of human DHODH was >200 µM for all listed compounds. Enzyme data
were collected with the DCIP assay. Growth inhibition by 7 was also tested
on additional P. falciparum cell lines. EC50 values for FCR3, K1, Dd2,
HB3, and D6 were 0.18 ( 0.018, 0.14 ( 0.008, 0.14 ( 0.05, 0.10 ( 0.044,
and 0.058 ( 0.001, respectively.
Scheme 1. Synthesis of the Triazolopyrimidine-Based Seriesa
Significance. Malaria is one of the most pressing medical
problems in the developing world. Target-based drug discovery
has been put forth as a promising mechanism for the discovery
of new drugs; however, it is often difficult to translate potency
on the enzyme target to activity in whole cell assays. Our
discovery of DHODH inhibitors by HTS that have potent
antimalarial activity provides a successful example of this
approach. The triazolopyrimidine-based series exhibits good
association between PfDHODH inhibitory activity and antima-
larial potency in the infected erythrocyte model, while showing
no appreciable activity against the human enzyme or a mouse
cell line. These data provide the first example of a DHODH
inhibitor with low nanomolar activity in malaria whole cell
assays, and the SAR analysis provides the best chemical
evidence to date validating PfDHODH as a target for the
discovery of new antimalarial compounds. Compound 7 is
druglike (as defined by Lipinski’s rule of five),31 simple, and
inexpensive to synthesize. Thus, our study has identified a highly
a Reagents and conditions: (i) AcOH, 3.5–8 h, reflux, 40–58%; (ii) POCl3,
30–60 min, reflux, 43–65%; (iii) R2R3NH, EtOH, 8–15 h, room temp,
80–87%.
against PfDHODH (0.05 to >200 µM), and importantly the
inhibitory activity against the enzyme shows strong association
with potency on the parasites in whole cell assays. These results
are consistent with DHODH being the cellular target of 7 and
its derivatives. The compound series also allowed us to establish

3652 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12
Brief Articles
inhibitory activity. General analytical and chemical methods, and
representative HPLC peaks for the most active compounds,
compounds 7 and 20, are provided as Supporting Information.
Physical Properties. 5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(4a). 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)+.
promising candidate for a lead optimization program to develop
an antimalarial drug that exploits this new target.
Experimental Section
HTS Screen. Compound 7 was identified by HTS based on a
colorimetric end point assay of PfDHODH activity. The details of
the HTS assay, the compound library, and the screen method have
been previously published.26
5-Trifluoromethyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4b). Mp
1
263 °C. H NMR (300 MHz, DMSO-d6): δ 8.40 (s, 1H), 8.04 (s,
1H, OH), 6.14 (s, 1H). MS m/z 202.9 (M – H)–.
Protein Purification and Steady-State Kinetic Analysis. P.
falciparum and human DHODH were expressed and purified and
steady-state kinetic assays were performed as previously described.26,32,33
Reaction details are provided in Supporting Information. Data were
fitted to eq 1 to determine IC50 values and to eq 2 to determine KI for
a tight-binding competitive inhibitor29 using Prism (GraphPad).
5-Ethyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4c). Mp 215 °C.
1H NMR (300 MHz, DMSO-d6): δ 8.18 (s, 1H), 5.82 (s, 1H), 2.60
(m, 2H), 1.21 (m, 3H). MS m/z 162.9 (M – H)–.
5,6-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4d). Mp 308
°C. 1H NMR (300 MHz, DMSO-d6): δ 8.15 (s, 1H), 2.29 (s, 3H),
1.92 (s, 3H). MS m/z 163.0 (M – H)–.
7-Chloro-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (5a). 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)+.
Vo
Vi )
(1)
[I]
IC50
1 +
7-Chloro-5-ethyl[1,2,4]triazolo[1,5-a]pyrimidine (5c). Mp 184 °C.
1H NMR (300 MHz, DMSO-d6): δ 8.52 (s, 1H), 7.13 (s, 1H), 3.04
(m, 2H), 1.40 (m, 3H). MS m/z 183.1 (M + H+).
[S]
IC50 ) Ki 1 +
+ 0.5[E]T
(2)
(
)
(7-Chloro-5,6-dimethyl[1,2,4]triazolo[1,5-a]pyrimidine (5d). Mp
Km
1
147 °C. H NMR (300 MHz, DMSO-d6): δ 8.59 (s, 1H), 2.63 (s,
3H), 2.40 (s, 3H). MS m/z 183.1 (M + H+).
Pre-Steady-State Kinetic Analysis by Stopped Flow Spectro-
scopy. The transition of FMN between the oxidized and reduced
state was monitored at 485 nm on a Bio-Logic SFM-3 stopped-
flow instrument as described.30
(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-2-
ylamine DSM1 (7). Mp 220 °C (lit.36 216–217 °C). 1H NMR (300
MHz, DMSO-d6): δ 10.35 (brs, NH, exchangeable), 8.50 (s, 1H),
7.85–8.05 (m, 4H), 7.45–7.60 (m, 3H), 6.50 (s, 1H), 2.40 (s, 3H).
MS m/z 276.1 (M + H+).
P. falciparum Cell Culture. Parasite clones were propagated34
and [3H]-hypoxanthine uptake was measured in drug-treated P.
falciparum infected erythrocytes and L1210 cells as described.15
Data were fitted to eq 3 to determine EC50.
(5-Trifluoromethyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphtha-
len-2-ylamine (8). Mp 239 °C. 1H NMR (300 MHz, DMSO-d6): δ
8.80 (s, 1H), 8.0–8.20 (m, 4H), 7.55–7.70 (m, 3H), 6.70 (s, 1H).
MS m/z 330.1 (M + H+).
(5-Ethyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-2-
ylamine (9). Mp 238 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.40
(brs, NH, exchangeable), 8.54 (s, 1H), 7.94–8.03 (m, 4H), 7.50–7.64
(m, 3H), 6.50 (s, 1H), 2.70 (m, 2H), 1.20 (m, 3H). MS m/z 290.2
(M + H+).
100%
% cell proliferation )
(3)
logEC -log[I] HillSlope
(
)
50
1 + 10
Curve Fitting and Error Analysis. Enzyme IC50 and parasite
EC50 data were determined over a range of inhibitor concentrations
using triplicate data points at each concentration. IC50 values were
determined using the graphing program Prism (GraphPad). Data
reported in Table 1 represent the average of at least two independent
experiments.
General Chemistry. 7-Amino substituted [1,2,4]triazolo[1,5-
a]pyrimidine compounds (Table 1) were prepared by Scheme 1.
Briefly, 3-amino-[1,2,4]triazole 3 was condensed with the substituted
ethyl acetoacetates 2a–d to form the substituted 7-hydroxy[1,2,4]tri-
azolo[1,5-a]pyrimidines 4a–d. Chlorination with phosphorous oxy-
chloride gave the corresponding 7-chloro-[1,2,4]triazolo[1,5-a]py-
rimidines 5a–d,35 which upon treatment with substituted aryl amines
in ethanol resulted in products (7–20).
(5,6-Dimethyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-2-
ylamine (10). Mp 262 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.0
(brs, NH, exchangeable), 8.82 (s, 1H), 7.75–7.95 (m, 3H), 7.35–7.60
(m, 4H), 2.65 (s, 3H), 2.05 (s, 3H). MS m/z 290.1 (M + H+).
Methyl-(5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphtha-
1
len-2-ylamine (11). Mp 158 °C. H NMR (300 MHz, DMSO-d6):
δ 8.23 (s, 1H), 7.82–7.93 (m, 3H), 7.69 (s, 1H), 7.52 (m, 2H),
7.40 (d, J ) 8.7 Hz, 1H), 6.64 (s, 1H), 3.73 (s, 3H), 2.51 (s, 3H).
MS m/z 290.2 (M + H+).
Benzyl-(5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-
2-ylamine (12). Mp 172 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.35
(s, 1H), 7.75–7.90 (m, 4H), 7.40–7.50 (m, 5H), 7.15–7.30 (m, 3H),
6.53 (s, 1H), 5.63 (s, 2H), 2.45 (s, 3H). MS m/z 366.3 (M + H+).
4-(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamino)benz-
Compounds 4a–d. A mixture of 3-amino-1,2,4-triazole 3 (20
mmol) and substituted ethyl acetoacetate 2a–d (20 mmol) was
heated under reflux in acetic acid (10 mL) for 3.5–8 h. The product
was then cooled to RT, filtered, washed with water, and dried under
vacuum to give a white solid with 40–58% yield.
1
amide (13). Mp 294 °C. H NMR (300 MHz, DMSO-d6): δ 8.60
(s, 1H), 7.90–8.04 (m, 2H and NH, exchangeable), 7.55 (m, 2H),
7.40 (brs, NH, exchangeable), 6.60 (s, 1H), 2.51 (s, 3H). MS m/z
269.1 (M + H+).
Compounds 5a–d. [1,2,4]triazolo[1,5-a]pyrimidin-7-ol (4a–d)
(6.5 mmol) was added to 1.82 ml (19.5 mmol) of phosphorous
oxychloride and heated under reflux for 30–60 min in a round
bottom flask, during which the solid dissolved and hydrogen
chloride was evolved. Excess phosphorous oxychloride was re-
moved 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 at a yield
of 43–65%. Compound 5b was used without purification.
Compounds 7–20. The appropriate aryl substituted amine (1
mmol) was added to compound 5a–d (1 mmol) in absolute ethanol
(10 mL) and stirred at RT for 8–15 h. Products were purified by
column chromatography with CH2Cl2/MeOH/NH4OH (23:1:1).
Yields ranged from 80–87%.
(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)quinolin-3-yl-
1
amine (14). Mp 243 °C. H NMR (300 MHz, CDCl3): δ 9.00 (s,
1H), 8.45 (s, 1H), 8.20–8.30 (m, 2H), 8.10 (brs, NH, exchangeable),
7.88 (d, J ) 7.5 Hz, 1H), 7.75 (m, 1H), 7.70 (m, 1H), 6.44 (s, 1H),
2.62 (s, 3H). MS m/z 277.1 (M + H+).
(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)quinolin-6-
ylamine (15). Mp 280 °C. 1H NMR (300 MHz, CDCl3): δ 9.00 (s,
1H), 8.45 (s, 1H), 8.20–8.25 (m, 2H), 8.12 (brs, NH, exchangeable),
7.80 (s, 1H), 7.75 (d, J ) 7.6 Hz, 1H), 7.54–7.60 (m, 1H), 6.54 (s,
1H), 2.55 (s, 3H). MS m/z 277.1 (M + H+).
3-(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ylamino)naphtha-
len-2-ol (16). Mp 301 °C. 1H NMR (300 MHz, DMSO-d6): δ 8.51
(s, 1H), 7.95 (s, 1H), 7.87 (m, 1H), 7.78 (m, 1H), 7.41–7.45 (m,
1H), 7.31–7.35 (m, 2H), 6.29 (s, 1H), 2.42 (s, 3H). MS m/z 292.1
(M + H+).
Analysis. As a test of purity, compounds were subjected to HPLC
analysis. Compounds eluted as a single peak and the activity of
the peak fraction was confirmed by demonstrating PfDHODH

Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 12 3653
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38, 476–480.
2-Methyl-7-(5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ylami-
no)chromen-4-one (17). Mp 306 °C. 1H NMR (300 MHz, CDCl3):
δ 8.41 (s, 1H), 8.30 (d, J ) 8.52 Hz, 1H), 8.15 (brs, NH,
exchangeable), 7.44 (s, 1H), 7.40 (d, J ) 8.0 Hz, 1H), 6.69 (s,
1H), 6.22 (s, 1H), 2.66 (s, 3H), 2.44 (s, 3H). MS m/z 308.1 (M +
H+).
(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalen-1-
ylamine (18). Mp 192 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.43
(brs, NH, exchangeable), 8.55 (s, 1H), 8.05 (m, 2H), 7.88 (m, 1H),
7.50–7.65 (m, 4H), 5.69 (s, 1H), 2.26 (s, 3H). MS m/z 276.1 (M +
H+).
(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)phenylamine (19).
Mp 187 °C (lit.37 188 °C). 1H NMR (300 MHz, CDCl3): δ 8.32 (s,
1H), 8.01 (brs, NH, exchangeable), 7.48–7.53 (m, 2H), 7.28–7.40
(m, 3H), 6.38 (s, 1H), 2.55 (s, 3H). MS m/z 226.1 (M + H+).
(5-Methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)anthracen-2-
ylamine (20). Mp 231 °C. 1H NMR (300 MHz, DMSO-d6): δ 10.45
(brs, NH, exchangeable), 8.61–8.55 (m, 3H), 8.08–8.20 (m, 4H),
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Acknowledgment. The authors gratefully acknowledge
Amgen for analytical chemistry support during the structural
identification of DSM1 (7). This work was supported by the
U.S. National Institutes of Health Grants AI053680 (to M.A.P.
and P.K.R.), MG00203 (to N.A.M.), and AI26912 and AI67670
(to P.K.R.). M.A.P. also acknowledges support from the Welch
Foundation (Grant I-1257), and P.K.R. acknowledges support
from a Senior Scholar Award in Global Infectious Diseases from
the Ellison Medical Foundation and from the UW Keck Center.
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Supporting Information Available: Detailed assay and analyti-
cal methods and HPLC traces for 7 and 20. This material is available
free of charge via the Internet at http://pubs.acs.org.
(26) Baldwin, J.; Michnoff, C. H.; Malmquist, N. A.; White, J.; Roth, M. G.;
Rathod, P. K.; Phillips, M. A. High-throughput screening for potent
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