Bioisosteric transformations and permutations in the triazolopyrimidine scaffold to identify the minimum pharmacophore required for inhibitory activity against plasmodium falciparum dihydroorotate dehydrogenase

Article abstract of DOI:10.1021/jm300351w

Plasmodium falciparum causes approximately 1 million deaths annually. However, increasing resistance imposes a continuous threat to existing drug therapies. We previously reported a number of potent and selective triazolopyrimidine-based inhibitors of P.

Full text of DOI:10.1021/jm300351w

Article
pubs.acs.org/jmc
Bioisosteric Transformations and Permutations in the
Triazolopyrimidine Scaffold To Identify the Minimum
Pharmacophore Required for Inhibitory Activity against Plasmodium
falciparum Dihydroorotate Dehydrogenase
Alka Marwaha,† John White,† Farah El_Mazouni,§ Sharon A Creason,‡ Sreekanth Kokkonda,†
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 Road, Dallas, Texas
75390-9041, United States
∥Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus),
Parkville, VIC 3052, Australia
ABSTRACT: Plasmodium falciparum causes approximately 1 million deaths annually.
However, increasing resistance imposes a continuous threat to existing drug therapies. We
previously reported a number of potent and selective triazolopyrimidine-based inhibitors of
P. falciparum dihydroorotate dehydrogenase that inhibit parasite in vitro growth with
similar activity. Lead optimization of this series led to the recent identification of a
preclinical candidate, showing good activity against P. falciparum in mice. As part of a
backup program around this scaffold, we explored heteroatom rearrangement and
substitution in the triazolopyrimidine ring and have identified several other ring
configurations that are active as Pf DHODH inhibitors. The imidazo[1,2-a]pyrimidines
were shown to bind somewhat more potently than the triazolopyrimidines depending on
the nature of the amino aniline substitution. DSM151, the best candidate in this series,
binds with 4-fold better affinity (Pf DHODH IC50 = 0.077 μM) than the equivalent
triazolopyrimidine and suppresses parasites in vivo in the Plasmodium berghei model.
reversed for pyrimidine biosynthesis where the parasite lacks
salvage enzymes for preformed pyrimidine bases and nucleo-
sides and thus relies on the de novo biosynthesis. Plasmodium
falciparum dihydroorotate dehydrogenase (PfDHODH), an
essential mitochondrial flavin mononucleotide (FMN)-depend-
ent enzyme, catalyzes the fourth and rate-limiting step in de
novo pyrimidine biosynthesis.16,17 Recent investigations have
revealed that the primary task of the mitochondrial electron
transport chain in the parasite is to supply oxidized ubiquinone
(CoQ) to DHODH for pyrimidine synthesis, confirming the
essential role of DHODH in parasite growth.18 The efficacy of
human DHODH inhibitors for the treatment of rheumatoid
arthritis has been well-documented in the literature, demon-
strating that DHODH is a druggable target.19−21 Finally, X-ray
structures of human dihydroorotate dehydrogenase
(hDHODH) and Plasmodium DHODH have highlighted an
extensive variation in amino acid sequence between the human
and the malarial enzymes,22−25 thereby providing the structural
basis for the identification of species-specific inhibitors.
INTRODUCTION
■
Malaria is a persistent and successful global disease that
threatens half of the world's population and kills up to a million
people each year.1,2 The developing world has urgently needed
new, safe, effective, and affordable antimalarial drugs since the
demise of chloroquine due to the emergence of resistant P.
falciparum strains.3,4 Strides have been made to develop an
array of antimalarial agents with artemisinin-based combination
therapies (ACTs) providing a major breakthrough, succeeding
in more than 90% of the malaria cases.5−8 However, artemisinin
effectiveness appears to be decreasing along the Thai−
Combodia border, threatening these recent gains.9,10 The
limitations of current antimalarial chemotherapy underscore the
need for novel drugs, ideally directed against innovative
therapeutic targets. The completion of the malaria genome
project11 has opened up channels to search for new targets in
the parasite. Despite the identification of many essential genes
and extensive efforts to understand the biology of the parasite,
very few unique validated targets have been identified. Hence,
the contemporary challenge is to blend our knowledge of
malaria genomics and drug discovery.
A high-throughput screen (HTS) of “drug-like” small
molecules, using a colorimetric enzyme assay, led to the
Nucleic acid biosynthesis in P. falciparum differs from that in
mammals. The parasite relies on purine salvage due to the
absence of de novo purine synthesis.12−15 The situation is
Received: March 13, 2012
© XXXX American Chemical Society
A
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identification of a highly potent and selective triazolopyrimi-
dine-based PfDHODH inhibitor 1 (DSM 1, Figure 1), (5-
rearrangements/replacements in the triazolopyrimidine ring
(6−11, Figure 3). We initially coupled each new ring system to
Figure 1. Triazolopyrimidines with active antimalarial activity. The
initial HTS hit (1), an analogue with improved plasma exposure (2),
and analogue with optimized plasma exposure and potency (3).
methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)naphthalene-2-yl-
amine.26−28 Compound 1 has potent activity against both
PfDHODH (IC50 = 0.047 μM) and P. falciparum parasites in
whole cell assays (EC50 = 0.079 μM for clone 3D7), without
inhibiting hDHODH. However, when administered orally to
mice infected with P. berghei, 1 had no antiparasite activity. The
lack of efficacy of 1 was traced to rapid and possible induced
metabolism and to species differences in inhibitor potency
between Pf DHODH and Plasmodium berghei dihydroorotate
dehydrogenase (PbDHODH).29 An integrated lead optimiza-
tion program combining medicinal chemistry, enzymology, and
pharmacologic evaluation was utilized to identify a modified
analogue 2 (DSM 74, Figure 1), 5-methyl-N-(4-
(trifluoromethyl)phenyl)-[1,2,4]triazolo[1,5-a]pyrimidin-7-
amine, with greater metabolic stability. This compound showed
good exposure in vivo after oral dosing and was able to
suppress parasitemia in the P. berghei model.29 However, 2
lacked the potency required of a clinical candidate. Subsequent
X-ray structure-guided lead optimization of the scaffold
identified 3 (DSM265, Figure 1), 2-(1,1-difluoroethyl)-5-
methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl] [1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine, which is a potent inhibitor of
PfDHODH that is able to kill parasites in vitro and showed
efficacy in a rodent model similar to chloroquine.30,31
Compound 3 is also metabolically stable and exhibits a long
half-life after oral administration in rodents. On the basis of
these strengths, 3 has recently been selected as a preclinical
drug development candidate.
Figure 3. (A) Bioisosterically transformed scaffolds 6−11. (B) A
virtual library comprising three types of rings with single isosteric
changes in 1. Arrows indicate the point of isosteric change. (C)
Various congeners with multiple isosteric changes in 1. Arrows
indicate the sites of structural diversity.
naphthyl amine (as in 1), but then, for active scaffolds, we
tested some additional aryl amines (12−16) to determine if the
optimal group in this position remained similar to what we have
observed for the triazolopyrimidines. Three additional heter-
oatom ring configurations were identified that showed
submicromolar Pf DHODH activity (6, 7, and 9), with the
best being the imidazo[1,2-a]pyrimidines (6 and 16, Table 1)
having equivalent to more potent binding affinity as compared
to the matched triazolopyrimidine. The imidazo[1,2-a]-
pyrimidine 16 was chosen for profiling of its in vivo properties,
including pharmacokinetic analysis and efficacy testing in the P.
berghei mouse model. Plasma exposure was lower than for the
matched triazolopyrimidine, leading to reduced efficacy. The in
vitro metabolism data suggested that the imidazo[1,2-a]-
pyrimidine scaffold is more extensively metabolized than the
triazolopyrimidine scaffold in mice, suggesting that additional
chemistry would be required to exploit the activity of this
scaffold. These studies have provided important additional
insight into the developing structure−activity relationships
(SARs) in this promising series of antimalarial compounds.
As part of our back up program to identify additional
compounds with clinical candidate potential, we extended our
explorations of the triazolopyrimidine scaffold by applying
bioisosteric morphing, a hit optimization strategy to replace the
core of a bioactive compound with groups of similar physical or
chemical properties without loss of activity. Using this strategy,
we have synthesized a series of compounds with either
modified bridging atoms between the triazolopyrimidine ring
and the aromatic moiety (4 and 5, Figure 2) or heteroatom
CHEMISTRY
■
Amide Mimics. To test the effects of modifying the linker
between the triazolopyrimidine ring and the naphthyl group,
the bridging nitrogen N1 was replaced with an amide linker
(Figure 2). The target inhibitors 4 and 5 were synthesized via a
three-step sequence as depicted in Scheme 1. The first two
steps were relatively straightforward, based on our reported
protocol.29 Our initial attempts to perform the coupling
Figure 2. Bioisterically transformed amide mimics 4 and 5.
B
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a
Table 1. Activity Profiles of Inhibitors 4−11
C
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Table 1. continued
a
The asterisks indicate that data for DSM1 and DSM74 were taken from refs 27 and 29. For enzyme data, the error represents the standard error of
the fit for n = 3 data points per concentration; for parasite data, the error displayed is the 95% confidence interval of the fit for n = 3 or 4 data points
per concentration.
a
targets, the reaction conditions were judiciously optimized
using transition metal-catalyzed Buchwald−Hartwig condi-
tions.32−35
Scheme 1. Synthetic Strategy for Inhibitors 4, 5, and 7
We tried a few combinations of 2,2′-bis(diphenylphosphino)-
1,1′-binaphthyl (BINAP) as the ligand, Pd(OAc)2 as a source of
palladium, and different reaction variables to determine the
efficient catalytic system and found that the amidation coupling
was optimal using a Pd(OAc)2 (5 mol %)/BINAP (5 mol %)
combination and sodium tertiary butoxide (5 mol equiv) as
base. Other stoichiometric bases like potassium carbonate or
cesium carbonate worked less well in terms of both yields and
reaction progress up to completion. A large excess of base,
although almost insoluble in toluene, was essential to obtain
high cross-coupling rates with dry toluene as the solvent. The
reaction progress was monitored by MS and thin-layer
chromatography (TLC) screening. From a complex reaction
mixture, the desired cross-coupled products 4 and 5 were
isolated in optimal yields (40−50%) after silica gel column
chromatographic purification.
a
Reagent and conditions: (i) RCONH2 (compounds 4 and 5) and
RNH2 (compound 7), Pd(OAc)2, BINAP NaOtBu, anhydrous
toluene, 24 h reflux, yield 40−50%.
reactions of amides with the key intermediate 7-chloro-5-
methyl-[1,2,4]triazolo[1,5-a]pyrimidine (17), using traditional
conditions like stirring at ambient temperature in absolute
ethanol with or without the p/o base, refluxing in toluene,
refluxing in absolute ethanol/dichloromethane/N,N-dimethyl
formamide, with and without the p/o bases like triethyl amine/
anhydrous potassium carbonate, were unsuccessful and rather
afforded either the hydroxy derivative or the ethoxy-substituted
products. To establish a robust synthetic procedure for the
Heteroatom Ring Rearrangements and Replace-
ments. To explore the SAR of the triazolopyrimidine core,
we synthesized a series of derivatives to test if the heteroatoms
(nitrogens) could be replaced with carbon, resulting in the
D
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corresponding heteroaromatic rings with comparable steric and
electronic characteristics (Figure 3A−C). Our prime interest
was the systematic evaluation of the effects of removal of
individual nitrogen atoms from the triazolopyrimidine core to
construct imidazo[1,2-a]pyrimidines (6), triazolo[1,5-a]-
pyridine (7), and pyrazolo[1,5-a]pyrimidine (8) ring systems
(Figure 3B), which were further extended to the related
congeneric purines (9), pyrazolo[3,4-d]pyrimidine (10), and
triazolo[4,3-a]pyridazine (11) (Figure 3C). Herein, we report
our approaches on the synthesis of these various bioisosteres.
Scaffold Hops 6−16. The imidazo[1,2-a]pyrimidine
analogues (6 and 16) were readily synthesized as depicted in
Scheme 2. The commercially available 2-amino-imidazole
(methylethoxymethylene)-aminoimidazole-5-carboxamide
(23a), which was then cyclized intramolecularly at elevated
temperatures to give 2-methylhypoxanthine (24a). Ensuing
chlorination and coupling of 25a with variously substituted
aromatic amines afforded 9 and 12−15.
Pyrazolo[3,4-d]pyrimidine (10) was synthesized from 5-
amino-1H-pyrazole-4-carboxamide hemisulfate (22b) as de-
lineated in Scheme 3. Refluxing 22b with triethyl orthoacetate
in anhydrous DMF afforded 5-(methylethoxymethylene)-
aminopyrazole-4-carboxamide (23b), intramolecular cyclization
of which at elevated temperature yielded 4-hydroxy-6-methyl-
1H-pyrazolo[3,4-d]pyrimidine (24b). Subsequent chlorina-
tion42 and coupling with 2-amino-naphthalene afforded 10.
The triazolopyridazine 11 was synthesized using the reaction
sequence highlighted in Scheme 2. 4-Amino-1,2,4-triazole
(19c) interacted with ethyl acetoacetate in refluxing glacial
acetic acid to produce 8-hydroxy-6-methyl-[1,2,4]triazolo[4,3-
b]pyridazine (20c), which on chlorination using phosphorus
oxychloride afforded 8-chloro-6-methyl-[1,2,4]triazolo[4,3-b]-
pyridazine (21c).43 Various conditions using a variety of
solvents, bases, and temperatures were attempted and found
unsuccessful. However, the reaction conditions were optimized
utilizing transition metal catalysis. As previously described (for
inhibitors 4 and 5, Scheme 1), the catalytic combination of
Pd(OAc)2 (5 mol %)/BINAP (5 mol %) was found to work
well with sodium tertiary butoxide (5 mol equiv) as the base
and anhydrous toluene as the solvent (Scheme 2). The reaction
temperature and the reaction time were 120 °C and 24 h.
a
Scheme 2. Synthesis of the Inhibitors 6, 8, 11, and 16
RESULTS AND DISCUSSION
■
Evaluation of Compound Activity against DHODH
and P. falciparum 3D7 Cells. The functional evaluation of
the target inhibitors 4−16 was carried out on the parasite and
host DHODH and on P. falciparum 3D7 cells in whole cell
assays (Table 1). The compounds showed a wide range of IC50
values against Pf DHODH (0.077 to >100 μM), and these
values showed a good correlation with activity against P.
falciparum 3D7 cells (Table 1 and Figure 4). All compounds
tested were inactive against human DHODH, showing that the
selectivity of the series was maintained by variations in the
triazolopyrimdine ring. Most compounds also showed better
activity against Pf DHODH than PbDHODH, similar to
previous reports for the series.27,29,31,44 We deduce the
following SAR trends by comparing the activity of these
compounds against the previously described triazolopyrimi-
dines 1 and 2.
1. Replacement of the −NH linker with amide −NHCO (4
and 5) resulted in the loss of activity when an aniline or
substituted aniline was adjacent to the amide bond.
2. Replacement of triazolopyrimidine core with imidazo[1,2-
a]pyrimidine was well tolerated and either slightly reduced or
slightly enhanced binding affinity depending on the nature of
the aromatic amine. Compound 6 was 3-fold less potent than 1
(IC50 = 0.15 vs 0.047 μM), but 16 was 4-fold more potent than
2.
a
Reagents and conditions: (i) CH3COCH2CO2C2H5, AcOH, reflux,
3.5−25 h, yield 70−85%. (ii) POCl3, reflux, 40 min−2 h, yield 60−
65%. (iii) RNH2, EtOH, 12−18 h, room temp., yield 65−80%
(compounds 6, 8, and 16); Pd(OAc)2, BINAP, NaOt-Bu, anhydrous
toluene, 24 h reflux, yield 45% (compound 11).
hemisulfate (19a) was condensed with ethyl acetoacetate in
refluxing glacial acetic acid to afford 5-hydroxy-7-
methylimidazo[1,2-a]pyrimidine (20a).36 Chlorination of 20a
using phosphorus oxychloride gave the intermediate 5-chloro-
7-methylimidazo[1,2-a]pyrimidine (21a). Final conjugation of
21a with 2-aminonaphthalene and 4-(trifluoromethyl)aniline
afforded 6 and 16, respectively.
The target inhibitor 7 was obtained by coupling 5-chloro-7-
methyl-[1,2,4]triazolo[1,5-a]pyridine (18, commercially avail-
able) with 2-aminonaphthalene utilizing transition metal-
catalyzed conditions (Scheme 1).
Compound 8 was prepared as outlined in Scheme 2. A
mixture of 1H-pyrazol-5-amine (19b) and ethyl acetoacetate
was refluxed in glacial acetic acid to yield 7-hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine (20b). Subsequent chlorina-
tion37,38 (21b) and coupling with 2-amino naphthalene
(Scheme 2) afforded the final adduct 8.
The congeners 9, 10, 11, and 12−15 were prepared as
depicted in the Schemes 2 and 3. Regarding purine analogues 9
and 12−15, condensation of 4-amino-1H-imidazole-5-carbox-
amide hydrochloride salt (22a) with triethyl orthoacetate in
refluxing anhydrous dimethylformamide39−41 afforded 4-
3. Replacement of the triazolopyrimidine core with triazolo-
[1,5-a]pyridine (7) resulted in a 100-fold reduction in binding
affinity, thereby showing that the pyrimidine nitrogen N5 plays
a pivotal role in the observed activity of the triazolopyrimidines.
4. Substitution of the core with pyrazolo-[1,5-a]pyrimidine
(8) reduced the activity by ∼8.5-fold, demonstrating the
importance of the N4 nitrogen in the five-membered ring to
both the enzyme inhibition and the antiparasitic activity.
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a
Scheme 3. Synthesis of Inhibitors 9, 10, and 12−15
a
Reagents and conditions: CH3C(OCH2CH3)3, anhydrous DMF, reflux, 15 min−1 h, 70−80%. (ii) Heat above 200 °C for 1 h, yield 65−70%. (iii)
POCl3, 30−45 min, reflux. (iv) RNH2, EtOH, 15−18 h, room temp., yield 68−80% (compounds 9−12) and 78% (compound 10).
7. Loss of N2 in analogues 9−11 is also correlated to a
significant reduction in activity, suggesting that N2 may also be
important to the potency of the compound class. However, in
all cases, these compounds also contained an additional
nitrogen in a position not found in the triazolopyrimidines,
so conformation of the role of N2 through additional synthesis
is still required.
Structural Basis of Activity Differences between the
Triazolopyrimidines and Variations in the Core. The X-
ray structure of key triazolopyrimidine Pf DHODH inhibitors,
including 1 and 2, was previously reported by our group.23
These structures showed that the triazolopyrimidine ring forms
a H-bond interaction between the pyrimidine nitrogen N5 and
the R265 and between the bridging nitrogen N1 and the H185
Figure 4. Comparison of the series activity on Pf DHODH vs P.
falciparum whole cell assays. The log of the PfDHODH IC50 data are
plotted vs the log of the P. falciparum EC50 data (in nanomolar). The
(Figure 5). These are the only polar interactions formed
between the inhibitor and the enzyme, and site-directed
mutagenesis confirmed that both interactions were essential
for high-affinity binding of these compounds. Analysis of the
structures described in this manuscript further confirms the
importance of these interactions. Compound 7, in which the
pyridine nitrogen N5 is replaced with carbon, is 100-fold less
potent than the corresponding triazolopyrimidine (1),
demonstrating that the interaction between the pyrimidine
nitrogen N5 and the R265 is essential for high-affinity binding.
In prior studies, we noted that introduction of heteratoms into
the naphthyl ring reduced activity for the triazolopyrimidine
series.26,27 This result holds true for the purine analogues (14
and 15 as compared to 9) and is explained by the X-ray
structure that shows that the naphthyl pocket is completely
hydrophobic with no potential to contribute to H-bond
interactions. Finally, the structure also provides insight into
the good activity of the imidazo[1,2-a]pyrimidines 6 and 16.
Nitrogen N3 is within 3.3 Å of NE1 of H185; yet, this
interaction cannot be productive because NE1 of H185 is an H-
bond acceptor and not an H-bond donor. Replacement of N3
with carbon may thus relieve a potentially negative interaction
between these groups. It also may lead to the displacement of
plotted data include compounds 1, 2, and 4−16 described in Table 1.
Compounds for which solubility limits prevented the quantitative
determination of either the IC50 or the EC50 have been left off the plot.
Data were fitted by linear regression analysis where r2 = 0.84 and slope
= 0.93.
5. The purine analogue 9 involving bioisosteric replacements
at two sites was ∼9-fold less active than the comparable
triazolopyrimidine compound. Additional derivatives of this
scaffold were also evaluated by replacing the naphthyl group
with anthracene, quinoline, isoquinoline, and substituted five-
membered pyrazole rings (12−15). Exchange of naphthyl with
larger moiety like anthracene (12) considerably lessened the
activity (∼87-fold), which is in contrast to what was observed
for this replacement in the triazolopyrimdine core structure.29
Furthermore, incorporation of heteroatoms in the naphthyl
system (13 and 14) reduced the activity as was previously
observed for the triazolopyrimidine scaffold.29 The five-
membered 4-cyanopyrazole (15) was inactive.
6. Bioisosteric modification involving four changes in the
purine-related analogues, that is, pyrazolo[3,4-d]pyrimidine 10
and triazolopyridazine 11, inactivated the compound.
F
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Figure 5. Binding site of 2 bound to PfDHODH. A limited set of
residues within the 4 Å shell of the bound inhibitor are displayed.
Compound 2 and orotate are displayed in pink, and FMN is displayed
in yellow. Hydrogen-bonding distances are displayed in Å. The
structure (3I6R.pdb) is displayed in PyMol.
Figure 6. Pharmacokinetics and in vivo efficacy of 16 in mice. (A)
Pharmacokinetic profile of 16 after administration of a single oral or ip
dose of 10 mg/kg. The drug concentration in micromolar is plotted vs
time postdose. (B) Mice were infected with P. berghei on day zero, and
16 was dosed orally at 50 mg/kg 1× daily for 4 days beginning 24 h
after infection. As a control, chloroquine was dosed at 10 mg/kg orally
using the same dosing schedule.
the structural water observed to form H-bond interactions with
N3, H185, and Y525. The entropic gain in releasing the water
could also contribute to greater binding affinity of 16 relative to
2. It is unclear why the effect of removing N3 on improving
potency was only evident when the imidazo[1,2-a]pyrimidine
core was coupled with the smaller para-CF3 aniline and not
when it was coupled to naphthyl amine (1 vs 6). These data
suggest that the relative contribution of N3 to the electronics of
the ring differs depending on the coupled aromatic amine and
that considerations beyond H-bond potential and displacement
of the bound water play roles in the potency of these
compounds.
In Vitro Metabolic Stability and PK in Mice. The most
active compound in the present series 16 was advanced to
metabolism and in vivo studies. In vitro metabolic analysis in
human liver microsomes for 16 was compared to previous
results collected for 2. Both 2 and 16 display low in vitro
intrinsic clearance values (Eh) in human liver microsomes, but
16 displayed intermediate clearance in mouse microsomes,
suggesting that plasma exposure in mice might limit the ability
of the compound to show good in vivo efficacy (Table 2). The
reason that 16 displays higher in vitro intrinsic clearance than 2
is unknown. To evaluate the pharmacokinetic profile of 16,
mice were dosed orally with 16 at a single dose of 10 mg/kg
(Figure 6A). Compound 16 reached peak plasma concen-
trations of 3.9 μM after oral dosing, but plasma levels were
maintained above 1 μM for only about 4 h, and by 16 h,
concentrations were below the limit of quantitation (0.003
μM). No adverse reactions were observed following oral
administration of 16 at the dose used in this study.
In Vivo Efficacy of 16 in P. berghei Mouse Model.
Compound 16 was evaluated for efficacy in the standard P.
berghei mouse model. Mice were infected with P. berghei on day
zero, and dosing began 24 h after infection. Mice were dosed
orally with 50 mg/kg 16 and 1× daily for 4 days, and parasite
blood levels were evaluated and compared to a chloroquine
control (Figure 6B). Compound 16 suppressed parasite levels
by 56% as measured 24 h after the final dose. In comparison, 2
was able to suppress parasite levels by 71% using the same
dosing scheme and end point.29 Thus, despite showing 2−4-
fold better potency against PbDHODH and Pf DHODH than
2, respectively, 16 is less efficacious than 2 in the mouse model.
The relatively poorer plasma exposure observed in mice for 16
most likely contributes to this finding. The finding that the
predicted in vitro clearance in mouse microsomes is
significantly higher than human microsomes suggests that this
compound would perform better against human infection than
for mice.
Table 2. In Vitro Metabolism in Human and Mouse
Microsomes
CONCLUSION
■
degradation
in vitro CLInt
predicted
putative
metabolites
In the quest for better Pf DHODH antagonists, we have
undertaken a study to systematically explore the importance of
the number and configuration of nitrogen atoms in the
triazolopyrimidine ring to the potency of the compound class.
The studies show that N1, N4, and N5 are functionally
important sites as the structural modification at these positions
render the molecule less or completely inactive (Figure 7). N3
can be replaced by carbon, leading to retained or improved
potency; however, the N3 nitrogen is a significant factor in the
metabolic stability and favorable pharmacokinetics of 2. In
particular, 16 was found to be 4-fold more potent than the
compd half-life (min) (mL/min/mg protein)
EH
human
a
2
230
368
0.0075
0.0047
0.29
0.21
P + 16
16
none
detected
mouse
0.034
16
51.8
0.59
none
detected
a
Data were previously reported in Gujjar et al.29 Data represent single
point measurements.
G
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acetonitrile. The relative loss of parent compound and formation of
metabolic products were monitored by LC-MS using a Waters/
Micromass QTOF mass spectrometer.
Test compound concentrations were determined by comparison to
a calibration curve prepared in microsomal matrix, and concentration
versus time data were fitted to an exponential decay function to
determine the first-order rate constant for substrate depletion. This
rate constant was then used to calculate the degradation half-life, in
vitro intrinsic clearance (CLint,in vitro), predicted in vivo intrinsic
clearance value (CLint), and predicted in vivo hepatic extraction ratio
(EH) as previously described,48 assuming predominantly hepatic
cytochrome P450-mediated clearance in vivo. The scaling parameters
used in these calculations included a human and mouse hepatic blood
flow of 20.7 and 90 mL/min/kg, respectively, liver mass of 25.7 and
87.5 g liver/kg body weight for human and mouse, respectively, and
microsomal content of 45 mg microsomal protein/g liver.49,50
Mouse Pharmacokinetics. All animal studies were performed in
accordance with the Australian and New Zealand Council for the Care
of Animals in Research and Training Guidelines, and the study
protocol was approved by the Monash University (Victorian College
of Pharmacy) Animal Experimentation Ethics Committee. The
pharmacokinetics of 16 were studied in nonfasted male Swiss outbred
mice weighing 23−33 g. Mice had access to food and water ad libitum
throughout the pre- and postdose sampling period. Compound was
administered orally by gavage (0.1 mL dose volume per mouse) in a
suspension of aqueous vehicle (0.5% w/v sodium caboxymethylcellu-
lose, 0.5% v/v benzyl alcohol, and 0.4% v/v Tween 80). Samples were
collected at 1, 2, 4, 7.5, 16, and 24 h postdosing with a single blood
sample from each mouse via cardiac puncture. Blood samples were
transferred to heparinised tubes and centrifuged immediately, and
supernatant plasma was removed and stored at −20 °C until analysis
(within 1−2 weeks). Plasma samples were assayed by LC-MS
following protein precipitation with acetonitrile, and the concentration
of drug in plasma was determined by comparison of the peak area to a
calibration curve prepared in plasma.
P. berghei Mouse in Vivo Efficacy Testing. All animal care and
experimental procedures were approved by the Institutional Animal
Care and Use Committee at the University of Washington. Eight to
ten week old female BALB/c mice were obtained from Jackson
Laboratories (Bar Harbor, ME) and maintained in a temperature/
humidity-controlled SPF facility with a 12 h light/dark cycle. P. berghei
isolate NK65 was obtained from MR4 (Malaria research and Reference
Resource Center, Monassas, VA). Erythrocytic stages of P. berghei
were harvested from infected donor mice via cardiac puncture and
diluted in RPMI 1640 media (ATCC, Monassas, VA) supplemented
with 20% heat-inactivated FBS (Altanta Biologicals, Atlanta, GA).
Recipient mice were infected with 2 × 107 infected erythrocytes, 200
μL, ip injection, 24 h prior to receiving the first drug dose. Tail vein
bleeds and weighing were performed daily for the duration of the
experiment. All infected mice were screened prior to receiving drug or
vehicle to verify an established minimum infection of 0.1% parasitemia.
Inhibition of parasite growth is determined microscopically by staining
a thin smear of blood obtained from the test animal using a Wright−
Giemsa stain (Fisher Scientific, Houston, TX). Compound 16 was
dosed in mice orally with vehicle or with 50 mg/kg/dose 1× daily for 4
consecutive days. The parasite blood levels were evaluated and
compared to the chloroquine control.
Figure 7. Structure−activity map. Structural drawing of the lead core
annotated to show where structural changes affect activity or potency.
equivalent triazolopyrimidine 2; however, the poorer plasma
exposure led to reduced efficacy against the parasite in vivo.
The addition of an amide linker between the triazolopyrimidine
core and the aniline also led to complete loss of activity.
Overall, the data have helped to define the minimal
pharmacophore of the triazolopyrimidine class of Pf DHODH
inhibitors and have highlighted the key importance of the
pyrimidine nitrogen N5.
EXPERIMENTAL SECTION
■
Protein Purification and Steady-State Kinetic Analysis.
PfDHODH, PbDHODH, and hDHODH enzymes were expressed
and purified as previously reported.27,28,45 Steady-state kinetic assays
were performed as previously described.27,28,45 Briefly, this colori-
metric assay monitors the reduction of 2,6-dichloroindophenol
(DCIP; 0.12 mM) at 600 nm (ε = 18.8 mM−1 cm−1), which is
coupled to the reoxidation of CoQD (coenzyme QD). The assay was
carried out using a solution containing 100 mM HEPES, pH 8.0, 150
mM NaCl, 10% glycerol, 0.1% Triton X-100, 20 μM CoQD, 200 μM L-
dihydroorotate, and 120 μM DCIP. Reactions were initiated by
addition of enzyme to a final concentration in the range of 5−10 nM,
while maintaining the temperature of the circulating water bath at 20
°C. The data obtained were fitted to eq 1 using GraphPad Prism, to
determine the IC50 values of the representative compounds. IC50 data
were determined over a range of inhibitor concentrations using
triplicate data points at each concentration, and errors represent the
standard error of the fit.
v
o
vi =
[I]
1 +
IC50
(1)
P. falciparum Cell Culture. The malarial parasite clone 3D7 was
passaged in Gibco-Invitrogen RPMI-1640 supplemented with 20%
human type A+ plasma and 2% (w/v) red blood cells (obtained from
Biochemed Services, Virginia).46 To study the inhibition of cell
proliferation, the standard [3H]-hypoxanthine uptake assay was used
to measure drug-treated P. falciparum-infected erythrocytes as
described previously.47 Data were fitted to eq 2 to determine EC50
(n = 3−4 data points at each concentration). Because the standard
error of the fit cannot be calculated for eq 2, the parasite data are
reported with the 95% confidence interval from the fit.
Molecular Modeling. Structures were displayed using the graphics
program PyMol (DeLano, W. L. The PyMOL Molecular Graphics
System (2002) on World Wide Web http://www.pymol.org).
Chemistry. General Methods. If not otherwise specified, reagents
and solvents were obtained from commercial suppliers and were used
without further purification. The reaction progress was monitored by
TLC using silica gel 60 F-254 (0.25 mm) plates. Visualization was
achieved with UV light and iodine vapor. Flash chromatography was
carried out with silica gel (32−63 μM). The procedures for all of the
synthesized compounds have been provided along with relevant
literature and their discussion as illustrated in the Schemes 1−3. 7-
Chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (12) was synthe-
sized as per the previously reported procedure.29 All aromatic amines
100%
% cell proliferation =
50−log[I])Hill slope
1 + 10(log EC
(2)
In Vitro Human Microsomal Metabolism. Metabolic stability
was evaluated by incubating test compounds (1 μM) individually at 37
°C with pooled human or mouse liver microsomes (BD Gentest,
Woburn, MA) at a microsomal protein concentration of 0.4 mg/mL.
The metabolic reaction was initiated by the addition of an NADPH-
regenerating buffer system containing 1 mg/mL NADP, 1 mg/mL
glucose-6-phosphate, 1 U/mL glucose-6-phosphate dehydrogenase,
and 0.67 mg/mL MgCl2, and reactions were quenched at various time
points over the incubation period by the addition of ice-cold
H
dx.doi.org/10.1021/jm300351w | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
or amides used in the reactions were obtained from commercial
sources except naphthamide, which was synthesized according to the
documented procedure.51
7-Methyl-N-(naphthalen-2-yl)imidazo[1,2-a]pyrimidin-5-amine
(6).52 2-Aminonaphthalene (490 mg, 3.42 mmol) was added to a well-
stirred solution of 21a (522 mg, 3.12 mmol) in excess of absolute
ethanol. The reaction mixture was heated intermittently yielding a
clear solution. Stirring was then continued overnight at ambient
temperature. Excess ethanol was removed by evaporation, and the
crude product was subjected to flash chromatographic purification (on
silica-gel with dichloromethane:methanol:ammonium hydroxide,
1
Analysis. H NMR spectra were recorded on dilute solutions in
CDCl3 or DMSO-d6 or MeOD-d4 at 300 MHz. Chemical shifts (δ) are
reported in ppm relative to TMS, and coupling constants (J) are
reported in Hz. Spin multiplicities are described as s (singlet), brs
(broad singlet), d (doublet), t (triplet), q (quartet), and m (multiplet).
Electronspray ionization mass spectra (ESI-MS) were acquired on a
Bruker Esquire liquid chromatograph-ion trap (LC-MS) mass
spectrometer. Melting points (Pyrex capillary) were determined on a
Mel-Temp apparatus and are not corrected. High-resolution mass
spectrometry (FABHRMS) data were obtained on JEOL HX-110 mass
spectrometer for compound 16. The purity for all compounds in Table
1 was assessed by LC-MS. Chromatography was performed on a
ZorbaxExtend C18 (80A) column using buffer A (water/5%
acetonitrile/1% acetic acid) and buffer B (acetonitrile/1% acetic
acid) with a 20 min gradient from 20 to 100% buffer B or on an
Agilent Exclipse XDB-C18 5 mm (dimensions 4.6 mm × 150 mm)
using acetonitrile/water (0.1%formic acid) and a step gradient of 20,
50, and 90% acetonitrile at 0, 10, and 20 min, respectively. The
absorbance was monitored at 254 nm. The compound purity was
confirmed to be >95% with the exception of compounds 5, 7, and 15,
where the purity was determined to be 86−90%. Given the poor
activity of these compounds, they were not of interest for further
development, and as the results would not be impacted by the
marginal improvement in purity to bring them to 95%, additional
purification was not performed.
Amide Mimics. General Procedure. To a suspension of Pd(OAc)2
(67 mg, 0.30 mmol), BINAP (185 mg, 0.30 mmol), and sodium
tertiary butoxide (2.8 g, 29.70 mmol) in anhydrous toluene (25 mL)
was added 7-chloro-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidine (17) (1
g, 5.93 mmol) and then aromatic amides [4-(trifluoromethyl)-
benzamide (1.3 g, 6.56 mmol) and benzamide (0.8 g, 6.54 mmol)].
The reaction mixture was stirred for 24 h at 120 °C, and the reaction
was monitored by LC-MS until no starting material remained. The
mixture was then filtered, and the residue was washed with
dichloromethane (2 × 10 mL). The dichloromethane parts were
dried over anhydrous sodium sulfate and subsequently concentrated in
vacuo. The crude material was purified by flash column chromatog-
raphy (on silica-gel with ethyl acetate:hexane, 3:2) to afford the
adducts [4 (646 mg, 43%) and 5 (918 mg, 48%)] as white solids.
N-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)benzamide (4).
mp (°C), 201. 1H NMR (300 MHz, MeOD-d4): δ 8.50 (s, 1H),
8.09 (s, 1H), 8.06 (m, 1H), 8.03 (s, 1H), 7.74−7.59 (m, 3H), 2.72 (s,
3H). ESI-MS (m/z): 254.0 (M+).
1
240:3:2) to yield 5 (546 mg, 70%). mp (°C), >250. H NMR (300
MHz, DMSO-d6): δ 7.98−7.91 (m, 5H), 7.62−7.41 (m, 4H), 5.72 (s,
1H), 2.31 (s, 3H). ESI-MS (m/z): 276.1 (M+).
7-Methyl-N-(4-(trifluoromethyl)phenyl)imidazo[1,2-a]pyrimidin-
5-amine (16).53 To a well-stirred solution of 21a (522 mg, 3.12
mmol) in excess absolute ethanol was added 4-(trifluoromethyl)aniline
(552 mg, 3.42 mmol) with intermittent heating yielding a clear
solution. Stirring was then continued for 24 h at room temperature,
excess ethanol was removed by evaporation, and the crude product
was purified by flash chromatography (on silica-gel with dichlor-
omethane:methanol:ammonium hydroxide, 240:3:2) to yield 16 (555
mg, 65%). mp (°C), 292. 1H NMR (300 MHz, DMSO-d6): δ 7.74 (d,
1H, J = 1.5 Hz), 7.67 (d, 2H, J = 8.7 Hz), 7.35 (d, 1H, J = 1.8 Hz),
7.26 (d, 2H, J = 8.4 Hz), 5.88 (s, 1H), 2.24 (s, 3H). ESI-MS (m/z):
293.2 (M+).
7-Methyl-N-(naphthalen-2-yl)-[1,2,4]triazolo[1,5-a]pyridin-5-
amine (7): General Procedure. To a suspension of Pd(OAc)2 (16.75
mg, 0.075 mmol), BINAP (46.44 mg, 0.075 mmol), and sodium
tertiary butoxide (715 mg, 7.45 mmol) in 20 mL of anhydrous toluene
was added 5-chloro-7-methyl-[1,2,4]triazolo[1,5-a]pyridine (18, com-
mercially obtained) (250 mg, 1.49 mmol) and then 2-amino-
naphthalene (235 mg, 1.64 mmol). The reaction mixture was refluxed
for 24 h, subsequently cooled to room temperature, and then
concentrated in vacuo. The crude material was purified by flash
column chromatography (on silica-gel using ethyl acetate:hexane, 3:2)
to afford 7 (172 mg, 42%) as a creamish white solid. mp (°C), 154. 1H
NMR (300 MHz, CDCl3): δ 8.41 (s, 1H), 7.97−7.82 (m, 4H), 7.73 (s,
1H), 7.61−7.45 (m, 3H), 7.17 (s, 1H), 6.60 (s, 1H), 2.46 (s, 3H). ESI-
MS (m/z): 275.2 (M+).
Pyrazolo[1,5-a]pyrimidine: General Procedure. 7-Hydroxy-5-
methylpyrazolo[1,5-a]pyrimidine (20b). A mixture of 1H-pyrazol-5-
amine (19b) (1.66 g, 20.00 mmol) and ethyl acetoacetate (2.7 mL, 2.8
g, 22.00 mmol) in glacial acetic acid (15 mL) was heated under reflux
for 20 h. After the reaction mixture cooled, the excess acetic acid was
removed in vacuo. The precipitated solid was then filtered, washed
with ethanol, and dried under vacuum to give the 20b (2.55 g, 85%) as
1
a white solid. mp (°C), 295. H NMR (300 MHz, DMSO-d6): δ 7.82
(s, 1H), 6.10 (s, 1H), 5.56 (s, 1H), 2.28 (s, 3H). ESI-MS (m/z): 150.1
N-(5-Methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-yl)-4-
(M+).
1
trifluoromethyl)benzamide (5). mp (°C), 187. H NMR (300 MHz,
7-Chloro-5-methylpyrazolo[1,5-a]pyrimidine (21b). A solution of
phosphorus oxychloride (2.0 mL, 21.40 mmol) and 20b (149 mg, 1.00
mmol) was heated under reflux for 40 min. The reaction mixture was
then brought to room temperature, excess reagent was removed in
vacuo, and the residue was triturated with ice−water. The chlorinated
product was extracted from the aqueous mixture using dichloro-
methane. The organic layer was separated, dried over anhydrous
sodium sulfate, and then filtered. The filtrate was concentrated and
purified by flash chromatography (silica-gel with ethyl acetate:hexane,
MeOD-d4): δ 8.51 (s, 1H), 8.24 (d, 2H, J = 8.1 Hz), 8.07 (s, 1H), 7.94
(d, 2H, J = 8.1 Hz), 2.74 (s, 3H). ESI-MS (m/z): 320.1 (M−) [ionizes
better in negative mode].
Imidazo[1,2-a]pyrimidine Derivarives: General Procedure. 5-
Hydroxy-7-methylimidazo[1,2-a]pyrimidine (20a). A solution of 2-
aminoimidazole hemisulfate (19a) (1 g, 7.58 mmol) and ethyl
acetoacetate (1.48 g, 11.37 mmol) in glacial acetic acid (15 mL) was
refluxed for 25 h. Upon cooling and subsequent evaporation of the
excess liquid under suction, a light brownish solid was obtained,
filtered, washed with dichloromethane several times, and dried under
1
3:2) to afford 21b (75 mg, 45%). mp (°C), 38. H NMR (300 MHz,
1
CDCl3): δ 8.18 (s, 1H), 6.87 (s, 1H), 6.69 (s, 1H), 2.62 (s, 3H). ESI-
vacuum to give the product 20a (0.790 g, 70%). mp (°C), 242. H
MS (m/z): 168.5 (M+).
NMR (300 MHz, DMSO-d6): δ 7.58 (d, 1H, J = 2.4 Hz), 7.36 (d, 1H,
J = 2.4 Hz), 5.76 (s, 1H), 2.26 (s, 1H). ESI-MS (m/z): 150.1 (M+).
5-Chloro-7-methylimidazo[1,2-a]pyrimidine (20a). A mixture of
the hydroxy-intermediate 20a (0.790 g, 5.30 mmol) and phosphorus
oxychloride (14 mL, 150.10 mmol) formed a clear red solution upon
refluxing for 2 h. The excess phosphorus oxychloride was removed in
vacuo, and the residue was precipitated using dichloromethane. The
precipitates were filtered, washed several times with dichloromethane,
and dried under vacuum to yield the chlorinated intermediate 21a
(577 mg, 65%), which was used without purification for subsequent
steps.
5-Methyl-N-(naphthalen-2-yl)pyrazolo[1,5-a]pyrimidin-7-amine
(8).52 2-Aminonaphthalene (57 mg, 0.40 mmol) was added to the
stirred solution of 21b (60 mg, 0.36 mmol) dissolved in absolute
ethanol (20 mL). Stirring was then continued for 8 h at room
temperature. Excess ethanol was removed by evaporation, and the
crude product was subjected to flash chromatographic purification (on
silica-gel using dichloromethane:methanol:ammonium hydroxide,
100:1:1) to yield 7 in quantitative yields (77 mg, 78%). mp (°C),
136. 1H NMR (300 MHz, DMSO-d6): δ 10.01 (brs, NH
exchangeable), 8.16 (s, 1H), 8.04−7.93 (m, 4H), 7.66−7.62 (m,
I
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Journal of Medicinal Chemistry
Article
1H), 7.59−7.48 (m, 2H), 6.43 (s, 1H), 6.34 (s, 1H), 2.38 (s, 3H). ESI-
MS (m/z): 275.1 (M+).
yield: 257 mg, 78%), N-(2-methyl-9H-purin-6-yl)quinolin-6-amine
(14; yield: 263 mg, 80%), and 3-(2-methyl-9H-purin-6-ylamino)-1H-
pyrazole-4-carbonitrile (15; yield: 195 mg, 68%) [recrystallization
solvents: ethanol (for 9 and 12) and dichloromethane:methanol 2:1
(for 13−15)].
Triazolo[4,3-b]pyridazine: General Procedure. 8-Hydroxy-6-
methyl-[1,2,4]triazolo[4,3-b]pyridazine (20c). A well-stirred mixture
of 4H-1,2,4-triazol-4-amine (500 mg, 5.95 mmol) and ethyl
acetoacetate (1.16 g, 8.93 mmol) in glacial acetic acid (20 mL) was
refluxed at 170 °C for 4 h. The reaction mixture was brought to room
temperature, and excess solvent was evaporated. The residue was
filtered using dichloromethane and was recrystallized from aqueous
acetic acid to give 20c as white solid (750 mg, 84%). mp (°C), 306. 1H
NMR (300 MHz, DMSO-d6): δ 9.42 (s, 1H, −OH), 6.36 (s, 1H), 2.41
(s, 3H). ESI-MS (m/z): 151.1 (M+).
8-Chloro-6-methyl-[1,2,4]triazolo[4,3-b]pyridazine (21c). A well-
stirred solution of 20c (500 mg, 3.34 mmol) in phosphorus
oxychloride (10 mL, 107.01 mmol) was refluxed for 45 min. After
completion, the reaction mixture was concentrated in vacuo to give a
red residue, which was precipitated using dichloromethane to give 21c
(340 mg, 62%). The chlorinated intermediate was used for the
subsequent step in the crude form. mp (°C), 181.
2-Methyl-N-(naphthalen-2-yl)-9H-purin-6-amine (9).52 mp (°C),
>300. 1H NMR (300 MHz, DMSO-d6): δ 10.45 (brs, NH
exchangeable), 8.54 (s, 1H), 8.43 (s, 1H), 7.94−7.87 (m, 4H),
7.54−7.42 (m, 2H), 2.64 (s, 3H). ESI-MS (m/z): 276.2 (M+).
N-(Anthracen-2-yl)-2-methyl-9H-purin-6-amine (12). mp (°C),
1
>320 (decomp). H NMR (300 MHz, DMSO-d6): δ 11.95 (bs, NH
exchangeable), 8.85 (s, 1H), 8.71 (s, 1H), 8.65−8.52 (m, 2H), 8.21−
7.93 (m, 4H), 7.65−7.43 (m, 2H), 2.79 (s, 3H). ESI-MS (m/z): 326.1
(M+).
N-(2-Methyl-9H-purin-6-yl)quinolin-3-amine (13). mp (°C), 290.
1H NMR (300 MHz, DMSO-d6): δ 11.98 (brs, NH exchangeable),
9.43 (s, 1H), 9.07 (s, 1H), 8.63 (s, 1H), 8.07−8.05 (m, 2H), 7.78−
7.67 (m, 2H), 2.71 (s, 3H). ESI-MS (m/z): 277.1 (M+).
1
N-(2-Methyl-9H-purin-6-yl)quinolin-6-amine (14). H NMR (300
6-Methyl-N-(naphthalen-2-yl)-[1,2,4]triazolo[4,3-b]pyridazin-8-
amine (11). To a suspenstion of Pd(OAc)2 (22.65 mg, 0.10 mmol),
BINAP (62.78 mg, 0.10 mmol), and sodium tertiary butoxide (968 mg,
10.00 mmol) in anhydrous toluene (20 mL) were added 20c (340 mg,
2.02 mmol) and then 2-naphthyl amine (318 mg, 2.22 mmol). The
mixture was stirred overnight at 120 °C and then concentrated in
vacuo. The crude material was purified by flash column chromatog-
raphy (on silica-gel with ethyl acetate:hexane, 11:9) to afford 11 (252
mg, 45%) as white solid. mp (°C), 195. 1H NMR (300 MHz, DMSO-
d6): δ 10.10 (brs, NH exchangeable), 9.44 (s, 1H), 8.00−7.91 (m,
4H), 7.67−7.63 (m, 1H), 7.56−7.46 (m, 2H), 6.60 (s, 1H), 2.38 (s,
3H). ESI-MS (m/z): 276.2 (M+).
Purine Analogues: General Procedure. 4-(Methylethoxymethy-
lene)-aminoimidazole-5-carboxamide (23a). A mixture of 5-amino-
1H-imidazole-4-carboxamide hydrochloride (22a) (1 g, 6.15 mmol)
and triethylorthoacetate (2 mL, 10.98 mmol) in anhydrous N,N-
dimethyl formamide (5 mL) was heated under reflux at 180 οC for 15
min yielding a clear solution that was cooled. Excess solvent was
removed under suction, and the filtration provided 23a as light brown
solid (0.949 g, 80%). mp (°C), 248. 1H NMR (300 MHz, DMSO-d6):
δ 12.6 (brs, NH exchangeable), 7.56 (s, 1H), 7.31 (s, 2H, −CONH2),
4.16 (q, 2H), 2.29 (s, 3H), 1.28 (t, 3H). ESI-MS (m/z): 197.1 (M+).
2-Methylhypoxanthine (24a). Intermediate 23a (1 g, 5.11 mmol)
was heated at 200 °C for 30 min, and the product was recrystallized
from water to afford 24a (612 mg, 80%) as a grayish solid. mp (°C),
>300. 1H NMR (300 MHz, DMSO-d6): δ 13.10 (brs, NH
exchangeable), 12.15 (s, 1H, −OH), 8.12 (s, 1H), 2.35 (s, 3H).
ESI-MS (m/z): 151.2 (M+).
6-Chloro-2-methyl-9H-purine (25a). N,N-Dimethylaniline (3.3
mL, 26.07 mmol) was added to a suspension of 24a (1 g, 6.67
mmol) in phosphorus oxychloride (15 mL, 214.00 mmol), and the
reaction mixture was refluxed for 1 h. Excess reagent was distilled off
under reduced pressure, and the syrupy residue was poured onto finely
crushed ice. The aqueous solution was extracted with diethyl ether in
order to remove N,N-dimethyl aniline. The ethereal solution was
washed 3−4 times with cold water, dried over anhydrous sodium
sulfate, and distilled to afford 25a (728 mg, 65%) (best results were
obtained using sufficient volume of ether and rapidly extracting it with
cold water).
Purine Analogues (9 and 12−15). General Procedure. Aromatic
amines [2-aminonaphthalene (186.86 mg, 1.31 mmol), 2-amino-
anthracene (252.10 mg, 1.31 mmol), 3-aminoquinoline (188.16 mg,
1.39 mmol), 6-aminoquinoline (188.16 mg, 1.39 mmol), and 3-amino-
1H-pyrazole-4H-carbonitrile (140.92 mg, 1.30 mmol)] were added to
a well-stirred solution of 25a (200 mg, 1.186 mmol) in absolute
ethanol (20 mL), and stirring was continued for 15−18 h at ambient
temperature. An excess of ethanol was removed by evaporation, and
the crude product were purified via recrystallization technique to
afford 2-methyl-N-(naphthalen-2-yl)-9H-purin-6-amine (9; yield: 246
mg, 75%), N-(anthracen-2-yl)-2-methyl-9H-purin-6-amine (12; yield:
310 mg, 80%), N-(2-methyl-9H-purin-6-yl)quinolin-3-amine (13;
MHz, DMSO-d6): δ 10.29 (brs, NH exchangeable), 8.98 (m, 1H), 8.92
(s, 1H), 8.73 (d, 1H, J = 8.1 Hz), 8.53 (s, 1H), 8.44 (m, 1H), 8.19 (d,
1H, J = 8.7 Hz), 7.77 (m, 1H), 2.70 (s, 3H, CH3). ESI-MS (m/z):
277.2 (M+).
3-(2-Methyl-9H-purin-6-ylamino)-1H-pyrazole-4-carbonitrile
(15). 1H NMR (300 MHz, DMSO-d6): δ 2.62 (s, CH3), 8.58 (s, 1H),
8.63 (s, 1H). ESI-MS (m/z): 241.1 (M+).
Pyrazolo[3,4-d]pyrimidin-4-amine: General Procedure. 5-(Meth-
ylethoxymethylene)-aminopyrazole-4-carboxamide (23b). A well-
stirred solution of 5-amino-1H-pyrazole-4-carboxamide hemisulfate
(22b) (1 g, 5.70 mmol) and triethylorthoacetate (1.72 mL, 9.44
mmol) in 5 mL of anhydrous dimethyl formamide was heated under
reflux for 1 h at 240 °C, and the resulting solution was cooled. The
excess solvent was removed by evaporation with subsequent filtration
providing 23b (1.38 g, 75%) as a light brown solid. mp (°C), 148
1
(decomp). H NMR (300 MHz, DMSO-d6): δ 8.01 (s, 1H), 7.25 (d,
2H, −CONH2), 4.25 (q, 2H), 2.21 (s, 3H), 1.35 (t, 3H). ESI-MS (m/
z): 197.1 (M+).
4-Hydroxy-6-methyl-1H-pyrazolo[3,4-d]pyrimidine (24b). The
intermediate 23b (1 g, 5.10 mmol) was heated at 250 °C for 2 h,
and the residue was filtered using dichloromethane. The crude product
was recrystallized from water to afford 24b (535 mg, 70%) as a grayish
1
solid. mp (°C), >300. H NMR (300 MHz, DMSO-d6): δ 7.96 (s,
1H), 2.34 (s, 3H). ESI-MS (m/z): 151.1 (M+).
4-Chloro-6-methyl-1H-pyrazolo[3,4-d]pyrimidine (25b). A mix-
ture of 24b (500 mg, 3.33 mmol), N,N-dimethylaniline (1.7 mL), and
phosphorus oxychloride (8 mL) was refluxed for 1 h until all of the
solid went into solution. The excess reagent was distilled under
reduced pressure, and the syrupy residue was poured slowly, with
vigorous stirring, onto finely crushed ice. The mixture was allowed to
stand for 20 min, and the aqueous suspension was extracted with ether.
The ethereal extract was washed well with water. After the extract was
dried over anhydrous sodium sulfate, the organic layer was distilled to
yield chlorinated adduct 25b (392 mg, 70%) as a yellow powder, used
in this form for the subsequent step.
6-Methyl-N-(naphthalen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-
amine (10). To a well-stirred solution of 25b (200 mg, 1.19 mmol) in
20 mL of absolute ethanol was added 2-aminonaphthalene (186.97
mg, 1.31 mmol). The stirring was continued for 12 h at ambient
temperature, the excess of ethanol was removed by evaporation, and
the crude product was purified via recrystallization using ethanol to
afford 10 (133 mg, 78%) as a light yellow powder. mp (°C), >300. 1H
NMR (300 MHz, DMSO-d6): δ 8.46 (s, 1H), 7.93−8.00 (m, 5H),
7.50−7.59 (m, 2H), 2.64 (s, 3H). ESI-MS (m/z): 276.1 (M+).
AUTHOR INFORMATION
■
Corresponding Author
*Tel: 214-645-6164. Fax: 214-645-6166. E-mail: Margaret.
Phillips@UTSouthwestern.edu (M.A.P.). Tel: 206-221-6069.
J
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We recognize the late Dr. Ian Bathurst from Medicines for
Malaria Venture (MMV) for his helpful support of the project.
We also thank Drs. David Floyd and David Matthews of the
(MMV) Expert Scientific Advisory Committee for helpful
discussions and Rajan K. Paranji and Martin Sadilek for
analytical support. This work was supported by the United
States National Institutes of Health Grants R01AI53680 (to
M.A.P. and P.K.R.) and U01AI075594 (to M.A.P., P.K.R., and
S.A.C.). M.A.P. acknowledges support of the Welch Founda-
tion (I-1257), and P.K.R. also acknowledges a 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 and the Beatrice and Miguel
Elias Distinguished Chair in Biomedical Science Beatrice and
Miguel Elias Distinguished Chair in Biomedical Science.
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ABBREVIATIONS USED
■
PfDHODH, Plasmodium falciparum dihydroorotate dehydro-
genase; PbDHODH, Plasmodium berghei dihydroorotate
dehydrogenase; hDHODH, human dihydroorotate dehydro-
genase; HTS, high-throughput screening; BINAP, 2,2′-bis-
(diphenylphosphino)-1,1′-binaphthyl; CoQ, ubiquinone; FMN,
flavin mononucleotide; ACTs, artemisinin-based combination
therapies; SAR, structure−activity relationships; DCIP, 2,6-
dichloroindophenol; TLC, thin-layer chromatography; HPLC/
MS, high-performance liquid chromatography/mass spectrom-
etry
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An immunomodulatory drug for the treatment of rheumatoid arthritis
and other autoimmune diseases. Immunopharmacology 2000, 47, 273−
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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.;
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