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

Article abstract of DOI:10.1021/jm200592f

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

Full text of DOI:10.1021/jm200592f

ARTICLE
pubs.acs.org/jmc
Structure-Guided Lead Optimization of Triazolopyrimidine-Ring
Substituents Identifies Potent Plasmodium falciparum Dihydroorotate
Dehydrogenase Inhibitors with Clinical Candidate Potential
Jose M. Coteron,§ María Marco,§ Jorge Esquivias,§ Xiaoyi Deng,† Karen L. White,|| John White,^
Maria Koltun,|| Farah El Mazouni,† Sreekanth Kokkonda,^ Kasiram Katneni,|| Ravi Bhamidipati,||
David M. Shackleford,|| I~nigo Angulo-Barturen,§ Santiago B. Ferrer,§ María Belꢀen Jimꢀenez-Díaz,§
Francisco-Javier Gamo,§ Elizabeth J. Goldsmith,‡ William N. Charman,|| Ian Bathurst,# David Floyd,#
David Matthews,# Jeremy N. Burrows,# Pradipsinh K. Rathod,^ Susan A. Charman,||
and Margaret A. Phillips*,†
†Department of Pharmacology and ‡Biochemistry, University of Texas Southwestern Medical Center at Dallas, 6001 Forest Park Rd,
Dallas, Texas 75390-9041, United States
§GlaxoSmithKline, Diseases of the Developing World (DDW)—Tres Cantos Medicines Development Campus, Madrid, Spain
Centre for Drug Candidate Optimisation, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville Campus),
Parkville, VIC 3052, Australia
^Departments of Chemistry and Global Health, University of Washington, Seattle, Washington 98195, United States
#Medicines for Malaria Venture, Geneva, Switzerland
S Supporting Information
b
ABSTRACT: Drug therapy is the mainstay of antimalarial therapy, yet current
drugs are threatened by the development of resistance. In an effort to identify new
potential antimalarials, we have undertaken a lead optimization program around our
previously identified triazolopyrimidine-based series of Plasmodium falciparum
dihydroorotate dehydrogenase (PfDHODH) inhibitors. The X-ray structure of
PfDHODH was used to inform the medicinal chemistry program allowing the
identification of a potent and selective inhibitor (DSM265) that acts through
DHODH inhibition to kill both sensitive and drug resistant strains of the parasite.
This compound has similar potency to chloroquine in the humanized SCID mouse
P. falciparum model, can be synthesized by a simple route, and rodent pharmaco-
kinetic studies demonstrated it has excellent oral bioavailability, a long half-life and
low clearance. These studies have identified the first candidate in the triazolopyr-
imidine series to meet previously established progression criteria for efficacy and
ADME properties, justifying further development of this compound toward clinical candidate status.
’ INTRODUCTION
widespread drug resistance to traditional therapies (e.g., chlor-
oquine and pyrimethamine/sulfadoxine), the introduction of
artemisinin-based combination therapies (ACTs) has become
the most powerful tool to combat the disease.6,7 ACTs are highly
effective for the treatment of Plasmodium falciparum malaria
leading to significant reduction in the mortality and morbidity of
the disease. Recent emergence of potential artemisinin resis-
tance along the ThaiꢀCambodia border threatens to derail these
successes, as well as any hope at global eradication.8 Few
clinically approved treatment options will remain if artemisinin
resistance becomes widespread.
Malaria is an ancient human enemy with descriptions of
malarial-like febrile illness documented over 2000 years ago in
the writings of the Greek physician Hippocrates.1 The identifica-
tion of the parasite and the link to mosquito-based transmission
in 1880 led to our modern understanding of the disease. Drugs to
treat malaria predate our knowledge of its etiology (e.g., quinine
was isolated from the cinchona bark in 1820), yet today malaria
still leads to 220 million cases and approximately 1 million
deaths per year, with over 2 billion people at risk for the
disease.2,3 The world community is currently involved in its
second attempt at global malaria eradication.4 While some
progress has been made in developing a vaccine, the best
candidate RTSS provides only partial immunity.5 Thus the
mainstay of antimalarial treatment and the key to successful
eradication continues to be chemotherapy. Against a backdrop of
A continual pipeline to identify and develop antimalarial
agents is required to combat the ability of the parasite to rapidly
Received: May 11, 2011
Published: June 22, 2011
r
2011 American Chemical Society
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’ CHEMISTRY
The focus of this study was to examine the SAR associated with
modifications of the substituent at C2 of the triazolopyrimidine
ring system. The development of a synthetic strategy for these
triazolopyrimidine derivatives was dictated by the nature of the
atom used as linker at the C2- position of the ring. The synthesis of
the 2-substituted analogues 11ꢀ55 shown in Table 1 proceeded
through the intermediate 7-hydroxy-triazolopyrimidines 9 shown
in Scheme 1, which was accessed by two routes. The most general
approach involved the condensation of ethyl 3-oxobutanoate with
aminoguanidine hydrochloride to afford diaminopyrimidone 7 in
moderate yield (11ꢀ42 and 45ꢀ55). This route had the advan-
tage that 7 served as a common intermediate from which a diverse
set of C2 alkyl analogues could be prepared. Reaction of 7 with a
variety of acid chlorides (R defined in Table 1) gave the C2
substituted triazolopyrimidones 9 in good to excellent yields, even
with sterically hindered R groups. This reaction apparently
proceeded by initial acylation of 7 followed by a slower cyclization
step yielding 9 and, in some cases, required more forcing condi-
tions to effect complete conversion to 9.23 Alternatively, to
generate 43, 44, and 100, we used our previously described
chemistry16,17,19 in which the 5-aminotriazoles 8 were either
commercially available or prepared initially (see Materials and
Methods). In these cases, the reaction of 8 with ethyl 3-oxobu-
tanoate proceeded through the acetoacetate ketone with forma-
tion of an aminocrotonate intermediate that cyclizes at the N-2 of
the triazole ring yielding the desired isomer 9.24,25 The limitation
of this approach was the availability of starting 3-substituted-5-
amino-1,2,4-triazole derivatives due to lengthy reaction times,
inefficiency and variable results during their preparation. Treat-
ment of 9 with phosphoryl chloride then gave the chloro
triazolopyrimidines 10, which could be converted to the desired
2-substituted 7-aminoaryl products 11ꢀ55, 100 by reaction with
the requisite aniline under a variety of conditions.
As shown in Scheme 2, the preparation of analogues with
either S- or O-linked substituents at C2 (58ꢀ84) started with the
commercially available triazolopyrimidone 56. Conversion of 56
to the chloride (57, R = SMe) under the standard conditions
followed by displacement of the chloro group with the requisite
aniline afforded compounds 58ꢀ61 in good yields. Oxidation
with hydrogen peroxide in the presence of catalytic Na2WO4 in
hot aqueous acetic acid proved to be very straightforward,
although competitive nitrogen oxidation lowered the yield of
this step. Nevertheless, compounds 62ꢀ65 were easily prepared
by this procedure. These compounds then served as intermedi-
ates for the preparation of compounds 66ꢀ84 by taking advan-
tage of the methylsulfonyl group to act as a leaving group. Thus,
treatment of these C2 methylsulfonyl analogues with a variety of
alkyl and functionalized alkyl alkoxides under microwave condi-
tions afforded the desired compounds (66ꢀ84) in good yields.
Analogues with amino (89ꢀ97) or hydroxyl (98, 99, 101)
functionality at the R position were obtained from specific
intermediates 45ꢀ47 (prepared as in Scheme 1) and further
derivatized as shown in Schemes 3 and 4. Compounds 89ꢀ97
were generated in moderate to good yields by treating the fully
elaborated chloromethyl (45a, 45b) or chloroethyl (46a, 46b)
analogues with the appropriate amine in methanol or THF in the
microwave at 120 ꢀC (Scheme 3). The hydroxyl alkyl analogues
98ꢀ99, 101 were prepared by hydrogenolysis (Scheme 4) of the
requisite benzyl ether (47a, 47b) using flow chemistry with an
H-cube instrument.
Figure 1. Structures of select triazolopyrimidines.
acquire resistance to chemotherapy in the field.9 The global effort
to identify new drugs is being fueled by not-for-profit organiza-
tions and the development of substantial portfolios of candidate
molecules ranging from identified hits to compounds in clinical
development.10ꢀ12 The completion of whole organism screens
of large chemical libraries has identified thousands of new
chemical species with antimalarial activity,13,14 while target-based
approaches are also being successfully used to find novel
chemotypes. The translation of these findings to clinical suc-
cesses still presents a formidable challenge to the development of
new drugs.
Dihydroorotate dehydrogenase (DHODH) has emerged as
the best validated new target for the development of novel
antimalarials since the identification of the bc1 complex as
the target of atovaquone, due to the discovery of several classes
of inhibitors with antimalarial activity.15 DHODH catalyzes the
fourth step in the de novo pyrimidine biosynthetic pathway, the
flavin mononucleotide (FMN)-dependent oxidation of dihy-
drorotate to orotic acid. P. falciparum DHODH (PfDHODH)
is localized to the inner mitochondrial-membrane and utilizes
ubiquinone (CoQ) as the final electron acceptor to regenerate
oxidized FMN. The malaria parasite is uniquely vulnerable to
inhibition of this pathway because it lacks the salvage enzymes
that serve as an additional source of pyrimidine nucleosides in
other organisms, including the human host. Several different
chemical series that are potent inhibitors of PfDHODH have
been identified by enzyme activity-based HTS and were subse-
quently found to have antimalarial activity both in vitro and
in vivo. These include the triazolopyrimidines (1) (Figure 1)
identified by our group16ꢀ19 and N-alkyl-5-(1H-benzimidazol-1-yl)-
thiophene-2-carboxamides identified by Genzyme and
colleagues.20,21 Structural analysis of the bound inhibitor enzyme
complexes has revealed that these different chemical classes have
overlapping but distinctly different binding modes owing to
conformational flexibility in the enzyme inhibitor binding
site.21,22 Most recently, metabolically stable triazolopyrimidines
(2ꢀ4) (Figure 1) have been identified that are able to suppress
parasites in a mouse model of infection, however these com-
pounds lacked the potency required in a clinical development
candidate.16,19 To improve the potency of the series, we have
utilized the X-ray structural information to guide additional
medicinal chemistry. The enzyme bound structure of 1 and 2
showed that the triazolopyrimidines filled most of the available
binding pocket but that a channel between the C2 position of the
triazolopyrimidine ring and the FMN cofactor was available that
could potentially accommodate additional functionality.22 In a
major advance forward, herein we describe the lead optimization
program to modify the C2 position of the triazolopyrimidine
scaffold that has yielded several potent compounds with phar-
macokinetic profiles and in vivo efficacy in mouse models that
meet previously established progression criteria.
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Table 1. StructureꢀActivity Relationships of the Triazolopyrimidine Compoundsa
compd
name
R
R1 R2 IC50 (μM) PfDHODH IC50 (μM) PbDHODH IC50 (μM) hDHODH EC50 (μM) Pf 3D7 cells
2
DSM74b
DSM161c
DSM89
DSM75
H
H
H
H
CF3
SF5
Cl
H
H
H
Cl
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
H
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
Cl
H
H
H
H
H
Cl
H
H
H
Cl
H
H
H
0.28
0.13
1.6
0.38
0.28
5.7
>100
>100
>100
>100
ND
0.34
1.3, 0.18*
12
3
5
6
H
1.4
31
8.5
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
38
39
40
41
42
43
44
48
49
50
51
52
53
54
55
58
DSM271 CH2CH3
DSM272 CH2CH3
DSM280 CH2CH3
DSM281 CH2CH3
DSM309 iso-Pr
Cl
0.28
0.24
0.087
0.15
0.33
0.12
0.11
0.10
>10
>10
2.0
2.3
0.79
3.3
H
ND
0.76
1.4
ND
CF3
SF5
Cl
>100
>100
ND
0.058
0.1
2.1
0.85
1.1
DSM308 iso-Pr
H
0.57
4.5
ND
DSM307 iso-Pr
CF3
SF5
Cl
>100
>100
ND
0.098
0.42
ND
DSM314 iso-Pr
3.8
DSM313 iso-Bu
ND
ND
ND
ND
15
DSM312 iso-Bu
H
ND
ND
DSM311 iso-Bu
CF3
SF5
CF3
SF5
CF3
SF5
Cl
ND
ND
DSM310 iso-Bu
>10
0.46
0.55
0.38
0.33
0.88
0.82
0.14
0.093
12
ND
ND
DSM357 tert-Bu
>100
>100
>100
>100
>100
>100
>100
>100
ND
0.29
2.3
DSM358 tert-Bu
16
DSM356 sec-Bu
4.6
0.27
0.51
6.8
DSM355 sec-Bu
4.5
DSM325 cycloPr
DSM324 cycloPr
DSM322 cycloPr
DSM323 cycloPr
DSM321 CH2cycloPr
DSM320 CH2cycloPr
DSM318 CH2cycloPr
DSM319 CH2cycloPr
DSM254 CF2CH3
DSM266 CF2CH3
DSM267 CF2CH3
DSM265 CF2CH3
DSM329 CF2CH2CH3
DSM328 CF2CH2CH3
DSM326 CF2CH2CH3
DSM327 CF2CH2CH3
DSM195 CF3
2.8
H
1.7
ND
CF3
SF5
Cl
2.6
0.15
0.50
ND
2.0
ND
ND
ND
ND
1.7
H
>10
1.7
ND
ND
CF3
SF5
Cl
ND
3.4
5.7
ND
>10
0.038
0.054
0.038d
0.033e
0.034
0.030
0.019
0.075
0.035
0.030
2.2
>100
ND
0.050
0.13
0.010f/0.0062*
0.046g/0.0078*
0.094
1.2
H
ND
2.4
CF3
SF5
Cl
>100
>100
>100
>100
>100
>100
>100
41
2.5
1.8
H
1.4
CF3
SF5
CF3
SF5
Cl
2.5
0.045
0.55
0.073
0.14
ND
4.7
1.3
DSM196 CF3
2.5
DSM299 CH2OCH3
DSM300 CH2OCH3
DSM305 CH2OCH3
DSM306 CH2OCH3
DSM301 CH2CH2OCH3
DSM302 CH2CH2OCH3
DSM303 CH2CH2OCH3
DSM304 CH2CH2OCH3
DSM257 SCH3
ND
ND
5.5
ND
H
1.7
ND
ND
CF3
SF5
Cl
0.41
0.39
>10
>10
2.5
ND
1.0
3.3
ND
2.1
ND
ND
ND
ND
ND
ND
ND
H
ND
ND
CF3
SF5
Cl
ND
4.2
2.5
ND
5.0
0.030
ND
0.15
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Table 1. Continued
compd
name
R
R1 R2 IC50 (μM) PfDHODH IC50 (μM) PbDHODH IC50 (μM) hDHODH EC50 (μM) Pf 3D7 cells
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
89
90
91
92
93
94
95
96
97
98
99
101
DSM258 SCH3
DSM261 SCH3
DSM262 SCH3
DSM259 SO2CH3
DSM260 SO2CH3
DSM263 SO2CH3
DSM264 SO2CH3
DSM227 OCH3
DSM241 OCH3
H
Cl
H
H
H
Cl
H
H
Cl
H
Cl
H
H
H
H
Cl
H
Cl
H
H
H
Cl
H
H
Cl
H
H
H
Cl
H
H
H
H
Cl
Cl
Cl
Cl
H
H
Cl
H
0.026
0.036
0.030
0.045
0.16
0.090
0.11
0.50
0.46
0.40
0.27
0.056
0.057
>10
4.9
ND
4.8
ND
ND
ND
ND
ND
ND
ND
ND
ND
>100
>100
>100
>100
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
0.15
0.0084
0.032
1.4
CF3
SF5
Cl
>10
ND
ND
ND
ND
1.3
H
>2.5
0.17
2.0
CF3
SF5
H
0.98
0.59
4.4
Cl
1.2
DSM245 OCH2CH3
H
1.9
DSM246 OCH2CH3
Cl
1.6
1.3
DSM293 OCH2CH3
CF3
SF5
Cl
2.7
0.058
0.10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
>2.5
ND
ND
ND
>2.5
ND
ND
ND
ND
>2.5
>10
0.48
DSM294 OCH2CH3
0.47
ND
>10
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
DSM298 OCH2CH2OCH3
DSM315 OCH2CH2OCH3
DSM274 O(CH2)2NHCH3
DSM273 O(CH2)2NHCH3
DSM275 O(CH2)2NHCH3
DSM276 O(CH2)2NHCH3
H
Cl
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
5.3
H
CF3
SF5
DSM287 O(CH2)2N(CH3)2 Cl
DSM285 O(CH2)2N(CH3)2
H
DSM289 O(CH2)2N(CH3)2 CF3
DSM291 O(CH2)2N(CH3)2 SF5
DSM295 O(CH2)2NH2
DSM296 O(CH2)2NH2
DSM297 O(CH2)2NH2
DSM288 dO
H
CF3
Cl
Cl
H
DSM286 dO
DSM253 (CH2)2NHCH3
DSM277 CH2NH2
Cl
Cl
Cl
Cl
H
DSM278 CH2NHCH3
DSM279 CH2NH(CH3)2
DSM256 (CH2)2NHCH3
DSM282 CH2NH(CH3)2
DSM283 CH2NH2
H
H
DSM284 CH2NHCH3
DSM292 CH2CH2NH2
DSM268 CH2OH
H
Cl
Cl
H
DSM255 CH2OH
6.1
DSM317 CH2CH2OH
CF3
0.93
a P. falciparum 3D7 results: data collected in media containing human serum, except * where Albumax was used instead. ND, not determined. b,c Previously
reported compounds.16,19 Values were determined based on triplicate data points at each test concentration. d Average of n = 4 independent experiments on
different synthetic batches (std dev = 0.012). e Average ofn = 4 independent experiments ondifferent synthetic batches (std dev = 0.0095). f Average ofn = 6
independent experiments on different synthetic batches (std dev = 0.0045). g Average of n = 6 independent experiments on different synthetic batches (std
dev = 0.013).
’ RESULTS
metabolically stable analogues (e.g., 2ꢀ4) that were able to
suppress parasite growth in the P. berghei mouse model16,19
(Figure 1). Additionally these studies identified the key anilines
(4-CF3 and 4-SF5) that provided the optimal balance between
parasite activity and metabolic stability. These compounds
(2ꢀ4) however were less potent than expected of a drug
candidate and required large doses for in vivo efficacy. The
X-ray structures of PfDHODH bound to 1 and 2 were deter-
mined, and this analysis identified a narrow channel leading from
Optimization of Inhibitor Potency. The original triazolopyr-
imidine lead compound (1) was identified by HTS in a
PfDHODH-based enzyme activity based screen.17 This com-
pound was a potent and selective inhibitor of PfDHODH,
however it was rapidly metabolized and showed no activity
against the parasite in vivo. Replacement of the naphthyl amine
with para-substituted anilines led to the identification of
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the C2 carbon of the triazolopyrimidine ring toward the bound
FMN cofactor.22 This channel is lined by several hydrophobic
side chains, the aliphatic side chain portion of Arg265 and by a
single polar group: the side chain hydroxyl of Tyr528. This
hydroxyl donates a hydrogen bond to the carbonyl oxygen of
Gly226. In the cocrystal structure of PfDHODH with 1, but not
with 2, a single-ordered water molecule is trapped between
bound inhibitor and protein on one side of the channel where
it donates hydrogen bonds to the hydroxyl of Tyr528 and to N1
Scheme 3. Synthesis of Triazolopyrimidines 89ꢀ97a
Scheme 1. Synthesis of the Triazolopyrimidine Compounds
11ꢀ55, 100a
a Reagents and conditions: (i) amine in MeOH or THF, MW, 120 ꢀC,
30ꢀ45 min, (40ꢀ70%).
Scheme 4. Synthesis of Triazolopyrimidines 98, 99, and 101a
a General conditions: (i) EtOH, reflux 5 h, overnight at RT; (ii) 9aꢀ9g,
9j, 9k, 9mꢀ9o; 1,4 dioxane, DMF, requisite acid chloride, reflux O/N or
9h, 9i; NaEtO, EtOH, 80 ꢀC, 30 min, appropriate difluoropropanoate or
difluorobutanoate, 30 min, RT, (1.5ꢀ3) h, 80 ꢀC; (R defined in
Table 1); (iii) 9l, 9p AcOH, reflux, 8 h; (iv) reflux in POCl3; (v)
requisite aniline (R1, and R2 defined in Table 1), EtOH, reflux 1ꢀ3 h.
a Reagents and conditions: (i) Pd/C, H2 35 psi for up to 40 h
(11ꢀ12%).
Scheme 2. Synthesis of the Triazolopyrimidine Compounds 66ꢀ84a
a General conditions: (i) POCl3, reflux, 10 h; (ii) requisite aniline (R1 and R2 defined in Table 1), EtOH, RT; (iii) AcOH, RT, H2O2, Na2WO4 (cat),
50 ꢀC; (iv) R3OH (R3 defined in Table 1), NaH, THF, microwave, 120 ꢀC, (0.5ꢀ1) h.
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of 1. The crystal structures indicate that the triazolopyrimidine in
2 penetrates about 0.3 Å more deeply into the narrow channel
than is the case with 1, which may force a slight movement of the
water molecule destabilizing its hydrogen bonding interactions
and disordering its binding. To improve the potency of the
compound series, we sought to exploit potential additional
binding energy by building functionality from the C2 position
of the ring. We reasoned that an unbranched alkyl substituent at
C2 would fit well into the allowed space in the pocket. But we
also recognized that protein conformational changes seen pre-
viously in PfDHODH as a response to binding different inhibitor
chemotypes22 could possibly alter the shape and electronic
characteristics of this narrow channel if probed by suitable C2
substituents.
To identify the most active and promising compound, we
undertook a systematic evaluation of modification of the triazolo-
pyrimidine ring at the C2 position including some larger and
more polar substituents that were designed to probe the sensi-
tivity of the channel to possible ligand induced conformational
changes. The chemistry was designed to allow study of different
structural effects including chain length effects, branch-group
effects at different distances in C2, electronic effects on the
5-membered ring of the triazolopyrimidine, heteroatom effects at
different distances in C2, polarity effects at different distances in
C2, or donor and acceptor hydrogen-bonding effects in C2
looking for an extra interaction with the FMN cofactor. To
evaluate whether the previously optimized anilines (4-CF3 and
4-SF5) remained appropriate for the various functional groups
that were coupled to C2, four different anilines (4-Cl, 3-Cl,
4-CF3, and 4-SF5) were tested within each series.
Figure 2. P. falciparum in vitro growth curves and genetic rescue with
yeast DHODH for 38. Data were collected in human serum and were
fitted to the four-parameter doseꢀresponse equation in Graphpad
Prism to obtain EC50 values. 38 (P. falciparum 3D7, EC50 = 0.019 μM
(0.016ꢀ0.022)); 3 (P. falciparum 3D7, EC50 = 1.1 μM (1.1ꢀ1.2)); 38
(P. falciparum D10, EC50 = 0.086 μM (0.066ꢀ0.11)); 38 (P. falciparum
D10 transfected with yeast DHODH, EC50 >20 μM). Values in
parentheses represent the 95% confidence interval. Addition of progua-
nil did not restore sensitivity to the D10_yeast DHODH cell line (EC50
>20 μM; data not shown).
(ꢀCH2OH (98, 99) or ꢀCH2CH2OH (101)). Addition of an
amine functionality into the R at C2 (74ꢀ84, 89ꢀ97) led to
complete loss in inhibitory activity independent of the substitu-
tion pattern on the N atom or its position in the chain, thus
confirming the lipophilic character of the channel. Transforma-
tion of C2 into a carbonyl moiety (85 and 86) also led to
complete loss in inhibitory activity.
Compounds were initially evaluated for potency against
PfDHODH and against the P. falciparum parasite in whole cell
assays (Table 1). Generally, we observed a good correspondence
between activity againstPfDHODH and inhibition ofP. falciparum
3D7 cell growth for the compounds in the series. Alkyl R-groups
at C2 were tested to explore the size of the pocket, including ethyl
(11ꢀ14), iso-propyl (15ꢀ18), iso-butyl (19ꢀ22), tert-butyl (23
and 24), sec-butyl (25 and 26), and cyclopropyl (27ꢀ30). All but
the iso-butyl substituents were tolerated and had similar activity
to that of the parent compounds (2ꢀ6), with a small (2ꢀ3-fold)
improvement in potency against PfDHODH observed for 13, 17,
and 29, or a larger 5ꢀ10-fold improvement observed for the 3-Cl
aniline (12 and 16) or the 4-Cl aniline (11 and 15) derivatives.
These data suggest the geometry of the channel is wide enough to
accept steric hindrance on the atom adjacent to the C2 carbon of
the triazolopyrimidine ring but that it narrows beyond the second
atom from C2. Compounds with a direct ether linkage to the
triazolopyrimidine ring, OMe (66 and 67) and OEt (68ꢀ71),
also showed similar to modestly improved potency when com-
pared to the parent compounds (2ꢀ6). In the context of 4-CF3ꢀ
or 4-SF5ꢀaniline, the addition of OEt at C2 (70 and 71) led to
4ꢀ8-fold improved potency against PfDHODH, however both
were less active than those containing a haloalkyl (37 and 38).
Addition of a linker carbon between C2 and cyclo-
propyl (31ꢀ34) led to reduced inhibitory activity as did
increasing the chain length of the ethyl ether group at C2
(ꢀOCH2CH2OCH3) (72 and 73), demonstrating that the
pocket is not able to accommodate these larger groups at the
C2 position. Compounds that contained an ether linkage in the R
group (48ꢀ55) that were not directly linked to the triazolopyr-
imidine ring showed in general lower activity than the parent
compounds (2ꢀ6), as did those containing an alcohol
The most potent inhibitory activity against PfDHODH was
from compounds that contained an unbranched haloalkyl R
group at C2, including ꢀCF2CH3 (35ꢀ38), ꢀCF2CH2CH3
(39ꢀ42), or ꢀCF3 (43 and 44), an unbranched alkyl sulfur or
oxygen containing group like ꢀSCH3 (58ꢀ61), or ꢀSO2CH3
(62ꢀ65). Of these compounds, 37 (4-CF3-aniline), 60 (4-CF3-
aniline), and 38 (4-SF5-aniline), demonstrated the best potency
against the parasite in whole cell assays (EC50s below 20 nM
against P. falciparum 3D7). For 35ꢀ38, the addition of CF2CH3
to the C2 position improved activity by 4ꢀ40-fold against
PfDHODH and of 25ꢀ240-fold against the parasite compared
to the matched analogues (2ꢀ6) containing a hydrogen at C2
(Figure 2). A similar improvement in potency was observed for
addition of SCH3 at C2 (60). Compounds containing 4-Cl
(35, 39) or 3-Cl (36, 40) aniline showed greater fold improve-
ment in potency against PfDHODH than the 4-CF3 (37) and
4-SF5 (38) anilines, but the larger fold improvement has its
origin in the poorer activity of the parent chloro compounds
(R = H, 5 and 6), and the absolute potency of these compounds
against PfDHODH is similar to those with 4-CF3 and 4-SF5
aniline.
Compounds 37 and 38 were also tested for whole cell activity
in media supplemented with Albumax instead of human serum
because we previously observed these two media gave different
results for compounds containing the 4-SF5 aniline.19 As ob-
served for compound 3, compound 38 also showed 6ꢀ7-fold
better activity against parasites cultured in media supplemented
with Albumax in comparison to human serum, whereas for 37,
results were similar in the two media (Table 1).
Physicochemical Properties and Plasma Protein Binding.
A selection of compounds were chosen for analysis of their
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Table 2. Physicochemical Properties and in Vitro Metabolisma
c
aqueous solubility
human plasma protein bindingb
(%)
CLint human/mouse
Eh
compd
log DpH 7.4
pH 6.5 (μM)
(μL/min/mg protein)
human/mouse
2
2.5
3.0
2.4
2.8
2.8
3.0
3.3
3.2
3.3
3.5
3.0
3.0
3.3
3.6
3.6
3.9
ND
3.7
2.5
2.8
2.7
3.0
3.2
3.5
3.1
3.4
2.2
21ꢀ43
71ꢀ142
ND
86.9b
95.4b
8/ND
0.29/ND
0.20/0.20
0.68/ND
0.42/0.73
0.81/0.85
<0.2/0.39
<0.2/0.42
0.44/0.87
<0.2/0.27
<0.2/0.36
0.47/0.73
0.71/0.76
<0.2/0.31
<0.2/0.26
<0.2/0.22
<0.2/0.23
<0.2/<0.2
<0.2/<0.2
0.27/0.37
0.24/0.38
0.34/0.38
0.39/0.38
0.51/0.77
0.56/0.82
0.28/0.38
<0.2/0.33
<0.2/0.23
3
<5/<6
6
ND
86.1b
86.7b
87.5b
96.4b
84.9b
92.8b
97.7b
87.9b
88.2b
90.4b; 98.8c
97.4b; 99.9c
93.2 b
97.7 b
ND
38.2/ND
12.8/63.1
78.3/125.4
<5/14.4
<5/16.7
14.1/157.7
<5/8.6
11
12
13
14
15
29
30
35
36
37
38
41
42
43
44
50
51
54
55
60
61
70
71
101
170ꢀ350
170ꢀ350
39ꢀ78
34ꢀ66
83ꢀ170
38ꢀ75
16ꢀ32
77ꢀ150
77ꢀ150
70ꢀ140
30ꢀ60
17ꢀ34
15ꢀ30
ND
<5/12.6
16/62.8
44.4/72.2
<5/10.1
<5/8.1
<5/6.5
<5/6.7
<5/<6
30ꢀ60
150ꢀ300
63ꢀ130
71ꢀ140
31ꢀ62
19ꢀ38
7.8ꢀ15
19ꢀ37
4ꢀ8
ND
<5/<6
75.6 b
91.7 b
73.7 b
87.7 b
91.8 b
97.7 b
92.4 b
97.7 b
67.0 b
6.7/13.3
5.6/14.3
9.0/14.3
11.5/14.1
18.7/76.9
22.4/107.5
7.0/14.4
<5/11.3
<5/6.9
19ꢀ37
a Data for 2, 3, and 6 were previously reported.16,19 b Chromatographic method. c Ultracentriguation method. Eh < 0.3 represents low predicted
metabolism, Eh 0.4ꢀ0.7 moderate predicted metabolism, and Eh >0.8 high predicted metabolism. ND, not determined.
physicochemical properties including log D, aqueous solubility,
and plasma protein binding (Table 2). The compounds in the
series all have physicochemical properties that are consistent
with good oral absorption (MW <430, H bond donors <2, and H
bond acceptors <6, polar surface area <90 Å2). The log D for the
series of compounds was found to range from 2.8 to 3.9 and was
typically highest for compounds containing a 4-SF5 aniline.
Aqueous solubility varied from poor to good, and compounds
with 4-SF5 aniline were the least soluble while those with 3- or
4-Cl aniline were among the best. The most potent compounds
(37 and 38) showed mid range solubility and, again, the 4-SF5
aniline (30ꢀ60 μM) derivative was less soluble than the 4-CF3
aniline (70ꢀ140 μM). Human plasma protein binding was
evaluated by two methods: (1) a chromatographic method that
allowed rapid evaluation of a larger number of compounds and
was useful for comparative purposes, and (2) a more rigorous
ultracentrifugation method that provided an accurate absolute
value for assessing total versus free drug concentrations in the
in vivo models. Using the chromatographic method, compounds
in the series typically ranged from 85 to 98% protein bound, with
those containing the 4-SF5 aniline having both the highest log D
and the highest plasma protein binding. For those compounds
(37 and 38) that were evaluated with both methods, the
ultracentrifugation method yielded a higher protein binding
value than the chromatographic method. Protein binding for
38 was found to be very high in both human and mouse plasma
(99.9 and 99.6%, respectively), whereas the binding of 37 was
somewhat lower (98.8 and 97.4%, respectively). For both 37 and
38, protein binding in rat plasma (94.2 and 97.3%, respectively)
was lower than that in either mouse or human plasma, but the
binding of 38 was still higher than that of 37. Protein binding in
the Albumax media used for in vitro activity assessment was not
measured but it is likely that the intrinsic “free” in vitro potency of
compound 38 is extremely high.
In Vitro Metabolic Stability. As an initial screen for good
in vivo pharmacokinetic properties, metabolic stability was
evaluated in vitro using human and mouse hepatic microsomes.
We have previously observed a good correlation between stabi-
lity in microsomal assays and exposure in rodents for the
triazolopyrimidine series.16,19 In this assay, in vitro intrinsic
clearance (CLint) values were measured and used to predict an
in vivo intrinsic clearance and, subsequently, an in vivo hepatic
extraction ratio (Eh) (fraction of compound predicted to be
metabolized on the first pass through the liver) with the
assumption that hepatic metabolism would represent the pri-
mary clearance mechanism. Compounds that contained
ꢀCF2CH3 (37ꢀ38), ꢀCF2CH2CH3 (41ꢀ42), or ꢀCF3
(43ꢀ44) at the C2 position showed low intrinsic clearance
(CLint <6 μL/min/mg microsomal protein in human micro-
somes and <10 μL/min/mg in mouse microsomes) in these
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assays, provided that these C2 R groups were coupled with the
4-CF3 (37, 41, 43) or 4-SF5 (38, 42, 44) aniline (Table 2). The
low intrinsic clearance values suggested that these compounds
would exhibit good metabolic stability in both mice and humans.
Compounds containing 4-Cl (11, 35) or 3-Cl (12, 36) aniline
had much higher intrinsic clearance values, suggesting that they
would be more rapidly metabolized in vivo. Compounds contain-
ing an ꢀCH2CH3 (13 and 14), an ꢀOCH2CH3 (70 and 71),
ꢀcyclopropyl (29 and 30), ꢀCH2OCH3 (50 and 51),
ꢀCH2CH2OCH3 (54 and 55), or ꢀCH2CH2OH (101) at C2
also showed good metabolic stability, again provided that these
C2 R groups were coupled to either 4-CF3 or 4-SF5 rather than to
3- or 4-Cl aniline. However, these compounds were all less
potent than those containing a haloalkyl (37 and 38) group.
Compounds that contained sulfur (60 and 61) were among the
most potent PfDHODH inhibitors in the series, however they
showed poor metabolic stability and were therefore not likely to
show sufficient exposure in vivo to provide efficacy. Thus, the
compounds that provided the best balance between potency and
metabolic stability were identified to be 37 and 38, and these
were selected as the front-runners for further characterization.
Species Selectivity and Evaluation of Cytotoxicity. Select
compounds were evaluated for activity against P. berghei, P. vivax,
and human DHODH (Table 1). The addition of groups larger
than hydrogen at C2 systematically reduced binding affinity to
the P. berghei enzyme; for example, 37 and 38 were 100-fold less
potent on P. berghei DHODH than on the P. falciparum enzyme.
In contrast to the results on PbDHODH, P. vivax DHODH was
inhibited with similar potency to PfDHODH by both 37 and 38
(P. vivax DHODH IC50 = 0.084 and 0.073 μM, respectively),
suggesting that both compounds will have good activity against
P. vivax parasites. Compounds in the series continued to show
excellent selectivity against the human enzyme, and the IC50 for
hDHODH was >100 μM (Table 1) for all of the evaluated
compounds, with the exception of 44, which showed weak
activity against the human enzyme (IC50 = 41 μM).
The two front-runner compounds 37 and 38 were tested
against an expanded panel of P. falciparum strains (Table 3). Both
compounds showed similar potency against drug-sensitive and
drug-resistant (chloroquine and atovaquone) strains. Cytotoxi-
city was assessed against both a mouse (L1210) and human
(HepG2) cell line, and no activity within the tested range (up to
50 μM) was observed for either compound against these
cell lines.
Evaluation of the Antimalarial Mode of Action. To confirm
that cell killing by the DHODH inhibitors results from inhibition
of PfDHODH, we tested the inhibitors against a P. falciparum
D10 cell line transfected with cytoplasmic yeast DHODH
(D10_yDHODH) that uses fumarate instead of mitochondrial
CoQ as the final electron acceptor.26 This cell line has previously
been demonstrated to rescue parasites from the toxicity of both
mitochondrial bc1 complex inhibitors such as atovaquone26 and
PfDHODH inhibitors such as our triazolopyrimidines.13,27 Addi-
tion of proguanil has been shown to restore sensitivity of
D10_yDHODH to bc1 complex inhibitors but not to specific
PfDHODH inhibitors. The D10_yDHODH cell line is resistant
to both 37 and 38, and this result was independent of the
presence of proguanil in the culture media, demonstrating that
the antimalarial effect of these compounds is due to inhibition of
PfDHODH. Representative data for 38 are shown in Figure 2.
X-Ray Structure Determination of PfDHODH in Complex
with 37. The X-ray structure of PfDHODH in complex with 37
Table 3. Comparison of P. falciparum Cell Lines and
Mammalian Cytotoxicity Dataa
compd
38 EC50 (μM)
37 EC50 (μM)
P. falciparum 3D7 (μM) 0.043 (0.034ꢀ0.055) 0.013 (0.012ꢀ0.014)
P. falciparum D6 (μM) 0.022 (0.014ꢀ0.034) 0.012 (0.011ꢀ0.013)
P. falciparum K1 (μM)
0.057 (0.048ꢀ0.068) 0.016 (0.014ꢀ0.017)
0.0079 (0.0072ꢀ0.0086)
P. falciparum HB3 (μM) 0.027 (23ꢀ31)
P. falciparum Dd2 (μM) 0.057 (0.041ꢀ0.079) 0.013 (0.012ꢀ0.014)
P. falciparum TM90C2B 0.015 (0.013ꢀ0.018) 0.0046 (0.0013ꢀ0.016)
P. falciparum TM90C2A 0.031 (0.023ꢀ0.033) 0.0079 (0.0076ꢀ0.0081)
mouse L1210
>50
>50
human Hep G2 (μM)
>50
>50
a Additional P. falciparum strains that were tested include drug sensitive
strains (D6, HB3, and NF54), chloroquine resistant strains (Dd2)
chloroquine and pyrimethamine resistant (K1), and atovoquone resis-
tant strains (TM90C2B and TM90C2A). Data were collect on strain
3D7 as a comparator. Data were collected in triplicate and values in
parentheses represent the 95% confidence interval. Data were collected
using human serum containing media.
was solved to 2.95 Å resolution (Supporting Information Table
S1, Figure S1). Both the triazolopyrimidine ring and the aniline
ring superimpose closely onto the position of 2 (pdb 3I6R),22
although there is a slight shift of both rings away from FMN and
toward Phe188 (rmsd = 0.4 Å) (Figure 3A). The key interactions
are preserved, including an H-bond between His185 and the
pendant nitrogen that bridges between the triazolopyrimidine
ring and the phenyl ring and an H-bond between Arg265 and the
pyrimidine nitrogen N4. The CF2CH3 R-substituent of 37 binds
into the channel that extends from the triazolopyrimidine ring
toward the FMN and makes good van der Waals interactions
throughout the pocket, which is composed of Ile263, Ile272, the
hydrophobic portion of the Arg265 side chain, and of the
hydroxyl and ring of Tyr528 (Figure 3B). Thus the addition of
the ꢀCF2CH3 R-substituent to the scaffold provides key hydro-
phobic interactions with the enzyme in the one significant space
that was left unoccupied by the previous analogues from the
series. Furthermore, the electron withdrawing effect of the
fluorine atoms will reduce electron density on the nitrogens
within the 5-membered triazole ring. This may, therefore, con-
tribute to the potency increase as a result of decreased desolva-
tion. These observations provide a structural explanation for the
improved potency of these analogues. The structure also pro-
vides insight as to why R groups that are larger than propyl are
inactive (e.g., 19ꢀ22), as the pocket has insufficient room to
accommodate a branched chain functionality as large as iso-butyl.
The complete inactivity of R groups containing an amine
functionality is explained by the hydrophobicity of the R-pocket
and the lack of H-bonding partners in the R-pocket except for
lone pair electrons of the Tyr528 side chain hydroxyl. Finally, the
X-ray structure provides insight into the poor binding of 37 to
PbDHODH. The replacement of Gly181 with Ser in
PbDHODH may lead to a restriction in the size of the R-group
that can be accommodated by this enzyme.
Pharmacokinetic Properties in Mice and Rats. To further
assess the potential for 37 and 38 to be selected as clinical
development candidates, their pharmacokinetic properties were
assessed in mice and rats. In noninfected mice, 37 and 38 showed
good exposure following single dose oral administration (Figure 4
and Table 4B). Maximum plasma concentrations (Cmax) and
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Figure 3. X-ray structure determination of PfDHODH in complex with 37. (a) Structural alignment of PfDHODHΔ384ꢀ413ꢀ37 (green) in comparison
to PfDHODH bound with 2 (PDB 3I6R) (pink). Residues within 4 Å of the bound inhibitor are displayed. L-Orotate and the FMN cofactor are also
displayed. (b) van der Waals surface representation of PfDHODH bound with 37. The enzyme surface is shown in pink, and orotate, FMN, and 37 are
shown in green as sticks. Structures were displayed using PyMol.
Figure 4. Pharmacokinetic analysis of (A) 38 and (B) 37 after oral dosing in mice (n = 1 mouse per time point). Plots represent plasma concentration
versus time after a single dose. Dose levels and route are shown in the graph legend.
AUC0ꢀinf values of 38 increased approximately in proportion
to the increase in dose over the range 0.4ꢀ10 mg/kg range,
however the relative increase was somewhat reduced at 50 mg/kg
dose. Compound 37 exhibited dose proportional increases in
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Table 4. In Vivo Pharmacokinetic Data for Key Triazolo-
pyrimidines in Micea
Table 5. In Vivo Pharmacokinetic Properties for 37 and 38 in
Rats (Average of n = 2 per Dose Level)
parameter
37
38
(A) compounds 37 and 38 evaluated after a single
oral dose of 0.4, 2, 10, or 50 mg/kg
iv dose (mg/kg)
1; 4.5
0.9; 2.6
3.6; 6
clearance (mL/min/kg)
t1/2 (h)
23.2; 12.6
6.1; 3.8
3.3; 2.9
2.0 ; 21.2
60; 195
0.9; 11.4
97; >100
6; 17
parameter
37
38
10.1; 12.6
po dose (mg/kg)
t1/2 (h)
0.4, 2, 10, 50
2.1, 1.9, 1.6, cnc
0.20, 1.85, 9.86, 57
0.5, 1, 4, 2
0.4, 2, 10b, 50
4.1, 3.5, 2.5, 3.2
0.06, 0.44, 3.1, 9.53
0.5, 1.0, 1.5, 2.0
0, 0, 8, 6
volume of distribution (L/kg)
po dose (mg/kg)
Tmax (min)
2.9; 5.9
2.1; 18.8; 46.7
195; 420; 1020
0.75; 3.9; 6.4
66; 57; 61
Cmax (μM)
Tmax (h)
Cmax (μM)
time TC>1 μM (h)
0, 6, 14, 14
bioavailability (F%)
t1/2 (h)
plasma AUC0ꢀinf (μM min) 40, 569, 4207, 14815c 27, 163, 1357, 2028
3
11.6; 15.5; 27.9
929; 7292; 19305
0.7 ꢀ 0.8
plasma protein binding (%) 97.4
99.6
AUC0ꢀinf (μM min)
233; 20492
0.7 ꢀ 0.8
94.2
3
blood:plasma
(B) compounds 13, 41, 70, and 71 after a single oral dose of 10 or 20 mg/kg
plasma protein binding (%)
97.3
parameter
13
20
41
70
20
71
38, 4 has similar activity against PfDHODH and PbDHODH,
and thus was able to be evaluated in both the SCID mouse and
the P. berghei mouse models. Chloroquine was also included as a
control to represent a standard antimalarial agent with known
behavior in the SCID mouse model.
po dose (mg/kg)
t1/2 (h)
10
3.1
3.5
30
4
20
2.7
1.1
60
3
8.4
12.6
60
1.4
5.8
60
Cmax (μM)
Tmax (min)
time TC>1 μM (h)
5
6
Compounds (4, 37, and 38) were dosed orally 1ꢁ daily for 4
days beginning 3 days after mice were infected with parasites.
The ED50s were determined 24 h after the final dose as
described.28 Both 37 and 38 markedly inhibited parasitemia in
peripheral blood of mice (ED90 = 25.9 and 8.1 mg/kg, re-
spectively), with 38 showing efficacy at doses in the order of
chloroquine (ED90 = 4.9 ( 0.5 mg/kg) and artesunate (12.7 (
1.3 mg/kg)28 (Table 6 and Figure 6). Compound 38 has a clear
potency advantage over 37, however both were more potent than
4 (by 7-fold and 2-fold respectively) and both meet our devel-
AUC0ꢀinf (μM min)
1624
652
1241
608
3
a cnc, could not calculate. b Values at 10 mg/kg are the average for
2 independent experiments. c AUC0ꢀ48 h
.
Cmax and AUC0ꢀinf across the full dose range (0.4ꢀ50 mg/kg).
Four other compounds (13, 41, 70, and 71) with good potency
(EC50 < 0.1 μM) against the parasite and good metabolic stability
were also tested to evaluate plasma exposure in mice (Table 4B).
Compounds 13 and 70 showed similar exposure to 37 and 38 as
evaluated by both Cmax and AUC0ꢀinf, while compounds 41 and
71 had somewhat lower exposure.
opment criteria for a preclinical candidate (defined as an ED90
<
30 mg/kg in this model). Interestingly, aberrant late tropho-
zoites/early schizonts were detected in peripheral blood of mice
treated effectively with compounds 37 and 38, which is consis-
tent with a mechanism of action affecting parasite metabolism.
The comparator compound 4 showed similar efficacy in the
SCID model (ED50 = 19 mg/kg) to what was observed pre-
viously in the P. berghei model (ED50 = 10 mg/kg).19
A pharmacokinetic study in uninfected and infected SCID
mice engrafted with human erythrocytes was performed in order
to interpret the efficacy results. The oral exposure of 4, 37, and 38
upon po administration at 10 mg/kg indicated that infection did
not significantly change compound disposition of compounds
(Table 6). Interestingly, the overall exposure in whole blood for
the three compounds studied was similar, which suggest that
differences in intrinsic potency rather than changes in pharma-
cokinetic parameters are most likely responsible for the differ-
ences in efficacy detected in vivo.
The pharmacokinetic properties of 37 and 38 in rats were
examined after both intravenous (iv) and oral (po) administration
(Table 5 and Figure 5). After iv dosing, 38 had low clearance
(4ꢀ6 mL/min/kg), moderately high volume of distribution (3ꢀ
6 L/kg), and a long half-life (10ꢀ13 h), whereas 37 exhibited
higher clearance (13ꢀ23 mL/min/kg), similar volume of distribu-
tion (3 L/kg), and a shorter half-life (4ꢀ6 h). Neither compound
was excreted unchanged in urine. After oraldosing, both 37 and 38
demonstrated good oral bioavailability and long half-lives (Table 5
and Figure 5), however the increase in both the time to reach the
maximum concentration (Tmax) and the half-life with increasing
dose suggested a slower rate of absorption with increasing dose.
Compound 38 showed dose-proportional kinetics over the oral
dose range of 2ꢀ50 mg/kg, while for 37, there was evidence for
dose-dependency over the range of 2ꢀ20 mg/kg.
In Vivo Efficacy of 37 and 38 in the P. falciparum SCID
Mouse Model. The observation of good bioavailability of 37 and
38 in mice and rats led us to test these compounds in an in
vivo efficacy model. The poor activity of 37 and 38 against
PbDHODH prohibited the use of the P. berghei mouse model to
evaluate their in vivo efficacy. To overcome this issue, 37 and 38
were tested against the human malaria parasite P. falciparum in
the humanized SCID mouse model.28 To provide a direct
comparator to our prior studies that used the P. berghei mouse
model, compound 4 (in vivo mouse P. berghei ED50 = 10 mg/kg
for 4)19 was included in the SCID mouse study. Unlike 37 and
’ DISCUSSION
The burden of malaria falls disproportionately on the world’s
poorest countries and, while effective treatments are in place, the
continual challenge is to stay ahead of the parasite’s unyielding
ability to select for drug resistance. The identification of new
chemical classes with different modes of action than existing
drugs and which also show good drug-like properties and efficacy
against the parasite is the key to treatment of the disease.
Through a structure-guided lead optimization program that
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Figure 5. Pharmacokinetic analysis of (A) 38 and (B) 37 after oral (po) or iv dosing in rats (n = 2 rats per dose route). Plots represent plasma
concentration versus time after a single dose. Dose levels and route are shown in the graph legend.
Table 6. In Vivo Efficacy and Pharmacokinetic Properties in
the SCID Mouse P. falciparum Model after Oral Dosing
(Average of n = 3 Mice Per Compound)a
and drug resistant strains of the parasite, all of which are
necessary to meet the target product profile.11 These compounds
represent a significant breakthrough in potency compared to
previously identified compounds from this series, and they
represent the first that are potent enough to be progressed for
clinical development.
4
37
38
ED50/ED90 (mg/kg)
po dose (mg/kg)
blood AUC(0ꢀt)#
19/59
10
9.6/26
5.7
2.8/8.1
7.0
The triazolopyrimidines were identified early in our program
to develop PfDHODH inhibitors for the treatment of malaria,
however previous compounds from the series have either lacked
metabolic stability (1) or have shown insufficient potency against
the parasite (2ꢀ4).16,17,19 The X-ray structure of 1 and 2 bound
to PfDHODH showed that a narrow channel leading from the C2
position of the triazolopyrimidine ring toward the FMN cofactor
afforded the best chance to build additional functionality into the
molecule to improve potency.22 We have systematically evalu-
ated a series of alkyl, haloalkyl, ether, and amine substituents in
this position, leading to the identification of several functional
groups that provided the desired boost in potency. Most of the
best functional groups at C2 are electron withdrawing and
hydrophobic, and the rank order of potency against both the
enzyme and the parasite in whole cell assays was ꢀCF2CH3,
ꢀSCH3 > ꢀCF3, ꢀCF2CH2CH3 > ꢀOCH2CH2 > ꢀCH2CH3.
Larger substituents and branched chain or cyclic groups were less
active. These results match well with the observation from the
X-ray structure of 37 bound to PfDHODH, which shows that the
C2 binding pocket is narrow and dominated by hydrophobic
residues. As was observed in prior compounds that contain
hydrogen at the C2 position,19 anilines substituted with 4-CF3
or 4-SF5 provided the best balance between potency and good
metabolic stability. Compounds with 4-Cl or 3-Cl showed
somewhat lower potency and were notably less metabolically
stable.
3848/3173 2521/2740 3469/3427
uninfected/infected (μM min)
3
C
max uninfected/infected (μM)
8.5/8.2
2.0/3.3
6.3/6.3
2.7/1.7
6.1/5.3
4/6.7
Tmax uninfected/infected (h)
a ED50 for chloroquine control 3.2 mg/kg. #t = 10 h after dosing for 4 and
37 and 24 h after dosing for 38. This time point represents the last time
point where compounds were detectable in whole blood of animals.
Figure 6. Efficacy of 38 in SCID mice infected with P. falciparum after
oral dosing. Mice were infected with parasites on day zero and a single
oral dose/day was given on days 3, 4, 5, and 6. The limit of detection of
the assay is 0.01% parasitemia. Dose levels are shown in the graph
legend.
In contrast to the good activity of the haloalkyl substituents, all
compounds containing amines at C2 were inactive. The X-ray
structure analysis shows that except for the hydroxyl oxygen of
Tyr528, there are no H bond acceptors in the pocket that would
bind a C2 substituent. In the absence of a major ligand induced
reorganization of the pocket, the Tyr528 side chain oxygen is
geometrically inaccessible to any potential hydrogen bonding
group such as an amine directly linked through a tetrahedral
carbon attached to the triazolopyrimidine at C2. The addition of
alcohols at C2 also led to reduced activity. These compounds
(particularly ꢀCH2CH2OH at C2) were designed to displace the
bound water molecule (W15) observed to bind between the
integrated pharmacokinetic and metabolism assessment into the
medicinal chemistry plan, we have identified two potent
PfDHODH inhibitors (37 and 38) from the triazolopyrimidine
series that meet previously established progression criteria. The
antiparasitic activity of these compounds in the humanized SCID
mouse P. falciparum model (ED90 < 30 mg/kg) suggests that
both compounds show good in vivo efficacy. Parallel pharmaco-
kinetic studies in mice and rats demonstrated sustained plasma
concentrations after oral dosing consistent with once daily
dosing, and both compounds are active against drug sensitive
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Tyr528 hydroxyl and N3 on the triazolopyrimidine in the X-ray
structure of PfDHODH bound to 122 and to test the hypothesis
that the resulting gain in entropy would improve binding affinity.
However, compounds with ꢀCH2OH (98 and 99) and
ꢀCH2CH2OH (101) at C2 were less active then the parent
compounds 2, 5, and 6, suggesting that even if the bound water
was displaced, this mechanism did not provide additional binding
energy possibly owing to the energetic cost of desolvating the
hydroxyl of 101 upon binding to PfDHODH.
The most potent of the analogues with ꢀCF2CH3 at C2
(37 and 38) were identified as the front-runners and evaluated in
a series of additional assays to determine their development
potential. These compounds showed 25ꢀ50-fold better activity
against P. falciparum 3D7 cells in whole cell assays than that of
compounds containing hydrogen at C2. This improvement in
potency was achieved while remaining on target, as parasites
transfected with yeast DHODH were resistant to both inhibitors
as would be expected if the cell killing occurred via DHODH
inhibition. Both compounds also showed good activity against
PvDHODH, suggesting that they will also be useful to treat
P. vivax infections, and both were fully active against drug
resistant strains of the parasite, including atovaquone and
chloroquine resistant strains.
Compounds 37 and 38 exhibited good metabolic stability
when evaluated in hepatic microsomal assays, and pharmacoki-
netic profiles confirmed low clearance and good oral bioavail-
ability in both mice and rats leading to high and extended plasma
exposure profiles. In rats, compound 37 showed significant dose
dependency in the kinetic profile even over a relatively low dose
range. The greater than proportional increase in AUC0ꢀinf for 37,
and the corresponding apparent bioavailability of >100% at the
higher dose, suggested saturation of the clearance pathway(s)
with increasing dose for this compound. In comparison, 38
exhibited reasonably dose proportional kinetics in rats over a
broad dose range. The delay in Tmax and prolongation of the half-
life with increasing dose for both 37 and 38 suggests a dose-
dependent reduction in the rate of absorption, possibly due to
dissolution rate-limited absorption resulting from the relatively
low aqueous solubility (Table 2). Importantly, the low solubility
did not appear to limit the extent of absorption based on the high
bioavailability for both compounds.
Compounds 37 and 38 required a significantly lower dose to
clear parasites in a SCID mouse model than prior compounds
from the triazolopyrimidine series, and this finding can be
attributed to the combination of the significantly improved
potency and the good plasma exposure profile. For 38, the dose
required (ED50/ED90 2.8/8.1 mg/kg) was similar to that for
chloroquine examined in the same study, and these values were
7-fold lower than for the previous most efficacious compound
from the series (4) when tested in the same SCID mouse model.
The matched analogue of 38 containing a hydrogen at C2 (3) has
not been tested in this model. However, because 3 and 4 have
similar activity against PbDHODH and PfDHODH, they were
both previously tested for efficacy in the P. berghei mouse
model.19 In this experiment the ED50 for 3 was 2-fold higher
than that for 4, suggesting that the addition of the CF2CH3 group
to C2 may have improved in vivo efficacy by up to 14-fold (e.g.,
38 was 7-fold more potent than 4 in the SCID model, and 4 is
2-fold more potent than 3 in the P. berghei model). These data
also differentiate these compounds from the reported N-alkyl-
5-(1H-benzimidazol-1-yl)thiophene-2-carboxamides where the
ED50 in the P. berghei in vivo model for the most potent of these
compounds (Genz-667348) was 2ꢀ6-fold higher (depending
on the parasite strain) and required BID dosing,21 in comparison
to the results obtained for 38 where QD dosing was used.
Results for the plasma protein binding studies with 37 and 38
indicate that the binding of 38 was higher than that of 37 in each
of the species tested (e.g., human, rat and mouse), meaning that
the free fraction of 38 would be lower than that of 37. While
binding in the Albumax media used for the in vitro cell-based
assay has not been assessed, the plasma protein binding trends
would suggest that 38 would also exhibit higher binding, and
lower free concentrations, in the Albumax media. Because 37 and
38 had similar activity in the P. falciparum cell-based assay, a
lower free fraction for 38 may indicate that it is actually more
potent than 37 if the free concentrations are considered. This
hypothesis, combined with the longer half-life and more ex-
tended exposure profile for 38 measured in normal mice, may
explain the difference in potency for the two compounds in the
SCID mouse model despite their similar measured whole blood
concentrations.
On the basis of the better in vivo efficacy and the observation
of more linear pharmacokinetics in rats, 38 has been chosen as
the lead compound for further workup toward clinical candidate
status. While none of the other compounds with potency in the
same range as 37 and 38 (EC50 < 20 nM in the whole cell
P. falciparum assay) were metabolically stable, compounds 13
and 70 were the next best compounds with respect to the
combination of potency and metabolic stability. Both com-
pounds also showed good exposure after oral dosing in non-
infected mice and thus might serve as backup compounds to the
identified front-runner (38).
’ CONCLUSION
We have identified a compound (38) from the triazolopyr-
imidine series of PfDHODH inhibitors that meets development
criteria, showing efficacy against P. falciparum in vivo in a murine
model, activity against drug resistant parasite strains, and activity
against PvDHODH. Importantly, the excellent in vivo efficacy
was obtained with QD dosing, attributable to the long half-life
and excellent oral exposure, and suggesting that 38 will be able to
meet the target product profile of cure in three consecutive daily
doses in patients. Final validation of the development potential of
38 will require toxicological studies in animals and the successful
clinical proof of concept of a DHODH inhibitor for the treat-
ment of malaria in man.
’ MATERIALS AND METHODS
Determination of Enzyme Inhibition Constants. The plas-
mids used for the E. coli expression of His-tagged PfDHODH,
PbDHODH, and hDHODH have been previously described.16ꢀ18
The P. vivax DHODH expression construct was generated as follows.
A 1266-bp DNA fragment of PvDHODH was amplified by PCR from
genomic P. vivax strain Belem DNA provided by John Barnwell (CDC)
using primers 50- GTA GTA GCT CTA TAC ATG TAT TTC GAG
TCC TAC GAC CCC G-30 and 50-CTC GAG GGC GGC CCG CCG
GTG GGC CCG CCC GAC GGC GT-30 and ligated into Zero Blunt
TOPO vector (Invitrogen). QuickChange site-directed mutagenesis kit
(Stratagene) was used to remove an internal NdeI site from PvDHODH
using primers 50-TGT CTA CTC ACA TGA TTT CTC AAA TG-30and
50-CAT TTG AGA AAT CAT GTG AGT AGA CA-30. The resultant
clone was used to PCR amplify the PvDHODH region introducing XhoI
and NdeI restriction sites, respectively, with primers 50-TGG AAT TCG
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CCC TCG AGG GCG GCC CGC CGG-30 and 50-GTA GTA GCT
CTA CAT ATG TAT TTC GAG TCC TAC GAC-30. This PCR
product was ligated into Zero Blunt TOPO vector (Invitrogen) and
digested with XhoI and NdeI to excise the DHODH coding sequence,
which was then ligated to pET22b expression vector (Novagen) to
generate the C-terminal 6xHis-tagged truncated PvDHODH construct
used for recombinant protein expression. All four proteins were
expressed as soluble truncated proteins in the absence of the N-terminal
domain that includes the membrane-spanning region of the protein.
Proteins were purified as previously described by Ni2+-agarose chroma-
tography and gel filtration.17,18
structures of PfDHODH bound to 37 versus 2 was calculated using
DaliLite.34
In Vitro Assay of P. falciparum. Parasites were grown in Gibco-
Invitrogen RPMI-1640 supplemented with 20% human type A+ plasma
and 2% (w/v) red blood cells35 or with Gibco-Invitrogen 0.5% Albumax I.
Before transferring cultures to media with in Albumax, cells were
washed three times in RPMI-1640 to remove plasma-supplemented
medium. The D10 cell line containing yeast DHODH was cultured in
the absence and in the presence of proguanil (5 μM). Low-passage
L1210 mouse leukemia cells (American Type Culture Collection) were
also grown in plasma-supplemented RPMI-1640, while HepG2 human
hepatocarcinoma cells (American Type Culture Collection) were grown
in F12/DMEM (1:1). Blood products were obtained from Biochemed
Services, Virginia. [3H]-hypoxanthine uptake was used to measure cell
growth, and incorporation of no-drug samples was compared to drug-
treated cells as described previously.36 Data were fitted to the log[I] vs
response ꢀ variable slope (four parameter) model in Graph Pad Prism
to determine the ED50’s for inhibition of parasite growth.
Steady-state kinetic assays to determine the IC50s for inhibitors were
performed as previously described.17,18 Briefly, a dye-based assay that
couples the final oxidation of CoQ to the reduction of 2,6-dichloroin-
dophenol (DCIP) was followed at 600 nm (ε = 18.8 mMꢀ1 cmꢀ1).
Enzyme stocks were diluted into assay buffer containing 0.1 mM BSA to
make a 100ꢁ working stock solution, which was kept on ice. Assays were
initiated by adding 5 μL of this stock solution to 500 μL of assay buffer
containing substrates and inhibitors. Conditions were as follows:
DHODH (ET = 5ꢀ10 nM), substrates (0.2 mM L-dihydroorotate and
0.02 mM CoQD), DCIP (0.12 mM), and assay buffer (100 mM HEPES,
pH 8.0, 150 mM NaCl, 10% glycerol, 0.1% Triton) at 20 ꢀC. Inhibitor
concentration was varied in a 3-fold dilution series (0.01ꢀ100 uM). The
percent inhibition relative to the no inhibitor control was determined
(vi/vo ꢁ 100), and data were fitted to the log[I] vs response (three
parameters) equation in Graph Pad Prism to determine the IC50.
X-Ray Structure Determination of PfDHODHΔ384ꢀ413ꢀ37.
Protein Purification. Protein for crystallization was expressed from a
different construct than that used for the inhibition studies. Deletion of a
surface loop (Δ384ꢀ413) was necessary to generate a protein that could
be crystallized, and both the construct and the purification procedure
have been previously described.21,22 These prior studies also showed
that deletion of this loop had no effect on enzyme activity. Preliminary
crystallization conditions were found using the random crystallization
screen AmSO4 suite (Nextal). The initial conditions were refined by
variation of pH, precipitant, and protein concentrations to find optimal
conditions. Crystallizations were set up using the hanging drop vapor
diffusion method at 20 ꢀC. Crystals of PfDHODHΔ384ꢀ413ꢀ37 com-
plex were obtained by mixing reservoir solution (1.64ꢀ1.74 M ammo-
nium sulfate, 0.1 M sodium acetate, pH 4.1, and 10 mM DTT) with an
equal volume of PfDHODHΔ384ꢀ413 (20 mg/mL) pre-equilibrated with
0.5 mM 37 and 2 mM dihydroorotate. Crystals typically grew in
4ꢀ7 days.
Physicochemical Properties. Partition coefficients (log DpH 7.4
)
were estimated by comparing their chromatographic retention proper-
ties to a set of standard compounds with known partition coefficients.
Data were collected using a Waters 2795 HPLC instrument with a
Waters 2487 dual channel UV detector with a Phenomenex Synergi
Hydro-RP 4 μm (30 mm ꢁ 2 mm) column. The mobile phase was
aqueous buffer (50 mM ammonium acetate, pH 7.4) and acetonitrile
with an acetonitrile gradient of 0ꢀ100% over 10 min. Compound
elution was monitored at 220 and 254 nm.
Aqueous solubility was estimated by nephelometry. Compound in
DMSO was spiked into either pH 6.5 phosphate buffer or 0.01 M HCl
(approximately pH 2.0), with the final DMSO concentration being 1%.
Samples were then analyzed by nephelometry to determine the
solubility range as described previously.37
Plasma protein binding was assessed using two different methods: (1)
chromatographic retention on a human albumin column, or (2) by
ultracentrifugation. The chromatographic method utilized a human
albumin column (ChromTech Chiral-HSA 50 mm ꢁ 3.0 mm, 5 μm,
Sigma-Aldrich) and compared retention characteristics to a series of
standard compounds with known human plasma protein binding values
using a modification of a published method.38 A Waters 2795 HPLC
system equipped with a Waters 2487 dual channel UV detector
(monitored at 220 and 254 nm) was used with a mobile phase comprised
of aqueous buffer (25 mM ammonium acetate buffer pH 7.4) and 30%
isopropyl alcohol in the same buffer. The isopropanol concentration was
varied over a 10 min gradient, and the column was reconditioned prior to
the next injection.
Measurement of plasma protein binding by ultracentrifugation was
performed using human, rat, and mouse plasma. Human blood was
obtained from the Australian Red Cross Blood Service, rat blood from
male SpragueꢀDawley rats, and mouse blood from male Swiss outbred
mice. Plasma was separated from whole blood by centrifugation and
stored frozen at ꢀ80 ꢀC (human) or ꢀ20 ꢀC (rat and mouse).
Compound stock solutions were prepared in DMSO (1 mg/mL) and
then further diluted in 50% (v/v) acetonitrile (20 and 200 μg/mL).
Plasma at 37 ꢀC was spiked with compound stock solutions to give
concentrations of 200 and 2000 ng/mL (maximum final DMSO and
acetonitrile concentrations were 0.2% and 0.5%, respectively), followed
by equilibration at 37 ꢀC for 1 h and ultracentrifugation (Beckman Rotor
type 42.2 Ti; 223000g and 37 ꢀC) for 4.2 h to separate plasma proteins
from plasmaꢀwater. An aliquot of supernatant (i.e., plasmaꢀwater) was
taken from each tube to determine free concentration. The total
concentration was determined in noncentrifuged plasma samples simi-
larly incubated. The concentration of test compound in samples was
determined by LC-MS using calibration standards prepared in the
respective matrices. The percentage of compound bound to plasma
Diffraction data were collected at 100K on beamline 19ID at
Advanced Photon Source (APS) using an ADSC Q315 detector. The
crystal of PfDHODHΔ384ꢀ413ꢀ37 diffracted to 2.95 Å and has a space
group of P64 with the cell dimension of a = b = 86.6, c = 138.2. The
structure contains only one molecule of PfDHODH in the asymmetric
unit. Diffraction data were integrated and intensities were scaled with
HKL2000 package.29
Crystallographic phases for PfDHODH inhibitors were solved by
molecular replacement with Phaser30 using the previously reported
structure of PfDHODHΔ384ꢀ413 (PDB ID 3I6522) with ligands removed
as the search model. Structures were rebuilt with COOT,31 refined with
REFMAC32 to R and Rfree of 0.236 and 0.285, respectively (Table S2 and
Figure S1, Supporting Information). The final structure contains
residues 161ꢀ347, 355ꢀ383, and 414ꢀ565 and 5 water molecules.
Electron density was not observed, for loop 348ꢀ354 is missing. All
residues in the final structure were within the allowed section of the
Ramachandran plot (Table S2, Supporting Information). Water mol-
ecules were added if the density was stronger than 3.4 σ and removed if
the density was weaker than 1 σ in the density map with ARP/warp.33
Molecular Modeling. Structures were displayed using PyMol
(DeLano Scientific LLC, San Carlos, CA). The rmsd between the
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proteins (% bound) was calculated according to the following equation:
%bound = 100(Cplasma ꢀ Cplasmaꢀwater)/Cplasma, where Cplasma was the
concentration observed in the noncentrifuged plasma 37 ꢀC sample and
Cplasmaꢀwater was the plasmaꢀwater phase following centrifugation.
In Vitro Human and Mouse Metabolism. Human and mouse
liver microsomes (BD Gentest, Discovery Labware Inc., Woburn,
Massachusetts) were suspended in 0.1 M phosphate buffer (pH 7.4)
at a final protein concentration of 0.4 mg/mL and incubated with com-
pounds (1 μM) at 37 ꢀC. An NADPH-regenerating system (1 mg/mL
NADP, 1 mg/mL glucose-6-phosphate, 1 U/mL glucose-6-phosphate
dehydrogenase) and MgCl2 (0.67 mg/mL) was added to initiate the
metabolic reactions, which were subsequently quenched with ice-cold
acetonitrile at time points ranging from 0 to 60 min. Samples were also
incubated in the absence of the NADPH-regenerating system to monitor
for noncytochrome P450-mediated metabolism in the microsomal
matrix. Samples were then subjected to centrifugation, and the amount
of parent compound remaining in the supernatant was monitored by
LC-MS. The first-order rate constant for substrate depletion was
determined by fitting the data to an exponential decay function, and
these values were used to calculate the in vitro intrinsic clearance value
(Clint) and the predicted in vivo intrinsic clearance value (CLint vivo) as
described.39 The predicted in vivo hepatic extraction ratio (Eh) was
calculated using the following relationship: Eh = CLint vivo/(Q +
CLint vivo), where Q is liver blood flow (20.7 mL/min/kg, 55.2 mL/
min/kg and 90 mL/min/kg in for humans, rats and mice, respectively).40
Pharmacokinetic Analysis. Animal studies were performed in
accordance with the Australian Code of Practice for the Care and Use of
Animals for Scientific Purposes, and the study protocol was approved by
the Monash Institute of Pharmaceutical Sciences Animal Ethics
Committee.
Systemic exposure was studied following oral administration of
compounds to nonfasted male Swiss outbred mice after oral adminis-
tration and to overnight fasted male SpragueꢀDawley rats after iv and
oral administration. Mice were given unrestricted access to food and
water throughout the pre- and postdose phases of the study. Rats were
given unrestricted access to water throughout the pre- and postdose
sampling period, and access to food was reinstated 4 h postdose. For oral
dosing, compounds were administered to mice and rats in an aqueous
suspension vehicle (0.5% w/v sodium caboxymethylcellulose, 0.5% v/v
benzyl alcohol, and 0.4% v/v Tween 80; 0.1 mL dose volume per mouse,
n = 1 mouse per time point; 1.0 mL per rat, n = 2 rats per compound) by
gavage. After the oral dose was administered to rats, the dosing tube was
rinsed with 1 mL of Milli-Q water to collect any residual formulation,
and the rinse was also administered. For intravenous dosing to rats,
compounds were administered in an aqueous solution vehicle as a 10
min constant rate infusion into the jugular vein at two dose levels
(1.0 mL per rat, n = 2 rats per dose level for each compound).
At specific time points following oral dosing to mice, animals
were anaesthetized with gaseous isoflurane and a single blood
sample was collected via cardiac puncture. For sample collection
from rats, samples of arterial blood were collected via an in-dwelling
carotid cannula. Blood samples collected from mice and rats were
immediately transferred to heparinized tubes that contained a
stabilization cocktail (Complete inhibitor cocktail, potassium fluor-
ide, and EDTA) to minimize the potential for ex vivo degradation of
the test compound. Plasma for analysis was then collected by
centrifugation.
Pharmacokinetic parameters were calculated using noncompartmen-
tal methods. The terminal elimination half-life (t1/2), the area under the
plasma concentration versus time profile from time zero to either the last
sample time point (AUC0ꢀ24 h) or extrapolated to infinity (AUC0ꢀinf),
plasma clearance, and volume of distribution at steady-state were
determined using WinNonlin software (version 5.2.1, Pharsight Cor-
poration, Mountain View, CA). The maximum plasma concentration
(Cmax), the time to reach the maximum concentration (Tmax), and the
time plasma concentrations remained above 1 μM (TC>1 μM) were taken
directly from the concentration versus time profiles. Oral bioavailability
(%F) was estimated by comparing the average dose-normalized
AUC0ꢀinf after oral administration to the average dose-normalized
AUC0ꢀinf after iv administration.
P. falciparum in Vivo Efficacy Model and Pharmacokinetic
Analysis. All the experiments were approved by the DDW Ethical
Committee on Animal Research, performed at the DDW Laboratory
Animal Science facilities accredited by AAALAC, and conducted accord-
ing to European Union legislation and GlaxoSmithKline policy on the
care and use of animals.
Compound efficacy was evaluated against P. falciparum Pf3D70087/N9
propagated in NOD-scid IL-2Rγnull mice that were engrafted with
human erythrocytes as previously described.41 Mice (n = 3) were
infected on day 0 with 2 ꢁ 107 parasites and compound was adminis-
tered QD on days 3ꢀ6 post infection by oral gavage in an aqueous
suspension vehicle (0.5% w/v sodium caboxymethylcellulose, 0.5% v/v
benzyl alcohol, and 0.4% v/v Tween 80) at 20 mL/kg. Control animals
received vehicle alone. ED50 and ED90 were estimated by fitting a four-
decade logistic equation for the log10 of parasitemia at day 7 versus log10
of the dose administered using GraphPad Prism 5.0 (GrahPad Soft-
ware). Parasitemia was assessed by FACS as previously described.28
In a parallel study, pharmacokinetic properties were also evaluated in
the SCID mouse model both in the presence and absence of infection.
Compound was administered in a single oral dose of 10 mg/kg and,
whole blood levels were followed for 24 h. Analytical methods for the
detection of compounds 37 and 38 were as described above. Groups of
3 animals received a single 10 mg/kg oral dose of compound formulated
in water/0.5% carboxymethylcellulose/0.4% Tween 80/0.5% benzyl
alcohol. Blood samples were collected over 48 h postdosing by puncture
of the lateral tail vein and mixed 1:1 with deionized water 0.1% saponin
and stored frozen at ꢀ70 ꢀC until analysis. After protein precipitation
and liquid/liquid extraction, the samples were assayed by LC/MS using
ESI conditions by selected ion monitoring in an API 4000 mass
spectrometer (Applied Biosystems Sciex, Foster City, CA) coupled to
a HPLC chromatograph (Agilent HP1100 series, Agilent Technologies,
Spain). Quantification was conducted by comparison to calibration
curves prepared in blood. Blood concentration versus time data were
analyzed by noncompartmental analysis (NCA) methods using Win-
Nonlin Professional Version 5.2.
Chemistry: Synthetic Methods and Experimental. General
Methods. Starting materials were obtained from commercial suppliers
and used without further purification unless otherwise stated. Flash
chromatography was carried out using prepacked Isolute Flash or
Biotage silica-gel columns as the stationary phase and analytical grade
solvents as the eluent unless otherwise stated.
NMR spectra were determined on a Varian Unity spectrometer.
Chemical shifts are reported as δ values (ppm) downfield from tetra-
methylsilane, used as an internal standard in the solvent indicated. All
coupling constants are reported in hertz (Hz), and multiplicities
are labeled br (broad), s (singlet), br s (broad singlet), d (doublet),
t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets),
td (triplet of doublets), ddd (doubleꢀdoubledoublet), and m
(multiplet).
Compound concentrations were quantitated in plasma samples by
LC-MS (on either a Micromass Quattro Premier or Micromass Xevo
TQ) using calibration standards that were prepared in blank plasma.
Plasma samples and standards were prepared by precipitation with
acetonitrile, followed by centrifugation and analysis of the supernatant.
The analytical lower limit of quantitation (LLQ) value for each
compound in plasma was typically 1 ng/mL or below.
Total ion current traces were obtained for electrospray positive and
negative ionization (ES+/ESꢀ) on a Waters ZMD 2000. Analytical
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chromatographic conditions used for the LC/MS analysis: Column,
ACE, 4.6 mm ꢁ 30 mm. The stationary phase particle size is 3 μm, and
the flow rate was 1 mL/min. Solvents were A, aqueous solvent = water +
0.1% formic acid; B, organic solvent = acetonitrile + 0.1% formic acid;
Methods, 5 min run time (0ꢀ0.2 min 10%B, 0.2ꢀ3.5 min 10ꢀ90% B,
3.5ꢀ4.01 min 90% B; 4.01ꢀ5 min 90ꢀ10% B). The following addi-
tional parameters were used: injection volume (5 μL), column tem-
perature (30 ꢀC), UV wavelength range (220ꢀ330 nm)
2-Cyclopropyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9f).
Eluting with DCM:MeOH mixtures, gradient from 100:0 to 85:15% to
yield the title compound as a white solid (22% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 13.26ꢀ12.77 (br, 1H), 5.75 (s, 1H), 2.27 (s, 3H), 2.07
(m, 1H), 1.02ꢀ0.97 (m, 2H), 0.9ꢀ0.86 (m, 2H). ESIMS m/z191 (MH)ꢀ.
2-(Cyclopropylmethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-
7-ol (9g). Eluting with DCM:MeOH mixtures, gradient from 100:0 to
90:10% to yield the title compound as a pale-yellow solid (24% yield).
1H NMR (300 MHz, DMSO-d6) δ ppm: 13.29ꢀ12.92 (br, 1H), 5.76 (s,
1H), 2.59 (d, J = 7.1 Hz, 2H), 2.29 (s, 3H), 1.14ꢀ1.03 (m, 1H),
0.50ꢀ0.46 (m, 2H), 0.24ꢀ0.21 (m, 2H). ESIMS m/z 205 (MH)+.
5-Methyl-2-[(methyloxy)methyl][1,2,4]triazolo[1,5-a]pyrimidin-
7-ol (9j). Eluting with DCM:MeOH mixtures, gradient from 100:0 to
The purity of all tested compounds was g95% using the analytical
method described above unless stated otherwise.
A table matching the synthetic intermediates to the final compounds
is provided as Table S1, Supporting Information.
Synthesis of Compounds 11ꢀ55, 100. These compounds
were synthesized following the route described in Scheme 1 through
intermediates 7, 8aꢀb, 9aꢀp, and 10aꢀp.
1
90:10% to yield the title compound as a white solid (87% yield). H
NMR (300 MHz, DMSO-d6) δ ppm: 13.01 (br s, 1H), 5.81 (s, 1H), 4.45
2,3-Diamino-6-methyl-4(3H)-pyrimidinone (7). To a solution of
NaEtO prepared from sodium (9.19 g, 400 mmol) and ethanol
(350 mL), aminoguanidine hydrochloride (44.2 g, 400 mmol) was
added and the reaction was heated at 50 ꢀC for 30 min. Then, the
reaction was filtered to remove NaCl and ethyl 3-oxobutanoate
(25.3 mL, 200 mmol) was added to the filtrate, the reaction mixture
was heated at reflux for 5 h, and then stirred at RT overnight. The
precipitate obtained was filtered and dried under vacuum to afford
(s, 2H), 3.32 (s, 3H), 2.30 (s, 3H). ESIMS m/z 195 (MH)+.
5-Methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo[1,5-a]pyrimidin-
7-ol (9k). Eluting with DCM:MeOH mixtures, gradient from 100:0 to
1
90:10% to yield the title compound as a white solid (58% yield). H
NMR (300 MHz, DMSO-d6) δ ppm: 13.04 (br s, 1H), 5.77 (s, 1H), 3.70
(t, J = 6.6 Hz, 2H), 3.23 (s, 3H), 2.90 (t, J = 6.6 Hz, 2H), 2.28 (s, 3H).
ESIMS m/z 209 (MH)+.
2-(2-Chloroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(9m). Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10%
to yield the title compound as a white solid (31% yield). 1H NMR (300
MHz, DMSO-d6) δ ppm: 13.12 (br s, 1H), 5.79 (s, 1H), 3.98 (t, J = 6.6 Hz,
2H), 3.16 (t, J = 6.6 Hz, 2H), 2.29 (s, 3H). ESIMS m/z 213 (MH)+.
2-(Chloromethyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(9n). Eluting with DCM:MeOH mixtures, gradient from 100:0 to
1
intermediate 7, a pale-pink solid (15.2 g, 54% yield). H NMR (400
MHz, DMSO-d6) δ ppm: 7.02 (br s, 2H), 5.51 (br, 1H), 5.29 (s, 2H),
1.98 (s, 3H). ESIMS m/z 141 (MH)+.
General Procedure for the Synthesis of Compounds 9aꢀ9g and
9jꢀ9k, 9mꢀ9o. A solution of 7 (2.5 mmol) and the appropriate alkyl
chloride (2.5 mmol) in a mixture of 1,4-dioxane (4 mL) and N,N-
dimethylformamide (DMF) (1 mL) was heated at reflux overnight. In
cases where the open, uncyclized intermediate was obtained, the
reaction mixture was concentrated under vacuum and the crude was
dissolved in acetic acid (5 mL) and heated at reflux overnight. The
reaction was then concentrated under vacuum, and the residue was
purified by flash chromatography on a silica gel column.
2-Ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9a). Eluting
with DCM:MeOH mixtures from 100:0 to 90:10% to yield the title
compound as a white solid (75% yield). 1H NMR (400 MHz, CDCl3) δ
ppm: 5.89 (s, 1H), 2.87 (q, J = 7.4 Hz, 2H), 2.50 (s, 3H), 1.41 (t, J = 7.5
Hz, 3H). ESIMS m/z 179 (MH)+.
1
90:10% to yield the title compound as a white solid (58% yield). H
NMR (300 MHz, DMSO-d6) δ ppm: 13.52ꢀ12.72 (br, 1H), 5.85 (s,
1H), 4.78 (s, 2H), 2.32 (s, 3H). ESIMS m/z 199 (MH)+.
5-Methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]triazolo[1,5-a]
pyrimidin-7-ol (9o). Eluting with DCM:MeOH mixtures, gradient from
100:0 to 90:10% to yield the title compound as a white solid (72% yield).
1H NMR (400 MHz, DMSO-d6) δ ppm: 7.27 (m, 5H), 5.84 (s, 1H),
4.76 (s, 2H), 4.55 (s, 2H), 2.45 (s, 3H). ESIMS m/z 271 (MH)+.
General Procedure for the Synthesis of Compounds 9h and 9i. To a
solution of NaEtO prepared from sodium (143 mmol) and ethanol
(150 mL), Intermediate 7 (71.4 mmol) was added, and the reaction
mixture was heated at 80 ꢀC for 30 min. Then, the reaction mixture was
cooled down to RT, and ethyl 2,2-difluoropropanoate (1.2 equiv) or
ethyl 2,2-difluorobutanoate (1.2 equiv) were added to synthesize 9h or
9i, respectively. The mixture was stirred at RT for 30 min before being
heated to 80 ꢀC for 1.5ꢀ3 h. The reaction mixture was concentrated to
dryness, and water (200 mL) was added. The reaction mixture pH was
adjusted to 4 by addition of 2N aq HCl solution while a white solid
precipitated. The solid was filtered off, washed with water, and dried
under vacuum to afford compounds 9h or 9i. The mother liquors were
further extracted with DCM (5 ꢁ 35 mL), and the combined organic
layers were dried over Na2SO4, filtered, and concentrated under vacuum
to yield additional desired product.
5-Methyl-2-(1-methylethyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9b).
Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to
1
yield the title compound as a white solid (45% yield). H NMR (300
MHz, DMSO-d6) δ ppm: 13.21ꢀ12.82 (br, 1H), 5.76 (s, 1H),
3.06ꢀ2.96 (m, 1H), 2.28 (s, 3H), 1.28 (d, J = 6.9 Hz, 6H). ESIMS
m/z 193 (MH)+.
5-Methyl-2-(2-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-ol (9c).
Eluting with DCM:MeOH mixtures, gradient from 100:0 to 90:10% to
1
yield the title compound as a white solid (46% yield). H NMR (300
MHz, DMSO-d6) δ ppm: 13.01 (br s, 1H), 5.76 (s, 1H), 2.54 (d, J = 7.1
Hz, 2H), 2.49 (s, 3H), 2.09- 2.04 (m, 1H), 0.92 (d, J = 6.6 Hz, 6H).
ESIMS m/z 207 (MH)+.
2-(1,1-Dimethylethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-
ol (9d). Eluting with DCM:MeOH mixtures, gradient from 100:0 to
85:15% to yield the title compound as a white solid (71% yield). H
2-(1,1-Difluoroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(9h). White solid (73% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm:
13.39 (br s, 1H), 5.91 (s, 1H), 2.33 (s, 3H), 2.06 (t, J = 19 Hz, 3H).
ESIMS m/z 215 (MH)+.
2-(1,1-Difluoropropyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-
7-ol (9i). White solid (53% yield). 1H NMR (400 MHz, DMSO-d6) δ
ppm: 13.36 (br s, 1H), 5.90 (s, 1H), 2.40ꢀ2.32 (m, 2H), 2.32 (s, 3H),
0.99 (t, J = 7.6 Hz, 3H). ESIMS m/z 229 (MH)+.
General Procedures for Compounds 9l, 9p (Scheme 1). 2-
(Trifluoromethyl)-5-methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(9l). A mixture of ethyl 3-oxobutanoate (20 mmol) and 5-(trifluoro-
methyl)-4H-1,2,4-triazol-3-ylamine 8a (20 mmol) (commercially
1
NMR (300 MHz, DMSO-d6) δ ppm: 13.26ꢀ12.85 (br, 1H), 5.76 (s,
1H), 2.28 (s, 3H), 1.33 (s, 9H). ESIMS m/z 207 (MH)+.
5-Methyl-2-(1-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidin-7-ol
(9e). Eluting with DCM:MeOH mixtures, gradient from 99:1 to 85:15%
to yield the title compound as a white solid (55% yield). 1H NMR (300
MHz, DMSO-d6) δ ppm: 13.33ꢀ12.78 (br, 1H), 5.76 (s, 1H),
2.85ꢀ2.76 (m, 1H), 2.28 (s, 3H), 1.79ꢀ1.68 (m, 1H), 1.65ꢀ1.55 (m,
1H), 1.25 (d, J = 6.8 Hz, 3H), 0.83 (t, J = 7.3 Hz, 3H). ESIMS m/z 207
(MH)+.
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dx.doi.org/10.1021/jm200592f |J. Med. Chem. 2011, 54, 5540–5561

Journal of Medicinal Chemistry
ARTICLE
available) was heated under reflux in acetic acid (10 mL) for 4ꢀ6 h and
then concentrated. The product was then cooled to RT, filtered, washed
with water, and dried under vacuum to give a pale-pink solid with 50ꢀ65%
yield; mp 172ꢀ174 ꢀC. 1H NMR (300 MHz, CDCl3): δ ppm: 6.5 (br s,
1H), 6.0 (s, 1H), 2.40 (s, 3H). MS m/z 219.2 [M + H]+.
δ ppm: 7.57 (s, 1H), 2.76 (d, J = 7.1 Hz, 2H), 2.660 (s, 3H), 1.24ꢀ1.12
(m, 1H), 0.52ꢀ0.48 (m, 2H), 0.28ꢀ0.24 (m, 2H). ESIMS m/z 223
(MH)+.
7-Chloro-2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo[1,5-a] pyr-
imidine (10h). White solid (86% yield). 1H NMR (400 MHz, CDCl3) δ
ppm: 7.17 (s, 1H), 2.75 (s, 3H), 2.18 (t, J = 18.7 Hz, 3H). ESIMS m/z
233 (MH)+.
7-Chloro-2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo[1,5-a] pyr-
imidine (10i). White solid (68% yield). 1H NMR (400 MHz, CDCl3) δ
ppm: 7.17 (s, 1H), 2.75 (s, 3H), 2.53ꢀ2.41 (m, 2H), 1.13 (t, J = 7.6 Hz,
3H). ESIMS m/z 247 (MH)+.
7-Chloro-5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo[1,5-a]pyri-
midine (10j). White solid (75% yield). 1H NMR (300 MHz, CDCl3) δ
ppm: 7.07 (s, 1H), 4.78 (s, 2H), 3.56 (s, 3H), 2.71 (s, 3H). ESIMS m/z
213 (MH)+.
3-{2-[(Phenylmethyl)oxy]ethyl}-1H-1,2,4-triazol-5-amine (8b). To
a solution of NaEtO prepared from sodium (0.237 g, 10.30 mmol) and
ethanol (10 mL), aminoguanidine hydrochloride (1.138 g, 10.30 mmol)
was added and the reaction was heated at 50 ꢀC for 30 min. The reaction
mixture was filtered to remove NaCl and methyl-3-benzyloxypropanoate
(1 g, 5.15 mmol) was added. The reaction mixture was then heated at reflux
for 5 h, the formation of product being observed by TLC (90:10 DCM/
MeOH) staining with KMnO4. The reaction mixture was concentrated
under vacuum, and product was purified by flash chromatography (silica
gel column, eluting with DCM/MeOH mixtures from 100:0 to 80:20%).
Upon collection of the appropriate fractions, the title compound was
7-Chloro-5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo[1,5-a]pyri-
midine (10k). White solid (85% yield). 1H NMR (300 MHz, CDCl3) δ
ppm: 7.03 (s, 1H), 3.93 (t, J = 6.6 Hz, 2H), 3.38 (s, 3H), 3.24 (t, J = 6.6
Hz, 2H), 2.69 (s, 3H). ESIMS m/z 227 (MH)+.
1
obtained as a white solid (0.320 g, 29% yield). H NMR (300 MHz,
CDCl3) δ ppm: 7.36ꢀ7.28 (m, 5H), 4.57 (s, 2H), 3.79 (t, J = 6.6 Hz, 2H),
2.95 (t, J = 6.6 Hz, 2H). ESIMS m/z 219 (MH)+.
7-Chloro-5-methyl-2-(trifluoromethyl)-[1,2,4]triazolo[1,5-a]pyri-
midine (10l). Melting point: 102ꢀ105 ꢀC. 1H NMR (300 MHz,
CDCl3): δ ppm: 7.29 (s, 1H), 2.80 (s, 3H). MS m/z 237.0 [M + H]+.
7-Chloro-2-(2-chloroethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimi-
dine (10m). White solid (67% yield). 1H NMR (300 MHz, CDCl3) δ
ppm: 7.06 (s, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 2.70
(s, 3H). ESIMS m/z 231 (MH)+.
7-Chloro-2-(chloromethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine
(10n). White solid (65% yield). 1H NMR (300 MHz, CDCl3) δ ppm:
7.11 (s, 1H), 4.82 (s, 2H), 2.73 (s, 3H). ESIMS m/z 217 (MH)+.
7-Chloro-5-methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]triazolo-
[1,5-a]pyrimidine (10o). White solid (87% yield). 1H NMR (400 MHz,
CDCl3) δ ppm: 7.44ꢀ7.35 (m, 5H), 7.07 (s, 1H), 4.84 (s, 2H), 4.77 (s,
2H), 2.71 (s, 3H). ESIMS m/z 289 (MH)+.
5-Methyl-2-{2-[(phenylmethyl)oxy]ethyl}[1,2,4]triazolo[1,5-a]pyri-
midin-7-ol (9p). A solution of Intermediate 8b (0.31 g, 1.42 mmol) and
ethyl 3-oxobutanoate (0.216 mL, 1.704 mmol) in acetic acid (5 mL) was
heated at 80 ꢀC overnight. The reaction mixture was concentrated under
vacuum to obtain a pale-yellow solid that was triturated in hexane to
remove the excess of ethyl 3-oxobutanoate. After drying the solid under
vacuum, the title compound was obtained as a pale-yellow solid (0.39 g,
97% yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 13.05 (br s, 1H),
7.33ꢀ7.24 (m, 5H), 5.77 (s, 1H), 4.49 (s, 2H), 3.81 (t, J = 6.6 Hz, 2H),
2.96 (t, J = 6.6 Hz, 2H), 2.28 (s, 3H). ESIMS m/z 285 (MH)+.
General Procedure for the Synthesis of Compounds 10aꢀ10p. A
suspension of Intermediate 9aꢀ9p (1.17 mmol) in phosphorus oxy-
chloride (3.5 mmol) was heated at reflux for 1 h. The reaction mixture
was added dropwise into iced water, neutralized with solid Na2CO3, and
product was extracted with DCM. The combined organic layers were
washed with brine and dried over anhyd Na2SO4. A brown oil was
obtained upon solvent removal in vacuo, which was purified by flash
chromatography on silica gel, eluting with hexanes:EtOAc mixtures from
100:0 to 40:60%.
7-Chloro-5-methyl-2-{2-[(phenylmethyl)oxy]ethyl}[1,2,4]triazolo-
1
[1,5-a]pyrimidine (10p). White solid (0.31 g, 75% yield). H NMR
(300 MHz, CDCl3) δ ppm: 7.33ꢀ7.31 (m, 5H), 7.02 (s, 1H), 4.58 (s,
2H), 4.01 (t, J = 6.6 Hz, 2H), 3.28 (t, J = 6.6 Hz, 2H), 2.69 (s, 3H).
ESIMS m/z 303 (MH)+.
General Procedure for the Synthesis of Compounds 11ꢀ55, 100.
To a suspension of Intermediate 10aꢀ10p (1 mmol) in ethanol (5 mL),
the corresponding aniline (1 mmol) was added and the mixture was
stirred at RT or at 50 ꢀC until the reaction reached completion.
Ammonia in methanol (1 mmol) was added and solvent was removed
in vacuo and the crude mixture was purified by flash chromatography
(silica gel, eluting with hexane:EtOAc mixtures from 75:25 to 25:75%)
to yield the title compound.
7-Chloro-2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine (10a).
White solid (76% yield). 1H NMR (400 MHz, CDCl3) δ ppm: 7.01 (s,
1H), 2.98 (q, J = 7.6 Hz, 2H), 2.68 (s, 3H), 1.43 (t, J = 7.6 Hz, 3H).
ESIMS m/z 197 (MH)+.
7-Chloro-5-methyl-2-(1-methylethyl)[1,2,4]triazolo[1,5-a] pyri-
midine (10b). White solid (65% yield). 1H NMR (300 MHz, CDCl3)
δ ppm: 7.01 (s, 1H), 3.35ꢀ3.25 (m, 1H), 2.68 (s, 3H), 1.45 (d, J = 7.1
Hz, 6H). ESIMS m/z 211 (MH)+.
N-(4-Chlorophenyl)-2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-
1
7-Chloro-5-methyl-2-(2-methylpropyl)[1,2,4]triazolo[1,5-a]pyri-
midine (10c). White solid (85% yield). 1H NMR (300 MHz, CDCl3) δ
ppm: 7.01 (s, 1H), 2.82 (d, J = 7.1 Hz, 2H), 2.68 (s, 3H), 2.34ꢀ2.29 (m,
1H), 1.45 (d, J = 6.6 Hz, 6H). ESIMS m/z 225 (MH)+.
7-Chloro-2-(1,1-dimethylethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyri-
midine (10d). Pale-yellow solid (58% yield). 1H NMR (300 MHz, CDCl3)
δ ppm: 6.99 (s, 1H), 2.67 (s, 3H), 1.50 (s, 9H). ESIMS m/z 225 (MH)+.
7-Chloro-5-methyl-2-(1-methylpropyl)[1,2,4]triazolo[1,5-a]pyrimidine
(10e). Colorless needles (56% yield). 1H NMR (300 MHz, CDCl3) δ
ppm: 7.01 (s, 1H), 3.12ꢀ3.03 (m, 1H), 2.68 (s, 3H), 2.02ꢀ1.91 (m,
1H), 1.81ꢀ1.7 (m, 1H), 1.42 (d, J = 7.1 Hz, 3H), 0.93 (t, J = 7.3 Hz, 3H).
ESIMS m/z 225 (MH)+.
7-amine (11). Pale-yellow solid (87% yield). H NMR (400 MHz,
DMSO-d6) δ ppm: 7.55 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.3 Hz, 2H),
6.48 (s, 1H), 2.86 (q, J = 7.6 Hz, 2H), 2.43 (s, 3H), 1.34 (t, J = 7.6 Hz,
3H). ESIMS m/z 288 (MH)+.
N-(3-Chlorophenyl)-2-ethyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-
1
7-amine (12). Pale-yellow solid (82% yield). H NMR (400 MHz,
DMSO-d6) δ ppm: 7.54ꢀ7.51 (m, 1H), 7.48ꢀ7.36 (m, 2H), 6.51 (s,
1H), 2.85 (q, J = 7.6 Hz, 2H), 2.44 (s, 3H), 1.34 (t, J = 7.6 Hz, 3H).
ESIMS m/z 288 (MH)+.
2-Ethyl-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-
a]pyrimidin-7-amine (13).42 White solid (90% yield). 1H NMR (400
MHz, DMSO-d6) δ ppm: 10.37 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.66 (d,
J = 8.5 Hz, 2H), 6.60 (s, 1H), 2.84 (q, J = 7.6 Hz, 2H), 2.42 (s, 3H), 1.33
(t, J = 7.6 Hz, 3H). ESIMS m/z 322 (MH)+.
7-Chloro-2-cyclopropyl-5-methyl[1,2,4]triazolo[1,5-a]pyrimidine
(10f). White solid (13% yield). 1H NMR (300 MHz, CDCl3) δ ppm:
6.97 (s, 1H), 2.66 (s, 3H), 2.28ꢀ2.22 (m, 1H), 1.26ꢀ1.22 (m, 2H),
1.15ꢀ1.10 (m, 2H). ESIMS m/z 209 (MH)+.
2-Ethyl-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]tria-
zolo[1,5-a]pyrimidin-7-amine (14).42 White solid (87% yield). 1H
NMR (400 MHz, DMSO-d6) δ ppm: 10.44 (br s, 1H), 7.95 (d, J = 8.3
7-Chloro-2-(cyclopropylmethyl)-5-methyl[1,2,4]triazolo[1,5-a]pyri-
midine (10g). Yellow solid (36% yield). 1H NMR (300 MHz, DMSO-d6)
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Journal of Medicinal Chemistry
ARTICLE
Hz, 2H), 7.66 (d, J = 8.3 Hz, 2H), 6.68 (s, 1H), 2.83 (q, J = 7.3 Hz, 2H),
2.43 (s, 3H), 1.32 (t, J = 7.3 Hz, 3H). ESIMS m/z 380 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-(1-methylethyl)[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (15). Colorless solid (35% yield). 1H NMR
(300 MHz, CDCl3) δ ppm: 7.77ꢀ7.63 (br, 1H), 7.49ꢀ7.45 (m, 2H),
7.36ꢀ7.31 (m, 2H), 6.27 (s, 1H), 3.29ꢀ3.19 (m, 1H), 2.58 (s, 3H), 1.46
(d, J = 7.1 Hz, 6H). ESIMS m/z 302 (MH)+.
7.97ꢀ7.95 (m, 2H), 7.68ꢀ7.66 (m, 2H), 6.67 (s, 1H), 2.98ꢀ2.89 (m,
1H), 2.43 (s, 3H), 1.9ꢀ1.79 (m, 1H), 1.73ꢀ1.62 (m, 1H), 1.32 (d,
J = 7.07 Hz, 3H), 0.88ꢀ0.84 (m, 3H). ESIMS m/z 408 (MH)+.
N-(4-Chlorophenyl)-2-cyclopropyl-5-methyl[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (27). White solid (69% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.66ꢀ10.25 (br, 1H), 7.55ꢀ7.52 (m, 2H), 7.48ꢀ
7.45 (m, 2H), 6.40 (s, 1H), 2.39 (s, 3H), 2.20ꢀ2.13 (m, 1H), 1.11ꢀ1.01
(m, 4H). ESIMS m/z 300 (MH)+.
N-(3-Chlorophenyl)-2-cyclopropyl-5-methyl-[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (28). Beige solid (82% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.53ꢀ10.26 (br, 1H), 7.52ꢀ7.48 (m, 2H), 7.45ꢀ
7.42 (m, 1H), 7.38ꢀ7.35 (m, 1H), 6.45 (s, 1H), 2.41 (s, 3H), 2.20ꢀ2.13
(m, 1H), 1.10ꢀ1.01 (m, 4H). ESIMS m/z 300 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-(1-methylethyl)[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (16). White solid (14% yield). H NMR
(300 MHz, CDCl3) δ ppm: 7.79ꢀ7.68 (br, 1H), 7.45ꢀ7.39 (m, 2H),
7.33ꢀ7.27 (m, 2H), 6.36 (s, 1H), 3.30ꢀ3.19 (m, 1H), 2.56 (s, 3H), 1.45
(d, J = 6.9 Hz, 6H). ESIMS m/z 302 (MH)+.
5-Methyl-2-(1-methylethyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (17).42 White solid (29% yield). 1H
NMR (300 MHz, CDCl3) δ ppm: 8.00ꢀ7.86 (br, 1H), 7.77 (d, J = 8.3
Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 6.47 (s, 1H), 3.32ꢀ3.22 (m, 1H), 2.58
(s, 3H), 1.46 (d, J = 6.9 Hz, 6H). ESIMS m/z 336 (MH)+.
2-Cyclopropyl-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (29).42 White solid (26% yield). 1H
NMR (300 MHz, DMSO-d6) δ ppm: 10.79ꢀ10.36 (br, 1H), 7.82 (d, J =
8.34 Hz, 2H), 7.67 (d, J = 8.34 Hz, 2H), 6.62 (s, 1H), 2.42 (s, 3H),
2.21ꢀ2.14 (m, 1H), 1.11ꢀ1.01 (m, 4H). ESIMS m/z 334 (MH)+.
2-Cyclopropyl-5-methyl-N-[4-(pentafluoro-l6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (30).42 Beige solid (60%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.89ꢀ10.35 (br,
1H), 8.01ꢀ7.95 (m, 2H), 7.66 (d, J = 8.8 Hz, 2H), 6.69 (s, 1H), 2.43 (s,
3H), 2.20ꢀ2.14 (m, 1H), 1.10ꢀ1.01 (m, 4H). ESIMS m/z 392 (MH)+.
N-(4-Chlorophenyl)-2-(cyclopropylmethyl)-5-methyl-[1,2,4]triazolo-
5-Methyl-2-(1-methylethyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (18).42 White solid (35%
yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.96ꢀ7.83 (br, 3H),
7.47 (d, J = 8.8 Hz, 2H), 6.50 (s, 1H), 3.30ꢀ3.20 (m, 1H), 2.58 (s, 3H),
1.45 (d, J = 6.9 Hz, 6H). ESIMS m/z 394 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-(2-methylpropyl)[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (19). White solid (87% yield). H NMR
1
(400 MHz, DMSO-d6) δ ppm: 10.15 (s, 1H), 7.50 (d, J = 8.3 Hz, 2H),
7.46 (d, J = 8.3 Hz, 2H), 6.36 (s, 1H), 2.66 (d, J = 7.1 Hz, 2H), 2.38 (s,
3H), 2.26ꢀ2.22 (m, 1H), 0.96 (d, J = 6.6 Hz, 6H). ESIMS m/z 316
(MH)+.
[1,5-a]pyrimidin-7-amine (31). White solid (60% yield). H NMR
(300 MHz, DMSO-d6) δ ppm: 10.65ꢀ10.29 (br, 1H), 7.56ꢀ7.53 (m,
2H), 7.49ꢀ7.45 (m, 2H), 6.44 (s, 1H), 2.76 (d, J = 6.8 Hz, 2H), 2.42 (s,
3H), 1.24ꢀ1.14 (m, 1H), 0.54ꢀ0.50 (m, 2H), 0.31ꢀ0.27 (m, 2H).
ESIMS m/z 314 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-(2-methylpropyl)[1,2,4]triazolo-
1
N-(3-Chlorophenyl)-2-(cyclopropylmethyl)-5-methyl-[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (20). White solid (88% yield). H NMR
[1,5-a]pyrimidin-7-amine (32). White solid (75% yield). H NMR
(400 MHz, DMSO-d6) δ ppm: 10.39 (s, 1H), 7.79 (d, J = 7.9 Hz, 2H),
7.67 (d, J = 7.3 Hz, 2H), 6.60 (s, 1H), 2.67 (d, J = 7.1 Hz, 2H), 2.42 (s,
3H), 2.21ꢀ2.17 (m, 1H), 0.96 (d, J = 6.6 Hz, 6H). ESIMS m/z 316
(MH)+.
(300 MHz, DMSO-d6) δ ppm: 10.65ꢀ10.28 (br, 1H), 7.53ꢀ7.49 (m,
2H), 7.46ꢀ7.43 (m, 1H), 7.38ꢀ7.36 (m, 1H), 6.48 (s, 1H), 2.75 (d, J =
71 Hz, 2H), 2.43 (s, 3H), 1.24ꢀ1.15 (m, 1H), 0.54ꢀ0.49 (m, 2H),
0.31ꢀ0.27 (m, 2H). ESIMS m/z 314 (MH)+.
5-Methyl-2-(2-methylpropyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (21).42 White solid (96% yield). 1H
NMR (400 MHz, DMSO-d6) δ ppm: 10.19 (s, 1H), 7.55ꢀ7.41 (m, 3H),
7.33ꢀ7.31 (m, 1H), 6.41 (s, 1H), 2.67 (d, J = 7.1 Hz, 2H), 2.40 (s, 3H),
2.20ꢀ2.17 (m, 1H), 0.96 (d, J = 6.6 Hz, 6H). ESIMS m/z 350 (MH)+.
5-Methyl-2-(2-methylpropyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (22).42 White solid (92%
2-(Cyclopropylmethyl)-5-methyl-N-(4-(trifluoromethyl)phenyl)-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (33). White solid (11%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.77ꢀ10.36 (br,
1H), 7.82 (d, J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H), 6.64 (s, 1H), 2.76
(d, J = 7.1 Hz, 2H), 2.44 (s, 3H), 1.25ꢀ1.14 (m, 1H), 0.54ꢀ0.49 (m,
2H), 0.30ꢀ0.27 (m, 2H). ESIMS m/z 348 (MH)+.
2-(Cyclopropylmethyl)-5-methyl-N-(4-(pentafluoro-λ6-sulfanyl)-
phenyl]-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (34). White solid
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm: 10.45 (s, 1H), 7.95
(d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 6.67 (s, 1H), 2.68 (d, J = 7.1
Hz, 2H), 2.43 (s, 3H), 2.22ꢀ2.17 (m, 1H), 0.96 (d, J = 6.6 Hz, 6H).
ESIMS m/z 408 (MH)+.
1
(14% yield). H NMR (300 MHz, DMSO-d6) δ ppm: 10.67ꢀ10.38
(br, 1H), 7.99ꢀ7.95 (m, 2H), 7.67 (d, J = 8.8 Hz, 2H), 6.70 (s, 1H), 2.75
(d, J = 7.1 Hz, 2H), 2.45 (s, 3H), 1.25ꢀ1.14 (m, 1H), 0.53ꢀ0.49 (m,
2H), 0.30ꢀ0.26 (m, 2H). ESIMS m/z 406 (MH)+.
2-(1,1-Dimethylethyl)-5-methyl-N-[4-(trifluoromethyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (23). White foam (66%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.15ꢀ10.04 (br,
1H), 7.81 (d, J = 8.6 Hz, 2H), 7.69ꢀ7.67 (m, 2H), 6.54 (s, 1H), 2.41 (s,
3H). 1.43 (s, 9H). ESIMS m/z 350 (MH)+.
N-(4-Chlorophenyl)-2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (35). Beige solid (91% yield). 1H NMR (400
MHz, DMSO-d6) δ ppm: 10.35 (s, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.46 (d,
J = 8.3 Hz, 2H), 6.45 (s, 1H), 2.42 (s,3H), 2.12 (t, J = 18.7 Hz, 3H).
ESIMS m/z 324 (MH)+.
2-(1,1-Dimethylethyl)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)-
phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (24). White solid
1
(49% yield). H NMR (300 MHz, DMSO-d6) δ ppm: 10.2ꢀ10.08
N-(3-Chlorophenyl)-2-(1,1-difluoroethyl)-5-methyl[1,2,4]triazolo-
1
(br, 1H), 7.96 (d, J = 9.1 Hz, 2H), 7.75ꢀ7.62 (m, 2H), 6.53 (s, 1H), 2.42
[1,5-a]pyrimidin-7-amine (36).42 Beige solid (91% yield). H NMR
(s, 3H), 1.43 (s, 9H). ESIMS m/z 408 (MH)+.
5-Methyl-2-(1-methylpropyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]-
(400 MHz, DMSO-d6) δ ppm: 10.39 (s, 1H), 7.53ꢀ7.47 (m, 3H),
7.45ꢀ6.36 (m, 1H), 6.52 (s, 1H), 2.44 (s, 3H), 2.13 (t, J = 18.8 Hz, 3H).
ESIMS m/z 324 (MH)+.
1
triazolo[1,5-a]pyrimidin-7-amine (25). White solid (35% yield). H
NMR (300 MHz, DMSO-d6) δ ppm: 10.41ꢀ10.3 (br, 1H), 7.8 (d, J =
8.3 Hz, 2H), 7.69ꢀ7.67 (m, 2H), 6.59 (s, 1H), 2.98ꢀ2.89 (m, 1H), 2.42
(s, 3H), 1.9ꢀ1.79 (m, 1H), 1.73ꢀ1.62 (m, 1H), 1.33 (d, J = 7.07 Hz,
3H), 0.86 (t, J = 7.3 Hz, 3H). ESIMS m/z 350 (MH)+.
2-(1,1-Difluoroethyl)-5-methyl-N-[4-(trifluoromethyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (37).42 White solid (81%
yield). H NMR (300 MHz, DMSO-d6) δ ppm: 10.58 (s, 1H), 7.83
1
(d, J = 8.5 Hz, 2H), 7.70 (d, J = 8.5 Hz, 2H), 6.73 (s, 1H), 2.47 (s, 3H),
5-Methyl-2-(1-methylpropyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (26). White solid (39% yield).
1H NMR (300 MHz, DMSO-d6) δ ppm: 10.49ꢀ10.31 (br, 1H),
2.14 (t, J = 18.8 Hz, 3H). ESIMS m/z 358 (MH)+.
2-(1,1-Difluoroethyl)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (38).42 White solid (81% yield).
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ARTICLE
1H NMR (400 MHz, DMSO-d6) δ ppm: 10.60 (br s, 1H), 7.97 (d, J = 9.3
Hz, 2H), 7.67 (d, J = 9.1 Hz, 2H), 6.79 (s, 1H), 2.47 (s, 3H), 2.13 (t, J = 18.9
Hz, 3H). ESIMS m/z 416 (MH)+.
(m, 9H), 6.54 (s, 1H), 4.75 (s, 2H), 4.65 (s, 2H), 2.46 (s, 3H). ESIMS
m/z 380 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo-
1
N-(4-Chlorophenyl)-2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (48). White solid (70% yield). H NMR
[1,5-a]pyrimidin-7-amine (39). White solid (88% yield). H NMR
(400 MHz, DMSO-d6) δ ppm: 10.28 (s, 1H), 7.52 (d, J = 8.3 Hz, 2H),
7.46 (d, J = 8.3 Hz, 2H), 6.41 (s, 1H), 4.59 (s, 2H), 3.37 (s, 3H), 2.40 (s,
3H). ESIMS m/z 304 (MH)+.
(400 MHz, DMSO-d6) δ ppm: 10.34 (s, 1H), 7.53 (d, J = 8.3 Hz, 2H),
7.48 (d, J = 8.3 Hz, 2H), 6.47 (s, 1H), 2.42ꢀ2.39 (m, 5H), 1.02 (t, J = 7.3
Hz, 3H). ESIMS m/z 338 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-[(methyloxy)methyl][1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (49). White solid (79% yield). H NMR
N-(3-Chlorophenyl)-2-(1,1-difluoropropyl)-5-methyl[1,2,4]triazolo-
1
(400 MHz, DMSO-d6) δ ppm: 10.32 (s, 1H), 7.51ꢀ7.45 (m, 3H),
7.35ꢀ7.32 (m, 1H), 6.46 (s, 1H), 4.60 (s, 2H), 3.37 (s, 3H), 2.42 (s,
3H). ESIMS m/z 304 (MH)+.
[1,5-a]pyrimidin-7-amine (40). White solid (77% yield). H NMR
(400 MHz, DMSO-d6) δ ppm: 10.38 (s, 1H), 7.54ꢀ7.48 (m, 3H),
7.38ꢀ7.36 (m, 1H), 6.52 (s, 1H), 2.44ꢀ2.39 (m, 5H), 1.02 (t, J = 7.3 Hz,
3H). ESIMS m/z 338 (MH)+.
5-Methyl-2-[(methyloxy)methyl]-N-[4-(trifluoromethyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (50). Pale-yellow solid (90%
yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 10.52 (s, 1H), 7.81 (d,
J = 8.5 Hz, 2H), 7.68 (d, J = 8.5 Hz, 2H, 6.66 (s, 1H), 4.61 (s, 1H), 3.38
(s, 3H), 2.44 (s, 3H). ESIMS m/z 338 (MH)+.
2-(1,1-Difluoropropyl)-5-methyl-N-[4-(trifluoromethyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (41).42 White solid (83%
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm: 10.58 (br s, 1H),
7.82 (d, J = 8.5 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 6.69 (s, 1H), 2.45 (s,
3H), 2.43ꢀ2.37 (m, 2H), 1.02 (t, J = 7.6 Hz, 3H). ESIMS m/z 372
(MH)+.
5-Methyl-2-[(methyloxy)methyl]-N-[4-(pentafluoro-λ6-sulfanyl)-
phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (51). White solid
1
2-(1,1-Difluoropropyl)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)-
phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (42).42 1H NMR
(400 MHz, DMSO-d6) δ ppm: 10.63 (br s, 1H), 7.97 (d, J = 9.0 Hz,
2H), 7.67 (d, J = 8.7 Hz, 2H), 6.78 (s, 1H), 2.47 (s, 3H), 2.45ꢀ2.37 (m,
2H), 1.02 (t, J = 7.3 Hz, 3H).ESIMS m/z 430 (MH)+; mp 132ꢀ134 ꢀC.
2-(Trifluoromethyl)-N-(4-(trifluoromethyl)phenyl)-5-
(92% yield). H NMR (400 MHz, DMSO-d6) δ ppm: 10.59 (s, 1H),
7.98 (d, J = 8.7 Hz, 2H), 7.69 (d, J = 8.7 Hz, 2H), 6.75 (s, 1H), 4.63 (s,
2H), 3.39 (s, 3H), 2.47 (s, 3H). ESIMS m/z 396 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo-
[1,5-a]pyrimidine-7-amine (52). White solid (93% yield). 1H NMR
(400 MHz, DMSO-d6) δ ppm: 10.17 (s, 1H), 7.51 (d, J = 7.3 Hz,
2H), 7.45 (d, J = 7.3 Hz, 2H), 6.37 (s, 1H), 3.78 (t, J = 6.6 Hz, 2H),
3.25 (s, 3H), 3.03 (t, J = 6.6 Hz, 2H), 2.38 (s, 3H). ESIMS m/z 318
(MH)+.
methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (43).42 Products
were purified by column chromatography with 15ꢀ20% EtOAc/hexane.
1
Yields ranged from 20ꢀ30%; mp 124ꢀ126 ꢀC. H NMR (300 MHz,
N-(3-Chlorophenyl)-5-methyl-2-[2-(methyloxy)ethyl][1,2,4]triazolo-
1
CDCl3): δ 7.93 (brs, NH, exchangeable), 7.82 (d, J = 8.1 Hz, 2H), 7.54
(d, J = 8.7 Hz, 2H), 6.61 (s, 1H), 2.65 (s, 3H). MS m/z 362.3 [MH]+.
2-(Trifluoromethyl)-N-(4-(sulfurpentafluoro)phenyl)-5-
[1,5-a]pyrimidin-7-amine (53). White solid (55% yield). H NMR
(400 MHz, DMSO-d6) δ ppm: 10.19 (s, 1H), 7.51ꢀ7.46 (m, 2H),
7.37ꢀ7.34 (m, 1H), 6.42 (s, 1H), 3.79 (t, J = 6.6 Hz, 2H), 3.25 (s, 3H),
3.03 (t, J = 6.6 Hz, 2H), 2.40 (s, 3H). ESIMS m/z 318 (MH)+.
5-Methyl-2-[2-(methyloxy)ethyl]-N-[4-(trifluoromethyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (54). White solid (86%
methyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (44).42 Products
were purified by column chromatography with 15ꢀ20% EtOAc/hexane.
1
Yields ranged from 20ꢀ30%; mp 178ꢀ180 ꢀC. H NMR (300 MHz,
CDCl3): δ 10.79 (brs, NH, exchangeable), 8.01 (d, J = 8.9 Hz, 2H), 7.70
(d, J = 9.2 Hz, 2H), 6.90 (s, 1H), 2.51 (s, 3H). MS m/z 420.3 [MH]+.
2-(Chloromethyl)-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (45a). Brown foam (82% yield). 1H NMR
(300 MHz, CDCl3) δ ppm: 7.97ꢀ7.63 (br, 1H), 7.51ꢀ7.46 (m, 2H),
7.35ꢀ7.30 (m, 2H), 6.33 (s, 1H), 4.80 (s, 2H), 2.56 (s, 3H). ESIMS m/z
308 (MH)+.
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm: 10.42 (s, 1H), 7.80
(d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.5 Hz, 2H), 6.61 (s, 1H), 3.79 (t, J = 6.6
Hz, 2H), 3.25 (s, 3H), 3.04 (t, J = 6.6 Hz, 2H), 2.42 (s, 3H). ESIMS m/z
352 (MH)+.
5-Methyl-2-[2-(methyloxy)ethyl]-N-[4-(pentafluoro-λ6-sulfanyl)-
phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (55). White solid
1
(72% yield). H NMR (400 MHz, DMSO-d6) δ ppm: 10.48 (s, 1H),
2-(Chloromethyl)-N-(3-chlorophenyl)-5-methyl-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (45b). Orange oil (91% yield). 1H NMR
(300 MHz, CDCl3) δ ppm: 7.96ꢀ7.67 (br, 1H), 7.48ꢀ7.39 (m, 2H),
7.36ꢀ7.27 (m, 2H), 6.41 (s, 1H), 4.80 (s, 2H), 2.59 (s, 3H); m/z 308
(MH)+.
7.96 (d, J = 8.7 Hz, 2H), 7.66 (d, J = 8.7 Hz, 2H), 6.69 (s, 1H), 3.79 (t, J =
6.6 Hz, 2H), 3.25 (s, 3H), 3.04 (t, J = 6.6 Hz, 2H), 2.43 (s, 3H). ESIMS
m/z 410 (MH)+.
Synthesis of Compounds 66ꢀ84. These compounds were
synthesized following the route described in Scheme 2 through inter-
mediates 56ꢀ65.
2-(2-Chloroethyl)-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (46a). White solid (81% yield). H NMR
7-Chloro-5-methyl-2-(methylthio)[1,2,4]triazolo[1,5-a]pyrimidine
(57). A suspension of commercially available 7-hydroxy-5-methyl-2-
methylthio-S-triazolo[1,5-a]pyrimidine (56) (2 g, 10.19 mmol) in
phosphorus oxychloride (5 mL, 53.6 mmol) was heated at reflux for
10 h, the starting material solubilizing and the mixture turning bright
orange. TLC analysis (hexane/EtOAC 1:1) showed a very messy
reaction that had not reached completion, but it was decided to stop
it to prevent further product degradation. Hence, the reaction mixture
was added dropwise to iced water. The solution was neutralized with aq
1N Na2CO3, and product was extracted with DCM. The aqueous layer
was further extracted with DCM, and the combined organic layers were
washed with brine and dried over anhyd Na2SO4. Solvent was removed
under reduced pressure, yielding a reddish solid which was purified by
flash chromatography (silica gel, eluting with hexane/EtOAc mixtures
from 95:5 to 40:60%). Upon collection of the appropriate fractions,
the title compound was obtained as a white solid (1.09 g, 47% yield).
(300 MHz, CDCl3) δ ppm: 7.69 (s, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.29
(d, J = 8.3 Hz, 2H), 6.29 (s, 1H), 4.04 (t, J = 6.6 Hz, 2H), 3.37 (t, J = 6.6
Hz, 2H), 2.53 (s, 3H). ESIMS m/z 322 (MH)+.
2-(2-Chloroethyl)-N-(3-chlorophenyl)-5-methyl[1,2,4]triazolo[1,5-a]-
1
pyrimidin-7-amine (46b). White solid (96% yield). H NMR (300
MHz, CDCl3) δ ppm: 7.76 (s, 1H), 7.43ꢀ7.28 (m 4 H), 6.38 (s, 1H),
4.04 (t, J = 6.6 Hz, 2H), 3.38 (t, J = 6.6 Hz, 2H), 2.56 (s, 3H). ESIMS m/z
322 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-{[(phenylmethyl)oxy]methyl}[1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (47a). Pale-yellow solid (96%
1
yield). H NMR (400 MHz, DMSO-d6) δ ppm: 7.56 (d, J = 8.3 Hz,
2H), 7.47 (d, J = 8.3 Hz, 2H), 7.36ꢀ7.20 (m, 5H), 6.50 (s, 1H), 4.75 (s,
2H), 4.65 (s, 2H), 2.45 (s, 3H). ESIMS m/z 380 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-{[(phenylmethyl)oxy]methyl}-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (47b). Pale-yellow solid
(97% yield). 1H NMR (400 MHz, DMSO-d6) δ ppm: 7.52ꢀ7.30
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ARTICLE
1H NMR (300 MHz, CDCl3) δ ppm: 6.99 (s, 1H), 2.74 (s, 3H), 2.68 (s,
3H). ESIMS m/z 215 (MH)+.
or sodium ethoxide (3 mmol) in ethanol (9 mL) (68ꢀ71), or to the
appropriate alcohol (1.2 mmol) with sodium hydride (2 mmol) in THF
(7 mL) (72ꢀ84). The mixture was heated under microwave irradiation
at 120 ꢀC for 0.5ꢀ1 h. Solvent was removed under vacuum, and the
crude mixture was purified by flash chromatography (silica gel, eluting
with DCM: MeOH mixtures from 100:0 to 90:10%) or preparative
HPLC (SunFire 30 mm ꢁ 250 mm, H2O 0.1% TFAꢀACN 0.1% TFA
gradient from 20 to 80%).
N-(3-Chlorophenyl)-5-methyl-2-(methyloxy)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (66). White solid (33% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.44ꢀ9.95 (br, 1H), 7.52ꢀ7.41 (m, 3H),
7.37ꢀ7.34 (m, 1H), 6.43 (s, 1H), 4.06 (s, 3H), 2.39 (s, 3H). ESIMS
m/z 290 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-(methyloxy)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (67). White solid (57% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.25ꢀ9.96 (br, 1H), 7.55ꢀ7.49 (m, 2H),
7.49ꢀ7.42 (m, 2H), 6.38 (s, 1H), 4.05 (s, 3H), 2.37 (s, 3H). ESIMS
m/z 290 (MH)+.
N-(3-Chlorophenyl)-2-(ethyloxy)-5-methyl[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (68). White solid (74% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.42ꢀ9.97 (br, 1H), 7.52ꢀ7.40 (m, 3H),
7.37ꢀ7.34 (m, 1H), 6.43 (s, 1H), 4.46 (q, J = 7.1 Hz, 2H), 2.39 (s,
3H), 1.39 (t, J = 7.1 Hz, 3H). ESIMS m/z 304 (MH)+.
General Procedures for the Synthesis of Compounds 58ꢀ61. To a
suspension of intermediate 57 (1 mmol) in ethanol (10 mL), the
appropriate aniline (1 mmol) was added and the mixture was stirred
at RT until reaching completion. Anhydrous ammonia (7 M solution in
MeOH, 1 mmol) was then added to the mixture and solvent was
removed in vacuo. The crude mixture was purified by flash chromatog-
raphy (silica gel, eluting with hexane/EtOAc mixtures from 95:5 to
40:60%).
N-(4-Chlorophenyl)-5-methyl-2-(methylthio)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (58). Beige solid (86% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.81ꢀ10.15 (br, 1H), 7.57ꢀ7.54 (m, 2H),
7.48ꢀ7.45 (m, 2H), 6.42 (s, 1H), 2.69 (s, 3H), 2.42 (s, 3H). ESIMS
m/z 306 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-(methylthio)[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (59). Pale-yellow solid (61% yield). 1H NMR (300
MHz, DMSO-d6) δ ppm: 11.26ꢀ10.10 (br, 1H), 7.55ꢀ7.5 (m, 2H),
7.45ꢀ7.39 (m, 2H), 6.48 (s, 1H), 2.69 (s, 3H), 2.44 (s, 3H). ESIMS m/z
306 (MH)+.
5-Methyl-2-(methylthio)-N-[4-(trifluoromethyl)phenyl][1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (60).42 White solid (86% yield). 1H
NMR (300 MHz, DMSO-d6) δ ppm: 10.33 (s, 1H), 7.81 (d, J = 8.3 Hz,
2H), 7.67 (d, J = 8.3 Hz, 2H), 6.62 (s, 1H), 2.68 (s, 3H), 2.42 (s, 3H).
ESIMS m/z 340 (MH)+.
N-(4-Chlorophenyl)-2-(ethyloxy)-5-methyl[1,2,4]triazolo[1,5-a]-
pyrimidin-7-amine (69). White solid (74% yield). 1H NMR (300 MHz,
DMSO-d6) δ ppm: 10.24ꢀ9.99 (br, 1H), 7.54ꢀ7.52 (m, 2H),
7.47ꢀ7.43 (m, 2H), 6.38 (s, 1H), 4.46 (q, J = 7.0 Hz, 2H), 2.37 (s,
3H), 1.39 (t, J = 7.0 Hz, 3H). ESIMS m/z 304 (MH)+.
5-Methyl-2-(methylthio)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (61)42. Light-yellow solid
1
(72% yield). H NMR (300 MHz, DMSO-d6) δ ppm: 10.39 (s, 1H),
2-(Ethyloxy)-5-methyl-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo-
1
7.98ꢀ7.96 (m, 2H), 7.66 (d, J = 8.8 Hz, 2H), 6.70 (s, 1H), 2.68 (s, 3H),
[1,5-a]pyrimidin-7-amine (70).42 Beige solid (66% yield). H NMR
2.44 (s, 3H). ESIMS m/z 398 (MH)+.
(300 MHz, CDCl3) δ ppm: 7.79ꢀ7.71 (br, 3H), 7.46 (d, J = 8.3 Hz,
2H), 6.47 (s, 1H), 4.56 (q, J = 7.1 Hz, 2H), 2.55 (s, 3H), 1.49 (t, J = 7.1
Hz, 3H). ESIMS m/z 338 (MH)+.
General Procedures for the Synthesis of Compounds 62ꢀ65
(Scheme 2). To a mixture of the corresponding derivative 58ꢀ61 (1
mmol) and acetic acid (7 mL), sodium tungstate dihydrate (0.034
mmol) was added at RT. The reaction mixture was vigorously stirred and
hydrogen peroxide (2 mmol) was added slowly at 40 ꢀC. The resulting
mixture was then heated at 50 ꢀC until reaching completion. The excess
hydrogen peroxide was destroyed by the addition of an aqueous solution
of sodium sulfite, product being extracted with DCM several times. The
combined organic layers were washed with brine and dried over anhydrous
Na2SO4. A white solid was obtained upon solvent removal in vacuo
which was purified by flash chromatography (silica gel, eluting with
hexane/EtOAc mixtures from 95:5% to 0:100%).
2-(Ethyloxy)-5-methyl-N-[4-(pentafluoro-λ6-sulfanyl)phenyl][1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (71).42 White solid (41% yield). 1H
NMR (300 MHz, CDCl3) δ ppm: 7.91ꢀ7.85 (m, 2H), 7.82ꢀ7.76 (br,
1H), 7.43 (d, J = 8.8 Hz, 2H), 6.51 (s, 1H), 4.56 (q, J = 7.1 Hz, 2H), 2.56
(s, 3H), 1.49 (t, J = 7.1 Hz, 3H). ESIMS m/z 396 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-{[2-(methyloxy)ethyl]oxy}[1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (72). White solid (46% yield). H
NMR (300 MHz, CDCl3) δ ppm: 7.51ꢀ7.49 (m, 2H), 7.33ꢀ7.31 (m,
2H), 6.26 (s, 1H), 4.63 (t, J = 4.6 Hz, 2H), 3.71 (d, J = 84.6 Hz, 2H), 3.45
(s, 3H), 2.5 (s, 3H). ESIMS m/z 334 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-(methylsulfonyl)[1,2,4]triazolo-
1
N-(3-Chlorophenyl)-5-methyl-2-{[2-(methyloxy)ethyl]oxy}[1,2,4]-
1
[1,5-a]pyrimidin-7-amine (62). White solid (36% yield). H NMR
triazolo[1,5-a]pyrimidin-7-amine (73). White solid (58% yield). H
(300 MHz, DMSO-d6) δ ppm: 10.58 (s, 1H), 7.57ꢀ7.54 (m, 2H),
7.50ꢀ7.47 (m, 2H), 6.57 (s, 1H), 3.48 (s, 3H), 2.46 (s, 3H). ESIMS m/z
338 (MH)+.
NMR (300 MHz, DMSO-d6) δ ppm: 10.26ꢀ9.98 (br, 1H), 7.51ꢀ7.47
(m, 2H), 7.43ꢀ7.40 (m, 1H), 7.36ꢀ7.33 (m, 1H), 6.43 (s, 1H),
4.53ꢀ4.51 (m, 2H), 3.71ꢀ3.69 (m, 2H), 3.3 (s, 3H), 2.38 (s, 3H).
ESIMS m/z 334 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-(methylsulfonyl)[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (63). White solid (35% yield). H NMR
N-(4-Chlorophenyl)-5-methyl-2-{[2-(methylamino)ethyl]oxy}-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (74). White solid (61%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.05ꢀ9.84 (br, 1H),
8.82ꢀ8.59 (br, 1H), 7.57ꢀ7.5 (m, 2H), 7.47ꢀ7.43 (m, 2H), 6.39 (s,
1H), 4.67ꢀ4.63 (m, 2H), 3.49ꢀ3.37 (br, 2H), 2.68ꢀ2.64 (m, 3H), 2.38
(s, 3H). ESIMS m/z 333 (MH)+.
N-(3-Chlorophenyl)-5-methyl-2-{[2-(methylamino)ethyl]oxy}-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (75). White solid (38%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.09ꢀ9.90 (br,
1H), 8.84ꢀ8.6 (br, 1H), 7.53ꢀ7.48 (m, 2H), 7.44ꢀ7.35 (m, 2H),
6.45 (s, 1H), 4.65 (t, J = 4.8 Hz, 2H), 3.56ꢀ3.33 (br, 2H), 2.68ꢀ2.64
(m, 3H), 2.40 (s, 3H). 1H NMR (300 MHz, CDCl3) δ ppm:
10.36ꢀ9.91 (br, 2H), 7.44ꢀ7.27 (m, 4H), 6.18 (s, 1H), 4.99ꢀ4.78
(br, 2H), 3.7ꢀ3.49 (br, 2H), 2.91 (s, 3H), 2.32 (s, 3H). ESIMS m/z
333 (MH)+.
(300 MHz, DMSO-d6) δ ppm: 10.62 (br s, 1H), 7.55ꢀ7.37 (m, 4H),
6.62 (s, 1H), 3.48 (s, 3H), 2.48 (s, 3H). ESIMS m/z 338 (MH)+.
5-Methyl-2-(methylsulfonyl)-N-[4-(trifluoromethyl)phenyl][1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (64). White solid (56% yield). H
NMR (300 MHz, CDCl3) δ ppm: 8.18 (br s, 1H), 7.81ꢀ7.79 (m, 2H),
7.53 (d, J = 8.3 Hz, 2H), 6.63 (s, 1H), 3.44 (s, 3H), 2.64 (s, 3H). ESIMS
m/z 372 (MH)+.
5-Methyl-2-(methylsulfonyl)-N-[4-(pentafluoro-λ6-sulfanyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (65). White solid (51%
1
yield). H NMR (300 MHz, CDCl3) δ ppm: 8.23ꢀ8.10 (br s, 1H),
7.93ꢀ7.91 (m, 2H), 7.50 (d, J = 8.6 Hz, 2H), 6.67 (s, 1H), 3.44 (s, 3H),
2.66 (s, 3H). ESIMS m/z 430 (MH)+.
General Procedures for the Synthesis of Compounds 66ꢀ86
(Scheme 2). The appropriate derivative 62ꢀ65 (0.15 g, 0.349 mmol)
was added to sodium methoxide (3 mmol) in methanol (9 mL) (66, 67),
5558
dx.doi.org/10.1021/jm200592f |J. Med. Chem. 2011, 54, 5540–5561

Journal of Medicinal Chemistry
ARTICLE
5-Methyl-2-{[2-(methylamino)ethyl]oxy}-N-[4-(trifluoromethyl)-
phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (76). White solid
General Procedures for the Synthesis of 89ꢀ97 (Scheme 3). The
corresponding amine (10 equiv) in MeOH or THF was added to a
solution of the corresponding 2-(chloromethyl) triazolopyrimidine
derivative (intermediates 45a,b or 46a,b) (1 mmol) in tetrahydrofuran
(THF) (6 mL). The mixture was heated under microwave irradiation at
120 ꢀC for 30ꢀ45 min. Whenever reaction completion had not been
reached, more amine in MeOH or THF (10 mmol) was added. The
mixture was microwave irradiated at 120 ꢀC until reaction completion
had been reached. The mixture was then filtered, and solvent was
removed under reduced pressure. The crude mixture was purified by
preparative HPLC (SunFire 19 m ꢁ 150 mm, H2O 0.1% TFAꢀACN
0.1% TFA gradient from 10 to 100%). Upon freeze-drying of the
corresponding fractions, desired product was obtained.
1
(48% yield). H NMR (300 MHz, DMSO-d6) δ ppm: 10.25ꢀ10.15
(br, 1H), 8.82ꢀ8.6 (br, 1H), 7.84ꢀ7.81 (m, 2H), 7.65 (d, J = 8.5 Hz,
2H), 6.64 (s, 1H), 4.68ꢀ4.64 (m, 2H), 3.64ꢀ3.26 (br, 2H), 2.68ꢀ2.64
(m, 3H), 2.42 (s, 3H). ESIMS m/z 367 (MH)+.
5-Methyl-2-{[2-(methylamino)ethyl]oxy}-N-[4-(pentafluoro-λ6-
sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (77). White
solid (25% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 9.92ꢀ9.67 (br,
2H), 7.88ꢀ7.86 (m, 2H), 7.55 (d, J = 8.6 Hz, 2H), 6.34 (s, 1H),
4.93ꢀ4.82 (m, 2H), 3.70ꢀ3.51 (br, 2H), 2.96ꢀ2.84 (br, 3H), 2.43 (s,
3H). ESIMS m/z 425 (MH)+.
N-(4-Chlorophenyl)-2-{[2-(dimethylamino)ethyl]oxy}-5-methyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (78). White solid (20% yield).
1H NMR (300 MHz, CDCl3) δ ppm: 13.26ꢀ11.23 (br, 1H), 7.52ꢀ7.46
(m, 2H), 7.36ꢀ7.34 (m, 2H), 6.26 (s, 1H), 4.9ꢀ4.88 (m, 2H), 3.66ꢀ3.64
(m, 2H), 3.00 (s, 6H), 2.51 (s, 3H). ESIMS m/z 347 (MH)+.
N-(3-Chlorophenyl)-2-{[2-(dimethylamino)ethyl]oxy}-5-methyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (79). White solid (38%
yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.46ꢀ7.4 (m, 2H),
7.36ꢀ7.27 (m, 2H), 6.34 (s, 1H), 4.9ꢀ4.88 (m, 2H), 3.66ꢀ3.64 (m,
2H), 2.97 (s, 6H), 2.53 (s, 3H), ESIMS m/z 347 (MH)+.
N-(4-Chlorophenyl)-5-methyl-2-[2-(methylamino)ethyl][1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (89). White solid (68% yield). H
NMR (400 MHz, DMSO-d6) δ ppm: 9.57 (br s, 1H), 7.53 (d, J = 7.3 Hz,
2H), 7.47 (d, J = 7.3 Hz, 2H), 6.42 (s, 1H), 3.33 (s, 3H), 3.32ꢀ3.27 (m,
2H), 2.61ꢀ2.33 (m, 4H). ESIMS m/z 317 (MH)+.
2-(Aminomethyl)-N-(4-chlorophenyl)-5-methyl-[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (90). White solid (16% yield). H NMR
(300 MHz, CDCl3) δ ppm: 9.52ꢀ8.38 (br, 2H), 7.35 (d, J = 8.6 Hz,
2H), 7.30ꢀ7.27 (m, 2H), 6.20 (s, 1H), 4.53 (br s, 2H), 2.46 (s, 3H).
ESIMS m/z 289 (MH)+.
2-{[2-(Dimethylamino)ethyl]oxy}-5-methyl-N-[4-(trifluoromethyl)-
phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (80). White solid
(7% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.76 (d, J = 8.6 Hz,
2H), 7.53 (d, J = 8.6 Hz, 2H), 6.47 (s, 1H), 4.93ꢀ4.91 (m, 2H), 3.63 (t,
J = 4.9 Hz, 2H), 2.99 (s, 6H), 2.55 (s, 3H). ESIMS m/z 381 (MH)+.
2-{[2-(Dimethylamino)ethyl]oxy}-5-methyl-N-[4-(pentafluoro-
λ6-sulfanyl)phenyl][1,2,4]triazolo[1,5-a]pyrimidin-7-amine (81). White
solid (13% yield). 1H NMR (300 MHz, CDCl3) δ ppm: 7.88 (d, J = 8.8 Hz,
2H), 7.51 (d, J = 8.6 Hz, 2H), 6.51 (s, 1H), 4.92 (t, J = 5.1 Hz, 2H), 3.61 (t,
J = 5.1 Hz, 2H), 2.97 (s, 6H), 2.56 (s, 3H). ESIMS m/z 439 (MH)+.
2-[(2-Aminoethyl)oxy]-N-(3-chlorophenyl)-5-methyl[1,2,4]triazolo-
N-(4-Chlorophenyl)-5-methyl-2-((methylamino)methyl)-[1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (91). White solid (71% yield). H
NMR (300 MHz, CDCl3) δ ppm: 10.61ꢀ9.65 (br, 1H), 7.53ꢀ7.44 (m,
4H), 6.24 (s, 1H), 4.40 (s, 2H), 2.97 (s, 3H), 2.49 (s, 3H). ESIMS m/z
303 (MH)+.
N-(4-Chlorophenyl)-2-((dimethylamino)methyl)-5-methyl-[1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (92). White solid (31% yield). H
NMR (300 MHz, CDCl3) δ ppm: 7.50ꢀ7.47 (m, 2H), 7.41ꢀ7.37 (m,
2H), 6.42 (s, 1H), 4.53 (s, 2H), 3.05 (s, 6H), 2.58 (s 3H). ESIMS m/z
317 (MH)+.
1
[1,5-a]pyrimidin-7-amine (82). White solid (54% yield). H NMR
N-(3-Chlorophenyl)-5-methyl-2-[2-(methylamino)ethyl][1,2,4]-
1
(300 MHz, DMSO-d6) δ ppm: 10 (s, 1H), 8.05 (br, 2H), 7.53ꢀ7.49 (m,
2H), 7.44ꢀ7.42 (m, 1H), 7.38ꢀ7.36 (m, 1H), 6.44 (s, 1H), 4.59 (t, J =
4.9 Hz, 2H), 3.35ꢀ3.31 (m, 2H), 2.39 (s, 3H). ESIMS m/z 319 (MH)+.
2-[(2-Aminoethyl)oxy]-5-methyl-N-[4-(trifluoromethyl)phenyl]-
[1,2,4]triazolo[1,5-a]pyrimidin-7-amine (83). White solid (46%
yield). 1H NMR (300 MHz, DMSO-d6) δ ppm: 10.35ꢀ10.08 (br,
1H), 8.06 (br, 2H), 7.83 (d, J = 8.6 Hz, 2H), 7.67ꢀ7.65 (m, 2H), 6.63 (s,
1H), 4.61ꢀ4.56 (m, 2H), 3.35- 3.31 (m, 2H), 2.41 (s, 3H). ESIMS m/z
353 (MH)+.
triazolo[1,5-a]pyrimidin-7-amine (93). White solid (53% yield). H
NMR (400 MHz, DMSO-d6) δ ppm: 9.57 (br s, 1H), 7.53ꢀ7.33 (m,
4H), 7.47 (d, J = 7.3 Hz, 2H), 6.48 (s, 1H), 3.33 (s, 3H), 3.23ꢀ3.20 (m,
2H), 2.64ꢀ2.34 (m, 4H). ESIMS m/z 317 (MH)+.
N-(3-Chlorophenyl)-2-((dimethylamino)methyl)-5-methyl-[1,2,4]-
triazolo[1,5-a]pyrimidin-7-amine (94). Pale-yellow solid (56% yield).
1H NMR (300 MHz, CDCl3) δ ppm: 7.46ꢀ7.42 (m, 2H), 7.35ꢀ7.32
(m, 2H), 6.48 (s, 1H), 4.49 (s, 2H), 3.03 (s, 6H), 2.59 (s 3H). ESIMS
m/z 317 (MH)+.
2-[(2-Aminoethyl)oxy]-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo-
1
2-(Aminomethyl)-N-(3-chlorophenyl)-5-methyl-[1,2,4]triazolo-
[1,5-a]pyrimidin-7-amine (95). Pale-yellow solid (11% yield). 1H
NMR (300 MHz, MeOH-d4) δ ppm: 7.53ꢀ7.50 (m, 2H), 7.43ꢀ7.40
(m, 2H), 6.50 (s, 1H), 4.42 (s, 2H), 2.52 (s, 3H). ESIMS m/z 289
(MH)+.
[1,5-a]pyrimidin-7-amine (84). White solid (18% yield). H NMR
(300 MHz, DMSO-d6) δ ppm: 9.97 (s, 1H), 8.04 (br, 2H), 7.54 (d, J =
8.6 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 6.39 (s, 1H), 4.59 (t, J = 4.9 Hz,
2H), 3.34ꢀ3.31 (m, 2H), 2.37 (s, 3H). ESIMS m/z 319 (MH)+.
General Procedures for the Synthesis of Compounds 85ꢀ86. These
compounds were obtained as a byproduct in the reaction yielding
78ꢀ79, probably due to the presence of NaOH in the reaction medium.
7-[(4-Chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyrimidin-
2(1H)-one (85). White solid (21% yield). 1H NMR (300 MHz, DMSO-
d6) δ ppm: 10.09 (br s, 1H), 7.51 (d, J = 8.8 Hz, 2H), 7.42 (d, J = 8.8 Hz,
2H), 6.44 (br s, 1H), 5.75 (s, 1H), 2.32 (s, 3H). 1H NMR (300 MHz,
DMSO-d6 + D2O) δ ppm: 7.51ꢀ7.49 (m, 2H), 7.42 (d, J = 8.8 Hz, 2H),
6.45 (s, 1H), 5.73 (s, 1H), 2.32 (s, 3H). ESIMS m/z 276 (MH)+.
7-[(3-Chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyrim-
N-(3-Chlorophenyl)-5-methyl-2-((methylamino)methyl)-[1,2,4]-
1
triazolo[1,5-a]pyrimidin-7-amine (96). White solid (21% yield). H
NMR (300 MHz, CDCl3) δ ppm: 10.7ꢀ10.08 (br, 1H), 7.39ꢀ7.32 (m,
2H), 7.28ꢀ7.27 (m, 2H), 6.31 (s, 1H), 4.39 (s, 2H), 2.97 (s, 3H), 2.50
(s, 3H). ESIMS m/z 303 (MH)+.
2-(2-Aminoethyl)-N-(4-chlorophenyl)-5-methyl[1,2,4]triazolo-
1
[1,5-a]pyrimidin-7-amine (97). White solid (40% yield). H NMR
(400 MHz, DMSO-d6) δ ppm: 7.77 (br s, 2H), 7.53 (d, J = 7.3 Hz, 2H),
7.47 (d, J = 7.3 Hz, 2H), 6.42 (s, 1H), 3.27 (m, 2H), 3.25 (m, 2H), 2.40
(s, 3H). ESIMS m/z 303 (MH)+.
General Procedures for the Synthesis of 98 and 99 (Scheme 4).
Palladium 10% on activated charcoal (330.06 mmol) was placed in a
hydrogenation flask under N2 atmosphere; intermediate 47a or 47b
(1 mmol) dissolved in acetic acid (10 mL) was added. The reaction
mixture was hydrogenated at 35 psi and checked at 16, 30, and 40 h. After
this time, LCMS showed a mixture of starting material, dehalogenated
1
idin-2(1H)-one (86). White solid (38% yield). H NMR (300 MHz,
DMSO-d6) δ ppm: 10.16 (br s, 1H), 7.50ꢀ7.46 (m, 2H), 7.41ꢀ7.39 (m,
1H), 7.35ꢀ7.33 (m, 1H), 6.49 (s, 1H), 5.76 (s, 1H), 2.34 (s, 3H). 1H
NMR (300 MHz, DMSO-d6 + D2O) δ ppm: 7.50ꢀ7.46 (m, 2H),
7.39ꢀ7.33 (m, 2H), 6.48 (s, 1H), 5.69 (s, 1H), 2.33 (s, 3H). ESIMS m/z
276 (MH)+.
5559
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Journal of Medicinal Chemistry
ARTICLE
starting material, desired product, and dehalogenated desired product in
ratio (35/5/50/10). The reaction mixture was concentrated under
vacuum and purified by preparative HPLC (SunFire, H2O 0.1%
TFAꢀACN 0.1% TFA gradient from 10 to 100%). After collection of
the appropriate fractions, solvent was evaporated under reduced pres-
sure and the product was dried under vacuum in presence of phosphorus
pentoxide overnight to give the desired product.
and Dr. Leonard D. Shultz and The Jackson Laboratory for
providing access to NOD-scid IL-2Rγnull for the in vivo evalua-
tion of compounds in the P. falciparum murine model through
collaboration with GlaxoSmithKline. The team also acknowl-
edges Emma Castro and Angel Pajares (Red Cross Blood Bank,
Madrid, Spain) for the supply of human erythrocytes to perform
the in vivo experiments in the P. falciparum murine model. This
work was supported by the United States National Institutes of
Health grants, U01AI075594 (to M.A.P., P.K.R., S.A.C. and I.B.)
and R01AI53680 (to M.A.P. and P.K.R.). M.A.P. acknowledges
support of the Welch Foundation (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.
{7-[(4-Chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyri-
1
midin-2-yl}methanol (98). White solid (11% yield). H NMR (400
MHz, DMSO-d6) δ ppm: 10.52 (br s, 1H), 7.53 (d, J = 8.3 Hz, 2H), 7.46
(d, J = 8.3 Hz, 2H), 6.45 (s, 1H), 4.66 (s, 2H), 2.42 (s, 3H). ESIMS m/z
290 (MH)+.
{7-[(3-Chlorophenyl)amino]-5-methyl[1,2,4]triazolo[1,5-a]pyri-
1
midin-2-yl}methanol (99). White solid (12% yield). H NMR (400
MHz, DMSO-d6) δ ppm: 10.36 (br s, 1H), 7.52ꢀ7.45 (m, 3H),
7.37ꢀ7.34 (m, 1H), 6.50 (s, 1H), 4.66 (s, 2H), 2.42 (s, 3H). ESIMS
m/z 290 (MH)+.
’ DEDICATION
Synthesis of Compound 101 (Scheme 4). 5-Methyl-2-{2-[(phenyl-
methyl)oxy]ethyl}-N-[4-(trifluoromethyl)phenyl][1,2,4]triazolo[1,5-
a]pyrimidin-7-amine (100). A suspension of Intermediate 10p (0.302
g, 0.997 mmol) and 4-(trifluoromethyl)aniline (0.125 mL, 0.997 mmol)
in ethanol (5 mL) was heated at 50 ꢀC for 1 h. The reaction mixture was
concentrated under vacuum, taken up with DCM (20 mL), and washed
with Na2CO3 (2 ꢁ 15 mL). The organic layer was dried over anhydrous
Na2SO4 filtered and concentrated under vacuum to afford a pale-yellow
We would like to dedicate this paper to the memory of our
colleague and dear friend Ian Bathurst, whose intelligence,
dedication and humor will be greatly missed by all who knew
him.
’ ABBREVIATIONS USED
PfDHODH, P. falciparum dihydroorotate dehydrogenase;
PbDHODH, P. berghei DHODH; PvDHODH, P. vivax DHODH;
hDHODH, human DHODH; CoQ, ubiquinone; FMN, flavin
mononucleotide;HTS, high throughput screen; ACTs, artemesinin-
based combination therapies; ADME, adsorption, distribution,
metabolism, excretion; SAR, structureꢀactivity relationships
1
solid (0.407 g, 95% yield). H NMR (300 MHz, DMSO-d6) δ ppm:
10.02 (s, 1H), 7.79 (d, J = 8.6 Hz, 2H), 7.67 (d, J = 8.6 Hz, 2H),
7.32ꢀ7.25 (m, 5H), 6.61 (s, 1H), 4.51 (s, 2H), 3.90 (t, J = 6.6 Hz, 2H),
3.10 (t, J = 6.6 Hz, 2H), 2.42 (s, 3H). ESIMS m/z 428 (MH)+.
2-(5-Methyl-7-{[4-(trifluoromethyl)phenyl]amino}[1,2,4]triazolo-
[1,5-a]pyrimidin-2-yl)ethanol (101). A solution of Intermediate 100
(0.25 g, 0.585 mmol) in methanol (3 mL) was hydrogenated using 10%
Pd/C as catalyst. The reaction mixture was concentrated, and the
residue was purified by flash chromatography (silica gel column, eluting
with DCM:MeOH mixtures from 100:0 to 90:10%). Upon collection of
the appropriate fractions, the title compound was obtained as a white
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’ ASSOCIATED CONTENT
S
Supporting Information. Table of synthetic intermedi-
b
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of the Fo ꢀ Fc difference map. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
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