Identification and optimization of inhibitors of trypanosomal cysteine proteases: Cruzain, rhodesain, and TbCatB

Article abstract of DOI:10.1021/jm901069a

Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas' disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival: cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A rec

Full text of DOI:10.1021/jm901069a

52 J. Med. Chem. 2010, 53, 52–60
DOI: 10.1021/jm901069a
Identification and Optimization of Inhibitors of Trypanosomal Cysteine Proteases: Cruzain,
Rhodesain, and TbCatB
Bryan T. Mott,‡,3 Rafaela S. Ferreira,§,#,^,3 Anton Simeonov,‡ Ajit Jadhav,‡ Kenny Kean-Hooi Ang, William Leister,‡
Min Shen,‡ Julia T. Silveira,‡ Patricia S. Doyle,# Michelle R. Arkin, James H. McKerrow,# James Inglese,‡
Christopher P. Austin,‡ Craig J. Thomas,‡ Brian K. Shoichet,§ and David J. Maloney*,‡
‡NIH Chemical Genomics Center, National Human Genome Research Institute, National Institutes of Health, 9800 Medical Center Drive, MSC
3370 Bethesda, Maryland, 20892-3370, §Department of Pharmaceutical Chemistry, Small Molecule Discovery Center, ^Graduate Program in
Chemistry and Chemical Biology and #Sandler Center for Basic Research in Parasitic Diseases, University of California San Francisco, 1700
Fourth Street, San Francisco, California, 94158-2550. 3Both of these authors contributed equally to this work.
Received July 20, 2009
Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas’ disease and African
sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival: cruzain
for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of
cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible
inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification
from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold,
while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel
of human cysteine and serine proteases to determine selectivity, and a cocrystal was obtained of our most
potent analogue bound to cruzain.
Introduction
Chagas’ disease is a neglected tropical disease, affecting
approximately 18 million people, predominately in Latin
America.1 Its infectious agent is the protozoan parasite Try-
panosoma cruzi (T. cruzi), with symptoms progressing from
mild swelling to intestinal disease and ultimately heart failure.
African sleeping sickness is a related disease caused by the
protozoan parasite Trypanosoma brucei (T. brucei). Current
chemotherapy for each disease is insufficient, as they exhibit
unacceptable side effects and often do not completely elimi-
nate the parasite despite chronic administration. Moreover,
resistance to these therapies has emerged.1 Given these defi-
ciencies, researchers have sought novel treatments of both
diseases. Several biological pathways and targets have been
explored in T. brucei including the cysteine protease rhodesain
(also known as brucipain) and more recently a cathepsin B-
like protease, TbCatB.2,3 Potential intervention for T. cruzi
includes the inhibition of the analogous cysteine protease
cruzain, which is an essential protease for the survival of the
T. cruzi.4,5 Recently, Kraus et al. also reported a strategy using
a rationally designed sterol 14R-demethylase inhibitor, which
displays potent activity against T. cruzi.6 Our primary focus at
the outset of this project was the discovery of nonpeptidic,
small molecule inhibitors of cruzain through the use of
quantitative high-throughput screening (qHTSa).7
Figure 1. Representative covalent inhibitors of cruzain.
pounds 1 and 2) eradicates infection of the parasite in cell
culture and animal models (Figure 1).4,5,8-10 Irreversible
inhibitors 1 and 2 are peptidic and nonpeptidic analogues,
respectively, that contain an electrophilic functional group
“warhead” (i.e., vinyl sulfone or 2,3,5,6-tetrafluorophenoxy-
methyl ketone) that can covalently bind to cruzain via nucleo-
philic attack of the active site cysteine.11 To date, only
irreversible inhibitors of cruzain have successfully cured para-
sitic infection,5 implying that tight binding to the enzyme may
be essential, although the relationship between reversible and
irreversible inhibition has not been fully explored. We sought
to identify and develop a small molecule reversible covalent
inhibitor of cruzain, which may offer the potential of fewer
off-target side-effects that are often associated with irrever-
sible enzyme inhibitors.12,13 The reagents developed here
provide new molecular tools for the study and comparison
of reversible and irreversible covalent inhibitors of cruzain.
Several groups have demonstrated that irreversible inhibi-
tion of cruzain by small molecules (examples include com-
Design
Quantitative high-throughput screening (qHTS) was con-
ducted at our center on 197,861 small molecules as part of
the NIH Molecular Libraries Probe Production Network
(MLPCN).7,14,15 Several diverse chemotypes emerged as
*To whom correspondence should be addressed. Phone: 301-217-
4381. Fax: 301-217-5736. E-mail: maloneyd@mail.nih.gov.
a Abbreviations: qHTS, quantitative high-throughput screening;
MLPCN, NIH Molecular Libraries Probe Production Network.
r
pubs.acs.org/jmc
Published on Web 11/12/2009
2009 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 53
Figure 4. Structural modification to the triazine core.
Scheme 1a
aReagents: (a) (iPr)2NEt, CH2Cl2, 0 ꢀC, various anilines or primary
and secondary amines; (b) (iPr)2NEt, CH2Cl2, 0 ꢀC, various anilines or
primary and secondary amines; (c) KCN, DMSO, 120 ꢀC.
Figure 2. (a) Top actives obtained from qHTS. (b) Lineweaver-
Burk plot for compound 4, showing competitive inhibition of
cruzain (Ki =180 nM).
plished by incorporating one of the distal nitrogens into a
heterocyclic ring, forming a purine core (Figure 4). Purine
nitriles are reported to be highly potent inhibitors of the
homologous cysteine proteases TbCatB3 and cathepsin K.22
It was hopeful that this trend would hold true for cruzain.
Chemistry
An initial round of analogues were pursued using com-
pound 3 as the core structure. This led to the discovery of
4-(ethylamino)-6-(phenylamino)-1,3,5-triazine-2-carbonitrile (9),
which had comparable in vitro potency yet is more amen-
dable to parallel synthesis, allowing rapid access to analogues
(Scheme 1).20 Early stage structure-activity relationships (SAR)
were sought through common functionality scans of the
aniline and alkyl-amino moieties. Equimolar amounts of either
alkyl/cycloalkyl-amines or anilines (depending on modification
strategy) were added to a solution of cyanuric chloride and
Hunig’s base in dichloromethane at 0 ꢀC. Depending on the
starting material used in the first step, either anilines or amines
were added in the second step, using the procedure described
above. Finally, the nitrile was then installed using potassium
cyanide (KCN) in DMSO at 120 ꢀC for 10 min. Importantly,
careful monitoring of the reaction was required as prolonged
reaction times led to decomposition products.
Previously reported studies of purine nitriles against
othercysteine proteasesrelieduponlarge hydrophobic groups
at the 6-position and alkyl substituents at N9.3,22 In target-
ing cruzain, the optimal regiochemistry of the aniline
and alkyl groups was not known. As such, two different
synthetic strategies were employed for the preparation of
isomers of type A and type B (Figure 4). Starting from 2,6-
dichloropurine, the 9-position was alkylated using the requi-
site alkyl halides in the presence of K2CO3 in DMF at 60 ꢀC
(Scheme 2).22 Two regioisomers were obtained, providing
alkylation primarily at N9. The ratio of the product distribu-
tion was dependent on the nature of the alkyl group, and the
two isomers were easily separable by column chromato-
graphy. Displacement of the 6-position chlorine was accom-
plished by heating various anilines in DMF at 160 ꢀC in the
presence of Hunig’s base. Finally, the nitrile was installed at
the 2-position using KCN in DMSO at 120 ꢀC. Of note,
performing both of these transformations in the microwave
greatly accelerated the reaction.
Figure 3. Triazine nitriles being explored for the potential treat-
ment of cancer (5),17 HIV (6),18 arthritis (7),19 atherosclerosis and
osteoporosis (8).20
promising chemically tractable hits, including the triazine
nitriles (Figure 2). In secondary assays performed at UCSF,
compounds such as 3 and 4 had midnanomolar IC50 values
with classical dose-response curves. The observed inhibition
was reversible by standard incubation-followed-by-dilution
assays16 and was competitive with substrate, with a Ki of
180 nM obtained for compound 4 (Figure 2). Compounds in
this class (triazine nitriles) have been explored for many years
as potential treatments for a variety of diseases, including
cancer,17 HIV,18 arthritis,19 atherosclerosis, and osteoporosis
(Figure 3).20 Because of the strong electron-withdrawing
nature of the triazine ring, the nitrile moiety in this class of
compounds is particularly electrophilic21 and is thought to
form a covalent but reversible bond with the active site
cysteine of a respective protease.13 Although the bond is
reversible, we hypothesized that the mechanism of covalent
modification would prove beneficial to the inhibition of
cruzain (see above). Moreover, the cysteine protease inhibi-
tory activity of triazine nitriles (Figure 3), coupled with the
privileged nature of this scaffold, should provide high hit rates
of active compounds, allowing for smaller libraries to be
synthesized. These factors, and the molecular simplicity of
this chemotype, prompted us to pursue the triazine nitriles as
inhibitors of cruzain.
After several rounds of optimization, and in order to
expand upon our chemotype diversity, the triazine core was
modified to adopt some structural rigidity. This was accom-

54 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mott et al.
Scheme 2a
Table 1. Alkyl Moeity Modifications
aReagents: (a) K2CO3, DMF, 60 ꢀC, various primary and secondary
amines; (b) (iPr)2NEt, DMF, 160 ꢀC (μW), various anilines; (c) KCN,
DMSO, 120 ꢀC (μW).
compd
R
IC50 (M)a
9
ethyl
0.71
0.71
0.79
0.89
0.89
0.89
0.89
0.89
1.26
1.41
1.58
Scheme 3a
10
11
12
13
14
15
16
17
18
19
cyclopentyl
propyl
isopropyl
methylcyclopropyl
isobutyl
butyl
4-hydroxybutyl
methyl
aReagents: (a) phenanthroline, Cu(OAc)2, 4 mol sieves, CH2Cl2, rt,
various aromatic boronic acids; (b) (iPr)2NEt, DMF, 120 ꢀC, various
primary and secondary amines; (c) KCN, DMSO, 120 ꢀC (μW).
cyclohexyl
2-hydroxyethyl
a IC50 values were determined at NIH Chemical Genomics Center
using the qHTS protocol and NCGC curve fitting software. Compounds
were run in duplicate with Log IC50 standard errors ranging from 0.001
to 0.05.
Analogues of type B (Figure 4) required a modified first
step. The aromatic ring was first installed at the 9-position
using a Buchwald-Hartwig-like copper catalyzed cross cou-
pling between the aromatic amine and desired boronic acids
(Scheme 3).23 Alkyl amines could then be installed at the
6-position in the presence of Hunig’s base in DMF at elevated
temperature (120 ꢀC). These analogues were also completed
by displacing the final 2-Cl with KCN in DMSO at 120 ꢀC in
the microwave.
Table 2. Second Round Modifications
Results and Discussion
Our initial strategy was to modify the alkyl amine chain
lengthandfunctionalityof the topactivefrom ourpreliminary
SAR studies, compound 9. While the SAR was generally flat
in this region, incorporation of the cyclopentyl group main-
tained potency of the lead compounds (Table 1). Also, some
hydrophilicity was tolerated, which, if necessary, could be
exploited at a later stage to improve pharmacokinetic proper-
ties (see below). As no appreciable improvements in potency
were observed for the amine substitutions, our efforts then
shifted to phenyl ring modifications. A library of compounds
was synthesized to explore the effects of electron-donating/
withdrawing groups in addition to incorporation of bulky
hydrophobic moieties around the phenyl ring. Electron with-
drawing groups at the 3-position on the phenyl ring proved to
be fruitful, increasing potency 10-fold (Table 2). Placement of
a nitro group at the 3-position (20) resulted in the most potent
analogue. 3-Fluoro and 3-chloro analogues displayed com-
parable potency (21 and 22, respectively), supporting the
notion that electron-withdrawing groups at this position were
most beneficial. Typically, electron-donating groups did not
substantially improve potency, and it appeared that larger
hydrophilic groups were also poorly tolerated. However, to
solidify the size requirements and tolerance around the tria-
zine scaffold, fused and pendant phenyl rings were incorpo-
rated. As anticipated, these changes resulted in decreased
potency.
compd
R1
R2
IC50 (μM)a
20
21
22
23
24
25
26
20
28
29
30
31
3-nitro
3-fluoro
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
cyclopentyl
2,2-difluoroethyl
0.063
0.079
0.079
0.1
3-chloro
3-bromo
4-fluoro
3-methyl
0.126
0.126
1.122
2.239
inactive
0.063
0.398
0.025
4-bromo
3-phenyl
3-trifluoromethyl
3,5-difluoro
3,5-dichloro
3,5-difluoro
a IC50 values were determined at NIH Chemical Genomics Center
using the qHTS protocol and NCGC curve fitting software. Compounds
were run in duplicate with Log IC50 standard errors ranging from
0 to 0.07.
Interestingly, 3,5-difluoro triazine nitrile 29 displayed com-
parable potency as the mono-m-nitro-substituted triazine 20
(both 63 nM IC50). However, 3,5-dichloro-triazine nitrile
30 was 5-fold less active than 3-chloro-triazine nitrile 22
(398 nM and 79 nM IC50, respectively), arguing again that
increased bulk is poorly tolerated on this phenyl moiety.
A final compound was synthesized and was designed to
incorporate electron withdrawing groups on the alkyl
amine. Compound 31 included the 2,2-difluoroethyl group,
To further explore the effect of 3-subsituted electron-
withdrawing substituents, a series of compounds were
synthesized including bis-meta-substituted anilines (Table 2).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 55
Table 3. In Vitro Potency of Purine Nitrile Analogues
Table 4. In Vitro and in Vivo Potency of Active Compounds against
Cysteine Protease Homologues
compd
R1
R2
IC50 (μM)a
cruzain IC50 rhodesain IC50 TbCatB IC50 Tbb MIC50
(μM)a
32
33
34
35
36
37
38
39
40
41
ethyl
3,5-difluorophenyl
3,5-difluorophenyl
3,5-difluorophenyl
3-chlorophenyl
3-chlorophenyl
2,2-difluoroethyl
ethyl
0.010
0.013
0.018
0.018
0.040
0.050
0.063
0.071
0.251
0.316
compd
(μM)a
(μM)a
(μM)
2,2-difluoroethyl
cyclopentyl
ethyl
3
0.09
0.07
0.3
>10
0.008
>1
>1
>1
>1
>100
>100
>1
6
>100
>100
100
100
6
4
20
21
22
23
24
25
26
27
28
29
30
31
32
35
36
0.001
0.01
cyclopentyl
0.02
0.006
0.006
0.04
3,5-difluorophenyl
3-chlorophenyl
3-chlorophenyl
3,5-difluorophenyl
3,5-difluorophenyl
0.002
0.002
0.03
6
2,2-difluoroethyl
ethyl
cyclopentyl
>100
25
0.03
0.06
0.05
0.1
>10
>10
>10
4
>100
25
25
a IC50 values were determined at NIH Chemical Genomics Center
using the qHTS protocol and NCGC curve fitting software. Compounds
were run in duplicate with Log IC50 standard errors ranging from
0 to 0.02.
0.05
0.04
0.03
0.07
0.004
0.01
0.005
0.03
6
25
>10
1
and this change further improved potency to 25 nM IC50
(Table 2).
0.001
0.0002
0.0004
0.0003
<0.006
<0.006
0.01
6
25
6
>100
>10
25
100
The SAR generated for the triazine nitrile class of com-
pounds greatly informed our efforts in exploring the purine
nitrile chemotype. The purine core provided an additional
6-fold increase in potency for structural analogues of type A
(Figure 4). The purine analogue containing our previously
most active substituents (3,5-difluoroaniline and cyclopentyl
amine) improved the potency from 63 to 18 nM (compounds
29 and 34, respectively, Tables 2 and 3). Incorporation of the
ethyl moiety (compound 32) gave a slight potency enhance-
ment (IC50 of 10 nM) as did the 2,2-difluoroethyl moiety
(compound 33) (IC50 of 13 nM). There was a noticeable
decrease in potency for analogues of type B. Incorporation
of the matching substitution patterns for this chemotype (for
instance, 3,5-difluoroaniline at position 9 and ethyl amine
substitution at position 6 in compound 40) gave a marked
decrease in potency (251 nM, Table 3). Overall, the trend in
potency for each substitution pattern correlated well across
the two different scaffolds, with the purine core generally
showing enhanced potency.
As a result of the homologous relationship between T. cruzi
and T. brucei, we tested selected compounds for inhibition of
rhodesain and TbCatB (Table 4). The activity of compounds
3, 20-32, 35, and 36 against rhodesain were quite comparable
to those observed for cruzain. The activities of these com-
pounds were consistently more potent in 96-well plate cruzain
assays than the corresponding 1536-well format, varying from
5- to 50-fold. Although the precise underlying mechanism for
these differences is presently unclear, they appear to be related
to the differences in enzyme availability in the two assay
conditions (96-well plate with large reaction volume and low
surface-to-volume ratio and 1536-format polystyrene well
with low reaction volume and very high surface-to-volume
ratio) and the method for compound delivery (pipetting into
96-well plate versus pintool transfer into a 1536-well plate),
ultimately resulting in different detection limits in the presence
of low concentration (∼1 nM) of enzyme. The difference in
apparent IC50s notwithstanding, there was consistency in the
ranking of compound activities between this assay platform
and the original screening technique throughout all analogue
production cycles. The similar inhibitory potential for these
agents against cruzain and rhodesain is not surprising given
the structural homology of these two enzymes. As we ex-
pected, on the basis of lower homology to cruzain, activity
against TbCatB was more modest. We were however encour-
aged by the observation that some compounds showed low
micromolar IC50 values against this enzyme, which has been
<0.006
a IC50 values were determined at UCSF. Curve fitting errors ranged
between 7% and 32%.
shown to be essential for T. brucei survival based on RNA
interference studies in vitro2 and in mice.24 In contrast,
knocking-downrhodesain produceda phenotype onlyinvivo,
when it prolonged life of infected mice but did not cure
parasitemia as observed for TbCatB knock-down. These
studies suggested that the relevant cysteine protease target
for cure of sleeping sickness is probably TbCatB, while
rhodesain seems to be a virulence factor, facilitating crossing
the blood-brain barrier24 of the host. While encouraged by
the subnanomolar in vitro potency of our lead compounds
against cruzain, and in some cases the low micromolar levels
of activity against TbCatB, we were eager to investigate the
activity against parasites in vivo.
To assess the in vivo activity of these agents, a subset of
optimized compounds and top actives from the primary
screen were tested against the T. brucei parasite (Tbb,
Table 4). The screening leads 3 and 4 showed no trypanocidal
activity up to 100 μM. Gratifyingly, several of the lead
triazines (22-23, 29) showed activity against T. brucei, with
MIC50 values of 6 μM. Furthermore, similar purine nitrile
analogues 32 and 35 were also active (MIC50 = 25 μM).
Among these compounds 29, 31, and 32 showed inhibition
of TbCatB at low micromolar levels (IC50 of 4, 1, and 6 μM,
respectively), at concentrations similar to the necessary for
antiparasitic activity. However, for some compounds such
as 22, 23 and 35, IC50 values against this enzyme are over
100 μM, despite trypanocidal activity.
Triazines 22, 23, 25, 27-30, and purine nitriles 32, 35, and
36 had no effect against the T. cruzi parasite in cell culture
despite the potent in vitro activity. The T. cruzi assay is
substantially more stringent than the T. brucei assay, as the
former measures parasite infection of human macrophages
over several weeks, demanding penetration of the host cell
membrane and stability over a long period of time,10 whereas
the T. brucei assay is conducted on free living parasites over a
few days. Given the disparity in activity of these compounds
in the in vitro and cell-based assays, it is likely that cell
permeability and solubility is lacking. Guy and co-workers
reported that incorporation of hydrophilic alkyl moieties at
N9 greatly improved activity against Tbb.3 As such, we are
hopeful that future studies directed toward the improvement

56 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mott et al.
of the pharmacokinetic properties of these molecules could
provide a platform for the treatment of T. cruzi utilizing
reversible covalent inhibitors.
To better understand the mechanism of action of these
covalent butreversible inhibitorsofcruzain, wedetermined an
˚
X-ray crystal structure of the cruzain/32 complex, to 1.1 A
resolution (Table 5). The ultrahigh resolution of this structure
allowed us to analyze the enzyme-inhibitor interactions in
detail. Even before fitting and refining compound 32, the
unbiased Fo - Fc electron density was unambiguous for the
inhibitor (Figure 5). The covalent adduct with the catalytic
Cys25 is clear, with the formally linear nitrile becoming a
planar imino-moiety, with a cysteine-sulfur to inhibitor
Table 5. Data Collection and Refinement Statistics (Molecular
Replacement) for the Cruzain/32 Complex Structure
Data Collection
space group
cell dimensions
P6522
˚
a, b, c (A)
82.97, 82.97, 101.90
90, 90, 120
41.56 (1.10) a
0.052 (0.407)
33.6 (2.8)
R, β, γ (deg)
resolution (A)
˚
carbon distance of 1.75 A and a sulfur-carbon-nitrogen
˚
angle of 125ꢀ, in agreement with standard values. Several
polar interactions are observed between nitrogens in the
purine ring in 32 and waters or cruzain residues. The nitrogen
in the newly formed iminothioether hydrogen-bonds to water
Rsym or Rmerge
I/σI
completeness (%)
redundancy
92.7 (65.1)
11.6 (4.3)
˚
˚
281 (3.17 A) and to the Nε in Gln19 (2.96 A), which is part
of the oxyanion hole in cysteine proteases of the papain
family, such as cruzain.26 The anilinic nitrogen and N7 are
solvent exposed and form hydrogen bonds to water 294
Refinement
˚
resolution (A)
1.10
no. reflections
R
73808
0.141/0.117
2075
work/Rfree
no. atoms
protein
˚
(distances 2.92 and 3.15 A, respectively). N7 is also involved
˚
in dipole-dipole interactions with waters 292 (3.14 A) and
1705
23
˚
368 (3.36 A). The inhibitor purine packs against the surface
ligand/ion
water
between S1 and S2 pockets of the cruzain active site, while
the aromatic ring off the 6-position extends into the S2 pocket
of the enzyme. Good van der Waals complementarity is
observed in this mostly hydrophobic pocket, which is com-
pletely filled by the 3,5-difluorophenyl ring from 32. This
supported our observation that larger, more hydrophobic
groups (i.e., 3,5-dichloro) in this region result in a decrease
in potency. Double conformation is observed for the terminal
carbon atom in the ethyl substituent at N9. This alkyl moiety
351
B factors
protein
8.29
10.43
22.34
ligand/ion
water
rms deviations
˚
bond lengths (A)
bond angles (deg)
0.020
2.006
a Values in parentheses are for highest-resolution shell.
˚
Figure 5. (A) Unbiased Fo - Fc 3σ electron density from the complex between molecule 32 and cruzain, determined to 1.1 A resolution, stereo
view. (B) Key interactions observed in the refined cruzain/32 X-ray structure, stereo view. Dashed lines represent hydrogen bonds (green) and
dipole-dipole interactions (salmon), waters are shown as red spheres. Protein carbon atoms colored gray, inhibitor carbon atoms colored
orange, oxygen, nitrogen, sulfur, and fluorine atoms colored red, blue, yellow, and cyan, respectively. Second conformation of terminal ethyl
carbon in 32 colored magenta. PDB entry 3I06. Images prepared with Pymol.25

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 57
Figure 6. Protease profiling of compounds 29, 22,32, and 40. IC50s were determined for all four compounds against a panel of cysteine
porteases, aspartyl proteases, peptidases, MMPs, and serine proteases. Black corresponds to inactives, in yellow are IC50s>1 μM, and in red
are IC50s < 1 μM. Data was determined by Reaction Biology, Inc., using a 10-dose, 3-fold serial dilution of compounds. The protease activities
were monitored as a time-course measurement of the increase in fluorescence signal from fluorescently labeled peptide substrate, and initial
linear portion slope (signal/min) was analyzed. (See Supporting Information for this data represented in tabular format.)
reaches toward the surface of the enzyme with no direct
interactions with the binding site being observed (Figure 5).
As such, a wide variety of substituents could be tolerated at
this position and ultimately exploited for the improvement of
pharmacokinetic properties (above).
Chemical Co. and used as received. Commercially available
starting materials and reagents were purchased from Aldrich,
Alfa Aesar, Acros, and Synquest and were used as received.
Analytical thin layer chromatography (TLC) was performed
with Sigma Aldrich TLC plates (5 cm ꢀ 20 cm, 60 A, 250 μm).
˚
Visualization was accomplished by irradiation under a 254 nm
UV lamp. Chromatography on silica gel was performed using
forced flow (liquid) of the indicated solvent system on Biotage
KP-Sil prepacked cartridges and using the Biotage SP-1 auto-
mated chromatography system. 1H- and 13C NMR spectra were
recorded on a Varian Inova 400 MHz spectrometer. Chemical
shifts are reported in ppm with the solvent resonance as the
internal standard (CDCl3 7.26 ppm, 77.00 ppm, DMSO-d6
2.49 ppm, 39.51 ppm for 1H, 13C, respectively). Data are
reported as follows: chemical shift, multiplicity (s = singlet,
d = doublet, t = triplet, q = quartet, br = broad, m = multi-
plet), coupling constants, and number of protons. Low resolu-
tion mass spectra (electrospray ionization) were acquired on an
Agilent Technologies 6130 quadrupole spectrometer coupled to
the HPLC system. High resolution mass spectral data was
collected in-house using and Agilent 6210 time-of-flight mass
spectrometer, also coupled to an Agilent Technologies 1200
series HPLC system. If needed, products were purified via a
Waters semipreparative HPLC equipped with a Phenomenex
Luna C18 reverse phase (5 μm, 30 mm ꢀ 75 mm) column having
a flow rate of 45 mL/min. The mobile phase was a mixture of
acetonitrile and H2O each containing 0.1% trifluoroacetic acid.
Samples were analyzed for purity on an Agilent 1200 series LC/
MS equipped with a Luna C18 reverse phase (3 μm, 3 mmꢀ
75 mm) column having a flow rate of 0.8-1.0 mL/min over a
3 min gradient and a 4.5 min run time. The mobile phase was a
mixture of acetonitrile (0.025% TFA) and H2O (0.05% TFA),
and a temperature was maintained at 50 ꢀC. Purity of final
compounds was determined to be >95%, using a 3 μL injection
with quantitation by AUC at 220 and 254 nm (Agilent diode
array detector).
Protease Activity Profiling
The lead compounds 29, 22, 32, and 40 were also screened
against a human protease panel (Figure 6) to identify possible
off-target activity. As expected, these analogues were active
against a few cathepsins that are highly homologous with
human papain cysteine proteases.27 These results, however,
are not necessarily adverse. For example, previously reported
vinyl sulfone inhibitors of cruzain, which were efficacious in
mice models of Chagas’ disease, were potent inhibitors of
cathepsins B, L, S. However, no toxicity was observed in mice,
rats, or dogs.28 Cathepsins are located in the lysosymes of
cells, whereas the parasites are located in the more accessible
cytoplasm. Potent compounds are likely to preferentially
inhibit the target parasite as a result. Aside from the cathe-
psins, the lead compounds gave little to no inhibition against a
panel of caspases, peptidases, matrix metalloproteinases
(MMPs), and serine proteases. This protease profile demon-
strates the specificity and broad utility of these triazine and
purine nitriles.
Conclusion
In summary, our initial screen focused on identification of
reversible, nonpeptidic small molecule cruzain inhibitors. The
screen identified the covalent reversible triazine nitriles,
among other classes of molecules (unpublished results), which
are known inhibitors of several human cysteine proteases. The
original hits, while modestly potent in biochemical assays
(down to midnanomolar IC50 values), were inactive against
T. brucei parasites. Modifications to the extended structure
both improved potency and achieved activity in vivo. The
determination of the crystal structure of the cruzain/32 com-
plex, at ultrahigh resolution, may help guide additional
medicinal chemistry efforts against this important parasite
target.
Experimental Procedures. Example Procedure for the Forma-
tion of 4,6-Dichloro-N-alkyl-1,3,5-triazine-2-amines (Step 1). 4,6-
Dichloro-N-cylopentyl-1,3,5-triazin-2-amine. To a solution of cy-
anuric chloride (1.25 g, 6.78 mmol) in CH2Cl2 (50 mL) at 0 ꢀC was
€
added Hunig’s base (1.12 mL, 6.78 mmol). After 5 min, cyclo-
pentylamine (0.67 mL, 6.78 mmol) was added and the reaction
mixture was allowed to stir for 15 min at 0 ꢀC, upon which time the
ice bath was removed. The reaction mixture was stirred at rt for 30
min, then concentrated under reduced pressure and directly
purified on silica column. Gradient elution with ethyl acetate
(2 f 40%) in hexanes provided 4,6-dichloro-N-cylopentyl-1,3,5-
triazin-2-amine as a colorless solid; yield(1.62 g, 5.85 mmol, 86%).
General Procedure for the Formation of 6-Chloro- N2-alkyl-N4-
phenyl-1,3-5-triazine-2,4-diamines (Step 2). 6-Chloro-N2-cyclo-
pentyl-N4-(3,5-difluorophenyl)-1,3,5-triazine-2,4-diamine. To a
solution of 4,6-dichloro-N-cyclopentyl-1,3,5-triazin-2-amine
Experimental Methods
Chemistry. Unless otherwise stated, all reactions were carried
out under an atmosphere of dry argon or nitrogen in dried
glassware. Indicated reaction temperatures refer to those of the
reaction bath, while room temperature (rt) is noted as 25 ꢀC. All
solvents were of anhydrous quality, purchased from Aldrich
€
(1.5 g, 6.44 mmol) in CH2Cl2 (50 mL) at 0 ꢀC was added Hunig’s

58 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mott et al.
base (1.12 mL, 6.44 mmol). After 5 min, 3,5-difluoroaniline
(0.83 g, 6.44 mmol) was added and the reaction mixture was
allowed to stir for 15 min at 0 ꢀC, upon which time the ice bath
was removed. The reaction mixture was stirred at rt for 30 min,
then concentrated under reduced pressure and directly purified
on silica column. Gradient elution with ethyl acetate (2 f 40%)
in hexanes provided 6-chloro-N2-cyclopentyl-N4-(3,5-difluoro-
phenyl)-1,3,5-triazine-2,4-diamine as a colorless solid; yield
(1.9 g, 5.83 mmol, 91%).
layer was separated, dried on magnesium sulfate, filtered, concen-
trated under reduced pressure, and directly purified on silica column.
Gradient elution with ethyl acetate (10 f 70%) in hexanes provided
35 as a colorless solid; yield (45 mg, 0.15 mmol, 93%). 1H (CDCl3) δ
1.60(t,J= 7.3 Hz, 3H), 4.34 (q, J= 7.4 Hz, 2H), 7.15 (ddd, J=8.1,
2.0, and 0.9 Hz, 1H), 7.35 (t, J= 8.1 Hz, 1H), 7.75 (ddd, J=8.2,2.2,
and 0.9 Hz, 1H), 7.87 (t, J = 2.1 Hz, 1H), 7.89 (s, 1H) and 8.03
(s, 1H). LC-MS: rt (min) = 3.67. LRMS (ESI) m/z = 299.0.
6-(3,5-Difluorophenylamino)-9-ethyl-9H-purine-2-carbonitrile
(32). The title compound was prepared using a procedure similar
to that detailed for 35, substituting 3,5-difluoroaniline in step 2,
providing 32 as a colorless solid; yield (35 mg, 90%). 1H NMR
(DMSO-d6) δ 1.46 (t, J = 7.3 Hz, 3H), 4.30 (q, J = 7.3 Hz, 2H),
6.94 (tt, J = 9.3 and 2.4 Hz, 1H), 7.63-7.83 (m, 2H), 8.67
(s, 1H), and 10.79 (s, 1H). HRMS (ESI) m/z = 301.1008 (M þ
H)þ (C14H11F2N6 requires 301.1006). LC-MS: rt (min) = 3.70.
LRMS (ESI) m/z = 301.0.
General Procedure for the Formation of 4-alkylamino-6-phe-
nylamino-1,3,5-triazine-2-carbonitriles (Step 3). 4-(Cyclopenty-
lamino)-6-(3,5-difluorophenylamino)-1,3,5-triazine-2-carbonitrile
(29). To a solution of 6-chloro-N2-cyclopentyl-N4-(3,5-difluoro-
phenyl)-1,3,5-triazine-2,4-diamine (1.0 g, 3.07 mmol) in DMSO
(25 mL) was added KCN (0.220 g, 3.38 mmol). The reaction
mixture was sealed and heated to 120 ꢀC for 10 min. Upon
completion, the reaction mixture was diluted with ethyl acetate
and washed several times with saturated sodium chloride solu-
tion. The organic layer was dried on magnesium sulfate, filtered,
and concentrated. The resulting residue was purified on silica
column. Gradient elution with ethyl acetate (2 f 40%) in hexanes
provided 4-(cyclopentylamino)-6-(3,5-difluorophenylamino)-
1,3,5-triazine-2-carbonitrile (29) as a colorless solid; yield (863
Example Procedure for the Formation of 2,6-Dichloro-9-phenyl-
9H-purines (Step 5). 2,6-Dichloro-9-(3,5-difluorophenyl)-9H-
purine. To a solution of 2,6-dichloro-9H-purine (0.6 g, 3.17
mmol) in CH2Cl2 (15 mL) was added 3,5-difluorophenylboronic
acid (1.0 g, 6.35 mmol), copper(II) acetate (1.15 g, 6.35 mmol),
˚
4 A molecular sieves (∼250 mg), and NEt3 (1.3 mL, 9.5 mmol).
1
mg, 2.73 mmol, 89%). H (DMSO-d6) δ 1.45-1.63 (m, 4H),
The reaction mixture was stirred at 100 ꢀC for 16 h. Upon
completion, the reaction mixture was diluted with ethyl acetate,
filtered through a pad of celite, washed with water and brine
(3 ꢀ 30 mL), and the solvent was removed under reduced
pressure. The remaining residue was directly purified on silica
column. Gradient elution with ethyl acetate (5 f 65%) in
hexanes provided 2,6-dichloro-9-(3,5-difluorophenyl)-9H-pur-
ine as a pale-yellow solid (200 mg, 0.66 mmol, 21%).
1.63-1.79 (m, 2H), 1.84-2.02 (m, 2H), 4.06-4.27 (m, 1H),
6.82-6.92 (m, 1H), 7.49 (d, J = 8.7 Hz, 1H), 7.56 (d, J = 8.1
Hz, 1H), 8.48 (d, J = 6.3 Hz, 1H), 8.61 (brs, 1H), 10.39 (brs, 1H)
and 10.52 (brs, 1H). 13C NMR (DMSO-d6) δ 23.82, 23.87, 32.15,
32.41, 52.57, 98.39, 103.43, 115.67, 141.85, 151.50, 151.93,
161.63, and 164.10. HRMS (ESI) m/z = 317.1319 (M þ H)þ
(C15H15F2N6 requires 317.1316). LC-MS: rt (min)=3.94. LRMS
(ESI) m/z = 317.1.
2-Chloro-9-(3,5-difluorophenyl)-N-ethyl-9H-purin-6-amine. The
title compound was prepared using a procedure similar to that
detailed in Step 2 above, substituting ethylamine, providing the
product as a pale-yellow solid; yield (60 mg, 83%).
Example Procedure for the Formation of 2,6-Dichloro-9-alkyl-
9H-purines (Step 1). 2,6-Dichloro-9-ethyl-9H-purine. To a solu-
tion of 2,6-dichloro-9H-purine (2.0 g, 10.6 mmol) in acetone
(45 mL) was added sodium carbonate (2.25 g, 21.2 mmol). The
reaction vessel was equipped with a reflux condenser, and the
mixture was heated under reflux conditions for 20 min. After
that time, iodoethane (0.86 mL, 10.6 mmol) was added in one
portion, and the reaction mixture was allowed to stir for 5 h.
Upon completion, the reaction mixture was concentrated under
reduced pressure and directly purified on silica column. Gradi-
ent elution with ethyl acetate (2 f 40%) in hexanes provided
regioisomers 2,6-dichloro-9-ethyl-9H-purine and 2,6-dichloro-
7-ethyl-7H-purine as pale-yellow solids; yield (1.5 g, 6.91 mmol,
82%; 0.4 g, 1.84 mmol, respectively).
9-(3,5-Difluorophenyl)-6-(ethylamino)-9H-purine-2-carbonitrile
(40). The title compound was prepared using a procedure
similar to that detailed in step 3 above, providing 40 as a colorless
1
solid; yield (23 mg, 48%). H NMR (CDCl3) δ 1.34-1.40 (m,
3H), 3.73 (brs, 2H), 6.02 (brs, 1H), 6.94 (tt, J = 8.7 and 2.3 Hz, 1
H), 7.39 (dd, J=7.2, 1.9 Hz, 2H) and 8.16 (s, 1H). 13C NMR
(CDCl3) δ 14.70, 35.94, 103.72, 103.97, 104.21, 106.34, 106.62,
116.59, 136.23, 138.93, 139.77, 155.00, 162.14, and 164.77.
HRMS (ESI) m/z = 301.1008 (M þ H)þ (C14H11F2N6 requires
301.1003).
Methods. Cruzain Enzymatic Assays in 1536-Well Format.
The compounds were initially prepared as 10 mM DMSO stock
solutions and were arrayed for testing as serial 2-fold dilutions at
5 μL per well in 1536-well Greiner polypropylene compound
plates following previously described protocols.29 Cruzain ac-
tivity was assayed in freshly prepared 100 mM acetate buffer pH
5.5, containing 5 mM dithiothreitol (DTT) and 0.01% Triton
X-100. Three μL of reagents (buffer as negative control and
cruzain at 1.5 nM final concentration in the remainder of the
plate) were dispensed by a Flying Reagent Dispenser (FRD)
(Beckman Coulter Inc., Fullerton, CA) into a black solid-
bottom 1536-well plate (Greiner Bio-One, Monroe, NC). In-
hibitors were delivered as 23 nL of DMSO solutions via pintool
transfer; vehicle-only control consisted of 23 nL DMSO. The
plate was incubated for 15 min at room temperature, and then
1 μL of cruzain fluorogenic substrate (Z-FR-AMC, Bachem,
2 μM final concentration) was added to start the reaction.
Following substrate dispense, the plate was immediately trans-
ferred into ViewLux high-throughput CCD imager (Perkin-
Elmer, Waltham, MA) for kinetic fluorescence data collection
utilizing standard 340 nm excitation and 450 nm emission filter
set. Percent inhibition was calculated relative to the no-enzyme
and uninhibited controls (48 wells averaged per condition) by
using the fluorescence intensity change recorded during the first
60 s of reaction.
Example Procedure for the Formation of 2-Chloro-N-phenyl-9-
ethyl-9H-purin-6-amines (Step 2). 2-Chloro-N-(3-chlorophenyl)-
9-ethyl-9H-purin-6-amine. To a solution of 2,6-dichloro-9-ethyl-
9H-purine (0.5 g, 2.30 mmol) in DMF (2 mL) was added
€
3-chloroaniline (0.29 g, 2.30 mmol) and Hunig’s base (0.40 mL,
2.30 mmol). The reaction mixture was sealed in a microwave tube
(2-5 mL) and heated to 110 ꢀC for 30 min at 90 W in a Biotage
Initiator microwave. Upon completion, the reaction mixture was
diluted with ethyl acetate and washed several times with 3 N
lithium chloride solution. The organic layer was separated, dried
on magnesium sulfate, filtered, concentrated under reduced
pressure, and directly purified on silica column. Gradient elution
with ethyl acetate (5 f 50%) in hexanes provided 2-chloro-N-(3-
chlorophenyl)-9-ethyl-9H-purin-6-amine as a pale-yellow solid;
yield (0.64 g, 2.08 mmol, 90%).
Example Procedure for the Formation of 6-Phenylamino-9-ethyl-
9H-purine-2-carbonitriles (Step 3). 6-(3-Chlorophenylamino)-9-
ethyl-9H-purine-2-carbonitrile (35). To a solution of 2-chloro-
N-(3-chlorophenyl)-9-ethyl-9H-purin-6-amine (0.05 g, 0.16 mmol)
in DMSO (1 mL) was added KCN (10.5 mg, 0.16 mmol). The
reaction mixture was sealed in a microwave tube (0.5-2 mL) and
heated in a microwave to 140 ꢀC for 1 h at 90 W. Upon completion,
the reaction mixture was diluted with ethyl acetate and washed
several times with saturated sodium chloride solution. The organic

Article
Cruzain, Rhodesain, and tbcatB Enzymatic Assays in 96-Well
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1 59
phages. After completion of the treatment period, cultures were
maintained for an additional 14 days to ensure “cure” of the
host cells. Under these experimental conditions, T. cruzi com-
pleted the intracellular cycle in 5 days in untreated controls,
while K777 cured cell infection and no free parasites were
observed. Seven triazine nitriles (compounds 22, 23, 25,
27-30) were tested at 1, 5, and 10 μM. All triazines were toxic
against macrophages at 10 μM, inactive against T. cruzi at 1 μM,
and either toxic (23 and 29) or inactive (22, 25, 27, 28, 30) at
5 μM. Purines 32, 35, and 36 were tested at 10 μM and were
inactive.
Cruzain Expression and Purification. Procruzain truncated at
the C-terminal30 was expressed and purified using a modified
protocol (Lee, Balouch, and Craik, unpublished results). A
0.5 mg/mL solution of procruzain (in 100 mM sodium acetate
pH 5.5, 10 mM EDTA, 5 mM DTT and 1 M NaCl, pH adjusted to
5.3) was activated at 37 ꢀC for 3 h. After activation, cruzain was
dialyzed in 10 mM Tris buffer pH 7.5, inhibited with the covalent
reversible inhibitor methyl methanethiosulfonate (MMTS), and
dialyzed again in the same buffer. Finally, the protein was purified
in a MONO-Q anion exchange column, using a 0 to 500 mM NaCl
gradient in 10 mM Tris buffer (pH 7.5).
Crystallography. MMTS inhibited cruzain was concentrated in
to 1 mg/mL in 2 mM bis tris pH 5.8. To reverse MMTS inhibition,
5 mM DTT was added, followed by addition of 400 μM of
compound 32. The solution was stirred for a few hours at 4 ꢀC
until cruzain was completely inhibited, and the protein was con-
centrated down to 7.5 mg/mL. Hanging drops for 384 crystallo-
graphy conditions (Joint Center Structure Genomics screens
I-IV, Qiagen) were set up using a Mosquito (TTP Labtech).
Each condition was screened in 1:1 and 2:1 ratio between protein
solution and mother liquor. After two weeks incubation at
18 ꢀC, a 200 μm crystal was obtained in 0.1 M Tris pH 8.5, 2.0
M NH4H2PO4, in the (200 nL protein solution):(100 nL mother
liquor) drop. Crystals were then reproduced in the same condi-
tions in 2-4 μL hanging drops, were transferred to a solution of
25% glycerol in mother liquor, and cryocooled in liquid nitrogen.
Data collection was performed on frozen crystals in beamline
7-1 in the Stanford Synchrotron Radiation Lightsource at the
SLAC National Accelerator Laboratory. Reflections were in-
dexed and integrated using Ipmosflm. PHASER31 was used for
the initially phasing, based on a previously reported structure of
cruzain bound to a noncovalent inhibitor (PDB entry 1ME3),32
with the ligand, water molecules and ions removed. The space
group was P6522. Data refinement was performed using the
REFMAC533 package and models were built and waters placed
using Coot.34
Plate Format. First, 100 μL per well of recombinant enzyme
cruzain, rhodesain, or tbcatB (in a buffer solution consists of
100 mM sodium acetate pH 5.5, 5 mM DTT and 0.001% Triton
X-100) was added to a 96-well black plate that contained 1 μL of
test compound (in DMSO). The enzyme-compound mixture
was incubated for 5 min at room temperature. Then 100 μL per
well of substrate Z-FR-AMC (in the same buffer solution as
above) was added to the enzyme-compound mixture to initiate
the reaction. The rate of increase in fluorescence (units/s),
resulting from the proteolytic cleavage of the substrate leading
to the release of fluorogenic AMC was monitored as with an
automated microtiter plate spectrofluorimeter (SpectraMax
M5, Molecular Devices) with fluorescence readout setting of
excitation at 355 nm and emission at 460 nm. The assay
concentrations of enzyme and substrate are 4 nM (cruzain),
4 nM (rhodesain), 258 nM (tbcatB), and 10 μM Z-Phe-Arg-
AMC, respectively. Positive control wells contained 1 μL of
DMSO. IC50 curve fitting was performed with Prism 4 software
(GraphPad, San Diego, CA).
Competition Assays and Ki Determination. Cruzain activity
was conducted in 96-well plates as described above for varying
concentrations of compound 5 and Z-FR-AMC in the presence
of 0.01% Triton. Each concentration of 5 (0, 25, 50, 100, 200,
and 500 nM) was tested in seven concentrations of substrate
(0.31 to 20 μM, in 2-fold increments). Compound was incubated
with cruzain for 15 min prior to addition of substrate and
enzyme activity was then monitored for 5 min. Given the
covalent reversible mechanism of this compound, initially an
increase in rates of activity was observed until equilibrium was
reached. Calculations of rates of cruzain activity were based
on time points after equilibrium, when a uniform rate was
observed with time. All assays were performed in duplicate.
A Lineweaver-Burk plot was built in Prism 4 .
Trypanosoma brucei brucei Assay. Trypanosoma brucei brucei
strain 221 was grown in complete HMI-9 medium containing
10% FBS, 10% Serum Plus medium (Sigma Inc. St. Louis MO)
and 1ꢀ penicillin/streptomycin. The trypanosomes were diluted
to 1 ꢀ 105 per mL in complete HMI-9 medium. Then 95 μL per
well of the diluted trypanosomes was added to sterile Greiner 96-
well flat white opaque culture plates that contained 5 μL of test
samples (in 10% DMSO). Control wells contained 95 μL of the
diluted trypanosomes and 5 μL of 10% DMSO while control
wells for 100% inhibition contained 95 μL of the diluted
trypanosomes and 5 μL of 1 mM thimerosal (in 10% DMSO).
Trypanosomes were incubated with test samples for 48 h at
37 ꢀC with 5% CO2 before monitoring viability. Trypanosomes
were then lysed in the wells by adding 50 μL of CellTiter-GloTM
(Promega Inc., Madison, WI). Lysed trypanosomes were placed
on an orbital shaker at room temperature for 2 min. The
resulting ATP-bioluminescence of the trypanosomes in the
96-well plates was measured at room temperature using an
Analyst HT plate reader (Molecular Devices, Sunnyvale, CA).
Each compound was evaluated in eight concentrations, in 4-fold
dilutions starting at 100 μM. The reported MIC50 is the lowest
tested compound concentration, which inhibited parasite
growth by at least 50%.
Trypanosoma cruzi Assay. Assays were conducted as pre-
viously described.10 Briefly, irradiated (1000 rad) J774 macro-
phages were plated overnight onto sterile tissue culture plates
prior to infection with Y strain T. cruzi trypomastigotes for 2 h.
Parasites were then removed and cells cultured with RPMI-1640
medium with the addition of compounds at the appropriate
concentration (n = 3 per compound). Untreated infected con-
trols and cultures treated with 10 μM K777 were included in
each independent experiment. Treatment was continued for 27
days and medium replaced every 48 h. Cultures were observed
daily by contrast phase microscopy (400ꢀ) for the appearance of
free trypomastigotes in the medium and or compound toxicity
(i.e., granulation, detachment, and death) for host macro-
Acknowledgment. The authors thank Jeremy Smith, Paul
Shinn, and Danielle van Leer for assistance with compound
management. We also thank Eaman Balouch for providing
the cruzain construct and information on cruzain expression
protocol, and Dr. Linda Brinen for helpful discussions and
suggestions. This research was supported by the Molecular
Libraries Initiative of the National Institutes of Health Road-
map for Medical Research and by NIH Grant GM59957 (to
BKS) and by the Sandler Center for Basic Research in
Parasitic Diseases (to JHM).
Supporting Information Available: Experimental procedure
and spectroscopic data (1H, 13C, LC/MS, and HRMS) are listed
for representative compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Chagas Disease; Special Programme for Research and Training in
Tropical Diseases (TDR), World Health Organization: Geneva, http://
www.who.int/tdr/diseases/chagas/direction.htm.

60 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 1
Mott et al.
(2) Mackey, Z. B.; OBrien, T. C.; Greenbaum, D. C.; Blank, R. B.;
McKerrow, J. H. A cathepsin B-like protease Is required for host
protein degradation in Trypanosoma brucei. J. Biol. Chem 2004,
279, 48426–48433.
(3) Mallari, J. P.; Shelat, A. A.; Obrien, T.; Caffrey, C. R.; Kosinksi,
A.; Connelly, M.; Harbut, M.; Greenbaum, D.; McKerrow, J. H.;
Guy, R. K. Development of potent purine-derived nitrile inhibitors
of the trypanosomal protease TbcatB. J. Med. Chem. 2008, 51, 545–
552.
(4) Barr, S. C.; Warner, K. L.; Kornreic, B. C.; Pisctelli, J.; Wolfe, A.;
Benet, L.; McKerrow, J. H. A cysteine protease inhibitor protects
dogs from cardiac damage during infection by Trypanosoma cruzi.
Antimicrob. Agents Chemother. 2005, 49, 5160–5161.
derivatives and related compounds with lysophphosphatidic acid
acyltransferase beta (LPAAT-BETA) inhibitory activity for use in
the treatment of cancer. Patent WO 03037346/2003, 2003.
(18) Kukla, M. J.; Ludovici, D. W.; Kavash, R. W.; De Corte, B. L. D.;
Heeres, J.; Janssen, P. A. J.; Koymans, L. M. H.; de Jonge, M. R.;
Van Aken, A.; Krief, A.; Gerardus, R.; Leenders, G. HIV replica-
tion inhibitors. Patent 0,171,374, 2003.
(19) Bailey, A.; Pairaudeau, G.; Patel, A.; Thom, S. Use of pyrimidine, or
triazine, 2 carbonitriles for treating diseases associated with cysteine
protease activity and novel pyrimidine-2-carbonitrile derivatives.
Patent 7,125,881, 2006.
(20) Rankovic, Z.; Cai, J.; Cumming, I. 2-Cyano-1,3,5-triazine-4,6-
diamine derivatives. Patent WO 2005/011703, 2005.
(5) Brak, K.; Doyle, P. S.; McKerrow, J. H.; Ellman, J. A. Identifica-
tion of a new class of nonpeptidic inhibitors of cruzain. J. Am.
Chem. Soc. 2008, 130, 6404–6410.
(6) Kraus, J. M.; Verlinde, C. L. J.; Karimi, M.; Lepesheva, G. I.; Gelb,
M. H.; Buckner, F. S. Rational modification of a candidate cancer
drug for use against Chagas disease. J. Med. Chem. 2009, 52, 1639–
1647.
(7) Inglese, J.; Auld, D. S.; Jadhav, A.; Johnson, R. L.; Simeonov, A.;
Yasgar, A.; Zheng, W.; Austin, C. P. Quantitative high-throughput
screening: a titration based approach that efficiently identifies
biological activities in large chemical libraries. Proc. Natl. Acad.
Sci. U.S.A. 2006, 11473–11478.
(8) Doyle, P. S.; Zhou, Y. M.; Engel, J. C.; McKerrow, J. H. A cysteine
protease inhibitor cures Chagas disease in an immunodeficient-
mouse model of infection. Antimicrob. Agents Chemother. 2007, 51,
3932–3939.
(9) Jacobsen, W.; Christians, U.; Benet, L. Z. In vitro evaluation of the
disposition of a novel cysteine protease inhibitor. Drug Metab.
Dispos. 2000, 28, 1343–1351.
(10) Engel, J. C.; Doyle, P. S.; Hsieh, I.; McKerrow, J. H. Cysteine
protease inhibitors cure an experimental Trypanosoma cruzi infec-
tion. J. Exp. Med. 1998, 188, 725–734.
(11) (a) Roush, W. R.; Gwaltney, S. L. II; Cheng, J.; Scheidt, K. A.;
McKerrow, J. H.; Hansell, E. Vinyl sulfonate esters and vinyl
sulfonamides: potent, irreversible inhibitors of cysteine proteases.
J. Am. Chem. Soc. 1998, 120, 10994–10995. (b) Roush, W. R.; Cheng,
J.; Knapp-Reed, B.; Alvarez-Hernandez, A.; McKerrow, J. H.; Hansell,
E.; Engel, J. C. Potent second generation vinyl sulfonamide inhibitors of
the typanosomal cysteine protease cruzain. Bioorg. Med. Chem. Lett.
2001, 11, 2759–2762.
(21) Oballa, R. M.; Truchon, J. F.; Bayly, C. I.; Chauret, N.; Day, S.;
Crane, S.; Berthelette, C. A generally applicable method for
assesing the electrophilicity and reactivity of diverse nitrile-con-
taining compounds. Bioorg. Med. Chem. Lett. 2007, 17, 998–
1002.
(22) Altmann, E.; Cowan-Jacob, S. W.; Missbach, M. Novel purine
nitrile derived inhibitors of the cysteine protease cathepsin K.
J. Med. Chem. 2004, 47, 5833–5836.
(23) Bakkestuen, A. K.; Gundersen, L. L. Regioselective N-9 arylation
of purines employing arylboronic acids in the presence of Cu(II).
Tetrahedron Lett. 2003, 44, 3359–3362.
(24) Abdulla, M. H.; O’Brien, T.; Mackey, Z. B.; Sajid, M.; Grab, D. J.;
McKerrow, J. H. RNA Interference of Trypanosoma brucei Cathe-
psin B and L Affects Disease Progression in a Mouse Model. PLoS
Neglected Trop. Dis. 2008, 2, e298.
(25) DeLano W. L .The Pymol Molecular Graphics System; DeLano
Scientific: San Carlos, CA, 2002.
ꢀ
(26) Menard, R.; Carriere, J.; Laflamme, P.; Plouffe, C.; Khouri, H. E.;
Vernet, T.; Tessier, D, C.; Thomas, D. Y.; Storer, A. C. Contribu-
tion of the glutamine 19 side chain to transition-state stabilization
in the oxyanion hole of papain. Biochemistry 1991, 30, 8924–8928.
(27) Despite the potent inhibition of several cathepsins (notably, Cat C,
L, and K), these analogues were essentially inactive across 60
relevant human cysteine proteases.
(28) McKerrow, J. H. Development of cysteine protease inhibitors as
chemotherapy for parasitic diseases: insights on safety, target
validation, and mechanism of action. Int. J. Parasitol. 1999, 29,
833–837.
(29) Yasgar, A.; Shinn, P.; Michael, S.; Zheng, W.; Jadhav, A.; Auld, D.
S.; Austin, C. P.; Inglese, J.; Simeonov, A. Compound management
for quantitative high-throughput screening. J. Assoc. Lab. Autom.
2008, 13, 79–89.
(12) K11777. This is despite the fact that compound 1 has not shown
significant off-target side effects to date.
(13) Hickey, E. R.; Bekkali, Y.; Patel, U. R.; Spero, D. M.; Thomson,
D. S.; Young, E. R. R. Nitriles useful as reversible inhibitors of
cysteine proteases. U.S. Patent 6,982,263, 2006.
(14) Austin, C. P.; Brady, L. S.; Insel, T. R.; Collins, F. S. NIH
Molecular Libraries Initiative. Science 2004, 306, 1138–1139.
(15) Jadhav, A.; Ferreira, R. S.; Klumpp, C.; Mott, B. T.; Austin, C. P.;
Inglese, J.; Thomas, C. J.; Maloney, D. J.; Shoichet, B. K.;
Simeonov, A. Quantitative Analyses of Aggregation, Autofluor-
escence, and Reactivity Arifacts in a Screen for Inhibitors of a Thiol
Protease. J. Med. Chem. 2009, 10.1021/jm901070c.
(30) Eakin, A. E.; McGrath, M. E.; McKerrow, J. H.; Fletterick, R. J.;
Craik, C. S. Production of crystallizable cruzain, the major cysteine
protease from Trypanosoma cruzi. J. Biol. Chem. 1993, 268, 6115–
6118.
(31) McCoy, A. J.; Grosse-Kunstleve, R. W.; Storoni, L. C.; Read, R. J.
Likelihood-enhanced fast translation functions. Acta Crystallogr.,
Sect. D: Biol. Crystallogr. 2005, 61, 458–464.
(32) Huang, L.; Brinen, L. S.; Ellman, J. A. Crystal structures of
reversible ketone-based inhibitors of the cysteine protease cruzain.
Bioorg. Med. Chem. 2003, 11, 21–29.
(16) McGovern, S. L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. A
common mechanism underlying promiscuous inhibitors from vir-
tual and high-throughput screening. J. Med. Chem. 2002, 45, 1712–
1722.
(33) Murshudov, G. N.; Vagin, A. A.; Dodson, E. J. Refinement of
macromolecular structures by the maximum-likelihood method.
Acta Crystallogr., Sect. D: Biol. Crystallogr 1997, 53, 240–255.
(34) Emsley, P; Cowtan, K. Coot: model-building tools for molecular
graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004,
60 (Pt 12 Pt 1), 2126–2132.
(17) Bhatt, R.; Gong, B.; Hong, F.; Jenkins, S. A.; Klein, P. J.; Kumar,
A. M.; Tulinsky, J. 6-Phenyl-N-phenyl-(1,3,5)-triazine-2,4-diamine