Nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitors as promising new leads for chagas disease chemotherapy

Article abstract of DOI:10.1021/jm901633v

A century after discovering that the Trypanosoma cruzi parasite is the etiological agent of Chagas disease, treatment is still plagued by limited efficacy, toxicity, and the emergence of drug resistance. The development of inhibitors of the major T. cruzi cysteine protease, cruzain, has been demonstrated to be a promising drug discovery avenue for this neglected disease. Here we establish that a nonpeptidic tetrafluorophenoxymethyl ketone cruzain inhibitor substantially ameliorates symptoms of acute Chagas disease in a mouse model with no apparent, toxicity. A high-resolution crystal structure confirmed the mode of inhibition and revealed key binding interactions of this novel inhibitor class. Subsequent structure-guided optimization then resulted in inhibitor analogues with improvements in potency despite minimal or no additions in molecular weight. Evaluation of the analogues in cell culture showed enhanced activity. These results suggest that nonpeptidic tetrafluorophenoxymethyl, ketone cruzain inhibitors have the potential to fulfill the urgent need for improved Chagas disease chemotherapy.

Full text of DOI:10.1021/jm901633v

J. Med. Chem. 2010, 53, 1763–1773 1763
DOI: 10.1021/jm901633v
Nonpeptidic Tetrafluorophenoxymethyl Ketone Cruzain Inhibitors as Promising New Leads for Chagas
Disease Chemotherapy†
Katrien Brak,‡ Iain D. Kerr,§ Kimberly T. Barrett,‡ Nobuhiro Fuchi,‡ Moumita Debnath,§ Kenny Ang, Juan C. Engel,
James H. McKerrow, Patricia S. Doyle,*, Linda S. Brinen,*,§ and Jonathan A. Ellman*,‡
‡Department of Chemistry, University of California, Berkeley, California 94720-1460, §Department of Cellular and Molecular Pharmacology,
University of California, San Francisco, California 94158-2330, and Department of Pathology, UCSF, San Francisco, California 94158-2330
Received November 4, 2009
A century after discovering that the Trypanosoma cruzi parasite is the etiological agent of Chagas
disease, treatment is still plagued by limited efficacy, toxicity, and the emergence of drug resistance. The
development of inhibitors of the major T. cruzi cysteine protease, cruzain, has been demonstrated to be a
promising drug discovery avenue for this neglected disease. Here we establish that a nonpeptidic
tetrafluorophenoxymethyl ketone cruzain inhibitor substantially ameliorates symptoms of acute
Chagas disease in a mouse model with no apparent toxicity. A high-resolution crystal structure
confirmed the mode of inhibition and revealed key binding interactions of this novel inhibitor class.
Subsequent structure-guided optimization then resulted in inhibitor analogues with improvements in
potency despite minimal or no additions in molecular weight. Evaluation of the analogues in cell
culture showed enhanced activity. These results suggest that nonpeptidic tetrafluorophenoxymethyl
ketone cruzain inhibitors have the potential to fulfill the urgent need for improved Chagas disease
chemotherapy.
Introduction
and a short circulating half-life.15,16 With this in mind, we
recently applied the substrate activity screening method to
cruzain to obtain novel and potent entirely nonpeptidic
inhibitors.17,18 In particular, the 1,2,3-triazole-based tetra-
fluorophenoxymethyl ketone irreversible inhibitor 2 was
found to completely eradicate the T. cruzi parasite in cell
culture (Figure 1). In addition to the nonpeptidic nature of
inhibitor 2, the tetrafluorophenoxymethyl ketone functiona-
lity represents a very promising mechanism-based pharmaco-
phore due to its high selectivity for cysteine protease in-
hibition,19-21 as well as the lack of toxicity in animal
studies, which was established for a tetrafluorophenoxy-
methyl ketone-based caspase inhibitor that has entered phase
II clinical trials.22
Herein we report an initial evaluation of inhibitor 2 in a
mouse model of Chagas disease. The promising results from
these animal studies motivated further development of the
tetrafluorophenoxymethyl ketone class of cruzain inhibitors.
A high resolution X-ray crystal structure of 2 complexed to
cruzain provided characterization of the binding mode of 2
and enabled the design of inhibitors that are approximately
4-fold more potent in addition to having more desirable
physicochemical properties. The nonpeptidic nature of these
compounds, coupled with their efficacy in cell-culture and
mice, makes this class of inhibitors promising candidates for
improved chemotherapy for Chagas disease.
Chagas disease, also known as American trypanosomiasis,
results from infection by the Trypanosoma cruzi parasite. It is
estimated that 15 million people are infected with the parasite,
resulting in more than 12000 deaths each year.1 Chagas
disease is the leading cause of cardiomyopathy in Latin
America.2 Current treatment consists of nitroaromatic drugs
that are not only toxic but also ineffective for the chronic stage
of the disease.3,4 These limitations of the existing drugs along
with emerging resistance have provided considerable impetus
for the development of novel chemotherapy for Chagas
disease.5,6 One approach consists of developing inhibitors of
cruzain, the primary cysteine protease expressed by T. cruzia
during infection.7-9 Cruzain, a cysteine protease of the pa-
pain-like family, plays a vital role at every stage of the
parasite’s life cycle; it is involved in protein degradation for
nutrition, host cell remodeling, and evasion of host defense
mechanisms.10
Vinyl sulfone 1, a peptidic irreversible inhibitor of cruzain,
has been shown to cure parasitic infections in both cell and
animal models (Figure 1).11-14 While inhibitor 1 is effective, it
is peptidic and consequently exhibits low oral bioavailability
†Data for the X-ray crystal structure of cruzain complexed to
inhibitor 2 has been deposited in the Protein Data Bank (PDB ID 3IUT).
*To whom correspondence should be addressed. For chemistry (J.A.
E.): phone, 510-642-4488; fax, 510-642-8369; E-mail, jellman@uclink.
berkeley.edu. For X-ray crystallography (L.S.B.): phone, 415-514-3426;
fax, 415-502-8193; E-mail, brinen@cmp.ucsf.edu. For biological studies
(P.S.D): phone, 415-476-0825; fax, 415-502-8193; E-mail, patricia.
doyle-engel@ucsf.edu.
Chemistry
The synthesis of 1,4-disubstituted-1,2,3-triazole cruzain
inhibitor analogues 3 with differing R1 and R2 substi-
tuents required the preparation of various aryloxymethyl
ketone azide and quinoline propargyl amine intermediates
a Abbreviations: T. cruzi, Trypanosoma cruzi; IC50, concentration of
inhibitor resulting in 50% inhibition; bid, twice daily; kinact/Ki, second-
order inactivation rate constant.
r
2010 American Chemical Society
Published on Web 01/20/2010
pubs.acs.org/jmc

1764 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Brak et al.
(Scheme 1). The bromomethyl ketone azides 4a-c were
obtained via a three-step, one-pot procedure from the corre-
sponding azido acids by preparation of the isobutyl mixed
anhydride, addition of diazomethane to form a diazomethyl
ketone, and final treatment with hydrobromic acid. Displace-
ment of the bromide by 2,3,5,6-tetrafluorophenol afforded
aryloxymethyl ketone azide intermediates 5a-c. Enantio-
merically pure propargyl amine intermediates 7a-f were
prepared by a two-step reductive amination of quinoline-
6-carboxyaldehyde with tertiary carbinamines 6a-f. 1,4-
Disubstituted-1,2,3-triazole inhibitor analogues 3a-i were
then synthesized via a regioselective Cu(I)-catalyzed 1,3-
dipolar cycloaddition. Formation of the triazole in the final
step enabled the rapid synthesis of a variety of inhibitors
resulting from various combinations of the azide and alkyne
intermediates.
Results and Discussion
Evaluation in a Mouse Model of Chagas Disease. Mice
infected with a large inoculum of T. cruzi parasites (1.2 ꢀ 106
trypomastigotes) were treated for 27 days with tetrafluoro-
phenoxymethyl ketone inhibitor 2 (Table 1). The treatment
consisted of 20 mg/kg inhibitor 2 in two daily doses via
intraperitoneal injection. The mice were monitored for a
total of 77 days, at which point they were sacrificed for
hemoculture and histopathology. Throughout the experi-
ment, the untreated control mice showed signs of Chagas
disease such as ascites (abdominal swelling), malaise, weak-
ness of the hind legs, and ruffled hair. Hemoculture and
histopathology revealed that all the untreated mice had
positive hemocultures and significant inflammation and
infection in heart and skeletal muscle tissue. The mice treated
with inhibitor 2, on the other hand, looked completely
normal when sacrificed 77 days postinfection. Importantly,
the treatment was well-tolerated by all the mice with no
apparent signs of toxicity. Two out of four mice had negative
hemocultures, implying animals had no detectable blood
parasitemia. Significantly, histopathology revealed that
two out of five mice had no inflammation in heart muscle.
All the treated mice did show some inflammation in skeletal
muscle suggestive of cryptic infection.
The substantial amelioration of acute Chagas disease
symptoms is highly significant because, outside of vinyl
sulfone 1, there are no other published reports of successful
treatment of Chagas disease with inhibitors of cysteine
proteases in animal models.12,14 An alternative treatment
regimen with 2 or treatment with a more potent analogue
could lead to a complete cure of T. cruzi infected mice.
Figure 1. Structures of potent irreversible cruzain inhibitors: di-
peptidyl vinyl sulfone 1 and 1,2,3-triazole-based tetrafluorophenoxy-
methyl ketone 2.
Scheme 1. Synthesis of 1,4-Disubstituted-1,2,3-triazole Cruzain Inhibitor Analoguesa
a Reagents: (a) isobutyl chloroformate, N-methylmorpholine, THF, -40 °C; (b) diazomethane, THF, 0 °C; (c) HBr, THF, 0 °C; (d) 2,3,5,6-
˚
tetrafluorophenol, KF, DMF, 0 °C; (e) 4 A molecular sieves, toluene, rt; (f) NaBH4, MeOH, 0 °C; (g) sodium ascorbate, CuSO4, 1:1 H2O:t-BuOH, rt.
Table 1. Treatment of T. cruzi-Infected C3H Mice with Tetrafluorophenoxymethyl Ketone Inhibitor 2
no. of mice
without T. cruzi
in tissuesa,b
no. of mice
with negative
hemoculturea,c
acute Chagas
disease symptoms
group
no. of mice
dose
untreated mice
treated mice
3
5
none
20 mg/kg bid for 27 days via ip
ascites, paralysis of hind legs, malaise
none
0
2
0
2d
a Mice were sacrificed 77 days postinfection. b Tissue analysis of sacrificed animals established the absence/presence of T. cruzi. c T. cruzi was cultured
from heart blood collected when animals were sacrificed. d Hemocultures were performed for 4/5 mice.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1765
Figure 2. Crystal structure of the cruzain 2 complex (PDB ID 3IUT) elucidates the binding mode of 2 in the cruzain substrate-binding site.
3
Cruzain residues are colored pale cyan and the inhibitor is colored gray. The unbiased mFo - DFc electron density for the inhibitor is shown in
violet, contoured at the 3σ level. Black dashed lines indicate hydrogen bond interactions between inhibitor 2 and amino acid residues in
cruzain’s catalytic pocket. The C(5) of the 1,2,3-triazole ring is labeled.
tetrafluorophenoxy moiety of the inhibitor. Taking into
account the high resolution of the data, there was no need
to include a specific restraint on the C Sγ distance while
3
ensuring overall good geometry for the model. As a result,
˚
the distance refines to 1.83 A, indicating the formation of a
covalent bond between Cys25 and the inhibitor. The pharma-
cophore is further stabilized in the S10 subsite through the
formation of hydrogen bonds with the peptide amide of
˚
˚
Cys25 (3.13 A) and Nε2 of Gln19 (3.13 A) (Figure 2).
A common feature of cruzain-small molecule structures is
hydrogen bonding with Gly66, stabilizing the inhibitor in the
substrate-binding cleft.26-30 In the cruzain 2 complex,
Gly66 forms a hydrogen bond with both the amine functiona-
˚
lity of the inhibitor (2.85 A) and the N(3) atom of the 1,2,3-
3
˚
triazole of the inhibitor (3.20 A) (Figure 2). The strong dipole
moment of 1,2,3-triazole rings polarize the C(5) proton to
such a degree that it can function as a hydrogen-bond
donor.31-34 Indeed, the hydrogen bond commonly observed
with the backbone carbonyl of Asp161 and amide protons of
small molecules is also observed with the C(5) proton of the
Figure 3. Proposed mechanism of inhibition for tetrafluorophenoxy-
methyl ketone inhibitor 2.
˚
triazole of inhibitor 2 (3.41 A) (Figure 2). Because of the
For this reason, a structure-guided design of second-genera-
tion inhibitors was carried out.
ability of 1,2,3-triazoles to function as rigid linking units that
mimic the atom placement and electronic properties of a
peptide bond without the susceptibility of hydrolysis, many
known 1,2,3-triazoles possess biological activity.35
Structural Insight Provided by the X-ray Crystal Structure.
The X-ray crystal structure of 2 complexed to cruzain
verified both the mode of binding and inactivation (PDB
ID 3IUT). Inhibitor 2 is bound in the general orientation that
was previously predicted by superimposition with a structure
of the homologous protein cathepsin S in complex with a
triazole inhibitor that is structurally similar to 2 (PDB ID
2H7J).17,23 In particular, the n-butyl group is located in the
S1 subsite, the methyl and isopropyl functionalities in the S2
subsite, and the quinoline ring in the S3 subsite (Figure 2).
The specific binding interactions between cruzain and
inhibitor 2 were elucidated by the high-resolution X-ray
Prior development of this inhibitor class focused on
optimizations of interactions in the S3 subsite.17 Planar
heterocycles were introduced to provide hydrophobic inter-
actions with the hydrophobic residues of the pocket. Nitro-
gen or oxygen heteroatoms were also positioned in the
heterocyle to take advantage of potential hydrogen-bonding
interactions with the serine residue present in the S3 pocket.
A quinoline substituent was found to provide the greatest
binding affinity. The crystal structure illustrates that efficient
binding is aided by the formation of a hydrogen bond
˚
crystal structure of cruzain 2. While aryloxymethyl ketone
between the quinoline nitrogen and Ser61 (2.79 A), in addi-
3
inhibitors are well-studied as caspase inhibitors,24,25 this
pharmacophore has not been structurally visualized for
cruzain or any members of the papain superfamily. The
proposed mechanism of inhibition of cysteine proteases by
activated ketones involves nucleophilic attack of the cysteine
thiolate on the carbonyl carbon, leading to the formation of a
thiohemiketal stabilized by the oxyanion hole (Figure 3).21,25
Breakdown of the tetrahedral intermediate then results in a
displacement of the leaving group and hence irreversible
inhibition. The crystal structure reveals that the thiol nucleo-
phile of the active site Cys25 has effectively displaced the
tion to nonpolar interactions of the quinoline with Gly65 and
Leu67 in the S3 subsite (Figure 2). Additional nonpolar
interactions also contribute toward binding, with the iso-
propyl moiety stabilized by Leu67 and the P2 methyl sub-
stituent stabilized by nonpolar regions of Asp161 oriented
toward the inhibitor.
The residue at the bottom of the S2 subsite is of particular
interest as it is crucial in determining the substrate specificity
of papain family cysteine proteases.30 With flexible Glu208
at this position, cruzain has evolved an S2 subsite that is
able to tolerate both basic and hydrophobic residues.

1766 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Brak et al.
An interesting feature of the cruzain 2 crystal structure is the
within 3 h under the assay conditions (pH=6.3, 37 °C).17
The P1 n-butyl substituent is less ordered than the rest of the
inhibitor and, accordingly, the B-factors in this region are
higher in comparison to the rest of the small molecule.
However, the crystal structure clearly establishes binding
of the epimer with the S-configuration at the alpha stereo-
center (Figure 2).
3
dual conformation of Glu208. In the crystallized complex,
one conformer points toward the inhibitor and interacts with
the amine functionality via two bridging water molecules
(Figure 2). A second conformer adopts a solvent-exposed
orientation, pointing out of the S2 subsite and into a nearby
solvent channel in the crystal. We were surprised to discover
that the latter appears to interact with a transient and low
occupancy second copy of 2 in a solvent channel of the
crystal (Figure 4). As the occupancy of the atoms appear low
(only the N-terminal portion is partially visible) and we were
unable to unequivocally assign atomic positions for all atoms
in the molecule, we have not included this second inhibitor
molecule in the final coordinates. Of import for this study,
while polar interactions with residues lining the solvent
channel are possible with this second molecule copy, there
is no evidence of covalent bond formation with the pharma-
cophore, in sharp contrast to what is experimentally
observed in the active site bound copy of 2.
Comparison of Cruzain Nonpeptidic Inhibitor Structures.
3
The coauthors recently determined the first crystal structure
of a nonpeptidic cruzain inhibitor, 8, complexed to cruzain
(PDB ID 3HD3).36 Least-squares superimposition with the
cruzain 2 complex matches 215 CR positions with root-
3
˚
mean-square distances (rmsd) of 0.36 A (3HD3, monomer
A) and 214 CR positions with root-mean-square distances
(rmsd) of 0.42 A (3HD3, monomer B). Inhibitor 8 is a
˚
moderately potent cruzain inhibitor (kinact/Ki=6020 ( 820
s-1 M-1) that carries the well-characterized vinyl sulfone
warhead at the P10 position (Figure 5a). Irreversible inhibi-
tion of cruzain is achieved by a Michael addition of the active
site thiolate to the β-carbon of the unsaturated vinyl group of
the inhibitor. The P2 and P3 substituents (cyclohexane and
chlorophenyl, respectively) differ in comparison with 2;
however, both inhibitors are capable of establishing non-
polar interactions at these positions with residues lining the
binding sites. Interestingly, both inhibitors share the n-butyl
group at the P1 position, and while this moiety is more
flexible than the other substituents in 2, the n-butyl moiety in
8 is well-ordered and unambiguous in the electron density.
Superimposition of the two models shows that inhibitor 2
is able to form polar interactions missing in the complex
with vinyl sulfone 8: specifically, the amine-Gly66 and
quinoline-Ser61 hydrogen bonds as well as the amine-
Glu208 indirect interaction through two bridging water
molecules (Figure 5b). The favorable binding resulting from
these interactions is likely the reason for the greater potency
of inhibitor 2 (kinact/Ki = 157000 ( 20000 s-1 M-1). The
amide of inhibitor 8 forms a hydrogen bond with the back-
bone carbonyl of Asp161, an interaction that is replaced in 2
by the C(5) hydrogen of the 1,2,3-triazole ring. With vinyl
sulfone 8 lying deeper in the substrate-binding cleft, the
n-butyl group is able to form nonpolar contacts with Gly23
that are not present in 2. Therefore, long alkyl functionalities
at P1 may not be desirable for the aryloxymethyl ketone
cruzain inhibitors. Indeed, only a modest reduction in kinact/Ki
is seen when the n-butyl is replaced by an ethyl in 3a (Table 2)
The stereocenter R to the aryloxymethyl ketone moiety
is configurationally unstable with complete racemization
Figure 4. Putative position of a second inhibitor 2 molecule, bound
at low occupancy in a solvent channel. Cruzain is colored pale cyan
and the inhibitor gray. The unbiased mFo - DFc electron density for
the inhibitor is shown in violet, contoured at the 3σ level.
Figure 5. Structural comparison of nonpeptidic inhibitors 2 and 8. (a) Chemical structures and second-order inactivation constants of
tetrafluorophenoxymethyl ketone inhibitor 2 and vinyl sulfone inhibitor 8. (b) A superimposition of cruzain 2 (PDB ID 3IUT) and cruzain 8
(PDB ID 3HD3) crystal structures. The cruzain 2 complex is colored pale cyan, while the cruzain 8 complex is colored yellow. Black dashed
3
3
3
3
lines indicate hydrogen bond interactions between the inhibitors and amino acid residues in cruzain’s catalytic pocket.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1767
Table 2. Inhibition Kinetics of Tetrafluorophenoxymethyl Ketone
Inhibitors Against Cruzain
Table 3. Evaluation of Efficacy and Toxicity of Inhibitors in Cell
Culture
T. cruzi IC50
(μM)b
host IC50
(μM)a,b
compd
2
5.1
8.3
4.3
3.1
5.7
4.1
5.8
4.2
>10
>10
>10
>10
>10
>10
>10
10
3a
3c
3d
3e
3f
3g
3h
a T. cruzi-infected BESM cells. b Values are an average of duplicate or
triplicate runs.
full advantage of the hydrophobic S2 pocket, a series of
inhibitor analogues with increasingly larger R2 substituents
were prepared. Replacement of the isopropyl group with the
isobutyl group in inhibitor 3d and the cyclobutyl group in
inhibitor 3f resulted in 4-fold improved potency with kinact/Ki
values of 657000 M-1 s-1 and 680000 M-1 s-1, respectively
(Table 2). Not surprisingly, the increase in kinact/Ki was a
direct result of a 4-fold increase in the binding affinity (lower
Ki) of these inhibitors for cruzain while the kinact remained
the same. Interestingly, inhibitor 3h with a cyclopentyl
substituent had a slightly lower kinact/Ki due to a slight
decrease in kinact. The cyclohexyl substituent of inhibitor 3i
was too large, resulting in a dramatic increase in Ki and hence
a drop in the overall second-order rate constant. These
results are consistent with modeling studies, which indicated
that the cyclobutyl and cyclopentyl ring were of optimal size,
while the cyclohexyl ring was sterically hindered by close
contacts with the S2 subsite. Inhibitor analogues combining
optimal R1 and R2 substituents, 3e (R1 = ethyl, R2 =
isobutyl) and 3g (R1 = ethyl, R2 = cyclobutyl), were also
prepared and showed a 3-fold increase in potency.
Cell-Culture Evaluation of the Inhibitor Analogues. Tetra-
fluorophenoxymethyl ketone inhibitor 2 has previously been
shown to completely eradicate the T. cruzi parasite in cell
culture.17 Macrophages infected with T. cruzi were treated
for 27 days with 10 μM inhibitor 2, and cells were monitored
for two more weeks to ensure complete elimination of the
parasite. The more potent inhibitor analogues 3a and 3c-h
were also evaluated for their effectiveness at eradicating the
T. cruzi parasite in cell culture. For these analogues as well as
inhibitor 2, a new high through-put assay that provides IC50
values as well as an evaluation of compound toxicity was
employed.38 In a 96-well plate, BESM cells infected with T.
cruzi were incubated with several concentrations of the
inhibitors (0, 0.3, 1, 3, and 10 μM) for 3 days at 37 °C. The
wells were fixed and stained with a DNA fluorescent dye and
then scanned with an automated fluorescence microscope.
Host nuclei (>150 μm2) and parasite kinetoplast (2-4 μm2)
were differentiated based on size. A reduction of the number
of T. cruzi per host cell provided a measure of parasite
growth inhibition and a decrease in the number of host
nuclei provided a quantitative measure of cytotoxicity.
All of the inhibitor analogues tested significantly de-
creased the number of intracellular parasites after the 3 day
period of incubation as compared to untreated controls
(Table 3). Curiously, even though the inhibitor derivatives
3a, 3e, and 3g with R1=ethyl had comparable second-order
inactivation rates to their n-butyl counterparts (Table 2),
they did not perform as well in cell culture. This is evidenced
a Kinetic assays were performed at least in triplicate (SD values
included).
(vide infra), suggesting that the additional two atoms do not
contribute appreciably to inhibitor potency.
Structure-Guided Design of Inhibitor Analogues. The crys-
tal structure allowed for the design of analogues that take
advantage of the specific contours of the hydrophobic
regions of the S1 and S2 pockets. Numerous studies on
cruzain substrates and inhibitors have established that a
variety of unbranched P1 residues are accommodated
because the S1 pocket is solvent-exposed and less well-
defined.29,37 As such, inhibitor analogues 3a (R1 = ethyl)
and 3b (R1=methyl) were synthesized to probe the relative
contributions of the aliphatic n-butyl chain while reducing
molecular weight and number of rotatable bonds of the
inhibitor. While decreasing the R1 side chain to a methyl
group resulted in a 13-fold decrease in potency, truncation to
the smaller ethyl chain did not significantly affect the in-
hibitor potency (Table 2). Analysis of the separate binding
constant Ki and rate constant for enzyme inactivation kinact
provided insight into how the structural changes are reflected
in these values. The reduction in kinact/Ki was largely a result
of increases in the binding constant Ki with an approximate
15- and 2-fold increase for inhibitor 3a and 3b, respectively.
Interestingly, the ethyl derivative 3a was only slightly less
potent despite a 2-fold decrease in binding affinity because
the kinact increased by almost 2-fold. The other sets of n-butyl
and ethyl analogues, 3d/3e and 3f/3g, displayed the same
trend: the ethyl derivatives had a slightly decreased binding
affinity (higher Ki) and an enhanced kinact resulting in
approximately the same second-order rate constants kinact/Ki.
Cruzain belongs to a family of enzymes where the inter-
action with the S2 pocket of the enzyme is the major
specificity determinant.30 The Connolly surface of the
hydrophobic S2 pocket indicated that the isopropyl sub-
stituent in inhibitor 2 did not entirely fill the cavity. To take

1768 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Brak et al.
in all three pairs of n-butyl/ethyl inhibitor analogues
(Table 3: 2/3a, 3d/3e, and 3f/3g). The more potent inhibitors
3d, 3f, and 3h with R1=n-butyl all had improved activity in
cell culture (3.1-4.2 μM) compared to the first-generation
inhibitor 2 (5.1 μM). Significantly, no apparent toxicity was
observed: the host cell IC50’s were greater than or equal to 10
μM for all of the tetrafluorophenoxymethyl ketone inhibi-
tors.
methylene chloride, and toluene were obtained from a Seca
solvent system by GlassContour (solvent dried over alumina
under an N2 atmosphere). Anhydrous DMF (water <50 ppm)
was purchased from Acros. p-Toluenesulfonylmethylnitrosa-
mide (Diazald) was purchased from Sigma-Aldrich. (S)-tert-
Butanesulfinamide was provided by AllyChem Co. Ltd.
(Dalian, China). Inhibitor 2 and intermediates 4a, 5a, and 7a
were synthesized as previously reported.17 Propargyl amines
6a-f were synthesized according to reported procedures.41 All
reactions were carried out in flame-dried glassware under an
inert N2 atmostphere. Normal-phase purification was carried
out with Merck 60 230-240 mesh silica gel. Reverse-phase
HPLC purification was conducted either with an Agilent 1100
series instrument or Biotage SP1 instrument (Charlotteville,
VA) equipped with a Biotage C18SH column. Reverse-phase
HPLC analysis was conducted with an Agilent 1100 series
instrument. 1H, 13C, 19F NMR spectra were obtained on a
Bruker AV-300, AVB-400, AVQ-400, or DRX-500 at room
temperature. Chemical shifts are reported in ppm, and coupling
Conclusion
The drugs available to treat Chagas disease are decades old
and limited in efficacy.3,4 Validation of cruzain, the major
cysteine protease of the T. cruzi parasite, as a target has
provided an opportunity for developing improved
chemotherapy.7-9 Proteases are a well-studied class of en-
zymes due to their involvement in many cellular processes and
importance as targets for small-molecule therapeutics.39 Pep-
tidic inhibitors, consisting of small peptides coupled to an
electrophilic pharmacophore, easily mimic the natural sub-
strates of the protease and hence result in potent inhibitors.
However, they do not possess the molecular properties re-
quired for drug candidates due to their susceptibility to
hydrolysis, metabolism, and rapid clearance.16,40 To address
this issue, we have developed a nonpeptidic class of triazole-
based cruzain inhibitors.17,18
1
constants are reported in Hz. H resonances are referenced to
CHCl3 (7.26 ppm) or DMSO-d6 (4.90 ppm), 13C resonances are
referenced to CHCl3 (77.23 ppm) or DMSO-d6 (39.50 ppm), and
19F resonances are referenced to CFCl3 (0 ppm). Combustion
analyses and high-resolution mass spectrometry analyses were
performed by the University of California at Berkeley Micro-
analysis and Mass Spectrometry Facilities. All of the reported
inhibitors displayed g95% purity as determined by either
combustion analysis or reverse-phase HPLC using two different
solvent systems.
In this study, nonpeptidic inhibitor 2 was evaluated in a
mouse model of Chagas disease by twice daily dosing (20 mg/
kg) via intraperitoneal injection for 27 days. After 77 days
postinfection, visible signs of Chagas disease such as abdom-
inal swelling, malaise, and weakness of the hind legs were not
observed for the treated mice. The treatment was also well-
tolerated with no apparent signs of toxicity. Additionally, two
mice had negative hemocultures and displayed no signs of
infection in heart tissue. For comparison, a similar treatment
regime with the vinyl sulfone inhibitor 1 rescued animals from
lethal infection at a higher daily dose (50 mg/kg).12,14 How-
ever, all of the treated mice did show some inflammation in
skeletal muscle suggesting unresolved cryptic infection, which
prompted the development of more potent inhibitors.
A high resolution crystal structure of inhibitor 2 complexed
with cruzain was obtained. The structural information re-
vealed how the triazole moiety of the inhibitor is able to
provide the same stabilizing interactions with the enzyme
active site as the amide bond in peptidic inhibitors. In addition
tocharacterizing the bindingmode ofinhibitor2, the structure
enabled the design of inhibitors with 4-fold increases in
inhibitory activity and, for select inhibitors, improved phys-
icochemical properties such as reduced molecular weight,
lower hydrophobicity, and a reduction in the number of
rotatable bonds. Several of these inhibitors also showed
comparable or modestly improved potency relative to 2 in
cell culture evaluation.
General Synthesis of 2,3,5,6-Tetrafluorophenoxymethyl Ke-
tone Azides 5b-c (Procedure A). Step 1: This procedure was
adapted from a prior publication.42 Isobutylchloroformate (1.1
equiv) was added to a 0.1 M solution of the azido acid43 (1 equiv)
and N-methyl morpholine (1.1 equiv) in THF at -40 °C. The
reaction mixture was stirred for 20 min and then cannula filtered
into a flask at 0 °C to remove the white solid. Excess diazo-
methane, prepared from Diazald (3.1 equiv), was introduced in
situ according to the literature procedure44 while the flask was
maintained at 0 °C. After addition of the diazomethane, the
reaction flask was stoppered and was maintained at 0 °C in a
refrigerator overnight. The reaction mixture was treated with
48% aqueous HBr (3.8 equiv) and stirred for 15 min at 0 °C.
After addition of the HBr, N2 gas evolution was observed. The
reaction mixture was diluted with EtOAc (50 mL) and was then
washed with 10 wt % citric acid (2 ꢀ 10 mL), saturated NaHCO3
(2 ꢀ 20 mL), and saturated NaCl (1 ꢀ 10 mL). The organic layer
was dried over Na2SO4, filtered, and concentrated under re-
duced pressure. Step 2: To a 0.6 M solution of 2,3,5,6-tetra-
fluorophenol (3.0-3.1 equiv) in DMF at 0 °C was added
potassium fluoride (3.0 equiv), and the reaction mixture was
stirred for 10 min. The appropriate bromomethyl ketone 4b or
4c (1.0 equiv) was then added in a small amount of DMF. The
reaction mixture was stirred at 0 °C for 3 h. The reaction mixture
was diluted with CH2Cl2 and washed with water (1ꢀ), saturated
NaHCO3 (1ꢀ), water (2ꢀ), and brine (1ꢀ). The organic layer
was dried over Na2SO4, filtered, and concentrated under re-
duced pressure. The crude reaction mixture was purified by
reverse-phase using 5-95% CH3CN in H2O with 0.1%
CF3CO2H.
The tetrafluorophenoxymethyl ketone inhibitor 2 and the
newly designed inhibitors 3 represent very promising drug
leads for the treatment of Chagas disease. Evaluation of the
pharmacokinetic properties of these compounds along with
their evaluation in animal models of Chagas disease via oral
administration is under active investigation.
(S)-3-Azido-1-(2,3,5,6-tetrafluorophenoxy)pentan-2-one (5b).
Procedure A, step 1, was followed using isobutylchloroformate
(0.663 mL, 5.11 mmol), 2-azidobutyric acid (0.600 g, 4.65
mmol), N-methyl morpholine (0.562 mL, 5.11 mmol), Diazald
(3.00 g, 13.95 mmol), and 48% aqueous HBr (1.05 mL) in THF
(50 mL) to afford the crude product as a pale-yellow oil.
Bromomethyl ketone 4b, which is unstable, was taken on
immediately without purification. 1H NMR (300 MHz, CDCl3):
δ 1.05 (t, 3H, J=7.4), 1.73-2.11 (m, 2H), 3.99-4.16 (m, 3H).
Procedure A, step 2, was followed using bromomethyl ketone 4b
(0.480 g, 2.33 mmol), 2,3,5,6-tetrafluorophenol (1.16 g, 6.99
Experimental Section
General Synthesis Methods. Unless otherwise noted, all re-
agents were obtained from commercial suppliers and used
without purification. Tetrahydrofuran (THF), diethyl ether,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1769
mmol), and potassium fluoride (0.406 g, 6.99 mmol) in DMF
(7.0 mL) to afford 0.380 g (56%) of 5b as a clear oil. 1H NMR
(400 MHz, CDCl3): δ 1.07 (t, 3H, J=7.4), 1.79-1.87 (m, 1H),
1.95-2.01 (m, 1H), 4.10 (dd, 1H, J=4.9, 8.0) 5.00 (d, 1H, J=
17.5), 5.05 (d, 1H, J=17.5), 6.78-6.85 (m, 1H). 13C NMR (100
MHz, CDCl3): δ 10.1, 24.1, 66.9, 75.5 (t, J=3.0), 99.9 (t, J=
23.0), 136.7-137.0 (m), 140.3 (dm, J=246), 146.3 (dm, J=246),
201.5. 19F NMR (376 MHz, CDCl3): δ -156.4 to -156.2 (m,
2F), -138.4 to -138.3 (m, 2F). HRMS-FAB (m/z): [MLi]þ
calcd for C11H9N3O2F4Li, 298.0785; found, 298.0787.
(sept, 1H, J=6.5), 2.40 (s, 1H), 3.99 (d, 1H, J=12.7), 4.06 (d, 1H,
J=12.7), 7.34 (dd, 1H, J=4.3, 8.2), 7.71 (d, 1H, J=8.8), 7.76 (s,
1H), 8.04 (d, 1H, J=8.7), 8.08 (d, 1H, J=8.4), 8.80-8.89 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 24.7, 24.80, 24.83, 27.7,
48.3, 50.5, 53.6, 71.5, 88.6, 121.3, 126.4, 128.4, 129.6, 130.7,
136.0, 139.4, 147.8, 150.2. HRMS-FAB (m/z): [MH]þ calcd for
C18H23N2, 267.1861; found, 267.1859.
(S)-2-Cyclobutyl-N-(quinolin-6-ylmethyl)but-3-yn-2-amine
(7d). Procedure B was followed using the HCl salt of propargyl
amine 6d (0.111 g, 0.700 mmol) and quinoline-6-carboxyalde-
hyde (0.110 g, 0.700 mmol) in toluene (3.0 mL), followed by
reduction with sodium borohydride (0.053 g, 1.400 mmol) in
methanol (4.0 mL) to afford 0.115 g (63%) of 7d as a clear oil. 1H
NMR (400 MHz, CDCl3): δ 1.26 (s, 3H), 1.72-1.88 (m, 2H),
1.90-1.97 (m, 2H), 2.06-2.16 (m, 2H), 2.49 (s, 1H), 2.51-2.57
(m, 1H), 3.99 (d, 1H, J=12.7), 4.06 (d, 1H, J=12.7), 7.38 (dd,
1H, J=4.2, 8.3), 7.72 (dd, 1H, J=1.8, 8.7), 7.78 (s, 1H), 8.05 (d,
1H, J=8.6), 8.12 (d, 1H, J=7.9), 8.87 (dd, 1H, J=1.5, 4.2). 13C
NMR (100 MHz, CDCl3): δ 17.1, 23.9, 24.0, 24.2, 44.9, 48.6,
57.2, 72.6, 86.2, 121.1, 126.3, 128.2, 129.4, 130.6, 135.8, 139.2,
147.6, 150.0. HRMS-ESI (m/z): [MH]þ calcd for C18H21N2,
265.1699; found, 265.1706. Anal. Calcd for C18H20N2: C, 81.78;
H, 7.63; N, 10.60. Found: C, 81.50; H, 7.77; N, 10.59.
(S)-2-Cyclopentyl-N-(quinolin-6-ylmethyl)but-3-yn-2-amine
(7e). Procedure B was followed using the HCl salt of propargyl
amine 6e (0.050 g, 0.29 mmol) and quinoline-6-carboxyaldehyde
(0.050 g, 0.32 mmol) in toluene (1.2 mL), followed by reduction
with sodium borohydride (0.022 g, 0.58 mmol) in methanol (1.5
mL) to afford 0.041 g (51%) of 7e as a clear oil. 1H NMR (400
MHz, CDCl3): δ 1.39 (s, 3H), 1.50-1.62 (m, 4H), 1.64-1.75 (m,
2H), 1.79-1.86 (m, 2H), 2.08-2.13 (m, 1H), 2.36 (s, 1H), 3.99 (d,
1H, J=12.8), 4.13 (d, 1H, J=12.8), 7.38 (dd, 1H, J=4.0, 8.0),
7.74 (dd, 1H, J=2.0, 8.4), 7.79 (s, 1H), 8.06 (d, 1H, J=8.4), 8.13
(d, 1H, J=8.4), 8.88 (dd, 1H, J=1.6, 4.4). 13C NMR (100 MHz,
CDCl3): δ 25.5, 25.8, 26.0, 27.8, 28.3, 48.4, 50.2, 57.5, 71.7, 87.0,
121.2, 126.3, 128.3, 129.6, 130.7, 135.9, 139.5, 147.8, 150.1.
HRMS-ESI (m/z): [MH]þ calcd for C19H23N2, 279.1856; found,
279.1859.
(S)-2-Cyclohexyl-N-(quinolin-6-ylmethyl)but-3-yn-2-amine
(7f). Procedure B was followed using the HCl salt of propargyl
amine 6f (0.145 g, 0.92 mmol) and quinoline-6-carboxyaldehyde
(0.129 g, 0.69 mmol) in toluene (2.8 mL), followed by reduction
with sodium borohydride (0.052 g, 1.38 mmol) in methanol (3.4
mL) to afford 0.080 g (40%) of 7f. 1H NMR (300 MHz, CDCl3):
δ 1.03-1.31 (m, 5H), 1.33 (s, 3H), 1.48-1.58 (m, 1H), 1.63-1.72
(m, 1H), 1.76-1.87 (m, 2H), 1.92-2.05 (m, 2H), 2.39 (s, 1H),
4.01 (d, 1H, J=12.6), 4.06 (d, 1H, J=12.6), 7.38 (dd, 1H, J=4.1,
8.1), 7.74 (dd, 1H, J=2.1, 8.7), 7.79 (s, 1H), 8.05 (d, 1H, J=8.7),
8.13 (d, 1H, J=8.1), 8.88 (dd, 1H, J=1.7, 4.3). 13C NMR (100
MHz, CDCl3): δ 23.8, 26.75, 26.81, 26.9, 27.0, 28.2, 46.6, 48.2,
57.1, 71.6, 88.3, 121.3, 126.4, 128.4, 129.6, 130.8, 136.0, 139.6,
147.8, 150.2. HRMS-FAB (m/z): [MH]þ calcd for C20H25N2,
293.2018; found, 293.2021.
(S)-3-Azido-1-(2,3,5,6-tetrafluorophenoxy)butan-2-one (5c).
Procedure A, step 1, was followed using isobutylchloroformate
(0.297 mL, 2.29 mmol), 2-azidopropionic acid (0.400 g, 2.08
mmol), N-methyl morpholine (0.251 mL, 2.29 mmol), Diazald
(1.34 g, 6.24 mmol), and 48% aqueous HBr (0.47 mL) in THF
(24 mL) to afford the crude product as a colorless oil. Bromo-
methyl ketone 4c, which is unstable, was taken on immediately
1
without purification. H NMR (300 MHz, CDCl3): δ 1.52 (d,
3H, J=7.0), 3.91 (d, 1H, J=7.0), 4.05 (d, 1H, J=13.0), 4.12 (d,
1H, J=13.0). Procedure A, step 2, was followed using bromo-
methyl ketone 4c (0.17 g, 0.89 mmol), 2,3,5,6-tetrafluorophenol
(0.442 g, 2.66 mmol), and potassium fluoride (0.155 g, 2.66
mmol) in DMF (2.2 mL) to afford 0.125 g (51%) of 5c as a clear
oil. 1H NMR (400 MHz, CDCl3): δ 1.54 (d, 3H, J=7.1), 4.26 (q,
1H, J = 7.1) 5.03 (d, 1H, J = 17.7), 5.07 (d, 1H, J = 17.7),
6.78-6.85 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 15.3, 60.9,
75.0 (t, J=3.0), 99.9 (t, J=23.0), 136.8-136.9 (m), 140.3 (dm, J=
251), 146.3 (dm, J=251), 201.6. 19F NMR (376 MHz, CDCl3): δ
-156.4 to -156.3 (m, 2F), -138.5 to -138.3 (m, 2F). HRMS-
FAB (m/z): [MLi]þ calcd for C10H7N3O2F4Li, 284.0629; found,
284.0631.
General Synthesis of Quinoline Propargyl Amines 7b-f (Pro-
cedure B). The HCl salt of propargyl amine 6b-f41 (1.0-1.4
equiv) was dissolved in water and basified to pH=11 with 1 M
NaOH. The aqueous layer was then extracted with toluene (3ꢀ)
and the organic layers were combined, dried over Na2SO4, and
filtered to provide a 0.25 M solution of the volatile free-based
amine 6b-f in toluene. To the solution of propargyl amine 6b-f
were added quinoline-6-carboxyaldehyde (1-1.1 equiv) and
˚
activated 4 A molecular sieves. The reaction mixture was stirred
for 16 h and then filtered through a plug of celite. The celite was
washed with CH2Cl2 (3ꢀ). The organic washes were combined
and concentrated to afford the crude imine. To a 0.2 M solution
of the propargyl imine (1 equiv) in methanol at 0 °C was added
sodium borohydride (2 equiv). After stirring the reaction mix-
ture at 0 °C for 1 h, it was diluted with water and extracted with
CH2Cl2 (3ꢀ). The organic layers were combined, dried over
Na2SO4, filtered, and concentrated. The crude reaction mixture
was purified by silica-gel column chromatography (hexanes/
ethyl acetate) to afford the pure product.
(S)-2-Cyclopropyl-N-(quinolin-6-ylmethyl)but-3-yn-2-amine
(7b). Procedure B was followed using the HCl salt of propargyl
amine 6b (0.105 g, 0.720 mmol) and quinoline-6-carboxyalde-
hyde (0.113 g, 0.720 mmol) in toluene (3.0 mL), followed by
reduction with sodium borohydride (0.054 g, 1.44 mmol) in
methanol (4.0 mL) to afford 0.088 g (49%) of 7b as a clear oil. 1H
NMR (400 MHz, CDCl3): δ 0.41-0.50 (m, 3H), 0.61-0.66 (m,
1H), 0.99-1.06 (m, 1H), 1.53 (s, 3H), 2.32 (s, 1H), 4.03 (d, 1H, J
=12.8), 4.18 (d, 1H, J=12.8), 7.39 (dd, 1H, J=4.0, 8.4), 7.74
(dd, 1H, J=2.0, 8.8), 7.81 (s, 1H), 8.06 (d, 1H, J=8.8), 8.13 (d,
1H, J=7.2), 8.89 (dd, 1H, J=1.5, 4.4). 13C NMR (100 MHz,
CDCl3): δ 0.7, 2.6, 20.1, 28.7, 48.7, 56.2, 72.1, 84.2, 121.1, 126.3,
128.2, 129.4, 130.6, 135.8, 139.2, 147.7, 150.0.
General Synthesis of 2,3,5,6-Tetrafluorophenoxymethyl Ke-
tone Inhibitors 3a-i (Procedure C). This procedure was adapted
from Sharpless.45 To a 0.25 M suspension of alkyne (1.0-1.2
equiv) and azide (1 equiv) in a 1:1 mixture of water and tert-butyl
alcohol was added an aqueous solution of sodium ascorbate (1
equiv of a freshly prepared 1.0 M solution in water), followed by
an aqueous solution copper(II) sulfate (0.1 equiv of a freshly
prepared 0.3 M solution in water prepared from copper(II)
sulfate pentahydrate). The heterogeneous mixture was stirred
vigorously overnight. Water was added and extracted with
EtOAc (3ꢀ). The organic layers were combined, washed with
saturated NaCl (1ꢀ), dried over NaSO4, filtered, and concen-
trated under reduced pressure. The crude reaction mixture was
purified by HPLC [preparatory reverse-phase C18 column (24.1
mm ꢀ 250 mm), CH3CN/H2O-0.1% CF3CO2H=5:95 to 95:5
over 55 min; 10 mL/min; 254 nm detection for 65 min] and
lyophilized to afford the CF3CO2H salt of the product. The free
(S)-3,5-Dimethyl-N-(quinolin-6-ylmethyl)hex-1-yn-3-amine
(7c). Procedure B was followed using the HCl salt of propargyl
amine 6c (0.066 g, 0.41 mmol) and quinoline-6-carboxyaldehyde
(0.048 g, 0.30 mmol) in toluene (1.6 mL), followed by reduction
with sodium borohydride (0.023 g, 0.60 mmol) in methanol (1.5
mL) to afford 0.053 g (66%) of 7c. 1H NMR (400 MHz, CDCl3):
δ 1.00 (d, 6H, J=6.8), 1.41 (s, 3H), 1.50-1.67 (m, 2H), 1.93

1770 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Brak et al.
amine of the product was obtained by dissolving the CF3CO2H
salt of the product in saturated aqueous NaHCO3 and extract-
ing with CH2Cl2 (4ꢀ). The organic layers were combined, dried
over Na2SO4, filtered, and concentrated under reduced pres-
sure.
1.5H, J = 6.6), 0.762 (d, 1.5H, J = 6.6), 0.83-0.92 (m, 6H),
1.09-1.23 (m, 1H), 1.23-1.45 (m, 3H), 1.61 (s, 1.5H), 1.62 (s,
1.5H), 1.68-1.80 (m, 2H), 1.82-1.88 (m, 2H), 1.98-2.12 (m,
1H), 2.24-2.36 (m, 1H), 3.61 (d, 0.5H, J=12.6), 3.62 (d, 0.5H,
J=12.6), 3.76 (d, 1H, J=12.6), 4.92 (s, 2H), 5.668 (dd, 0.5H, J=
4.7, 10.4), 5.673 (dd, 0.5H, J=4.7, 10.4), 6.75-6.85 (m, 1H), 7.38
(dd, 1H, J=4.3, 8.2), 7.59 (s, 0.5H), 7.60 (s, 0.5H), 7.64-7.69 (m,
1H), 7.72 (s, 1H), 8.02 (d, 0.5H, J=8.7), 8.03 (d, 0.5H, J=8.7),
8.09-8.14 (m, 1H), 8.85-8.89 (m, 1H). 19F NMR (376 MHz,
CDCl3): δ -156.2 to -156.0 (m, 2F), -138.0 to -137.8 (m, 2F).
HRMS-FAB (m/z): [MH]þ calcd for C31H36N5O2F4, 586.2796;
found, 586.2805. Anal. Calcd for C31H35N5O2F4: C, 63.58; H,
6.02; N, 11.96. Found: C, 63.53; H, 6.18; N, 11.96.
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3e. Proce-
dure C was followed using propargyl amine 7c (0.018 g, 0.069
mmol), azide 5b (0.020 g, 0.069 mmol), 1 M aqueous sodium
ascorbate (0.069 mL, 0.069 mmol), and 0.3 M aqueous copper-
(II) sulfate (0.023 mL, 0.0069 mmol) in 1:1 tBuOH:H2O (0.28
mL) to afford 26.0 mg (68%) of a 1:1 mixture of diastereomers of
3e as a pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ 0.76 (d,
3H, J=6.6), 0.88-0.99 (m, 6H), 1.60 (br s, 1H), 1.61 (s, 1.5H),
1.62 (s, 1.5H), 1.70-1.80 (m, 1H), 1.82-1.88 (m, 2H), 2.04-2.15
(m, 1H), 2.30-2.42 (m, 1H), 3.62 (d, 1H, J=12.7), 3.76 (d, 1H,
J=12.7), 4.93 (s, 2H), 5.68 (dd, 1H, J=4.8, 10.3), 6.74-6.85 (m,
1H), 7.37 (dd, 1H, J=4.1, 8.2), 7.59 (s, 0.5H), 7.60 (s, 0.5H),
7.64-7.66 (m, 0.5H), 7.66-7.69 (m, 0.5H), 7.72 (s, 1H), 8.02 (d,
0.5H, J = 8.7), 8.03 (d, 0.5H, J = 8.7), 8.09-8.14 (m, 1H),
8.84-8.90 (m, 1H). 19F NMR (376 MHz, CDCl3): δ -156.2 to
-156.1 (m, 2F), -138.0 to -137.9 (m, 2F). HRMS-FAB (m/z):
[MH]þ calcd for C29H32N5O2F4, 558.2485; found, 558.2487.
Anal. Calcd for C29H31N5O2F4: C, 62.47; H, 5.60; N, 12.56.
Found: C, 62.37; H, 5.78; N, 12.35.
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3f. Proce-
dure C was followed using propargyl amine 7d (0.023 g, 0.086
mmol), azide 5a (0.027 g, 0.086 mmol), 1 M aqueous sodium
ascorbate (0.086 mL, 0.086 mmol), and 0.3 M aqueous copper-
(II) sulfate (0.026 mL, 0.0086 mmol) in 1:1 tBuOH:H2O (0.34
mL) to afford 25.7 mg (51%) of a 1:1 mixture of diastereomers of
3f as a pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ 0.84 (t,
1.5H, J = 7.2), 0.86 (t, 1.5H, J = 7.2), 1.09-1.23 (m, 1H),
1.23-1.45 (m, 4H), 1.537 (s, 1.5H), 1.542 (s, 1.5H), 1.67-2.00
(m, 7H), 2.00-2.14 (m, 1H), 2.24-2.37 (m, 1H), 2.81 (quint, 1H,
J=8.8), 3.63 (d, 1H, J=12.8), 3.788 (d, 0.5H, J=12.8), 3.794 (d,
0.5H, J=12.8), 4.92 (s, 2H), 5.661 (dd, 0.5H, J=4.8, 10.4), 5.665
(dd, 0.5H, J=4.8, 10.4), 6.75-6.81 (m, 1H), 7.37 (dd, 1H, J=4.2,
8.4), 7.58 (s, 0.5H), 7.59 (s, 0.5H), 7.686 (dd, 0.5H, J=4.4, 8.8),
7.691 (dd, 0.5H, J=4.4, 8.8), 7.74 (s, 1H), 8.02 (d, 0.5H, J=8.8),
8.03 (d, 0.5H, J=8.8), 8.09-8.14 (m, 1H), 8.84-8.89(m, 1H).
19F NMR (376 MHz, CDCl3): δ -156.2 to -156.1 (m, 2F),
-138.0 to -137.9 (m, 2F). HRMS-ESI (m/z): [MH]þ calcd for
C31H34N5O2F4, 584.2643; found, 570.2649. The purity of the
inhibitor was determined by HPLC-MS analysis (C18 column
(2.1 mm ꢀ 150 mm); 0.4 mL/min; 254 nm detection in two
solvent systems: CH3CN/H2O-0.1% CF3CO2H, 5:95 to 95:5
over 16 min, 95:5 for 2 min: 99%; CH3OH/H2O, 5:95 to 95:5
over 20 min, 95:5 for 10 min: 96%).
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3g. Pro-
cedure C was followed using propargyl amine 7d (0.023 g,
0.086 mmol), azide 5b (0.025 g, 0.086 mmol), 1 M aqueous
sodium ascorbate (0.086 mL, 0.086 mmol), and 0.3 M aqueous
copper(II) sulfate (0.026 mL, 0.0086 mmol) in 1:1 tBuOH:H2O
(0.34 mL) to afford 32.3 mg (68%) of a 1:1 mixture of diaster-
eomers of 3g as a clear oil. 1H NMR (400 MHz, CDCl3): δ 0.93
(t, 1.5H, J=7.2), 0.95 (t, 1.5H, J=7.2), 1.53 (s, 1.5H), 1.54 (s,
1.5H), 1.64-1.99 (m, 7H), 2.02-2.17 (m, 1H), 2.30-2.43 (m,
1H), 2.80 (quint, 1H, J=8.8), 3.627 (d, 0.5H, J=12.8), 3.631 (d,
0.5H, J=12.8), 3.785 (d, 0.5H, J=12.8), 3.788 (d, 0.5H, J=12.8),
4.93 (s, 2H), 5.57 (dd, 0.5H, J=4.8, 9.6), 5.58 (dd, 0.5H, J=4.8,
9.6), 6.75-6.85 (m, 1H), 7.37 (dd, 1H, J=4.4, 8.4), 7.58 (s, 0.5H),
7.59 (s, 0.5H), 7.688 (dd, 0.5H, J=3.6, 8.6), 7.693 (dd, 0.5H,
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3a. Pro-
cedure C was followed using propargyl amine 7a (0.030 g, 0.12
mmol), azide 5b (0.032 g, 0.11 mmol), 1 M aqueous sodium
ascorbate (0.024 g, 0.012 mmol), and 0.3 M aqueous copper(II)
sulfate (0.003 g, 0.012 mmol) in 1:1 tBuOH:H2O (0.5 mL) to
afford 33.6 mg (52%) of a 1:1 mixture of diastereomers of 3a as a
pale-yellow oil. 1H NMR (300 MHz, CDCl3): δ 0.80 (d, 3H, J=
6.6), 0.93 (t, 1.5H, J=7.5), 0.96 (t, 1.5H, J=7.5), 1.02 (d, 3H, J=
6.6), 1.50 (s, 1.5H), 1.51 (s, 1.5H), 2.03-2.21 (m, 2H), 2.33-2.37
(m, 1H), 3.59 (d, 1H, J=12.9), 3.77 (d, 1H, J=12.9), 4.92 (s, 2H),
5.57 (dd, 1H, J=5.1, 10.2), 6.73-6.85 (m, 1H), 7.37 (dd, 1H, J=
4.5, 8.4), 7.57 (s, 0.5H), 7.58 (s, 0.5H), 7.67- 7.69 (m, 1H), 7.73 (s,
1H), 8.02 (d, 0.5H, J=8.7), 8.03 (d, 0.5H, J=8.7), 8.11 (m, 1H),
8.84-8.88 (m, 1H). 19F NMR (376 MHz, CDCl3): δ -156.3 to
-156.2 (m, 2F), -138.0 to -137.9 (m, 2F). HRMS-ESI (m/z):
[MH]þ calcd for C28H30N5O2F4, 544.2330; found, 544.2336.
Anal. Calcd for C28H29N5O2F4: C, 61.87; H, 5.38; N, 12.88.
Found: C, 62.18; H, 5.40; N, 12.62.
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3b. Pro-
cedure C was followed using propargyl amine 7a (0.030 g, 0.12
mmol), azide 5c (0.031 g, 0.11 mmol), 1 M aqueous sodium
ascorbate (0.024 g, 0.12 mmol), and 0.3 M aqueous copper(II)
sulfate (0.003 g, 0.012 mmol) in 1:1 tBuOH:H2O (0.5 mL) to
afford 27.1 mg (43%) of a 1:1 mixture of diastereomers of 3b as a
pale-yellow oil. 1H NMR (300 MHz, CDCl3): δ 0.81 (d, 3H, J=
6.9), 1.02 (d, 3H, J=6.9), 1.51 (s, 3H), 1.84 (d, 1.5H, J=5.1), 1.86
(d, 1.5H, J=5.1), 2.17-2.22 (m, 1H), 3.61 (d, 1H, J=12.9), 3.78
(d, 1H, J=12.9), 4.87 (d, 1H, J=16.8), 4.95 (d, 1H, J=16.8),
5.74-5.80 (m, 1H), 6.73-6.84 (m, 1H), 7.37 (dd, 1H, J=4.5,
8.4), 7.54 (s, 0.5H), 7.55 (s, 0.5H), 7.66- 7.73 (m, 2H), 8.02 (d,
0.5H, J = 8.4), 8.04 (d, 0.5H, J = 8.4), 8.09-8.13 (m, 1H),
8.85-8.88 (m, 1H). 19F NMR (376 MHz, CDCl3): δ -156.3 to
-156.2 (m, 2F), -138.0 to -137.9 (m, 2F). HRMS-ESI (m/z):
[MH]þ calcd for C27H28N5O2F4, 530.2174; found, 530.2176.
The purity of the inhibitor was determined by HPLC-MS
analysis (C18 column (2.1 mm ꢀ 150 mm); 0.4 mL/min; 254
nm detection in two solvent systems: CH3CN/H2O-0.1%
CF3CO2H, 5:95 to 95:5 over 16 min, 95:5 for 2 min: 98%;
CH3OH/H2O, 5:95 to 95:5 over 20 min, 95:5 for 10 min: 95%).
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3c. Proce-
dure C was followed using propargyl amine 7b (0.030 g, 0.12
mmol), azide 5a (0.032 g, 0.10 mmol), 1 M aqueous sodium
ascorbate (0.024 g, 0.12 mmol), and 0.3 M aqueous copper(II)
sulfate (0.003 g, 0.012 mmol) in 1:1 tBuOH:H2O (0.5 mL) to
afford 33.4 mg (49%) of a 1:1 mixture of diastereomers of 3c as a
pale-yellow oil. 1H NMR (300 MHz, CDCl3): δ 0.18-0.22 (m,
1H), 0.41-0.50 (m, 3H), 0.85 (t, 1.5H, J=7.0), 0.87 (t, 1.5H, J=
7.0), 1.15-1.22 (m, 1H), 1.25-1.40 (m, 4H), 1.54 (s, 3H),
2.02-2.08 (m, 1H), 2.20-2.33 (m, 1H), 3.74 (d, 1H, J=12.6),
3.87 (d, 1H, J=12.6), 4.93 (s, 2H), 5.63-5.68 (m, 1H), 6.76-6.84
(m, 1H), 7.37 (dd, 1H, J=4.5, 8.4), 7.61 (s, 0.5H), 7.62 (s, 0.5H),
7.65- 7.70 (m, 1H), 7.73 (s, 1H), 8.02 (d, 0.5H, J=8.4), 8.05 (d,
0.5H, J=8.4), 8.09-8.13 (m, 1H), 8.84-8.88 (m, 1H). 19F NMR
(376 MHz, CDCl3): δ -156.2 to -156.1 (m, 2F), -138.0 to
-137.9 (m, 2F). HRMS-ESI (m/z): [MH]þ calcd for
C30H32N5O2F4, 570.2492; found, 570.2488. Anal. Calcd for
C30H31N5O2F4: C, 63.26; H, 5.49; N, 12.30. Found: C, 62.93;
H, 5.50; N, 11.90.
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3d. Pro-
cedure C was followed using propargyl amine 7c (0.017 g, 0.065
mmol), azide 5a (0.021 g, 0.065 mmol), 1 M aqueous sodium
ascorbate (0.065 mL, 0.065 mmol), and 0.3 M aqueous copper-
(II) sulfate (0.022 mL, 0.0065 mmol) in 1:1 tBuOH:H2O (0.26
mL) to afford 30.0 mg (79%) of a 1:1 mixture of diastereomers of
3d as a pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ 0.760 (d,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1771
J=3.6, 8.6), 7.74 (s, 1H), 8.02 (d, 0.5H, J=8.6), 8.03 (d, 0.5H, J=
8.6), 8.09-8.14 (m, 1H), 8.85-8.89 (m, 1H). 19F NMR (376
MHz, CDCl3): δ -156.3 to -156.1 (m, 2F), -138.0 to -137.8
(m, 2F). MS (ESI): m/z 556 [MH]þ. Anal. Calcd for
C29H29N5O2F4: C, 62.69; H, 5.26; N, 12.61. Found: C, 62.27;
H, 5.05; N, 12.29.
least in triplicate, and the average and standard deviation of the
assays is reported.
T. cruzi Cell Culture Assay. Mammalian cells were cultured in
RPMI-1640 medium supplemented with 5% heat-inactivated
fetal calf serum (FCS) at 37 °C in 5% CO2. The Y strain of T.
cruzi was maintained by serial passage in bovine embryo skeletal
muscle (BESM) cells. Infectious trypomastigotes were collected
from culture supernatants. A BESM cell suspension was dis-
pensed into sterile 96-well black plates with clear bottom
(Greiner Bio-One). Following cell attachment, cultures were
infected with T. cruzi trypomastigotes. After infection, the
culture medium was removed and replaced with 200 μL of fresh
culture medium containing several concentrations of the test
inhibitors (0, 0.3, 1, 3, and 10 μM). Culture plates were incu-
bated for 72 h, washed once with PBS, and fixed for 2 h with 4%
paraformaldehyde. Following a PBS wash to remove the fixa-
tive, host cell and parasite DNAs were labeled with 0.1 μg/mL
DAPI. Plates were kept in the dark at 4 °C until used for image
acquisition by the IN Cell Analyzer 1000 imager (GE
Healthcare) with a 10ꢀ objective. Ten image fields comprising
200-300 host cells were acquired per well. The IN Cell Work-
station 3.5 multitarget analysis module was used for image
analysis. Image segmentation parameters were set to identify
host nuclei segmented with a minimum area of 150 μm2 and
intracellular parasite nuclei with an area size of 2-4 μm2.
Parasite nuclei/ host nuclei ratios were selected as measurement
output and the average of duplicate or triplicate runs were used
to generate IC50 plots (Figure S1, Supporting Information).
Efficacy Studies in Mice. Female C3H mice (Jax) (mean
weight 22 g; n = 8) were inoculated by intraperitoneal (ip)
injection with 1.2 ꢀ 106 T. cruzi tissue culture trypomastigotes
resuspended in 100 μL of RPMI medium with 5% heat inacti-
vated horse serum. Treatment (n=5) was initiated 24 h post
infection. A control group (n=3) of similarly infected animals
was left untreated. Compound 2 was resuspended daily in 100
μL of 70% DMSO:30% ddH2O, and injected b.i.d. via ip for 27
days at a dose of 20 mg/kg weight. Animals were sacrificed 77
days postinfection. Heart, skeletal muscle, liver, spleen, and
colon were collected for histopathological studies. Blood (50
μL) was collected by heart puncture and cultured in BHT
medium at 27 °C. Hemocultures were maintained for 90 days
and observed weekly for live parasites by contrast phase micro-
scopy. Detailed histopathology and hemoculture results are
listed in Table S1 (Supporting Information).
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3h. Pro-
cedure C was followed using propargyl amine 7e (0.020 g, 0.072
mmol), azide 5a (0.025 g, 0.079 mmol), 1 M aqueous sodium
ascorbate (0.072 mL, 0.072 mmol), and 0.3 M aqueous copper-
(II) sulfate (0.022 mL, 0.0072 mmol) in 1:1 tBuOH:H2O (0.29
mL) to afford 29.0 mg (67%) of a 1:1 mixture of diastereomers of
3h as a pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ 0.85 (t,
1.5H, J = 7.2), 0.87 (t, 1.5H, J = 7.2), 1.08-1.22 (m, 1H),
1.22-1.53 (m, 9H), 1.57 (s, 1.5H), 1.58 (s, 1.5H), 1.64-1.81
(m, 3H), 1.99-2.12 (m, 1H), 2.23-2.35 (m, 1H), 2.42 (quint, 1H,
J=8.8), 3.64 (d, 1H, J=12.8), 3.81 (d, 1H, J=12.8), 4.91 (s, 2H),
5.65 (dd, 1, J=4.8, 10.4), 6.76-6.84 (m, 1H), 7.38 (dd, 1H, J=
4.0, 8.0), 7.58 (s, 0.5H), 7.59 (s, 0.5H), 7.69 (dd, 0.5H, J=4.4,
8.8), 7.70 (dd, 0.5H, J=4.4, 8.8), 7.74 (s, 1H), 8.02 (d, 0.5H, J=
8.8), 8.03 (d, 0.5H, J=8.8), 8.08-8.14 (m, 1H), 8.83-8.89 (m,
1H). 19F NMR (376 MHz, CDCl3): δ -156.2 to -156.0 (m, 2F),
-138.0 to -137.8 (m, 2F). HRMS-ESI (m/z): [MH]þ calcd for
C32H36N5O2F4, 598.2800; found, 598.2808. Anal. Calcd for
C32H35N5O2F4: C, 64.31; H, 5.90; N, 11.72. Found: C, 63.99;
H, 6.15; N, 11.72.
2,3,5,6-Tetrafluorophenoxymethyl Ketone Inhibitor 3i. Proce-
dure C was followed using propargyl amine 7f (0.024 g, 0.082
mmol), azide 5a (0.026 g, 0.082 mmol), 1 M aqueous sodium
ascorbate (0.082 mL, 0.082 mmol), and 0.3 M aqueous copper-
(II) sulfate (0.027 mL, 0.0082 mmol) in 1:1 tBuOH:H2O (0.33
mL) to afford 33.5 mg (67%) of a 1:1 mixture of diastereomers of
3i as a pale-yellow oil. 1H NMR (400 MHz, CDCl3): δ 0.85 (t,
1.5H, J = 7.2), 0.87 (t, 1.5H, J = 7.2), 0.91-1.11 (m, 2H),
1.12-1.43 (m, 6H), 1.51 (s, 1.5H), 1.52 (s, 1.5H), 1.56-1.73
(m, 5H), 1.74-1.86 (m, 2H), 2.01-2.12 (m, 2H), 2.24-2.37 (m,
1H), 3.58 (d, 1H, J=13.0), 3.77 (d, 1H, J=13.0), 4.92 (s, 2H),
5.62-5.69 (m, 1H), 6.75-6.85 (m, 1H), 7.37 (dd, 1H, J=4.2,
8.3), 7.55 (s, 0.5H), 7.56 (s, 0.5H), 7.65- 7.70 (m, 1H), 7.72 (s,
1H), 8.01 (d, 0.5H, J=8.8), 8.02 (d, 0.5H, J=8.8), 8.09-8.14 (m,
1H), 8.85-8.89 (m, 1H). 19F NMR (376 MHz, CDCl3): δ
-156.2 to -156.1 (m, 2F), -138.0 to -137.9 (m, 2F). HRMS-
FAB (m/z): [MH]þ calcd for C33H38N5O2F4, 612.2971; found,
612.2962. Anal. Calcd for C33H37N5O2F4: C, 64.80; H, 6.10; N,
11.45. Found: C, 64.51; H, 6.46; N, 11.14.
Crystallization and Data Collection. Cruzain was recombi-
nantly expressed and purified (see Supporting Information for
experimental details). Crystallization conditions were screened
with the Mosquito drop-setting system (TTP Labtech) against a
number of commercially available kits. Conditions yielding
crystals (20% PEG 3000, 0.1 M sodium acetate pH 4.5) were
reproduced in a 24-well format using 500 μL of crystallization
solution per well and hanging drops consisting of l μL of protein
and l μL of well solution. Crystals were flash-cooled in well
solution supplemented with 30% ethylene glycol and mounted
in a cassette for the Stanford Auto Mounter (SAM) system.47
Diffraction data were collected at the Stanford Synchrotron
Cruzain Inhibition Assay. The kinact/Ki for inhibitors were
determined under pseudo-first-order conditions using the pro-
gress curve method.46 The proteolytic cleavage of N-acyl ami-
nocoumarins by cruzain was conducted in Dynatech Microfluor
fluorescence 96-well microtiter plates. Assay wells contained a
mixture of inhibitor and 1.0 μM of Cbz-Phe-Arg-AMC (Km=
1.1 μM; purchased from Bachem) in buffer (100 mM solution of
pH 6.3 sodium phosphate buffer with 400 mM of sodium
chloride, 5 mM of DTT, 10 mM of EDTA, and 0.025%
Triton-X 100). Aliquots of cruzain were added to each well to
initiate the assay. The final enzyme concentration was 0.1 nM.
Hydrolysis of the AMC substrate was monitored fluorometri-
cally for 25 min using a Molecular Devices Spectra Max Gemini
SX instrument. The excitation wavelength was 350 nm and the
emission wavelength was 450 nm, with a cutoff of 435 nm. To
determine the inhibition parameters, time points for which the
control ([I]=0) was linear were used. For each inhibitor, a kobs
was calculated for at least four different concentrations of
inhibitors via a nonlinear regression of the data according to
the equation P = (vi/kobs)[1 - exp(-kobst) (where product
formation=P, initial rate=vi, time=t, and the first-order rate
constant=kobs) using Prism 5 (GraphPad). For all inhibitors,
kobs varied hyperbolically with [I] and nonlinear regression
analysis was performed with Prism to determine kinact/Ki using
kobs=kinact[I]/([I] þ Ki*(1 þ [S]/Km)). Inhibition was measured at
Radiation Lightsource (SSRL). Cruzain 2 data were collected
3
˚
˚
to 1.20 A on BL7-1 at a temperature of 100 K and λ=0.98 A
after selecting an optimal crystal from screening performed with
the robotic SAM system. Reflections were indexed and inte-
grated in the primitive monoclinic setting using MOSFLM48
and scaled and merged in space group P21 with SCALA.49
Intensities were then converted to structure factor amplitudes
in TRUNCATE.50 The cruzain 2 structure was solved by
3
molecular replacement in MOLREP51 using a high resolution
structure of cruzain bound to a hydroxymethyl ketone inhibitor
(PDB ID 1ME3). A single, clear rotation function solution was
obtained at greater than 3 ꢀ peak height/σ of the second highest
solution, suggesting one molecule of cruzain in the asymmetric
unit. The translation function yielded a clear solution for the

1772 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Brak et al.
monomer with a score of 0.67 and initial Rwork of 34.6%. Rigid
body refinement, followed by simulated annealing and grouped
B factor refinement in CNS52 gave an Rfree of 28.56% an Rwork
of 26.88% and yielded electron density maps of excellent
quality. The inhibitor molecule was placed into mFo - DFc
difference electron density contoured at the 3σ level using
COOT.53 The resulting coordinates were refined through a
combination of simulated annealing, positional, and isotropic
B factor refinement in CNS. The model was completed by
interspersing rounds of positional and anisotropic B factor
refinement in REFMAC554 with manual adjustments in COOT.
The structure has excellent stereochemistry, as determined by
MOLPROBITY,55 with 96.6% of residues in the favored re-
gions, 100% in allowed regions, and no outliers. The final model
contains 1 molecule of mature cruzain, 1 molecule of 2, 265
water molecules, and 4 molecules of ethylene glycol. Statistics
for data collection and refinement are given in Table S2
(Supporting Information). The coordinates and observed struc-
ture factors amplitudes for each model have been deposited in
the Protein Data Bank under accession code 3IUT.
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Acknowledgment. J.A.E. and K.B. gratefully acknowledge
the NIH (GM0545051) for support of this work. K.B. greatly
appreciates an American Chemical Society Division of
Organic Chemistry Graduate Student Fellowship sponsored
by Wyeth Pharmaceuticals. J.H.M., L.S.B., J.C.E., P.S.D.,
and I.D.K. gratefully acknowledge National Institutes of
Health grant AI35707 and the Sandler Family Foundation
for support of this work. Part of this research was performed
attheStanfordSynchrotron Radiation Lightsource (SSRL), a
national user facility operated by Stanford University on
behalf of the U.S. Department of Energy, Office of Basic
Energy Sciences. We thank Rafaela Ferreira for kindly provid-
ing the pPICZR C construct with the cruzain insert. We also
thank Clifford Bryant and Dr. Adam Renslo (Small Molecule
Discovery Center, UCSF) for providing inhibitor 8 for kinetic
analyses.
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Supporting Information Available: T. cruzi cell culture IC50
plots, detailed histopathology and hemoculture results, experi-
mental details for the expression and purification of recombi-
nant cruzain, and crystallographic data collection and refine-
ment statistics. This material is available free of charge via the
Internet at http://pubs.acs.org.
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Article
peptide surrogate in the rapid synthesis of HIV-1 protease inhibi-
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