Crystal structures of reversible ketone-Based inhibitors of the cysteine protease cruzain

Article abstract of DOI:10.1016/S0968-0896(02)00427-3

The crystal structures of two hydroxymethyl ketone inhibitors complexed to the cysteine protease cruzain have been determined at 1.1 and 1.2 A resolution, respectively. These high resolution crystal structures provide the first structures of non-covalent inhibitors bound to cruzain. A series of compounds were prepared and tested based upon the structures providing further insight into the key binding interactions.

Full text of DOI:10.1016/S0968-0896(02)00427-3

Bioorganic & Medicinal Chemistry 11 (2003) 21–29
Crystal Structures of Reversible Ketone-Based Inhibitors of
the Cysteine Protease Cruzain
Lily Huang,a Linda S. Brinenb,*,y and Jonathan A. Ellmana,*
aCenter for New Directions in Organic Synthesis, Department of Chemistry,
University of California, Berkeley, CA, 94720, USA
bDepartment of Pathology, University of California, San Francisco, CA 94143, USA
Received 28 May 2002; accepted 8 August 2002
Abstract—The crystal structures of two hydroxymethyl ketone inhibitors complexed to the cysteine protease cruzain have been
determined at 1.1 and 1.2 A resolution, respectively. These high resolution crystal structures provide the first structures of non-
covalent inhibitors bound to cruzain. A series of compounds were prepared and tested based upon the structures providing further
insight into the key binding interactions.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
alkylated with Cbz-Phe-Ala-fluoromethyl ketone. In a
later study, Gillmor et al. reported the structures of
cruzain complexed to Cbz-Arg-Ala-fluoromethyl ketone
and Cbz-Tyr-Ala-fluoromethyl ketone at 2.2 and 2.1 A
resolution, respectively.5 These structures provided a
structural basis for the binding specificity of the P2
substituent, which is often a critical specificity determi-
nant for cysteine proteases. In particular, these struc-
tures revealed that cruzain is able to accommodate both
hydrophobic and basic residues in the P2 position by a
rotation of the Glu205 residue, which is found at the
bottom of the S2 pocket. More recently, four high-reso-
lution crystal structures of cruzain bound to vinyl sul-
fone derivatives were reported (resolution ranged from
1.6 to 2.15 A).6 As expected, the mode of inhibition was
a Michael addition between the active site Cys25 and
the vinyl sulfone.
Infection by the protozoan parasite Trypanosoma cruzi
results in Chagas’ disease, which is the principal cause
of heart disease in South America.1 The only currently
available treatment is insufficient due to considerable
toxicity.2 The lack of effective treatment has stimulated
efforts to identify novel drug targets against this para-
site. One target that has received significant attention is
cruzain, the major cysteine protease found in T. cruzi.
Recently, McKerrow et al. have shown that irreversible
inhibitors of cruzain can cure parasitic infections in
mouse models.3 These results demonstrate the ther-
apeutic promise of inhibitors of cruzain for the treat-
ment of Chagas’ disease.
Knowledge of the high-resolution structure of a target
protease significantly facilitates the design of potent and
selective inhibitors. Previous crystal structures of cruzain
complexed with irreversible inhibitors have provided
useful information about the binding interactions of
inhibitors with cruzain. McGrath et al. reported the first
crystal structure of cruzain in 1995.4 In this 2.35 A struc-
ture, the active-site cysteine of cruzain was irreversibly
We have recently reported a series of highly potent and
selective reversible inhibitors of cruzain.7 We focused on
developing reversible inhibitors in order to minimize the
toxicity that can be observed with irreversible inhibi-
tors.3b Our efforts have also led to the identification of
hydroxymethyl ketone analogues that are moderately
potent inhibitors of cruzain. Herein, we report a 1.1 A
crystal structure of hydroxymethyl ketone 1 complexed
to cruzain and a 1.2 A crystal structure of hydroxymethyl
ketone 2 complexed to cruzain. These high-resolution
results represent the first reported X-ray structures of
cruzain bound to non-covalent inhibitors.
*Corresponding author. Fax:+1-510-642-8369; e-mail: jellman@
uclink.berkeley.edu
yPresent address: Joint Center for Structural Genomics, Stanford
University, 2575 Sand Hill Road, SSRL MS69, Menlo Park, CA
94025, USA.
0968-0896/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00427-3

22
L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
Crystal Structures Results and Discussion
high degree of similarity to one another (Table 1 and
Fig. 2). In both structures, no covalent bond is formed
between the active-site cysteine and the ketone pharma-
cophore, as is evidenced by a clear gap in the experi-
mental electron density between the two moieties. In
fact, the ketone moiety and Cys25 are oriented in con-
formations that are incompatible with covalent bond
formation (Fig. 3). In contrast, the ketone carbonyl is
stabilized through a set of hydrogen-bonding interac-
tions with Gln19 and Cys25. Contacts between
Gln19 Ne and the carbonyl oxygen are 3.11 and 3.15 A
for cruz-1 and cruz-2, respectively. Contacts between the
S of Cys25 and the carbonyl oxygen are 3.11 and 3.08 A
for cruz-1 and cruz-2, respectively.
The crystal structures of the two inhibitors 1 and 2 (Fig.
1), respectively, show very similar modes of binding.
When the structures of 1 bound to cruzain and 2 bound
to cruzain (from here on abbreviated as cruz-1 and cruz-2,
respectively) are aligned and superimposed, they show a
Figure 1. Hydroxymethyl ketone inhibitor structures and Ki values.
Table 1. Data collection and refinement statistics
Both inhibitors contained a phenylalanine residue in the
P2 position. Similar to previously published crystal
structures, the S2 pocket was defined by the hydro-
phobic residues Leu67, Ala133 and Leu157. As expec-
ted, the Glu205 residue at the bottom of the S2 pocket
was rotated away from the hydrophobic phenylalanine
side chain.
Crystal and diffraction data
cruz-1
cruz-2
Unit-cell dimensions:
a (A)
b (A)
c (A)
b (ꢀ)
Space group
42.52
51.71
46.04
116.53
P21
42.57
51.59
45.95
116.62
P21
The inhibitor backbone is stabilized by a series of
hydrogen bonds with the Gly66 residue. In the cruz-1
structure, the distance between the amide NH of Gly66
and the carbonyl of the P2 phenylalanine residue is 3.08
A. The amide NH of the P2 residue shows a distance of
3.18 A with a nearby water molecule. This amide NH
Data Collection Statistics
Resolution limit (A)
Total number of reflections collected
1.1
440603
1.2
252261
43377
0.030
0.065
Number of unique reflections (F>0s|F|) 53954
0.034
Rmerge (%)a
Rmerge on highest resolution bin of data
Completeness
Overall (%)
0.13
96.4
91.1
99.1
97.8
Highest resolution shell (%)
Refinement statistics
Resolution range (A)
Rcryst
30–1.2
30–1.2
b
0.0969 for
52264 refl
0.130 for
1631 refl
0.0939 for
42575 refl
0.135 for
4792 refl
c
Rfree
Rmsd
Figure 2. Cruzain’s active site, shown in cartoon, with superimposed 1
(salmon) and 2 (aqua) inhibitor molecules. Hydrogen bond interac-
tions described in the text are shown. Figure prepared with PyMOL.
Bond lengths (A)
Bond angles (ꢀ)
From flat planes
0.016
0.031
0.011
0.029
0.0295 for
0.0297 for
1309 atoms 1309 atoms
Water molecules
Average B factor (A2)
All atoms
Protein
Inhibitor
Water
484
551
15.01
8.44
14.48
35.15
13.45
8.04
16.10
28.44
RMSD of superimposed complex
(cruzain+inhibitor) structures
All backbone atoms (860atoms)
All atoms (1591 atoms)
0.11 A
0.41 A
0.59 A
All side-chain atoms (792 atoms)
ꢁ
ꢁ
ꢁ
ꢀ
ꢀ
D Eꢂ ꢂ
ꢃ
P
P P
ꢁ
aRmerge
¼
I À
I
hI where Ih is the mean structure—
iꢁ
ꢁ
h
hi
h
factor intensity of i observations of symmetry-related reflections with
Bragg intensity index h.
ꢄ
ꢅꢃ
P P
bRcryst
=
ikFobsjÀjFcalc
k
jjFobsjÞ where Fobs and Fcalc are the
Figure 3. Electron density at 1.0sigma (3Fo-2Fc) is shown for inhi-
bitor 1 and the side chain of Cys25. A gap between the protein and
inhibitor is visible. The high B-factor values for the Phe of 1 result in
noncontinuous electron density for this moiety. Figure prepared with
PyMOL.
h
observed and the calculated structure factor magnitudes.
ꢆ
ꢆ
ꢁ
ꢁ
ꢅ
ꢃ
P
P
cRfree
=
FobsðhklÞ jÀkjF
ꢁ ꢁ
ðhklÞꢀ FobsðhklÞ where the t set of
ðhklÞ
ðhklÞꢀ
reflections is omitted from the refinement process; 10% of the data
were included in the t set.

L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
Table 2. Cruzain inhibition data
23
also shows a potential interaction with the cruzain main
chain carbonyl of Asp158. In addition, this NH is 2.81
A away from the carbonyl of Gly66. The carbamate
carbonyl interacts with two water molecules at distances
of 2.75 and 3.14 A away. In the cruz-2 structure, con-
tacts between the amide N of Gly66 and the carbonyl of
the P2 phenylalanine residue is 3.07 A. The amide NH
of the P2 residue is 3.0A away from a nearby water
molecule. In addition, this NH is 2.74 A away from the
carbonyl of Gly66. The carbamate carbonyl interacts with
a water molecule at 2.74 A and the pyridinyl N shows a
potential interaction with the hydroxyl group in Ser61.
Compd
Structure
Cruzain Kl (nM)a
3
4.4ꢁ0.9
4
5
6
>10,000
>10,000
>5000
Interestingly, both crystal structures showed a strong
hydrogen bond between His159 (part of the canonical
catalytic triad, Cys25, His159 and Asn175, of this family
of cysteine proteases) and the hydroxyl group in the
inhibitor. These hydrogen bond distances are 2.68 and
2.50A for the 1 and 2 inhibitors, respectively. This type
of hydrogen bond interaction has not been previously
observed in similar cysteine protease crystal structures.
aKi values determined at four to eight concentrations in duplicate
using the linear regression analysis of Williams and Morrison.14
The homophenylalanine (hPhe) residue in the P1 position
of 2 extends into the solvent and does not make pro-
ductive interactions with the enzyme. The hPhe residue
was initially chosen since researchers observed that
incorporation of the hPhe side chain at P1 resulted in
inhibitors that were not degraded by human proteases,
in contrast to the corresponding inhibitors that incor-
porate natural amino acids in this position.8 This repla-
cement also reduced the toxicity of vinyl sulfone
inhibitors in animal studies.9 The high B-factor seen in
this position shows that this is a good position for fur-
ther modification. Preliminary docking studies suggest
that changing the hPhe side chain to a butyl or hydro-
xypropyl group may improve the interactions with the
enzyme while reducing the molecular weight of the
inhibitors.
thiol does not form a covalent bond with the ketone
pharmacophore. We therefore chose to replace the
ketone carbonyl with a methylene group to provide
inhibitor 4 (Table 2), since the carbonyl has the poten-
tial to result in toxicity and unfavorable pharmacoki-
netics. No inhibition was observed even at 10 mM
concentrations of 4, clearly indicating the importance of
the hydrogen bonding interactions with the ketone
pharmacophore.
A series of compounds was prepared in an attempt to
improve the inhibitory activity of compounds lacking
the ketone pharmacophore. First, inhibitor 5 was syn-
thesized with a carboxylic acid group in place of the
hydroxyl group, since the carboxylic acid should
provide a stronger hydrogen bonding interaction with
the active site imidazole. However, no improvement
in the inhibitory activity of 5 was observed. Inhibitor
6 with a thiol group in place of the hydroxyl group
was also prepared, since the corresponding substitu-
tion to provide the ketone inhibitor 3 resulted in a
dramatic improvement in activity (vide infra). Unfor-
tunately, inhibitor 6 showed only a modest 5 mM
inhibition.
Evaluation of inhibitor with a thiol group replacing the
hydroxyl group
To probe the importance of the hydrogen bonding
interaction between the hydroxyl group of inhibitors 1
and 2 and the active site imidazole, the hydroxyl group
was replaced with a thiol group. Surprisingly, inhibitor
3 showed greater than 15-fold improvement in inhibi-
tory activity despite the generally reduced hydrogen
bonding potential of a thiol versus an alcohol. The
potency of inhibitor 3 could possibly result from dis-
ulfide bond formation with the active site cysteine thiol.
To test this possibility the assay was performed under
different redox conditions by varying the concentration
of DTT (1–10mM). However, the inhibitory activity of
inhibitor 3 was independent of DTT concentration
implying that the potency of this compound is not due
to disulfide bond formation.
Inhibitor synthesis
The N-Cbz-Phe-Phe hydroxymethyl ketone (1) was syn-
thesized as summarized in Scheme 1. The commercially
available N-Cbz-Phe-Phe dipeptide 7 was converted to
the bromomethyl ketone dipeptide 8 in a one-pot proce-
dure.10 Displacement of the bromomethyl ketone with
benzoylformic acid,11 followed by hydrolysis of the for-
mate ester 9 with aqueous potassium bicarbonate affor-
ded the desired hydroxymethyl ketone 1.
Evaluation of inhibitors lacking the ketone pharmacophore
The crystal structures of complexes of inhibitors 1 and 2
show that the enzyme makes a number of hydrogen
bonds to the ketone carbonyl. However, the active site
The 3-pyridinylmethoxycarbonyl-Phe-hPhe hydroxy-
methyl ketone 2 was synthesized as shown in Scheme 2.

24
L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
Scheme 1. Synthesis of hydroxymethyl ketone 1 and mercaptomethyl ketone 3.
Scheme 2. Synthesis of hydroxymethyl ketone 2.
The acyloxymethyl ketone analogue 15 was synthesized
using a previously reported solid-phase strategy.12 The
benzoate was then hydrolyzed under basic conditions to
afford the hydroxymethyl ketone 2.
oxygen-free and moisture-free conditions to prevent
oxidation and decomposition of the thiol.
The analogues lacking the ketone pharmacophore were
synthesized using tert-butanesulfinamide 16 as a chiral
auxiliary (Scheme 3). tert-Butanesulfinamide was con-
densed with hydrocinnamaldehyde to give the tert-
butanesulfinyl imine 17. Addition of a titanium enolate
The sulfhydryl ketone 3 was synthesized by displacement
of 8 with potassium thiolate to afford the thioester 10
(Scheme 1). The thioester was hydrolyzed under rigorous
Scheme 3. Synthesis of inhibitors lacking the ketone pharmacophore.

L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
25
of methyl acetate to 17 gave the chiral product 18.
Cleavage of the N-tert-butanesulfinyl group followed by
acylation with N-Cbz-Phe-OH gave the methyl ester
intermediate 19. Reduction with LiBH4 resulted in
selective reduction of the methyl ester to the primary
alcohol 4 without reduction of the amide bond.
under a N2 atmosphere. To this solution was added
sequentially 4-methyl morpholine (0.69 mL, 6.3 mmol,
1.4 equiv) and isobutyl chloroformate (0.75 mL, 5.8
mmol, 1.3 equiv). The latter was added dropwise and
resulted in the immediate formation of white solid. The
reaction mixture was stirred vigorously, and the bath
maintained at a temperature around À25 ꢀC. After 1 h,
the reaction mixture was filtered and the solid was
rinsed with ice-cold THF. Rinses were added to the
solution, which was stirred again at À25 ꢀC. Additional
THF for rinsing was added until the concentration was
no lower than 0.2 M. Diazomethane was introduced in
situ using Diazald (2.97 g, 13.9 mmol, 3.1 equiv), and
the reaction flask was maintained at À25 ꢀC for 30min
once addition was complete. The solution was allowed
to warm to rt for 30min before immersion in an ice
water bath. A mixture of 1:1 48% HBr/glacial acetic
acid was added dropwise until bubbling ceased. The
solvent was evaporated and the pale yellow/orange
crude product was dissolved in ethyl acetate. The
organic layer was washed with water and then with
saturated NaHCO3 until rinses were no longer acidic.
The organic layer was dried, the solvent removed and
the crude product was recrystallized in absolute ethanol.
The analogous sulfhydryl inhibitor 6 was synthesized
from alcohol 4 (Scheme 3). A Mitsunobu reaction of
thioacetic acid and alcohol 4 provided an intermediate
thioester that was hydrolyzed under rigorous oxygen-free
conditions to prevent oxidation of the liberated thiol.
Finally, the carboxylic acid inhibitor 5 could be pre-
pared by saponification of the methyl ester intermediate
19.
Conclusions
We have reported two very high resolution crystal
structures of two hydroxymethyl ketones 1 and 2 bound
to cruzain. These crystal structures provide valuable
insight into the binding of these mechanism-based inhi-
bitors to the enzyme active site. In particular, these
crystal structures show that the active site cysteine thiol
does not form a covalent bond with the ketone phar-
macophore. In addition, the structures show a strong
hydrogen bond between the active site imidazole and
the hydroxyl group of the inhibitor. A series of second
generation compounds were prepared and tested pro-
viding further insight into the key binding interactions.
To a solution of bromomethyl ketone 8 (0.100 g, 0.191
mmol) in 2 mL of DMF was added benzoyl formic acid
(0.034 g, 0.229 mmol, 1.2 equiv) and potassium fluoride
(0.017 g, 0.287 mmol, 1.5 equiv). This yellow solution
was stirred overnight at rt. This solution was diluted
with 5 mL of EtOAc and 2.5 mL of water. The organic
layer was washed with saturated NaCl, dried with
MgSO4 and concentrated to form a yellow-white solid.
To a solution of unpurified 9 in 10mL of THF was
Experimental
added 10mL of a 1 M solution of NaHCO . This reac-
3
General methods
tion mixture was stirred overnight, resulting in a cloudy
white solution. This solution was diluted in EtOAc and
washed with water, saturated NaCl, dried with MgSO4
and concentrated to give the desired product 1 as a
white solid 65.6 mg (75%). A portion of the product
was chromatographed (ꢂ2 mL of silica and a solvent
gradient of 4:1 hexanes/ethyl acetate to 1:1 hexanes/
ethyl acetate) to ensure complete purity, providing 13
mg of product 1 for biochemical analysis. IR: 1658,
Unless otherwise noted, all reagents were obtained from
commercial suppliers and used without further purifica-
tion. THF was distilled under N2 from sodium/benzo-
phenone, and CH2Cl2 and i-Pr2EtN were distilled over
CaH2 immediately prior to use. ArgoGel-OH resin was
purchased from Argonaut Technologies (San Carlos,
CA, USA). Reaction progress was monitored through
thin-layer chromatography on Merck 60F
0.25 mm
1687, 1726, 3292 cmÀ1 1H NMR (500 MHz): d
.
254
silica plates. Unless otherwise specified, extracts were
dried over MgSO4 and solvents were removed with a
rotary evaporator at aspirator pressure. All compounds
were purified by flash chromatography using Merck 60
230–400 mesh silica gel. Infrared spectra were recorded
with a Perkin-Elmer 1600 series Fourier transform
spectrometer as thin films on NaCl plates and only
2.89–3.01 (m, 4H), 3.94 (d, 1H, J=19.5 Hz), 4.07 (d,
1H, J=19.5 Hz), 4.35–4.40(m, 1H), 4.75 (m, 1H), 5.07
(s, 2H), 5.22 (d, 1H, J=7.5 Hz), 6.33 (d, 1H, J=7.0
Hz), 6.95–7.38 (m, 15H). 13C NMR (125 MHz): d 30.3,
37.4, 38.0, 56.0, 56.1, 67.4, 127.3, 127.4, 128.1, 128.3,
128.6, 128.8, 128.9, 129.0, 129.2, 135.0, 135.9, 135.9,
155.9, 170.8, 208.0. FABHRMS: 461.2076 (MH+,
C27H28N2O5 requires 461.2081).
1
partial data are listed. H and 13C NMR spectra were
obtained with Bruker AMX-300 and DRX 500 spectro-
meters. Unless otherwise specified, all spectra were
obtained in CDCl3; chemical shifts are reported in parts
per million relative to TMS, and coupling constants are
reported in Hertz. HRMS analysis was performed at the
UC Berkeley Mass Spectrometry Facility.
Compound 2. Using a previously reported solid-phase
strategy,12 an acyloxymethyl ketone derivative of N-[(3-
pyridinyl)methoxycarbonyl]-Phe-hPhe (15) was synthe-
sized. Acyloxymethyl ketone 15 was purified via column
chromatography (ꢂ2 mL of silica and a solvent gra-
dient from 4:1 hexanes/ethyl acetate to 100% ethyl ace-
tate) in a 60% yield. The acyloxymethyl ketone was
subsequently hydrolyzed under the following basic con-
ditions.13 In a Schlenk flask, 15 (78 mg, 0.13 mmol) was
Compound 1. A solution of N-Cbz-Phe-Phe-OH dipep-
tide (2.00 g, 4.47 mmol) in 6 mL of THF was immersed
in a dry ice/acetonitrile bath at À25 ꢀC with stirring

26
L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
dissolved in 4 mL of a 20:10:1 solvent mixture of
CH2Cl2/MeOH/H2O and subjected to three freeze—
pump—thaw cycles. Oxygen-free conditions were
required to prevent oxidation of the resulting hydro-
xymethyl ketone 2. Under positive N2 pressure, K2CO3
(0.10 g, 0.72 mmol) was added to the reaction mixture
and stirred under N2 for 3.5 h. The resulting solution
was diluted with 10mL of EtOAc and 5 mL of water in
a separatory funnel. The layers were separated and the
organic layer was further washed with sat. NaCl, dried
with MgSO4 and concentrated. Purification by column
chromatography (ꢂ2 mL of silica and a solvent gra-
dient of 4:1 hexanes/ethyl acetate to 100% ethyl acetate)
afforded the white solid in a 49% unoptimized yield
was dissolved in 2 mL of CH2Cl2 and stirred under N2.
To this solution was added Ti(OEt)4 (0.60 mL, 2.6
mmol, 2.3 equiv) and freshly distilled hydro-
cinnamaldehyde (0.16 mL, 1.2 mmol, 1.1 equiv). This
solution was stirred at rt and under N2 for 1.5 h. The
solution was diluted threefold with CH2Cl2 and incu-
bated with a 2:1 finely powdered mixture of sand/
.
Na2SO4 H2O (5 g/mmol imine) for 1 h with occasional
shaking by hand. Filtration, rinsing, and concentration
led to the imine product 17 (0.27 g, 100%) as a light
yellow oil. Imine 17 was generally used immediately in
the next step.
Compound 18. In a 25-mL flame-dried round-bottom
flask, diisopropyl amine (0.35 mL, 2.5 mmol, 2.2 equiv)
was dissolved in THF (5.7 mL) and stirred at 0 ꢀC under
N2. To this solution, n-BuLi (1.3 mL, 2.39 mmol, 2.1
equiv) was added dropwise. This solution was stirred at
0 ꢀC for 20min and then cooled to À78 ꢀC. Anhydrous
methyl acetate (0.18 mL, 2.3 mmol, 2 equiv) was added
dropwise and the pale yellow reaction mixture was stir-
red for an additional 30–45 min. To this mixture was
added a solution of TiCl(O-i-Pr)3 (1.1 mL, 4.8 mmol,
4.2 equiv) in THF (1.1 mL), giving an opaque orange
solution. After this solution was stirred at À78 ꢀC for 35
min, a solution of imine 17 (1.1 mmol) in 2 mL of THF
was added dropwise. This reaction mixture was then
stirred at À78 ꢀC for 2 h, followed by quenching with 2
mL of saturated NH4Cl at À78 ꢀC. A white precipitate
immediately formed and the reaction mixture was
allowed to warm up to rt before it was diluted with
water and extracted with EtOAc (3ꢃ5 mL). The com-
bined organic extracts were washed with saturated
NaCl, dried with MgSO4 and concentrated to give a
yellow oil. The enolate addition product was purified by
column chromatography (ꢂ20mL of silica and a gra-
dient column from 3:1 hexanes/ethyl acetate to 100%
ethyl acetate) to afford 0.23 g (67%) of 18.
1
(31.5 mg). IR: 1658, 1690, 1723, 3293 cmÀ1. H NMR
(300 MHz): d 1.56–1.87 (m, 1H), 1.94–2.17 (m, 1H),
2.34–2.39 (m, 1H), 2.49–2.54 (m, 1H), 3.03–3.07 (m,
2H), 4.18–4.27 (m, 2H), 4.42–4.52 (m, 1H), 4.57 (m,
1H), 4.99–5.10(m, 2H), 5.61–5.88 (m, 1H), 6.71–6.86
(m, 1H), 7.02–7.33 (m, 11H), 7.58–7.61 (m, 1H),
8.51–8.54 (m, 2H). 13C NMR (125 MHz): d 31.3, 32.6,
38.1, 54.8, 56.4, 64.4, 66.6, 123.5, 126.4, 127.2, 128.3,
128.5, 128.8, 129.2, 131.8, 131.9, 136.1, 140.1, 140.2,
149.0, 155.7, 171.2, 208.1. FABHRMS: 476.2185
(MH+, C27H29N3O5 requires 476.2176).
Compound 3. To a solution of bromomethyl ketone 8
(0.100 g, 0.191 mmol) in 2 mL of DMF was added
potassium thioacetate (0.024 g, 0.210 mmol, 1.1 equiv).
This cloudy orange-yellow solution was stirred over-
night at rt. This solution was diluted with 10mL of
EtOAc and 5 mL of water. The organic layer was
washed with saturated NaCl, dried with MgSO4 and
concentrated to form 73.6 mg (74%) of the thioester 10.
In a 25 mL flame-dried Schlenk flask, reagent grade
acetone (5 mL) and 3 N NaOH (5 mL) were subjected to
four freeze—pump—thaw cycles to form strict oxygen-
free conditions. Under positive N2 pressure, the crude
compound 10 (0.1 g, 0.2 mmol) was added as a solid to
the Schlenk flask. This reaction mixture was stirred
under N2 for 2 h. A solution of 1 M HCl was added to
neutralize the solution, resulting in the formation of a
brown precipitate. The aqueous layer was extracted
with CH2Cl2 (3ꢃ5 mL) and the combined extracts were
dried with MgSO4 and concentrated to give 82.2 mg of
brown solid. This solid was purified by column chro-
matography (ꢂ2 mL of silica and a solvent gradient
from 4:1 hexanes:ethyl acetate to 100% ethyl acetate) to
give 38.2 mg of product 3. The major diasteromer is
reported.
1H NMR (500 MHz): d 1.26 (s, 9H), 1.82–1.96 (m, 2H),
2.60–2.87 (m, 4H), 3.54–3.60 (m, 1H), 3.68 (s, 3H), 4.27
(d, 1H, J=8.8 Hz), 7.16–7.30(m, 5H). 13C NMR
(125 MHz): d 22.7, 32.2, 37.3, 40.2, 51.7, 53.4, 56.0,
126.0, 128.4, 128.5, 141.3, 172.4.
Compound 19. The sulfinyl group in sulfinamide 18 (0.23
g, 0.76 mmol) was cleaved by addition of 4 N HCl
solution in dioxane (0.4 mL, 1.5 mmol, 2 equiv) and an
equal volume of MeOH, while stirring under N2 in a
cold water bath. After 30min of stirring, the reagents/
solvents were removed by passing N2 over the solution.
The resulting amine salts were re-dissolved in CH2Cl2
and free-based with Et3N (0.21 mL, 1.5 mmol, 2 equiv).
The organic layers were washed with water, saturated
NaCl, dried with MgSO4 and concentrated to give the
free amine as a yellow oil.
40% yield. IR: 1654, 1700, 1702 cmÀ1 1H NMR
.
(500 MHz): d 2.82–3.18 (m, 6H), 4.37 (m, 1H), 4.92 (m,
1H), 5.08 (s, 2H), 5.19 (m, 1H), 6.34 (m, 1H), 7.01–7.36
(m, 15H). 13C NMR (125 MHz): d 32.8, 37.6, 38.2, 56.2,
57.7, 67.2, 127.1, 127.3, 127.3, 129.1, 128.3, 128.5, 128.8,
128.9, 129.1, 129.2, 135.4, 136.0, 155.9, 170.7, 203.5.
FABHRMS: 477.1848 (MH+, C27H28N2O4S requires
477.1842).
The crude amine was dissolved in CH2Cl2 (3.8 mL,
0.2 M) and stirred in an ice bath. To this solution was
added HOBt (0.21 g, 1.5 mmol, 2 equiv), N-Cbz-Phe-
OH (0.25 g, 0.84 mmol, 1.1 equiv) and EDC (0.20 g, 1.1
mmol, 1.4 equiv). This reaction mixture was stirred for 2
h at 0 ꢀC and then slowly warmed to rt overnight. After
Compound 17. In a flame dried 50-mL round-bottom
flask, (R)-tert-butanesulfinamide (0.125 g, 1.14 mmol)

L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
27
this incubation period, the solution was diluted in
CH2Cl2 and washed with 1 N HCl, saturated NaHCO3
(2ꢃ5 mL), water, 1 N HCl (2ꢃ5 mL), and saturated
NaCl. The organic layer was dried with MgSO4, con-
centrated and purified through recrystallization in
absolute ethanol to afford 0.23 g (61%) of 19.
FABHRMS: 475.2233 (MH+, C28H30N2O5 requires
475.2238).
Compound 6. To a stirred solution of PPh3 (162 mg,
0.617 mmol) in 1 mL of THF was added diethyl azodi-
carboxylate (0.97 mL, 0.617 mmol) dropwise. This
solution was stirred under N2 at 0 ꢀC and shielded from
light. To this orange solution was added a mixture of
alcohol 4 (142 mg, 0.308 mmol) and thioacetic acid
(0.088 mL, 1.23 mmol) in 6 mL of THF. This reaction
mixture was stirred at 0 ꢀC for 1 h and then gradually
warmed up to rt overnight, giving a light pink solution.
This solution was concentrated and purified by column
chromatography (ꢂ2 mL of silica and a solvent gradient
6:1 to 2:1 hexanes/ethyl acetate) to give the thioester in
70% yield (0.112 g).
1H NMR (500 MHz): d 1.63–1.72 (m, 2H), 2.41–2.48
(m, 4H), 3.00–3.10 (m, 2H), 3.61 (s, 3H), 4.19 (m, 1H),
4.37 (m, 1H), 5.05–5.14 (m, 2H), 5.30 (m, 1H), 6.26 (d,
1H, J=9 Hz), 7.08–7.33 (m, 15H). FABHRMS:
489.2387 (MH+, C29H32N2O5 requires 489.2389).
Compound 4. Ester 19 (0.16 g, 0.32 mmol) was dissolved
in 1.6 mL of THF and stirred under N2. LiBH4 (0.48
mL, 0.96 mmol, 3 equiv) was added dropwise to this
solution and it was stirred overnight. Following this
incubation period, the reaction mixture was cooled in
an ice bath and acidified with 1 N HCl until the pH
reached 0. The reaction mixture was concentrated and
then dissolved in water. This aqueous solution was then
extracted with CH2Cl2. The combined organic extracts
were washed with 1 N HCl and saturated NaCl, dried
with MgSO4, and concentrated to give a white solid. The
product was isolated in 69% yield (0.10 g) after column
chromatography (ꢂ20mL of silica and a solvent gra-
dient 4:1 hexanes/ethyl acetate to 1:1 ethyl acetate).
In a 25 mL Schlenk flask, reagent-grade acetone (3 mL)
and 3 M NaOH (3 mL) were subjected to four freeze—
pump—thaw cycles to provide strict oxygen-free condi-
tions. Under positive N2 pressure, the thioester (50mg,
0.1 mmol) was added as a solid to the Schlenk flask.
This reaction mixture was stirred under N2 for 2 h. A
solution of 1 M HCl was added to neutralize the solu-
tion. The aqueous layer was extracted with CH2Cl2
(3ꢃ5 mL) and the combined extracts were dried with
MgSO4 and concentrated. This solid was purified
through column chromatography (ꢂ2 mL of silica and
a solvent gradient from 6:1 to 1:1 hexanes/ethyl acetate)
to give 28% (13 mg) of desired product.
IR: 1650, 1693, 3292 cmÀ1 1H NMR (500 MHz): d
.
1.27–1.32 (m, 1H), 1.52–1.60(m, 1H), 1.62–1.69 (m,
1H), 1.76–1.81 (m, 1H), 2.37 (m, 2H), 3.08 (m, 2H),
3.45–3.62 (m, 3H), 4.01 (m, 1H), 4.42 (m, 1H), 5.06 (app
s, 2H), 5.76 (br s, 1H), 6.32 (br s, 1H), 7.06–7.33 (m,
15H). 13C NMR (125 MHz): d 32.2, 36.8, 37.9, 38.4,
46.6, 56.8, 58.4, 67.1, 126.0, 127.1, 127.9, 128.2, 128.3,
128.4, 128.5, 128.7, 129.3, 136.0, 136.3, 141.3, 156.2,
172.1. Anal. calcd for C28H32N2O4: C, 73.02; H, 7.00;
N, 6.08. Found: C, 73.15; H, 7.07; N, 6.21.
1
IR: 1649, 1686 cmÀ1. H NMR (500 MHz): d 1.47–1.55
(m, 2H), 1.62–1.68 (m, 2H), 1.70–1.77 (m, 1H), 2.39–
2.42 (m, 4H), 3.03 (dd, 1H, J=7.9, 13.6 Hz), 3.15 (dd,
1H, J=6.1, 13.6 Hz), 4.04 (m, 1H), 4.35 (dd, 1H,
J=7.9, 14.1), 5.06–5.14 (m, 2H), 5.30 (br s, 1H), 5.56 (d,
1H, J=9.3 Hz), 7.07–7.35 (m, 15H). 13C NMR
(125 MHz): d 32.0, 36.7, 38.1, 39.4, 48.1, 56.7, 67.2, 67.9,
125.9, 127.2, 128.1, 128.26, 128.28, 128.4, 128.6, 128.8,
129.3, 136.0, 136.3, 141.3, 156.2, 170.5. FABHRMS:
477.2211 (MH+, C28H32N2O3S requires 477.2200).
Compound 5. Ester 19 (50mg, 0.10mmol) was dissolved
in dioxane (0.37 mL) and flushed under N2. To this
solution was added 0.25 M LiOH (0.4 mL) which had
been flushed with N2. A white precipitate immediately
formed and additional dioxane (0.4 mL) and LiOH (0.4
mL) was added to facilitate stirring. After 2 h of stirring
at rt, 0.5 N HCl (0.2 mL) was added to quench the
reaction. The reaction mixture was diluted in EtOAc
and the aqueous layer was extracted further with EtOAc
(3ꢃ5 mL). The combined organic extracts were dried
with MgSO4, and concentrated to give the desired pro-
duct, which was purified through column chromato-
graphy (ꢂ2 mL of silica and a solvent gradient from 4:1
to 1:1 hexanes/ethyl acetate) to give 35% yield (17 mg)
of analytically pure product.
High throughput cruzain assay
A fluorometric assay for activity toward cruzain was
performed in 96-well microtiter plates. The assay was
performed in Dynatech Microfluor fluorescence micro-
titer plates (opaque white plates), and readings were
taken on a Molecular Devices SPECTRAmax Gemini
XS Dual scanning microplate spectrofluorometer. The
excitation wavelength was 355 nm and the emission
wavelength was 450nm. A 430nm cutoff filter for
emission was used. The peptide substrate Z-Phe-Arg-
AMC (Bachem, Km=1 mM) concentration was 2.5 mM
and the cruzain concentration was 0.1 nM. The buffer
consisted of a 100 mM solution of pH 5.5 sodium ace-
tate buffer and 1 mM of DTT.
1
IR: 1647, 1661, 1697, 3295 cmÀ1. H NMR (500 MHz,
acetone-d6): d 1.80–1.84 (m, 2H), 2.47–2.60 (m, 4H),
2.94–2.99 (m, 1H), 3.16–3.19 (m, 1H), 4.21 (m, 1H), 4.42
(dd, 1H, J=14.4, 8.3 Hz), 5.02 (m, 3H), 6.51 (d, 1H,
J=7.9 Hz), 7.13–7.32 (m, 15H). 13C NMR (125 MHz,
acetone-d6): ꢁ 32.0, 35.8, 38.1, 38.7, 46.2, 56.7, 65.7,
125.6, 126.4, 127.5, 127.6, 128.15, 128.18, 128.2, 128.3,
129.3, 137.2, 137.7, 141.9, 155.8, 170.7, 171.8.
The fluorescent unit readings were taken at ten time
points within the linear region of the substrate cleavage,
and percentage activity of the enzyme was determined
by comparing the change of fluorescent units (FU) for
each well against the average change in FU for eight

28
L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
control wells without inhibitor. All compounds were
assayed in duplicate.
inhibitor.6 Crystals appeared within 7–10days of
growth at 18 ꢀC.
Sample procedure. In each well was placed 25 mL of
enzyme solution, 125 mL of buffer solution and 10 mL of
the inhibitor in DMSO. The compounds were placed
only in rows 2–11, with row 12 used for the control
wells. Following a 5-min preincubation of the enzyme
and inhibitor, 50 mL of the substrate solution was added
to each well. The plate was immediately placed into the
plate reader and fluorescent readings were taken. The
data were analyzed by transferring each plate’s readings
from the spectrophotometer to EXCEL spreadsheets.
An EXCEL macro was written to obtain the slope for
each well compared to the slope of the controls. Inhibi-
tors were screened in 2-fold dilutions from 10 mM to 78
nM.
Data collection,structure solution and crystallographic
refinement
All diffraction data were collected at Stanford Synchro-
tron Radiation Laboratory (SSRL), BL9–1, using
monochromatic radiation of 0.98 A. A MAR345 image
plate was used with low temperature conditions of 100
K at the crystal position. Crystals of cruz-1 and cruz-2
were flash cooled in liquid nitrogen and mounted in the
experimental coldstream for data collection. The struc-
tures were solved via molecular replacement using a 1.6
A model of cruzain (PDB ID 1F2A), without inhibitor
or waters. The molecular replacement solution
(AmoRe17) for cruz-1 yielded an Rfactor of 0.377 with a
correlation coefficient of 58.0. Similarly, for cruz-2, the
molecular replacement Rfactor was 0.381 with a corre-
lation coefficient of 55.7. These statistics indicated that
correct solutions had been found. Rigid body refine-
ments, followed by iterative cycles of positional and
individual B-factor refinement were completed with
XPLOR.16 Topology, parameter and starting coordi-
nate files for both 1 and 2 inhibitors were generated with
the program Moloc.17 After fitting the inhibitor mole-
cules to their difference electron density in the cruzain
active site cleft, water molecules were added auto-
matically using QUANTA (Accelrys, San Diego, CA).
Final rounds of high-resolution refinement were per-
formed with SHELX-97,18 including modeling of ani-
sotropic thermal parameters for all non-hydrogen
atoms.
Ki determination
For all inhibitors, the data was fit by nonlinear regres-
sion analysis to the equation derived by Williams and
Morrison:14
vo
v ¼
2Et
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
8
"
#
u
u
t
ꢀ
ꢀ
ꢁ
ꢁ
ꢀ
ꢁ
þ It ꢀ Et 2þ4Ki 1 þ
Et
<
S
S
Ki 1 þ
:
Km
Km
ꢂ
ꢀ
ꢁ
ꢃꢄ
S
ꢀ Ki 1 þ
þ It ꢀ Et
Km
Accession numbers
For cruzain, the Km for the substrate was determined to
be 1 mM by using a Lineweaver Burke Plot and Eadie—
Hofstee Plot. The variables S, Et, and It are the con-
centrations of substrate, active enzyme, and inhibitor,
respectively.
The coordinates for cruz-1 and cruz-2 have been depos-
ited in the Protein Data Bank with accession numbers
1me4 (cruz1) and 1me3 (cruz2), respectively.
Acknowledgements
Preparation,purification and crystallization of cruzain
bound to inhibitors
L. H. and J. A. E. acknowledge support from the NIH
(Grant GM 54051) and the Burroughs Wellcome Fund
(New initiatives in Malaria Award). L. S. B. acknowledges
support from the NIH (AI 35707). Dr. J. H. McKerrow
and E. Hansell are also gratefully acknowledged for sup-
plying cruzain, as is Thomas J. Stout for assistance with
high-resolution refinement. The Center for New Direc-
tions in Organic Synthesis is supported by Bristol-Myers
Squibb as a Sponsoring Member and Novartis as a Sup-
porting Member. Portions of this research were carried
out at the Stanford Synchrotron Radiation Laboratory, a
national user facility operated by Stanford University on
behalf of the US Department of Energy, Office of Basic
Energy Sciences. The SSRL Structural Molecular Biol-
ogy Program is supported by the Department of Energy,
Office of Biological and Environmental Research, and by
the National Institutes of Health, National Center for
Research Resources, Biomedical Technology Program,
and the National Institute of General Medical Sciences.
Cruzain was expressed and purified as described pre-
viously5,15 [activated cruzain, temporarily ‘blocked’ with
methyl methane thiosulfonate (MMTS) was incubated
on ice in solution with 5 mM DTT. The protein solution
was buffered in 20mM bis tris (pH 5.8) with some NaCl
(from chromatographic purification]. An excess (1–2
mg) of inhibitor was dissolved in DMSO and added to
the protein solution. After stirring for 60min, the solu-
tion was concentrated via vacuum dialysis against a
buffer of 2 mM bis tris (pH 5.8) to a final concentration
of ꢂ10mg/mL. Crystals of either cruz-1 or cruz-2 were
obtained via the hanging drop method against a grid of
0.6–1.0 M sodium citrate (pH 6.6–7.0). Equal volumes
of 10mg/mL protein and sodium citrate solution were
mixed and suspended over reservoirs of sodium citrate
solution. These drops were allowed to equilibrate at
18 ꢀC for 24 h and subsequently microseeded with
existing crystals of cruzain bound to a vinyl sulfone

L. Huang et al. / Bioorg. Med. Chem. 11 (2003) 21–29
29
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