Identification of potent and selective mechanism-based inhibitors of the cysteine protease cruzain using solid-phase parallel synthesis

Article abstract of DOI:10.1021/jm010333m

Targeted libraries of ketone-based cysteine protease inhibitors were synthesized and screened against cruzain, a cysteine protease implicated in Chagas' disease. A number of single digit nanomolar, low molecular weight inhibitors were identified and optimized for solubility and potency. Specifically, the best inhibitors identified have K_i values of 0.9-10 nM and molecular weights between 499 and 609 Da. The most effective inhibitor was also found to be greater than 1000-fold selective for cruzain relative to cathepsin B and 100-fold selective for cruzain relative to cathepsin L.

Full text of DOI:10.1021/jm010333m

676
J . Med. Chem. 2002, 45, 676-684
Id en tifica tion of P oten t a n d Selective Mech a n ism -Ba sed In h ibitor s of th e
Cystein e P r otea se Cr u za in Usin g Solid -P h a se P a r a llel Syn th esis
Lily Huang, Alice Lee, and J onathan A. Ellman*
Department of Chemistry, University of California, Berkeley, California 94720
Received J uly 18, 2001
Targeted libraries of ketone-based cysteine protease inhibitors were synthesized and screened
against cruzain, a cysteine protease implicated in Chagas’ disease. A number of single digit
nanomolar, low molecular weight inhibitors were identified and optimized for solubility and
potency. Specifically, the best inhibitors identified have Ki values of 0.9-10 nM and molecular
weights between 499 and 609 Da. The most effective inhibitor was also found to be greater
than 1000-fold selective for cruzain relative to cathepsin B and 100-fold selective for cruzain
relative to cathepsin L.
Ch a r t 1
In tr od u ction
Chagas’ disease, the leading cause of heart disease
and inflammation in South America, is caused by the
protozoan parasite Trypanosoma cruzi.1 The only cur-
rently available treatment is unsatisfactory due to
significant toxicity.2 The lack of effective treatments has
stimulated efforts to identify novel drug targets against
this parasite. One target that has received considerable
attention is cruzain, the major cysteine protease found
in T. cruzi. Recently, McKerrow and co-workers have
shown that irreversible inhibitors of cruzain can cure
parasitic infections in mouse models.3 These results
demonstrate the therapeutic promise of inhibitors of
cruzain for the treatment of Chagas’ disease.
Previous design of inhibitors of cruzain has exclu-
sively focused on irreversible inhibitors such as fluo-
romethyl ketones and vinyl sulfones. Although animal
studies have established that vinyl sulfone inhibitors
(1a and 1b) (Chart 1) are not toxic at therapeutic doses,
the poor selectivity of these irreversible inhibitors for
cruzain over human cysteine proteases3 remains a
significant concern. Highly potent and selective revers-
ible inhibitors have been identified for other cysteine
proteases, with the ketone carbonyl acting as one of the
most promising pharmacophores.4 In particular, Veber
and co-workers have identified potent and highly selec-
tive reversible ketone-based inhibitors of cathepsin K,
such as 2 and 3, that are efficacious in the treatment of
osteoperosis in animal models of this disease.5 Herein
we report the application of targeted library synthesis
to identify potent, ketone-based inhibitors of cruzain (Ki
e 0.9-10 nM), the most promising of which shows
greater than 1000-fold selectivity for cruzain over hu-
man cathepsin B and 100-fold selectivity for cruzain
over human cathepsin L.
(Alloc)-protected chloromethyl ketone, prepared in a
one-pot procedure7 from the corresponding N-Alloc
amino acid, introduces the R1 side chain and provides
sites for further functionalization on both sides of the
ketone carbonyl (Scheme 1). The chloromethyl ketone
scaffold is attached to support using a hydrazine linker
4 to provide support-bound hydrazone 5. The hydrazone
not only provides the site of attachment to the solid
support but also protects the carbonyl from nucleophilic
attack as well as racemization at the R-stereocenter.
Displacement of the chlorine with nucleophiles, such as
amines and thiols, introduces functionality on one side
of the ketone. Removal of the Alloc group under Pd(0)-
mediated conditions, followed by acylation and cleavage
from support, gives the fully functionalized ketone-based
inhibitors. This synthetic strategy allows the rapid
parallel synthesis of mercaptomethyl (7) and amidom-
ethyl (9) classes of inhibitors in moderate to high yields.
Design of In h ibitor s. Our initial design was based
upon previously developed diazomethyl ketone8 and
vinyl sulfone9 inhibitors. These inhibitors established
effective binding interactions N-terminal to the site of
peptide cleavage (P side) but provided little information
regarding binding interactions C-terminal to the site of
peptide cleavage (P′ side). We therefore chose to display
diverse functionality at the P′ side10 of the ketone
carbonyl (Figure 1) while fixing the P side as N-Cbz Phe-
Phe, which is present in a number of known potent
irreversible inhibitors of cruzain.8,9 A 192-compound
library of amidomethyl ketones (9) was first synthesized
using a diverse set of 12 amines and 16 carboxylic acids
Resu lts
Syn th etic Str a tegy. We have previously reported
the development of a solid-phase synthesis approach to
prepare ketone-based inhibitors.6 An allyloxycarbonyl
* Address correspondence to this author. Phone: (510) 642-4488.
Fax: (510) 642-8369. E-mail: jellman@uclink4.berkeley.edu.
10.1021/jm010333m CCC: $22.00 © 2002 American Chemical Society
Published on Web 12/29/2001

Identification of Mechanism-Based Inhibitors of Cruzain
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 677
Ta ble 1. Cbz-Phe-Phe Mercaptomethyl Ketones
Sch em e 1a
inhibitor
R
% yielda
Ki (nM)b
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
-CH2(4-Cl Ph)
-CH2Ph
62
89
67
37
42
90
48
75
97
96
46
60
63
50
51
64
82
49
63
82.7 ( 6.4
44.0 ( 2.6
37.0 ( 6.6
34.3 ( 1.6
34.3 ( 1.5
32.4 ( 2.3
31.2 ( 1.5
30.1 ( 3.2
22.0 ( 1.6
15.0 ( 0.9
10.4 ( 0.6
7.5 ( 0.5
5.8 ( 0.5
5.0 ( 0.7
4.6 ( 0.4
4.2 ( 0.2
2.9 ( 0.3
2.5 ( 0.2
2.0 ( 0.2
-CH(CH3)CO2Et
-CH2CH2NHCOCH3
-c-(C5H9)
-CH2(4-OCH3 Ph)
-CH2CO2Et
-(CH2)2CH(CH3)2
-CH2(4-C(CH3)3 Ph)
-CH2CONHCH3
-C(CH3)2CH2C(CH3)3
-c-(C6H11
)
-CH(CH3)CH2CH3
-(C(CH3)(CH2C(CH3)3)2
-CH(CH3)2
-CH2CH2Ph
-(CH2)2CO2Et
-C(CH3)3
-(CH2)3Ph
a
b
Overall yield of purified product for five steps. Ki values
determined at four to eight concentrations in duplicate using the
linear regression analysis of Williams and Morrison.11
a
(a) Chloromethyl ketone derivative of N-Alloc amino acid,
THF, 4 h, rt; (b) R2SH, i-Pr2EtN, DMF; (c) NH2R2, DMF or (1)
NaN3, MeOH (2) SnCl2, PhSH, i-Pr2EtN, THF; (d) R3CO2H,
PyBOP, HOAt, i-Pr2EtN, DMF; (e) Pd(PPh3)4, CH2Cl2, TMSN3,
TBAF‚3H2O; (f) R4CO2H, PyBOP, HOAt, i-Pr2EtN, DMF; (g) 1:4:
15 TFA/H2O/trifluoroethanol.
Ta ble 2. Cbz-Leu-Phe Mercaptomethyl Ketones
inhibitor
R
% yielda
Ki (nM)b
11a
11b
11c
11d
-(CH2)2CO2Et
-CH(CH3)2
-C(CH3)3
44
52
51
44
8.0 ( 0.8
7.8 ( 0.6
1.3 ( 0.1
1.0 ( 0.1
F igu r e 1. Standard nomenclature for protease cleavage.
(not shown). The most active amidomethyl inhibitors
had IC50 values between 500 nM and 1 µM against
cruzain.
-(CH2)3Ph
a
b
Overall yield of purified product for five steps. Ki values
determined at four to eight concentrations in duplicate using the
linear regression analysis of Williams and Morrison.11
More promising was a second library of 20 mercap-
tomethyl ketones (10a -s), which showed improved
potency over the amidomethyl ketones with most in-
hibitors having Ki values below 50 nM (Table 1).
Evaluation of the SAR data suggests that cruzain can
accommodate large bulky substituents or long hydro-
phobic hydrocarbon chains in the P′ side of the inhibitor
structure. The most potent compounds contained iso-
propyl mercaptan (10o), phenethyl mercaptan (10p ),
ethyl 3-mercaptopropionate (10q), tert-butyl mercaptan
(10r ), and 3-phenyl propyl mercaptan (10s).
To reduce the number of aromatic rings present in
the inhibitor structure while concomitantly reducing the
molecular weight and improving the solubility,12 we
explored replacing phenylalanine with leucine at the P2
position, affording compounds 11a -d . Both aromatic
and aliphatic hydrophobic amino acids are known to be
accommodated by the enzyme at this site.13 As shown
in Table 2, replacement of Phe with Leu at the P2
position resulted in comparable or improved Ki values.
Op tim iza tion of Solu bility. The Cbz inhibitors
proved to be poorly soluble in aqueous solution due to
the number of hydrophobic phenyl rings in the inhibitor
structures. The Cbz group was therefore replaced with
a morpholinecarbonyl group. Bromme and co-workers
demonstrated the use of this functionality as an alter-
native to Cbz in several vinyl sulfone inhibitors target-
ing cruzain.9 Incorporation of the morpholinecarbonyl
group was achieved on support via acylation with the
appropriate N-morpholinecarbonyl amino acid prior to
cleavage from resin. The N-morpholinecarbonyl amino
acids were easily synthesized by addition of 4-morpho-
line carbonyl chloride to the desired R-amino acid using
Schotten-Bauman conditions. With the three mercaptyl
side chains that provided the highest activity, six
morpholinecarbonyl dipeptide mercaptomethyl ketones
were synthesized, with Phe or Leu in the P2 position.
While the previously reported morpholine-derived vinyl
sulfones were very potent inhibitors of cruzain, our
morpholine-derived ketones (12a -f) were poor to mod-
est inhibitors (Table 3).
To improve the solubility of the inhibitors while still
maintaining potency, we next replaced the Cbz group
with a pyridinylmethylcarbonyl group. Kempf and co-
workers had previously found that pyridinyl analogues
of Cbz promote higher bioavailability and increased

678 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3
Ta ble 3. Replacement of Cbz with Morpholinecarbonyl
Huang et al.
Ta ble 5. Aqueous Solubility Measurements
aqueous
inhibitor
Cap
P2
P1
R2
solubility (mM)a
%
inhibitor
R1
R2
yielda
Ki (nM)b
10r
16i
16j
Cbz
3-Pyr
3-Pyr
Phe
Phe
Leu
Phe
Phe
Phe
C(CH3)3
C(CH3)3
C(CH3)3
0.070
0.288
0.378
12a
12b
12c
12d
12e
12f
-C(CH3)3
-C(CH3)3
-CH2CH(CH3)2
-CH2Ph
70
55
76
75
54
70
59.7 ( 6.4
54.6 ( 6.3
-(CH2)2CO2Et -CH2CH(CH3)2
-(CH2)2CO2Et -CH2Ph
69.6 ( 5.6
a
Solubility determined using UV analysis. Inhibitor was pre-
incubated for 2 h at 37 °C.
59.1 ( 6.5
-(CH2)3Ph
-(CH2)3Ph
-CH2CH(CH3)2
-CH2Ph
130.7 ( 13.3
216.3 ( 18.9
Ta ble 6. Selectivity against Other Cysteine Proteases
a
b
Overall yield of purified product for five steps. Ki values
determined at four to eight concentrations in duplicate using the
linear regression analysis of Williams and Morrison.11
cruzain
cathepsin B
cathepsin L
inhibitor
Ki (nM)a
Ki (nM)a
Ki (nM)a
10q
10r
10s
11c
11d
16e
16f
16h
16k
16l
2.9 ( 0.3
2.5 ( 0.2
2.0 ( 0.2
1.3 ( 0.1
1.0 ( 0.1
0.9 ( 0.1
1.1 ( 0.1
14.1 ( 0.9
1.4 ( 0.1
2.0 ( 0.1
42.7 ( 7.1
45.0 ( 7.1
45.4 ( 10.5
111.8 ( 12.8
135.9 ( 18.2
700 ( 100
1.5 ( 0.1
1.7 ( 0.1
Sch em e 2
2.8 ( 0.4
7.9 ( 1.5
4.7 ( 1.1
28.8 ( 2.3
144.4 ( 16.0
28.8 ( 2.3
9.6 ( 0.8
1700 ( 300
4800 ( 300
600 ( 200
1300 ( 400
44.0 ( 5.0
a
Ki values determined at four to eight concentrations in
duplicate using the linear regression analysis of Williams and
Morrison.11
Ta ble 4. Pyridinylmethyl Carbamate Mercaptomethyl Ketones
16h (Ki ) 14.1 ( 0.9). To quantify the increase in
solubility, aqueous solubility measurements were de-
termined for the lead compound 10r and the more
soluble pyridinyl analogues 16i and 16j (Table 5).
Replacement of the Cbz group with the pyridinylmethyl
carbamate resulted in a 4-fold increase in aqueous
solubility, and further replacement of Phe in the P2
position with Leu resulted in a modest increase in
solubility.
To further enhance the potency and to reduce the
potential for proteolytic degradation of the inhibitors,
the homophenylalanine side chain was used in place of
the phenylalanine side chain at the P1 position. For
irreversible inhibitors, incorporation of the homophe-
nylalanine side chain at P1 has resulted in inhibitors
that are not degraded by human proteases, in contrast
to the corresponding inhibitors that incorporate natural
amino acid side chains at this position.15 The inhibitors
(16e, 16f, 16k , and 16l) with homophenylalanine at the
P1 position showed a 2- to 4-fold increase in potency.
%
inhibitor Cap
P2
P1
R2
yielda Ki (nM)b
16a
16b
16c
16d
16e
16f
16g
16h
16i
16j
16k
16l
2-Pyr Phe Phe
3 Pyr Phe Phe
3 Pyr Phe Phe
3 Pyr Leu Phe
3 Pyr Phe hPhe (CH2)3Ph
3 Pyr Leu hPhe (CH2)3Ph
2 Pyr Leu Phe
4-Pyr Leu Phe
3-Pyr Phe Phe
3-Pyr Leu Phe
3-Pyr Phe hPhe C(CH3)3
3-Pyr Leu hPhe C(CH3)3
(CH2)2CO2Et
(CH2)2CO2Et
(CH2)3Ph
65
64
50
63
73
64
52
65
58
60
51
50
30.4 ( 2.4
22.7 ( 2.0
5.5 ( 0.4
5.4 ( 0.2
0.9 ( 0.1
1.1 ( 0.1
13.8 ( 0.8
14.1 ( 0.9
4.6 ( 0.2
4.8 ( 0.2
1.4 ( 0.1
2.0 ( 0.1
(CH2)3Ph
C(CH3)3
C(CH3)3
C(CH3)3
C(CH3)3
a
b
Overall yield of purified product for five steps. Ki values
determined at four to eight concentrations in duplicate using the
linear regression analysis of Williams and Morrison.11
aqueous solubility for a series of HIV protease inhibi-
tors.14 The pyridinyl capped inhibitors were synthesized
on support by acylation with the N-pyridinylmethyloxy-
carbonyl-protected R-amino acids 15. Acids 15 were
prepared by reaction of the 2-, 3-, or 4-substituted
hydroxymethylpyridine with an amino acid ester iso-
cyanate 14 followed by ester hydrolysis (Scheme 2).
The pyridinyl inhibitors (16a -l) showed improved
solubility while maintaining the potency observed for
the Cbz-protected mercaptomethyl ketones (Table 4).
Incorporation of the 3-pyridinyl group typically resulted
in more potent inhibitors than incorporation of the 2-
and 4-substituted pyridinyl groups. For example, 3-py-
ridinyl inhibitor 16j (Ki ) 4.8 ( 0.2) is 3 times more
potent than either the corresponding 2-pyridinyl inhibi-
tor 16g (Ki ) 13.8 ( 0.8) or the 4-pyridinyl inhibitor
Sp ecificity of In h ibitor s. To probe the selectivity
of these inhibitors for cruzain over human cysteine
proteases, the 10 best inhibitors against cruzain were
screened against human cathepsin B and human cathe-
psin L. These compounds are very poor inhibitors of
cathepsin B (Table 6), especially the inhibitors incor-
porating the pyridinylmethyl carbamate. For cathepsin
L, the Cbz-protected inhibitors share similar Ki values
with cruzain. However, the inhibitors incorporating the
pyridinylmethyl carbamate show a 5- to 100-fold selec-
tivity for cruzain over cathepsin L. The data demon-
strates that both the P2 side chain and the mercaptyl
functionality are also important for conferring selectiv-
ity with Phe at P2 and the 3-phenyl propyl mercaptan
providing the greatest selectivity. This level of selectiv-
ity is quite promising, since cruzain is classified as a

Identification of Mechanism-Based Inhibitors of Cruzain
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 679
pressure. Flash chromatography was carried out with Merck
60 230-400 mesh silica gel according to the procedure
described by Still.21 Infrared spectra were recorded with a
Perkin-Elmer 1600 series Fourier transform spectrometer as
thin films on NaCl plates or as KBr pellets, and only partial
cathepsin L-like enzyme with the central domain of
cruzain showing a 56.8% homology with cathepsin L.13
Tim e Dep en d en ce of In h ibitor s. In the enzyme
inhibition assays, some of the identified inhibitors
showed a very modest time dependence. Specifically, the
enzyme and inhibitor were preincubated for 5 min, 1 h,
and 3 h, followed by addition of the substrate to begin
the reaction. The Cbz-protected inhibitors exhibited the
same IC50 values for each preincubation time. However,
the pyridinyl inhibitors exhibited some time-dependent
behavior, with a 2- to 4-fold increase in potency after a
3 h incubation.
For the most potent compounds, time-dependent
behavior was also evaluated for activity against cathe-
psin B and cathepsin L. The substrate was added only
after a 5 min and 1 h preincubation, due to instability
of these enzymes over prolonged incubation times. For
cathepsin B, none of the inhibitors showed any change
in IC50, except for 16j. Interestingly, for cathepsin L,
the IC50s of the Cbz-protected inhibitors showed a 2- to
3-fold time dependence after 1 h, while the IC50s of the
pyridinyl inhibitors were unchanged, which is the
opposite trend as observed for cruzain.
1
data are listed. H and 13C NMR spectra were obtained with
Bruker AMX-300, AMX 400, AM-400, and DRX 500 spectrom-
eters. 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.
Melting points were determined on a MelTemp apparatus and
are reported uncorrected. Solubility measurements were per-
formed by Robertson Microlit Laboriatories (Madison, NJ ).
Solubility was quantitated using UV analysis (λ ) 211 nm),
and inhibitors were preincubated at pH 7.4 aqueous buffer for
2 h at 37 °C. M-H-W Laboratories (Phoenix, AZ) performed
elemental analyses. For most compounds, combustion analysis
data are reported. For the remaining compounds, purity was
evaluated by HPLC analysis using two different solvent
systems. The first solvent system (I) was acetonitrile/water
with a gradient of 40% to 80% in 15 min. The second solvent
system (II) was methanol/water with a gradient of 40% to 80%
in 10 min. The first system was recorded on a Rainin Microsorb
C18 column (100 Å, 5 µM, 25 cm). The second system was
recorded on an LCMS (Hewlett-Packard, C18 column).
Gen er a l Meth od s for Solid -P h a se Syn th esis. Solvents
were distilled for resin reactions. Unless otherwise stated,
reactions were conducted in 12 mL polypropylene cartridges
with 70 mm PE frits attached to Teflon stopcocks. Cartridges
and stopcocks were obtained from Applied Separations (Allen-
town, PA). Syringe plungers from 10 mL disposable syringes
were used as stoppers for the cartridges. Capped syringes were
gently rocked on an orbital shaker table during solid-phase
reactions. Solvents were removed by taking off the syringe
barrel and opening the stopcock. Resin was incubated with
wash solvent for 2-5 min before filtration.
The modest time dependence suggests that the in-
hibitors are either slow binding or very slowly inactivat-
ing. Alkyl mercaptomethyl ketones have previously been
reported to show time-dependent inhibition of cathepsin
K16 and the caspases.17 There are no reports of alkylm-
ercaptomethyl ketone inhibitors that are irreversible
inactivators.18
Con clu sion s
Gen er a l Meth od s for Clea va ge of Com p ou n d s fr om
Resin a n d for Yield Qu a n tita tion . To 0.2 g of derivatized
resin was added a 5 mL solution of 1:4:15 TFA/H2O/trifluoro-
ethanol. After the mixture sat at room temperature for 4 h,
the solution was removed. The resin was washed with THF
(3 × 5 mL), and the washings were combined and concentrated.
Toluene was added to form an azeotrope with the residual
water and TFA. After concentration, the resultant crude oil
or solid was quantitated for yield calculation by 1H NMR
calibration with p-xylene (0.02 mmol). Analytically pure
samples were obtained by purification by column chromatog-
raphy or recrystallization.
Gen er a l P r oced u r e for Syn th esis of Mer ca p tom eth yl
Keton es. A solution of N-allyloxycarbonyl chloromethyl ketone
(4 equiv) in 0.2 M of THF was added to carbazate linker 4
presolvated in THF. After 4 h of gentle rocking on a shaker
table, the solution was removed, and the resin was rinsed with
THF (5 × 5 mL). The support-bound ketone was used im-
mediately in order to prevent decomposition through azodiene
formation. A solution of the thiol (4 mmol, 50 equiv) and i-Pr2-
EtN (4.8 mmol, 60 equiv) in a 2 M solution of DMF was added
to the resin. The cartridge was gently rocked on the shaker
table for 12 h. After filtration of the solution, the resin was
rinsed with DMF (5 × 5 mL) and CH2Cl2 (5 × 5 mL). A solution
of trimethylsilyl azide (0.09 mL, 0.7 mmol, 8 equiv), tetrabu-
tylammonium fluoride (0.063 g, 0.24 mmol, 3 equiv), and Pd-
(PPh3)4 (0.018 g, 0.16 mmol, 0.2 equiv) in 5 mL of CH2Cl2 was
added to the resin in a glovebag under N2 atmosphere. The
resin was gently rocked for 4 h and was protected from light.
After removal of the solution, the resin was rinsed with CH2-
Cl2 (5 × 5 mL) and DMF (5 × 5 mL).
We have described a series of potent mercaptomethyl
ketones against cruzain (Ki e 1 nM) that are selective
for cruzain over cathepsin B and cathepsin L. These
inhibitors were synthesized rapidly using a solid-phase
combinatorial strategy. Substititution at the P1′ position
has provided insight into the size of the binding pocket,
showing a preference for large bulky substituents and
long hydrophobic side chains.
Studies to elucidate the binding mode of these inhibi-
tors are currently being undertaken by analyzing the
crystal structure of cruzain bound to the ketone inhibi-
tors. Knowledge of the binding interactions between the
enzyme and inhibitor should provide valuable insight
toward the rational design of future inhibitors.
Exp er im en ta l Section
Gen er a l Meth od s. Unless otherwise noted, all reagents
were obtained from commercial suppliers and used without
further purification. THF was distilled under N2 from sodium/
benzophenone, and CH2Cl2 and i-Pr2EtN were distilled over
CaH2 immediately prior to use. Pd(PPh3)4 was prepared
according to literature procedure.19 ArgoGel-OH resin was
purchased from Argonaut Technologies (San Carlos, CA).
Diazomethane was generated in situ using the following
literature procedure.20 To a 0.7 M solution of p-toluenesulfo-
nylmethylnitrosamide (Diazald) in absolute ethanol was added
small aliquots of 1.5 N KOH until the Diazald solution became
white. Diazomethane gas was produced and transferred by
cannula (two fire-polished pipets connected by rubber tubing)
under positive N2 flow into the stirring THF solution of the
mixed anhydride.
An acylation stock solution of the desired carboxylic acid
(0.4 mmol, 5 equiv), i-Pr2EtN (0.8 mmol, 10 equiv), HOAt (0.4
mmol, 5 equiv), and PyBOP (0.4 mmol, 5 equiv) in 0.2 M of
DMF was prepared immediately before addition. The acylation
solution was added to the resin, and the cartridge was gently
rocked for 4 h. Upon removal of the solution, the resin was
rinsed with DMF (5 × 5 mL) and the acylation reaction
Reaction progress was monitored through thin-layer chro-
matography on Merck 60 F254 0.25 mm silica plates. Unless
otherwise specified, extracts were dried over MgSO4, and
solvents were removed with a rotary evaporator at aspirator

680 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3
Huang et al.
sequence was repeated. After excess reagents were filtered,
the resin was rinsed with DMF (5 × 5 mL) and THF (5 × 5
mL) and dried overnight in vacuo. The material was cleaved
and quantified as previously described.
128.8, 129.1, 129.2, 135.8, 136.1, 155.8, 169.6, 170.5, 202.0.
Anal. (C31H34N2O6S) C, H, N.
1
10h : 75% yield. IR: 1658, 1687, 1717 cm-1. H NMR (500
MHz): δ 0.86 (d, J ) 3.0 Hz, 3H), 0.87 (d, J ) 3.0 Hz, 3H),
1.34-1.39 (m, 2H), 1.60 (septet, J ) 6.6 Hz, 1H), 2.32 (t, J )
7.8 Hz, 2H), 2.93-3.08 (m, 6H), 4.38 (m, 1H), 5.02 (m, 1H),
5.08 (s, 2H), 5.18 (m, 1H), 6.42 (d, J ) 7.3 Hz, 1H), 7.04-7.36
(m, 15H). 13C NMR (125 MHz): δ 22.1, 22.2, 27.3, 30.0, 37.6,
37.8, 38.1, 38.7, 56.1, 57.1, 67.2, 127.06, 127.14, 128.1, 128.2,
128.5, 128.6, 128.8, 129.16, 129.23, 135.9, 136.0, 155.8, 170.4,
202.1. Anal. (C32H38N2O4S) C, H, N.
Syn th esis of N-Cbz-P h e-P h e Keton e In h ibitor s. Load-
ing, displacement, and deprotection of the N-allyloxycarbonyl
phenylalanine chloromethyl ketone were performed according
to the general procedure. Acylation using N-Cbz phenylalanine
as the desired carboxylic acid resulted in the formation of the
desired inhibitor in 39-97% yields, as determined by NMR
calibration with p-xylene (0.02 mmol).
10i: 97% yield. IR: 1652, 1700, 1715 cm-1 1H NMR (500
.
10a : 62% yield. IR: 1654, 1688, 1715 cm-1 1H NMR (500
.
MHz): δ 1.43 (s, 9H), 2.88-3.09 (m, 6H), 3.48 (d, J ) 13.4 Hz,
1H), 3.53 (d, J ) 13.4 Hz, 1H), 4.38 (m, 1H), 4.98 (m, 1H),
5.06 (s, 2H), 5.20 (m, 1H), 6.40 (d, J ) 7.2 Hz, 1H), 6.98-7.33
(m, 19H). 13C NMR (125 MHz): δ 30.3, 31.3, 34.5, 35.4, 37.6,
37.6, 38.2, 56.1, 57.3, 67.1, 125.5, 127.07, 127.14, 128.1, 128.2,
128.5, 128.6, 128.78, 128.84, 129.16, 129.23, 133.7, 135.7,
135.9, 136.0, 150.3, 155.8, 170.4, 202.1. Anal. (C38H42N2O4S)
C, H, N.
MHz): δ 2.87-3.02 (m, 6H), 3.45 (d, J ) 13.6, 1H), 3.49 (d, J
) 13.6 Hz, 1H), 4.38 (m, 1H), 4.98 (m, 1H), 5.06 (d, J ) 12.3
Hz, 1H), 5.09 (d, J ) 12.3 Hz, 1H,), 5.17 (m, 1H), 6.38 (d, J )
6.8 Hz, 1H), 7.01-7.34 (m, 19H). 13C NMR (125 MHz): δ 35.0,
37.3, 37.6, 38.1, 56.1, 57.2, 67.2, 127.1, 127.2, 128.0, 128.3,
128.5, 128.65, 128.67, 128.8, 129.1, 129.2, 130.5, 133.1, 135.4,
135.7, 135.9, 136.0, 155.8, 170.4, 202.0. Anal. (C34H33ClN2O4S)
C, H, N.
10j: 96% yield. IR: 1655, 1702, 1714 cm-1 1H NMR (300
.
1
10b: 89% yield. IR: 1658, 1691, 1709 cm-1. H NMR (500
MHz): δ 2.75-3.10 (m, 9H), 3.23 (d, J ) 15.5 Hz, 1H), 4.38
(m, 1H), 4.86 (app q, J ) 7.2 Hz, 1H), 5.06 (s, 2H), 5.21 (d, J
) 7.5 Hz, 1H), 6.54 (d, J ) 7.3 Hz, 1H), 6.98-7.38 (m, 15H).
13C NMR (125 MHz): δ 26.5, 35.6, 37.2, 37.9, 39.7, 56.1, 57.8,
67.2, 127.2, 127.2, 127.3, 128.0, 128.3, 128.6, 128.8, 129.1,
129.2, 135.5, 135.8, 136.0, 155.8, 168.6, 170.8, 202.6. LCMS
(EI): m/z 548.7 (M + 1)+; tr ) 4.44 min (95% purity) using
solvent system I, tr ) 2.70 min (95% purity) using solvent
system II.
MHz): δ 2.89-3.07 (m, 6H), 3.50 (d, J ) 13.5 Hz, 1H), 3.55
(d, J ) 13.5 Hz, 1H), 4.38 (m, 1H), 4.97 (m, 1H), 5.06 (s, 2H),
5.17 (m, 1H), 6.38 (d, J ) 7.2 Hz, 1H), 7.02-7.33 (m, 20H).
13C NMR (125 MHz): δ 35.8, 37.5, 37.6, 38.1, 56.1, 57.3, 67.2,
127.1, 127.2, 127.3, 128.1, 128.2, 128.5, 128.6, 128.8, 129.15,
129.17, 129.23, 135.8, 136.0, 136.0, 155.8, 170.4, 202.1. Anal.
(C34H34N2O4S) C, H, N.
10c: Since racemic mercaptan was used, an approximate
1:1 mixture of diastereomers was produced. 67% yield. IR:
1658, 1686, 1714, 1730 cm-1. 1H NMR (500 MHz): δ 1.27 (t, J
) 7.1 Hz, 3H), 1.35 (d, J ) 7.2 Hz, 3H), 2.90-3.08 (m, 4H),
3.20-3.43 (m, 3H), 4.16 (q, J ) 7.1 Hz, 2H,), 4.38 (m, 1H),
4.89-4.97 (m, 1H), 5.07 (s, 2H), 5.20 (m, 1H), 6.46-6.41 (m,
1H), 7.05-7.36 (m, 15H). 13C NMR (125 MHz): δ 14.1, 16.7,
16.8, 37.3, 37.4, 38.1, 38.75, 38.76, 40.97, 41.04, 56.1, 57.6, 57.9,
61.3, 67.1, 67.9, 127.11, 127.15, 128.0, 128.22, 128.23, 128.5,
128.66, 128.67, 128.74, 128.8, 129.13, 129.14, 129.2, 155.8,
170.50, 170.52, 172.3, 172.4, 202.3, 202.5. Anal. (C32H36N2O6S)
C, H, N.
10k : 46% yield. IR 1653, 1699, 1715 cm-1 1H NMR (500
.
MHz): δ 1.01 (s, 9H), 1.31 (s, 6H), 1.56 (s, 2H), 2.93-3.07 (m,
4H), 3.18 (d, J ) 15.1 Hz, 1H), 3.24 (d, J ) 15.0 Hz, 1H), 4.40
(m, 1H), 5.02-5.09 (m, 3H), 5.23 (d, J ) 7.1 Hz, 1H), 6.41 (d,
J ) 7.0 Hz, 1H), 7.05-7.36 (m, 15H). 13C NMR (125 MHz): δ
30.1, 31.6, 32.7, 37.6, 37.9, 38.2, 48.2, 54.6, 56.0, 57.5, 67.1,
127.10, 127.13, 128.0, 128.2, 128.5, 128.7, 129.1, 129.3, 135.8,
136.1, 155.8, 170.3, 204.4. Anal. (C35H44N2O4S) C, H, N.
10l: 60% yield. IR: 1653, 1699, 1715 cm-1 1H NMR (300
.
MHz): δ 1.17-1.43 (m, 5H), 1.60-1.88 (m, 5H), 2.43 (m, 1H),
2.92-3.16 (m, 6H), 4.38 (m, 1H), 5.01-5.16 (m, 4H), 6.43 (d,
J ) 7.2 Hz, 1H), 7.05-7.34 (m, 15H). 13C NMR (125 MHz): δ
25.6. 25.8, 32.8, 37.3, 37.8, 43.3, 56.1, 57.1, 67.2, 127.0, 127.1,
128.1, 128.2, 128.5, 128.6, 128.8, 129.18, 129.22, 135.93,
135.99, 136.01, 155.8, 170.4, 202.7. Anal. (C33H38N2O4S) C, H,
N.
1
10d : 37% yield. IR: 1653, 1700, 1716 cm-1. H NMR (500
MHz): δ 1.97 (d, J ) 5.9 Hz, 3H,), 2.47-2.56 (m, 2H), 2.85-
3.16 (m, 6H), 3.21-3.33 (m, 2H), 4.37 (m, 1H), 5.01-5.10 (m,
3H), 5.19 (m, 1H), 6.18 (m, 1H), 6.47 (m, 1H), 6.98-7.34 (m,
15H). 13C NMR (125 MHz): δ 30.3, 32.2, 36.0, 37.4, 38.0, 38.9,
56.0, 57.2, 67.2, 127.3, 128.0, 128.3, 128.6, 128.77, 128.79,
128.9, 129.1, 129.2, 135.6, 135.9, 155.8, 170.4, 170.7, 203.1.
Anal. Calcd for C31H35N3O5S: C, 66.29; H, 6.28; N, 7.48. LCMS
(EI): m/z 562.3 (M + 1)+; tr ) 10.78 min (95% purity) using
solvent system I, tr ) 9.08 min (99% purity) using solvent
system II.
10m : Since racemic sec-butyl mercaptan was used, an
approximate 1:1 mixture of diastereomers was produced. 63%
1
yield. IR: 1654, 1701, 1716 cm-1. H NMR (500 MHz): δ 0.92
(m, 3H), 1.14-1.26 (m, 3H), 1.41-1.55 (m, 2H), 2.53 (m, 1H),
2.94-3.14 (m, 6H), 4.38 (m, 1H), 5.03-5.13 (m, 3H), 5.17 (m,
1H), 6.40 (m, 1H), 6.98-7.36 (m, 15H). 13C NMR (125 MHz):
δ 10.9, 11.2, 19.9, 20.1, 28.9, 29.1, 37.6, 37.78, 37.88, 37.9, 38.1,
41.6, 41.7, 56.1, 57.1, 67.1, 127.06, 127.13, 128.1, 128.2, 128.5,
128.6, 128.8, 129.15, 129.23, 135.9, 136.0, 155.8, 170.3, 202.8.
Anal. (C31H36N2O4S) C, H, N.
10e: 42% yield. IR: 1658, 1691, 1714 cm-1 1H NMR (500
.
MHz): δ 1.34-1.42 (m, 2H), 1.55 (m, 2H), 1.70 (m, 2H), 1.86-
1.93 (m, 2H), 2.86 (app quintet, J ) 7.0 Hz, 1H), 2.94-3.11
(m, 6H), 4.38 (m, 1H), 5.04-5.17 (m, 3H), 5.18 (m, 1H), 6.42
(m, 1H), 7.04-7.36 (m, 15H). 13C NMR (125 MHz): δ 24.6, 24.7,
33.1, 33.4, 37.8, 38.1, 39.2, 43.7, 56.1, 57.1, 67.1, 127.0, 127.1,
128.1, 128.2, 128.5, 128.6, 128.8, 129.17, 129.23, 135.9, 136.0,
155.8, 170.3, 202.6. Anal. (C32H36N2O4S) C, H, N.
10n : 50% yield. IR 1653, 1699, 1716 cm-1 1H NMR (500
.
MHz): δ 0.81-1.44 (m, 25H), 2.92-3.20 (m, 6H), 4.40 (m, 1H),
5.07 (m, 3H), 5.23 (m, 1H), 6.41 (m, 1H), 6.99-7.36 (m, 15H).
13C NMR (125 MHz): δ 28.4, 29.4, 30.3, 37.1, 37.8, 38.2, 56.0,
57.5, 67.1, 127.1, 128.01, 128.02, 128.2, 128.5, 128.68, 128.73,
10f: 90% yield. IR: 1655, 1689, 1715 cm-1 1H NMR (500
.
MHz): δ 2.89-3.05 (m, 6H), 3.46 (d, J ) 13.4 Hz, 1H), 3.50
(d, J ) 13.4 Hz, 1H), 3.78 (s, 3H), 4.38 (m, 1H), 4.97 (m, 1H),
5.07 (s, 2H), 5.19 (m, 1H), 6.39 (d, J ) 6.7 Hz, 1H), 6.83 (d, J
) 8.7 Hz, 2H), 7.01 (d, J ) 6.2 Hz, 2H), 7.14-7.34 (m, 15H).
13C NMR (125 MHz): δ 35.3, 37.4, 37.7, 38.2, 55.3, 56.1, 57.3,
67.2, 114.0, 127.1, 127.2, 128.1, 128.3, 128.6, 128.7, 128.78,
128.83, 129.2, 129.3, 130.4, 135.9, 136.1, 155.8, 158.9, 170.4,
202.2. Anal. (C35H36N2O5S) C, H, N.
129.1, 129.2, 135.8, 136.0, 155.8, 170.4, 204.2. Anal. (C39H52
N2O4S) C, H, N.
-
10o: 51% yield. IR: 1653, 1699, 1716 cm-1 1H NMR (500
.
MHz): δ 1.16 (d, J ) 6.7 Hz, 3H), 1.19 (d, J ) 6.6 Hz, 3H),
2.70 (septet, J ) 6.7 Hz, 1H), 2.93-3.15 (m, 6H), 4.38 (m, 1H),
5.05-5.13 (m, 1H), 5.08 (s, 2H), 5.18 (m, 1H), 6.41 (m, 1H),
6.92-7.36 (m, 15H). 13C NMR (125 MHz): δ 22.65, 22.71, 35.0,
37.8, 37.9, 38.1, 56.1, 57.1, 67.2, 127.06, 127.13, 128.1, 128.2,
128.5, 128.6, 128.8, 129.15, 129.22, 135.9, 136.0, 155.8, 170.4,
202.6. Anal. (C30H34N2O4S) C, H, N.
10g: 48% yield. IR: 1655, 1701, 1717 cm-1 1H NMR (500
.
MHz): δ 1.27 (t, J ) 7.1 Hz, 3H), 2.90-3.13 (m, 6H), 3.25 (m,
2H), 4.17 (q, J ) 7.1 Hz, 2H), 4.37 (m, 1H), 4.94 (m, 1H), 5.07
(s, 2H), 5.17 (m, 1H), 6.40 (d, J ) 7.5 Hz, 1H), 7.04-7.36 (m,
15H). 13C NMR (125 MHz): δ 14.1, 33.1, 37.3, 38.1, 38.9, 56.1,
57.6, 61.5, 67.1, 127.10, 127.14, 128.0, 128.2, 128.5, 128.7,
1
10p : 64% yield. IR: 1655, 1700, 1715 cm-1. H NMR (500
MHz): δ 2.59 (m, 2H), 2.79 (m, 2H), 2.90-3.11 (m, 6H), 4.38
(m, 1H), 5.00 (m, 1H), 5.07 (s, 2H), 5.19 (m, 1H), 6.40 (d, J )

Identification of Mechanism-Based Inhibitors of Cruzain
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 681
7.0 Hz, 1H,), 6.99-7.34 (m, 20H). 13C NMR (125 MHz): δ 33.2,
35.3, 37.7, 38.1, 38.7, 56.1, 57.1, 67.2, 126.4, 127.1, 127.2, 128.0,
128.2, 128.5, 128.5, 128.5, 128.6, 128.8, 129.15, 129.20, 135.8,
136.0, 138.2, 139.8, 155.8, 170.4, 202.0. Anal. (C35H36N2O4S)
C, H, N.
tion of the N-allyloxycarbonyl phenylalanine chloromethyl
ketone were performed according to the general procedure.
Acylation using N-morpholinecarbonyl phenylalanine or N-
morpholinecarbonyl leucine as the desired carboxylic acid
resulted in the formation of the desired inhibitor in 54-76%
yields, as determined by 1H NMR calibration with p-xylene
(0.02 mmol).
1
10q: 82% yield. IR: 1653, 1699, 1715, 1732 cm-1. H NMR
(300 MHz): δ 1.26 (t, J ) 7.1 Hz, 3H), 2.47-2.63 (m, 4H),
2.93-3.10 (m, 6H), 4.14 (q, J ) 7.1 Hz, 2H,), 4.37 (m, 1H),
4.97 (m, 1H), 5.07 (s, 2H), 5.16 (m, 1H), 6.38 (d, J ) 7.9 Hz,
1H,), 7.03-7.36 (m, 15H). 13C NMR (125 MHz): δ 14.1, 26.7,
34.0, 37.7, 38.7, 56.1, 57.2, 60.7, 61.2, 127.1, 127.2, 128.1, 128.2,
128.5, 128.7, 128.8, 129.1, 129.2, 135.8, 136.0, 155.8, 170.4,
171.4, 202.0. Anal. (C32H36N2O6S) C, H, N.
12a : 70% yield. IR: 1622, 1654, 1719 cm-1 1H NMR (300
.
MHz): δ 0.89 (d, J ) 6.2 Hz, 3H,), 0.91 (d, J ) 6.3 Hz, 3H),
1.25 (s, 9H), 1.27-1.68 (m, 3H), 3.02 (dd, J ) 7.3 Hz, 14.0,
1H), 3.13 (dd, J ) 6.7 Hz, 14.0, 1H), 3.27-3.34 (m, 6H), 3.67
(app t, J ) 4.9 Hz, 4H), 4.27-4.34 (m, 1H), 4.75 (d, J ) 7.7
Hz, 1H), 5.09 (app q, J ) 7.2 Hz, 1H), 6.74 (d, J ) 7.5 Hz,
1H), 7.16-7.32 (m, 5H). 13C NMR (125 MHz): δ 22.2, 22.8,
24.8, 30.6, 37.8, 37.8, 41.4, 43.2, 43.9, 52.8, 57.5, 66.4, 127.0,
128.6, 129.2, 136.2, 157.2, 172.9, 205.0. Anal. (C25H39N3O4S)
C, H, N.
1
10r : 49% yield. IR: 1655, 1701 cm-1. H NMR (300 MHz):
δ 1.24 (s, 9H), 2.90-3.08 (m, 4H), 3.16 (d, J ) 15.2 Hz, 1H),
3.22 (d, J ) 15.3 Hz, 1H), 4.38 (m, 1H), 5.02-5.09 (m, 3H),
5.21 (d, J ) 7.9 Hz, 1H), 6.40 (d, J ) 7.5 Hz, 1H), 6.98-7.34
(m, 15H). 13C NMR (125 MHz): δ 30.6, 37.7, 37.9, 38.2, 56.0,
57.3, 67.1, 127.1, 128.0, 128.2, 128.5, 128.69, 128.74, 129.1,
129.2, 135.7, 136.0, 155.8, 170.4, 204.5. Anal.(C31H36N2O4S)
C, H, N.
1
12b: 55% yield. IR: 1621, 1654, 1718 cm-1. H NMR (300
MHz): δ 1.25 (s, 9H), 2.89-3.10 (m, 4H), 3.18-3.38 (m, 6H),
3.61-3.64 (m, 4H), 4.49-4.56 (m, 1H), 4.86 (d, J ) 7.1 Hz,
1H), 4.99-5.06 (m, 1H), 6.49 (d, J ) 7.5 Hz, 1H), 7.02-7.32
(m, 10H). 13C NMR (125 MHz): δ 30.7, 37.65, 37.68, 38.3, 43.3,
43.9, 55.3, 57.4, 66.3, 126.99, 127.03, 128.6, 128.7, 129.1, 129.3,
136.0, 136.6, 156.8, 171.3, 204.5. Anal. (C28H37N3O4S) C, H,
N.
10s: 63% yield. IR: 1653, 1700, 1716 cm-1 1H NMR (500
.
MHz): δ 1.81 (m, 2H), 2.34 (t, J ) 7.3 Hz, 2H), 2.65 (t, J ) 7.5
Hz, 2H), 2.92-3.09 (m, 6H), 4.37 (m, 1H), 5.01 (m, 1H), 5.08
(s, 2H), 5.15 (m, 1H), 6.36 (m, 1H), 7.03-7.34 (m, 20H). 13C
NMR (125 MHz): δ 30.2, 31.3, 34.6, 37.8, 38.1, 38.7, 56.1, 57.1,
67.2, 126.0, 127.08, 127.10, 128.0, 128.2, 128.4, 128.5, 128.6,
128.8, 129.15, 129.22, 131.8, 135.8, 136.0, 141.1, 155.8, 170.4,
202.1. Anal. (C36H38N2O4S) C, H, N.
1
12c: 76% yield. IR: 1626, 1660, 1713, 1729 cm-1. H NMR
(400 MHz): δ 0.87-0.90 (m, 6H), 1.24 (t, J ) 7.2 Hz, 3H),
1.40-1.44 (m, 1H), 1.51-1.59 (m, 2H), 2.49-2.52 (m, 2H),
2.62-2.66 (m, 2H), 3.00 (dd, J ) 7.5 Hz, 13.9, 1H), 3.10-3.34
(m, 7H), 3.65 (m, 4H), 4.12 (q, J ) 7.1 Hz, 2H), 4.28 (m, 1H),
4.83 (d, J ) 7.5 Hz, 1H), 4.96 (m, 1H), 6.91 (d, J ) 7.6 Hz,
1H), 7.15-7.28 (m, 5H). 13C NMR (101 MHz): δ 14.2, 22.2,
22.8, 24.8, 26.9, 34.1, 37.6, 39.0, 41.2, 44.0, 52.8, 57.4, 60.8,
66.4, 127.1, 128.7, 129.2, 136.2, 157.3, 171.6, 173.0, 202.8.
Anal. (C26H39N3O6S) C, H, N.
Syn th esis of N-Cbz-Leu -P h e Keton e In h ibitor s. Load-
ing, displacement, and deprotection of the N-allyloxycarbonyl
phenylalanine chloromethyl ketone were performed according
to the general procedure. Acylation using N-Cbz leucine as the
desired carboxylic acid resulted in the formation of the desired
1
inhibitor in 44-52% yields, as determined by H NMR calibra-
tion with p-xylene (0.02 mmol).
1
12d : 75% yield. IR: 1624, 1659, 1729 cm-1. H NMR (500
1
11a : 44% yield. IR: 1655, 1708 cm-1. H NMR (500 MHz):
MHz): δ 1.17 (t, J ) 7.1 Hz, 3H), 2.40-2.47 (m, 2H), 2.50-
2.52 (m, 2H), 2.82-3.04 (m, 6H), 3.14-3.17 (m, 4H), 3.51-
3.53 (m, 4H), 4.05 (q, J ) 7.1 Hz, 2H), 4.44 (m, 1H), 4.80-
4.94 (m, 2H), 6.60 (d, J ) 7.6 Hz, 1H), 6.94 (m, 2H), 6.96-
7.20 (m, 8H). 13C NMR (125 MHz): δ 14.1, 26.7, 33.9, 37.4,
38.1, 38.6, 43.8, 55.4, 57.2, 60.6, 66.2, 126.9, 127.0, 128.5, 128.6,
129.1, 129.2, 136.0, 136.5, 156.7, 171.4, 171.4, 202.1. Anal.
(C29H37N3O6S) C, H, N.
δ 0.90 (d, J ) 6.2 Hz, 6H), 1.25 (t, J ) 7.1 Hz, 3H), 1.39-1.43
(m, 1H), 1.56-1.62 (m, 2H), 2.51-2.53 (m, 2H), 2.65 (m, 2H),
2.98-3.01 (m, 1H), 3.12-3.26 (m, 3H), 4.14 (m, 3H), 5.00-
5.09 (m, 4H), 6.65 (d, J ) 7.1 Hz, 1H), 7.14-7.36 (m, 10H).
13C NMR (125 MHz): δ 14.2, 22.8, 24.6, 26.8, 34.0, 37.6, 38.9,
41.0, 53.5, 57.2, 60.7, 67.2, 127.1, 128.1, 128.3, 128.5, 128.7,
129.2,135.95,136.0,156.1,171.5,171.8,202.6.Anal.(C29H38N2O6S)
C, H, N.
1
12e: 54% yield. IR: 1621, 1658, 1713 cm-1 1H NMR (500
.
11b: 52% yield. IR: 1656, 1701 cm-1. H NMR (500 MHz):
MHz): δ 0.90 (m, 6H), 1.41-1.47 (m, 1H), 1.55-1.65 (m, 2H),
1.78-1.87 (m, 2H), 2.39 (t, J ) 7.3 Hz, 2H), 2.66 (t, J ) 7.6
Hz, 2H), 3.01 (dd, J ) 7.4, 14.0 Hz, 1H), 3.12-3.21 (m, 3H),
3.27-3.36 (m, 4H), 3.67 (app t, J ) 4.9 Hz, 4H), 4.28-4.33
(m, 1H), 4.78 (d, J ) 7.6 Hz, 1H), 5.03 (app q, J ) 7.2 Hz, 1H),
6.87 (d, J ) 7.7 Hz, 1H), 7.15-7.29 (m, 10H). 13C NMR (125
MHz): δ 22.2, 22.8, 24.8, 30.3, 31.4, 34.6, 37.7, 39.0, 41.3, 43.9,
52.8, 57.1, 66.3, 126.0, 127.0, 128.36, 128.37, 128.6, 129.2,
136.2, 141.1, 157.2, 172.8, 202.8. Anal. (C30H41N3O4S) C, H,
N.
δ 0.90 (d, J ) 6.2 Hz, 6H), 1.19 (d, J ) 6.8 Hz, 3H), 1.21 (d, J
) 6.6 Hz, 3H), 1.38-1.43 (m, 1H), 1.56-1.66 (m, 2H), 2.76
(septet, J ) 6.7 Hz, 1H,), 2.99-3.03 (m, 1H), 3.15 (d, J ) 6.7
Hz, 13.9, 1H), 3.22 (s, 2H), 4.13 (m, 1H), 5.04 (d, J ) 7.2 Hz,
1H), 5.08-5.16 (m, 3H), 6.61 (d, J ) 7.2 Hz, 1H), 7.05-7.37
(m, 10H). 13C NMR (125 MHz): δ 22.7, 22.8, 24.6, 35.1, 37.9,
38.1, 41.1, 53.5, 57.1, 67.2, 127.1, 128.1, 128.2, 128.5, 128.7,
129.2, 136.0, 156.0, 171.7, 203.2. Anal. (C27H36N2O4S) C, H,
N.
1
11c: 51% yield. IR: 1657, 1705 cm-1. H NMR (500 MHz):
12f: 70% yield. IR: 1623, 1657, 1712 cm-1 1H NMR (500
.
δ 0.90 (d, J ) 6.1 Hz, 6H), 1.23-1.61 (m, 12H), 2.99-3.03 (m,
1H), 3.10-3.15 (m, 1H), 3.29-3.30 (m, 2H), 4.14 (m, 1H), 5.04
(d, J ) 7.2 Hz, 1H,), 5.09-5.15 (m, 3H), 6.58 (d, J ) 7.3 Hz,
1H,), 6.91-7.37 (m, 10H). 13C NMR (125 MHz): δ 22.8, 24.6,
30.6, 37.7, 37.9, 41.2, 43.3, 53.4, 57.3, 67.1, 127.1, 128.1, 128.2,
128.5, 128.7, 129.2, 135.9, 136.0, 156.0, 171.7, 204.9. Anal.
(C28H38N2O4S) C, H, N.
MHz): δ 1.77-1.88 (m, 2H), 2.36 (t, J ) 7.3 Hz, 2H), 2.66 (t,
J ) 7.6 Hz, 2H), 2.91-3.19 (m, 6H), 3.20-3.29 (m, 4H), 3.58-
3.65 (m, 4H), 4.53 (m, 1H), 4.86 (d, J ) 7.1 Hz, 1H), 4.98 (m,
1H), 6.57 (d, J ) 7.7 Hz, 1H), 7.02-7.04 (m, 2H), 7.15-7.29
(m, 13H). 13C NMR (125 MHz): δ 30.2, 31.3, 34.6, 37.6, 38.2,
38.7, 43.9, 55.4, 57.1, 66.3, 126.0, 127.0, 127.1, 128.4, 128.5,
128.7, 129.2, 129.3, 136.1, 136.6, 141.1, 156.8, 171.3, 202.2.
Anal. (C33H39N3O4S) C, H, N.
1
11d : 44% yield. IR: 1657, 1702 cm-1. H NMR (500 MHz):
δ 0.90 (d, J ) 6.2 Hz, 6H), 1.39-1.43 (m, 1H), 1.56-1.63 (m,
2H), 1.80-1.88 (m, 2H), 2.40 (t, J ) 7.2 Hz, 2H), 2.66 (t, J )
7.6 Hz, 2H), 2.98-3.02 (m, 1H), 3.12-3.22 (m, 3H), 4.13 (m,
1H), 5.02 (d, J ) 7.1 Hz, 1H), 5.05-5.10 (m, 3H), 6.61 (d, J )
7.4 Hz, 1H,), 7.14-7.35 (m, 15H). 13C NMR (125 MHz): δ 22.8,
24.6, 30.3, 31.3, 34.6, 37.8, 38.9, 41.1, 53.5, 57.1, 67.2, 126.0,
127.1, 128.1, 128.3, 128.37, 128.39, 128.5, 128.7, 129.2, 136.0,
141.1, 156.0, 171.7, 202.6. Anal. (C33H40N2O4S) C, H, N.
Syn th esis of Keton e In h ibitor s Th a t In cor p or a te th e
N-Mor p h olin e Ur ea . Loading, displacement, and deprotec-
Syn th esis of N-[(n -P yr id in yl)m eth oxy]ca r bon yl] Ke-
ton e In h ibitor s. Loading, displacement, and deprotection of
the N-allyloxycarbonyl-protected phenylalanine or homophen-
ylalanine chloromethyl ketones were performed according to
the general procedure. Acylation using N-[(n-pyridinyl)-
methoxy] carbonyl] phenylalanine or leucine as the desired
carboxylic acid resulted in the formation of the desired
1
inhibitor in 50-73% yields, as determined by H NMR calibra-
tion with p-xylene (0.02 mmol).

682 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3
Huang et al.
1
16a : 65% yield. IR: 1670, 1726 cm-1. H NMR (300 MHz):
(dd, J ) 6.7, 14.0 Hz, 1H), 3.25 (s, 2H), 4.10-4.15 (m, 1H),
5.09 (m, 1H), 5.26 (d, J ) 14.7 Hz, 1H), 5.46 (d, J ) 14.7 Hz,
1H), 6.01 (d, J ) 7.8 Hz, 1H), 7.03 (d, J ) 7.8 Hz, 1H), 7.16-
7.33 (m, 5H), 7.59 (d, J ) 7.5 Hz, 2H), 8.07 (t, J ) 7.6 Hz,
1H), 8.84 (m, 1H). 13C NMR (125 MHz): δ 21.6, 22.7, 24.6,
30.6, 37.6, 37.8, 41.0, 43.1, 54.2, 57.3, 64.1, 123.1, 124.4, 127.0,
128.6, 129.2, 136.1, 141.3, 145.8, 153.9, 155.0, 171.9, 204.9.
LCMS (EI): m/z 500.3 (M + 1)+; tr ) 11.20 min (96% purity)
using solvent system I, tr ) 9.87 min (96% purity) using solvent
system II.
δ 1.25 (m, 3H), 2.45-2.57 (m, 4H), 2.87-3.11 (m, 6H), 4.13 (q,
J ) 7.1 Hz, 2H), 4.34-4.36 (m, 1H), 4.95-5.03 (m, 1H), 5.20
(d, J ) 14.3 Hz, 1H), 5.37 (d, J ) 14.2 Hz, 1H), 5.84 (m, 1H),
6.79 (d, J ) 7.5 Hz, 1H), 7.09-7.30 (m, 10H), 7.44-7.49 (m,
2H), 7.96 (m, 1H), 8.74 (m, 1H). 13C NMR (125 MHz): δ 14.2,
26.7, 34.0, 37.4, 38.0, 38.8, 56.6, 57.2, 60.7, 64.1, 122.7, 123.9,
127.0, 127.1, 128.6, 128.7, 129.2, 136.00, 136.01, 146.8, 154.4,
155.1, 170.6, 171.5, 202.3. LCMS (EI): m/z 578.3 (M + 1)+; tr
) 10.10 min (94% purity) using solvent system I, tr ) 9.31
min (97% purity) using solvent system II.
1
16h : 65% yield. IR: 1668, 1720 cm-1. H NMR (500 MHz):
1
16b: 64% yield. IR: 1670, 1722 cm-1. H NMR (500 MHz):
δ 0.90 (d, J ) 6.7 Hz, 3H), 0.92 (d, J ) 6.6 Hz, 3H), 1.24 (s,
9H), 1.45-1.65 (m, 3H), 3.02-3.06 (m, 1H), 3.09-3.14 (m, 1H),
3.26 (d, J ) 15.4 Hz, 1H), 3.31 (d, J ) 15.0 Hz, 1H), 4.16 (m,
1H), 5.16-5.31 (m, 3H), 5.66 (m, 1H), 6.68 (d, J ) 6.9 Hz, 1H),
7.14-7.28 (m, 5H), 7.67 (app s, 2H), 8.76 (app s, 2H). 13C NMR
(125 MHz): δ 21.8, 22.7, 24.7, 30.6, 37.8, 37.8, 41.5, 43.3, 53.8,
57.4, 64.0, 123.3, 127.2, 128.7, 129.1, 135.7, 143.6, 155.0, 155.2,
171.5, 205.0. LCMS (EI): m/z 500.3 (M + 1)+; tr ) 10.64 min
(>99% purity) using solvent system I, tr ) 9.72 min (96%
purity) using solvent system II.
δ 1.25 (t, J ) 7.1 Hz, 3H), 2.48-2.51 (m, 2H), 2.55-2.61 (m,
2H), 2.93-3.09 (m, 6H), 4.14 (q, J ) 7.1 Hz, 2H), 4.38 (m, 1H),
5.00 (m, 1H), 5.15 (s, 2H), 5.56 (s, 1H), 6.51 (d, J ) 5.5 Hz,
1H), 7.05-7.30 (m, 10H), 7.60 (s, 1H), 7.98 (d, J ) 7.9 Hz,
1H), 8.67 (s, 1H), 8.73 (s, 1H). 13C NMR (125 MHz): δ 14.1,
26.8, 34.0, 37.6, 38.3, 38.8, 56.3, 57.2, 60.8, 63.3, 125.2, 127.1,
127.2, 128.7, 128.8, 129.1, 129.2, 134.6, 135.7, 135.9, 140.1,
144.8, 155.1, 170.3, 174.5, 202.1. LCMS (EI): m/z 578.3 (M +
1)+; tr ) 9.88 min (>99% purity) using solvent system I, tr )
9.04 min (98% purity) using solvent system II.
1
16i: 58% yield. IR: 1668, 1720 cm-1. H NMR (500 MHz):
1
16c: 50% yield. IR: 1670, 1719 cm-1. H NMR (500 MHz):
δ 1.26 (s, 9H), 2.93-3.07 (m, 4H), 3.01-3.07 (m, 1H), 3.20 (d,
J ) 15.1 Hz, 1H), 3.24 (d, J ) 15.0 Hz, 1H), 4.38 (m, 1H), 5.10
(app q, J ) 7.1 Hz, 1H), 5.15 (s, 2H), 5.58 (d, J ) 7.3 Hz, 1H),
6.50 (d, J ) 6.8 Hz, 1H), 7.07-7.66 (m, 10H), 7.66 (m, 1H),
8.04 (d, J ) 7.3 Hz, 1H), 8.69 (br s, 1H), 8.76 (br s, 1H). 13C
NMR (125 MHz): δ 30.6, 37.7, 37.8, 38.4, 43.3, 56.2, 57.4, 63.1,
125.3, 127.1, 127.2, 128.67, 128.73, 129.1, 129.2, 134.9, 135.7,
135.9, 140.5, 144.31, 144.34, 155.0, 170.1, 204.5. LCMS (EI):
m/z 534.2 (M + 1)+; tr ) 11.25 min (>99% purity) using solvent
system I, tr ) 9.99 min (96% purity) using solvent system II.
δ 1.78-1.84 (m, 2H), 2.34 (m, 2H), 2.65 (t, J ) 7.7 Hz, 2H),
3.06-2.93 (m, 6H), 4.37 (m, 1H), 5.03 (m, 1H), 5.15 (s, 2H),
5.48 (m, 1H), 6.43 (d, J ) 7.2 Hz, 1H), 7.05-7.29 (m, 15H),
7.59 (br s, 1H), 7.96 (d, J ) 7.7 Hz, 1H), 8.67-8.73 (m, 2H).
13C NMR (125 MHz): δ 30.2, 31.3, 34.5, 38.1, 38.3, 38.8, 56.2,
57.1, 63.3, 125.9, 126.0, 127.1, 127.2, 128.38, 128.4, 128.6,
128.8, 129.1, 129.2, 135.8, 135.9, 140.0, 141.0, 144.8, 155.1,
170.2, 202.1. LCMS (EI): m/z 596.3 (M + 1)+; tr ) 14.09 min
(>99% purity) using solvent system I, tr ) 11.95 min (>99%
purity) using solvent system II.
1
16j: 60% yield. IR: 1663, 1715 cm-1. H NMR (500 MHz):
1
16d : 63% yield. IR: 1669, 1716 cm-1. H NMR (300 MHz):
δ 0.88-0.91 (m, 6H), 1.24 (s, 9H), 1.43-1.61 (m, 3H), 3.00-
3.04 (m, 1H), 3.09-3.13 (m, 1H), 3.25 (d, J ) 15.4 Hz, 1H),
3.30 (d, J ) 15.3 Hz, 1H), 4.10-4.16 (m, 1H), 5.15 (m, 1H),
5.20 (app s, 2H), 5.45 (d, J ) 7.7 Hz, 1H), 6.67 (d, J ) 7.1 Hz,
1H), 7.15-7.30 (m, 5H), 7.67 (m, 1H), 8.10 (d, J ) 7.9 Hz, 1H),
8.69 (s, 1H), 8.81 (s, 1H). 13C NMR (125 MHz): δ 21.8, 22.7,
24.6, 30.6, 37.77, 37.8, 41.4, 43.3, 53.7, 57.3, 63.1, 125.4, 127.1,
128.7, 129.1, 135.8, 140.8, 144.00, 144.01, 155.2, 171.1, 205.0.
LCMS (EI): m/z 500.3 (M + 1)+; tr ) 10.49 min (>99% purity)
using solvent system I, tr ) 8.48 min (95% purity) using solvent
system II.
δ 0.86-0.90 (m, 6H), 1.43-1.67 (m, 3H), 1.75-1.85 (m, 2H),
2.38 (t, J ) 7.3 Hz, 2H), 2.63 (t, J ) 7.6 Hz, 2H), 2.96-3.03
(m, 1H), 3.09-3.22 (m, 3H), 4.10-4.18 (m, 1H), 5.07 (app q, J
) 7.3 Hz, 1H), 5.20 (s, 2H), 5.59 (d, J ) 7.8 Hz, 1H), 6.79 (d,
J ) 7.7 Hz, 1H), 7.13-7.30 (m, 10H), 7.65-7.69 (m, 1H), 8.11
(d, J ) 7.9 Hz, 1H), 8.68 (br s, 1H), 8.83 (br s, 1H). 13C NMR
(125 MHz): δ 21.8, 22.7, 24.6, 30.3, 31.4, 34.5, 37.6, 39.0, 41.2,
53.8, 57.1, 63.2, 125.3, 126.0, 127.10, 127.11, 128.4, 128.6,
129.2, 135.0, 135.9, 140.5, 140.5, 141.1, 144.3, 155.3, 171.7,
202.7. LCMS (EI): m/z 562.3 (M + 1)+; tr ) 13.58 min (>99%
purity) using solvent system I, tr ) 11.69 min (>99% purity)
using solvent system II.
1
16k : 51% yield. IR: 1667, 1717 cm-1. H NMR (500 MHz):
δ 1.26 (s, 9H), 1.85 (m, 1H), 2.24 (m, 1H), 2.52 (m, 2H), 3.00-
3.11 (m, 2H), 3.29 (d, J ) 15.1 Hz, 1H), 3.33 (d, J ) 14.9 Hz,
1H), 4.42 (m, 1H), 4.94 (m, 1H), 5.14 (d, J ) 13.3 Hz, 1H),
5.18 (d, J ) 13.7 Hz, 1H), 5.54 (m, 1), 6.58 (d, J ) 6.8 Hz, 1H),
7.09-7.30 (m, 10H), 7.58 (m, 1H), 7.99 (d, J ) 7.4 Hz, 1H),
8.64 (s, 1H), 8.74 (s, 1H). 13C NMR (125 MHz): δ 30.6, 31.3,
33.0, 36.5, 38.3, 43.5, 56.3, 56.5, 63.2, 125.2, 126.3, 127.2, 128.3,
128.5, 128.8, 129.2, 134.9, 135.9, 140.4, 140.6, 144.4, 155.1,
170.4, 171.2, 204.8. LCMS (EI): m/z 548.3 (M + 1)+; tr ) 12.25
min (>99% purity) using solvent system I, tr )10.60 min (95%
purity) using solvent system II.
1
16e: 73% yield. IR: 1670, 1719 cm-1. H NMR (500 MHz):
δ 1.83-1.89 (m, 3H), 2.2 (m, 1H), 2.44 (t, J ) 7.2 Hz, 2H),
2.52 (m, 2H), 2.68 (t, J ) 7.5 Hz, 2H), 3.00-3.19 (m, 4H), 4.44
(m, 1H), 4.83 (m, 1H), 5.16 (s, 2H), 5.63 (m, 1H), 6.66 (d, J )
6.4 Hz, 1H), 7.08-7.28 (m, 15H), 7.56 (s, 1H), 8.00 (d, J ) 7.6
Hz, 1H), 8.26 (s, 1H), 8.75 (s, 1H). 13C NMR (125 MHz): δ
30.3, 31.5, 33.1, 34.5, 37.9, 38.3, 56.4, 56.4, 63.1, 125.4, 126.0,
126.3, 127.2, 128.3, 128.37, 128.38, 128.5, 128.8, 129.2, 135.1,
135.9, 140.3, 140.8, 141.1, 144.0, 155.2, 162.4, 170.6, 202.7.
LCMS (EI): m/z 610.3 (M + 1)+; tr ) 14.95 min (>99% purity)
using solvent system I, tr ) 12.34 min (>99% purity) using
solvent system II.
1
16l: 50% yield. IR: 1668, 1720 cm-1. H NMR (500 MHz):
δ 0.93 (m, 6H), 1.25 (s, 9H), 1.50-1.53 (m, 1H), 1.60-1.64 (m,
2H), 1.89 (m, 1H), 2.25 (m, 1H), 2.59 (m, 2H), 3.39 (m, 2H),
4.18 (m, 1H), 4.96 (m, 1H), 5.18 (d, J ) 13.4 Hz, 1H), 5.24 (d,
J ) 13.4 Hz, 1H), 5.52 (m, 1), 6.73 (d, J ) 7.2 Hz, 1H), 7.12-
7.33 (m, 5H), 7.59 (m, 1H), 8.07 (d, J ) 7.7 Hz, 1H), 8.63 (d, J
) 4.2 Hz, 1H), 8.80 (s, 1H). 13C NMR (125 MHz): δ 21.8, 22.8,
24.7, 30.6, 31.5, 33.2, 36.6, 41.3, 43.5, 53.8, 56.5, 63.1, 126.3,
128.4, 128.6, 129.9, 135.2, 140.6, 140.7, 143.9, 155.4, 171.8,
205.2. LCMS (EI): m/z 514.2 (M + 1)+; tr ) 11.69 min (>99%
purity) using solvent system I, tr ) 10.38 min (96% purity)
using solvent system II.
Gen er a l P r oced u r e for F or m a t ion of N-[[(n -P yr i-
d in yl)m eth oxy]ca r bon yl]a m in o Acid s. In a 250 mL round-
bottomed flask, 50 mL of CH2Cl2 and 50 mL of saturated
sodium bicarbonate were added to 5 mmol of the amino acid
ester hydrochloride. This suspension or solution was stirred
at 0 °C for 10 min. A solution of phosgene (1.93 M in toluene,
1
16f: 64% yield. IR: 1668, 1713 cm-1. H NMR (500 MHz):
δ 0.92-0.95 (m, 6H), 1.51-1.54 (m, 1H), 1.60-1.65 (m, 2H),
1.85-1.93 (m, 3H), 2.24 (m, 1H), 2.47 (t, J ) 7.2 Hz, 2H), 2.59
(t, J ) 7.6 Hz, 2H), 2.67 (t, J ) 7.4 Hz, 2H), 3.25 (d, J ) 14.5
Hz, 1H), 3.32 (d, J ) 14.5 Hz, 1H), 4.19 (m, 1H), 4.87 (m, 1H),
5.18-5.30 (m, 2H), 5.54 (s, 1H), 6.76 (d, J ) 6.1 Hz, 1H), 7.11-
7.28 (m, 10H), 7.59 (s, 1H), 8.09 (d, J ) 7.4 Hz, 1H), 8.62 (s,
1H), 8.82 (s, 1H). 13C NMR (125 MHz): δ 21.8, 22.8, 24.7, 30.3,
31.53, 31.54, 33.2, 34.5, 38.0, 41.2, 53.9, 56.4, 63.1, 126.0, 126.3,
128.4, 128.6, 132.6, 135.4, 140.6, 140.9, 141.1, 143.6, 143.7,
155.4, 171.9, 203.1. LCMS (EI): m/z 476.3 (M + 1)+; tr ) 14.62
min (>99% purity) using solvent system I, tr ) 12.15 min
(>99% purity) using solvent system II.
1
16g: 52% yield. IR: 1670, 1721 cm-1. H NMR (300 MHz):
δ 0.86 (d, J ) 6.3 Hz, 3H), 0.89 (d, J ) 6.4 Hz, 3H), 1.21 (s,
9H), 1.26-1.61 (m, 3H), 2.96 (dd, J ) 7.6, 14.0 Hz, 1H), 3.12

Identification of Mechanism-Based Inhibitors of Cruzain
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3 683
5.2 mL, 10 mmol) was added by syringe to the organic layer.
The solution was stirred an additional 20 min. After the
aqueous layer was extracted with CH2Cl2 (3 × 50 mL), the
extracts were dried over MgSO4, filtered, and concentrated in
vacuo to afford the crude isocyanate as a milky-white oil. A
solution of the hydroxymethylpyridine in 0.2 M of CH2Cl2 was
added to the crude isocyanate and stirred overnight. The
methyl ester was purified by silica gel chromatography to give
66-100% of the product ester. To hydrolyze the ester, a
solution of the phenylalanine methyl ester (2.5 g, 7.96 mmol)
in dioxane (29 mL, 0.27 M) was treated with aqueous LiOH
(32 mL, 0.5 M). After being stirred for 2h, the solution was
quenched with HCl (14 mL, 1.0 M). The solution was extracted
with EtOAc (3 × 25 mL), dried over MgSO4, and concentrated
to provide 55-69% of the desired N-protected amino acid as a
white solid.
for Alloc chloroformate. These amino acids were used without
purification in the solid-phase synthesis.
17a (Mu Leu OH): 24% yield. IR: 1643, 1662 cm-1. 1H NMR
(500 MHz, DMSO): δ 0.82 (d, J ) 6.5 Hz, 3H), 0.86 (d, J )
6.6 Hz, 3H), 1.43-1.62 (m, 3H), 3.11 (m, 2H), 3.21-3.30 (m,
2H), 3.48-3.54 (m, 4H), 4.00 (m, 1H), 6.50 (m, 1H). 13C NMR
(125 MHz, DMSO): δ 21.9, 23.5, 24.8, 44.4, 47.2, 66.30, 66.35,
158.1, 163.5. Anal. (C11H20N2O4) C, H, N.
17b (Mu P h eOH): 56% yield. IR: 1630, 1727 cm-1. 1H NMR
(300 MHz, DMSO): δ 2.83-2.91 (m, 1H), 3.00 (m, 1H), 3.09-
3.21 (m, 4H), 3.45 (m, 4H), 4.15-4.22 (m, 1H), 6.73 (d, J ) 8.2
Hz, 1H), 7.14-7.38 (m, 5H). 13C NMR (125 MHz, DMSO): δ
37.0, 44.3, 47.2, 55.8, 66.26, 66.29, 126.7, 128.5, 129.5, 138.8,
157.8, 174.6. Anal. (C14H18N2O4) C, H, N.
High Th r ou gh p u t Cr u za in Assa y. A fluorometric high
throughput assay for activity toward cruzain was performed
in 96-well microtiter plates. The assay was performed in
DYNATECH Microfluor fluorescence microtiter plates (opaque
white plates), and readings were taken on a Perkin-Elmer LS-
50B with an attached 96-well plate reader. The excitation
wavelength was 355 nm, and the emission wavelength was
450 nm. A 430 nm cutoff filter for emission was used. The
peptide substrate Z-Phe-Arg-AMC (Bachem, Km ) 1 µM)
concentration was 2.5 µM, and the cruzain concentration was
0.1 nM. The buffer consisted of a 100 mM solution of pH 5.5
sodium acetate buffer and 1 mM of DTT.
The fluorescent unit readings were taken at four time points
within the linear region of the substrate cleavage, and
percentage activity of the enzyme was determined by compar-
ing the change of fluorescent units (FU) for each well against
the average change in FU for eight control wells without
inhibitor. All compounds were assayed in duplicate.
Sample procedure: In each well was placed 25 µL of enzyme
solution, 125 µL of buffer solution, and 10 µL 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 preincu-
bation of the enzyme and inhibitor, 50 µL 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. Inhibitors were screened
at 1 µM, 330 nM, 110 nM, and 55 nM. Potent inhibitors were
screened in 2-fold dilutions from 55 nM to 1.7 nM.
15a (2-P yr P h eOH): 68% yield. IR: 1660, 3436 cm-1 1H
.
NMR (500 MHz, DMSO): δ 2.81-2.86 (m, 1H), 3.06-3.09 (m,
1H). 4.16-4.20 (m, 1H), 5.00 (s, 2H), 7.17-7.30 (m, 7H), 7.74-
7.78 (m, 2H), 8.49 (d, J ) 4.1 Hz, 1H). 13C NMR (125 MHz,
DMSO): δ 36.0, 56.0, 66.4, 121.3, 123.2, 126.8, 128.6, 129.5,
137.2, 138.3, 149.1, 156.2, 157.0, 173.4. LCMS (EI): m/z 301.1
(M + 1)+; tr ) 4.04 min (>99% purity) using solvent system I,
tr ) 0.84 min (>99% purity) using solvent system II.
15b (3-P yr P h eOH): 60% yield. IR: 1655, 3425 cm-1 1H
.
NMR (500 MHz, DMSO): δ 2.79-2.84 (m, 1H), 3.02-3.08 (m,
1H), 4.14-4.18 (m, 1H), 4.98 (d, J ) 12.9 Hz, 1H), 5.02 (d, J
) 12.8 Hz, 1H), 7.19-7.37 (m, 6H), 7.64-7.69 (m, 2H), 8.50
(s, 2H). 13C NMR (125 MHz, DMSO): δ 36.9, 56.0, 63.4, 123.9,
126.8, 128.6, 129.5, 133.0, 135.9, 138.3, 149.35, 149.43, 156.2,
173.7. Anal. (C16H16N2O4S) C, H, N.
15c (4-P yr P h eOH): 68% yield. IR: 1661, 1713, 3436 cm-1
.
1H NMR (500 MHz, DMSO): δ 2.84 (m, 1H), 3.10 (dd, J )
4.0, 14.0 Hz, 1H), 4.18-4.22 (m, 1H), 5.01 (s, 2H), 7.10-7.29
(m, 7H), 7.83 (d, J ) 8.6 Hz, 1H), 8.49-8.50 (m, 2H). 13C NMR
(125 MHz, DMSO): δ 36.9, 56.0, 66.8, 121.7, 126.8, 128.6,
129.5, 138.3, 146.8, 149.9, 156.1, 173.7. LCMS (EI): m/z 301.1
(M + 1)+; tr ) 3.91 min (95% purity) using solvent system I, tr
) 0.68 min (>99% purity) using solvent system II.
15d (2-P yr Leu OH): 69% yield. IR: 1655, 3425 cm-1 1H
.
NMR (500 MHz, DMSO): δ 0.84 (d, J ) 6.6 Hz, 3H), 0.88 (d,
J ) 6.6 Hz, 3H), 1.44-1.58 (m, 2H), 1.62-1.69 (m, 1H), 3.95-
4.00 (m, 1H), 5.07 (s, 2H), 7.29-7.31 (m, 1H), 7.37 (d, J ) 7.8
Hz, 1H), 7.69 (d, J ) 8.2 Hz, 1H), 7.80 (td, J ) 7.7, 1.8 Hz,
1H), 8.52 (m, 1H). 13C NMR (125 MHz, DMSO): δ 21.5, 23.3,
24.7, 52.7, 66.6, 121.5, 123.2, 137.3, 149.4, 156.4, 157.0, 174.8.
LCMS (EI): m/z 267.1 (M + 1)+; tr ) 3.88 min (>99% purity)
using solvent system I, tr ) 0.82 min (>99% purity) using
solvent system II.
In h ibit ion Assa y for Ca t h ep sin B a n d Ca t h ep sin L.
Continuous fluorometric inhibition assays for cathepsin B and
cathepsin L were performed as described in the literature.22
Cathepsin B (10 nM) and cathepsin L (6.9 nM) were assayed
at room temperature with the substrate Z-Phe-Arg-AMC (for
cathepsin B 10 µM, Km ) 220 µM; for cathepsin L 4 µM, Km
)
15e (3-P yr Leu OH): 55% yield. IR: 1651, 1712 cm-1 1H
.
2.9 µM). The assay is the same as performed above for cruzain,
except that the buffer was composed of 0.25 M solution of pH
5.5 sodium acetate buffer containing 2 mM EDTA, 0.015% Brij-
35, and 1 mM DTT.
NMR (500 MHz, DMSO): δ 0.83 (d, J ) 6.5 Hz, 3H), 0.86 (d,
J ) 6.6 Hz, 3H), 1.42-1.55 (m, 2H), 1.58-1.65 (m, 1H), 3.93-
3.98 (m, 1H), 5.05 (d, J ) 12.7 Hz, 1H), 5.08 (d, J ) 12.7 Hz,
1H), 7.38 (dd, J ) 4.8, 7.8 Hz, 1H), 7.62 (d, 1, J ) 8.2), 7.76 (d,
J ) 7.8 Hz, 1H), 8.51-8.56 (m, 2H). 13C NMR (125 MHz,
DMSO): δ 21.5, 23.3, 24.7, 52.6, 63.6, 123.9, 133.0, 136.0,
149.5, 149.5, 156.4, 174.7. LCMS (EI): m/z 267.1 (M + 1)+; tr
) 3.75 min (>99% purity) using solvent system I, tr ) 0.71
min (>99% purity) using solvent system II.
Ki Deter m in a tion . For potent inhibitors, the data was fit
by nonlinear regression analysis to the equation derived by
Williams and Morrison:11
vo
2
S
Km
S
Km
v )
Ki 1 +
+ It - Et + 4Ki 1 +
Et
-
{
x
15f (4-P yr Leu OH): 56% yield. IR: 1649, 3401 cm-1
.
1H
( (
)
)
[
(
)
[
]
2Et
NMR (500 MHz, DMSO): δ 0.84 (d, J ) 6.6 Hz, 3H), 0.88 (d,
J ) 6.6 Hz, 3H), 1.45-1.49 (m, 1H), 1.52-1.58 (m, 1H), 1.64-
1.67 (m, 1H), 3.94-4.00 (m, 1H), 5.05 (d, J ) 14.3 Hz, 1H),
5.10 (d, J ) 14.3 Hz, 1H), 7.26-7.35 (m, 2H), 7.73 (d, J ) 8.2
Hz, 1H), 8.53-8.54 (m, 2H). 13C NMR (125 MHz, DMSO): δ
21.5, 23.3, 24.7, 52.7, 64.0, 121.9, 146.7, 150.0, 156.3, 174.7.
LCMS (EI): m/z 267.1 (M + 1)+; tr ) 3.80 min (97% purity)
using solvent system I, tr ) 0.70 min (>99% purity) using
solvent system II.
S
Ki 1 +
+ It - Et
}
(
)
]
Km
For cruzain, the Km for the substrate was determined to be 1
µM by using a Lineweaver Burke Plot and Eadie-Hofstee Plot.
The variables S, Et, and It are the concentrations of substrate,
active enzyme, and inhibitor, respectively.
Ack n ow led gm en t. The authors acknowledge sup-
port from the NIH (Grant GM 54051) and the Bur-
roughs Wellcome Fund (New Initiatives in Malaria
Award). We are also grateful to Dr. J . H. McKerrow and
Gen er a l P r oced u r e for F or m a tion of N-Mor p h olin -
eca r bon yl Am in o Acid . These N-protected amino acids were
synthesized using a modified procedure by Stevens and
Watanabe.19 4-Morpholinecarbonyl chloride was substituted

684 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 3
Huang et al.
Med. Chem. 1995, 38, 3193-3196. (b) Roush, W. R.; Gwaltney,
S. L., II; Chen, J .; Scheidt, K. A.; McKerrow, J . H.; Hansell, E.
Vinyl sulfonate esters and vinyl sulfonamides: Potent, irrevers-
ible inhibitors of cysteine proteases. J . Am. Chem. Soc. 1998,
120, 10994-10995.
E. Hansell for providing cruzain and for their technical
assistance and insight in inhibitor design.
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J M010333M