Peptidyl allyl sulfones: A new class of inhibitors for clan CA cysteine proteases

Article abstract of DOI:10.1016/j.bmc.2004.07.016

A new series of peptidyl allyl sulfone inhibitors was discovered while trying to synthesize epoxy sulfone inhibitors from vinyl sulfones using basic oxidizing conditions. The various dipeptidyl allyl sulfones were evaluated with calpain I, papain, cathepsins B and L, cruzain and rhodesain and found to be potent inhibitors. In comparison to the previously developed class of vinyl sulfone inhibitors, the novel dipeptidyl allyl sulfones were more potent inhibitors than the corresponding dipeptidyl vinyl sulfones. It was observed that the stereochemistry of the vinyl sulfone precursor played a role in the potency of the dipeptidyl allyl sulfone inhibitor.

Full text of DOI:10.1016/j.bmc.2004.07.016

Bioorganic & Medicinal Chemistry 12 (2004) 5203–5211
Peptidyl allyl sulfones: a new class of inhibitors for clan CA
cysteine proteases
Marion G. Go¨tz,a Conor R. Caffrey,b Elizabeth Hansell,b James H. McKerrowb
and James C. Powersa,
*
aSchool of Chemistry and Biochemistry and the Petit Institute of Bioscience and Bioengineering, Georgia Institute of Technology,
Atlanta, GA 30332-0400, USA
bSandler Center for Basic Research in Parasitic Diseases, Box 0511, University of California San Francisco, San Francisco,
CA 94143, USA
Received 17 March 2004; revised 5 July 2004; accepted 7 July 2004
Available online 7 August 2004
Abstract—A new series of peptidyl allyl sulfone inhibitors was discovered while trying to synthesize epoxy sulfone inhibitors from
vinyl sulfones using basic oxidizing conditions. The various dipeptidyl allyl sulfones were evaluated with calpain I, papain, cathep-
sins B and L, cruzain and rhodesain and found to be potent inhibitors. In comparison to the previously developed class of vinyl
sulfone inhibitors, the novel dipeptidyl allyl sulfones were more potent inhibitors than the corresponding dipeptidyl vinyl sulfones.
It was observed that the stereochemistry of the vinyl sulfone precursor played a role in the potency of the dipeptidyl allyl sulfone
inhibitor.
ꢀ 2004 Elsevier Ltd. All rights reserved.
1. Introduction
Calpains are a family of cysteine proteases in clan CA,
which are activated by calcium. So far, eight homologs
of human calpain have been identified, two of which
have received most attention: calpain I (or l-calpain)
and calpain II (or m-calpain). Their activity in vitro is
dependent on a calcium concentration in micromolar
amounts for calpain I and millimolar amounts for cal-
pain II.3–6 Both forms are present in the cytosol of mam-
malian cells and are involved in physiological processes
that control the degradation of the cytoskeleton as well
as hormone receptors.7,8 Enhanced calpain activity has
been associated with cell injury due to ischemic stroke,9
physical damage,10 and hypoxia.11 Thus, calpains are
attractive targets for the development of inhibitors,
which should be useful for treatment of a variety of
diseases.
Proteases or proteolytic enzymes catalyze the hydrolysis
of a wide variety of peptides and proteins. Proteases can
be divided into classes of cysteine, serine, aspartate,
threonine, and metallopeptidases depending on the cat-
alytic nucleophile.1,2 Cysteine proteases employ a thio-
late residue, which performs a nucleophilic attack on
the amide bond of the peptide backbone to form a tetra-
hedral intermediate. The intermediate collapses to re-
lease the first product and the resulting acyl enzyme
then undergoes hydrolysis. Based on their sequence
homology, cysteine proteases are divided into several
clans and families. Clan CA and clan CD contain the
majority of cysteine proteases. Clan CD contains several
important enzymes including caspases, legumains, gingi-
pain and clostripain. The papain family of cysteine pro-
teases belongs to the clan CA and includes cathepsins,
calpains, and the parasite cysteine proteases cruzain
and rhodesain.
The clan CA proteases, cruzain and rhodesain, are
essential for the development and survival of the proto-
zoan parasites Trypansoma cruzi and T. brucei, respec-
tively. T. cruzi causes Chagasꢀ disease in humans in
South and Central America, whereas T. brucei causes
sleeping sickness in humans in large areas of central
and southern Africa. Current drug therapies are accom-
panied by serious side effects and widespread resistance.
Thus, the need of new medicinal agents is urgent.12,13
Keywords: Cysteine protease inhibitor; Vinyl sulfone; Cathepsins;
Cruzain.
*
Corresponding author. Tel.: +1-404-894-4038; fax: +1-404-894-
2295; e-mail: james.powers@chemistry.gatech.edu
0968-0896/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.07.016

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M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
Cathepsins comprise a large family of lysosomal cysteine
proteases and are involved in the degradation of host
connective tissues, the generation of bioactive proteins
and antigen processing.14,15 They have been implicated
in a variety of disease states such as rheumatoid arthri-
tis,16 muscular dystrophy,17,18 and tumor metastasis.19
The high similarity in the substrate specificity among
the individual cathepsins presents a challenge in finding
selective and thus effective cathepsin inhibitors.
acid 5 using standard mixed anhydride conditions fur-
nished the dipeptidyl vinyl sulfone precursors 6.
In order to synthesize the epoxy sulfone, we decided to
epoxidize the double bond moiety in the peptidyl vinyl
sulfone precursor using basic peroxidizing conditions.
When we tried to epoxidize the vinyl sulfone using tBuLi
and nBuOOH in freshly distilled THF at ꢀ78ꢁC, the
formation of a new compound was observed by TLC.
The vinyl sulfone starting material was consumed within
20min. The new compound was purified by column
chromatography and carefully characterized using sev-
eral spectroscopic methods. We found that the conju-
gated double bond of the vinyl sulfone moiety was no
Clan CA cysteine proteases are effectively inhibited by
several classes of peptidyl inhibitors including transition
state, reversible and irreversible inhibitors.20 Examples
of reversible transition state inhibitors are peptide alde-
hydes,21–24 a-diketones,25–28 a-ketoesters,29,30 a-keto-
amides,29,31 and a-ketoacids.29 Clan CA proteases are
also irreversibly inhibited by peptidyl diazomethyl
ketones and fluoromethyl ketones,32,33 peptidyl epoxides
(E-64, E-64-c, E-64-d),34 and vinyl sulfones.35 However,
the potency and specificity of many of the irreversible
inhibitors could be improved.
1
longer present in the H NMR. However, according to
the mass spectrum (MS), the molecular weight of the
newly formed product was unchanged. Elemental analy-
sis also confirmed that the new compound was a consti-
tutional isomer of the vinyl sulfone starting material.
The characteristic sulfone stretch was still present in
the FT-IR spectrum. Since the new compound was
formed by isomerization and not oxidation, the critical
reagent was the tBuLi base and not the oxidizing nBu-
OOH. Treatment of the vinyl sulfone with nBuLi with-
out the peroxide tBuOOH gave the same product.
However, the reaction was much more sluggish, and
required several hours at room temperature.
In order to develop new functional groups suitable for
irreversible inhibition of cysteine proteases, we at-
tempted the synthesis of peptidyl epoxy sulfones. The
design of the peptidyl epoxy sulfone structure was based
on vinyl sulfones, which are a class of promising irre-
versible peptidyl inhibitors for clan CA cysteine prote-
ases.35 Vinyl sulfones contain a Michael acceptor
functional group and react irreversibly with the active
site cysteine. Another widely used warhead in cysteine
protease inhibitors is the epoxide moiety of the natural
product inhibitor E-64. We recently reported aza-pep-
tide epoxides as potent and selective inhibitors for clan
CD cysteine proteases including caspases and legu-
mains.36,37 Here, we propose that by combining the
reactivity of the E-64 epoxide electrophile with the selec-
tivity of the vinyl sulfone moiety will result in both a po-
tent and specific new class of inhibitors for cysteine
proteases. We initially focused on the clan CA cysteine
proteases calpain and papain, and thus decided to attach
this novel warhead to an optimal dipeptide sequence for
effective calpain and papain inhibition. We chose the
Leu-Phe, Ala-Phe, and Val-Phe sequences with the N-
terminal Cbz-protecting group (Cbz = Ph–CH2–OCO–,
benzyloxycarbonyl).25
We considered several possible isomeric structures for
the nBuLi reaction product (Scheme 2). Two likely pos-
sibilities were a substituted oxazoline (7d) formed by
cyclization or an allyl sulfone (AS), with the double
bond isomerized toward the adjacent a-carbon. Oxazo-
lines have characteristic 13C NMR signals at 170ppm
for the C-2 carbon atom.39 Oxazolines have previously
been synthesized, and we prepared the parent compound
11 following the synthetic method described by Wipf
and Miller.39 This oxazoline could potentially acylate
the active site cysteine of a cysteine protease, but the
1H NMR spectrum was not identical to our isomeriza-
tion product. The 13C NMR spectrum of our product
has a new peak at 106ppm and not one at 170ppm
for the oxazoline. In addition, we observed the presence
of an NH in the 1H NMR. This ruled out the formation
of the oxazoline in the isomerization reaction.
The allyl sulfone structure (7a–c) was supported by all
the spectroscopic data. The methylene of the phenylala-
nine side chain shifts substantially from 2.9 to 3.8ppm in
the 1H NMR spectrum, and the shift is explained by the
close proximity to the double bond in the newly formed
isomerization product. The distinct triplet at 4.9ppm is
consistent with a vinylogous proton. Upon decoupling
the vinyl proton triplet at 4.9ppm, the methylene group
next to the sulfone moiety at 3.83ppm appears as a sin-
glet, indicating that the protons involved are located on
adjacent carbons. Supporting information was also pro-
vided by a series of COSY (correlation spectroscopy)
and NOESY (nuclear Overhauser effect spectroscopy)
experiments for Cbz-Val-Phe-AS-Ph (7b, Fig. 1). We
have determined NOE (nuclear Overhauser effect) corre-
lations between the vinyl proton and the ortho ring pro-
tons of the phenylalanine side chain. These results
2. Results and discussion
2.1. Synthesis
Our strategy for the synthesis of epoxy sulfones began
with the synthesis of the appropriate peptidyl vinyl sul-
fone (Scheme 1). Vinyl sulfones have been previously
synthesized and prepared by coupling the chiral amino
acid aldehydes 1 and the corresponding sulfonyl phos-
phonate 2 using Wadsworth–Emmons chemistry.35
The stereochemistry of the vinyl sulfone is primarily E,
but the presence of small amounts of the Z isomer can-
not be excluded.38 The Boc-group (Boc = butyloxycar-
bonyl) was then removed using 4N HCl in ethyl
acetate. Coupling of the salt 4 to a Cbz-protected amino

M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
5205
Scheme 1. Synthesis of dipeptidyl vinyl sulfones.
Scheme 2. Isomerization of dipeptidyl vinyl sulfones to allyl sulfones.
indicate that the allyl sulfones derived from the L-Phe
vinyl sulfone precursor assume Z stereochemistry after
isomerization. It has previously been reported that treat-
ment of vinyl sulfones with strong base results in the for-
mation of allyl sulfones (Scheme 2), as allyl sulfones are
thermodynamically more stable than vinyl sulfones.40,41
The stereochemistry of the isomerized double bond was
not determined, however, according to the 1H NMR, we
are certain that only one isomer is present. Both E and Z
or an E/Z mixture have been obtained upon base cata-
lyzed isomerization of vinyl sulfones.42,43
methylcoumarin) as the fluorogenic substrate.44 Cathep-
sin B was tested using commercially available enzyme
and Cbz-Arg-Arg-AMC as the substrate.45 The sub-
strate Cbz-Phe-Arg-AMC was used with the cathepsin
L inhibition assays.46 Papain inhibition was measured
using commercially available papain and Cbz-Arg-Phe-
pNA (pNA = para-nitroaniline) as the substrate.47 The
dipeptidyl allyl sulfones do not inhibit cathepsin B and
show limited rates with both calpain I and papain.
In contrast are the striking second-order inhibition
rate constant (kobs/[I]) values of 564Mꢀ1 sꢀ1 and
1062Mꢀ1 sꢀ1 for Cbz-Leu-Phe-AS-Ph (isomer B) with
calpain I and cathepsin L, respectively. This dipeptidyl
allyl sulfone was synthesized from the dipeptidyl vinyl
sulfone precursor Cbz-Leu-D-Phe-VS-Ph. When the
double bond isomerizes, the original Z stereochemistry
of the vinyl sulfone is lost and the product has an
unknown ratio of E to Z isomers at the new allyl double
bond. There are several factors, which can contribute to
a mixed ratio of isomers, such as the so-called ꢁsyn-
effectꢀ42,43 and the chelating lithium metal. This explains
the difference in inhibitor potency between Cbz-Leu-
Phe-AS-Ph (isomer A), which is derived from the
vinyl sulfone precursor Cbz-Leu-L-Phe-VS-Ph, and
2.2. Inhibition of clan CA cysteine proteases
The novel, newly synthesized peptidyl allyl sulfones had
the potential of being cysteine protease inhibitors and
were tested along with the oxazoline 11 with papain, cal-
pain I, cathepsin B and L, cruzain and rhodesain (Table
1). This new class of compounds shows moderate inacti-
vation rates with calpain and papain, and faster rates of
inactivation with rhodesain and cruzain. The oxazoline
was also a slow inhibitor of calpain I. Kinetic inhibition
assays were performed using calpain I from bovine ery-
throcytes and Suc-Leu-Tyr-AMC (AMC = 7-amino-4-

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M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
the allyl sulfone inhibitor. The difference in potency of
the two isomers is also observed with inhibition of cruz-
ain and rhodesain. However, in this case the ranking is
reversed. The Cbz-Leu-Phe-AS-Ph (isomer A), which
is derived from the vinyl sulfone precursor Cbz-Leu-L-
Phe-VS-Ph, is more potent (IC50 = 0.06lM for cruzain
and 0.04lM for rhodesain) than isomer B (IC50
=
0.3lM for cruzain and 0.18lM for rhodesain).
We were surprised to find the novel allyl sulfone inhibi-
tors Cbz-Leu-Phe-AS-Ph (both isomers) to be more po-
tent than the corresponding vinyl sulfone Cbz-Leu-Phe-
VS-Ph. Vinyl sulfones have so far been the most effective
inhibitors for cruzain and rhodesain.48 It has been
shown that the peptide sequence and the nature of the
substituent on the prime side of the vinyl sulfone func-
tionality plays an important role in the potency of the
vinyl sulfone inhibitor towards the target enzyme.35,49
Using extensive SAR studies, vinyl sulfones have been
improved in potency by factors of 103. Therefore, it is
likely that with further modifications of the peptide
sequence and the P0 sulfonyl substituent, this new class
of dipeptidyl allyl sulfone inhibitors has the potential
of becoming as effective a class of inhibitors as the vinyl
sulfones.
Figure 1. NOESY spectrum of Cbz-Val-Phe-AS-Ph (7b) at 500MHz in
CDCl3 showing the correlation of the vinyl proton at 4.90ppm with
the ortho ring proton of the Phe side chain at 7.20ppm. We have
designated the allyl sulfone ꢁASꢀ. Thus, an allyl sulfone derivative will
be abbreviated AA-AS-R, where R is the phenyl substituent on the
sulfone functional group; Cbz = Ph–CH2–OCO–.
One mechanism for the inhibition reaction could involve
re-isomerization of the peptidyl allyl sulfone to the vinyl
sulfone. This isomerization could be catalyzed by the
active site histidine and would be reflected by a higher
inhibition rate (kobs/[I]) for the parent vinyl sulfone
inhibitor. However, as the parent vinyl sulfone Cbz-
Leu-Phe-VS-Ph was less potent with papain (kobs
/
Cbz-Leu-Phe-AS-Ph (isomer B). The initial stereochem-
istry of the phenylalanine side chain probably plays a
significant role in the final ratio of Z to E isomers in
[I] = 10Mꢀ1 sꢀ1) than the corresponding allyl sulfone
Cbz-Leu-Phe-AS-Ph (isomer A) (kobs/[I] = 49Mꢀ1 sꢀ1),
we conclude that the mechanism of inhibition does not
Table 1. Inhibition of various cysteine proteases by dipeptidyl allyl sulfones
Inhibitor
kobs/[I] (Mꢀ1 sꢀ1 a
)
IC50 (lM)b
Cruzain Rhodesain
Calpain Ic
Papaind
Cathepsin Be
Cathepsin Le
7a
7b
7c
8
Cbz-Ala-Phe-AS-Ph
3
3
0
0
4
9
6
0
1
6
1
0
N.I.f
N.I.
N.I.
N.I.
N.I.
183 10g
310 52g
700 43g
1060 121g
219 96g
>10
6
>10
5
Cbz-Val-Phe-AS-Ph
Cbz-Leu-Phe-AS-Ph (isomer A)h
Cbz-Leu-Phe-AS-Ph (isomer B)i
Cbz-Leu-Phe-VS-Ph
23
49
15
10
0.06
0.3
2
0.04
0.18
0.5
564 32g
550 2g
6c
Z-Leu
O
11
13
1
N.I.
N.I.
N.I.
>10
>10
N
H
N
a kobs is the pseudo-first-order rate constant obtained from plots of ln vt/v0 versus time unless indicated otherwise.
b Cruzain (2nM) or rhodesain (3nM) was incubated with 0.0001 to 1lM inhibitor in 100mM sodium acetate buffer, pH5.5 and 5mM DTT (buffer
A), for 5min at room temperature prior to substrate addition.
c Calpain I assay conditions: Irreversible kinetic assays were performed by the incubation method with calpain I from porcine erythrocytes.
Enzymatic activities of calpain I were measured at 23ꢁC in 50mM Hepes buffer (pH7.5) containing 10mM cysteine and 5mM CaCl2.
d Papain assay conditions: Incubation kinetics were measured using an enzyme stock solution for the papain assays, which was freshly prepared from
330lL of enzyme storage solution (1.19mg/mL) diluted with 645lL papain buffer (50mM Hepes, and 2.5mM EDTA at pH7.5) and 25lL of DTT
(0.1M).
e Cathepsin B and L assay conditions: Enzymatic activities of cathepsin B and L were measured in 0.1M KHPO4, 1.25mM EDTA, 0.01% Brij, pH6.0
buffer and at 23ꢁC.
f N.I.=no inhibition after 20min of incubation.
g kobs is obtained by the progress curve method and corrected for substrate.
h Allyl sulfone is derived from the L-isomer of phenylalanine.
i Allyl sulfone is derived from the D-isomer of phenylalanine.

M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
5207
Scheme 3. Proposed mechanisms of inhibition of cysteine proteases by allyl sulfones.
involve re-isomerization of the allyl sulfone to the vinyl
sulfone with a subsequent attack of the cysteine thiol on
the Michael acceptor double bond. This was even more
pronounced with cruzain and rhodesain, where the same
vinyl sulfone was over 30- and 12-fold less potent with
cruzain (IC50 = 2lM) and rhodesain (IC50 = 0.5lM),
respectively, than the corresponding allyl sulfone Cbz-
Leu-Phe-AS-Ph. With calpain I it is possible that the
isomerization mechanism is occurring, since the vinyl
sulfone is a much more effective inhibitor of calpain.
zation of the double bond. This new class of inhibitors
contains a novel cysteine protease warhead and shows
moderate rates of inactivation of calpain, papain and
cathepsin L. The inhibitors are potent inactivators of
the parasitic proteases cruzain and rhodesain.
We observed that the allyl sulfones were generally more
effective inhibitors in comparison to the vinyl sulfone
precursor. We have not yet determined the optimum
sequences for the peptidyl allyl sulfones. Therefore, we
would expect substantial improvement in the potency
of allyl sulfones using an SAR study in order to deter-
mine the optimum peptide sequence and the nature of
the prime side substituent. However, the two classes of
inhibitors have different inhibition mechanisms and dif-
ferent points of attack. Therefore, it remains to be seen
whether the peptidyl allyl sulfones can be improved as
much as peptidyl vinyl sulfones, which have been highly
optimized by SAR studies.
Two other mechanisms can be envisaged for enzyme
inhibition by allyl sulfones (Scheme 3). The active site
cysteine could directly displace phenyl sulfinic acid in
an SN2 reaction (pathway b) or could attack the allylic
double bond with loss of phenyl sulfinic acid (pathway
a). Both pathways result in alkylation of the active site
cysteine residue. It has previously been reported that
allyl sulfones when reacted with nucleophiles undergo
a tosyl elimination or displacement process, in which
the allyl sulfone moiety undergoes alkylation at either
the a- or c-carbon.41,50,51 An alternative possibility for
enzyme inactivation follows a mechanism-based path-
way, which is initiated by removal of the amide nitrogen
proton by a base or the active site histidine, followed by
the elimination of phenyl sulfinic acid and the formation
of the imine 9. The active site cysteine then reacts with 9
in a Michael addition, which also irreversibly alkylates
the enzyme. Several PLP-dependent enzymes (PLP =
pyridoxal phosphate) are irreversibly inhibited by
mechanism-based inactivators, where the pathway also
follows displacement and b-elimination reactions. A
vinylic imine forms as an intermediate before undergo-
ing attack by a nucleophile in the active site of the
enzyme.52
We envision achievement of even greater potency with
both cruzain and rhodesain by attaching the allyl sul-
fone warhead to a cruzain and rhodesain specific peptide
sequence in combination with the optimization of the P0
sulfonyl substituent. It would be of interest to extend
this warhead to other cysteine proteases and investigate
the effect of the stereochemistry of the double bond on
the potency of the inhibitor. It would also be of interest
to determine the mechanism of inhibition since allyl sulf-
ones are likely mechanism-based inhibitors.
4. Experimental
4.1. Materials and methods
Materials were obtained from Acros, Bachem Biosci-
ence Inc., or Sigma Aldrich and used without further
purification. The purity of each compound was con-
3. Conclusions
1
In summary, we report the synthesis of a new class
of inhibitors, peptidyl allyl sulfones. In an effort to syn-
thesize epoxy sulfone inhibitors, allyl sulfones were
unexpectedly produced from vinyl sulfones by isomeri-
firmed by TLC, H NMR, MS, and elemental analysis.
Chemical shifts are reported in ppm relative to an inter-
nal standard (trimethylsilane). Thin layer chromatogra-
phy (TLC) was performed on Sorbent Technologies

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M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
(250lm) silica gel plates. The 1H NMR spectra were ob-
tained on a Varian Mercury 400MHz spectrometer. The
NOESY and COSY spectra were obtained on a Bruker
DRX 500MHz spectrometer. Electrospray ionization
(ESI), fast-atom-bombardment (FAB) and high-resolu-
tion mass spectrometry were obtained using Micromass
Quattro LC and VG Analytical 70-SE instruments. Ele-
mental analysis was carried out by Atlantic Microlab
Inc., Norcross, GA.
solution (16mM) in DMSO (2% final concentration).
Activity was monitored by following the change in fluo-
rescence for 20min at 465nm. The kobs values were ob-
tained by nonlinear regression analysis and corrected for
substrate (1 + [S]/KM = 1.274). All assays were run in
duplicate and the standard deviation was determined.
4.1.4. Cruzain and rhodesain IC50 determinations. Inhib-
itors were screened for effectiveness against purified re-
combinant cruzain53 and rhodesain.48 Cruzain (2nM)
or rhodesain (3nM) was incubated with 0.0001 to 1lM
inhibitor in 100mM sodium acetate buffer, pH5.5 and
5mM DTT (buffer A), for 5min at room temperature.
4.1.1. Papain and cathepsin B assays. The incubation
method was used to measure the irreversible inhibition
of papain and cathepsin B. With cathepsin B, 30lL of
a stock inhibitor solution was added to 300lL of a
0.1M potassium phosphate buffer containing 1.25mM
EDTA, 0.01% Brij 35 at pH6.0, followed by the addi-
tion of 30lL of a freshly prepared cathepsin B solution
(approximate concentration 6.98 · 10ꢀ3 lg/lL) in
the same potassium phosphate buffer containing 1mM
DTT (freshly prepared). Aliquots (50lL) from the inhi-
bition mixture were withdrawn at various time intervals
and added to 200lL of a 0.1M potassium phosphate
buffer containing 1.25mM EDTA, 0.01% Brij 35 at
pH6.0, and the substrate Cbz-Arg-Arg-AMC
(499lM). The release of 7-amino-4-methylcoumarin
was monitored (kex = 360nm, kem = 465nm) using a
Tecan Spectra Fluor microplate reader. Pseudo first-
order inactivation rate constants were obtained from
plots of ln vt/vo versus time.
Buffer
A containing Cbz-Phe-Arg-AMC (Bachem,
KM = 1lM, AMC = 7-amino-4-methylcoumarin) was
added to the enzyme and inhibitor to give 20 or 10lM
substrate concentration for cruzain and rhodesain,
respectively, in 200lL. The increase in fluorescence
(excitation at 355nm and emission at 460nm) was fol-
lowed with an automated microtiter plate spectrofluo-
rimeter (Molecular Devices, Flex Station). Inhibitor
stock solutions were prepared at 20mM in DMSO and
serial dilutions were made in DMSO (0.7% DMSO in
assay). Controls were performed using enzyme alone,
and enzyme with DMSO. IC50 values were determined
graphically using inhibitor concentrations in the linear
portion of a plot of enzyme activity versus log[I] (7 con-
centrations tested with at least 2 in the linear range).
4.1.5. Diethyl phenylsulfonylmethanephosphonate (2,
PhSO2CH2PO(OEt)2). A mixture of chloromethyl
The incubation method was also used for commercially
available papain. The inhibition incubation buffer for
papain was 50mM Hepes buffer at pH7.5, containing
2.5mM DTT (dithiothreitol) and 2.5mM EDTA. The
assay used the substrate Cbz-Phe-Arg-pNA (53.7lM)
in the same buffer. The approximate concentration of
papain added to the incubation buffer was 0.29mg/
mL. The release of p-nitroanilide was monitored at
405nm with a Molecular Devices Thermomax micro-
plate reader. All assays were run in duplicate.
phenyl sulfide (3.2g, 20mmol) and triethyl phosphite
(3.3g, 20mmol) was heated at 130–140ꢁC for 5h. The
resultant mixture was distilled under reduced pressure,
and the starting materials were distilled out. The oily
residue was diethyl phenylmercaptomethanephospho-
1
nate, yield 3.4 g (65%). H NMR (CDCl3) d 1.2–1.3 (t,
6H, 2 · CH3), 3.1–3.2 (d, 2H, S–CH2), 4.0–4.2 (m, 4H,
2 · CH2), 7.2–7.4 (m, 5H, Ph). Diethyl phen-
ylsulfonylmethanephosphonate was prepared by oxida-
tion of diethyl phenylmercaptomethanephosphonate
with potassium permanganate, yield 55%. 1H NMR
(CDCl3) d 1.2–1.3 (t, 6H, 2 · CH3), 3.7–3.8 (d, 2H,
SO2–CH2), 4.1–4.2 (m, 4H, 2 · CH2), 7.5–7.7 (m, 3H,
Ph), 7.9–8.0 (d, 2H, Ph). MS m/z 293 (M+1).
4.1.2. Calpain assays. Calpain I was purchased from
Calbiochem (La Jolla, CA) in a solution of 30% glycerol
at a concentration of 6.96lM and stored at ꢀ20ꢁC prior
to use. The calpain I assay was conducted with 235lL of
a solution of 50mM Hepes, 0.5M CaCl2, 0.5M cysteine,
at pH7.5 (calpain I buffer), 6.5lL of Suc-Leu-Tyr-AMC
substrate solution in DMSO and 4.2lL of the enzyme
solution (6.96lM) at 23ꢁC. The enzymatic activity was
monitored by following the change in fluorescence for
10min at 465nm. The k2 values were obtained by non-
linear regression analysis and corrected for substrate
(1 + [S]/KM = 1.274). All assays were run in duplicate
and the standard deviation was determined.
4.1.6. General procedure for mixed anhydride coupling. 2-
(tert-butoxycarbonylamino)-3-phenylpropionaldehyde (1,
Boc-Phe-H). N-Methylmorpholine (2mmol) was added
to Boc-Phe-OH in CH2Cl2 at ꢀ15ꢁC followed by isobu-
tyl chloroformate. N-Methylmorpholine (2mmol) was
added to a cooled solution (ꢀ15ꢁC) of N,O-dim-
ethylhydroxylamine hydrochloride (2mmol) in CH2Cl2.
This solution was added to the Boc-Phe-OH mixture,
which had been stirring at ꢀ15ꢁC. The mixture was con-
tinued to stir at ꢀ15ꢁC for 30min, then warmed to room
temperature and continued to stir over night. The
amount of solvent was doubled, then washed with citric
acid (10%, 3 · 50mL), saturated NaHCO3 (3 · 50mL),
and brine (3 · 50mL), and finally dried (MgSO4). The
solvent was evaporated to give Boc-Phe-N(OCH3)CH3
4.1.3. Cathepsin L assays. Cathepsin L was purchased
from Athens Research Technology (Athens GA) in a
20mM malonate buffer solution at pH5.5 with 1mM
EDTA and 400mM NaCl with a specific activity of
4.133U/mg. The progress of cathepsin L inhibition
was conducted with 243lL of a solution of 0.25M
NaAc, 2mM EDTA, 0.015% Brij, 5mM DTT (dithio-
threitol) at pH5.5, 4lL of Cbz-Phe-Arg-AMC substrate
1
as a pure white solid in good yield (93%). H NMR
(CDCl3) d 1.4 (s, 9H, Boc), 2.8–2.9 (m, 1H, CH2–Phe),

M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
5209
3.0–3.1 (m, 1H, CH2–Phe), 3.2 (s, 3H, N-CH3), 3.6 (s,
3H, O–CH3), 4.9 (m, 1H, a-H), 5.2 (b, 1H, NH), 7.1–
7.3 (m, 5H, Ph). MS (FAB+) m/z 309 (M+1, 30%), 253
Leu-Phe-VS-Ph (3.00g, 5.61mmol) in dry THF (30mL)
was added dropwise. The reaction was continued to stir
at ꢀ20ꢁC for 45min (TLC hexane/EtOAc 1:1). The
reaction mixture was quenched with saturated aqueous
ammonium chloride (50mL) and allowed to warm to
room temperature. The organic layer was separated,
and the aqueous layer was extracted with ethyl acetate
(3 · 30mL). The extracts were then washed with aque-
ous sodium sulfite (10%, 3 · 20mL). The combined or-
ganic layers were dried (MgSO4) and evaporated to
give Cbz-Leu-Phe-AS-Ph as a white powder, yield
63%. 1H NMR (CDCl3) d 0.8–0.9 (2d, 6H, 2 · Leu–
CH3), 1.4–1.6 (m, 2H, Leu–CH2), 3.8 (s, 2H, CH2–
Phe), 3.9 (d, 2H, CH2–SO2), 4.1 (m, 1H, a-H), 4.8 (t,
1H, CH@), 4.9–5.0 (b, 1H, NH), 5.1 (m, 2H, Cbz),
7.1–7.4 (m, 10H, 2 · Ph), 7.5–7.7 (m, 5H, SO2–Ph), 8.4
(b, 1H, NH). 13C NMR (400MHz, CDCl3) d 172.1,
156.3, 144.4, 140.8, 135.7, 134.2, 133.6, 131.2, 129.5,
129.4 x 2, 129.3, 128.8 · 2, 128.7, 128.6, 128.4, 128.3,
127.8, 127.3, 126.9, 106.3, 67.6, 56.0, 55.6, 41.7, 40.4,
22.1, 21.0, 18.4. MS (FAB+) m/z 535 (M+1, 100%).
Anal. Calcd for C30H34N2O5S: C, 67.39; H, 6.56; N,
5.43. Found: C, 67.41; H, 6.56; N, 5.43.
(MꢀtBu+1,
100%).
Reduction
of
Boc-Phe-
N(OCH3)CH3 with lithium aluminum hydride accord-
ing to a previously described method54 gave Boc-Phe-
1
H, yield 88%. H NMR (CDCl3) d 1.4 (s, 9H, Boc),
3.1 (d, 2H, CH2–Phe), 4.4 (m, 1H, a-H), 5.0 (b, 1H,
NH), 7.1–7.3 (m, 5H, Ph), 9.6 (s, 1H, CHO). MS
(FAB+) m/z 250 (M+1, 15%), 150 (MꢀBoc+1, 100%).
4.1.7. Phenyl (3S)-3-amino-4-phenylbut-1-enyl sulfone
hydrochloride (4, Phe-VS-Ph Æ HCl). Boc-Phe-VS-Ph
was prepared by reaction of Boc-Phe-H with diethyl
phenylsulfonylmethanephosphonate in the presence of
1
2N sodium methoxide, yield 85%. H NMR (CDCl3)
1.3–1.4 (s, 9H, Boc), 2.9 (d, 2H, CH2–Phe), 4.4–4.5 (b,
1H, a-H), 4.6–4.7 (b, 1H, NH), 6.3 (d, 1H, CH@),
6.9–7.0 (dd, 1H, CH@), 7.1–7.3 (m, 5H, Ph), 7.5–7.8
(m, 5H, SO2–Ph). MS (FAB+) m/z 388 (M+1, 15%),
288 (MꢀBoc+1, 100%). Boc-Phe-VS-Ph was deblocked
with 6.7N HCl in EtOAc to give Phe-VS-Ph Æ HCl, yield
88%. 1H NMR (DMSO-d6) d 2.9–3.0 (m, 1H, CH2–Phe),
3.1–3.2 (m, 1H, CH2–Phe), 4.2 (b, 1H, a-H), 6.7–6.8 (m,
2H, CH@), 7.1–7.3 (m, 6H, CH@ and Ph), 7.6–7.8 (m,
5H, SO2–Ph), 8.6–8.8 (b, 2H, NH2). MS (FAB+) m/z
288 (MꢀCl, 100%).
4.1.11. Phenyl (3R)-3-(N-carbobenzyloxyleucyl)amino-4-
phenylbut-1-enyl sulfone (6c, Cbz-Leu-D-Phe-VS-Ph).
Cbz-Leu-D-Phe-VS-Ph was prepared from Cbz-Leu-
OH and D-Phe-VS-Ph Æ HCl using standard mixed
anhydride coupling method, yield 81%. 1H NMR
(CDCl3) d 0.8–0.9 (2d, 6H, 2 · Leu–CH3), 1.4–1.6 (m,
2H, Leu–CH2), 2.06 (m, 1H, Leu–CH), 2.9–3.0 (m,
2H, CH2–Phe), 3.9–4.0 (m, 1H, a-H), 4.8–4.9 (b, 1H,
a-H), 4.9–5.0 (m, 1H, NH), 5.1 (m, 2H, Cbz), 6.3–6.4
(d and b, 2H, NH and CH@), 6.9–7.0 (dd, 1H, CH@),
7.1–7.4 (m, 10H, 2 · Ph), 7.5–7.7 (m, 5H, SO2–Ph).
MS (ESI) m/z 535.
4.1.8. Phenyl (3R)-3-amino-4-phenylbut-1-enyl sulfone
hydrochloride (D-Phe-VS-Ph Æ HCl). Boc-D-Phe-VS-Ph
was prepared by reaction of Boc-D-Phe-H with diethyl
phenylsulfonylmethanephosphonate in the presence of
1
2N sodium methoxide, yield 85%. H NMR (CDCl3)
1.3–1.4 (s, 9H, Boc), 2.9 (d, 2H, CH2–Phe), 4.4–4.5 (b,
1H, a-H), 4.6–4.7 (b, 1H, NH), 6.3 (d, 1H, CH@),
6.9–7.0 (dd, 1H, CH@), 7.1–7.3 (m, 5H, Ph), 7.5–7.8
(m, 5H, SO2–Ph). MS (FAB+) m/z 388 (M+1, 15%),
288 (MꢀBoc+1, 100%). Boc-D-Phe-VS-Ph was de-
blocked with 6.7N HCl in EtOAc to give D-Phe-VS-
Ph Æ HCl, yield 88%. 1H NMR (DMSO-d6) d 2.9–3.0
(m, 1H, CH2–Phe), 3.1–3.2 (m, 1H), 4.2 (b, 1H, a-H),
6.7–6.8 (m, 2H, CH@), 7.1–7.3 (m, 6H, CH@ and Ph),
7.6–7.8 (m, 5H, SO2–Ph), 8.6–8.8 (b, 2H, NH2). MS
(FAB+) m/z 288 (MꢀCl, 100%).
4.1.12. Phenyl 3-(N-carbobenzyloxyleucyl)amino-4-phen-
ylbut-2-enyl sulfone (7c, Cbz-Leu-Phe-AS-Ph) (isomer
B). Cbz-Leu-D-Phe-VS-Ph was treated with butyllithium
and tert-butylhydroperoxide in freshly distilled THF as
described with Cbz-Leu-Phe-VS-Ph above to give Cbz-
Leu-Phe-AS-Ph (isomer B) as a white powder, yield
15%. 1H NMR (CDCl3) d 0.8–0.9 (2d, 6H, 2 · Leu–
CH3), 1.4–1.6 (m, 2H, Leu–CH2), 2.06 (m, 1H, Leu–
CH), 3.8 (s, 2H, CH2–Phe), 3.9 (d, 2H, CH2–SO2), 4.1
(m, 1H, a-H), 4.8 (t, 1H, CH@), 4.9–5.0 (b, 1H, NH),
5.1 (m, 2H, Cbz), 7.1–7.4 (m, 10H, 2 · Ph), 7.5–7.7 (m,
5H, SO2–Ph); 8.4 (b, 1H, NH). MS (ESI) m/z 535
(M+1, 100%). Anal. Calcd for C30H34N2O5S: C, 67.39;
H, 6.56; N, 5.43. Found: C, 67.12; H, 6.61; N, 5.33.
4.1.9. Phenyl (3S)-3-(N-carbobenzyloxyleucyl)amino-4-
phenylbut-1-enyl sulfone (6c, Cbz-Leu-Phe-VS-Ph).
Cbz-Leu-Phe-VS-Ph was prepared from Cbz-Leu-OH
and Phe-VS-Ph Æ HCl using standard mixed anhydride
1
coupling method, yield 82%. H NMR (CDCl3) d 0.8–
0.9 (2d, 6H, 2 · Leu–CH3), 1.4–1.6 (m, 2H, Leu–CH2),
2.06 (m, 1H, Leu–CH), 2.9–3.0 (m, 2H, CH2–Phe),
3.9–4.0 (m, 1H, a-H), 4.8–4.9 (b, 1H, NH), 4.9–5.0 (m,
1H, a-H), 5.1 (m, 2H, Cbz) 6.3–6.4 (d and b, 2H, NH
and CH@), 6.9–7.0 (dd, 1H, CH@), 7.1–7.4 (m, 10H,
2 · Ph), 7.5–7.7 (m, 5H, SO2–Ph). MS (ESI) m/z 535.
4.1.13. Phenyl (3S)-3-(N-carbobenzyloxyvalyl)amino-4-
phenylbut-1-enyl sulfone (6b, Cbz-Val-Phe-VS-Ph).
Cbz-Val-Phe-VS-Ph was prepared from Cbz-Val-OH
and Phe-VS-Ph Æ HCl using standard mixed anhydride
coupling method, yield 98%. 1H NMR (CDCl3) d
0.70–0.84 (2d, 6H, Val-CH3), 2.06 (m, 1H, Val-CH),
2.91 (m, 2H, CH2–Phe), 3.82 (m, 1H, a-H), 4.80–4.90
(m, 1H, a-H), 5.10 (s, 2H, Cbz), 5.96 (d, 1H, NH) 6.34
(d, 1H, CH@), 6.9–7.0 (dd, 1H, CH@), 7.1–7.4 (m,
10H, 2 · Ph), 7.5–7.7 (m, 5H, SO2–Ph). MS (ESI) m/z
521 (M+1, 100%).
4.1.10. Phenyl 3-(N-carbobenzyloxyleucyl)amino-4-phe-
nylbut-2-enyl sulfone (7c, Cbz-Leu-Phe-AS-Ph). Butylli-
thium (3.63mL, 6.17mmol, 1.7M in pentane) was
added dropwise to a solution of tert-butylhydroperoxide
(2.55mL, 8.42mmol, 3.3M in toluene) in freshly distilled
THF (80mL) at ꢀ78ꢁC under argon. A solution of Cbz-

5210
M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
4.1.14. Phenyl 3-(N-carbobenzyloxyvalyl)amino-4-phen-
ylbut-2-enyl sulfone (7b, Cbz-Val-Phe-AS-Ph). Cbz-Val-
Phe-VS-Ph was treated with butyllithium and tert-butyl-
hydroperoxide in freshly distilled THF as described with
Cbz-Leu-Phe-VS-Ph above to give Cbz-Val-Phe-AS-Ph
as a white powder, yield 21%. 1H NMR (CDCl3)
d 0.70–0.85 (2d, 6H, Val–CH3), 2.1 (m, 1H, Val–CH),
3.80 (d, 2H, CH2–Phe), 3.95 (m, 3H, CH2–SO2 and a-
H), 4.90 (t, 1H, CH@), 5.12 (m, 3H, NH and Cbz),
7.1–7.4 (m, 10H, 2 · Ph), 7.55 (t, 2H, SO2–Ph), 7.65 (t,
1H, SO2–Ph), 7.91 (d, 2H, SO2–Ph), 8.42 (b, 1H, NH).
13C NMR (300MHz, CDCl3) d 172.1, 156.3, 144.4,
140.8, 135.7, 134.2, 133.6, 131.2, 129.5, 129.4 · 2,
129.3, 128.8 · 2, 128.7, 128.6, 128.4, 128.3, 127.8,
127.3, 126.9, 106.3, 67.6, 56.0, 55.6, 41.7, 22.0, 21.0,
18.4. MS (FAB+) m/z 521 (M+1, 100%). Anal. Calcd
for C29H32N2O5S Æ 1/10 H2O: C, 66.67; H, 6.21; N,
5.36. Found: C, 66.31; H, 6.23; N, 5.26.
4.1.18. N-Benzyloxycarbonylleucylalanine (Cbz-Leu-Ala-
OH). Cbz-Leu-Ala-OMe (4.23g, 12.2mmol) was
suspended in methanol at room temperature. Aqueous
sodium hydroxide (14.69mL, 1N, 14.69mmol) was
added. The reaction was stirred for 1h (TLC Hex/
EtOAc 1:1). The reaction mixture was cooled to 0ꢁC
and acidified with 1N HCl (pH2). The mixture was ex-
tracted with ethyl acetate (3 · 50mL). The organic layer
was washed with water (3 · 20mL) and brine
(3 · 20mL) and dried (MgSO4). The solvent was evapo-
rated to give Cbz-Leu-Ala-OH as a white powder, yield
1
85%. H NMR (CDCl3) d 0.90 (d, 6H, Leu–CH3), 1.41
(d, 3H, Ala–CH3), 1.59 (m, 1H, Leu–CH), 1.63 (m,
2H, Leu–CH2), 4.30 (m, 1H, a-H), 4.59 (m, 1H, a-H),
5.11 (s, 2H, Cbz), 5.60 (b, 1H, NH), 6.97 (b, 1H, NH),
7.31 (m, 5H, Ph).
4.1.19. N-(N-Benzyloxycarbonylleucylalanyl)-N-2-etha-
nolamine (Cbz-Leu-Ala-NH(CH2)2OH). Cbz-Leu-Ala-
OH was coupled to ethyl hydroxyl amine using standard
mixed anhydride coupling to give crude Cbz-Leu-Ala-
NH(CH2)2OH, which was recrystallized from cold ethyl
4.1.15. Phenyl (3S)-3-(N-carbobenzyloxyalanyl)amino-4-
phenylbut-1-enyl sulfone (6a, Cbz-Ala-Phe-VS-Ph). Cbz-
Ala-Phe-VS-Ph was prepared from Cbz-Ala-OH and
Phe-VS-Ph Æ HCl using standard mixed anhydride cou-
1
acetate to give a white powder, yield 63%. H NMR
1
pling method, yield 88%. H NMR (CDCl3) d 1.25 (d,
(CDCl3) d 0.90 (d, 6H, Leu–CH3), 1.41 (d, 3H, Ala–
CH3), 1.58 (m, 1H, Leu–CH), 1.62 (m, 2H, Leu–CH2),
2.92 (b, 1H, OH), 3.38 (m, 1H, NHCH2), 3.41 (m, 1H,
NHCH2), 3.69 (d, 2H, CH2OH), 4.13 (m, 1H, a-H),
4.43 (m, 1H, a-H), 5.11 (s, 2H, Cbz), 5.19 (b, 1H,
NH), 6.45 (b, 1H, NH), 6.79 (b, 1H, NH), 7.31 (m,
5H, Ph).
3H, Ala-CH3), 2.91 (dq, 2H, CH2–Phe), 4.10 (m, 1H,
a-H), 4.80–4.90 (m, 1H, a-H), 5.10 (s, 2H, Cbz), 6.34
(b, 1H, NH), 6.39 (d, 1H, CH@), 6.90–6.98 (dd, 1H,
CH@), 7.10–7.39 (m, 10H, 2 · Ph), 7.55 (t, 2H, SO2–
Ph), 7.65 (t, 1H, SO2–Ph), 7.91 (d, 2H, SO2–Ph). 13C
NMR (400MHz, CDCl3) d 172.1, 156.3, 144.4, 140.8,
135.7, 134.2, 133.6, 131.2, 129.5, 129.4 · 3, 129.3,
128.8 · 2, 128.7, 128.6, 128.4, 128.3, 127.8, 127.3,
126.9, 67.6, 51.7, 51.6, 40.2, 19.4.
4.1.20. 2-(1-Benzyloxycarbonylleucylaminoethyl)oxazo-
line
(11,
Cbz-Leu-Ala-oxazoline).
Cbz-Leu-Ala-
NH(CH2)2OH (1.15g, 3.02mmol) was dissolved in
THF (20mL). Triphenyl phosphine (1.19g, 4.54mmol)
was added followed by dropwise addition of diisopropyl-
azadicarboxylate (0.92g, 4.54mmol). The reaction mix-
ture was stirred for 2h at room temperature. The
solvent was evaporated, and the crude oil was subjected
to column chromatography (silica, MeOH/CH2Cl2 7%)
to give Cbz-Leu-Ala-oxazoline as a white powder, yield
4.1.16. Phenyl 3-(N-carbobenzyloxyalanyl)amino-4-phen-
ylbut-2-enyl sulfone (7a, Cbz-Ala-Phe-AS-Ph). Cbz-Ala-
Phe-VS-Ph was treated with butyllithium and tert-butyl-
hydroperoxide in freshly distilled THF as described
with Cbz-Leu-Phe-VS-Ph above to give Cbz-Ala-Phe-
AS-Ph as a white powder, yield 93%. 1H NMR (CDCl3)
d 1.21 (d, 3H, Ala–CH3), 3.80 (m, 4H, CH2–Phe and
CH2–SO2), 4.12 (q, 1H, a-H), 4.88 (t, 1H, CH@), 5.11
(m, 3H, NH and Cbz), 7.30 (m, 10H, 2 · Ph), 7.50 (t,
2H, SO2–Ph), 7.64 (t, 1H, SO2–Ph), 7.83 (d, 2H, SO2–
Ph), 8.40 (b, 1H, NH). 13C NMR (400MHz, CDCl3) d
172.1, 156.3, 144.4, 140.8, 135.7, 134.2, 133.6, 131.2,
129.5, 129.4 · 2, 129.3, 128.8 · 2, 128.7, 128.6, 128.4,
128.3, 127.8, 127.3, 126.9, 107.2, 67.6, 51.7, 51.6, 40.2,
19.4. MS (FAB+) m/z 493 (M+1, 100%). HRMS calcd
for C27H29N2O5S: 493.17972. Obsd. 493.17663. Anal.
Calcd for C27H28N2O5S: C, 65.83; H, 5.73; N, 5.69.
Found: C, 65.73; H, 5.82; N, 5.68.
1
75%. H NMR (CDCl3) d 0.90 (d, 6H, Leu–CH3), 1.41
(d, 3H, Ala-CH3), 1.58 (m, 1H, Leu–CH), 1.69 (m,
2H, Leu–CH2), 3.83 (t, 2H, NCH2), 4.19 (m, 1H, a-
H), 4.36 (t, 2H, OCH2), 4.62 (m, 1H, a-H), 5.11 (s,
2H, Cbz), 5.19 (b, 1H, NH), 6.62 (b, 1H, NH), 7.36
(m, 5H, Ph). MS (FAB+) m/z 362 (M+1, 100%). HRMS
calcd for C19H28N3O4: 362.20813. Obsd. 362.20798.
Anal. Calcd for C19H27N3O4 Æ 1/4 H2O: C, 62.36; H,
7.57; N, 11.48. Found: C, 62.22; H, 7.55; N, 11.64.
Acknowledgements
4.1.17. N-Benzyloxycarbonylleucylalanine methyl ester
(Cbz-Leu-Ala-OMe). Cbz-Leu-Ala-OMe was prepared
from Cbz-Leu-OH and Ala-OMe using standard mixed
anhydride coupling, yield 93%. 1H NMR (CDCl3) d 0.90
(d, 6H, Leu–CH3), 1.41 (d, 3H, Ala–CH3), 1.58 (m, 1H,
Leu–CH), 1.62 (m, 2H, Leu–CH2), 3.88 (s, 3H, OCH3),
4.21 (m, 1H, a-H), 4.59 (m, 1H, a-H), 5.11 (s, 2H,
Cbz), 5.23 (b, 1H, NH), 6.45 (b, 1H, NH), 7.31 (m,
5H, Ph).
This work was supported by grants from the National
Institute of General Medical Sciences (GM54401 and
GM61964) and the Sandler Family Support Foundation
(UCSF). M.G. acknowledges the Graduate Assistance
in Areas of National Need (GAANN) fellowship from
the US Department of Education. We would like to
thank Leslie Gelbaum for assistance with the NOESY
and COSY NMR spectra.

M. G. Go¨tz et al. / Bioorg. Med. Chem. 12 (2004) 5203–5211
5211
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