Strategy in inhibition of cathepsin B, a target in tumor invasion and metastasis

Article abstract of DOI:10.1021/ja0489240

Cathepsin B, a cysteine protease, is an important target in fighting cancer. This enzyme has been implicated in enhancing tumor invasiveness and metastasis, therefore inhibitors for cathepsin B are highly sought as potential anticancer and antimetastatic

Full text of DOI:10.1021/ja0489240

Published on Web 08/03/2004
Strategy in Inhibition of Cathepsin B, A Target in Tumor
Invasion and Metastasis
In Taek Lim,† Samy O. Meroueh,† Mijoon Lee,† Mary Jane Heeg,‡ and
Shahriar Mobashery*,†
Contribution from the Department of Chemistry and Biochemistry and Walther Cancer Research
Center, UniVersity of Notre Dame, Notre Dame, Indiana 46556 and Department of Chemistry,
Wayne State UniVersity, Detroit, Michigan 48202
Received February 25, 2004; E-mail: mobashery@nd.edu
Abstract: Cathepsin B, a cysteine protease, is an important target in fighting cancer. This enzyme has
been implicated in enhancing tumor invasiveness and metastasis, therefore inhibitors for cathepsin B are
highly sought as potential anticancer and antimetastatic agents. A structure-based design effort was pursued
in arriving at a template for inhibition of cathepsin B. Focused compound libraries were synthesized based
on this template, which were screened for cathepsin B inhibitory properties. Compound 2, 1-(2(R)-{1(S)-
acetoxy-2-[2(S)-(2,4-difluoro-benzoylamino)-3-phenyl-propionylaminooxy]-2-oxo-ethyl}-pentanoyl)-pyr-
rolidine-2(S)-carboxylic acid benzyl ester, is the prototype of this novel class of cysteine protease inhibitor
that emerged from the search. The molecule modifies the active site of cathepsin B covalently, irreversibly,
and efficiently, a process for which the kinetic parameters were evaluated. A set of three judiciously altered
variants of compound 2 was also synthesized to explore the details of the proposed mechanism of action
by this inhibitor. Compound 2 and its analogues may prove useful tools in reversing the deleterious effect
of cathepsin B in fighting cancer.
Malignant tumors have a tendency to invade the surround-
ing tissue and gain access to the vasculature.1 The tumor sheds
cells into the vasculature, which in turn transports the cells to
secondary sites for reestablishment of the tumor. The occurrence
of these events, referred to as tumor metastasis, bodes poorly
for the patients and leads to death in more than 90% of the
cases.2 Although processes involved in tumor invasion and
metastasis are complex, a prerequisite for these events is the
degradation of the extracellular matrix by specific proteases.3-8
Cathepsin B, an important cysteine protease of the papain
superfamily has been documented to be important in many
metastatic tumors.9-20 The linking of cathepsin B to tumor
progression and invasion is based on observations that its levels
of activity and secretion are increased in tumors.9-24 The highest
level of cathepsin B activity is seen in the most malignant
tumors, and particularly so in cells that are at the invasive edge
of these tumor masses.25-27 Therefore, cathepsin B is considered
an important target in cancer intervention and inhibitors for it
are highly sought.28-33
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† Department of Chemistry and Biochemistry and Walther Cancer
Research Center, University of Notre Dame.
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Kopitar-Jerala, N.; Turk, V.; Lah, T. T. Clin. Exp. Metastasis 1997, 15,
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‡ Department of Chemistry, Wayne State University.
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J. AM. CHEM. SOC. 2004, 126, 10271-10277
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A R T I C L E S
Lim et al.
Scheme 1
Scheme 2
It is believed that Cys-29 and His-199 exist as an ion pair in
the native cathepsin B.34-36 Cathepsin B undergoes acylation
by its substrates at Cys-29 and the acyl-enzyme species
undergoes hydrolysis in the second part of catalysis. We
conceived of a strategy in inhibition of cathepsin B by tying up
the two catalytic residues in a manner that they would not be
available for the catalytic processes. We envisioned that a
molecule could be designed such that it would undergo
interaction with the reactive Cys-29 thiolate, and in the process
it would makes a hydrogen bond to His-199, such that it cannot
perform its proton transfer function in the normal turnover events
of the enzyme. Such a molecule may conceivably result in either
a stable acyl-enzyme species or a species en route to the acyl-
enzyme entity, either of which could result in irreversible
inhibition of cathepsin B.
4, which would be expected to hydrogen bond with the
unprotonated His-199 and attenuate its basicity. By so doing,
not only the histidine will be a poorer base for promotion of
the water molecule for the “second step” of catalysis, but also
the hydroxyl moiety that interacts with it is predisposed at the
position of the incoming hydrolytic water molecule. Other
strategies in inhibition that tie up catalytic residues in serine-
dependent enzymes have been described.39-46
Either species 3 or 4 could account for the mechanism of
inhibition by compound 2, both of which are unprecedented
and novel for inhibition of cysteine proteases.47,48
The design process started with the X-ray structure of
cathepsin B32 and that of an inhibitor bound to its active site
cysteine.37 The original concept went through a number of
iterations by extensive use of molecular docking based on a
genetic algorithm,38 and several analogues were synthesized and
tested in in vitro experiments with the purified enzyme. The
lead structural template that emerged out of these efforts (1)
was refined by syntheses of several focused libraries of 80
compounds that created diversity for groups R1 to R4 (see
structure 1), which were individually tested for cathepsin B
inhibition. The culmination of these efforts was compound 2.
Compound 2 was expected to modify Cys-29 to give species 3
(Scheme 1). Molecular modeling argues that the potential for
hydrogen bonding between the acetyl moiety and the protonated
His-199 exits and this structure may be stabilized within the
active site. Alternatively, species 3 may fragment to give species
Synthesis of compound 2 is depicted in Scheme 2. 2(R)-Allyl-
3(S)-hydroxysuccinic acid diisopropyl ester 6 was prepared from
(S)-malic acid diisopropyl ester49 and was hydrolyzed to give
the diacid 7. The absolute stereochemistry of compound 7 was
determined by X-ray crystallography, and the ORTEP presenta-
tion of the crystal structure is given as Supporting Information.
The acetonide group was introduced for protection of the
R-hydroxycarboxylic acid (8). Catalytic hydrogenation of the
allyl group of 8 led to the derivative 9. Compound 9 was
elaborated with L-proline benzyl ester to give 10. After
deprotection of the acetonide, the hydroxyl group was acetylated
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Inhibition of Cathepsin B
A R T I C L E S
Table 1. Kinetic Parameters for Inhibition of Cathepsin B from
Bovine Spleen by the Synthetic Inhibitors
To explore if species 4 plus fragment 5 are formed on
exposure of cathepsin B to 2, we prepared a 13C version of
inhibitor 2 in which the label was at the carbonyl carbon of the
acetyl moiety. The synthesis was the same as in Scheme 2,
except labeled acetic anhydride was used.
inhibitor
K (µM)
i
k
inact (s-1) × 103
k
inact/K (M-1s-1) × 10-2
i
2a
4.4 ( 0.6
7.3 ( 0.8
34 ( 2
150 ( 110
3.4 ( 0.7
3.6 ( 0.4
0.057 ( 0.005
> 300
360 ( 210
7.7 ( 1.9
15
16
17
4.9 ( 0.8
We incubated 13C-2 (500 µM) with bovine cathepsin B (160
µM) in 300 µL of 50 mM MES buffer, 200 mM NaCl, 5 mM
EDTA, 0.15% BRIJ 35, 1 mM DTT, and 1.5% DMSO-d6, pH
6.0 at 4 °C in an NMR tube. A HMBC experiment was carried
out,52,53 which revealed the presence of two acetyl signals, one
at 13C NMR δ 172.6 and 1H NMR δ 1.9 corresponding to 13C-2
and another at 13C NMR δ 180.8 and 1H NMR δ 1.7
corresponding to 5. The former showed 13C coupling to the
methine (1H NMR δ 4.4) three bonds away in the inhibitor and
the latter showed 13C coupling to the hydroxamate NH (1H NMR
δ 8.2), also three bonds away in species 5. Therefore, it would
appear that the interaction of inhibitor 2 with the cathepsin B
active site does generate species 3 to give immediate inhibition
of the enzyme, and over time release of compound 5 gives rise
to species 4, which is the ultimate inhibitory species.
The proposed mechanism of action of inhibitor 2 (Scheme
1) envisions that the acetoxyl moiety will interact with His-
199. We evaluated the importance of the acetoxyl group for
inhibition by synthesizing derivatives 15 and 16 (see Supporting
Information).
0.078 ( 0.011
ND
0.046 ( 0.004
0.16 ( 0.01
NI
810 ( 47
a Kinetic parameters for inactivation of cathepsin B from human liver
by inactivator 2: Ki ) 3.2 ( 0.4 µM, kinact ) (4.7 ( 1.6) × 10-2 s-1 and
(3.9 ( 2.3) × 10-3 s-1, kinact/Ki (1.5 ( 0.5) × 104 M-1s-1 and (1.2 ( 0.7)
× 103 M-1s-1. ND stands for “not determined”. NI stands for “no
inactivation”.
to give 11. A standard coupling of L-phenylalanine methyl ester
with difluorobenzoyl chloride afforded compound 13, which in
turn resulted in 14 after the reaction with hydroxylamine. The
coupling of 11 with 14 in the presence of PYBOP produced
the desired compound 2. That the acylation took place at the
hydroxamic acid oxygen and not the nitrogen was confirmed
by the ferric chloride test (5% FeCl3‚6H2O in 0.5 N HCl).50
Compound 2 inhibited cathepsin B in a time-dependent
manner, typical of covalent enzyme modifiers (Table 1). The
kinetics of enzyme modification were biphasic, a rapid phase
(kinact ) 0.15 ( 0.11 s-1) preceded a slower one [kinact ) (3.4
( 0.7) × 10-3 s-1]. The dissociation constant for the nonco-
valent pre-acylation complex of the inhibitor and enzyme, a
measure of the affinity of the enzyme for the ligand, was
determined at Ki ) 4.4 ( 0.6 µM by a Dixon plot. The values
for kinact/Ki were calculated at (3.6 ( 2.1) × 104 M-1s-1 and
(7.7 ( 1.9) × 102 M-1s-1 for the first and second phases,
respectively. The process of inhibition is clearly favorable. We
attempted to measure a rate constant for a potential recovery of
enzyme activity (i.e., reversal of inhibition; krec), but such a
measurement was not possible as recovery of activity was not
detectable under these conditions. We allowed a portion of the
enzyme to be inhibited completely and then dialyzed the sample
overnight. Under these conditions, 5-6% of activity was
recovered over the long duration. Therefore, the enzyme-
inhibitor complex is stable. Furthermore, we investigated
whether the process of enzyme inhibition also entailed any
turnover of inhibitor 2 by measuring the partition ratio51 for
the inhibitor. This experiment revealed that essentially one
molecule of inhibitor 2 resulted in inhibition of one molecule
of enzyme.
It was interesting that both compounds 15 and 16 gave
biphasic time-dependent inhibition of cathepsin B. However,
they were both substantially poorer inhibitors compared to
compound 2 (Table 1). They were also irreversible inhibitors
of cathepsin B, which showed recoveries of activity of less than
2% after overnight dialysis of the inhibited enzyme samples.
Both compounds also had poorer dissociation constants (Ki)
compared to 2. Since the hydroxyl moiety in 15 should still be
able to hydrogen bond with His-199, its Ki was only 2-fold
higher than that of 2. In terms of kinact/Ki, compound 2 was
superior to both 15 and 16 by 2 to 4 orders of magnitude.
Therefore, the acetoxyl group was clearly important for efficient
inhibition of cathepsin B (effects on both Ki and kinact), but not
critical for the mode of inhibition.
The hydroxamate species of 2 would contribute to the
favorable interactions with the active site in stabilizing the
tetrahedral species 3. To explore the contribution of the
hydroxamate group to the inhibition process, we synthesized
the carbon analogue, compound 17 (see Supporting Informa-
tion). Compound 17 has all the features of inhibitor 2, except
its hydroxamate nitrogen was replaced by a carbon. Compound
17 was merely a poor noncovalent competitive inhibitor of
cathepsin B, with a Ki of 810 ( 47 µM.
We also investigated the nature of the inhibited enzyme
species by electrospray ionization mass spectrometry. Mass
spectrometry revealed the formation of a 28 274 Da species on
inhibition of the bovine cathepsin B, consistent with the
incorporation of the entire structure of the inhibitor to the
enzyme active site (an increase in mass of 708 Da; M+H). This
finding is consistent with species 3 as the likely inhibited form
of the enzyme. However, if the noncovalent fragment 5 were
to be retained by the enzyme, the mass spectrometric result
cannot preclude the formation of 4, as the molecular mass for
species 3, and for 4 plus the retained fragment 5 are the same.
(50) Surman, M. D.; Mulvihill, M. J.; Miller, M. J. J. Org. Chem. 2003, 67,
4115-4121.
(51) Silverman, R. B. Mechanism-Based Enzymes InactiVators: Chemistry and
Enzymology; CRC Press: Boca Raton, FL, 1988.
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A R T I C L E S
Lim et al.
Figure 2. Distance from Nδ of His-199 to (A) the ester oxygen carbonyl
and (B) the sp3 hybridized oxygen atom of the ester moiety and (C) to the
charged oxygen of the tetrahedral species over the course of the 960 ps
trajectory.
Figure 1. (A) A stereoview of the computational model for compound 2
bound to the active site of cathepsin B. A solvent-accessible surface was
constructed for the active site. The ligand is shown in capped-sticks
representation and color-coded according to atom types (white, blue, red,
and green correspond to carbon, nitrogen, oxygen, and fluorine, respec-
tively). (B) A schematic illustrating the various hydrogen bonding interac-
tions of compound 2 with the enzyme. Distances correspond to mean values,
which were determined by averaging over all the snapshots collected during
the 960-ps simulation.
to be the opportunity for protonation of the tetrahedral species
by His-199. The proline side chain binds into the S2′ subsite,
whereas the benzyl ester moiety points toward the S3′ pocket,
with its carbonyl forming a hydrogen bond with Nδ of His-
110. These two residues are thought to be the basis for efficient
binding of the C-termini in substrates to the active site of
cathepsin B. As shown in the schematic in Figure 1B, the
oxygen of the tetrahedral species is ensconced into the oxyanion
hole, forming hydrogen bonds with the backbone nitrogen of
Cys-29 and the side-chain amide nitrogen of Gln-23, with mean
distances of 2.8 and 3.0 Å, respectively.
The mechanism of inhibition of cathepsin B by nitrile-based
inhibitors, which consist of the formation of a thioimidate as a
result of proton transfer from the charged His-199 to the
thioimide,54 inspired the investigation of a similar event for
species 3. The distance from the charged oxygen of the
tetrahedral species to the hydrogen at Nδ was monitored over
the course of the 960-ps trajectory and is shown in Figure 2. In
a fleeting moment during the course of the trajectory (in the
first 100 ps), the charged oxygen comes within hydrogen-
bonding distance of Nδ of His199, making the proton migration
from His-199 to the tetrahedral intermediate a possible event.
The resulting neutral species in the formation of species 3 may
be plausible and might serVe to stabilize the tetrahedral
intermediate, which ultimately leads to species 4 in due course.
In this manuscript, we have disclosed a novel class of
irreversible inhibitors for cathepsin B, which is an important
target for intervention of tissue invasiveness and metastasis of
To further elucidate the structural bases for inhibition, a 960-
ps molecular dynamics simulation of species 3 was carried out.
The system was fully solvated and PME electrostatics were used
to describe long-range electrostatics. The Cartesian coordinates
from the last snapshot in the trajectory were used to illustrate
the binding mode of 2 in the tetrahedral species, as shown in
Figure 1A. The compound effectively exploits the primed and
unprimed subsites of the active site. The propyl moiety at R2 is
pointing toward the S1′ pocket of the enzyme, sharing the subsite
with the acetoxy moiety of 2. It is of interest to note that the
acetoxy moiety forms hydrogen bonds with the Nδ of His-199;
both ester oxygens seem to be interacting with Nδ of His-199
over the course of the trajectory, with mean distances of 3.2
and 2.9 Å, respectively (Figure 2A,B). Figure 2A shows that
the hydrogen bond between Nδ and the carbonyl oxygen
experiences large fluctuations in the first 250 ps, but remains
stable over the remainder of the trajectory. The other hydrogen
bondswith the sp3 hybridized oxygen of the ester moietysis
more stable as evidenced by the lack of large fluctuations in
the distance (Figure 2B). These hydrogen bonds are thought to
be the basis for the increase in efficiency of cathepsin B
inactivation by compound 2, compared to compounds 15 and
16. It is interesting to note that in the period that the hydrogen
bond to the carbonyl breaks (Figure 2A), there would appear
9
10274 J. AM. CHEM. SOC. VOL. 126, NO. 33, 2004

Inhibition of Cathepsin B
A R T I C L E S
1H NMR (500 MHz, CDCl3) δ 4.49 (d, J ) 4.5 Hz, 1H, CHOR), 2.91
(m, 1H, CHCO2H), 1.84 (m, 1H), 1.75 (m, 1H), 1.60 (s, 3H), 1.54 (s,
3H), 1.43 (m, 2H), 0.95 (t, J ) 7.3 Hz, 3H, CH2CH3); 13C NMR (125
MHz, CDCl3) δ 176.7, 172.4, 111.3, 74.2, 47.1, 29.9, 26.8, 26.4, 20.7,
14.1; HRMS (FAB) calcd for C10H17O5 (MH+) 217.1076, found
217.1058.
cancer. The research benefited from computational methods and
from syntheses of focused libraries of compounds that provided
structural diversity. Compound 2 is the prototype of this type
of cysteine protease inhibitor that should prove a new and useful
tool in fighting cancer.
1-[2(R)-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4(S)-yl)-pentanoyl]-
proline Benzyl Ester (10). The acid 9 (1.54 g, 7.1 mmol), PYBOP
(3.64 g, 7.0 mmol) and 1-hydroxy-7-azabenzotriazole (0.95 g, 7.0
mmol) were dissolved in anhydrous DMF (15 mL). L-ProOBn‚HCl
(1.72 g, 7.1 mmol) was added to the reaction mixture at -10 °C,
followed by dropwise addition of DIPEA (2.47 mL, 14.2 mmol) over
5 h. After 18 h, the residue was diluted with EtOAc (40 mL). The
organic layer was washed with brine (20 mL) and saturated aqueous
NaHCO3 (20 mL), dried over MgSO4, filtered, and evaporated. The
crude product was purified by column chromatograph (hexane:EtOAc
) 2:1 ∼ 3:2) to afford 10 (2.21 g, 78%) as an oil; 1H NMR (500 MHz,
CDCl3) δ 7.33 (m, 5H, Ph), 5.15 (q, J ) 12.0 Hz, 2H, OCH2Ph), 4.63
(dd, J ) 8.5, 4.0 Hz, 1H, CHCO2Bn), 4.52 (d, J ) 9.5 Hz, 1H, CHOR),
3.64 (m, 2H, CH2NR2), 2.95 (td, J ) 9.5, 4.5 Hz, 1H, CHPr), 2.20 (m,
1H, CH2CHCO2Bn), 1.98 (m, 4H), 1.77 (m, 1H, CH2Et), 1.60 [s, 3H,
C(CH3)2], 1.51 [s, 3H, C(CH3)2], 1.48 (m, 1H, CH2CH3), 1.28 (m, 1H,
CH2CH3), 0.83 (t, J ) 7.3 Hz, 3H, CH2CH3); 13C NMR (125 MHz,
CDCl3) δ 172.4, 172.1, 171.1, 135.9, 128.8, 128.4, 128.3, 110.7, 74.5,
67.0, 59.1, 47.6, 46.4, 30.2, 29.4, 27.3, 26.1, 25.0, 20.0, 14.4.
1-[2(R)-((S)-Acetoxy-carboxy-methyl)-pentanoyl]-proline Benzyl
Ester (11). A solution of 10 (0.57 g, 1.4 mmol) in THF (2 mL), t-BuOH
(1 mL) and 1 N HCl (2 mL) was stirred at 4 °C overnight. The solvent
was removed to dryness under reduced pressure. The residue was
dissolved in dichloromethane (10 mL) and was treated with pyridine
(178 mL. 2.2 mmol) and acetic anhydride (198 mL, 2.1 mmol). The
resulting solution was stirred at room temperature for 6 h. The solvent
was concentrated to dryness to give a crude product, which was
subjected to column chromatography (CH2Cl2:MeOH:AcOH ) 120:
5:1 ∼ 60:5:1) giving the pyridinium salt of 11 as an oil (0.63 g, 93%);
1H NMR (500 MHz, CDCl3) δ 8.64 (d, J ) 4.5 Hz, 2H, pyr), 7.83 (m,
1H, pyr), 7.42 (m, 2H, pyr), 7.30 (m, 5H, Ph), 5.13 (dd, J ) 38.5, 12.0
Hz, 2H, OCH2Ph), 5.13 (d, J ) 6.0 Hz, 1H, CHOAc), 4.60 (dd, J )
8.5, 4.0 Hz, 1H, CHCO2Bn), 3.74 (m, 1H, CH2NR2), 3.64 (m, 1H,
CH2NR2), 3.04 (m, 1H, CHPr), 2.22 (m, 1H), 2.04 (s, 3H, Ac), 1.99
(m, 3H), 1.68 (m, 1H), 1.60 (m, 1H), 1.43 (m, 1H), 1.32 (m, 1H), 0.83
(t, J ) 12.5 Hz, 3H, CH2CH3); 13C NMR (125 MHz, CDCl3) δ 173.2,
171.5, 170.9, 170.1, 147.3, 138.9, 135.6, 128.8, 128.6, 128.4, 124.9,
73.4, 67.3, 59.5, 48.1, 45.0, 31.0, 29.4, 25.0, 20.9, 20.4, 14.3; HRMS
(FAB) calcd for C21H28NO7 (MH+) 406.1866, found 406.1891.
(S)-2,4-Difluorobenzoylphenylalanine Methyl Ester (13). The title
compound was prepared by a modified method of Fitt and co-workers.55
Sodium 2-ethylhexanoate (6.65 g, 40 mmol) was added to a suspension
of phenylalanine methyl ester hydrochloride (4.31 g, 20 mmol) in THF
(40 mL). After being stirred at room temperature for 1 h, 2,4-
difluorobenzoyl chloride (2.58 mL, 21 mmol) was added to the solution
at -10 °C. After 3 h, the residue was diluted with EtOAc (200 mL)
and was washed with saturated NaHCO3 (100 mL × 3) and brine (60
mL). The organic layer was dried over MgSO4, filtered, and concen-
trated. The product was purified by silica gel chromatography (hexane:
Experimental Section
(2R,3S)-Diisopropyl 2-allyl-3-hydroxysuccinate (6). A modified
procedure described by Seebach was applied.49 A 2.5 M solution of
n-butyllithium (40 mL, 100 mmol) in hexane was added dropwise to
a stirred solution of N-isopropylcyclohexylamine (16.8 mL, 102 mmol)
and 2,2′-dipyridyl (107 mg, 0.69 mmol) in anhydrous THF (250 mL)
at -75 °C. The reaction temperature was raised to -50 °C over 30
min. Diisopropyl (S)-malate (10.9 g, 50 mmol) in THF (20 mL) was
added dropwise at -75 °C to the reaction mixture. The reaction flask
was warmed to -50 °C over 1 h and then was cooled to -75 °C. A
solution of 3-bromo-1-propene (8.65 mL, 100 mmol) in THF (10 mL)
was added dropwise. Stirring was continued at -50 °C for 1 h, and
then overnight while the temperature was raised to -5 °C. The reaction
was quenched by addition of a solution of glacial acetic acid (12 g,
200 mmol) in diethyl ether (20 mL) at -50 °C and then poured into a
mixture of ether (500 mL) and water (100 mL). The organic layer was
washed with saturated NaHCO3 and brine and dried over MgSO4. The
solvent was concentrated to dryness to give a crude product which was
subjected to column chromatography (hexane:EtOAc ) 10:1 ∼ 6:1)
yielding 6 as an oil (10.6 g, 82%); 1H NMR (400 MHz, CDCl3) δ 5.79
(m, 1H), 5.12 (m, 2H), 5.01 [m, 2H, OCH(CH3)2], 4.21 (dd, J ) 6.4,
3.2 Hz, 1H, CHOH), 3.20 (d, J ) 6.4 Hz, 1H, OH), 2.89 (m, 1H,
CHCO2R), 2.60 (m, 1H), 2.40 (m, 1H), 1.27 [dd, J ) 6.0, 2.0 Hz, 6H,
CH(CH3)2], 1.20 [d, J ) 6.8 Hz, 6H, CH(CH3)2]; 13C NMR (100 MHz,
CDCl3) δ 173.3, 171.7, 135.3, 118.0, 70.5, 70.0, 68.8, 48.3, 32.5, 22.0,
21.9; HRMS (FAB) calcd for C13H23O5 (MH+) 259.1545, found
259.1540.
(2R,3S)-2-Allyl-3-hydroxysuccinic Acid (7). 2 N KOH (20 mL,
40 mmol) was added to a solution of 6 (3.20 g, 12.3 mmol) in 1:1
THF-MeOH (20 mL). The reaction mixture was stirred at room
temperature for 24 h. The volume was reduced by approximately 60%
under reduced pressure. The residue was acidified to pH 1 with 6 N
HCl and was saturated with NaCl. The resultant aqueous solution was
washed with EtOAc (5×). The combined organic layers were dried
over MgSO4, filtered, and concentrated to dryness. Recrystallization
from ether/pentane gave 7 (1.88 g, 88%) as a white solid. mp 91-93
°C; 1H NMR (500 MHz, CD3OD) δ 5.84 (m, 1H), 5.09 (m, 2H), 4.90
(br, 3H), 4.25 (d, J ) 5.0 Hz, 1H, CHOH), 2.90 (m, 1H, CHCO2R),
2.50 (m, 1H), 2.35 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 175.2,
174.3, 135.5, 116.4, 70.4, 48.8, 32.2; MS (EI) 197.04 (M + Na+),
192.08, 175.04, 129.04.
2(R)-[2,2-Dimethyl-5-oxo-(1,3)dioxolan-4(S)-yl]-pent-4-enoic Acid
(8). Compound 7 (1.81 g, 10.4 mmol) in dimethoxypropane (10 mL)
was treated with TsOH (100 mg, 0.6 mmol). The reaction mixture was
stirred at room-temperature overnight, after which it was concentrated
to dryness. The residue was diluted with EtOAc (40 mL) and was
washed with water and brine. The organic layer was dried over MgSO4,
filtered, and concentrated, giving a crude product which was subjected
to column chromatograph (CH2Cl2:MeOH:AcOH ) 300:5:1) to afford
1
EtOAc ) 5:1 ∼ 3:1) to afford 13 (6.13 g, 96%) as an oil; H NMR
1
(500 MHz, CDCl3) δ 8.08 (q, J ) 8.2 Hz, 1H), 7.27 (m, 3H), 7.16 (dd,
J ) 3.5, 1.5 Hz, 2H), 7.09 (m, 1H, NHCO), 6.97 (m, 1H), 6.46 (ddd,
J ) 11.0, 8.5, 2.5 Hz, 1H), 5.06 (qd, J ) 8.0, 2.0 Hz, 1H, CHCO2Me),
3.75 (s, 3H, OCH3), 3.23 (qd, J ) 14.0, 6.0 Hz, 2H, CHCH2Ph); 13C
8 (1.71 g, 77%) as an oil; H NMR (400 MHz, CDCl3) δ 5.78 (m,
1H), 5.17 (m, 2H), 4.51 (d, J ) 4.0 Hz, 1H, CHOR), 3.05 (m, 1H,
CHCO2H), 2.70 (m, 1H), 2.49 (m, 1H), 1.59 (s, 3H), 1.53 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ 176.8, 172.1, 134.2, 118.9, 111.5, 73.2,
46.5, 32.0, 26.7, 26.3.
(52) Bax, A.; Summers, M. F. J. Am. Chem. Soc. 1986, 108, 2093-2094.
(53) Martin, G. E.; Zektzer, A. S. Two-Dimensional NMR Methods for
Establishing Molecular ConnectiVity; VCH: Weinheim, 1988.
(54) Brisson, J. R.; Carey, P. R.; Storer, A. C. J. Biol. Chem. 1986, 261, 9087-
9089.
(55) Fitt, J.; Prasad, K.; Repic, O.; Blacklock, T. J. Tetrahedron Lett. 1998, 39,
6991-6992.
2(R)-(2,2-Dimethyl-5-oxo-[1,3]dioxolan-4(S)-yl)-pentanoic Acid
(9). A solution of 8 (0.88 g, 4.11 mmol) in MeOH (20 mL) was charged
with activated carbon (150 mg, 10%). The mixture was stirred at room-
temperature overnight under an atmosphere of hydrogen. After filtration,
the filtrate was concentrated to dryness to give 9 (0.89 g, quantitative);
9
J. AM. CHEM. SOC. VOL. 126, NO. 33, 2004 10275

A R T I C L E S
Lim et al.
NMR (125 MHz, CDCl3) δ 171.9, 165.2 (dd, J ) 256.2, 13.1 Hz, CF),
162.1 (d, J ) 2.8 Hz, CO), 161.3 (dd, J ) 251.6, 13.1 Hz, CF), 135.9,
134.0 (dd, J ) 10.2, 3.8 Hz), 129.5, 128.9, 127.5, 117.2 (dd, J ) 11.2,
3.8 Hz, q), 112.6 (dd, J ) 21.4, 2.8 Hz), 104.6 (dd, J ) 27.9, 26.0
Hz), 54.2, 52.6, 38.1; HRMS (FAB) calcd for C17H16F2NO3 (MH+)
320.1098, found 320.1112.
6 µM) and the concentrations of inhibitors were varied from 0 to 80
µM. Enzyme concentration in the assays was 3 nM.
Initial rates of inactivation (kinact) were measured as described by
Silverman.51 A series of inactivation experiments were carried out with
inactivator concentrations within the range of 1 to 80 µM. Typically,
a solution of inactivator in DMF was added to an enzyme solution in
the above-mentioned assay mixture to afford a final concentration of
0.70 µM enzyme in 1.2% DMF for a given inactivator concentration.
The solutions were incubated at 0 °C. Every few minutes, 30 µL
samples of the inactivation mixture were removed and added to 570
µL of the assay buffer containing 6 µM of Z-Phe-Arg-AMC and the
increase in the emission fluorescence at 460 nm was monitored for the
first 30 s of the reaction immediately.
The partition ratio was evaluated by the titration method.51 A series
of solutions (200 µL each), containing 0.4 µM enzyme and various
molar equivalents of inhibitor 2, were prepared to give [I]0/[E]0 ratios
of 0.01 to 5 in 50 mM MES buffer, 200 mM NaCl, 5 mM EDTA, 1
mM DTT, and 1% DMSO, pH 6.0 and the solutions were incubated at
4 °C for 15-17 h. Subsequently, 50 µL samples of each solution were
added to 550 µL of the above-mentioned assay mixture containing 10
µM of Z-Phe-Arg-AMC (Sigma) and the activities were measured
immediately. Control experiments were carried out in the same manner
in the absence of the inhibitor.
(S)-2,4-Difluorobenzoyl-phenylalanine N-Hydroxyamide (14). The
synthesis was carried out according to a modified procedure of Ritter.56
A solution of 13 (3.46 g, 10.8 mmol) in methanol (20 mL), cooled in
an ice bath, was charged with a preformed slurry of hydroxylamine
hydrochloride (1.39 g, 20 mmol) and KOH (2.24 g, 40 mmol). The
reaction mixture was stirred under argon overnight and then was
acidified to an apparent pH of 4 with concentrated HCl. The solvent
was removed under reduced pressure to give a white solid. The solid
was repeatedly boiled in ethyl acetate and filtered until TLC analysis
revealed that no ferric chloride-positive component remained in the
filtrate. The combined filtrate was concentrated under reduced pressure
1
to yield 14 (3.42 g, 99%) as a white powder; H NMR (500 MHz,
CD3OD) δ 7.70 (q, J ) 8.0 Hz, 1H), 7.27 (m, 3H), 7.21 (m, 2H), 7.03
(m, 2H), 4.86 (s, 3H), 4.73 (t, J ) 7.5 Hz, 1H, CHCH2Ph), 3.16 (dd,
J ) 13.5, 7.0 Hz, 1H, CHCH2Ph), 3.04 (dd, J ) 14.0, 8.0 Hz, 1H,
CHCH2Ph); 13C NMR (125 MHz, CD3OD) δ 168.8, 164.9 (dd, J )
253.5, 12.1 Hz, CF), 164.0, 160.9 (dd, J ) 252.5, 12.1 Hz, CF), 136.8,
132.3 (dd, J ) 10.2, 3.6 Hz), 129.2, 128.4, 126.8, 118.9 (d, J ) 14.7
Hz, q), 111.7 (dd, J ) 22.2, 3.8 Hz), 104.3 (t, J ) 27.0 Hz), 53.2,
38.0; HRMS (FAB) calcd for C16H15F2N2O3 (MH+) 321.1051, found
321.1074.
Sample preparation for Mass Spectrometry of the Enzyme-
Inhibitor Complex. Cathepsin B from bovine spleen (Sigma, 75 µg,
2.73 nmol) was dissolved in 200 µL of an assay buffer containing 50
mM MES, 200 mM NaCl, 5 mM EDTA, 0.15% BRIJ 35, 1mM DTT,
pH 6.0 at 4 °C, and then a 32 mM DMF solution of inhibitor 2 (4 µL,
128 nmol) was added to the mixture. After 2 h, when the enzyme was
inhibited completely, the enzyme-inhibitor complex was dialyzed at
4 °C against distilled water (4 × 800 mL) over 24 h. The solution was
concentrated to 40 µL using centrifugal filtration (Ultrafree-4, Millipore)
to give 68-µM enzyme. A control experiment was carried out in the
exact same manner, except no inhibitor was added to the enzyme
solution.
1-(2(R)-{1(S)-acetoxy-2-[2(S)-(2,4-difluoro-benzoylamino)-3-phenyl-
propionylaminooxy]-2-oxo-ethyl}-pentanoyl)-pyrrolidine-2(S)-car-
boxylic Acid Benzyl Ester (2). Diisopropylethylamine (409 µL, 2.35
mmol) was added dropwise to a mixed solution of acid 11 (940 mg,
2.32 mmol), hydroxyamide 14 (288 mg, 0.90 mmol), and PYBOP (1.21
g, 2.32 mmol) in DMF (20 mL) at -10 °C over 8 h. The reaction
mixture was stirred at room-temperature overnight. An additional
amount of diisopropylethylamine (208 µL, 1.20 mmol) was added
dropwise to the reaction mixture over 4 h. After an additional 3 h, the
solvent was evaporated under reduced pressure. Column chromatog-
raphy (CHCl3:EtOH:AcOH ) 60:1:0.2) afforded the title compound
Computational Procedures. The X-ray structure of an acyl-enzyme
complex provided the initial coordinates for the molecular dynamics
simulations (RCSB code 1GMY). Crystallographic waters were retained
and hydrogen atoms were added to the protein using the “Protonate”
program, which is part of the AMBER 759 suite of programs. AMBER
force field parameters and atomic charges were assigned to all atoms
using the parm99 set of parameters. The Sybyl program (Tripos Inc.,
St. Louis, MO) was used for the manipulation and visualization of all
structures and for the protonation of the bound ligand. The atomic
charges of the bound ligand were determined using the RESP
methodology.60 This consisted of first optimizing the molecules using
HF/6-31G*, followed by a HF/6-31G* single-point energy calculation
to determine the electrostatic potential around the molecule, which was
subsequently used in the two-stage RESP fitting procedure. The
1
(1.29 g, 78%); H NMR (500 MHz, CDCl3) δ 8.03 (m, 1H), 7.37-
7.21 (m, 11H), 7.11 (dd, J ) 12.5, 7.0 Hz, 1H), 6.97 (m, 1H), 6.84 (m,
1H), 5.16 (q, J ) 13.5 Hz, 2H, OCH2Ph), 5.10 (d, J ) 10.0 Hz, 1H,
CHOAc), 4.96 (m, 1H, CHCH2Ph), 4.61 (dd, J ) 8.5, 4.5 Hz, 1H,
CHCO2Bn), 3.76 (m, 1H, CH2NR2), 3.68 (m, 1H, CH2NR2), 3.30 (dd,
J ) 14.0, 6.5 Hz, 1H, CHCH2Ph), 3.19 (dd, J ) 14.5, 8.5 Hz, 1H,
CHCH2Ph), 3.15 (m, 1H, CHPr), 2.22 (m, 1H), 2.08-1.95 (m, 3H),
2.05 (s, 3H, ROCOCH3), 1.75 (m, 2H, CH2Et), 1.48 (m, 1H, CH2-
CH3), 1.27 (m, 1H, CH2CH3), 0.87 (t, J ) 7.3 Hz, 3H, CH2CH3); 13C
NMR (125 MHz, CDCl3) δ 171.9, 170.6, 170.2, 168.5, 167.7, 165.5
(dd, J ) 256.2, 12.1 Hz, CF), 163.3, 161.4 (dd, J ) 251.6, 12.9 Hz,
CF), 135.8, 134.1 (dd, J ) 10.2, 3.3 Hz), 129.6, 129.0, 128.8, 128.5,
128.4, 127.5, 116.5 (d, J ) 11.2 Hz, q), 112.8 (d, J ) 21.4 Hz), 104.7
(dd, J ) 28.8, 26.0 Hz), 73.4, 67.1, 59.3, 52.7, 47.7, 44.9, 37.5, 30.2,
29.5, 25.1, 20.6, 19.9, 14.3; HRMS (FAB) calcd for C37H40F2N3O9
(MH+) 708.2733, found 708.2759.
Kinetic Determinations. The fluorometric activity assays were
carried out by monitoring hydrolysis of Z-Phe-Arg-7-amido-4-meth-
ylcoumarin hydrochloride (Z-Phe-Arg-AMC, Sigma) at room temper-
ature for cathepsin B from bovine spleen (Sigma), with excitation
wavelength of 380 nm and emission wavelength of 460 nm. The assay
mixture was comprised of 50 mM MES, 200 mM NaCl, 5 mM EDTA,
0.15% BRIJ 35, 1mM DTT, pH 6.0 at 20 °C. These experiments were
performed under conditions of excess substrate concentrations.57
The dissociation constants (Ki) were calculated by the method of
Dixon.58 Two concentrations of Z-Phe-Arg-AMC were used (2 µM and
(58) Dixon, M. Biochem. J. 1953, 55, 170-171.
(59) Case, D. A.; Pearlman, D. A.; Caldwell, J. W.; Cheatham III, T. E.; Wang,
J.; Ross, W. S.; Simmerling, C. L.; Darden, T. A.; Merz, K. M.; Stanton,
R. V.; Cheng, A. L.; Vincent, J. J.; Crowley, M.; Tsui, V.; Gohlke, H.;
Radmer, R. J.; Duan, Y.; Pitera, J.; Massova, I.; Seibel, G. L.; Singh, U.
C.; Weiner, P. K.; Kollman, P. A.; AMBER 7 ed.; University of
California: San Francisco, 2002.
(60) Bayly, C. I.; Cieplak, P.; Cornell, W. D.; Kollman, P. A. J. Phys. Chem.
1993, 97, 10 269-10 280.
(61) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M.
A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery Jr., J. A.; Stratmann,
R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.; Daniels, A. D.; Kudin,
K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.; Barone, V.; Cossi, M.; Cammi,
R.; Mennucci, B.; Pomelli, C.; Adamo, C.; Clifford, S.; Ochterski, J.;
Petersson, G. A.; Ayala, P. Y.; Cui, Q.; Morokuma, K.; Malick, D. K.;
Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz,
J. V.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.;
Gomperts, R.; Marti, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng,
C. Y.; Nanayakkara, A.; Gonzalez, C.; Challacombe, M.; Gill, P. M. W.;
Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez, C.; Head-
Gordon, M.; Replogle, E. S.; Pople, J. A.; Gaussian Inc.: Pisttsburgh, PA,
1998.
(56) Ritter, A. R.; Miller, M. J. J. Org. Chem. 1994, 59, 4602-4611.
(57) Koerber, S. C.; Fink, A. L. Anal. Biochem. 1987, 165, 75-87.
9
10276 J. AM. CHEM. SOC. VOL. 126, NO. 33, 2004

Inhibition of Cathepsin B
A R T I C L E S
Gaussian 98 package60 was used to carry out all ab initio calculations.
The acyl-enzyme complex was immersed in a box of TIP3P62 water
molecules such that no atom in the acyl-enzyme complex was within
12 Å from any side of the box; after solvation of the acyl-enzyme
complex, the system consisted of a total of 65071 atoms. All bonds
involving hydrogen atoms were constrained using the SHAKE algorithm
and a 2-fs time step was used. The particle mesh Ewald63 method was
used to treat long-range electrostatics. Water molecules were first
energy-minimized and equilibrated by running a short simulation with
the acyl-enzyme species fixed using Cartesian restraints. This was
followed by a series of energy minimizations, where the Cartesian
restraints were gradually relaxed from 500 kcal/A2 to 0 kcal/A2 and
the system was subsequently slowly heated to 300 K via a 300 ps
molecular dynamics run. Another 50 ps simulation was carried out at
300 K for further equilibration. A 960-ps simulation at constant pressure
and temperature (1 atm and 300 K, respectively) was then carried out
on a 1.533 GHz Athlon processor cluster with a Myrinet switch.
Snapshots were collected every 0.2 ps.
As compounds were designed over the course of this study, they
were systematically docked into the active site of cathepsin B using a
Lamarckian genetic algorithm as implemented in the AutoDock 3.0438
program. Sybyl was used to protonate the protein and charges were
assigned using Sybyl’s Kollman charges. AM1-BCC charges were
assigned to the ligand using the antechamber module of the AMBER
package. Parameters for the docking runs were similar to those used
previously, except for the following differences:38 the quaternion step,
the translation step, and the torsion step were set to 0.2, 5, and 5,
respectively. The number of evaluations was increased to 2.5 × 106
and the ligand was fully flexible during the docking runs.
Acknowledgment. This work was supported by Grant No.
DAMD17-00-1-0496 from the U.S. Army.
Supporting Information Available: Supporting Information
Available: Synthetic protocols and characterization of inter-
mediates leading to compounds 15, 16, and 17 (PDF). This
material is available free of charge via the Internet at
http://pubs.acs.org.
(62) Jorgensen, W. L.; Chandraseklar, J.; Maduar, J. D.; Impey, R. W.; Klein,
M. L. J. Chem. Phys. 1983, 79, 926-935.
(63) Darden, T. A.; York, D. M.; Pedersen, L. G. J. Chem. Phys. 1993, 98,
10 089-10 092.
JA0489240
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