Development of cell-active non-peptidyl inhibitors of cysteine cathepsins

Article abstract of DOI:10.1016/j.bmc.2013.03.062

Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular processes, and are implicated in various types of human diseases. However, small molecule inhibitors that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and development of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the efficacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demonstrates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast carcinoma MDA-MB-231 cells.

Full text of DOI:10.1016/j.bmc.2013.03.062

Bioorganic & Medicinal Chemistry 21 (2013) 2975–2987
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of cell-active non-peptidyl inhibitors of cysteine
cathepsins
Dibyendu Dana a, Anibal R. Davalos a, Shatarupa De a, Pratikkumar Rathod a,b, Ranjith K. Gamage a,
Juliana Huestis a, Nisar Afzal a, Yuriy Zavlanov a, Suneeta S. Paroly a, Susan A. Rotenberg a,
Gopal Subramaniam a, Kevin J. Mark b, Emmanuel J. Chang b, Sanjai Kumar a,
⇑
a Department of Chemistry and Biochemistry, Queens College and the Graduate Center of The City University of New York, Queens, NY 11367-1597, USA
b Department of Chemistry, York College of The City University of New York, Jamaica, NY 11451, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Cysteine cathepsins are an important class of enzymes that coordinate a variety of important cellular
processes, and are implicated in various types of human diseases. However, small molecule inhibitors
that are cell-permeable and non-peptidyl in nature are scarcely available. Herein the synthesis and devel-
opment of sulfonyloxiranes as covalent inhibitors of cysteine cathepsins are reported. From a library of
compounds, compound 5 is identified as a selective inhibitor of cysteine cathepsins. Live cell imaging
and immunocytochemistry of metastatic human breast carcinoma MDA-MB-231 cells document the effi-
cacy of compound 5 in inhibiting cysteine cathepsin activity in living cells. A cell-motility assay demon-
strates that compound 5 is effective in mitigating the cell-migratory potential of highly metastatic breast
carcinoma MDA-MB-231 cells.
Received 16 January 2013
Revised 15 March 2013
Accepted 23 March 2013
Available online 2 April 2013
Keywords:
Cysteine protease inhibitor
Cathepsin inhibitor
Sulfonyloxiranes
Cancer metastasis
Cell migration
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
It is now well established that over-expression and aberrantly
regulated activity of cysteine cathepsins play a major role in pro-
Precisely controlled proteolysis of cellular and extracellular pro-
teins is a critically important cellular event that is elegantly
orchestrated by a large family of 561 human proteases.1 Among
these are the fifteen members of cathepsin proteases that are pri-
marily housed in membrane-bound organelles called lysosomes.
Members of the cathepsin family, classified in terms of their cata-
lytic nucleophilic residue, consist of 11 cysteine proteases (cathep-
sin B, C/J/dipeptidyl peptidase I, F, H, L, K, O, S, W, V and Z/X/P), two
serine proteases (cathepsin A and G), and two aspartyl proteases
(cathepsin D and E).2 Recent decades of research strongly suggest
that cysteine cathepsins play critically important and non-redun-
dant roles in many physiological processes, such as cell death,
the immune response, collagen degradation, and neurobiology.3,4
Furthermore, either gain or loss of function as a result of their
mis-regulation has been directly associated with a variety of hu-
man pathologies, such as cancer, osteoporosis, and autoimmune
and metabolic disorders.5–8 In addition to their importance in hu-
man biology, cysteine cathepsins are also implicated in many other
types of diseases involving lower organisms such as parasites.9,10
Consequently, they are important therapeutic targets for drug
development.
moting many hallmarks of cancer.5,11 The expression of cysteine
cathepsins (e.g., B, F, and L) has indeed been found to be signifi-
cantly upregulated in solid tumors from various origins, such as
breast, skin, colorectal, pancreatic, ovarian, brain, head and
neck.11,12 More importantly, a well-defined role for cysteine
cathepsins in promoting cell invasion and cancer metastasis has
recently emerged.13,14 Indeed high level expression of cysteine
cathepsin B was observed in invasive tumor cells derived from
metastatic cancer patients of distinct origins.15,16 Deletion of either
cysteine cathepsins, B, L or S led to decreased tumor invasion with
tumors progressively returning to a more benign form.12 In a sep-
arate study, cysteine cathepsin X was able to compensate in part
for the absence of cathepsin B in a mouse model of mammary
cancer.13 In this study, inhibition of cysteine cathepsin X with a
neutralizing antibody resulted in a drastic loss of invasive behavior
in cathepsin B null cultured cells. E-cadherin, an important adher-
ens junction protein that maintains cell–cell adhesion in epithelial
cells, was recently identified as a direct proteolytic target of
cysteine cathepsins B, L and S.12 Since it is well established that
functional loss of E-cadherin promotes cell invasion and metastatic
behavior of cancer cells, an effective inhibition of this family of
enzymes is anticipated to provide significant therapeutic benefit
in reducing metastatic potential of solid tumors.17 These studies
⇑
Corresponding author. Tel.: +1 718 997 4120; fax: +1 718 997 5531.
E-mail address: Sanjai.Kumar@qc.cuny.edu (S. Kumar).
have led to an interesting hypothesis that promotes
a
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.03.062

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D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
poly-pharmacological approach to target pro-tumorigenic mem-
bers of cysteine cathepsins collectively for the development of
effective anti-cancer agents.
CA030-cathepsin B complex,21 and (c) the sulfonyl group will fur-
ther enhance the electrophilicity of the oxiranyl carbon, compared
to a carbonyl group, thereby facilitating the entrapment of the ac-
tive site Cys residue. Upon successful demonstration of the afore-
mentioned hypotheses, this class of molecules could serve as a
new type of chemical motif on which selective cysteine cathepsin
inhibitors could be developed in future, exploiting the surrounding
S3 and S0 pockets. Such a strategy has indeed been utilized to de-
velop potent and selective inhibitors of cysteine cathepsins.25–30
A small library of sulfonyloxirane compounds was therefore
synthesized. Cysteine cathepsin B, a promoter of cancer metastasis,
was chosen as the initial inhibitory target to evaluate the efficacy
by following a time-dependent loss of enzyme activity (Table 1).
The initial screening was performed by incubating a fixed concen-
tration of compounds 1–13 with cathepsin B under a pseudo-first
While numerous studies of cysteine cathepsins have clearly
established them as important therapeutic targets for the develop-
ment of anti-cancer agents, progress towards development of
small molecule inhibitory agents that are non-peptidyl in nature
remains slow. Currently, a majority of the reported inhibitory
agents of cysteine cathepsins are peptidyl or peptidomimetic in
nature, making them poorly cell permeable (reviewed by Turk
et al.).18 Small molecule inhibitory agents of cysteine cathepsins
that are uncharged and non-peptidyl in nature are therefore desir-
able so that they can be further developed as suitable biological re-
agents, in addition to serving as lead chemotypes for drug
development. Furthermore, if the mechanism of inactivation could
be designed to produce a covalent and irreversible chemistry with
the catalytic active site Cys residue, then the inhibitors could also
be developed as activity-based probes (ABPs) for the functional
analysis of cysteine proteases in complex proteomes. Activity-
based probes have found extensive applications in functional
annotation of enzymes in their native biological environments.19
order condition.
A small aliquot of the incubation mixture
was withdrawn at a fixed time interval and the remaining
cathepsin B activity was measured in a large volume of assay mix-
ture (17.5-fold dilution) containing cathepsin B substrate. The IC50
values were deliberately not estimated for this library, since they
are not considered a good yardstick for measuring a time-depen-
dent irreversible loss of enzyme activity due to a non-equilibrium
binding mechanism.31
2. Results and discussion
Compounds 1 and 2 which bear a non-polar 4-fluorophenyl and
a propyl substituent respectively at the second carbon of the oxi-
rane ring were non-inhibitory. This result indicated that a second
substitution at the oxirane moiety in this class of molecules was
perhaps detrimental to their inhibitory efficacies. Furthermore,
compound 12, which incorporates a methylene linker between
the arylsulfonyl group and oxirane moiety, was also inert. We sur-
mised that the lack of reactivity of 12 could presumably be due to
lack of assistance from a general acid appropriately positioned in
the cathepsin B active site to promote nucleophilic opening of oxi-
In this study, we synthesized a library of compounds containing
sulfonyloxirane moiety in the context of a small non-peptidyl mol-
ecule and screened for their efficacy as time-dependent inhibitors
of cysteine cathepsins. Compound 5 was identified as the lead
inhibitory agent of cysteine cathepsin B. Enzymology and mass
spectrometry-based experiments were performed to demonstrate
that this class of molecules inhibited cysteine cathepsin B in a
covalent and irreversible manner. Importantly, 5 was shown to
be effective in inhibiting intracellular activity of cysteine
cathepsin B, and the cell migratory behavior of highly metastatic
human breast carcinoma MDA-MB-231 cells.
rane ring by Cys29-S residue. So, a thiirane analogue, 13, that
c
potentially contains a better leaving group thiolate (compared to
alkoxide in 12), was synthesized, and was also found to be ineffec-
tive. This again indicated that appropriate positioning of arylsulfo-
nyl group with respect to the reactive oxirane group within the
cysteine cathepsin active site was critically important for its inhi-
bition. Efforts then became focused on synthesizing analogues of
2-(arylsulfonyl)oxiranes where functional variations of the aryl
ring was further investigated.
Among the synthesized library, compound 5 was found to be
most effective in inhibiting cysteine cathepsin B activity. The effec-
tiveness of compound 5 was evident from a progressive loss of en-
zyme activity when it was incubated with active cathepsin B
(Fig. 1A). No gain in cathepsin B activity was observed even after
dilution of 5–inactivated cathepsin B complex, thereby indicating
that the mechanism of inhibition was perhaps covalent and irre-
versible in nature (data not shown). To assess the inhibitory effi-
cacy, inactivation experiments were performed at appropriate
concentrations of compound 5 under pseudo-first order conditions,
and the experimental data thus obtained were analyzed using a
simple two-step inhibition model (Fig. 1B):
2.1. Design and synthesis of compounds 1–13 and time-
dependent inhibitory evaluation
The advent of E-64 from Aspergillus japonicus, an epoxysuccinyl-
derived inhibitory agent, was a critically important stepping stone
for deciphering the undocumented function of cysteine cathepsins
family of enzymes.20 Although, E-64 is a generic non-selective
inhibitor of cysteine cathepsins, structural investigation of the
E-64-inhibited complex have subsequently led to the development
of selective cysteine cathepsin B inhibitors (e.g., CA030 and
CA074).21,22 The marked success of E-64 and its subsequent ana-
logues as effective inhibitors of cysteine cathepsins relies on the
exclusive reactivity of an active site cysteine residue with the elec-
trophilic ‘warhead’ epoxy group. The catalytic mechanism of cys-
teine cathepsins involves participation of this invariant and
reactive cysteine residue.23 A close visual inspection of the struc-
tures of CA030 and CA074 in complex with cysteine cathepsins re-
vealed that there exist well-defined binding pockets, S2 and S10,
surrounding the S1 site (Schechter and Berger nomenclature)24
that prefer to accommodate aromatic hydrophobic groups.21,22 It
was therefore hypothesized that an arylsulfonyl-substituted oxira-
nyl moiety could serve as a small molecule covalent inactivator of
cysteine cathepsins, targeting the key residues of the core enzyme
active site. This approach envisions that (a) an appropriate binding
of inhibitory compounds within the active site of cysteine cathep-
sin will allow the reactive Cys residue to be entrapped by the
activated sulfonyloxirane group, (b) the sulfonyl group will
likely mimic the potential H-bonding interactions that the car-
Ki
ki
E þ I ꢀ E ꢀ I ! E ꢁ I
where Ki represents the reversible equilibrium binding constant for
the first step, and ki is the first order inactivation rate constant for
the second irreversible step. To obtain these two parameters, the
following procedure was adopted. The pseudo-first order rate con-
stants of inactivation (kobs) were obtained at appropriate concentra-
tion of 5 ([I] ꢂ [cathepsin B]), and a Kitz–Wilson analysis was
performed to fit the experimentally obtained data (Fig. 1C).32 This
procedure yielded the equilibrium binding constant (Ki = 86
bonyl group
a to the epoxy moiety exhibits in the inhibited
3 lM) for the first equilibrium binding step, and the first order

D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
2977
Table 1
General chemical structure and time-dependent inhibitory screen of human liver cathepsin B by the synthesized sulfonyloxirane library compounds
R
X
)
H
C
(
O
S
Ar
O
General Chemical Structure
Compound ID
Chemical structure (name)
% Inhibition
F
O2
S
1
O
NIa
NIa
44
2-(4-fluorophenyl)-3-(phenylsulfonyl) oxirane
O2
S
O
2
3
2-(phenylsulfonyl)-3-propyloxirane
O2
S
O
Cl
2-((4-chlorophenyl)sulfonyl)oxirane
O2
S
O
4
5
30
F3CO
2-(4-(trifluoromethoxy)phenylsulfonyl)oxirane
O2
S
O
>98
2-(2-ethylphenylsulfonyl)oxirane
O2
S
O
6
7
40
42
2-(m-tolylsulfonyl)oxirane
O
O S O
2-(2,5-dimethylphenylsulfonyl)oxirane
O2
S
O
8
9
35
OCH3
2-(3-methoxyphenylsulfonyl)oxirane
O2
H3CO
S
O
25
OCH3
2-(2,5-dimethoxyphenylsulfonyl)oxirane
(continued on next page)

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D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
Table 1 (continued)
Compound ID
Chemical structure (name)
% Inhibition
O2
S
Cl
O
10
50
Cl
2-(3,5-dichlorophenylsulfonyl)oxirane
O2
S
O
11
12
13
18
2-(naphthalen-3-ylsulfonyl)oxirane
O2
S
O
NIa
2-((phenylsulfonyl)methyl)oxirane
O2
S
S
NIa
2-((phenylsulfonyl)methyl)thiirane
The initial screening for time-based inactivation was performed at 500
l
M inhibitory concentrations under pseudo-first order condition ([I] ꢂ [E]; cathepsin B = 175 nM). The
enzyme activity was measured using a standard cathepsin B substrate after 10 min of incubation, as described in the enzyme assay protocol section.
a
NI: No measurable time-dependent inhibition.
inactivation rate constant (ki = 0.21 0.01 minꢁ1) for the second
interactions (2.8 Å) with a tightly-bound active site water molecule
irreversible step. We further investigated if the time-dependent
inhibition of cathepsin B by 5 could be alleviated in the presence
of a known active site-directed cathepsin B inhibitor, leupeptin
(Ki = 5 nM).33 This is mainly because if inhibitor 5 were to be an ac-
tive-site directed agent, the rate of 5-mediated cathepsin B inactiva-
tion reaction would be retarded in presence of a competitive
inhibitor competing for the binding to the same active site.34 A pro-
tection from inhibition of cathepsin B was indeed observed in pres-
ence of leupeptin, thereby indicating that 5 occupied the core
cathepsin B active site that harbors the low pKa nucleophilic
Cys29 residue (Fig. 1D). A MALDI-based mass spectrometric analy-
sis of the 5–inhibited cathepsin B complex showed the expected
mass increase of 212.1 amu in the cathepsin B protein. No further
mass increase was observed even after prolonged incubation of 5
with cathepsin B. This result indicated that the mode of inhibition
by 5 was indeed covalent and irreversible in nature and the stoichi-
ometry of inhibition was likely 1:1 (Fig. 1E). This was important to
establish since most cysteine cathepsins do contain multiple cyste-
ines, in addition to the nucleophilic active site cysteine residue.18
(Wtr12). The OH group of inhibitor was also stabilized with two
h-bond interactions, one with the backbone carbonyl of a S2 site
residue Gly198 (3.1 Å) and the other with an active site water
(Wtr56) molecule (2.5 Å). The phenyl moiety of the inhibitor was
found to be involved in a hydrophobic interaction with a non-polar
patch containing Met196, Gly197, and Gly198 residues of S2
pocket. In addition, the ortho-ethyl group interacted with CH–
p
interactions with the aromatic side chains of Trp221 and His110
from S20 pocket, albeit weakly.35 A conclusive design of future
inhibitory ligands that are selective towards individual members
of the cysteine cathepsin family will require a structural elucida-
tion of 5-inhibited cysteine cathepsin complex using X-ray crystal-
lography. Our modeling study suggests that ortho-substituted aryl
groups containing a negatively charged group could prove to be
selective inhibitory agents towards cathepsin B. Notably,
cathepsin B is a unique member of cysteine cathepsins in that it
exhibits endopeptidase, peptidyldipeptidase and carboxypeptidase
activities.36–38 This broad substrate specificity of cathepsin B has
been ascribed to its unique structural feature that contains a flex-
ible occluding loop (residues 104–126). Since His110 and His111
are part of this occluding loop, securing potential interactions from
this loop could result in enhanced selectivity and potency towards
cathepsin B. Such features could be exploited in designing selective
inhibitors of cathepsin B.
2.2. Computational analysis of 5–inhibited cysteine cathepsin B
complex
To understand the potential molecular interactions of 5 with
cysteine cathepsin B in the covalently inhibited complex, computa-
tional modeling studies were performed. The CA073-inhibited
complex of cathepsin B (PDB id: 2DC6) was appropriately modified
2.3. Compound 5 is inert to external nucleophile and inhibits
cysteine cathepsins selectively
to incorporate the inhibitor 5 covalently linked to Cys29-S of
c
cathepsin B active site. A molecular dynamic simulation (100 ps)
was performed on the inhibitory complex and the trajectories of
the molecular dynamics simulation were analyzed. Several interac-
tions between the inhibitor 5 and cathepsin B active site were
noteworthy (Fig. 2). While the O3 of sulfonyl oxygen formed a
To evaluate if the presence of an external nucleophile
could compromise the inhibitory efficacy of 5, we carried out
cathepsin B inactivation kinetics in the presence of a strong nucleo-
phile, azide ion (1 mM). No measurable change in inactivation kinet-
ics was evident thereby indicating that compound 5 is relatively
inert to an external nucleophile and unleashes its covalent reactivity
only upon binding to the cathepsin B active site (data not shown).
h-bond (3.4 Å) with the side chain of Gln23-N —a key oxyanion
e
hole residue, its O2 atom was engaged in a robust hydrogen bond

D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
2979
Figure 1. The mechanistic investigation of 5-mediated cathepsin B inhibition. [A] Progress curves indicating a time-dependent loss of cathepsin B activity. Compound 5 was
incubated with cathepsin B under pseudo-first order condition, and the progress curves were obtained at appropriate times of inactivation (red: 0 min, blue: 11 min, green:
17 min, black: 25 min, and pink: 41 min) using a chromogenic substrate Z-RR-para-nitroanilide. [B] Concentration-dependent loss of Cathepsin B activity with time. The
points are experimental, and the lines joining them are fitted to obtain the pseudo-first order inactivation rate constants (kobs). [C] Kitz–Wilson plot of 5-mediated cathepsin B
inactivation kinetics. The data points are fitted to a line that yields (a) Ki = 86
3 l
M for the first equilibrium step of inhibitor binding, and (b) ki = 0.21 0.01 minꢁ1 for the
first order inactivation rate constant for the second step. [D] The rate of 5-mediated cathepsin B inhibition is decreased in the presence of leupeptin (40 nM; blue line), a
known competitive inhibitor of cathepsin B, compared to the control (5% DMSO; red line). This experiment suggests that 5 is an active site-directed competitive inhibitor. [E]
A mass spectrometric analysis of 5–inhibited cathepsin B complex. A mass difference of 212.1 amu, consistent with the molecular weight of 5 (212.1), is observed in singly
charged 5–inhibited cathepsin B complex (lower panel) compared to uninhibited cathepsin B enzyme (upper panel). This experiment demonstrates that the mechanism of
5-mediated cathepsin B inhibition is covalent and irreversible in nature.

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D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
Figure 2. Stereoscopic view of the energy-minimized structure of covalently modified 5–cathepsin B complex. The low pKa active site Cys29 residue is the site for 5-mediated
covalent modification. For clarity, only the inhibitor 5 (black) and the key cathepsin B active site residues are shown. The key H-bonding distances of 5 with cathepsin B are
shown in green. The intramolecular H-bonding interactions among cathepsin B active site residues near the inhibitor binding site are depicted in red (heavy atom h-bond
donor and acceptor atoms cut off is 3.6 Å). The position of the primed and non-primed binding pockets on cathepsin B surface is shown in green text with underline.
The selectivityof inhibition ofcompound 5 towards cysteinecathep-
sins was investigated next. To do so, a panel of distinct family of en-
zymes were chosen and their inhibition by 5 was assessed (Table 2).
While inhibition of cathepsin B by 5 was about 1.9-fold more selec-
tive compared to other two prominent members of cysteine cathep-
sins, namely cathepsin F and cathepsin S, an aspartyl-utilizing
cathepsin D was only weakly (1/38th fold compared to cysteine
cathepsin B) inhibited. Cathepsin G and trypsin protease that use a
Ser nucleophile for catalysis remained inert to inhibition. These
experiments suggested that compound 5 is selective towards cys-
teine cathepsins that utilize the uniformly conserved and reactive
Cys residue for catalysis. The ability of compound 5 to inhibit protein
tyrosine phosphatases (PTPs) was also evaluated. This enzyme uti-
lizes a low pKa Cys residue in nucleophilic catalysis. Although 2-
(arylsulfonyl)-oxiranes were previously reported to be modest
inhibitors of PTPs,39 this enzyme also remained inert to inhibition
by 5. No apparent inhibition was observed with the prototypical
PTP, hPTP1B, even at a concentration as high as 1 mM and after a pro-
longed incubation of one hour. While a conclusive analysis of this
selectivity pattern towards cysteine cathepsins will require struc-
tural elucidation of the 5–inhibited cathepsin B complex, it appears
that the presence of an ethyl group at the ortho position of 2-(phen-
ylsulfonyl)oxirane is significantly favored for binding at the cysteine
cathepsin active site.
2.4. Compound 5 is cell permeable and inhibits intracellular
cysteine cathepsin activity in metastatic human breast
carcinoma MDA-MB-231 cells
Before proceeding to evaluate the cellular efficacy of 5 to inhibit
cysteine cathepsins in human cells, it was important to establish
its hydrolytic stability at physiological pH. This consideration
was prompted by the oxirane functionality in 5 which could be-
come hydrolytically unstable as a result of the adjacent electron-
withdrawing sulfonyl moiety. This could render the inhibitor
unsuitable for biological applications. Using NMR-based experi-
ments, the half-life of 5 at physiological pH (pH 7.4) was found
to be 3.6 h (Supplementary data, Fig. S1). This study suggested that
compound 5 was relatively stable at physiological pH, and there-
fore could be used as an important tool in biological applications.
Cysteine cathepsins play important functional roles in both
intracellular and extracellular signaling events.40 Contrary to pop-
ular belief, recent studies strongly suggest that within the cell, ac-
tive cysteine cathepsins play regulatory roles in distinct cellular
compartments other than lysosomes, such as nucleus, mitochon-
drial matrix, cytoplasm, and plasma memebrane.4,41,42 For exam-
ple, a mis-regulated and an enhanced proteolytic activity of
cytoplasmic cysteine cathepsins have shown to be linked to tumor
invasion.40 Indeed, the invasion potential of both human mela-
noma and prostate carcinoma cells was found to be significantly
reduced only when these cells were subjected to cell membrane
permeable cysteine cathepsin inhibitors, thereby suggesting the
importance of intracellular cathepsins and their effective inhibi-
tion.43 Similarly, a study by Sloane and co-workers in human
breast carcinoma BT549 cells suggested that only cell-permeable
cysteine cathepsin inhibitors were able to reduce the intracellular
proteolysis in these cells.44 These investigations together under-
score the importance of developing non-peptidyl and cell perme-
able small molecule inhibitors of cysteine cathepsins, so that
their precise function can be annotated in both normal physiology
and human diseases. Unfortunately, cell-permeable and non-
peptidyl inhibitory agents of cysteine cathepsins remain scarcely
available today.
Table 2
Selectivity of inhibition of compound 5 towards cysteine cathepsins as assessed by
the 2nd order enzyme inactivation rate constants
Enzyme (enzyme family)
2nd Order inactivation
Rate constant (Mꢁ1 minꢁ1
)
Cathepsin B (cysteine cathepsin)
Cathepsin F (cysteine cathepsin)
Cathepsin S (cysteine cathepsin)
Cathepsin D (aspartyl cathepsin)
Cathepsin G (serine cathepsin)
Trypsin (serine protease)
2441 202a
1300 208b
1196 96b
65 5b
NIc
NIc
hPTP1B (protein tyrosine phosphatase)
NIc
a
Calculated from the ki and Ki parameters obtained using Kitz–Wilson analysis.
Determined by measuring pseudo-first order rate constant of inactivation kobs
Thus in an initial approach, we assessed whether 5 had the po-
tential to inhibit intracellular cysteine cathepsin B activity in living
cells. The metastatic MDA-MB-231 human breast cell line was cho-
sen for this study since it has been shown that cysteine cathepsin B
b
first and dividing the kobs by the inhibitor concentration.
c
NI: No apparent time-dependent inhibition even at a concentration as high as
1 mM during a 1-h inactivation reaction.

D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
2981
activity is upregulated in these cells.45 A highly efficient and spe-
cific cathepsin B substrate, Z-RR-AMC (kcat/Km = 105 Mꢁ1 sꢁ1), pre-
viously employed in cell-based experiments, was chosen for this
study.46,47 While efficient proteolysis of the substrate was evident
in control MDA-MB-231 cells, a significant loss of intracellular
cathepsin B activity was observed in 5-treated cells (Fig. 3A). Fur-
thermore, intracellular cathepsin B activity was shown to decrease
in a dose-dependent manner (Fig. 3B).
In a second approach, the usefulness of 5 to inhibit cellular cys-
teine cathepsin activity in cells was also investigated using a phys-
iologically relevant proteolytic protein target of cysteine cathepsin
B and S, namely E-cadherin, an adherens junction protein.12 The
integrity of this epithelial adhesion protein is critically important
in maintaining both intercellular contacts and cellular morphol-
ogy.48–50 While it has been shown that a mere loss of cell–cell
contacts alone is not sufficient to promote metastasis, a loss of
E-cadherin alone has been shown to be sufficient to induce this
behavior through epithelial-to-mesenchymal transition (EMT),
enhanced motility, resistance to apoptosis, and invasiveness.51
The majority of human tumors are carcinomas that originate from
cells of epithelial tissues where a complete or a partial loss of
E-cadherin occurs as the tumor progressively becomes malig-
nant.52–54 Thus, the loss of E-cadherin has been directly linked to
invasive migratory behavior of cancer cells.51,55 In a study involv-
ing metastatic MDA-MB-231 breast cancer cells which expresses
very low levels of E-cadherin,54 the re-expression of E-cadherin
led to the restoration of normal E-cadherins-mediated cell–cell
junctions, and a reversion to a normal epithelial morphology.56
We therefore evaluated the levels of E-cadherin in MDA-MB-231
cells using an E-cadherin antibody in the presence and absence
of inhibitor 5. As anticipated, a dose-dependent increase in the lev-
els of E-cadherin was observed in a population of 5-treated MDA-
MB-231 cells, as compared to control cells treated only with DMSO
(Fig. 4A and B). It should also be noted that the overall cellular
morphology of metastatic MDA-MB-231 cells also changed upon
inhibitory treatment (Figs. 3A and 4A). This perhaps is not too
Figure 3. Effect of inhibitor 5 on cathepsin B activity in breast cancer MDA-MB-231 cells. [A] Cells were incubated with vehicle (DMSO 0.1%) and 20
followed by an additional 10 min of incubation with the substrate Z-RR-AMC (25 M net) of 5. Images show a significant loss of signal intensity (green) from the enzymatic
turnover of the fluorophore cathepsin B substrate Z-RR-AMC (shown by red arrow in the bottom panel) at 20 M of 5. The control experiment without 5 indicates that a high
level of substrate turnover occurs in these cells, thereby indicating a robust cathepsin B activity (white arrow—top panel). Scale bar = 10 m. [B] The dose-dependent
lM of 5 for 20 min,
l
l
l
inhibition of intracellular cysteine cathepsin B activity with inhibitor 5 as measured by average fluorescence intensity over the entire cell. Note: The fluorescent intensity
values are mean S.D. calculated over a population of at least 5–8 cells for each condition. ⁄Statistical significance was evaluated by the Student’s t-test.

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D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
Figure 4. A physiological substrate of cysteine cathepsins, E-cadherin, is enriched in 5-treated MDA-MB-231 breast cancer cells. [A] E-cadherin expression levels as detected
by immunofluorescence in MDA-MB-231 cells. Cells were treated with control (0.1% DMSO) and cysteine cathepsin-B inhibitor 5 (20 M) for 20 min, fixed, and then stained
l
for E-cadherin. Bright field and merged fluorescence images of E-cadherin (red) and nuclear stain Hoechst 33342 (blue signal) are shown. Green and yellow arrows show
enhanced E-cadherin levels (red) near the cell periphery (green arrow) and perinuclear region (yellow arrow) respectively in 5-treated cell, compared to control (0.1% DMSO).
Scale bar = 10 lm. [B] A dose-dependent increase of E-cadherin expression levels from immunocytochemistry as measured by average fluorescence intensity over the cell-
periphery. The fluorescence intensity values are mean S.D. calculated over a population of 28 cells for each condition. ⁄Statistical significance was evaluated by the Student’s
t-test.
surprising since cellular enrichment of E-cadherin is known to
have a direct impact on cellular morphology.48,49 Further experi-
ments will be required to investigate the molecular basis of altered
cellular morphology in MDA-MB-231 cells upon inhibitory treat-
ment. A Western blot analysis of 5-treated MDA-MB-231 cells
was also performed that exhibited an enrichment of E-cadherin
levels in 5-treated cells, as compared to the control cells (see Sup-
plementary data, Fig. S2). Collectively, these studies demonstrated
that 5 was effective in inhibiting the intracellular activity of cys-
teine cathepsins in the invasive MDA-MB-231 breast cancer cells.
Finally to demonstrate that inhibition of cysteine cathepsins by
5 has a functional relevance in metastatic MDA-MB-231 breast
cancer cells, a study involving cell migration behavior was planned.
An enrichment of E-cadherin levels, as a result of inhibition of cys-
teine cathepsins by 5, is anticipated to exhibit a reduced cell migra-
tory behavior in a metastatic cancer cell line.57,58 Thus, a cell
MDA-MB-231 cells. As anticipated, a dose-dependent decrease in
cell migratory behavior of metastatic MDA-MB-231 cells was
observed (Fig. 5). This experiment supported the notion that the
compound 5 was effective in inhibiting the cell migratory potential
of these highly metastatic cells.
3. Conclusions
We have developed a new class of small molecule inhibitors of
cysteine cathepsins. A detailed kinetic and mass spectrometric
analysis was performed to determine the kinetic parameters and
mode of inhibition. The covalent inhibition of compound 5 was
found to be selective towards cysteine cathepsins. The mechanisti-
cally distinct enzymes, such as aspartyl cathepsin D, serine cathep-
sin G, serine protease trypsin and a low pKa cysteine-containing
enzyme protein tyrosine phosphatase 1B, were not inhibited
by 5. Molecular modeling studies of the 5–inhibited cysteine
cathepsin B complex indicated that 5 interacted favorably with
motility assay was carried out in presence (10 and 20
lM) and
absence (control, 0.05% DMSO) of inhibitor 5 using metastatic

D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
2983
involved a 1-h long reaction of 4-fluorobenzaldehyde with (chloro-
methylsulfonyl)benzene at room temperature in the presence of a
phase transfer catalyst, tetra-n-butylammonium bromide
(10 mol %), in toluene and potassium hydroxide solution. The final
purification was performed using silica gel-based flash chromatog-
raphy procedure that yielded compound 1 as a white solid.
Rf = 0.28 Hexane:EtOAc (9:1). Yield: 90%. 1H NMR (400 MHz,
CDCl3) d ppm: 4.16 (d, J = 1.4 Hz, 1H), 4.58 (d, J = 1.4 Hz, 1H), 7.06
(m, 2H), 7.24 (m, 2H), 7.64 (m, 2H), 7.75 (m, 1H), 7.99 (m, 2H).
13C NMR (125 MHz, CDCl3); d ppm 56.94, 70.95, 116.11 (d,
JC–F = 22.3 Hz, 2C intensity), 128.00 (d, JC–F = 8.5 Hz, 2C intensity),
128.53 (d, JC–F = 3.3 Hz, 1C), 128.88 (2C intensity), 129.56 (2C
intensity), 134.70, 136.77, 163.49 (d, JC–F = 250 Hz, 1C intensity).
LRMS [ESI-MS +ve] calculated for (C14H11FO3S+Na): 301.03, ob-
served: 301.25.
4.1.3. 2-(Phenylsulfonyl)-3-propyloxirane (2)
This compound was synthesized using the same synthetic pro-
tocol as outlined in the synthesis of 1. The final purification was
performed using silica gel-based flash chromatography procedure
that yielded compound 2 as a viscous oil. Rf = 0.22 Hexane:EtOAc
Figure 5. The effect of cysteine cathepsin inhibitor 5 on the cell-migratory behavior
of metastatic human breast cancer MDA-MB-231 cells. The cell migration pheno-
type was reduced upon treatment of cells with compound 5 (10 and 20 lM),
compared to control (0.05% DMSO). Each reported change in area value is the
average of triplicate measurements with the corresponding standard deviation
value.
(9:1). Yield: 85%. 1H NMR (500 MHz, CDCl3)
d ppm: 0.94
(t, J = 7.4 Hz, 3H) 1.47 (m, 2H), 1.57 (m, 1H), 1.66 (m, 1H), 3.63
(m, 1H), 3.90 (d, J = 1.6 Hz, 1H), 7.58 (m, 2H), 7.68 (m, 1H), 7.91
(m, 2H). 13C NMR (125 MHz, CDCl3) d ppm: 13.63, 18.93, 32.22,
57.79, 68.30, 128.69 (2C intensity), 129.41 (2C intensity), 134.43,
137.08. LRMS [ESI-MS +ve] calculated for (C11H14O3S+Na):
249.06, observed: 249.25.
the key active site residues surrounding S1 and S2 pockets. The
inhibitor 5 was shown to be stable to spontaneous hydrolysis,
cell-permeable, and effective in inhibiting intracellular cysteine
cathepsin B activity in metastatic human breast carcinoma MDA-
MB-231 cells. We also demonstrated that compound 5 was effec-
tive in reducing the migratory potential of the metastatic MDA-
MB-231 breast cancer cell lines. This class of inhibitory agents
has potential applications in the construction of selective activ-
ity-based cysteine cathepsin probe, and towards the development
of specific inhibitory agents for the individual members of cysteine
cathepsins.
4.1.4. 2-(4-Chlorophenylsulfonyl)oxirane (3)
This compound was synthesized by following an earlier pub-
lished protocol.39 The final purification was performed using silica
gel-based flash chromatography procedure that yielded compound
3 as a white powder. Rf = 0.50 (hexane/EtOAc = 9/1). Yield: 63%. 1H
NMR (400 MHz, CDCl3) d ppm: 3.07 (dd, J = 5.4 and 3.7 Hz, 1H),
3.37 (dd, J = 5.4 and 2.1 Hz, 1H), 4.05 (dd, J = 3.7 and 2.1 Hz, 1H),
7.52 (d, J = 8.6 Hz, 2H), 7.81 (d, J = 8.6 Hz, 2H). 13C NMR
(100 MHz, CDCl3) d ppm: 45.54, 63.25, 129.84 (2C intensity),
130.31 (2C intensity), 135.25, 141.55. LRMS [ESI-MS +ve] calcu-
lated for (C8H7ClO3S+H): 218.99, observed: 219.00.
4. Experimental section
4.1. Synthesis
4.1.1. General
4.1.5. 2-((4-Trifluoromethoxy)phenylsulfonyl)oxirane (4)
This compound was synthesized by following a published pro-
tocol.39 The final purification was performed using silica gel-based
flash chromatography procedure that yielded compound 4 as
white semi-solid. Rf = 0.29 (hexane/EtOAc = 19/1). Yield: 40%. 1H
NMR (400 MHz, CDCl3) d ppm: 3.16 (dd, J = 5.3 and 3.6 Hz, 1H),
3.46 (dd, J = 5.3 and 2.0 Hz, 1H), 4.14 (dd, J = 3.6 and 2.0 Hz, 1H),
7.43 (d, J = 8.8 Hz, 2H), 8.01 (d, J = 8.8 Hz, 2H). 13C NMR
(100 MHz, CDCl3) d ppm: 45.58, 63.24, 120.20 (q, JC–F = 283 Hz),
121.10 (2C intensity), 131.22 (2C intensity), 134.99, 153.71. LRMS
[ESI-MS +ve] calculated for (C9H7F3O4S+H): 269.01, observed:
269.00.
13C NMR and 1H spectra were recorded on a Brucker DPX
400 MHz FT NMR with automatic sample changer. Chemical shifts
(d) are reported in parts per million (ppm) and referenced to resid-
ual solvent peaks in the deuterated solvent, CDCl3 (7.26 ppm for 1H
and 77.0 ppm for 13C), DMSO (2.50 ppm for 1H and 39.5 ppm for
13C), (CD3)2CO (2.05 ppm for 1H and 29.9, 206.7 for 13C) and CD3OD
(4.87, 3.31 for 1H and 49.1 for 13C). Coupling constants (J) are re-
ported in Hertz (Hz) and multiplicities are abbreviated as singlet
(s), doublet (d), doublet of doublets (dd), triplet (t), triplet of dou-
blets (td), and multiplet (m). The following materials were pur-
chased from Matrix Scientific (Columbia, SC, USA) m-thiocresol,
3-methoxybenzenethiol, 3,5-dichlorothiophenol, 2,5-dimethylthi-
ophenol, 4-(trifluoromethoxy)thiophenol, 2-ethylthiophenol and
2,5-dimethoxythiophenol. Thiophenol, 2-naphthalenethiol, chloro-
methyl phenyl sulfone, 4-fluorobenzaldehyde, butyraldehyde,
disodium salt of silica gel (Merck, grade9385, 230–400 mesh,
60 Å) were purchased from Sigma–Aldrich (St. Louis, MO, USA). So-
dium hydroxide, hydrochloric acid, sodium chloride, sodium sul-
fate, ammonium chloride, and solvents were purchased from
Fisher Scientific Inc. (Pittsburgh, PA, USA).
4.1.6. 2-(2-Ethylphenylsulfonyl)oxirane (5)
This compound was synthesized by following a published pro-
tocol.39 The final purification was performed using silica gel-based
flash chromatography procedure that yielded compound 5 as a
pale yellow viscous oil. Rf = 0.28 (hexane/EtOAc = 19/1). Yield:
72%. 1H NMR (400 MHz, CDCl3) d ppm: 1.32 (t, J = 7.4 Hz, 3H),
3.09 (q, J = 7.4 Hz, 2H), 3.11 (dd, J = 5.4 and 3.7 Hz, 1H), 3.40 (dd,
J = 5.4 and 2.1 Hz, 1H), 4.19 (dd, J = 3.7 and 2.1 Hz, 1H), 7.42 (m,
2H), 7.61 (td, J = 7.7 and 1.2 Hz, 1H), 7.99 (dd, J = 8.0 and 1.2 Hz,
1H). 13C NMR (100 MHz, CDCl3) d ppm: 16.15, 26.59, 44.87,
63.65, 126.64, 130.81, 131.33, 134.57, 134.92, 145.86. LRMS [ESI-
MS +ve] calculated for (C10H12O3S+H): 213.06, observed: 213.08.
4.1.2. 2-(4-Fluorophenyl)-3-(phenylsulfonyl)oxirane (1)
The synthesis of this compound was achieved in a single step by
appropriate modification of a published protocol.59 The procedure

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D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
4.1.7. 2-(m-Tolylsulfonyl)oxirane (6)
1.8 Hz, 1H), 7.96 (d, J = 8.6 Hz, 1H), 8.03 (dd, J = 8.4 and 1.0 Hz, 1H),
8.06 (d, J = 8.4 Hz, 1H), 8.53 (d, J = 1.0 Hz, 1H).13C NMR (100 MHz,
CDCl3) d ppm: 45.43, 63.39, 123.09, 127.85, 128.06, 129.60,
129.71, 129.76, 131.02, 132.20, 133.69, 135.75. LRMS [ESI-MS +ve]
calculated for (C12H10O3S+H): 235.05, observed: 235.08.
This compound was synthesized by following a published pro-
tocol.39 The final purification was performed using silica gel-based
flash chromatography procedure that yielded compound 6 as a col-
orless viscous oil. Rf = 0.39 (hexane/EtOAc = 9/1). Yield: 70%. 1H
NMR (400 MHz, CDCl3) d ppm: 2.46 (s, 3H), 3.12 (dd, J = 5.4 and
3.7 Hz, 1H), 3.41 (dd, J = 5.4 and 2.0 Hz, 1H), 4.13 (dd, J = 3.7 and
2.0 Hz, 1H), 7.49 (m, 2H), 7.75 (s, 2H). 13C NMR (100 MHz, CDCl3)
4.1.13. 2-((Phenylsulfonyl)methyl)oxirane (12)
This compound was synthesized by following the literature-re-
ported protocol60 and its characterization was found to be consis-
tent with the previously reported NMR spectra.61
d
ppm: 21.34, 45.35, 63.25, 125.92, 129.06, 129.33, 135.39,
136.66, 139.82. LRMS [ESI-MS +ve] calculated for (C9H10O3S+H):
199.05, observed: 199.25.
4.1.14. 2-((Phenylsulfonyl)methyl)thiirane (13)
This compound was synthesized by following the literature-re-
ported protocol60 and its characterization was found to be consis-
tent with the previously reported NMR spectra.62
4.1.8. 2-(2,5-Dimethylphenylsulfonyl)oxirane (7)
This compound was synthesized by following an earlier pub-
lished protocol.39 The purification using silica gel column chroma-
tography yielded 7 as colorless viscous oil. Rf = 0.32 (hexane/
EtOAc = 9/1). Yield: 68%. 1H NMR (400 MHz, CDCl3) d ppm: 2. 39
(s, 3H), 2.67 (s, 3H), 3.10 (dd J = 5.3 and 3.7 Hz, 1H), 3.88 (dd,
J = 5.3 and 2.1 Hz, 1H), 4.18 (dd, J = 3.7 and 2.1 Hz, 1H), 7.25 (d,
J = 7.5 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H), 7.79 (s, 1H). 13C NMR
(100 MHz, CDCl3) d ppm: 20.33, 20.80, 44.68, 63.36, 131.03,
132.83, 134.87, 135.22, 136.31, 136.78. LRMS [ESI-MS +ve] calcu-
lated for (C10H12O3S+H): 213.06, observed: 213.08.
4.2. Enzymology, physiochemical, and mass spectrometric
studies
4.2.1. General
Following enzymes and their substrates were purchased from
Enzo Life Sciences (Farmingdale, NY, USA): cathepsin B (BML-
SE198), cathepsin
D (BML-SE199), cathepsin S (BML-SE453),
cathepsin F (BML-SE568), cathepsin G (BML-SE283), human PTP1B
(BML-SE332), Z-Arg-Arg-pNA (BML-P138), Z-Arg-Arg-AMC (BML-
P137), Z-Phe-Arg-AMC (BML-P139), Suc-Ala-Ala-Pro-Phe-pNA
4.1.9. 2-(3-Methoxyphenylsulfonyl)oxirane (8)
This compound was synthesized by following a published pro-
tocol.39 The purification using silica gel column chromatography
yielded 8 as a brownish viscous oil. Rf = 0.27 (hexane/EtOAc =
9/1). Yield: 70%. 1H NMR (400 MHz, CDCl3) d ppm: 3.13 (dd,
J = 5.4 and 3.7 Hz, 1H), 3.42 (dd, J = 5.4 and 2.0 Hz, 1H), 3.89 (s,
3H), 4.13 (dd, J = 3.7 and 2.0 Hz, 1H), 7.23 (ddd, J = 7.6, 2.5, and
1.9 Hz, 1H), 7.42 (dd, J = 1.9 and 1.6 Hz, 1H), 7.52 (m, 2H). 13C
(BML-P141), Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-D-
Arg-NH2 (BML-P145), Z-Val-Val-Arg-AMC (BML-P199). N-alpha-
Benzoyl-DL-arginine-p-nitroanilide (BAPNA) (B4875), trypsin
(T1426), leupeptin, para-nitrophenylphosphate and bovine serum
albumin (A2153) were purchased from Sigma–Aldrich (St. Louis,
MO, USA). All the biological grade buffers were used and were pur-
chased from Sigma–Aldrich (St. Louis, MO, USA). All enzyme kinet-
ics experiments (unless otherwise stated) were carried out at 30 °C
in appropriate buffer condition with 5% DMSO concentration. The
mass spectra were acquired using a MALDI-microMX mass spec-
trometer (Waters Corp., Milford, MA) instrument equipped with
an N2 laser operating at 10 Hz, using delayed extraction and posi-
tive ion linear mode analysis. For measuring the initial rates of en-
zyme catalyzed reaction, a temperature-controlled steady-state arc
lamp fluorometer equipped with Felix 32 software (Photon Tech-
nology Instrument, Birmingham, NJ, USA) and a UV–Vis spectro-
photometer (Model k25; Perkin Elmer Inc., Waltham, MA, USA)
was used.
NMR (100 MHz, CDCl3)
d ppm: 45.31, 55.78, 63.25, 112.93,
120.94, 121.27, 130.54, 137.94, 160.15. LRMS [ESI-MS +ve] calcu-
lated for (C9H10O4S+H): 215.04, observed: 214.83.
4.1.10. 2-(2,5-Dimethoxyphenylsulfonyl)oxirane (9)
This compound was synthesized by following a published pro-
tocol.39 The purification using silica gel column chromatography
yielded 9 as a white semi-solid. Rf = 0.24 Hexane:EtOAc (9:1).
Yield: 78%. 1H NMR (500 MHz, (CD3)2CO) d ppm: 3.19 (dd, J = 5.6
and 3.7 Hz, 1H), 3.31 (dd, J = 5.6 and 2.0 Hz, 1H), 3.83 (s, 3H),
3.97 (s, 3H), 4.75 (q, J = 3.7 and 2.0 Hz, 1H), 7.28 (m, 3H).13
C
NMR (125 MHz, (CD3)2CO)
d
ppm: 44.56, 55.46, 56.47,
62.31, 114.07, 114.71, 121.83, 126.84, 151.82, 153.37. LRMS
[ESI-MS +ve] calculated for (C10H12O5S+H): 245.05, observed:
245.08.
4.2.2. Methodologies for assessing time-dependent inactivation
of cysteine cathepsin B and inhibitor screening procedure
The inactivation reaction of human liver cathepsin B (net
175 nM) with inhibitor was performed under pseudo first order
condition ([I] ꢂ [E]) in a 0.5 ml eppendorf tube maintained at
30 °C in a temperature controlled bath. Freshly made inhibitor
stock solution (in DMSO) was used for each experiment. A previ-
ously reported cathepsin B assay procedure was employed.63,64
Cathepsin B enzyme was first activated for 10 min in sodium phos-
phate buffer (100 mM, pH 6.0) containing 5 mM DTT and 1 mM
Na2EDTA. The inactivation reaction was initiated by the addition
of inhibitor where DMSO concentration was maintained at 5%.
4.1.11. 2-(3,5-Dichlorophenylsulfonyl)oxirane (10)
This compound was synthesized by following a published pro-
tocol.39 The purification using silica gel column chromatography
yielded 10 as a white powder. Rf = 0.67 (hexane/EtOAc = 9/1).
Yield: 45%. 1H NMR (400 MHz, CDCl3) d ppm: 3.18 (dd, J = 5.4
and 3.7 Hz, 1H), 3.48 (dd, J = 5.4 and 2.0 Hz, 1H), 4.16 (dd, J = 3.7
and 2.0 Hz, 1H), 7.69 (t, J = 1.8 Hz, 1H), 7.82 (d, J = 1.8 Hz, 2H).13
C
NMR (100 MHz, CDCl3) d ppm: 45.54, 63.19, 127.18 (2C intensity),
134.61, 136.54 (2C intensity), 139.72. LRMS [ESI-MS +ve] calcu-
lated for (C8H6 Cl2O3S+Na): 274.93, observed: 275.00.
After suitable time intervals, an aliquot of 10
ture was withdrawn and added to an assay mixture (net assay vol-
ume 175 l) that contained 3 mM of Z-RR-pNA (Km = 0.9 mM)
ll of incubation mix-
l
4.1.12. 2-(Naphthalen-3-ylsulfonyl)oxirane (11)
substrate at 30 °C. A progress curve was recorded at 405 nM, and
the enzyme activity was determined by measuring the initial rates
of substrate turnover. For initial screening experiments, the assay
protocol remained the same except that the concentration of all
This compound was synthesized by following a published pro-
tocol.39 The purification using silica gel column chromatography
yielded 11 as a white powder. Rf = 0.27 (hexane/EtOAc = 19/1).
Yield: 78%. 1H NMR (400 MHz, CDCl3) d ppm: 3.15 (dd, J = 5.4
and 3.7 Hz, 1H), 3.47 (dd, J = 5.4 and 2.1 Hz, 1H), 4.19 (dd, J = 3.7
and 2.1 Hz, 1H), 7.66 (m, 1H), 7.72 (m, 1H), 7.91 (dd, J = 8.6 and
inhibitors was fixed at 500
lM and the residual enzyme activity
was measured after 10 min of reaction.

D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
2985
4.2.3. Method for estimation of kinetic parameters for 5-
mediated cathepsin B inhibition
To obtain Ki and ki parameters for the two step inhibition mod-
el, the following procedure was adopted. A sequence of indepen-
dent cathepsin B inactivation experiments were performed at 25,
Mca-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(Dnp)-
D
-Arg-NH2
[Mca = (7-methoxycoumarin-4-yl)acetyl] cathepsin D substrate
(Km = 3.7 M). Enzyme activity was monitored by measuring
l
fluorescent enhancement over the time.
50, 75, 100, and 150
l
M of 5 under pseudo-first order condition
4.2.8. Serine protease (trypsin) inhibition assay
([I] ꢂ [Cathepsin B]) and enzyme activities were monitored at
The assay procedure was adopted from an earlier published
protocol.70 Trypsin enzyme solution was freshly made using
1 mM HCl just before the inactivation reaction. Inactivation reac-
tion was initiated by the addition of inhibitor to a pre-activated
appropriate time intervals. The data thus obtained at each concen-
tration were plotted and fitted to the following equation
ꢃ t)
At = Af ꢁ (Af ꢁ A0)e(ꢁk
where At = enzyme activity at time t
obs
of cathepsin B inactivation, Af = final cathepsin B activity at infinite
time of inactivation, A0 = initial enzyme activity at zero time of
inactivation, and kobs = pseudo-first order rate constant of inactiva-
tion. Kitz–Wilson analysis is then performed to estimate the
reversible equilibrium binding constant Ki for the first step, and
the first order inactivation rate constant (ki) for the second irre-
versible step, as described elsewhere.32 For, demonstration that
compound 5 is active site-directed, the inactivation reaction of 5
with cathepsin B was performed in presence of an established
competitive inhibitor of cathepsin B, leupeptin (40 nM) and the
residual cathepsin B activity was measured, as described above.
Such a methodology has been previously utilized to demonstrate
the active site-directed nature of inhibition for previously unchar-
acterized covalent and irreversible inhibitory agents.34,65
trypsin (net concentration of 190–200 U/
intervals, an aliquot of 2 l incubation mixture were withdrawn
and added to a 67 mM sodium phosphate assay buffer (net assay
volume of 175 l) which contained 3 mM N-alpha-benzoyl-DL-argi-
ll). After suitable time
l
l
nine-p-nitroanilide (BAPNA, Km = 0.94 mM) as a substrate. Enzyme
activity was monitored by measuring the initial rate of substrate
consumption at 410 nm.
4.2.9. Protein tyrosine phosphatase 1B (PTP1B) inhibition assay
The time-dependent inactivation kinetics of 5 with PTP1B was
performed as described before.39
4.2.10. Mass-spectral studies
Cathepsin B was activated and incubated with inhibitor 5
(800 lM) for 10 min under the conditions similar to those used
4.2.4. Cathepsin F inhibition assay
for kinetics studies. Both 5–inhibited cathepsin B inhibitory com-
plex and control (5% DMSO) were prepared for matrix-assisted la-
ser desorption/ionization mass spectrometry using the ultra-thin
layer essentially as published.71,72 Samples were saturated in a
A previously reported enzyme assay procedure was adopted
with appropriate modifications.66 The enzyme was activated for
10 min in 50 mM potassium phosphate buffer at pH 6.5 containing
2.5 mM DTT, 2.5 mM Na2EDTA, and 1.0 mg/ml BSA at 25 °C. The
inactivation reaction was initiated by addition of inhibitor 5 to
activated human cathepsin F (net 10.8 nM). After suitable time
1:9 ratio with a saturated solution of recrystallized
a-cyano-4-
hydroxycinnamic acid in 50% formic acid/33% isopropyl alcohol/
17% water (v/v/v; Fisher Scientific-all HPLC grade). Acceleration
potential of 12,000 V and an extraction pulse of 1950 V with a de-
lay time for 410 ns were used. Spectra were smoothed and baseline
corrected using MassLynx (Waters Corp.) and calibrated pseudo-
internally using equine apomyoglobin.
intervals, an aliquot of 10
and the enzyme activity was measured in an assay buffer (net
175 l) containing of Z-FR-AMC fluorogenic substrate
(Km = 0.44 M) at 25 °C.
ll of incubation mixture was withdrawn
l
3 lM
l
4.2.5. Cathepsin S inhibition assay
For assaying cathepsin activity,
4.2.11. Hydrolytic stability of inhibitor 5
S
a
previously published
To study the hydrolytic stability of compound 5, NMR experi-
ment was carried out in following manner. First, sodium phosphate
buffer (10 mM) of pH 7.4 was prepared. A portion (5 ml) of this
buffer was lyophilized and re-dissolved in deuterium oxide. This
step was repeated thrice so as to ensure a complete H/D exchange.
The buffer components were re-dissolved in deuterium oxide
(5 ml). The hydrolysis experiment was carried out under pseudo
first order reaction condition. A stock solution of 5, freshly made
in deuterated DMSO was added to deuterated phosphate buffer
protocol was followed with appropriate modification.67 Thus,
cathepsin S enzyme was activated first in the 100 mM potassium
phosphate buffer (pH 6.5) containing 5 mM DTT, 5 mM Na2EDTA
for 10 min. The inactivation reaction of human cathepsin S (net
134.6 nM) with inhibitor 5 (75
time intervals, an aliquot of 3
drawn and the enzyme activity was measured in assay buffer
lM) was initiated. After suitable
ll of incubation mixture was with-
(175
>100
l
l
l; 8% DMSO) containing 250
lM of Z-VVR-AMC (Km
M;68 Ex/Em: 365/440 nm) fluorogenic substrate.
(total volume 500 ll; net concentration of 5 at 2.5 mM) and NMR
spectra were recorded after appropriate time intervals (see Supple-
mentary data, Fig. S1). The rate of hydrolysis was measured by
comparing the integration values of initial and final compounds.
The rate constant of hydrolysis was obtained by fitting the data
to a single exponential decay curve (It = I0eꢁk ꢃ t) and the half
was calculated using the following equation: t1/2 = 0.693/k.
4.2.6. Cathepsin G inhibition assay
A previously reported assay conditions were followed.69 The en-
zyme was activated in 20 mM Tris–HCl buffer (pH 7.4) containing
500 mM NaCl for 15 min. The inactivation reaction of human
cathepsin G (net 7.66 nM) with inhibitor 5 was initiated. After suit-
able time intervals, an aliquot of 10
ll of incubation mixture was
withdrawn and the enzyme activity was measured in assay buffer
4.3. Molecular modeling
containing 3.4 mM Suc-AAPF-pNA cathepsin
G
substrate
(Km = 1.7 mM).
The molecular modeling studies were performed on a Dell Pow-
erEdge 6000 server operating on Red Hat Enterprise Linux ES re-
lease 4. The CH3CONHCysCONHCH3 peptide covalently attached
to inhibitor 5 at the terminal aliphatic carbon was built with open
oxirane ring in Builder module of Maestro 7.5 software and partial
charges on each atoms were determined in Jaguar (Schrodinger
Inc., New York, NY, USA) using density-functional theory (DFT) cal-
culation with B3LYP/6-31G⁄ basis set. The coordinate file of
cathepsin B (PDB id: 2DC6) was downloaded and appropriately
4.2.7. Cathepsin D inhibition assay
The enzyme was activated in 50 mM sodium acetate buffer
(pH 4) containing 5 mM DTT and 1 mg/ml bovine serum albumin.
The inactivation reaction of human Cathepsin D (net 20 nM) with
inhibitor was initiated. After suitable time intervals, an aliquot of
1
l
l of incubation mixture was withdrawn and added to an assay
mixture (net assay volume of 175 l) that contained 40 M of
l
l

2986
D. Dana et al. / Bioorg. Med. Chem. 21 (2013) 2975–2987
modified at pH 7.0 to include inhibitor 5 covalently linked to
Cys29S residue using Insight II software (Accelrys Inc., SanDiego
CA, USA). The partial charges on the atoms of inhibitor were as-
immersion objective lens using a Zeiss Axio Imager-M2 upright
fluorescence microscope (Zeiss, Heidelberg, Germany). Results for
every condition are the average of three independent experiments
in each of which 28 cells were analyzed by ImageJ software.
c
signed from DFT calculations and the complex was soaked with a
15 Å spherical shell of water with Cys29Sc as the center. The
resulting complex was energy minimized (10,000 steps of Steepest
Descent). A molecular dynamics run (100 ps) was then carried out
on the energy-minimized complex using Consistent Valence Force-
field (CVFF) force field in Discover standalone program (Accelrys
Inc., San Diego CA, USA). The trajectories from molecular dynamic
run were saved every 2 ps and analyzed. During the energy mini-
mation procedure and the molecular dynamics simulations, no
constratins were imposed on 5–cathepsin B inhibitor complex.
4.4.4. Cell motility assay
The effect of inhibitor 5 on cell migratory behavior of MDA-MB-
231 cells was evaluated by following a slight modification of an
earlier published protocol.76 Thus MDA-MB-231 cells were plated
as concentric circles on a 10-well glass slide pre-coated with 1%
BSA through a 10-hole cell sedimentation manifold (CSM, Inc.,
Phoenix, AZ) followed by incubation at 37 °C and 5% CO2. After re-
moval of the manifold, cells were treated with control vehicle
(0.05% DMSO) or appropriate concentrations of inhibitor 5, and
incubated for 6 h at 37 °C. Images were acquired by a digital cam-
era (Moticam 2000) attached to an inverted Nikon Diaphot micro-
scope both before (t0) and after inhibitory treatment (t6). Motility
of cells was calculated by the change in total area (in square
micrometers) by Image J software. Each reported value is the aver-
age of triplicate measurements with corresponding standard devi-
ation value (S.D.).
4.4. Biology
4.4.1. Cell culture
All the cell culture reagents were purchased from Invitrogen
(Carlsbad, CA). MDA-MB-231 cells (American Type Culture Collec-
tion, Manassas, VA) were cultured on 10-cm plates (BD Falcon™) at
37 °C and 5% CO2 in Iscove’s Modified Dulbecco’s medium with
L-glutamine (IMDM), 10% fetal bovine serum, and antibiotics (1%
penicillin/streptomycin and 0.5
lg/ml fungizone) as described ear-
Acknowledgments
lier.73 Cells were passaged at 1:3 ratio every 3–4 days and subcul-
tured by 0.25% trypsin–EDTA treatment. Cells were re-plated one
day prior to experiment so that the cell density was 70–80% on
the day of the experiment.
The authors wish to gratefully acknowledge the funding sup-
port provided by the PSC-CUNY Research Award Program, and
Queensborough Community College (QCC)/Queens College NSF-
STEP and NIH Bridges programs.
4.4.2. Confocal microscopy
For live-cell imaging, cells were seeded to poly-D-lysine coated
Supplementary data
35 mm-glass bottom dishes (MatTek; Ashland, MA) and grown
overnight in complete medium. Prior to imaging, cells were treated
in 15 mM HEPES containing phenol-red (PR)-free complete med-
ium. To reduce photo-bleaching, 0.6u of Oxyrase enzyme per ml
(Oxyrase Inc.) was added to the PR-free medium containing
10 mM sodium-lactate, as previously described.74 Cells were incu-
bated with either dimethylsufoxide (DMSO, 0.1% v/v) as vehicle
control, or varying concentrations of 5 for 20 min at 37 °C, followed
by an additional 10 min of incubation with the substrate Z-RR-
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.03.062.
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