Design, synthesis, and evaluation of inhibitors of cathepsin L: Exploiting a unique thiocarbazate chemotype

Article abstract of DOI:10.1016/j.bmcl.2008.04.065

Recently, we identified a thiocarbazate that exhibits potent inhibitory activity against human cathepsin L. Since this structure represents a novel chemotype with potential for activity against the entire cysteine protease family, we designed, synthesized, and assayed a series of analogs to probe the mechanism of action, as well as the structural requirements for cathepsin L activity. Molecular docking studies using coordinates of a papain-inhibitor complex as a model for cathepsin L provided useful insights.

Full text of DOI:10.1016/j.bmcl.2008.04.065

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3646–3651
Design, synthesis, and evaluation of inhibitors of cathepsin L:
Exploiting a unique thiocarbazate chemotype
Michael C. Myers,a,b Parag P. Shah,a,c Mary Pat Beavers,a,c Andrew D. Napper,a,c
a,b,
Scott L. Diamond,a,c Amos B. Smith, IIIa,b, and Donna M. Huryn
*
*
aPenn Center for Molecular Discovery, University of Pennsylvania, 1024 Vagelos Research Laboratories,
Philadelphia, PA 19104-6383, USA
bDepartment of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA
cInstitute for Medicine and Engineering, University of Pennsylvania, 1024 Vagelos Research Laboratories,
Philadelphia, PA 19104-6383, USA
Received 29 February 2008; revised 16 April 2008; accepted 21 April 2008
Available online 1 May 2008
Abstract—Recently, we identified a thiocarbazate that exhibits potent inhibitory activity against human cathepsin L. Since this
structure represents a novel chemotype with potential for activity against the entire cysteine protease family, we designed, synthe-
sized, and assayed a series of analogs to probe the mechanism of action, as well as the structural requirements for cathepsin L activ-
ity. Molecular docking studies using coordinates of a papain–inhibitor complex as a model for cathepsin L provided useful insights.
Ó 2008 Elsevier Ltd. All rights reserved.
Human cathepsin L is an endosomal cysteine protease
that has been implicated in a variety of physiological
and pathophysiological processes.1–3 Cathepsin L is
widely distributed, and plays key roles in bone remodel-
ing and the immune response, as well as in disease states
such as cancer,4 rheumatoid arthritis5 and osteo-arthri-
tis.6,7 Furthermore, a number of infectious agents (e.g.,
Ebola, SARS, and Leishmania) have been reported to
require cathepsin L or cathepsin L-like activity for viral
processing and infectivity.8–12 As such, the identification
of inhibitors of cathepsin L would provide valuable
tools to probe the role of this enzyme in biological sys-
tems, as well as to provide potential starting points for
drug discovery efforts.
ases.14 This effort recently led to the identification15
and characterization16 of (ꢀ)-1, a novel and potent
inhibitor of human cathepsin L (Fig. 1).17
Most cysteine protease inhibitors require the presence
of an electrophilic warhead that provides a site of reac-
tion (either reversible or irreversible) for the active site
thiolate. Selectivity and potency are often dictated by
the reactivity of the warhead in conjunction with addi-
tional binding interactions of the molecule across the
enzyme active site. Classic warheads include epoxides,
nitriles, activated carbonyls, vinyl sulfones, oxocarbaz-
2,18–20
ates, and aza-peptides.
Indeed, incorporation of
such warheads has led to cathepsin K and cathepsin
S inhibitors currently in clinical trials.3 Potent inhibi-
The Penn Center for Molecular Discovery (PCMD), a
member of the Molecular Libraries Screening Center
Network (MLSCN), has conducted a series of High-
throughput Screening (HTS) campaigns of the Molecu-
lar Libraries Small Molecular Repository (MLSMR) to
identify inhibitors of cysteine (cathepsins B,13 L, and S)
and serine (cathepsin G, Factor XIa, and XIIa) prote-
O
O
H
N
BocHN
S
N
H
N
H
O
Et
HN
(-)-1
Keywords: MLSCN; Cathepsin L inhibitor; Cysteine protease; Thioc-
arbazate; Oxocarbazate.
Figure 1. Thiocarbazate cathepsin L inhibitor (ꢀ)-1.
*
Corresponding authors; E-mail: huryn@sas.upenn.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.065

M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3646–3651
3647
tors of cathepsin L that incorporate azepanones and
cyanamides have also been described recently.21,22 To
the best of our knowledge, thiocarbazates and their
corresponding biological activity have not been de-
scribed prior to our original report.15 Since the thiocar-
bazate core embodies the potential for broad utility as
a cysteine protease inhibitor scaffold, we sought to
understand further the requirements for activity within
this substructure.
ments of the indole side chain, the –NHBoc, and the
2-ethylphenyl anilide of (ꢀ)-1 were targeted. Thiocar-
bazates for this study were prepared from the requisite
hydrazides exploiting our previously developed chemis-
try.15 In a one-pot reaction, hydrazides were treated
with carbonyl sulfide gas followed by an appropriate
electrophile (i.e., R2–Br). Preparative reverse phase
HPLC was employed to purify the final products,25
which were assayed for their ability to inhibit cathepsin
L.26
In an effort to evaluate the potential binding mode of
(ꢀ)-1 with cathepsin L, we performed docking studies
using the publicly available X-ray coordinates for pa-
As illustrated by the results listed in Table 1, occupation
of the S2 subsite is essential for cathepsin L inhibition.
Partial occupation, as in (ꢀ)-2 where the indole side
chain is replaced with the smaller phenyl group, results
in less potent activity (IC50 = 115 vs 56 nM). Thiocar-
bazate 3, in which the entire indole side chain has been
eliminated, exhibits no inhibition. Also pronounced are
the –NHBoc group’s contributions to potency, as illus-
trated by thiocarbazate 4’s significantly reduced activity
(IC50 = 22 lM). In this case, we reason that the loss of a
key hydrogen bond between the –NHBoc group and the
Asp158 residue leads to diminished activity. These re-
sults support the importance of maintaining hydropho-
bic and hydrogen bonding interactions in the active
site, consistent with the mode of docking proposed.
pain complexed to
a succinyl epoxide inhibitor
(1cvz.pdb).23 The papain model was a relevant model
for cathepsin L due to the high degree of sequence
homology between the binding sites of these two cys-
teine proteases. In these studies we observed the simulta-
neous occupation of the S2, S3, and S10 subsites by
hydrophobic and aromatic functionalities of thiocarbaz-
ate (ꢀ)-1 as shown in Fig. 2; the indole side chain occu-
pies the S2 subsite; the –NHBoc group occupies the S3
subsite, and the 2-ethylphenyl aniline occupies the S10
subsite. A key hydrogen bond is observed between the
Gly66 backbone NH and the amino acid derived car-
bonyl of the diacyl hydrazine. In other inhibitor systems,
the absence of a hydrogen bonding interaction between
Gly66 and inhibitor has been reported to lead to a loss
of inhibition in numerous cathepsins, including cathep-
sin L.22 Details of the molecular docking studies are
reported elsewhere;24 however, they suggest that (a)
the thiocarbazate carbonyl is in sufficient proximity to
the active site Cys25 to permit nucleophilic thiolate
addition and (b) significant binding interactions (both
hydrogen bonding and van der Waals) are present be-
tween the inhibitor and protease subsites. These obser-
vations support our hypothesis that specific binding
interactions as well as appropriate reactivity of (ꢀ)-1
are essential for the observed inhibitory properties.
From the docking studies of thiocarbazate (ꢀ)-1,16,24 we
hypothesized that additional room for structural modifi-
cations and ring constraints was available in the anilide
portion of this thiocarbazate (S10 subsite). Based on this
observation, a tetrahydroquinoline anilide (ꢀ)-5 was
substituted for the 2-ethylphenyl anilide moiety (Table
1).27 An improvement in potency was observed
(IC50 = 41 nM), further supporting our hypothesis. To
explore this area further, two additional analogs were
prepared: thiocarbazate 6, in which constraints were im-
posed by incorporation of an N-phenyl pyrrolidinone
group, and a methyl ester thiocarbazate (ꢀ)-7. 28,29 Both
analogs 6 and (ꢀ)-7 exhibited reduced activity against
cathepsin L with IC50 values of 110 and 201 nM,
respectively.
The docking studies were validated by the synthesis of
analogs in which key residues occupying the S2, S3,
and S10 subsites were modified. Specifically, replace-
Our thiocarbazates are structurally related to oxocar-
bazates (e.g., A) and aza-peptides (e.g., B), known pro-
tease inhibitors (Fig. 3)30–32 that are active by the
virtue of their activated carbonyl groups. Depending
on the nature of the leaving group present, these inhib-
itors often bind and react with the active site serine or
cysteine, resulting in the formation of a stable acyl–en-
zyme complex, which then undergoes slow hydroly-
sis.30,31 Alternatively, oxocarbazate and aza-peptide
inhibitors with poor leaving groups are believed to form
stable tetrahedral intermediates without acyl–enzyme
18,33,34
adduct formation.
To further understand the
cathepsin L inhibitory activity of thiocarbazates, (ꢀ)-1
was incubated in the presence of stoichiometric amounts
of cysteine or cathepsin L over prolonged time periods
in the presence of assay buffer. The reactions were mon-
itored by LC–MS for the disappearance of (ꢀ)-1 as well
Figure 2. Thiocarbazate (ꢀ)-1 (IC50 = 56 nM) in the binding subsite of
papain. The indole forms hydrophobic contacts within the S2 subsite,
the –NHBoc group forms hydrophobic contacts within the S3 subsite,
and the 2-ethylphenyl anilide occupies the S10 subsite.
as the appearance of reaction products such as cysteine
35
adducts and products of hydrolysis.
In both
experiments, thiocarbazate (ꢀ)-1 was found to remain

3648
M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3646–3651
Table 1. Synthesis and cathepsin L inhibitory activity of thiocarbazates
a) SCO (gas), KOH, EtOH
H
N
R1
S
R1
NH2
23 oC, 15 h
N
H
R2
N
H
b) R2-Br, 23 oC, 1 h
O
Hydrazides
Thiocarbazates
Thiocarbazate
R1
R2
IC50 (lM)
(ꢀ)-1
Boc-L-Trp
0.056 0.001
O
(ꢀ)-2
3
Boc-L-Phe
Boc-Gly
0.115 0.006
Inactive
N
H
Et
O
4
21.797 1.836
0.041 0.002
O
O
HN
(ꢀ)-5
Boc-L-Trp
N
6
Boc-L-Trp
Boc-L-Trp
0.110 0.003
0.201 0.012
N
O
(ꢀ)-7
OMe
unchanged after greater than 24 h, and no evidence of
newly formed reaction products were detected. As the
formation of a stable acyl–enzyme complex would have
resulted in the generation of a new product based on the
leaving group embedded in (ꢀ)-1, we hypothesize that a
stable acyl–enzyme adduct is not formed. These data are
consistent with detailed kinetic analysis16 indicating that
(ꢀ)-1 is a slowly reversible inhibitor of cathepsin L.
sponding diacyl hydrazine (ꢀ)-9, aza-peptide (ꢀ)-11,
and oxocarbazate (ꢀ)-13 were prepared as illustrated
in Scheme 1.
Diacyl hydrazine (ꢀ)-9 was prepared from succinanilic
acid36 8 via an EDC-mediated coupling reaction with
Boc-L-Trp-NHNH2. Aza-peptide (ꢀ)-11 was generated
via the reaction of preformed isocyanate 10 with Boc-
L-Trp-NHNH2.37 Oxocarbazate (ꢀ)-13 was synthesized
using a three-component protocol using Boc-L-Trp-
NHNH2, a-bromoanilide 12, and carbon dioxide.38,39
While yields for these analogs were modest (33-65%), 1
mmol scale reactions yielded material of >99% purity
for biological assay. When tested for inhibition of
cathepsin L, diacyl hydrazine (ꢀ)-9 was found to be
To probe the necessity of the thiocarbazate core in
inhibitors such as (ꢀ)-1 and (ꢀ)-5, a series of analogs
were prepared in which a carbon, oxygen, or nitrogen
atom replaced the sulfur atom. This series was designed
to incorporate the preferred S10 subsite substituent, a
tetrahydroquinoline anilide. Toward this end, the corre-
O
H
O
O
H
N
H
N
AcHN
N
O
AcHN
N
H
N
H
OMe
Me
O
O
A
B
Me
on = 30500 M-1s-1, papain
on = 310 M-1s-1, cathepsin B
Hanzlik and Xing 1998
k
on = 770 M-1s-1, papain
Abeles et al., 1996
k
k
,
Figure 3. Examples of known oxocarbazate A and aza-peptide B inhibitors.

M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3646–3651
3649
O
HO
O
O
N
H
N
BocHN
O
8
N
H
N
O
EDC, DMAP, DMF
HN
(-)-9
Inactive
O
O
O
OCN
H
N
H
N
N
BocHN
N
H
N
10
O
Boc-L-Trp-NHNH2
HN
(-)-11
pyridine, THF
IC50 = 2.998 0.355 μM
O
O
H
BocHN
N
O
a. TBAI, Cs2CO3, DMF
N
N
H
O
b. CO2,
Br
O
HN
(-)-13
N
12
IC50 = 0.007 0.004 μM
Scheme 1. Synthesis of tetrahydroquinoline-derived diacyl hydrazine (ꢀ)-9, aza-peptide (ꢀ)-11, and oxocarbazate (ꢀ)-13 analogs starting from Boc-
L-Trp-NHNH2.
inactive and aza-peptide analog (ꢀ)-11 displayed only
modest potency. In these cases, the presence of the pre-
ferred tetrahydroquinoline anilide could not compensate
for the lack of an activated carbonyl [e.g., (ꢀ)-9] or
unoptimized carbonyl reactivity (e.g., (ꢀ)-11]. In con-
trast, oxocarbazate (ꢀ)-13, in which an oxygen atom
was substituted for the sulfur atom of (ꢀ)-5, was the
most potent cathepsin L inhibitor identified within this
study (IC50 = 7 nM).40,41
Based on these studies we conclude that full occu-
pancy of the S2, S3, and S10 subsites is required for
potent inhibition. With these requirements met, the
activated carbonyl group is positioned in close prox-
imity to the Cys25 active site residue. Although we
observed no evidence of reaction between the protein
and the inhibitors, changes in the functionality adja-
cent to the putative reactive carbonyl (i.e., sulfur, car-
bon, oxygen, and nitrogen analogs) strongly influenced
potency. In the course of these studies, we designed
and synthesized a highly potent cathepsin L inhibitor,
oxocarbazate (ꢀ)-13, that contains a preferred tetrahy-
droquinoline anilide group. Future efforts in our labo-
ratory will focus on the thiocarbazate chemotype and
its potential to exhibit broad cysteine protease inhibi-
tory activity.
As in the case of (ꢀ)-1, oxocarbazate (ꢀ)-13 and
thiocarbazate (ꢀ)-5 were found to be unreactive to
transesterification by cysteine and DTT nucleophiles.
Furthermore, (ꢀ)-13 remained intact for greater than
24 h when incubated with cathepsin L under stoichi-
ometric conditions, and in the presence of assay buf-
fer.26 Electrostatic potential calculations were also
conducted to determine the relative electrophilicities
of the carbonyls within the tetrahydroquinoline
substituted inhibitors; however the results from these
studies gave no clear correlation between electrophi-
licity and activity, further supporting our hypothesis
that both reactivity and binding interactions dictate
potency.
Acknowledgments
Financial support for this work was provided by the
NIH (5U54HG003915-02 and 5U54HG003915-03). We
thank Dr. Carlo E. Ballatore and Mr. Onur Atasoylu
for electrostatic potential calculations and Professor
Barry S. Cooperman for helpful discussions. Finally
we thank Dr. G. T. Furst and Dr. R. Kohli at the Uni-
versity of Pennsylvania for assistance in obtaining NMR
and high-resolution mass spectra.
In summary, through the design, synthesis, and assay
of a series of thiocarbazates we have characterized the
activity of a novel family of cathepsin L inhibitors.

3650
M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3646–3651
25. General procedure to form amino acid-substituted thiocar-
References and notes
bazates: Boc-protected amino acid hydrazide (1.0 mmol,
1.0 equiv) was added to a 25-mL round-bottomed flask
followed by a solution of KOH in 95% EtOH (0.25 M,
4.4 mL, 1.1 equiv). After stirring for 5 min at 23 °C, a
balloon of carbonyl sulfide gas was attached to the flask.
The flask was purged with the gas (5 s) and a full balloon
was reattached. The reaction was stirred for 15 h at 23 °C.
After stirring overnight, the a-bromo anilide (1.1 mmol,
1.1 equiv) was added in one portion and the reaction was
monitored by LC–MS. The a-bromo anilides were typi-
cally consumed within 20–60 min, and the reaction mix-
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ferred into a 96-well Corning 3686 assay microplate to give
16 dilutions ranging from 50 lM to 1.5 nM. Human liver
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35. The following standards were utilized as potential reaction
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mercapto-acetamide, and 2-ethyl-phenylamine.
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M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3646–3651
3651
38. For a general procedure used to prepare oxocarbazates
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Alford, K. L., Salvatore, R. N. Tetrahedron Lett. 2004, 45,
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J = 8.0 Hz, 1H), 7.20 (t, J = 7.0 Hz, 2H), 7.20 to 7.17
(m, 1H), 7.12 (t, J = 7.5 Hz, 1H), 7.06 (t, J = 7.0 Hz, 1H),
6.98 (t, J = 7.5 Hz, 1H), 6.32 (br s, 1H), 4.82 (br s, 2H),
4.31 (br m, 1H), 3.69 (t, J = 6.5 Hz, 2H) 3.16 to 3.12 (m,
1H) 2.97 to 2.92 (m, 1H), 2,74 (t, J = 6.5 Hz), 1.92 (pentet
J = 6.5 Hz), 1.30 (br s, 9H); 13C NMR (125 MHz, DMSO-
d6, major rotamer) d 171.9, 166.5, 155.6, 155.1, 137.7,
136.0, 132.0, 128.7, 127.3, 125.9, 125.0, 124.9, 123.8, 120.8,
118.5, 118.1, 111.2, 109.9, 77.9, 62.1, 53.4, 42.8, 28.1, 27.6,
26.1, 23.2; high resolution mass spectrum (ES+)
m/z 558.2325 [(M+Na)+; Calcd for C28H33N5O6Na:
558.2329].
40. PubChem substance number for (ꢀ)-13 is SID 46493575.
24
41. Characterization data for (ꢀ)-13 mp 157 °C; ½aꢁD ꢀ 36:4 (c
0.1, AcOH); IR (KBr) 3413, 2926, 1685, 1654, 1162, 1121,
1
1061 cmꢀ1; H NMR (500 MHz, DMSO-d6, VT-350K) d
10.62 (br s, 1H), 9.76, (br s, 1H), 9.10 (br s, 1H), 7.61 (d,
J = 8.0 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H), 7.33 (d,