Design, synthesis and biological evaluation of a library of thiocarbazates and their activity as cysteine protease inhibitors

Article abstract of DOI:10.2174/138620710791054303

Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead. Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity against a panel of proteases indicated a significant preference for members of the papain family of cysteine proteases over serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several potent inhibitors of cathepsin L and S were identified. The SAR data were employed in docking studies in an effort to understand the structural elements required for cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of the papain family of cysteine proteases.

Full text of DOI:10.2174/138620710791054303

Combinatorial Chemistry & High Throughput Screening, 2010, 13, 337-351
337
Design, Synthesis and Biological Evaluation of a Library of Thiocarbazates
and Their Activity as Cysteine Protease Inhibitors
Zhuqing Liu1,2, Michael C. Myers1,2, Parag P. Shah2,3, Mary Pat Beavers2,3, Phillip A. Benedetti1,2,
Scott L. Diamond2,3, Amos B. Smith, III1,2 and Donna M. Huryn*,1,2
1Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-6323, USA
2Penn Center for Molecular Discovery, University of Pennsylvania, 1024 Vagelos Research Laboratories, Philadelphia,
PA 19104-6383, USA
3Institute for Medicine and Engineering, University of Pennsylvania, 1024 Vagelos Research Laboratories,
Philadelphia, PA 19104-6383, USA
Abstract: Recently, we identified a novel class of potent cathepsin L inhibitors, characterized by a thiocarbazate warhead.
Given the potential of these compounds to inhibit other cysteine proteases, we designed and synthesized a library of
thiocarbazates containing diversity elements at three positions. Biological characterization of this library for activity
against a panel of proteases indicated a significant preference for members of the papain family of cysteine proteases over
serine, metallo-, and certain classes of cysteine proteases, such as caspases. Several potent inhibitors of cathepsin L and S
were identified. The SAR data were employed in docking studies in an effort to understand the structural elements
required for cathepsin S inhibition. This study provides the basis for the design of highly potent and selective inhibitors of
the papain family of cysteine proteases.
Keywords: Thiocarbazates, cathepsin B, cathepsin S, cathepsin L, cysteine protease inhibitor, library.
INTRODUCTION
azepanones [13], aziridines and azodicarboxamides [14, 15],
vinyl sulfones [16], and aza peptides [17].
Cysteine proteases, ubiquitous in Nature, are frequent
targets of drug discovery efforts due to roles they play in a
number of physiological and pathophysiological processes.
In mammals, three classes of cysteine proteases have been
characterized: papain-like (such as the cysteinyl cathepsins),
calpains, and caspases [1, 2]. Papain-like cysteine proteases
play a role in protein turnover, and their overexpression or
disregulation has been implicated in certain inflammatory
diseases, in cancer, and in osteoporosis, and arthritis, among
other diseases. Inappropriate activity of calpains has also
been associated with a number of disease conditions
including neurodegeneration, muscular dystrophy and
diabetes. Caspases play a role in the inflammatory process
and in apoptosis; their inhibition has been suggested as an
approach to degenerative diseases such as arthritis and
stroke. A number of infectious agents (bacteria, viruses, and
protozoa) also utilize either host or their own cysteine
proteases for infectivity, virulence and/or replication
processes, and targeting these proteases has been a popular
strategy for anti-infective drug discovery efforts [3].
Recently, we disclosed a novel series of cathepsin L
inhibitors characterized by a unique thiocarbazate warhead
[18]. Through further optimization, potent and selective
inhibitors of cathepsin L were developed; Compound 1 was
characterized as a competitive inhibitor that exhibited slow
binding and reversible kinetics [19-21]. Since this warhead
had not been described, prior to our publication, we explored
its potential to inhibit other proteases. Our study entailed the
design and synthesis of a library of thiocarbazates and their
biological characterization in a series of protease assays to
define the potential of this chemotype to exhibit broad
protease inhibition. Based on the results of these studies, the
library was tested against specific cathepsins; docking
studies to rationalize our findings were also carried out. The
results from these efforts are reported herein.
O
O
H
N
A
BocHN
S
N
N
H
H
C
Inhibitors of cysteine proteases typically rely on the
presence of a “warhead” to provide a site for nucleophilic
attack by the active site cysteine thiolate [4]. Examples of
warheads include peptidyl halomethyl ketones [5], peptidyl
diazomethanes [6], peptide aldehydes [7], acyloxymethyl
ketones [8], epoxysuccinyl derivatives [9], epoxyketones
[10], ꢀ-aminoalkyl epoxides [11], ꢀ-keto-aldehydes [12],
O
Et
HN
1
B
Fig. (1). Design of thiocarbazate library.
*Address correspondence to this author at the Department of Chemistry,
University of Pennsylvania, 231 South 34th Street, Philadelphia, PA 19104-
6323, USA; Tel: 215-746-3567; E-mail: huryn@sas.upenn.edu
1386-2073/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

338 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
Table 1. Physical Properties of Thiocarbazate Library
DESIGN OF THE THIOCARBAZATE LIBRARY
Our strategy involved the design of a library containing a
thiocarbazate scaffold incorporating a variety of functional
groups at three different positions A, B and C (Fig. 1). From
the outset, we sought to optimize diversity of the final
products in terms of size, shape and functionality. At the
same time, we focused on maintaining optimal physical
properties to ensure solubility, permeability and other “drug-
like” properties. Finally, we maintained a strict requirement
for an expedient synthesis that would produce a minimum of
10 mg of final product in purities of at least 95% as
determined by LC-MS (liquid chromatography/mass
spectrometry) analysis. Modifications at the A position
involved changes in size, as well as replacement of the t-
butyloxycarbonyl group. Previous work in our laboratories
identified an issue of instability with the presence of the free
amine at this position; therefore all modifications took that
possibility into consideration. Position B underwent the most
extensive modifications, where changes in size, polarity,
acidity, and functionality were incorporated. Thiocarbazates
derived from natural amino acids, such as methionine,
valine, alanine, glutamic acid, leucine, proline,
phenylalanine, tyrosine, threnonine, serine, glutamic acid,
lysine, arginine and histine, along with unnatural amino
Average
Range
Median
Mode
AlogP
3.5
4.8
1.0-6.0
3-7
3.5
5.0
4.1
5.0
H-Bond Acceptors
H-Bond Donors
3.9
2-7
4.0
4.0
Lipinski Violations
Molecular Weight
Polar Surface Area (Å)
Rotatable Bonds
1.0
0-3
1.0
1.0
526.1
134.3
16
367.2-706.6
95.5-210.8
9-23
536.6
132.6
16
539.6
125.6
16
treated with an alkylating agent to form the thiocarbazate
products. The thiocarbazates were isolated and purified by
HPLC to at least 95% purity. All compounds were
characterized by high resolution mass spectrometry
(HRMS), and a subset further characterized by 1H NMR, 13
C
NMR and IR. The general procedure for the preparation of
thiocarbazates was amenable to all of the thiocarbazates
produced in the library (Table 2).
acids were prepared. Modifications at
C
involved
incorporation of ring constraints, removal of the amide bond
and exploration of size requirements; a variety of acetamides
derived from aniline, primary amines, and methyl esters
were included. Examples of substituents at position C
include differentially substituted anilines, quinolines and
isoquinolines, non-aromatic amines, morpholines, indolines,
and pyridinones.
To ensure that the library contained compounds with a
range of acceptable physical properties such as logP,
molecular weight, polar surface area, etc., we relied on the
cheminformatics tool, Leadscope [22]. Table 1 below details
this analysis. The aggregated library exhibited excellent
properties according to Lipinski-like [23] parameters:
average LogP = 3.5, average H-bond acceptors = 4.8,
average H-bond donors = 3.9. The average molecular weight
(526) is typical of small molecule protease inhibitors, but
slightly above the Lipinski target of <500. Consequently, the
number of Lipinski violations on average was 1, which is
reflective of the molecular weight characteristics. Polar
surface area and rotable bonds were on average 134 Å and
16, respectively. While the PSA was within the range
suggested by Veber [24] for orally available drugs, the
rotatable bond count was somewhat higher than suggested as
optimal.
Scheme 1. General synthesis of thiocarbazates.
While many of the amino acid and acid starting materials
were commercially available, several required preparation.
Hydrazides derived from commercially available R or S-2-
methyl-3-hydroxypropionate were generated directly from
the ester as shown in Scheme 2, then converted to the
corresponding thiocarbazates (e.g., 67-70). Subsequent
etherification of 69 afforded the tert-butyldimethylsilyl
(TBS) [26], and para-methoxyl benzyl (PMB) [27] ether
analogs 71 and 72. Beta amino acids, such as those
incorporated into thiocarbazates 74-86, were prepared using
a modified Arndt-Eistert protocol to furnish the desired ꢀ-
amino acid in yields of 70-95% [28].
H
NH2-NH2
HO
N
HO
OMe
NH2
O
O
SYNTHESIS OF THE THIOCARBAZATE LIBRARY
O
TBSCl, imidazole
H
HO
67-70
N
C
Based on chemistry developed in our laboratory [18, 19]
we designed a versatile synthetic strategy to prepare the
thiocarbazate library as illustrated in Scheme 1. A variety of
acids were treated with ethylchloroformate to form the
corresponding mixed anhydride, which were not isolated.
Treatment with hydrazine monohydrate then furnished the
hydrazides which were isolated after aqueous workup.
Reaction with carbonyl sulfide gas in ethanol [25] formed
the thiosemicarbazide intermediates that were directly
N
S
or
NO2
O
H
O
N
O
H
RO
N
C
OMe
N
S
H
O
71: R = TBS
72: R = PMB
Scheme 2. Preparation of thiocarbazates 67-72.

Library of Thiocarbazates and Their Activity
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4 339
Table 2. Thiocarbazates Synthesized and Assayed Against Cathepsins B, L, and S
HRMS (m/z)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Observed
Theoretical
Error (ppm)
Thiocarbazate
Structure
O
H
O
[M+Na]+
562.2126
562.2100
4.62
BocHN
HN
N
N
S
N
1
4.45
0.056
0.96
H
H
O
Et
R
[M+Na]+
574.2102
574.2100
0.35
O
O
H
N
BocHN
S
5
6
7
4.46
>25.0
6.59
0.096
>25.0
0.029
0.93
>25.0
0.32
N
H
N
O
R
[M+H]+
526.2488
526.2483
0.95
O
H
N
BocHN
R
S
N
H
N
H
O
Et
[M+Na]+
562.2094
562.2100
0.96
O
O
H
N
BocHN
S
N
H
N
H
O
R
CO2Et
[M+Na]+
606.1995
606.1998
0.5
O
O
H
8
>25.0
0.062
0.68
BocHN
R
N
S
N
N
H
H
O
CO2Et
[M+Na]+
606.1995
606.1998
0.5
O
O
H
BocHN
R
N
S
9
>25.0
3.23
0.057
0.22
0.11
0.019
1.03
1.05
0.58
.95
N
N
H
H
O
[M+Na]+
576.1874
576.1893
0.33
O
O
H
N
BocHN
S
10
11
12
13
14
N
N
H
O
O
R
[M+Na]+
560.1943
560.1944
0.2
O
O
H
N
BocHN
R
S
N
H
N
2.60
1.65
0.53
2.90
2.07
O
[M+Na]+
574.2109
574.2100
1.50
O
O
H
BocHN
R
N
S
N
N
3.25
H
O
[M+Na]+
728.1520
728.1518
0.27
O
O
Ph
H
BocHN
N
S
Br
N
H
N
>25.0
14.04
O
R
[M+Na]+
560.1924
560.1944
3.50
O
O
H
BocHN
R
N
S
N
N
H
O

340 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
[M+Na]+
473.1458
473.1471
2.75
O
O
H
BocHN
R
N
S
N
OMe
15
16
17
18
19
20
21
22
23
24
25
26
6.33
>25.0
2.03
0.098
>25.0
1.29
0.57
>25.0
0.69
0.63
0.41
0.31
3.74
4.24
7.24
0.62
>25.0
>25.0
H
O
[M+Na]+
562.2126
562.2100
4.62
O
O
H
BocHN
N
S
N
N
H
H
O
Et
R
[M+Na]+
507.1713
507.1712
0.19
O
O
H
N
BocHN
MeS
S
N
H
N
H
O
Et
[M+Na]+
519.1699
519.1712
0.59
O
O
H
N
BocHN
MeS
S
2.21
1.34
N
H
N
O
[M+Na]+
519.1709
519.1712
0.96
O
O
H
BocHN
MeS
N
S
2.31
0.52
N
N
H
O
[M+Na]+
546.1807
546.1821
2.56
NH
O
O
H
N
BocHN
MeS
S
2.42
0.33
N
H
N
H
O
[M+Na]+
475.2003
475.1991
2.5
O
O
H
BocHN
BocHN
BocHN
N
S
0.87
0.66
N
N
H
H
O
Et
[M+Na]+
487.1994
487.1991
0.62
O
O
H
N
S
0.63
0.68
N
H
N
O
[M+Na]+
473.1838
473.1835
0.63
O
O
H
N
S
0.44
0.45
N
N
H
O
[M+Na]+
501.2155
501.2148
0.14
O
O
H
BocHN
N
S
>25.0
>25.0
>25.0
0.11
N
H
N
H
O
[M+Na]+
447.1685
447.1678
1.46
O
O
H
N
BocHN
S
>25.0
>25.0
N
H
N
H
O
Et
[M+Na]+
459.1674
459.1678
1.31
O
O
H
N
BocHN
S
N
H
N
O

Library of Thiocarbazates and Their Activity
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4 341
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
[M+Na]+
505.1733
505.1733
0
O
O
H
N
BocHN
HO2C
S
27
28
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
N
H
N
H
O
Et
[M+Na]+
517.1725
517.1733
1.54
O
O
H
N
BocHN
HO2C
S
N
H
N
O
O
O
[M+H]+
467.2318
467.2328
0.21
H
BocHN
BocHN
BocHN
N
S
N
N
29
30
31
2.63
>25.0
2.72
1.09
>25.0
1.64
1.65
>25.0
1.64
H
H
O
Et
O
[M+H]+
453.2516
453.2536
0.44
H
N
S
N
H
N
H
O
Et
O
O
[M+H]+
479.2327
479.2328
0.28
H
N
S
N
H
N
O
[M+Na]+
473.1819
473.1835
3.34
O
O
Boc
N
H
N
S
32
33
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
N
N
H
H
H
O
Et
[M+H]+
463.2014
463.2015
0.22
O
O
Boc
N
H
N
S
N
H
N
H
O
O
O
[M+Na]+
523.1977
523.1991
2.67
H
BocHN
N
S
N
N
H
34
3.06
0.12
0.93
H
O
Et
O
[M+H]+
487.2383
487.2379
0.82
H
N
BocHN
S
N
H
N
H
35
36
>25.0
5.08
>25.0
0.83
>25.0
2.83
O
Et
O
O
H
N
[M+Na]+
629.2419
629.2410
1.43
BocHN
S
N
H
N
H
O
Et
BnO

342 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
O
O
H
N
[M+Na]+
619.2406
619.2410
0.64
BocHN
S
N
H
N
37
3.94
0.63
3.34
O
BnO
O
O
H
N
[M+Na]+
646.1946
646.1948
0.31
BocHN
BocHN
BocHN
S
N
H
N
H
38
39
40
2.77
3.19
3.12
0.59
0.086
0.70
2.08
1.31
8.00
O
NO2
BnO
O
H
[M+Na]+
641.2401
641.2410
1.43
N
S
N
N
H
O
BnO
O
O
H
[M+Na]+
627.2245
627.2253
1.27
N
S
N
N
H
O
BnO
BnO
BnO
O
O
H
N
[M+Na]+
643.2224
643.2202
3.42
BocHN
S
N
H
N
41
42
3.03
1.56
0.50
3.98
1.90
O
O
O
H
[M+Na]+
655.2554
655.2566
1.83
BocHN
N
S
N
N
H
H
>25.0
O
CO2Et
O
O
[M+Na]+
673.2322
673.2308
2.10
H
BocHN
N
S
N
H
N
H
43
>25.0
0.14
1.08
O
BnO
[M+Na]+
567.2239
567.2253
2.47
O
O
O
O
H
BocHN
N
S
S
S
44
45
46
3.67
4.35
14.3
13.7
>25.0
1.27
3.32
5.15
2.15
N
H
N
H
O
O
O
Et
OBn
[M+Na]+
579.2236
579.2253
2.93
O
H
N
BocHN
N
H
N
OBn
[M+Na]+
553.2101
553.2097
2.53
O
H
N
BocHN
BnO
N
H
N
H
Et

Library of Thiocarbazates and Their Activity
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4 343
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
[M+Na]+
565.2097
565.2097
0
O
O
O
O
O
O
H
N
BocHN
BnO
S
S
S
47
48
49
50
51
52
53
54
55
56
57
58
13.2
>25.0
>25.0
9.28
1.51
15.4
17.9
0.37
0.51
0.58
0.38
1.85
0.70
0.43
>25.0
1.27
2.64
4.42
5.89
0.95
0.65
0.91
0.92
3.00
1.44
1.64
5.15
2.15
N
H
N
O
O
O
[M+Na]+
553.2087
553.2097
1.80
H
N
BocHN
BnO
N
H
N
H
Et
[M+Na]+
565.2088
565.2097
1.26
H
N
BocHN
BnO
N
H
N
[M+Na]+
565.2083
565.2097
1.23
O
O
H
BocHN
BnO
N
S
N
N
H
O
[M+Na]+
553.2082
553.2097
2.7
O
O
H
N
BocHN
BnO
S
N
H
NH
>25.0
>25.0
>25.0
13.7
O
[M+H]+
609.1385
609.1382
0.49
O
O
H
BocHN
N
S
Br
N
NH
H
O
BnO
[M+Na]+
592.2198
592.2206
1.16
O
O
H
BocHN
BnO
N
S
N
NH
NH
H
O
[M+Na]+
553.2094
553.2097
0.54
O
H
N
BocHN
S
N
N
H
O
O
BnO
[M+Na]+
570.1649
570.1635
1.05
O
O
H
N
BocHN
BnO
S
5.07
N
H
N
H
O
NO2
[M+Na]+
597.1998
597.1995
0.50
O
O
H
N
BocHN
S
>25.0
4.35
N
H
N
CO2Et
H
O
BnO
[M+H]+
628.2802
628.2805
0.50
O
O
H
N
BocHN
S
N
H
N
CbzHN
O
[M+H]+
616.2786
616.2805
3.1
O
O
H
BocHN
N
S
14.3
N
H
N
H
CbzHN
O
Et

344 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
O
O
O
O
H
BocHN
O
N
S
S
S
N
H
N
N
N
[M+H]+
662.2664
662.2648
0.60
O
O
O
59
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
NH
O
O
H
N
BocHN
O
N
H
[M+H]+
674.2639
674.2648
1.33
60
NH
O
O
H
N
BocHN
O
N
H
[M+H]+
662.2669
662.2648
3.17
61
NH
O
O
O
H
[M+Na]+
577.2145
577.2169
4.16
BocHN
HN
HN
N
S
N
N
H
H
62
63
>25.0
3.67
2.15
3.07
O
Et
NHNO2
O
O
H
N
[M+H]+
565.2214
565.2193
3.71
BocHN
HN
HN
S
N
H
N
19.7
>25.0
O
NHNO2
[M+Na]+
433.1513
433.1522
2.08
O
O
H
BocHN
N
S
64
65
>25.0
13.7
>25.0
>25.0
>25.0
>25.0
N
N
H
H
O
Et
O
O
[M+H]+
491.2057
491.2077
4.07
H
N
BocHN
N
S
N
H
N
H
O
Et
NH
O
O
[M+H]+
503.2071
503.2077
1.19
H
N
BocHN
N
S
N
H
N
66
11.6
>25.0
>25.0
O
NH

Library of Thiocarbazates and Their Activity
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4 345
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
[M+Na]+
362.1144
362.1150
1.66
O
O
H
N
S
67
68
69
70
71
72
73
74
75
76
77
78
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
11.5
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
5.52
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
2.41
HO
N
H
N
H
O
Et
[M+Na]+
374.1152
374.1150
0.53
O
O
H
N
S
HO
N
H
N
O
[M+Na]+
362.1144
362.1150
1.70
O
O
H
N
S
HO
N
H
N
H
O
Et
[M+Na]+
374.1152
374.1150
0.53
O
O
H
N
S
HO
N
H
N
O
[M+Na]+
476.2003
476.2015
2.52
O
O
H
N
S
TBSO
N
N
H
H
O
Et
[M+Na]+
482.1727
428.1726
0.21
O
O
H
N
S
O
N
N
H
H
O
Et
MeO
[M+H]+
447.1485
447.1467
4.92
O
O
H
N
S
N
N
H
H
O
Et
HN
[M+H]+
439.2004
439.2015
2.5
O
O
H
N
S
BocHN
N
N
H
H
O
Et
[M+H]+
451.2012
451.2015
0.67
O
O
H
N
S
BocHN
N
H
N
O
[M+H]+
499.2047
499.2049
0.40
MeS
O
O
H
N
S
BocHN
N
H
N
H
O
Et
[M+H]+
511.2044
511.2049
0.98
MeS
O
O
H
N
S
6.95
3.01
BocHN
N
H
N
O
[M+Na]+
489.2125
489.2148
4.70
O
O
H
N
S
7.52
>25.0
BocHN
N
H
N
H
O
Et

346 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
(Table 2) contd…..
HRMS (m/z)
Observed
Theoretical
Error (ppm)
IC50 (ꢀM)
Cat B
IC50 (ꢀM)
Cat L
IC50 (ꢀM)
Cat S
Thiocarbazate
Structure
[M+H]+
479.2319
479.2328
1.88
O
O
H
N
S
79
80
81
82
83
84
85
15.8
>25.0
>25.0
>25.0
>25.0
2.12
>25.0
>25.0
>25.0
4.47
BocHN
N
N
H
O
[M+H]+
465.2172
465.2172
0
O
O
H
H
N
S
>25.0
>25.0
>25.0
>25.0
>25.0
>25.0
N
N
N
Boc
H
H
O
Et
[M+H]+
477.2177
477.2172
1.05
O
O
H
H
N
S
N
N
H
N
Boc
O
[M+Na]+
567.2276
567.2253
4.05
BnO
O
O
H
N
S
BocHN
N
N
H
H
O
Et
[M+H]+
557.2435
557.2434
0.18
BnO
O
O
H
N
S
5.95
BocHN
N
H
N
O
[M+H]+
545.2438
545.2434
0.73
BnO
O
O
H
N
S
>25.0
>25.0
15.2
BocHN
N
H
N
H
O
Et
[M+H]+
557.2454
557.2434
3.59
BnO
O
O
H
N
S
>25.0
BocHN
N
H
N
O
HN
[M+Na]+
576.2249
576.2257
1.39
O
O
H
86
>25.0
>25.0
>25.0
N
S
BocHN
N
N
H
H
O
Et
CHARACTERIZATION OF THIOCARBAZATE LIBRARY
activity was detected against any other protease family
member (e.g., serine, aspartyl, metallo-). Second, even
among the cysteine protease family, a preference for the
papain family is observed, as only modest activity against
representatives of the calpain or caspase families is seen.
Finally, several thiocarbazates exhibit potent activity (>95%
inhibition at 10 μM) against cathepsins L, S, V, K, and
papain.
As neither thiocarbazates nor their activity as protease
inhibitors have been described prior to our publication, we
profiled
a
subset of twenty-two compounds at
a
concentration of 10 μM for inhibitory activity against 75
different proteases [29] (Fig. 2). The proteases chosen
covered a broad spectrum of classes including serine
proteases, metallo-proteases, aspartyl proteases and cysteine
proteases. The aim of this study was to determine quickly
whether thiocarbazates as a class displayed selectivity
towards different families of proteases, or displayed broad
protease inhibition properties.
Based on this study, we focused further attention on a
more thorough biological characterization of this library
against cathepsins B, L and S. This choice was based on the
potential for potent inhibition, as well as selectivity. In
addition, all three cathepsins represent important targets for
drug discovery efforts. Towards this end, IC50 values were
generated for all 82 members of the thiocarbazate library
(Table 1) against cathepsin B, L and S.
The heatmap illustrated in Fig. (2) allows us to draw
several broad conclusions. First, the thiocarbazates as a class
exhibit selectivity towards cysteine proteases. No significant

Library of Thiocarbazates and Their Activity
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4 347
Fig. (2). Protease profiling heatmap of 22 thiocarbazates at 10 μM against 75 proteases.
preference for hydrophobic amino acids at the P2 position
Based on these data, we offer several generalizations for
broad cathepsin inhibition. First, the substitution patterns
displayed at positions A and B are key determinants for
inhibitory activity. Alpha amino acid derived thiocarbazates
are the preferred substituents, while those prepared from beta
amino acid derivatives (74-86) and acid precursors (67-73)
are devoid of activity. Among the thiocarbazates that
incorporate alpha amino acids, the size of substituent B had a
profound effect on potency. Thiocarbazates containing large
groups (such as 3-indolemethylene, benzyl, 4-
benzyloxybenzyl) or medium sized substituents (such as
isopropyl, methyl thioethyl) were more potent than those
incorporating smaller groups [e.g., hydrogen (64), methyl
(25)]. Incorporation of the specific amino acids proline (32,
33, 80, 81) or glutamic acid (27, 28) proved detrimental to
activity. Based on our earlier modeling studies on a
prototypical thiocarbazate (1) [21], we presume the B
substituent occupies the S2 site of the active site binding
pocket. The structure activity relationships (SAR) trends for
the B position are consistent with substrate specificity
profiling studies on cysteine proteases that indicate a
[30]. Stereochemistry preferences at position B were also
explored through the preparation of key enantiomeric pairs
such as 1 and 16; 46 and 48; and 47 and 49. In those
examples, the thiocarbazate derived from the L-amino acid
was more potent than that derived from the D isomer.
Structural requirements for activity at position C involved a
strong preference for a carbonyl group (6 vs 5; 30 vs 29; 35
vs 34). In all cases where the direct comparison could be
made, removal of the carbonyl group significantly
diminishes the inhibitory activity against all three cathepsins.
In contrast, the specific nature of the amine side chain
influenced activity far less.
CATHEPSIN B
Inhibitors of cathepsin B have been proposed for the
treatment of cancer, osteoporosis, arthritis, and viruses [2].
As such, there is an interest in identifying potent and
selective inhibitors of this member of the papain family. As a
class,
a number of thiocarbazates effectively inhibit

348 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
cathepsin B with potencies between 500nM and 10 μM, a
range lower than the potencies associate with cathepsin L
and S inhibition. The B position generally tolerated a wider
array of substitutents (aliphatic, aromatic, and histidine) than
the other cathepsins. This observation is consistent with
substrate profiling studies indicating a broader tolerance at
the P2 position [30]. Unlike other cathepsins, cathepsin B
contains a glutamic acid lining the S2 pocket which may
explain its unique tolerance of an imidazole moiety
occupying the S2 site and the selectivity observed in
compounds 65 and 66. The most potent thiocarbazates, with
IC50 values in the sub-micromolar range, incorporate leucine
in the A/B area (e.g., 21, 22, 23) and a variety of substituents
at C, but are non-selective.
48, 49) analogs exhibit >2-fold selectivity over cathepsin L.
These suggestions of selectivity may find their basis on the
presence of a hydrophobic phenylalanine residue at the S2
subsite, rather than the smaller alanine in cathepsin L which
prefers larger residues.
CATHEPSIN S DOCKING STUDIES
The publicly available x-ray crystal structure of cathepsin
S complexed to a small molecule inhibitor (2hh5.pdb) was
selected to predict the protein/ligand binding interactions of
thiocarbazates 69 and 20. Both inhibitors are presumed to
bind within the catalytic triad binding site of cathepsin S.
These two compounds were selected for docking studies
given their differences in inhibitory activity: 69 has an IC50
of only 25 μM, whereas 20 has an IC of 310 nM. Each
50
CATHEPSIN L
compound was docked independently in the binding site of
Cathepsin S (Fig. 3) [33]. As expected, a significant
difference between the total energy scores for 69 and 20 was
calculated. The total energy score for the potent inhibitor 20
was –6.13 kcal/mol, while the score for the weakly active 69
was only –1.54 kcal/mol. It is presumed that each of the two
compounds binds covalently to the active site Cys25 thiolate
in the protein with the electrophilic carbon adjacent to the
sulfur in the thiocarbazates. The distance between the Cys25
sulfur and the electrophilic carbon of the inhibitors is in the
range of 2.9-3.3 Å for both compounds, further supporting
covalent binding between the inhibitor and the protein.
The role of cathepsin L in bone and cartilage remodeling,
as well as in infectivity by certain infectious organisms, has
focused interest on identifying inhibitors of this protease. A
number of potent inhibitors of cathepsin L were identified
among the thiocarbazate library. The most potent (<100 nM)
and selective (>10-fold over cathepsin B and S) examples
(e.g., 1, 5, 7, 8, 9, 12, 39) contained an aromatic group at
position B, with a preference for 3-indolemethyl over benzyl
and substituted benzyl analogs. The high potency afforded
when an aromatic moiety can occupy the S2 site is consistent
with substrate specificity studies showing cathepsin L’s
strong preference for aromatic amino acid residues over
aliphatic residues at the P2 position. This preference has been
attributed to a small residue (Ala205) at the bottom of the S2
site, which results in a relatively large and open-ended
pocket compared to the analogous larger residues, and
therefore small pockets, found in cathepsin B and S [31].
There is a tolerance for a broad array of C substituents with
anilides (1, 8, 9), constrained anilides (5), alkyl amides (7,
12), amines (39) and esters (15) exhibiting potent inhibition.
This position, which according to our previous modeling
studies [21] likely occupies the S1’ site, does not appear to
be a strong determinant of selectivity, an observation
consistent with the high similarity of the cathepsin B, S and
L S1’ binding pockets [32].
Examination of the non-covalent interactions between 20
and the catalytic binding site residues of cathepsin S reveals
strong hydrophobic interactions within the S2 subsite,
specifically between Phe 211 of cathepsin S and the indole
of 20. In the S1’ subsite, there is also a strong hydrophobic
pocket formed by the interaction of the O-tert-butyl group of
20 and Trp 186. Non-covalent binding interactions of the
weakly active inhibitor were also noted. However, docking
studies reveal that 69 does not provide any side-chain
hydrophobic group for interaction within the S2 subsite (Fig.
3), as seen with 20. This observation could explain the lack
of potency of 69. The overlay of 69 and 20 in the binding
site of cathepsin S illustrates this void in the S1’ pocket
around Trp 186 for 69.
CATHEPSIN S
CONCLUSIONS
Inhibitors of cathepsin S have garnered significant
attention as potential treatment for several autoimmune
diseases such as psoriasis, rheumatoid arthritis, multiple
sclerosis and asthma. Among the thiocarbazate library, a
number of sub-micromolar inhibitors have been identified.
Unlike cathepsins B and L, cathepsin S shows a broad
tolerance for the nature of the substituent at B. Strong
inhibition (< 1 μM) was observed among thiocarbazates
containing aromatic (7, 12, 15), branched alkyl (24),
thioethers (17, 18, 19, 20), and benzyl ethers (51, 52, 53).
This hydrophobic preference was observed in substrate
specificity profiling [30]. Similar to the cathepsin B and L
SAR, tolerances for diverse size and functionalities at
position C were observed. The most potent cathepsin S
inhibitors typically were even more potent cathepsin L
inhibitors (e.g., 7, 9, 12). However certain trends suggest that
selectivity over cathepsin L is possible. For example, several
alkyl thioether (e.g., 17, 18) and benzyl ether (e.g., 44, 45,
Based on our earlier finding that thiocarbazates can act as
inhibitors of proteases, we designed and synthesized a
library with structural modifications at three different
positions. The library was designed to incorporate diverse
size, shapes and functionalities in order to profile
thiocarbazates as a class of inhibitors against a series of
representative proteases. Preliminary profiling indicated that
thiocarbazates are selective for the papain-like cysteine
protease; exhibiting limited or no activity against serine,
aspartyl, metallo-proteases, nor members of the calpain or
caspase families of cysteine proteases. Complete IC50
characterization against cathepsins B, L and S identified a
number of potent and selective inhibitors of cathepsin L. In
addition, trends to direct the design of new thiocarbazates
exhibiting potent and selective inhibitory activity against
cathepsins B and S were also developed. Many of these SAR
trends are consistent with previous studies detailing the

Library of Thiocarbazates and Their Activity
Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4 349
substrate specificity of cathepsins B, L and S. and can be
rationalized on the basis of residues in the enzyme binding
pocket. Further characterization of the cathepsin L inhibitors
disclosed, as well as the development of improved inhibitors
of Cathepsin B and L will be reported in due course.
Boc-phenylalanine; 36-43 from N-Boc-Try(Bzl); 44-45 from
N-Boc-Thr(Bzl); 46-47, 50-56 and 74-75 from N-Boc-
serine; 48-49 from D-N-Boc-serine; 57-58 from N-Boc-
Lys(Z); 59-61 from N-Boc-Glu(Xan); 62-63 from N-Boc-
Arg(NO2); 64 from N-Boc-glycine; 65-66 from N-Boc-
histine; 62-72 from 2-methyl-3-hydroxypropionate.
Synthesis of Thiocarbazates Derived from 2-Methyl-3-
Hydroxypropionate
Synthesis of 67-70: To a solution of 2-methyl-3-
hydroxypropionate (1 mL, 7.94 mmol) in THF (10 mL) was
added hydrazine hydrate (1.16 mL, 11.91 mmol). The
mixture was stirred at room temperature overnight and
concentrated to give hydrazide (820 mg, 88%). The hydroxyl
thiocarbazates 67-70 were obtained following the procedure
described above.
Synthesis of 71: To the solution of 69 (34 mg, 0.1 mmol)
in CH2Cl2 (8 mL) was added imidazole (13.6 mg, 0.20
mmol) and TBSCl (17 mg, 0.11 mmol) at 0º C. The resulting
mixture was warmed and stirred at room temperature for
48h. The mixture was washed with 1M HCl, saturated
NaHCO3 and brine solution and extracted with CH2Cl2. The
combined organic layers were dried and concentrated. The
residue was purified by HPLC to give 71 (22mg, 50%).
Fig. (3). Docking poses of 20 and 69 into cathepsin S Model.
EXPERIMENTAL SECTION
Synthesis of 72: To the solution of 69 (34 mg, 0.1 mmol)
in CH2Cl2 (7 mL) was added 2-(4-methoxylbenzyoxy)-3-
nitropyridine (39 mg, 0.15 mmol) and CSA (5 mg). The
resulting mixture was stirred at room temperature for 3 h.
The mixture was washed with saturated NaHCO3 and
extracted with CH2Cl2. The combined organic layers were
dried and concentrated. The residue was purified by HPLC
to give 72 (7 mg, 16%).
General Procedure for the Synthesis of Thiocarbazates
To a solution of acid (10.0 mmol) in THF (15 mL) was
added NEt3 (1.45 mL). After stirring at room temperature for
5 min, the mixture was cooled to -10ºC followed by the
addition of the solution of ethyl chloroformate (1.0 mL) in
THF (15 mL). After stirring 10 min, the mixture was filtered
and washed with THF (2 x 5 mL). The combined filtrate was
slowly added to a solution of hydrazine (2 mL) in methanol
(26 mL) at 0ºC. The mixture was allowed to warm to room
temperature and was concentrated after stirring 1 h. The
residue was diluted with ethyl acetate (80 mL) and washed
successively with saturated NaHCO3 (12 mL) and brine (12
mL). The organic layer was dried over Na2SO4 and filtered.
After removal of volatile solvents, the residue was used
without further purification.
NMR of Representative Thiocarbazates
Compound 1: 1H NMR (500 MHz, DMSO-d6, VT-
350K) ꢀ 10.61 (br s, 1H), 10.04 (br s, 1H), 9.88 (br s, 1H),
9.14 (br s, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.43 (d, J = 7.4 Hz,
1H), 7.32 (d, J = 8.2 Hz, 1H), 7.21 (d, J = 7.3 Hz, 1H), 7.16-
7.11 (m, 3H), 7.05 (t, J = 7.1 Hz, 1H), 6.97 (t, J = 7.4 Hz,
1H), 6.47 (br s, 1H), 4.31 (br s, 1H), 3.73 (br s, 2H), 3.17
(dd, J = 14.7, 4.1 Hz, 2H), 2.96 (m, 2H), 2.59 (q, J = 7.5 Hz,
2H), 1.29 (br s, 9H), 1.13 (t, J = 7.5 Hz, 3H).
The hydrazide (1.0 mmol, 1.0 equiv.) was dissolved into
a solution of KOH in 95% EtOH (0.25 M, 4.4 mL, 1.1
equiv.) in a 25 mL round bottom flask at room temperature.
After stirring for 5 min, 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 mixture
was stirred for 15 h at 23°C, followed by the addition of a
bromide (1.1 equiv.). The reaction was monitored by LC-
MS, and the alkylating agent was typically consumed within
20 to 60 min. The reaction mixture was filtered and the
filtrate was concentrated in vacuo. The residue was purified
by preparative reverse phase HPLC and identified by
HRMS.
1
Compound 5: H NMR (500 MHz, DMSO-d6,) ꢀ 9.79-
10.80 (m, 3H), 7.67 (m, 1H), 7.46 (m, 1H), 7.33 (m, 1H),
6.93-7.18 (m, 5H), 6.78 & 6.31-6.40 (br s, 1H), 4.23 (br s,
1H), 4.04 (br, s, 1 H), 3.87 (br s, 1H), 3.70 (br s, 2H), 3.12
(m, 1H), 2.90 (m, 1H), 2.69 (m, 2H), 1.88 (m, 2H), 1.29 &
1.13 (br s, 9H).
1
Compound 14: H NMR (500 MHz, DMSO-d6,) ꢀ9.93-
10.87 (m, 3H), 7.65 (m, 2H), 7.31 (m, 2H), 7.13 (m, 2H),
7.04 (m, 2H), 6.87(m, 1H), 4.28 (m, 1H), 3.86 (m, 2H), 3.12
(m, 1H), 2.90 (m, 1H), 2.69 (m, 2H), 2.10 (s, 3 H), 2.15-1.81
(m, 2H), 1.28 & 1.14 (br s, 9H).
Using this procedure compounds 1, 5-15 and 73 were
prepared from L-N-Boc trytophan; 16 from D-N-Boc-
tryptophan; 17-20 and 76-77 from N-Boc methionine; 21-24
from N-Boc valine; 25-26 and 78-79 from N-Boc alanine;
27-28 from N-Boc-Asp(OBzl); 29-31 from N-Boc-
leucine;32-33 and 80-81from N-Boc-proline; 34 from N-
1
Compound 19: H NMR (500 MHz, CDCl3) ꢀ 8.94 (br
s, 1H), 7.21-7.11 (m, 5H), 5.33 (br s, 1H), 4.72 (m, 2H), 4.42
(d. J = 5.4 Hz, 1H), 3.93 (br s, 1H), 3.91 (br s, 1H), 3.82 (t, J
= 5.8 Hz, 1H), 3.75 (t, J = 5.9 Hz, 1H), 2.94 (t. J = 5.8 Hz,
1H), 2.85 (t, J = 5.9 Hz, 1H), 2.63-2.58 (m, 2H), 2.12 (br s,
1H), 1.96 (ddd, J = 14.2, 7.1, 7.1 Hz, 1H), 1.43 (s, 9H).

350 Combinatorial Chemistry & High Throughput Screening, 2010, Vol. 13, No. 4
Liu et al.
1
Compound 24: H NMR (500 MHz, CDCl3) ꢀ 8.82 (br
s, 1H), 8.35 (br s, 1H), 7.21(s, 1H), 7.18(d, J = 8.3 Hz, 2H),
6.96 (d, J 8.3 Hz, 2H), 5.15 (br s, 1H), 4.07-4.01 (m, 1H),
3.62 (br s, 2H), 2.70 (s, 2H), 2.69 (s, 2H), 2.05 (br s, 1H),
1.75 (t, J = 3.0 Hz, 4H), 1.41 (s, 9H), 1.00 (d, J = 6.7 Hz,
3H), 0.97 (d. J = 6.7 Hz, 3H).
ACKNOWLEDGEMENTS
Financial support for this work was provided by the NIH
(5U54HG003915). We thank Dr. G.T. Furst (NMR Facility)
and Dr. R. Kohli (Mass Spectrometry Facility) at the
University of Pennsylvania for assistance in obtaining NMR
and high-resolution mass spectra.
1
Compound 37: H NMR (500 MHz, CDCl3) ꢀ7.41 (d, J
= 6.9 Hz, 2H), 7.38-7.35 (m, 2H), 7.32-7.30 (m, 1H), 7.20-
7.17 (m, 2H), 7.16-7.11 (m, 4H), 6.89 (dd, J = 8.6, 3.2 Hz,
2H), 5.06 (br s, 1H), 5.01 (s, 2H), 4.70 (d, J = 14.5 Hz, 2H),
4.44 (br s, 1H), 3.90 (d, J = 9.5 Hz, 2H),3.81 (t, J = 6.0 Hz,
1H), 3.73 (t, J = 6.0 Hz, 1H), 3.10 (d, J = 14.1, 6.3 Hz, 1H),
2.97 (t, J = 6.9 Hz, 1H), 2.93 (t, J = 5.8 Hz, 1H), 2.84 (t, J =
5.8 Hz, 1H), 1.37 & 1.37 (s, 9H).
Compound 38: 1H NMR (500 MHz, CDCl3) ꢀ 8.67 (d, J
= 8.6 Hz, 2H), 8.61 (br s, 1H), 8.15 (d, J = 8.4 Hz, 2H), 7.61
(t, J = 8.1 Hz, 1H), 7.40-7.36 (m, 4H), 7.32-7.29(m, 1H),
7.16 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 8.4 Hz, 2H), 6.88 (d, J =
8.4 Hz, 2H), 5.15 (br s, 1H), 4.99 (s, 2H), 4.45 (br, d, J = 6.0
Hz, 1H), 3.77 (br s, 2H), 3.11-3.08 (m, 1H), 2.97 (br s, 1H),
1.37 (s, 9H).
Compound 72: 1H NMR (500 MHz, DMSO-d6,) ꢀ 9.37-
10.16 (m, 3H), 7.40 (d, J = 7.5 Hz, 1H), 7.11 (m, 5H), 6.89
(d, J = 8.0 Hz, 2H), 4.48 & 4.38 (br s, 2H), 3.80 (m, 1H),
3.73 (s, 3H), 3.69 (s, 1H), 3.56 (m, 2H), 2.58 (m, 3H), 1.11
(t, J = 7.5 Hz, 3H), 1.03 (m, 3H).
Compound 85: 1H NMR (500 MHz, CDCl3) ꢀ 7.47-7.27
(m, 5H), 7.20-7.05 (m, 4H), 5.37 (br s, 1H), 4.52 (s, 2H),
4.14-4.11 (m, 1H), 3.92 (br s, 2H), 3.80 (t, J = 6.4 Hz, 2H),
3.63-3.56 (m, 1H), 3.54-3.51(m, 1H), 2.74-2.58 (m, 2H),
2.66-2.53(m, 2H), 1.99-1.91 (m, 2H), 1.42 (s, 9H).
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Received: January 20, 2009
Revised: June 17, 2009
Accepted: July 21, 2009