Identification and synthesis of a unique thiocarbazate cathepsin L inhibitor

Article abstract of DOI:10.1016/j.bmcl.2007.10.107

Library samples containing 2,5-disubstituted oxadiazoles were identified as potent hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) directed at discovering inhibitors of cathepsin L. However, when synthesized in pure form, the putative actives were found to be devoid of biological activity. Analyses by LC-MS of original library samples indicated the presence of a number of impurities, in addition to the oxadiazoles. Synthesis and bioassay of the probable impurities led to the identification of a thiocarbazate that likely originated via ring opening of the oxadiazole. Previously unknown, thiocarbazates (-)-11 and (-)-12 were independently synthesized as single enantiomers and found to inhibit cathepsin L in the low nanomolar range.

Full text of DOI:10.1016/j.bmcl.2007.10.107

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 210–214
Identification and synthesis of a unique
thiocarbazate cathepsin L inhibitor
Michael C. Myers,a,b Parag P. Shah,a,c Scott L. Diamond,a,c,
*
a,b,
Donna M. Huryna,b, and Amos B. Smith, III
*
*
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 6 October 2007; revised 22 October 2007; accepted 25 October 2007
Available online 1 November 2007
Abstract—Library samples containing 2,5-disubstituted oxadiazoles were identified as potent hits in a high throughput screen (HTS)
of the NIH Molecular Libraries Small Molecule Repository (MLSMR) directed at discovering inhibitors of cathepsin L. However,
when synthesized in pure form, the putative actives were found to be devoid of biological activity. Analyses by LC–MS of original
library samples indicated the presence of a number of impurities, in addition to the oxadiazoles. Synthesis and bioassay of the prob-
able impurities led to the identification of a thiocarbazate that likely originated via ring opening of the oxadiazole. Previously
unknown, thiocarbazates (ꢀ)-11 and (ꢀ)-12 were independently synthesized as single enantiomers and found to inhibit cathepsin
L in the low nanomolar range.
Ó 2007 Elsevier Ltd. All rights reserved.
The cathepsins comprise a family of lysosomal protease
enzymes whose primary function (i.e., protein degrada-
tion) plays a critical role in normal cellular homeosta-
sis.1 Overexpression of cathepsin L and/or abnormal
activity has been implicated in a number of disease
states.2 For example, cathepsin L is responsible for bone
resorption through degradation of collagen type I; this
disregulation is believed to lead to osteo- and rheuma-
toid arthritis.3 In addition, several infective organisms,
such as SARS and Ebola viruses, utilize cathepsin L-like
proteins for replication in human cells.4 The large num-
ber of disease states associated with cathepsin L calls for
an understanding of its biological function.2
including cathepsins B, L, and S.6 In this letter, we detail
our continuing efforts to create a comprehensive, pub-
licly available profile of small-molecule inhibitors of
the cysteine protease class, and herein describe the iden-
tification of a novel class of potent cathepsin L
inhibitors.
Previously reported inhibitors of cathepsin L include the
peptides, leupeptin and aprotinin, and the fluoromethyl
ketone, Z-LLL-FMK.3,7 The few known, potent small-
molecule inhibitors are either peptidic and therefore
likely to suffer from physiological instability and poor
permeability, or are non-selective for cathepsin L.3,8,9
The identification of potent, selective, stable, and cell
permeable small-molecule inhibitors would therefore
provide valuable tools to interrogate cathepsin L and
cathepsin L-like function, as well as potential starting
points for drug discovery and development.10–15
Recently, the Penn Center for Molecular Discovery
(PCMD),5 completed a high throughput screening
(HTS) campaign of the NIH Molecular Libraries Small
Molecule Repository (MLSMR) to identify inhibitors of
members of the papain-like cysteine protease family,
Initial HTS results of our cathepsin L screen indicated
that several structurally related oxadiazoles exhibited
potent inhibitory activity (Table 1).16–18 The most po-
tent hit, cataloged in the MLSMR as disubstituted oxa-
diazole 1, exhibited an IC50 of 0.13 lM.17 Wells
containing related analogs (2–5), differing only in the
Keywords: Thiocarbazate; Cathepsin L inhibitor; Cysteine protease
inhibitor; MLSCN.
*
Corresponding authors. E-mail addresses: sld@seas.upenn.edu;
huryn@sas.upenn.edu; smithab@sas.upenn.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.107

M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 210–214
211
Table 1. Cathepsin L inhibitory activity of oxadiazole-containing
library samplesa
to a-amino acid substituted oxadiazolethiones could
provide an entry to this class of sulfur-based 2,5-disub-
stituted oxadiazoles. Toward this end, commercially
available L-Boc-Trp-OH was converted in high yield to
the corresponding hydrazide (+)-6, on a 10-g scale
(Scheme 1).20 Cyclization with carbon disulfide in etha-
nol at reflux afforded the expected substituted thione
(+)-7 as a stable solid in excellent yield, which upon
chemoselective alkylation with 2-bromo-N-(2-ethyl-phe-
nyl)-acetamide21 (9) efficiently generated (+)-8 (94%
yield). Further study indicated that isolation of (+)-7
was unnecessary, and (+)-8 could be prepared directly
from (+)-6, in a single flask. Both sequences furnished
(+)-8 in high yield. In similar fashion, the antipode,
(ꢀ)-8, was prepared starting from D-Boc-Trp-OH. High
enantiomeric purities (>99%) were demonstrated via
chiral supercritical fluid chromatography.
HN
NH2 HCl
O
O
S
R1
+ Impurities
N
(S)
R2
N
N
b
PubChem SID
R1
2-Ethylbenzene
2,4-Dimethylbenzene
R2
IC50 (lM)
861540 (1)
861087 (2)
861840 (3)
861542 (4)
861992 (5)
H
0.13 0.01
0.16 0.05
0.17 0.02
0.30 0.04
0.51 0.02
H
Me
2,3-Dimethylbenzene
Et
Me
H
Et
a Library samples containing the parent oxadiazole with impurities.
b IC50 values are reported as means standard deviation (number of
determinations = 3).
Initial attempts to remove the Boc-group utilizing HCl
in dioxane (4 N) or HCl in water (6 N) were compro-
mised by the poor solubility of (+)-8. However, small
quantities of the HCl salt of (+)-1 could be obtained
by treatment with 6 N HCl over 45 min. Upon LC–
MS analysis of synthetic (+)-1, we recognized that the
impurities formed during the acid-promoted Boc-depro-
tection step were identical to those found in the library
screening samples (vide infra). Optimal conditions to re-
move the Boc-group were eventually developed. Specif-
ically, treatment of (+)-8 with 25% water in TFA for
15 min, followed in turn by adjustment of the pH to
8.0 with NaHCO3 and aqueous extraction with methy-
lene chloride-furnished the free-base of (+)-1 in 89%
yield and 99% purity after column chromatography. In
the end, oxadiazole (+)-1 was prepared on gram scale
from L-Boc-Trp-OH; the overall yield was 76%.
amide nitrogen substitution, displayed a range of activ-
ities (IC50 = 0.16–0.51 lM), suggesting a nascent struc-
ture activity relationship. As is our practice, library
samples containing the putative active oxadiazoles were
evaluated for both purity and integrity by LC–MS anal-
ysis. This analysis indicated that the primary constituent
of each sample was indeed the expected oxadiazole, in
up to 60% purity, however numerous impurities were
also present. To confirm the biological activity attrib-
uted to 1, a synthetic sequence was developed to gener-
ate the oxadiazoles in pure form.
Although little literature precedence exists for the con-
struction of compounds such as (+)-1, we realized that
modification of the Woodward–Confalone19 approach
O
O
a) Et3N, EtOCOCl
THF, -10 oC, 15 min
BocHN
HN
BocHN
NHNH2
OH
b) NH2-NH2, MeOH
0 oC, 45 min, (96%)
(+)-6
HN
NHBoc
O
S
2-bromo-N-(2-ethyl-
phenyl)-acetamide (9)
a) CS2, KOH, EtOH
reflux, 2 h;
N
NH
Et3N, CHCl3, 23 oC, 1 h
(94%)
b) HCl (1 N) (95%)
HN
(+)-7
NR1R2
O
O
S
N
H
6 N HCl (aq)
N
N
Et
23 oC, 45 min (10%
plus recovered (+)-8
or
HN
(+)-8; R1 = H, R2 = Boc
(+)-1
TFA:H2O (3:1)
23 oC, 15 min (89%)
Scheme 1. Synthesis of 2,5-disubstituted oxadiazoles.

212
M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 210–214
HN
NHBoc
O
4
2
O
S
Inactive Series
N
H
N
N
Et
(+)-8
O
O
H
2
HCl H2N
HN
N
S
N
N
4
HCl
H2O
H
H
Et
O
10
Active Series
Figure 1. Conversion of oxadiazole (+)-8 to thiocarbazate 10.
To our surprise, the free-base of (+)-1 was found to be
completely devoid of activity when assayed against
cathepsin L.18 This result suggested that an impurity
present in the original library sample was responsible
for the observed activity. Based on LC–MS analyses
of the biologically active samples, we hypothesized that
the active component was likely the ring-opened product
10, formed via acid-promoted addition of H2O to (+)-8
(Fig. 1). The presence of a molecular [M+1] ion equal to
440 amu in the LC–MS analysis of impure samples was
consistent with the presence of 10, thereby providing
strong support for the hypothesis.
Thiocarbazates such as 10 have not been described pre-
viously in the literature. However, these compounds do
bear structural resemblance to aza-peptides22–24 (e.g., A;
Fig. 2),25 examples, of which have been reported to ex-
hibit cysteine protease inhibitory activity through a
mechanism involving attack by the active site cysteine
on the carbamate carbonyl.26
To test the hypothesis that 10 was indeed the active spe-
cies, an expedient synthesis of this structural class was
devised. We envisioned that introduction of the C2 car-
bonyl unit of 10 could be achieved via chemistry parallel
to that used to prepare (+)-1, beginning with hydrazide
(+)-6. We began the synthesis by converting tryptophan
hydrazide (+)-6 to an intermediate thiosemicarbazide
(not shown) employing carbonyl sulfide (S@C@O) gas
dissolved in ethanol (Scheme 2).27 The intermediate thi-
osemicarbazide did not precipitate from solution, there-
fore 2-bromo-N-(2-ethyl-phenyl)-acetamide21 (9) was
added to the reaction flask to generate thiocarbazate
(ꢀ)-11.28 Pleasingly, the yield for the two-step, one-flask
operation was 62%. Deprotection of (ꢀ)-11, employing
our previously developed conditions (25% water in
TFA), generated the free-base (ꢀ)-12 in 98% yield with
O
H
AcHN
N
OPh
N
H
O
A
kon = >11000 M-1s-1, papain
Abeles and Magrath (1992)
Figure 2. Known aza-peptide cathepsin inhibitor A.
O
a) SCO (gas), KOH, EtOH
BocHN
23 oC, 15 h
NHNH2
b) 2-bromo-N-(2-ethyl-phenyl)-
acetamide (9), 23 oC, 20 min
(62%)
HN
(+)-6
O
O
H
R2R1N
N
S
N
N
H
H
O
Et
HN
(-)-11; R1 = H, R2 = Boc
(-)-12; R1, R2 = H
TFA:H2O (3:1)
23 oC, 20 min
(98%)
Scheme 2. Synthetic procedure to prepare thiocarbazates.

M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 210–214
213
O
O
H
2
H2N
N
S
(-)-12
4
N
N
H
H
O
Et
HN
decomposition
in DMSO
O
O
O
HN
HN
NH
N
H
N
2
4
H
NH
SH
Et
S
Et
Et
S
H
14
15
13
O
N
O
Figure 3. Decomposition products of (ꢀ)-12 in DMSO.
>95% purity. This three-step sequence permitted con-
struction of (ꢀ)-12 on gram scale, starting from L-Boc-
Trp-OH; the overall yield was 58%.
the C2 carbonyl group. Reactivity at the anilide car-
bonyl is also a possibility and cannot be ruled out. How-
ever, we observe no evidence of side-products resulting
from reaction at this position (cf. Fig. 3). A complete
biochemical characterization of this novel chemotype
is underway.31
Both (ꢀ)-11(S) and (ꢀ)-12(S) exhibited potent inhibi-
tory activity against cathepsin L with IC50 values of
56 nM and 133 nM, respectively.29 The R-enantiomer,
(+)-11(R),30 was only modestly active against cathepsin
L (IC50 = 34 lM).29 Some variability in IC50 values
determined for (ꢀ)-12 prompted us to explore the stabil-
ity of both (ꢀ)-11 and (ꢀ)-12 in solvents relevant to the
bioassay. While (ꢀ)-11 was found to be completely sta-
ble in DMSO, as well as in the buffer employed in the
cathepsin L assay [NaOAc (20 mM), pH 5.5; EDTA
(1 mM); cysteine (5 mM)], the free-base (ꢀ)-12 proved
unstable in DMSO, generating decomposition products
13, 14, and 15 after only 1 h at room temperature
(Fig. 3).
In summary, samples from the NIH MLSMR revealed
promising cathepsin L inhibitory activity attributed to
a series of 2,5-disubstituted oxadiazoles (1–5). Analyti-
cal analyses (LC–MS) of the library samples, however,
indicated numerous impurities, thus requiring the devel-
opment of an efficient synthesis for the putative active
2,5-disubstituted oxadiazoles. Synthetic samples of pure
2,5-disubstituted oxadiazoles were found to be com-
pletely devoid of cathepsin L inhibitory activity. Careful
LC–MS investigation of both the library and synthetic
samples revealed a thiocarbazate to be the active com-
ponent. Bioassay of the synthetic thiocarbazates con-
firmed the hypothesis: (ꢀ)-11 and (ꢀ)-12 display
potent inhibitory activity against cathepsin L, with
IC50 values of 56 nM and 133 nM, respectively, however
instability of (ꢀ)-12 was also noted. The design, synthe-
ses, and biological evaluation of analogs of the highly
potent lead thiocarbazate class are now ongoing in our
laboratories, and will be reported in due course.
Presumably 13–15 are formed from (ꢀ)-12 via intramo-
lecular attack of the primary amine on the C2 thiocar-
bazate moiety, with release of 13. These products,
prepared either via synthesis (13 and 14) or by HPLC
purification of decomposed material (15), were assayed
for cathepsin L activity and found to be inactive. Due
to the instability of (ꢀ)-12 under the assay conditions,
IC50 values vary somewhat, rendering interpretation of
the bioassay data difficult. We are, however, confident
in the accuracy of the bioassay results for (ꢀ)-11, as this
compound is stable under all conditions evaluated.
Thus, future efforts will be focused on the more stable
thiocarbazate (ꢀ)-11 and related congeners.
Acknowledgments
Financial support for this work was provided by the
NIH (5U54HG003915-02 and 5U54HG003915-03). We
thank Professor Barry S. Cooperman (University of
Pennsylvania) and Dr. Mary Pat Beavers (University
of Pennsylvania) for helpful discussions. We also thank
Christina Kraml (Lotus Separations) for chiral SFC
analysis. Finally we thank Dr. G.T. Furst (NMR Facil-
ity) and Dr. R. Kohli (Mass Spectrometry Facility) at
the University of Pennsylvania for assistance in obtain-
ing NMR and high-resolution mass spectra.
Since most cysteine protease inhibitors contain an elec-
trophilic ‘warhead,’ and work through a mechanism
involving reaction with the active site cysteine,7,26 we
strongly believe these novel thiocarbazates behave simi-
larly, and are active by virtue of their electrophilic car-
bonyl moiety. The formation of 15 supports this
mechanism and is indicative of the electrophilicity of

214
M. C. Myers et al. / Bioorg. Med. Chem. Lett. 18 (2008) 210–214
eter logistic model (IDBS XLfit equation 205) to obtain
IC50 values in triplicate.
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28. Characterization data for (ꢀ)-11 (PubChem SID
23
26681509) mp = 131 °C; ½aꢁD ꢀ14.8 (c 0.5, AcOH); IR
1
(KBr) 3412, 3343, 2971, 1680, 1529, 1167 cmꢀ1; H NMR
(500 MHz, DMSO-d6, VT-350K) d 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); 13C NMR
(125 MHz, DMSO-d6, major rotamer) d 172.7, 171.6,
166.9, 155.2, 137.3, 136.0, 135.4, 128.5, 127.1, 125.7, 125.5,
125.2, 124.0, 120.9, 118.5, 118.1, 111.3, 109.9, 78.0, 53.6,
33.1, 28.1, 27.7, 23.6, 14.2; high-resolution mass spectrum
(ES+) m/z 562.2126 [(M+Na)+; Calcd for C27H33N5O5S-
Na: 562.2100].
13. Zhou, N. E.; Guo, D.; Thomas, G.; Reddy, A. V. N.;
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Singh, R. Bioorg. Med. Chem. Lett. 2003, 13, 139.
´
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17. PubChem web address for PCMD cathepsin L hits: http://
pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=460.
18. HTS and initial followup (cherry picks) studies were
conducted with the following assay buffer: 20 mM sodium
acetate, 1 mM EDTA, and 5 mM DTT, pH 5.5. Confir-
matory results were obtained utilizing the following assay
conditions, replacing DTT with cysteine in the assay
buffer: compounds were serially diluted in DMSO and
transferred into a 96-well Corning 3686 assay microplate
to give 16 dilutions ranging from 50 lM to 1.5 nM.
Human liver cathepsin L (Calbiochem 219402) was
activated by incubating with assay buffer for 30 min.
Assay buffer consisted of 20 mM sodium acetate, 1 mM
EDTA, and 5 mM cysteine, pH 5.5. Upon activation,
cathepsin L (300 pM) was incubated with 1 lM Z-Phe-
Arg-AMC substrate and test compound in 100 lL of assay
buffer for 1 h at room temperature. Fluorescence of AMC
released by enzyme-catalyzed hydrolysis of Z-Phe-Arg-
AMC was read on a Perkin-Elmer Envision microplate
reader (excitation 355 nm, emission 460 nm). Data were
scaled using internal controls and fitted to a four-param-
29. IC50 values as means standard deviations: (ꢀ)-11(S)
56 nM 4 nM; (ꢀ)-12(S) 133 nM
2 nM; (+)-11(R)
34 lM 2 lM.
23
30. Optical rotation for (+)-11: ½aꢁD +12.8. The enantiomeric
purity of both (ꢀ)-11 and (+)-11 was assayed using an
OD-RH chiral column with the following LC parameters:
1.0 mL/min with a linear gradient of 90% water in
acetonitrile to 10% water in acetonitrile over 15 min.
Using this method, baseline separation was obtained for
the enantiomers and retention times for (ꢀ)-11 and (+)-11
were 14.01 min and 13.02 min, respectively. The synthesis,
outlined in Scheme 2, produced both enantiomers in >99%
enantiomeric purity.
31. Shah, P. P.; Myers, M. C.; Beavers, M. P.; Purvis, J. E.;
Jing, H.; Grieser, H. J.; Sharlow, E. R.; Huryn, D. M.;
Cooperman, B. S.; Smith, A. B., III.; Diamond, S. L., in
preparation.