Small-molecule inhibitors of cathepsin L incorporating functionalized ring-fused molecular frameworks

Article abstract of DOI:10.1016/j.bmcl.2012.12.025

Cathepsin L is a cysteine protease that is upregulated in a variety of malignant tumors and plays a significant role in cancer cell invasion and migration. It is an attractive target for the development of small-molecule inhibitors, which may prove beneficial as treatment agents to limit or arrest cancer metastasis. We have previously identified a structurally diverse series of thiosemicarbazone-based inhibitors that incorporate the benzophenone and thiochromanone molecular scaffolds. Herein we report an important extension of this work designed to explore fused aryl-alkyl ring molecular systems that feature nitrogen atom incorporation (dihydroquinoline-based) and carbon atom exclusivity (tetrahydronaphthalene-based). In addition, analogues that contain oxygen (chromanone-based), sulfur (thiochroman-based), sulfoxide, and sulfone functionalization have been prepared in order to further investigate the structure-activity relationship aspects associated with these compounds and their ability to inhibit cathepsins L and B. From this small-library of 30 compounds, five were found to be strongly inhibitory (IC₅₀ <500 nM) against cathepsin L with the most active compound (7-bromodihydroquinoline thiosemicarbazone 48) demonstrating an IC₅₀ = 164 nM. All of the compounds evaluated were inactive (IC₅₀ >10,000 nM) as inhibitors of cathepsin B, thus establishing a high degree (>20-fold) of selectivity (cathepsin L vs. cathepsin B) for the most active cathepsin L inhibitors in this series.

Full text of DOI:10.1016/j.bmcl.2012.12.025

Accepted Manuscript
Small-Molecule Inhibitors of Cathepsin L Incorporating Functionalized Ring-
Fused Molecular Frameworks
Jiangli Song, Lindsay M. Jones, Gustavo E. Chavarria, Amanda K. Charlton-
Sevcik, Adam Jantz, Audra Johansen, Liela Bayeh, Victoria Soeung, Lindsey
K. Snyder, Shawn D. Lade, David J. Chaplin, Mary Lynn Trawick, Kevin G.
Pinney
PII:
S0960-894X(12)01609-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2012.12.025
BMCL 19910
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
9 October 2012
30 November 2012
10 December 2012
Please cite this article as: Song, J., Jones, L.M., Chavarria, G.E., Charlton-Sevcik, A.K., Jantz, A., Johansen, A.,
Bayeh, L., Soeung, V., Snyder, L.K., Lade, S.D., Chaplin, D.J., Trawick, M.L., Pinney, K.G., Small-Molecule
Inhibitors of Cathepsin L Incorporating Functionalized Ring-Fused Molecular Frameworks, Bioorganic &
Medicinal Chemistry Letters (2012), doi: http://dx.doi.org/10.1016/j.bmcl.2012.12.025
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Small-Molecule Inhibitors of Cathepsin L Incorporating Functionalized Ring-Fused
Molecular Frameworks
Jiangli Songa, Lindsay M. Jonesa, Gustavo E. Chavarriaa, Amanda K. Charlton-Sevcika,
Adam Jantza, Audra Johansena, Liela Bayeha, Victoria Soeunga, Lindsey K. Snydera,
Shawn D. Lade, Jr. a, David J. Chaplinb, Mary Lynn, Trawicka, and Kevin G. Pinneya,*
aDepartment of Chemistry and Biochemistry, Baylor University, Waco, TX 76798, USA
bOXiGENE Inc., 701 Gateway Blvd., Suite 210, South San Francisco, CA 94080, USA
*Corresponding Author: Kevin G. Pinney
Telephone: 1-254-710-4117
Fax: 1-254-710-4272
Email: Kevin_Pinney@baylor.edu
Submitted to: Bioorganic and Medicinal Chemistry Letters
Page 1 of 16

Abstract
Cathepsin L is a cysteine protease that is upregulated in a variety of malignant tumors
and plays a significant role in cancer cell invasion and migration. It is an attractive target
for the development of small-molecule inhibitors, which may prove beneficial as
treatment agents to limit or arrest cancer metastasis. We have previously identified a
structurally diverse series of thiosemicarbazone-based inhibitors that incorporate the
benzophenone and thiochromanone molecular scaffolds. Herein we report an important
extension of this work designed to explore fused aryl-alkyl ring molecular systems that
feature nitrogen atom incorporation (dihydroquinoline-based) and carbon atom
exclusivity (tetrahydronaphthalene-based). In addition, analogues that contain oxygen
(chromanone-based), sulfur (thiochroman-based), sulfoxide, and sulfone
functionalization have been prepared in order to further investigate the structure activity
relationship aspects associated with these compounds and their ability to inhibit
cathepsins L and B. From this small-library of thirty compounds, five were found to be
strongly inhibitory (IC50 < 500 nM) against cathepsin L with the most active compound
(7-bromodihydroquinoline thiosemicarbazone 48) demonstrating an IC50 = 164 nM. All
of the compounds evaluated were inactive (IC50 > 10,000 nM) as inhibitors of cathepsin
B, thus establishing a high degree (> 20 fold) of selectivity (cathepsin L versus cathepsin
B) for the most active cathepsin L inhibitors in this series.
Page 2 of 16

The powerful cysteine protease cathepsin L is overexpressed in many cancer cell lines
and in tumor tissue.1-4 There is a growing body of literature that describes the essential
role of cathepsin L in the dissemination of cancer cells from the primary tumor to distant
locations.5-8 Cathepsin L has been implicated in the differentiation of endothelial
progenitor cells and their homing to specific sites in the development of new blood
vessels,9 and in the differentiation of mouse embryonic stem cells.10 Elevated levels of
cathepsin L are predictive of progression to metastasis.5,8 Treatment of several cancer
cell lines with broad spectrum cathepsin L inhibitors reduced cell line invasiveness.5
Cathepsin L is also biosynthesized and targeted to the cell lysosome and secreted from
cells as a precursor where it is activated by extracellular biomolecules and the acidic pH
associated with growing tumors.11,12 As the activated enzyme, cathepsin L can degrade
extracellular proteins such as collagen, laminin, and fibronectin, and participate in the
metastasis of primary tumor cells to distant sites.13,14 Therefore, effective inhibition of
cathepsin L by small-molecule therapeutic agents holds great promise to limit or arrest
cancer metastasis7,15 and to disrupt the recruitment of progenitor cells necessary for
tumors to expand their blood supply through angiogenesis.9,16,17 Although small-
molecule inhibitors of cathepsin L have been previously investigated (Fig. 1),18-32 to the
best of our knowledge there are no such agents in human clinical trials. Support for
cathepsin cysteine proteases as targets for drug development is provided by the
continuing success (in clinical trials) of odanacatib, a specific inhibitor of cathepsin
K.33,34 Cathepsin K is an enzyme that is involved in degradation of the extracellular
matrix proteins associated with bone resorption, thus odanacatib demonstrates efficacy
against osteoporosis, and cathepsin K inhibitors are also being investigated for their
potential role in relationship to bone cancer. 35-38 Cathepsin L is a unique biomolecular
target for anticancer drug development, and it is especially significant due to the dual role
that it appears to play in both cancer metastasis and the angiogenic process.
Page 3 of 16

Figure 1. Non-Inclusive, Representative Sampling of Cathepsin L Inhibitors: E-64,18,19
JMP-OEt,20 CLIK-195,21 CLIK-148,19,21 thiocarbazate,22 oxocarbazate,23 azepanone,24
and purine nitrile25
Our own investigation of small-molecule inhibitors of cathepsin L followed a logical and
yet somewhat circuitous pathway beginning with the discovery and development of
potent inhibitors of cruzain39 for the potential treatment of Chagas disease.40 In order to
demonstrate selectivity for cruzain versus related human cysteine proteases, we screened
our original privileged small-library of thiosemicarbazone analogues against cathepsin L
and identified several promising hits, which ultimately translated into lead compounds for
further investigation.41-43 The most potent inhibitors of cathepsin L to emerge from our
previous studies (Fig. 2) function in the low nanomolar range. These inhibitors also
demonstrate selectivity for cathepsin L versus cathepsin B, a related cysteine protease
that is also involved in intracellular and extracellular protein degradation. It is important
Page 4 of 16

to note that thiosemicarbazone-based inhibitors of cathepsin L have also been reported by
McKerrow and Guy.26
Figure 2. Representative Inhibitors of Cathepsin L from Previous Studies by Pinney and
Trawick (note: squiggly bond between nitrogen atoms denotes either an E/Z isomer
mixture or a single isomer with stereochemistry not determined).41-44
In order to expand the structure activity relationship (SAR) platform related to small-
molecule inhibitors of the cathepsins, a variety of new analogues containing
thiochromanone, benzothiepine, indanone, and 3-dihydroquinolin-4-one molecular
scaffolds functionalized as either thiosemicarbazone (TSC) or hydrazinecarboximidamide
(HZCI) derivatives were prepared by chemical synthesis as detailed in schemes 1-5. In
brief, the synthesis of the 6-methylsulfonyl TSC analogue 13 mirrored our previously
described methodology.43 Carboxylic acid derivative 10, prepared through an SN2
reaction, was cyclized in the presence of PPA to afford 6-thiomethy-thiochromanone 11
through an acid-catalyzed, intramolecular electrophilic aromatic substitution reaction.
Hydrogen peroxide mediated oxidation of both sulfur atoms followed by condensation of
ketone 11 with thiosemicarbazide afforded thiosemicarbazone analogue 13 (Scheme 1).
Page 5 of 16

Scheme 1. Synthesis of 6-methylsulfonyl-1,1-dioxo-thiochromanone thiosemicarbazone
Synthesis of the 7-nitro-HZCI analogue 21 and its sulfone counterpart 23, along with the
7-nitro-TSC sulfoxide 19 and the 7-amino-TSC sulfone 17 each originated with 6-
nitrothiochroman-4-one (Scheme 2) and involved standard functional group manipulation.
Scheme 2. Synthesis of 6-nitro and 6-amino analogues
Oxidation of 6-nitrothiochroman-4-one using hydrogen peroxide in the presence of
trifluoroacetic acid afforded sulfoxide 18, which was treated with thiosemicarbazide to
generate thiosemicarbazone 19. The synthetic route to thiosemicarbazone 17 was
Page 6 of 16

initiated by the reduction of 6-nitrothiochroman-4-one with tin/HCl to yield 6-amino
thiochromanone 14. Selective protection (Fmoc) of the amino group followed by
oxidation (H2O2/AcOH) to the corresponding sulfone, subsequent cleavage of the Fmoc
group, and treatment with thiosemicarbazide afforded thiosemicarbazone 17. The
synthesis of the aminoguanidine derivatives 21 and 23 was achieved by condensation of
6-nitrothiochroman-4-one and 6-nitro-1,1-dioxothiochromanone 22 with
aminoguanidinenitrate, respectively.
Scheme 3. Synthesis of 3,4-dihydro-1-benzothiepin-5-one thiosemicarbazone analogues
(Note: NOESY experiments45 indicate the E-configuration for compounds 28 and 29)
The fused 6,7-membered benzothiepinone ring (analogues 26 and 27) was constructed
through an intramolecular electrophilic substitution reaction of functionalized butanoic
acids 24 and 25, respectively (Scheme 3). The butanoic acids were prepared by
alkylation of the appropriate thiophenol with 4-bromobutyric acid. Cyclic ketones 26 and
27 were separately condensed with thiosemicarbazide to give the corresponding
Page 7 of 16

thiosemicarbazone derivatives 28 and 29, respectively. Alternatively, oxidation of
ketones 26 and 27 (separately) with hydrogen peroxide afforded sulfone derivatives 30
and 31, which were converted to their corresponding thiosemicarbazone derivatives 32
and 33 by condensation with thiosemicarbazide.
Scheme 4. Synthesis of indanone thiosemicarbazone analogue
The synthesis of the fused 5,6-membered TSC analogues is described in scheme 4.
Trimethylsilyl enol ether 35 (prepared from 6-bromoindanone) was subjected to Saegusa
oxidation conditions,46,47 employing palladium acetate (Pd(OAc)2), to afford α,β-
unsaturated indenone 36. A Michael addition of aniline followed by condensation with
thiosemicarbazide afforded the thiosemicarbazone derivative 38.
The synthetic route to 2,3-dihydroquinolin-4-one TSC analogues is depicted in scheme 5.
Commercially available aniline derivatives were subjected to methyl acrylate thus
affording carboxylic acids 39-42 via a Michael addition. Cyclization (PPA mediated)
generated the corresponding ketones 43-46 which were converted to their
thiosemicarbazone derivatives 47, 48,40b 49, 50 by condensation with thiosemicarbazide.
Page 8 of 16

For the nitro analogues 54, 56, and 59, 60, a single nitration step was necessary prior to
condensation. The nitration was performed under two sets of reaction conditions. In one
case, nitric acid and acetic acid were utilized to form the regioisomeric mono-nitro 2,3-
dihydroquinolin-4-ones 51 and 53. Separately, treatment with a nitronium salt afforded
the dinitro-2,3-dihydroquinolin-4-ones 57 and 58. Thiosemicarbazone derivatives 1-9, 20,
and 34 (not depicted in a scheme) were prepared by condensation of the appropriate
commercially available ketone with thiosemicarbazide (see supporting information).
Scheme 5. Synthesis of 2,3-dihydroquinolin-4-one thiosemicarbazone analogues (note:
NOESY experiments45 indicate the E-configuration for compounds 48, 54, 56, and 59)
Each of the thirty TSC and NZCI analogues prepared by chemical synthesis were
evaluated for their ability to inhibit cathepsin L and cathepsin B in separate assays. The
Page 9 of 16

most active compound (7-bromodihydroquinoline thiosemicarbazone 48) demonstrated
an impressive IC50 of 164 nM against cathepsin L (Table 1). We previously reported43
that the corollary 7-bromothiochromanone thiosemicarbazone is also a potent cathepsin L
inhibitor (IC50 = 152 nM) while its complementary sulfone analogue is 3.5-fold less
active (IC50 = 574 nM). Incorporation of an NO2 group at position 7 of the
dihydroquinolinone system (compound 56) or NO2 groups (compounds 59 and 60)
resulted in analogues that were strongly inhibitory against cathepsin L (449, 228, and 338
nM, respectively). Therefore, the SAR around these heteroatom incorporated, fused 6-
membered ring systems is extended by the replacement of NH (compounds 47-48, 49
(NCH3), 50, 54 (NNO2), 55 (NCOCH3), 56, 59 (NNO2), and 60) for S and SO2 at
position-1 in the heteroatom-alkyl ring.
The mostly likely mechanism of inactivation involves an attack of the cysteine-25
thiolate of cathepsin L to the carbon-sulfur double-bond of the thiosemicarbazone
resulting in a covalent or transiently covalent bond. It was envisioned that replacement
of the thiosemicarbazone moiety with a hydrazinecarboximidamide group might be
beneficial (potentially increasing water solubility). Accordingly, a 7-
nitrothiochromanone hydrazinecarboximidamide analogue (21) and its corresponding
sulfone derivative (23) were prepared, however both proved to be inactive (IC50 > 10,000
nM) against cathepsin L. This is in sharp contrast to the 7-nitrothiochromanone
thiosemicarbazone and sulfone counterparts that we previously identified43 as potent
inhibitors of cathepsin L (IC50 = 68 nM and 112 nM, respectively).
Each of the exclusively carbon-bearing, 6-membered fused ring system analogues (1-7)
and the two 5-membered, fused ring counter-parts (34 and 38) were inactive (IC50 >
10,000 nM) against cathepsin L. Extension of the SAR to include the chromanone
thiosemicarbazone functionality resulted in a 7-chloro derivative (8) that was active
against cathepsin L (IC50 = 369 nM) and a non-chlorinated, 3-phenyl analogue (9) that
was inactive. In addition, a preliminary exploration of 6,7-fused ring systems bearing
ring-atom incorporation of sulfur (28, 29) and sulfone (32, 33) identified the 7-nitro-
substituted analogue (28) as a cathepsin L inhibitor with modest activity (IC50 = 658 nM).
Page 10 of 16

All of the compounds evaluated were inactive (IC50 > 10,000 nM) as inhibitors of
cathepsin B, thus establishing a high degree (> 20 fold) of selectivity (cathepsin L versus
cathepsin B) for the most active cathepsin L inhibitors in this series.
Table 1. Inhibitory Activity of Thiosemicarbazone and Hydrazinecarboximidamide
Derivatives against Cathepsins L and B
IC50 (nM)a
compound
R1
R2
R3
n
X
Y
Cat L
Cat B
1
OCH3
H
H
H
H
H
H
Cl
H
OCH3
H
H
NH2
H
H
H
H
H
H
H
H
H
H
H
F
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
S
S
S
S
S
S
S
S
S
S
S
S
S
N
N
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
369
>10,000
>10,000
>10,000
1740
>10,000
>10,000
>10,000
658
≥10,000
>10,000
>10,000
>10,000
>10,000
>10,000
164
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
2
1
1
1
1
1
2
1
1
1
1
1
0
1
1
2
2
2
2
0
0
1
1
1
1
1
1
1
1
1
3
4
5
6b
7
8
9c
13
17
19
20d
21
23
28
29
32
33
34
38
47
48
49
50
54
55
56
59
60
H
O
CH3SO2
NH2
NO2
NO2
NO2
NO2
NO2
F
NO2
F
Br
Br
H
Br
Br
H
H
H
SO2
SO2
SO
Fused Aryl
S
SO2
S
S
SO2
SO2
CH2
CHNHC6H5
NH
NH
NCH3
NH
NNO2
NCOCH3
NH
NNO2
NH
H
H
H
H
H
H
CH2C6H5
>1,000
>10,000
>10,000
>10,000
449
H
H
H
H
NO2
NO2
NO2
NO2
228
338
Note: a. Each assay utilized 2% DMSO with a five minute pre-incubation
and 50 µM substrate (cat. L) or 60 µM substrate (cat. B)
b. H2NNHCSNHN at C-2 position
c. C6H5 at C-3 position
d. fused m-Nitro phenyl
Page 11 of 16

In summary, our investigation of a diverse assembly of synthetic, fused-ring
thiosemicarbazone analogues and related derivatives has identified a
bromodihydroquinoline-based compound that is strongly inhibitory against cathepsin L
and has significantly extended the known SAR associated with small-molecule inhibitors
of cathepsin L, especially in regard to nitro and halogen group incorporation. Inhibitors
of cathepsin L have the potential to disrupt cancer metastasis, thus highlighting the
urgency for enhancing drug discovery and development efforts in this emerging area of
potential future therapeutic intervention.
Acknowledgments
The authors express their appreciation to OXiGENE Inc. (grants to K.G.P. and M.L.T)
for their financial support of this project, and to the National Science Foundation for
funding the Varian 500 MHz NMR spectrometer (grant no. CHE-0420802). The authors
are grateful to Dr. James Karban and Dr. Michelle Nemec (Director) for use of the shared
Molecular Biosciences Center at Baylor University.
Supplementary data
Supplementary data (including details regarding synthetic and biochemical experimental
procedures and compound characterization) associated with this article can be found in
the online version (doi to be added upon publication).
Page 12 of 16

References and notes
1. Chauhan, S. S.; Goldstein, L. J.; Gottesman, M. M. Cancer Res. 1991, 51, 1478.
2. Gocheva, V.; Zeng, W.; Ke, D.; Klimstra, D.; Reinheckel, T.; Peters, C.; Hanahan, D.;
Joyce, J. A. Gene Dev, 2006, 20, 543.
3. Herszenyi, L.; Farinati, F.; Cardin, R.; Istvan, G.; Molnar, L. D.; Hritz, I.; De Paoli,
M.; Plebani, M.; Tulassay, Z. BCM Cancer 2008, 8, 194.
4. Goulet, B.; Sansregret, L.; Leduy, L.; Bogkyo, M.; Weber, E.; Chauhan, S. S.;
Nepveu, A. Mol. Cancer Res. 2007, 5, 899.
5. Bell-McGuinn, K. M.; Garfall, A. L.; Bogyo, M.; Hanahan, D.; Joyce, J. A. Cancer
Res. 2007, 67, 7378.
6. Frade, R.; Rousselet, N.; Jean, D. Cancer Gene Ther. 2008, 15, 591.
7. Gocheva, V.; Joyce, J. A. Cell Cycle 2007, 6, 60.
8. Jagodic, M.; Vrhovec, I.; Borstnar S.; Cufer T. Neoplasma 2005, 52, 1.
9. Urbich, C.; Heeschen, C.; Aicher, A.; Sasaki, K-i.; Bruhl, T.; Farhadi, M. R.;
Vajkoczy, P.; Hofmann, W. K.; Peters, C.; Pennacchio, L. A.; Abolmaali, N. D.;
Chavakis, E.; Reinheckel, T.; Zeiher, A. M.; Dimmeler, S. Nature Med. 2005, 11, 206.
10. de Azevedo-Pereira, R. L.; Lima, A. P. C. A.; de Carvalho Rodrigues, D.; Rondinelli,
E.; Medei, E. H.; Goldenberg, R. C.; de Carvalho, A. C. C.; Mendez-Otero, R. Stem Cells
and Devel. 2011, 20, 1859.
11. Gal, S.; Gottesman, M. M. J. Biol. Chem. 1986, 261, 1760.
12. Jordans, S.; Jenko-Kokalj, S.; Kuhl, N. M.; Tedelind, S.; Sendt, W.; Bromme, D.;
Turk, D.; Brix, K. BMC Biochem. 2009, 10.
13. Blondeau, X.; Vidmar, S. L.; Emod, I.; Pagano, M.; Turk, V.; Keil-Dlouha, V. Biol.
Chem. Hoppe-Seyler. 1993, 374, 651.
14. Guinec, N.; Dalet-Fumeron, V. Pagano, M. Biol. Chem. Hoppe-Seyler. 1993, 374,
1135.
15. Lankelma, J. M.; Voorend, D. M.; Barwari, T.; Koetsveld, J.; Van der Spek, A. H.;
De Porto, A. P. N. A.; Rooijen, G. V.; Van Noorden, C. J. F. Life Sci. 2010, 86, 225.
16. Urbich, C.; Dernbach, E.; Rossig, L.; Zeiher, A. M.; Dimmeler, S. J. Mol. Cell.
Cardiol. 2008, 45, 429.
Page 13 of 16

17. Rebbaa, A.; Chu, F.; Sudha, T.; Gallati, C.; Dier, U.; Dyskin, E.; Yalcin, M.;
Bianchini, C.; Shaker, O.; Mousa, S. A. Anticancer Res. 2009, 29, 4473.
18. Pealermo, C.; Joyce, J. A. Trends Pharmacol. Sci. 2007, 29, 22.
19. Tsuge, H.; Nishimura, T.; Tada, Y.; Asao, T.; Turk, D.; Turk, V.; Katunuma, N.
Biochem. Bioph. Res. Co. 1999, 266, 411.
20. Joyce J. A.; Baruch, A.; Chehade, K.; Meyer-Morse, N.; Giraudo, E.; Tsai, F. Y.;
Greenbaum, D. C.; Hager, J. H. ; Bogyo, M.; Hanahan, D. Cancer Cell. 2004, 5, 443.
21. Katunuma, N. Proc. Jpn. Acad. Ser. B. 2011, 87(2), 29.
22. Myers, M. C.; Shah, P. P.; Diamond, S. L.; Huryn, D. M.; Smith III, A. B. Bioorg.
Med. Chem. Lett. 2008, 18, 210.
23. Shah, P. P.; Wang, T.; Kaletsky, R. L.; Myers, M. C.; Purvis, J. E.; Jing, H.; Huryn, D.
M.; Greenbaum, D. C.; Smith III, A. B.; Bates, P; Diamond, S. L. Mol. Pharmacol. 2010,
78, 319.
24. Marquis, R. W.; James, I; Zeng, J.; Trout, R. E.; Thompson, S.; Rahman, A.;
Yamashita, D. S.; Xie, R.; Ru, Y.; Gress, C. J.; Blake, S.; Lark, M. A.; Hwang, S. M.;
Tomaszek, T.; Offen, P.; Head, M. S.; Cummings, M. D.; Veber, D. F. J. Med. Chem.
2005, 48, 6870.
25. Mallari, J. P.; Shelat, A. A.; Kosinski, A.; Caffrey, C. R.; Connelly, M.; Zhu, F. Y.;
McKerrow, J. H.; Kiplin Guy, R. J. Med. Chem. 2009, 52, 6489.
26. Mallari, J. P.; Shelat, A.; Kosinski, A.; Caffrey, C. R.; Connelly, M.; Zhu, F.;
McKerrow, J. H.; Guy, R. K. Bioorg. Med. Chem. Lett. 2008, 18, 2883.
27. Asaad, N.; Bethel, P. A.; Coulson, M. D.; Dawson, J. E.; Ford, S. J.; Gerhardt, S.;
Grist, M.; Hamlin, G. A.; James, M. J.; Jones, E. V.; Karoutchi, G. I.; Kenny, P. W.;
Morley, A. D.; Oldham, K.; Rankine, N.; Ryan, D.; Wells, S. L.; Wood, L.; Augustin,
M.; Krapp, S.; Simader, H.; Steinbacher, S. Bioorg. Med. Chem. Lett. 2009, 19, 4280.
28. Yasuma, T.; Oi, S.; Choh, N.; Nomura, T.; Furuyama, N.; Nishimura, A.; Fujisawa,
Y.; Sohda, T. J. Med. Chem. 1998, 41, 4301.
29. Rydzewski, R. M.; Bryant, C.; Oballa, R.; Wesolowski, G.; Rodan, S. B.; Bass, K. E.;
Wong, D. H. Bioorg. Med. Chem. 2002, 10, 3277.
Page 14 of 16

30. Chowdhury, S. F.; Sivaraman, J.; Wang, J.; Devanathan, G.; Lachance, P.; Qi, H.;
Ménard, R.; Lefebvre, J.; Konishi, Y.; Cygler, M.; Sulea, T.; Purisima, E. O. J. Med.
Chem. 2002, 45, 5321.
31. Marquis, R. W.; James, I.; Zeng, J.; Lee Trout, R. E.; Thompson, S.; Rahman, A.;
Yamashita, D. S.; Xie, R.; Ru, Y.; Gress, C. J.; Blake, S.; Lark, M. M.; Hwang, S.-M.;
Tomaszek, T.; Offen, P.; Head, M. S.; Cummings, M. D.; Verber, D. F. J. Med.
Chem. 2005, 48, 6870.
32. Falgueyret, J. P.; Oballa, R. M.; Okamoto, O.; Wesolowski, G.; Aubin, Y.;
Rydzewski, R. M.; Prasit, P.; Riendeau, D.; Rodan, S.; Percival, M. D. J. Med. Chem.
2001, 44, 94.
33. Williams, S. C. P. Nature Medicine 2012, 18, 1158.
34. Mazziotti, G.; Bilezikian, J.; Canalis, E.; Cocchi, D.; Giustina, A. Endocrine 2012,
41, 58.
35. Jensen, A. B.; Wynne, C.; Ramirez, G.; He, W.; Song, Y.; Berd, Y.; Wang, H.;
Mehta, A.; Lombardi, A. Clinical Breast Cancer. 2010, 10, 452.
36. Le Gall, C.; Bellahcene, A.; Bonnelye, E.; Gasser, J. A.; Castronovo, V.; Green, J.;
Zimmermann, J.; Clezardin, P. Cancer Res. 2007, 67, 9894.
37. Le Gall, C.; Bonnelye, E.; Clezardin, P. Curr. Opin. Support. Palliat. 2008, 2, 218.
38. Quintilla-Dieck, M. J.; Codriansky, K.; Keady, M.; Bhawan, J.; Runger, T. M. J.
Invest. Dermatol. 2008, 128, 2281.
39. Fujii, N.; Mallari, J. P.; Hansell, J. E.; Mackey, H. Z.; Doyle, P.; Zhou, Y. M.; Gut, J.;
Rosenthal, P. J.; McKerrow, J. H.; Guy, R. K. Bioorg. Med. Chem. Lett. 2005, 15, 121.
40. (a) Siles, R.; Chen, S.-E.; Zhou, M.; Pinney, K. G.; Trawick, M. L. Bioorg. Med.
Chem. Lett. 2006, 16, 4405. (b) Siles, R.; Zhou, M.; Ackley, J. F.; Pinney, K. G.; Chen,
S-E.; Arispe-Angulo, W. M.; Trawick, M. L. U.S. Pat. Appl. Publ. 2009, US
20090076076 A1 20090319.
41. (a) Kishore, G. D. K.; Chavarria, G. E.; Carlton-Sevick, A. K.; Arispe, W. M.;
MacDonough, M. T.; Strecker, T. E.; Chen, S.-E.; Siim, B. G.; Chaplin, D. J.; Trawick,
M. L.; Pinney, K. G. Bioorg. Med. Chem. Lett. 2010, 20, 1415. (b) Trawick, M. L.;
Chen, S.; Arispe, W. M.; Siles, R. E.; Zhou, M.; Pinney, K. G. American Society for
Page 15 of 16

Biochemistry and Molecular Biology (ASBMB), Experimental Biology meeting, San
Francisco, CA, 2006 (poster presentation).
42. Kishore Kumar, G. D.; Chavarria, G. E.; Charlton-Sevcik, A. K.; Yoo, G. K.; Song,
J.; Strecker, T. E.; Siim, B. G.; Chaplin, D. J.; Trawick, M. L.; Pinney, K. G. Bioorg.
Med. Chem. Lett. 2010, 20, 6610.
43. Song, J.; Jones, L. M.; Kishore Kumar, G. D.; Conner, E. S.; Bayeh, L.; Chavarria, G.
E.; Charlton-Sevcik, A. K.; Chen, S-E.; Chaplin, D. J.; Trawick, M. L.; Pinney, K. G.
ACS Med. Chem. Lett. 2012, 3, 450.
44. Chavarria, G. E.; Horsman, M. R.; Arispe, W. M.; Kishore Kumar, G. D.; Chen, S-E.;
Strecker, T. E.; Parker, E. N.; Chaplin, D. J.; Pinney, K. G.; Trawick, M. L. Eur. J. Med.
Chem. 2012, 58, 568.
45. While it is established that molecules containing the thiosemicarbazone moiety can
interconvert between their E and Z isomers, the propensity for this to occur is dependent
on the particular substrate itself. With the exception of compound 6, all of the
thiosemicarbazone and hydrazinecarboximidamide analogues reported in this study
appeared to be one isomer (as evidenced by NMR and, in most instances, HPLC), and
nuclear Overhauser effect spectroscopy (NOESY) experiments (see supplementary data)
suggested the presence of only the E isomer in compounds 28, 29, 48, 54, 56, and 59).
46. Ito, Y.; Hirao, T.; Saegusa, T. J. Org. Chem. 1978, 43, 1011.
47. Wu, Y-J. Tetrahedron Lett. 2006, 47, 8459.
Page 16 of 16

Captions for Figures, Schemes, and Table 1
Figure 1. Non-Inclusive, Representative Sampling of Cathepsin L Inhibitors: E-64,18,19
JMP-OEt,20 CLIK-195,21 CLIK-148,19,21 thiocarbazate,22 oxocarbazate,23 azepanone,24 and
purine nitrile25
Figure 2. Representative Inhibitors of Cathepsin L from Previous Studies by Pinney and
Trawick (note: squiggly bond between nitrogen atoms denotes either an E/Z isomer
mixture or a single isomer with stereochemistry not determined).41-44
Scheme 1. Synthesis of 6-methylsulfonyl-1,1-dioxo-thiochromanone thiosemicarbazone
Scheme 2. Synthesis of 6-nitro and 6-amino analogues
Scheme 3. Synthesis of 3,4-dihydro-1-benzothiepin-5-one thiosemicarbazone analogues
(Note: NOESY experiments45 indicate the E-configuration for compounds 28 and 29)
Scheme 4. Synthesis of indanone thiosemicarbazone analogue
Scheme 5. Synthesis of 2,3-dihydroquinolin-4-one thiosemicarbazone analogues (note:
NOESY experiments45 indicate the E-configuration for compounds 48, 54, 56, and 59)
Table 1. Inhibitory Activity of Thiosemicarbazone and Hydrazinecarboximidamide
Derivatives against Cathepsins L and B

O
H
NH
NH2
O
H
H
OEt
H
N
N
N
H
N
N
HOOC
H
O
H
O
O
O
O
HO
E-64
JMP-OEt
Ph
O
Ph
CH3
O
O
CH3
N
N
O
O
H
N
N
CH3
N
N
CH3
H
H
H
O
H3C
H
H
H
O
O
N
CH3
CLIK-195
CLIK-148
O
O
O
O
H
H
H
H
O
N
N
S
O
N
N
O
N
N
N
N
H
H
H
O
O
O
O
HN
HN
Oxocarbazate
Thiocarbazate
H
N
O
NH
O
H
N
N
N
N
N
N
S
O
O
H
O
N
N
N
Azepanone
Purine Nitrile
HO

S
NH2
H
S
NH2
H
S
NH2
H
N
N
N
N
N
N
OH
F
Br
Br
Br
Cat L, IC50 = 30.5 nM
Br
CF3
Cat L, IC50 = 126.1 nM
Cat L, IC50 = 46.5 nM
S
N
NH2
H
S
NH2
H
S
NH2
H
N
N
N
N
N
F
F
O2N
S
S
Br
OH
Cat L, IC50 = 46 nM
Cat L, IC50 = 68 nM
Cat L, IC50 = 131.4 nM

Scheme 1
O
S
O
S
S
S
PPA
1) NaOH, H2O
H3C
H3C
H3C
O
+
Br
OH
yield: 37%
2) Na2CO3, H2O
yield 68%
SH
S
OH
10
11
H2N
N
S
NH
O
S
O
S
O
H2NNHCSNH2
TsOH, MeOH
S
H2O2,AcOH
yield: 56%
H3C
H3C
O
O
yield: 29%
S
O
O
O
O
13
12

Scheme 2
H
N
NH2
O
S
N
S
H2NNHCNHNH2•HNO3
MeOH, H2SO4
NH
O2N
O2N
yield: 69%
O
22
O
O
O
23
H2O2, AcOH
CH2Cl2
yield: 52%
H2N
N
S
NH
H
N
NH2
NH
O
S
N
S
O
H2NNHCSNH2
TsOH, MeOH
H2O2/TFA
CH2Cl2
H2NNHCNHNH2•HNO3
MeOH, H2SO4
O2N
O2N
O2N
O2N
yield: 50%
yield: 61%
yield: 29%
S
S
O
21
O
18
Sn/HCl
THF
19
yield: 27%
H2N
N
S
NH
O
S
1) H2O2/AcOH
CH2Cl2
Fmoc
HN
O
S
Fmoc-Cl
1,4-dioxane
H2N
H2N
2) piperidine
3) H2NNHCSNH2
yield: 57%
yield: 30%
S
O
O
14
15
17

scheme 3
H2N
N
S
OH
O
NH
Br
R1
R2
R1
R2
PPA
R1
R2
R1
R2
O
H2NNHCSNH2
TsOH, MeOH
110 O
C
OH
K2CO3,DMF
SH
S
S
S
O
24 R1= NO2, R2= H yield:31%
25 R1= F, R2= F yield:51%
28 R1= NO2,R2= H yield:14%
29 R1= F, R2= F yield: 19%
26 R1= NO2, R2= H yield:22%
27 R1= F, R2= F yield: 82%
H2O2/AcOH
CH2Cl2
H2N
N
S
NH
R1
R2
O
R1
H2NNHCSNH2
TsOH, MeOH
R2
S
S
O
O
O
O
32 R1= NO2, R2= H yield:31%
33 R1= F, R2= F yield:33%
30 R1= NO2, R2= H yield:68%
31 R1= F, R2= F yield:60%

Scheme 4
O
OTMS
O
Pd(OAc)2
yield:29%
Br
Br
Br
TMSOTf, Et3N
yield: 74%
35
36
aniline,THF
r.t. 2 days
yield:69%
H2N
N
S
NH
O
Br
Br
H2NNHCSNH2
TsOH, MeOH
HN
HN
yield:32%
37
38

scheme 5
H2N
N
S
NH
O
H2NNHCSNH2
MeOH, TsOH
R1
R1
N
N
R2 R3
R2 R3
59 R1= NO2, R2= H, R3= NO2, yield: 33%
60 R1= R2= NO2, R3= H, yield: 48%
57 R1= NO2, R2= H, R3= NO2, yield: 5%
58 R1= R2= NO2, R3= H, yield: 19%
.
BF4 • NO2, CH3NO2
-78 0C, CH2Cl2
H2N
N
S
R1
O
NH
R3
R1
R1
N
H
2NNHCSNH2
O
H
R1
PPA
R2
1) MW,120 0
2) NaOH, MeOH
C
MeOH, TsOH
N
OH
N
or Eaton's reagent
+
R2 R3
N
R2 R3
for cmpd.43
OMe
R2 R3
O
43 R1= H, R2= H, R3= H, yield: 49%
44 R1= Br, R2= H, R3= H, yield: 3%
45 R1= Br, R2= H, R3= CH3, yield: 13%
47 R1= H, R2= H, R3= H, yield: 97%
48 R1= Br, R2= H, R3= H, yield: 76%
49 R1= Br, R2= H, R3= CH3, yield: 17%
39 R1= H, R2= H, R3= H, yield: 9%
40 R1= Br, R2= H, R3= H
41 R1= Br, R2= H,R3= CH3, yield:63%
42 R1= H, R2= C6H5CH2, R3= H
46 R1= H, R2= C6H5CH2, R3= H, yield:13% 50 R1= H, R2= C6H5CH2, R3= H, yield: 36%
HNO3, AcOH
O
H2N
N
S
NH
H2NNHCSNH2
MeOH, TsOH
R1
R1
N
N
R2 R3
R2 R3
54 R1= H, R2= H, R3= NO2, yield: 37%
55 R1= H, R2= H, R3= CH3CO, yield: 34%
56 R1= NO2, R2= H, R3= H, yield: 7%
51 R1= H, R2= H, R3= NO2, yield: 65%
52 R1= H, R2= H, R3= CH3CO, yield: 25%
53 R1= NO2, R2= H, R3= H, yield: 12%

Table 1. Inhibitory Activity of Thiosemicarbazone and Hydrazinecarboximidamide
Derivatives against Cathepsins L and B
IC50 (nM)a
compound
R1
R2
R3
n
X
Y
Cat L
Cat B
1
2
3
4
OCH3
H
H
H
H
H
H
Cl
H
CH3SO2
NH2
NO2
NO2
NO2
NO2
NO2
F
NO2
F
Br
Br
H
H
OCH3
H
H
NH2
H
H
H
H
H
H
H
H
H
H
H
F
H
F
H
H
H
H
H
H
H
H
H
H
H
H
H
OCH3
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1
CH2
CH2
CH2
CH2
CH2
CH2
CH2
O
S
S
S
S
S
S
S
S
S
S
S
S
S
N
N
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
369
>10,000
>10,000
>10,000
1740
>10,000
>10,000
>10,000
658
≥10,000
>10,000
>10,000
>10,000
>10,000
>10,000
164
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
>10,000
1
1
1
1
1
2
1
1
1
1
1
0
1
1
2
2
2
2
0
0
1
1
1
1
1
1
1
1
1
5
6b
7
8
9c
13
17
19
20d
21
23
28
29
32
33
34
38
47
48
49
50
54
55
56
59
60
O
SO2
SO2
SO
Fused Aryl
S
SO2
S
S
SO2
SO2
CH2
CHNHC6H5
NH
NH
NCH3
NH
NNO2
NCOCH3
NH
NNO2
NH
Br
Br
H
H
>1,000
>10,000
>10,000
>10,000
449
CH2C6H5
H
H
H
H
H
NO2
NO2
NO2
228
338
NO2
Note: a. Each assay utilized 2% DMSO with a five minute pre-incubation
and 50 μM substrate (cat. L) or 60 μM substrate (cat. B)
b. H2NNHCSNHN at C-2 position
c. C6H5 at C-3 position
d. fused m-Nitro phenyl

S
NH2
NH
S
NH2
NH
N
N
Br
Cl
S
NH2
NH
N
O
H
IC50 = 164 nM
IC50 = 369 nM
NH2
N
Optimization
R1
R2
S
NH2
NH
S
N
NH2
S
n
NH
NH
X
N
N
R3
O2N
O2N
O2N
N
N
H
N
NO2
H
NO2
IC50 = 449 nM
IC50 = 338 nM
IC50 = 228 nM