Synthesis and biological evaluation of a water-soluble phosphate prodrug salt and structural analogues of KGP94, a lead inhibitor of cathepsin L

Article abstract of DOI:10.1016/j.bmcl.2016.12.039

The magnitude of expression of cathepsin L, often upregulated in the tumor microenvironment, correlates with the invasive and metastatic nature of certain tumors. Inhibition of cathepsin L represents an emerging strategy for the treatment of metastatic ca

Full text of DOI:10.1016/j.bmcl.2016.12.039

Accepted Manuscript
Synthesis and Biological Evaluation of a Water-Soluble Phosphate Prodrug Salt
and Structural Analogues of KGP94, a Lead Inhibitor of Cathepsin L
Erica N. Parker, Samuel O. Odutola, Yifan Wang, Tracy E. Strecker, Rajeswari
Mukherjee, Zhe Shi, David J. Chaplin, Mary Lynn Trawick, Kevin G. Pinney
PII:
S0960-894X(16)31303-8
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.12.039
BMCL 24528
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
5 October 2016
13 December 2016
14 December 2016
Please cite this article as: Parker, E.N., Odutola, S.O., Wang, Y., Strecker, T.E., Mukherjee, R., Shi, Z., Chaplin,
D.J., Trawick, M.L., Pinney, K.G., Synthesis and Biological Evaluation of a Water-Soluble Phosphate Prodrug Salt
and Structural Analogues of KGP94, a Lead Inhibitor of Cathepsin L, Bioorganic & Medicinal Chemistry Letters
(2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.12.039
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Graphical Abstract
To create your abstract, type over the instructions in the template box below.
Fonts or abstract dimensions should not be changed or altered.
Synthesis and Biological Evaluation of a
Water-Soluble Phosphate Prodrug Salt and
Structural Analogues of KGP94, a Lead
Inhibitor of Cathepsin L
Erica N. Parker,a Samuel O. Odutola,b Yifan Wang,a Tracy E. Strecker,a Rajeswari Mukherjee,a Zhe Shi,a David
J. Chaplin,a, c Mary Lynn Trawick,a, b, c,* and Kevin G. Pinneya, b, c,*

Bioorganic & Medicinal Chemistry Letters
Synthesis and Biological Evaluation of a Water-Soluble Phosphate Prodrug Salt
and Structural Analogues of KGP94, a Lead Inhibitor of Cathepsin L
Erica N. Parker,a Samuel O. Odutola,b Yifan Wang,a Tracy E. Strecker,a Rajeswari Mukherjee,a Zhe Shi,a
David J. Chaplin,a, c Mary Lynn Trawick,a, b, c,* and Kevin G. Pinneya, b, c,*
a Department of Chemistry and Biochemistry Baylor University One Bear Place #97348 Waco, TX 76798-7348
b Institute of Biomedical Studies Baylor University One Bear Place #97224 Waco, TX 76798-7224
c Mateon Therapeutics, Inc. 701 Gateway Blvd, Suite 210 South San Francisco, CA 94080
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The magnitude of expression of cathepsin L, often upregulated in the tumor microenvironment,
correlates with the invasive and metastatic nature of certain tumors. Inhibition of cathepsin L
represents an emerging strategy for the treatment of metastatic cancer. A potent, small-molecule
inhibitor (referred to as KGP94) of cathepsin L, and new KGP94 analogues were synthesized.
(3,5-Dibromophenyl)-(3-hydroxyphenyl) ketone thiosemicarbazone (22), with an IC50 value of
202 nM, exhibited similar inhibitory activity against cathepsin L compared to KGP94 (IC50
=
Keywords:
189 nM). Due to limited aqueous solubility of KGP94, a water-soluble phosphate salt (KGP420)
was prepared in order to facilitate future in vivo studies. Enzymatic hydrolysis with alkaline
phosphatase (ALP) demonstrated that the phosphate prodrug, KGP420, was readily converted to
the parent compound, KGP94.
Cysteine protease inhibitor
Anti-metastatic agent
Thiosemicarbazone
Benzophenone
2016 Elsevier Ltd. All rights reserved.
Phosphate Prodrug
The discovery and development of effective therapeutic
regimens that target cancer metastasis remains an urgent and
largely unmet need. Metastasis is a significant contributing factor
in over ninety percent of deaths attributed to cancer.1 The
sequence of steps involved in the metastatic process associated
with tumor cells includes invasion of these cells from the primary
tumor into the surrounding tissue, intravasation into the
circulatory system, extravasation from the circulatory system,
and establishment of a secondary tumor.1–3 One promising
strategy towards the development of anti-metastatic agents
involves targeting one or more members of the papain family of
cysteine protease cathepsins (comprised of 11 members: B, C, F,
H, K, L, O, S, V, W, and X/Z) with small-molecule inhibitors.4–8
Cathepsins aid in the invasion and migration of tumor cells
through the degradation of proteins comprising the extracellular
neck cancers.18,19 Decreased tumor volume and increased survival
rates were observed in a mouse model upon treatment with the
pan-cathepsin inhibitor JPM-OEt in combination with
cyclophosphamide, an established chemotherapeutic agent.20
In addition to the role that these enzymes play in metastasis
and migration of cancer cells through degradation of components
of the extracellular matrix, cysteine cathepsin proteases have
been implicated as targets for osteoporosis,21 rheumatoid
arthritis,22 atherosclerosis,23 and diseases of the immune system.24
Cathepsin inhibitors as drug candidates that have advanced in the
pharmaceutical pipeline for the treatment of various diseases
include VBY-825 (Virobay),25,26 Odanacatib (Merck),27–29
LY3000328 (Eli Lilly),30,31 (Figure 1).
matrix
including
collagen,9–15
fibronectin,9,10,16,17
and
laminin.9,10,17 Elevated levels of cathepsins L, B, H, X, and S
have been detected in several cancer types including breast,
prostate, brain, colorectal, and lung cancers.18,19 Moreover,
increased expression of these cathepsins correlates to poor
prognosis in breast, ovarian, colorectal, brain, lung, and head and
———
Abbreviations: CatL, cathepsin L; HUVECs, human umbilical vein
endothelial cells; SAR, structure-activity relationship; MMP, matrix
metalloprotease; ALP, alkaline phosphatase.
Corresponding author. Tel: 254-710-4117. Fax: 254-710-4272. E-mail:
Kevin Pinney@baylor.edu, E-mail: Mary_Lynn_Trawick@baylor.edu
Figure 1. Sampling of cathepsin inhibitors recently described in the pharma-
ceutical pipeline

VBY-825 (Virobay), a pan cysteine protease inhibitor
targeting the treatment of liver fibrosis, incorporates a carbonyl
warhead into a peptide-like backbone allowing for reversible
inhibition of targeted cathepsins.25,26 Odanacatib (Merck), a
nitrile based inhibitor, targets cathepsin K for suppression of
bone resorption in osteoporosis.27–29 LY3000328 (Eli Lilly), a
A lead compound (referred to as KGP94), which is a slowly
reversible, time-dependent inhibitor of cathepsin L, emerged
from this small library of structurally diverse thiosemicarbazone
analogues.43,46,47 Low cytotoxicity against human umbilical vein
endothelial cells (HUVECs), the ability to inhibit the invasive
and migratory potential of both PC-3ML (prostate cancer cell
line) and MDA-MB-231 (breast cancer cell line) in vitro, and the
ability to reduce metastatic tumor burden and increase survival
rate in PC-3ML tumor bearing mice has led to the identification
of KGP94 as a pre-clinical candidate for potential development
as an anti-metastatic agent, functioning through a potent
inhibition of cathepsin L.43,47,52 Since KGP94 has limited
solubility in water, it proved desirable to prepare a water-soluble
prodrug salt to further the pre-clinical development of this
promising agent. Fortunately, KGP94 bears a phenolic hydroxyl
group which provides a convenient molecular handle for the
introduction of a bioreversible ester linkage in order to improve
aqueous solubility. Recently, we have reported the synthesis of
the phosphate prodrug OXi8007, a vascular disrupting agent
currently in pre-clinical studies for the treatment of cancer.53
Installation of a phosphate prodrug salt for the purpose of
increasing aqueous solubility of FDA approved drugs intended
for oral or parental administration has been successfully
noncovalent cathepsin S inhibitor, targets the treatment of
30,31
abdominal aortic aneurysm.
Although significant progress
has been achieved towards the development of cathepsin
inhibitors as therapeutic treatment options, no FDA approved
drugs currently exist in this area. Additionally, small-molecule
agents that specifically target the inhibition of cathepsin L for the
treatment of pathological processes currently in clinical trials
remains an unmapped frontier open to exploration.
demonstrated
by
fosfpropofol,
fosamprenavir,
and
fosfluconazole.54
In addition to the synthesis of a water-soluble phosphate
prodrug of KGP94, several analogues of this parent, lead
compound were also prepared. Previous SAR studies related to
thiosemicarbazone inhibitors based on the benzophenone
molecular scaffold highlighted the importance of the 3-
bromophenyl moiety.45,46 The extended series of benzophenone-
based thiosemicarbazone inhibitors incorporated both m-hydroxy
and m-bromo substituents. Our initial synthetic route46 to KGP94
(Scheme 1) utilized the addition of 3-bromophenylmagnesium
bromide to the corresponding Weinreb amide XIV to afford (3-
bromophenyl)(3-hydroxyphenyl)methanone (XV), which was
condensed with thiosemicarbazide followed by deprotection of
silyl ether XVI. However, HPLC analysis of the final product
revealed the presence of a trace amount of an impurity in which
the bromine atom had been replaced by a hydrogen atom.46
Replacement of bromine by hydrogen likely occurred during the
halogen-metal exchange reaction since excess magnesium was
Figure 2. Representative potent covalent inhibitors of CatL.
Small-molecule inhibitors of cathepsin
L have been
synthesized that incorporate a variety of electrophilic moieties
(warheads) capable of interacting with the catalytic site residue
Cys25 (Figure 2). Warheads which undergo covalent bonding
with the Cys25 thiolate of cathepsin L include the epoxide in
Clik 148 (I),32 the carbonyl of thiocarbazate II,33 the epoxide in
JPM-OEt III,34,35 the nitrile in the triazine analogue IV,36 the
cyclic carbonyl in azepanone V,37 the α,β-unsaturated amide of
gallinamide A (VI),38,39
and the aldehyde of the N-(1-
naphthalenylsulfonyl) peptide derivative VII40 (Figure 2).
Cruzain, cathepsin L-like cysteine protease found in
a
Trypanosoma cruzi, is targeted for the treatment of Chagas’
disease. Initial studies towards the development of cruzain41,42
inhibitors led to the discovery of cathepsin L inhibitors bearing
the thiosemicarbazone moiety which contain an electrophilic
thiocarbonyl warhead. Building on these results, we embarked on
a structure-activity relationship (SAR) guided program designed
to incorporate the thiosemicarbazone moiety within appropriately
functionalized benzophenone, pyridine, thiophene, fluorene,
thiochromanone, benzothiepine, and dihydroquinoline molecular
scaffolds. A focused small library of cathepsin inhibitors resulted
from these studies,43–51 from which a sub-set of 42 molecules
demonstrated IC50 values below 500 nM (Figure 3).
used
to
prepare
benzophenone
(3-bromophenyl)-(3-
hydroxyphenyl)-methanone (XV).
Scheme 1. Previously reported synthetic route towards KGP9446
In an effort to avoid impurities in which the Br atom was
replaced by a hydrogen atom, KGP94 and analogues were
synthesized through a revised route (Scheme 2). Instead of using
1,3-dibromobenzene as the precursor to the organometallic
Figure 3. Representative examples of previously described
thiosemicarbazone based inhibitors of CatL.

Scheme 2. Improved synthetic route towards KGP94 and analogues 20, 22, 23-26
reagent for the synthesis of KGP94 (18), a protected m-
bromophenol 1 was reacted with n-butyllithium to form the
intermediate organolithium reagent, which was reacted with
hydrogenation led to multiple products and with longer reaction
times, in addition to removal of the benzyl groups, the carbonyl
group was reduced to its corresponding methylene.
Weinreb amide
5 to afford the desired functionalized
benzophenone 9. Benzophenone intermediates 10 and 12 were
synthesized in a similar manner by reacting the appropriately
substituted aromatic ring with n-butyllithium followed by the
addition of Weinreb amide 6 to afford ketone 10 or the addition
of aldehyde 7 followed by oxidation with PCC to afford ketone
12. Condensation of benzophenone intermediates 9, 10, and 12
(separately) with thiosemicarbazide followed by desilylation with
TBAF afforded target thiosemicarbazone analogues KGP94 (18),
20, and 22. HPLC analysis indicated no trace amount of the
impurity in which bromine was replaced by hydrogen in the final
product for KGP94 (18).
Synthesis of dimethylresorcinol and resorcinol analogues 23-
26 utilized commercially available 1-bromo-3,5-dimethoxy
benzene as a starting material to form an intermediate
organolithium reagent which upon reaction with Weinreb 5 or 8
afforded ketones 13 and 15, respectively (Scheme 2).
Demethylation
of
these
3,5-dimethoxybenzophenone
intermediates 13 and 15 with boron tribromide afforded 3,5-
dihydroxybenzophenones 14 and 16. Subsequent condensation of
ketones 13-16 with thiosemicarbazide (separately) under
microwave irradiation generated target thiosemicarbazone
analogues 23-26.
In order to increase the aqueous solubility and possibly the
bioavailability of KGP94 (18), a water-soluble phosphate
prodrug salt was prepared through phosphorylation of the
phenolic moiety (Scheme 3). Initial attempts to incorporate the
dual thiosemicarbazone and phosphate moieties were met with
Scheme 3. Prodrug derivatization of KGP94 (18) to form water-soluble salt
KGP420 (31).
Successful completion of the phosphate prodrug was
accomplished through phosphorylation of 3-bromophenyl-3-
hydroxyphenyl methanone 27 with dibenzyl chlorophosphate
(prepared in situ)55 to afford the corresponding dibenzyl
phosphate ester 28 (Scheme 3). Subsequent deprotection of the
benzyl groups with 33% HBr in AcOH generated phosphoric
acid ester 29. Successful completion of the synthesis of the
difficulty
and
included
phosphorylation
of
(3-
hydroxyphenyl)(phenyl)ketone thiosemicarbazone (used as a
model system) which led to multiple products including
benzylated and debenzylated side products. Attempts to deprotect
dibenzyl(3-(3-bromobenzoyl)phenyl) phosphate using catalytic

phosphate salt of benzophenone thiosemicarbazone KGP420 (31)
was accomplished by the condensation of phosphoric acid ester
29 with thiosemicarbazide to afford benzophenone
thiosemicarbazone 30, which upon reaction with sodium
carbonate generated the desired disodium phosphate salt KGP420
(31). Interestingly, KGP420 (31) represents the second known
The isomer ratio was affected to the greatest degree by the
replacement of hydrogen with a hydroxyl substituent in the meta
position as demonstrated by the decrease of Z-isomer present at
equilibrium as exemplified by the comparison of analogues 18
(23% Z-isomer) and 24 (13% Z-isomer) as well as analogues 22
(18% Z-isomer) and 26 (9% Z-isomer). The replacement of a
hydrogen with a bromo substituent in the meta position on the
ring trans to the –NH of the thiosemicarbazone moiety of the
major isomer led to a decrease of Z-isomer present at equilibrium
as demonstrated by the comparison of analogues 18 and 22, 23
and 25, 24 and 26. Although this is a limited data set, it is
interesting to note correlations between isomer ratios and
substituent effects. For further analysis see supplementary
material.
example of
a
reported scaffold incorporating both
a
thiosemicarbazone and phosphate moiety.56
One challenge inherent to the synthesis of thiosemicarbazone
based inhibitors is the propensity for isomerization about the
imine bond.57–59 As a notable example, 2-formylpyridine-4’,4’-
dimethyl thiosemicarbazone, isolated in the Z configuration,
isomerized in solution generating varying Z/E equilibrium
isomeric ratios which were dependent on the capability of the
particular solvent to disrupt the intramolecular hydrogen bonding
interaction between the pyridine nitrogen and N-H hydrogen of
the thiosemicarbazone.60 The benzophenone thiosemicarbazone
analogues reported herein, except KGP420 (31) (60:40 isomeric
mixture in D2O), exist predominately in the E configuration in
solution (Figure 4). Previous molecular modeling of cathepsin L
and KGP94 showed the thiosemicarbazone moiety oriented in the
E configuration47 which correlates with the orientation of the
major isomer found in solution.
(3,5-Dibromophenyl)-(3-hydroxyphenyl)
ketone
thiosemicarbazone (22) exhibited comparable activity to KGP94
(18)47 against cathepsin L with an IC50 value of 202 nM (Table
2). Using Morrison's equation for tight binding (or covalent,
reversible) inhibitors we obtained a Kiapp of 8.4 nM (5 min pre-
incubation) compared to a value of 3.7 nM for compound 18. The
progress curves for compound 22 demonstrated the time
dependence of inhibition (see supplementary material). With
cathepsin L inhibition of 40% - 52% at 10 μM, activity of the
symmetrical,
dimethylresorcinol,
and
resorcinol
thiosemicarbazone analogues 20, 23, 25, 26 bordered the internal
cutoff threshold in our laboratory (percent inhibition ≤ 50% at 10
μM). While the presence of one m-hydroxyl group on the
aromatic ring opposing the 3-bromophenyl substituent in the
thiosemicarbazone analogues was important for activity against
cathepsin L, for this group of compounds, the presence of two m-
hydroxyl or two m-dimethoxy substituents diminished inhibitory
activity against cathepsin L. KGP94 (18) did not display
significant activity against other proteases such as matrix
metalloprotease MMP-9 or the cysteine protease caspase-3 (See
supplementary material).
Table 2. Inhibitory activity of benzophenone thiosemicarbazone
analogues.
Figure 4. ROESY and COSY correlations for analogue 22 as a representative
example of the major isomer observed in DMSO-d6.
Isomerization of KGP94 (18) and benzophenone
thiosemicarbazone analogues in DMSO-d6 occurred over a period
of time (Table 1). Analogue 26 isomerized the least with only 9%
of the minor isomer present after standing in DMSO-d6 for one
week. Increasing the number of m-bromo substituents on the ring
trans to the –NH of the thiosemicarbazone moiety of the major
isomer, and increasing the number of m-hydroxy substituents on
the ring cis to the –NH of the thiosemicarbazone moiety in the
major isomer led to an increase in the concentration of E-isomer
present at equilibrium.
IC50a Values (nM)
Cmpd
18
R1
H
R2
Br
R3
H
R4
OH
CatL
189b
CatB
>10000 b
>10000
>10000
>10000
>10000
>10000
>10000
NDd
Table 1. Isomerization of benzophenone thiosemicarbazone
analogues in DMSO-d6
20
Br
Br c
H
OH
Br c
Br
Br
OH
>10000
202
22
H c
OHc
OCH3
OH
Percent Z isomer present a
23
OCH3
OH
OCH3
OH
H
>10000
~10000
>10000
>10000
Cmpd
18
0 hours
3%
48 hours
23%
17%
12%
13%
10%
8%
1 week
23%
18%
20%
13%
14%
9%
24
H
Br
25
Br
Br
H
Br
OCH3
OH
22
1.5%
4%
26
Br
23
31
Br
O-P(O)O2Na2 >10000
24
11%
2%
a These values are averages of a minimum of a triplicate of experiments.
Each assay utilized 2% DMSO with a 5 min pre-incubation period. Standard
error values can be found in the supplementary material. b Previously reported
25
26
0.2%
by us.47 Assigned R groups in Table 2 do not correspond to assigned R
c
a
Isomerization of KGP94 and analogues were monitored by 1H NMR in
groups in Scheme 2. R groups were arranged in this manner to provide clarity
for the SAR study. d Not Determined.
DMSO-d6 as solvent.

The phosphate prodrug KGP420 (31) demonstrated aqueous
solubility greater than 400 mg per mL in comparison to the anti-
cancer agent KGP94 (18) which demonstrated aqueous solubility
less than 0.67 mg per mL (see supplementary material). The
stability of phosphate prodrug KGP420 (31) was evaluated in
aqueous solution. KGP420 (31) underwent very minor
spontaneous hydrolysis over 48 h incubation at 37 °C in 10 mM
glycine buffer solution (pH 8.6) without alkaline phosphatase
(ALP) (Figure 5A). Additionally, it was not hydrolyzed when
stored in water at 4 °C for one week. Enzymatic cleavage of
prodrug KGP420 (31) occurred with nearly 100% conversion to
the anticipated parent drug KGP94 (18) when treated with 1 unit
of ALP over the course of 48 hours (Figure 5B). Enzymatic
cleavage of KGP420 (31) yielded the active product KGP94 (18)
which inhibited cathepsin L by 88% at 10 µM.
resulted in a desirable water soluble analogue. In vitro studies
demonstrated that KGP420 was hydrolyzed to the parent
compound, KGP94, in the presence of alkaline phosphatase.
Additionally, KGP420 favorably displayed low cytotoxicity to
HUVECs, which were used as a model for normal cells. The in
vitro cleavage studies coupled with the desirable property of low
cytotoxicity positions KGP420 for future in vivo studies and pre-
clinical evaluation as a prodrug.
Acknowledgments
The authors are grateful to Mateon Therapeutics, Inc. (grant to
KGP and MLT) and the Vice Provost for Research at Baylor
University [Faculty Research Investment Program (FRIP), grant
to KGP] for their financial support of this project. The authors
express their appreciation to Dr. Kevin K. Klausmeyer and Dr.
Marissa Penney for X-ray crystallography studies, Dr. Alejandro
Ramirez (Mass Spectrometry Core Facility, Baylor University)
and Dr. Michelle Nemec (Director of the Molecular Biosciences
Center at Baylor University) for use of shared facilities, and Mr.
Ricardo Francis and Ms. Amy Boylan for technical assistance.
References and notes
(1)
Iiizumi, M.; Liu, W.; Pai, S. K.; Furuta, E.; Watabe, K. Drug
Development against Metastasis-Related Genes and Their
Pathways: A Rationale for Cancer Therapy. Biochim.
Biophys. Acta BBA - Rev. Cancer 2008, 1786 (2), 87–104.
Sleeman, J.; Steeg, P. S. Cancer Metastasis as a Therapeutic
Target. Eur. J. Cancer 2010, 46 (7), 1177–1180.
Steeg, P. S. Tumor Metastasis: Mechanistic Insights and
Clinical Challenges. Nat. Med. 2006, 12 (8), 895–904.
Lah, T. T.; Durán Alonso, M. B.; Van Noorden, C. J. F.
Antiprotease Therapy in Cancer: Hot or Not? Expert Opin.
Biol. Ther. 2006, 6 (3), 257–279.
(2)
(3)
(4)
Figure 5. Cleavage of KGP420 (31) with alkaline phosphatase (ALP)
analyzed by HPLC. (A) Control, 31 (tR = 1.6 min) was incubated for 48 h
without ALP; (B) 31 was treated with 1 unit of ALP for 48 h, with a single
product peak 18 (tR = 5.4 min) observed.
(5)
(6)
Sudhan, D. R.; Siemann, D. W. Cathepsin L Targeting in
Cancer Treatment. Pharmacol. Ther. 2015.
Lankelma, J. M.; Voorend, D. M.; Barwari, T.; Koetsveld,
J.; Van der Spek, A. H.; De Porto, A. P. N. A.; Van Rooijen,
G.; Van Noorden, C. J. F. Cathepsin L, Target in Cancer
Treatment? Life Sci. 2010, 86 (7–8), 225–233.
Gocheva, V.; Joyce, J. A. Cysteine Cathepsins and the
Cutting Edge of Cancer Invasion. Cell Cycle 2007, 6 (1),
60–64.
Fonović, M.; Turk, B. Cysteine Cathepsins and Extracellular
Matrix Degradation. Biochim. Biophys. Acta BBA - Gen.
Subj. 2014, 1840 (8), 2560–2570.
Guinec, N.; Dalet-Fumeron, V.; Pagano, M. “In Vitro” study
of Basement Membrane Degradation by the Cysteine
Proteinases, Cathepsins B, B-like and L. Digestion of
Collagen IV, Laminin, Fibronectin, and Release of
Gelatinase Activities from Basement Membrane
Fibronectin. Biol. Chem. Hoppe. Seyler 1993, 374 (12),
1135–1146.
Buck, M. R.; Karustis, D. G.; Day, N. A.; Honn, K. V.;
Sloane, B. F. Degradation of Extracellular-Matrix Proteins
by Human Cathepsin B from Normal and Tumour Tissues.
Biochem. J. 1992, 282 ( Pt 1), 273–278.
KGP94 (18) and the corresponding prodrug KGP420 (31)
were evaluated for cytotoxicity toward normal primary cells.
HUVECs were used a model for normal primary cells. Both
KGP94 (18) and KGP420 (31) did not exhibit significant
cytotoxicity against HUVECs especially compared to FDA
approved cancer therapeutics doxorubicin and paclitaxel (Table
3). Aggressive cell migration is associated with a metastatic
phenotype, and compounds 22, 18, and 31 significantly inhibited
the migration of MDA-MB-231 breast cancer cells compared to
the vehicle treated control cells in a scratch assay (see
supplementary material).
(7)
(8)
(9)
Table 3. Cytotoxicity against HUVECs
Compound Doxorubicin Paclitaxel KGP94 (18) KGP420 (31)
Cytotoxicity
0.0268a
0.00148a
26.9
20.2
GI50 (µM)
a Previously reported51
b For additional data see reference47
(10)
Improved methodology resulted in an alternative synthesis of
KGP94, which successfully circumvented unwanted byproduct
formation. Benzophenone thiosemicarbazone-based analogues
which exhibited a lower threshold for isomerization compared to
KGP94 were prepared and evaluated for inhibitory activity
against cathepsin L. The most potent of these, [(3,5-
dibromophenyl)(3-hydroxyphenyl) ketone] thiosemicarbazone
exhibited comparable activity to KGP94 with an IC50 value of
202 nM. Advancement of the pre-clinical candidate, KGP94,
through phosphate prodrug derivatization to generate KGP420
(11)
(12)
Kirschke, H.; Kembhavi, A. A.; Bohley, P.; Barrett, A. J.
Action of Rat Liver Cathepsin L on Collagen and Other
Substrates. Biochem. J. 1982, 201 (2), 367–372.
Maciewicz, R. A.; Wotton, S. F.; Etherington, D. J.;
Duance, V. C. Susceptibility of the Cartilage Collagens
Types II, IX and XI to Degradation by the Cysteine
Proteinases, Cathepsins B and L. FEBS Lett. 1990, 269 (1),
189–193.
(13)
Garnero, P.; Borel, O.; Byrjalsen, I.; Ferreras, M.; Drake, F.
H.; McQueney, M. S.; Foged, N. T.; Delmas, P. D.;

Delaissé, J. M. The Collagenolytic Activity of Cathepsin K
Is Unique among Mammalian Proteinases. J. Biol. Chem.
1998, 273 (48), 32347–32352.
Sameni, M.; Dosescu, J.; Moin, K.; Sloane, B. F. Functional
Imaging of Proteolysis: Stromal and Inflammatory Cells
Increase Tumor Proteolysis. Mol. Imaging 2003, 2 (3), 159–
175.
Rothberg, J. M.; Bailey, K. M.; Wojtkowiak, J. W.; Ben-
Nun, Y.; Bogyo, M.; Weber, E.; Moin, K.; Blum, G.;
Mattingly, R. R.; Gillies, R. J.; Sloane, B. F. Acid-Mediated
Tumor Proteolysis: Contribution of Cysteine Cathepsins.
Neoplasia N. Y. N 2013, 15 (10), 1125–1137.
Blondeau, X.; Vidmar, S. L.; Emod, I.; Pagano, M.; Turk,
V.; Keil-Dlouha, V. Generation of Matrix-Degrading
Proteolytic System from Fibronectin by Cathepsins B, G, H
and L. Biol. Chem. Hoppe. Seyler 1993, 374 (8), 651–656.
Ishidoh, K.; Kominami, E. Procathepsin L Degrades
Extracellular Matrix Proteins in the Presence of
Glycosaminoglycans in Vitro. Biochem. Biophys. Res.
Commun. 1995, 217 (2), 624–631.
(29)
(30)
Mullard, A. Merck & Co. Drops Osteoporosis Drug
Odanacatib. Nat. Rev. Drug Discov. 2016, 15 (10), 669–669.
Jadhav, P. K.; Schiffler, M. A.; Gavardinas, K.; Kim, E. J.;
Matthews, D. P.; Staszak, M. A.; Coffey, D. S.; Shaw, B.
W.; Cassidy, K. C.; Brier, R. A.; Zhang, Y.; Christie, R. M.;
Matter, W. F.; Qing, K.; Durbin, J. D.; Wang, Y.; Deng, G.
G. Discovery of Cathepsin S Inhibitor LY3000328 for the
Treatment of Abdominal Aortic Aneurysm. ACS Med.
Chem. Lett. 2014, 5 (10), 1138–1142.
Payne, C. D.; Deeg, M. A.; Chan, M.; Tan, L. H.; LaBell, E.
S.; Shen, T.; DeBrota, D. J. Pharmacokinetics and
Pharmacodynamics of the Cathepsin S Inhibitor,
LY3000328, in Healthy Subjects: PK/PD and Safety and
Tolerability of Cathepsin S Inhibitor. Br. J. Clin.
Pharmacol. 2014, 78 (6), 1334–1342.
Shah, P. P.; Wang, T.; Kaletsky, R. L.; Myers, M. C.;
Purvis, J. E.; Jing, H.; Huryn, D. M.; Greenbaum, D. C.;
Smith, A. B.; Bates, P.; Diamond, S. L. A Small-Molecule
Oxocarbazate Inhibitor of Human Cathepsin L Blocks
Severe Acute Respiratory Syndrome and Ebola Pseudotype
Virus Infection into Human Embryonic Kidney 293T Cells.
Mol. Pharmacol. 2010, 78 (2), 319–324.
(14)
(15)
(31)
(32)
(16)
(17)
(18)
(19)
Berdowska, I. Cysteine Proteases as Disease Markers. Clin.
Chim. Acta 2004, 342 (1–2), 41–69.
Ullah, M. F.; Aatif, M. The Footprints of Cancer
Development: Cancer Biomarkers. Cancer Treat. Rev. 2009,
35 (3), 193–200.
(33)
Liu, Z.; Myers, M. C.; Shah, P. P.; Beavers, M. P.;
Benedetti, P. A.; Diamond, S. L.; Smith, A. B.; Huryn, D.
M. Design, Synthesis and Biological Evaluation of a Library
of Thiocarbazates and Their Activity as Cysteine Protease
Inhibitors. Comb. Chem. High Throughput Screen. 2010, 13
(4), 337–351.
Meara, J. P.; Rich, D. H. Mechanistic Studies on the
Inactivation of Papain by Epoxysuccinyl Inhibitors. J. Med.
Chem. 1996, 39 (17), 3357–3366.
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. Cathepsin Cysteine Proteases Are
Effectors of Invasive Growth and Angiogenesis during
Multistage Tumorigenesis. Cancer Cell 2004, 5 (5), 443–
453.
Ehmke, V.; Winkler, E.; Banner, D. W.; Haap, W.;
Schweizer, W. B.; Rottmann, M.; Kaiser, M.; Freymond, C.;
Schirmeister, T.; Diederich, F. Optimization of Triazine
Nitriles as Rhodesain Inhibitors: Structure-Activity
Relationships, Bioisosteric Imidazopyridine Nitriles, and X-
Ray Crystal Structure Analysis with Human Cathepsin L.
ChemMedChem 2013, 8 (6), 967–975.
Marquis, R. W.; James, I.; Zeng, J.; Trout, R. E. L.;
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. Azepanone-Based Inhibitors of Human
Cathepsin L. J. Med. Chem. 2005, 48 (22), 6870–6878.
Linington, R. G.; Clark, B. R.; Trimble, E. E.; Almanza, A.;
Ureña, L.-D.; Kyle, D. E.; Gerwick, W. H. Antimalarial
Peptides from Marine Cyanobacteria: Isolation and
Structural Elucidation of Gallinamide A. J. Nat. Prod. 2009,
72 (1), 14–17.
Miller, B.; Friedman, A. J.; Choi, H.; Hogan, J.;
McCammon, J. A.; Hook, V.; Gerwick, W. H. The Marine
Cyanobacterial Metabolite Gallinamide A Is a Potent and
Selective Inhibitor of Human Cathepsin L. J. Nat. Prod.
2014, 77 (1), 92–99.
Yasuma, T.; Oi, S.; Choh, N.; Nomura, T.; Furuyama, N.;
Nishimura, A.; Fujisawa, Y.; Sohda, T. Synthesis of Peptide
Aldehyde Derivatives as Selective Inhibitors of Human
Cathepsin L and Their Inhibitory Effect on Bone Resorption
1. J. Med. Chem. 1998, 41 (22), 4301–4308.
Du, X.; Guo, C.; Hansell, E.; Doyle, P. S.; Caffrey, C. R.;
Holler, T. P.; McKerrow, J. H.; Cohen, F. E. Synthesis and
Structure−Activity Relationship Study of Potent
Trypanocidal Thio Semicarbazone Inhibitors of the
(20)
Bell-McGuinn, K. M.; Garfall, A. L.; Bogyo, M.; Hanahan,
D.; Joyce, J. A. Inhibition of Cysteine Cathepsin Protease
Activity Enhances Chemotherapy Regimens by Decreasing
Tumor Growth and Invasiveness in a Mouse Model of
Multistage Cancer. Cancer Res. 2007, 67 (15), 7378–7385.
Rodan, S. B.; Duong, L. T. Cathepsin K – A New Molecular
Target for Osteoporosis. IBMS BoneKEy 2008, 5 (1), 16–24.
Yasuda, Y.; Kaleta, J.; Brömme, D. The Role of Cathepsins
in Osteoporosis and Arthritis: Rationale for the Design of
New Therapeutics. Adv. Drug Deliv. Rev. 2005, 57 (7), 973–
993.
(34)
(35)
(21)
(22)
(23)
Cheng, X. W.; Huang, Z.; Kuzuya, M.; Okumura, K.;
Murohara, T. Cysteine Protease Cathepsins in
(36)
(37)
Atherosclerosis-Based Vascular Disease and Its
Complications. Hypertension 2011, 58 (6), 978–986.
Zavasnik-Bergant, T.; Turk, B. Cysteine Cathepsins in the
Immune Response. Tissue Antigens 2006, 67 (5), 349–355.
Pan cathepsin inhibitor: VBY-825.
(24)
(25)
http://www.virobayinc.com/VBY-825.php (accessed Sep 2,
2015).
(26)
Elie, B. T.; Gocheva, V.; Shree, T.; Dalrymple, S. A.;
Holsinger, L. J.; Joyce, J. A. Identification and Pre-Clinical
Testing of a Reversible Cathepsin Protease Inhibitor
Reveals Anti-Tumor Efficacy in a Pancreatic Cancer Model.
Biochimie 2010, 92 (11), 1618–1624.
Gauthier, J. Y.; Chauret, N.; Cromlish, W.; Desmarais, S.;
Duong, L. T.; Falgueyret, J.-P.; Kimmel, D. B.;
Lamontagne, S.; Léger, S.; LeRiche, T.; Li, C. S.; Massé, F.;
McKay, D. J.; Nicoll-Griffith, D. A.; Oballa, R. M.; Palmer,
J. T.; Percival, M. D.; Riendeau, D.; Robichaud, J.; Rodan,
G. A.; Rodan, S. B.; Seto, C.; Thérien, M.; Truong, V.-L.;
Venuti, M. C.; Wesolowski, G.; Young, R. N.; Zamboni, R.;
Black, W. C. The Discovery of Odanacatib (MK-0822), a
Selective Inhibitor of Cathepsin K. Bioorg. Med. Chem.
Lett. 2008, 18 (3), 923–928.
Stoch, S. A.; Zajic, S.; Stone, J. A.; Miller, D. L.; van
Bortel, L.; Lasseter, K. C.; Pramanik, B.; Cilissen, C.; Liu,
Q.; Liu, L.; Scott, B. B.; Panebianco, D.; Ding, Y.;
Gottesdiener, K.; Wagner, J. A. Odanacatib, a Selective
Cathepsin K Inhibitor to Treat Osteoporosis: Safety,
Tolerability, Pharmacokinetics and Pharmacodynamics -
Results from Single Oral Dose Studies in Healthy
Volunteers: Odanacatib Pharmacokinetics and
Pharmacodynamics. Br. J. Clin. Pharmacol. 2013, 75 (5),
1240–1254.
(27)
(38)
(39)
(40)
(41)
(28)

Trypanosomal Cysteine Protease Cruzain. J. Med. Chem.
2002, 45 (13), 2695–2707.
4 with Application as a Vascular Disrupting Agent. J. Nat.
Prod. 2013, 76 (9), 1668–1678.
(42)
(43)
Siles, R.; Chen, S.-E.; Zhou, M.; Pinney, K. G.; Trawick, M.
L. Design, Synthesis, and Biochemical Evaluation of Novel
Cruzain Inhibitors with Potential Application in the
Treatment of Chagas’ Disease. Bioorg. Med. Chem. Lett.
2006, 16 (16), 4405–4409.
Sudhan, D. R.; Siemann, D. W. Cathepsin L Inhibition by
the Small Molecule KGP94 Suppresses Tumor
Microenvironment Enhanced Metastasis Associated Cell
Functions of Prostate and Breast Cancer Cells. Clin. Exp.
Metastasis 2013, 30 (7), 891–902.
Siles, R.; Zhou, M.; Ackley, J. F.; Pinney, K. G.; Chen, S.-
E.; Arispe-Angulo, W. M.; Trawick, M. L. Inhibitors of
Cysteine Proteases and Methods of Use Thereof.
US8173696 B2, May 8, 2012.
Kishore Kumar, G. D.; Chavarria, G. E.; Charlton-Sevcik,
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. Design, Synthesis, and Biological Evaluation
of Potent Thiosemicarbazone Based Cathepsin L Inhibitors.
Bioorg. Med. Chem. Lett. 2010, 20 (4), 1415–1419.
Kumar, G. D. K.; 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. Functionalized
Benzophenone, Thiophene, Pyridine, and Fluorene
Thiosemicarbazone Derivatives as Inhibitors of Cathepsin
L. Bioorg. Med. Chem. Lett. 2010, 20 (22), 6610–6615.
Chavarria, G. E.; Horsman, M. R.; Arispe, W. M.; Kumar,
G. D. K.; Chen, S.-E.; Strecker, T. E.; Parker, E. N.;
Chaplin, D. J.; Pinney, K. G.; Trawick, M. L. Initial
Evaluation of the Antitumour Activity of KGP94, a
Functionalized Benzophenone Thiosemicarbazone Inhibitor
of Cathepsin L. Eur. J. Med. Chem. 2012, 58, 568–572.
Song, J.; Jones, L. M.; Kumar, G. D. K.; Conner, E. S.;
Bayeh, L.; Chavarria, G. E.; Charlton-Sevcik, A. K.; Chen,
S.-E.; Chaplin, D. J.; Trawick, M. L.; Pinney, K. G.
Synthesis and Biochemical Evaluation of Thiochromanone
Thiosemicarbazone Analogues as Inhibitors of Cathepsin L.
ACS Med. Chem. Lett. 2012, 3 (6), 450–453.
(54)
(55)
Stella, V. J. Prodrugs: Some Thoughts and Current Issues. J.
Pharm. Sci. 2010, 99 (12), 4755–4765.
Silverberg, L. J.; Dillon, J. L.; Vemishetti, P. A Simple,
Rapid and Efficient Protocol for the Selective
Phosphorylation of Phenols with Dibenzyl Phosphite.
Tetrahedron Lett. 1996, 37 (6), 771–774.
Li, J.; Luo, X.; Wang, Q.; Zheng, L. M.; King, I.; Doyle, T.
W.; Chen, S. H. Synthesis and Biological Evaluation of a
Water Soluble Phosphate Prodrug of 3-Aminopyridine-2-
Carboxaldehyde Thiosemicarbazone (3-AP). Bioorg. Med.
Chem. Lett. 1998, 8 (22), 3159–3164.
Karabatsos, G. J.; Vane, F. M.; Taller, R. A.; Hsi, N.
Structural Studies by Nuclear Magnetic Resonance. VIII.
Ring-Substituted Phenylhydrazones, Semicarbazones, and
Thiosemicarbazones. J. Am. Chem. Soc. 1964, 86 (16),
3351–3357.
Carradori, S.; Cirilli, R.; Dei Cicchi, S.; Ferretti, R.; Menta,
S.; Pierini, M.; Secci, D. 3-Methylcyclohexanone
Thiosemicarbazone: Determination of E/Z Isomerization
Barrier by Dynamic High-Performance Liquid
Chromatography, Configuration Assignment and
Theoretical Study of the Mechanisms Involved by the
Spontaneous, Acid and Base Catalyzed Processes. J.
Chromatogr. A 2012, 1269, 168–177.
Tenchiu (Deleanu), A.-C.; Kostas, I. D.; Kovala-Demertzi,
D.; Terzis, A. Synthesis and Characterization of New
Aromatic Aldehyde/Ketone 4-(β-D-
Glucopyranosyl)thiosemicarbazones. Carbohydr. Res. 2009,
344 (11), 1352–1364.
(56)
(57)
(58)
(44)
(45)
(46)
(47)
(48)
(49)
(59)
(60)
Pessôa, M. M. .; Andrade, G. F. .; Paoli Monteiro, V. R.;
Temperini, M. L. . 2-Formylpyridinethiosemicarbazone and
Methyl Derivatives: Spectroscopic Studies. Polyhedron
2001, 20 (26–27), 3133–3141.
Supplementary Data
Supplementary data (Experimental details regarding synthesis,
characterization (1H NMR, 13C NMR, 31P NMR, COSY, ROESY,
HRMS, HPLC, and X-ray data (KGP94 (18) CDCC deposition
number: 1445273), and biological assays) associated with this
article can be found, in the online version, at
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. Bioorg. Med. Chem. Lett. 2013, 23 (9), 2801–
2807.
(50)
(51)
Chaplin, D. J.; Devanna, K. K. G.; Parker, E.; Pinney, K. G.;
Song, J.; Trawick, M. L. Compositions and Methods for
Inhibition of Cathepsins. US8877967 B2, November 4,
2014.
Parker, E. N.; Song, J.; Kishore Kumar, G. D.; Odutola, S.
O.; Chavarria, G. E.; Charlton-Sevcik, A. K.; Strecker, T.
E.; Barnes, A. L.; Sudhan, D. R.; Wittenborn, T. R.;
Siemann, D. W.; Horsman, M. R.; Chaplin, D. J.; Trawick,
M. L.; Pinney, K. G. Synthesis and Biochemical Evaluation
of Benzoylbenzophenone Thiosemicarbazone Analogues as
Potent and Selective Inhibitors of Cathepsin L. Bioorg. Med.
Chem. 2015.
(52)
(53)
Sudhan, D. R.; Siemann, D. W. Abstract 4030: In-Vivo
Evaluation of the Anti-Metastatic Efficacy of Small
Molecule Cathepsin L Inhibitor KGP94 in a Prostate Cancer
Model. Cancer Res. 2014, 74 (19 Supplement), 4030–4030.
Hadimani, M. B.; MacDonough, M. T.; Ghatak, A.;
Strecker, T. E.; Lopez, R.; Sriram, M.; Nguyen, B. L.; Hall,
J. J.; Kessler, R. J.; Shirali, A. R.; Liu, L.; Garner, C. M.;
Pettit, G. R.; Hamel, E.; Chaplin, D. J.; Mason, R. P.;
Trawick, M. L.; Pinney, K. G. Synthesis of a 2-Aryl-3-
Aroyl Indole Salt (OXi8007) Resembling Combretastatin A-