Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L

Article abstract of DOI:10.1016/j.bmc.2015.09.036

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed, synthesized, and evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC₅₀ values of 9.9 nM, 14.4 nM, and 8.1 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10 μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5 μM. The most active cathepsin L inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells (HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L. Active members of this structurally diverse group of benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-metastatic, pre-clinical drug candidates.

Full text of DOI:10.1016/j.bmc.2015.09.036

Accepted Manuscript
Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarba-
zone analogues as potent and selective inhibitors of cathepsin L
Erica N. Parker, Jiangli Song, G.D. Kishore Kumar, Samuel O. Odutola,
Gustavo E. Chavarria, Amanda K. Charlton-Sevcik, Tracy E. Strecker, Ashleigh
L. Barnes, Dhivya R. Sudhan, Thomas R. Wittenborn, Dietmar W. Siemann,
Michael R. Horsman, David J. Chaplin, Mary Lynn Trawick, Kevin G. Pinney
PII:
S0968-0896(15)30061-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.09.036
BMC 12583
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
15 July 2015
11 September 2015
22 September 2015
Please cite this article as: 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, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.09.036
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Synthesis and biochemical evaluation of
benzoylbenzophenone thiosemicarbazone analogues
as potent and selective inhibitors of cathepsin L
a
a
a
b
Erica N. Parker , Jiangli Song , G. D. Kishore Kumar , Samuel O. Odutola , Gustavo E.
a
a
a
a
Chavarria , Amanda K. Charlton-Sevcik , Tracy E. Strecker , Ashleigh L. Barnes , Dhivya R.
c
e
c,d
e
Sudhan , Thomas R. Wittenborn , Dietmar W. Siemann , Michael R. Horsman , David J.
f
a,b,*
a,b,
Chaplin , Mary Lynn Trawick , Kevin G. Pinney *
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-
7
224
c
Department of Radiation Oncology, University of Florida Health Cancer Center, Gainesville,
FL 32610
d
Department of Pharmacology and Therapeutics, College of Medicine, University of Florida,
Gainesville, FL 32610
e
Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark
f
OXiGENE, Inc. 701 Gateway Blvd, Suite 210 South San Francisco, CA 94080
*
Co-corresponding Authors: Tel: 254-710-4117, E-mail: Kevin_Pinney@baylor.edu; Tel: 254-
7
10-6857, E-mail: Mary_Lynn_Trawick@baylor.edu
Abbreviations used
HUVECs, human umbilical vein endothelial cells; ECM, extracellular matrix; TSC,
thiosemicarbazide; SAR, structure-activity relationship; Z-FR-AMC, N-carbobenzyloxy-L-Phe-
L-Arg-7-amino-4-methylcoumarin; FBS, fetal bovine serum; SRB, sulforhodamine B.
ABSTRACT
1

Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell
invasion and migration through degradation of the extracellular matrix suggest that cathepsin L
is a promising biological target for the development of anti-metastatic agents. Based on
encouraging results from studies on benzophenone thiosemicarbazone cathepsin inhibitors, a
series of fourteen benzoylbenzophenone thiosemicarbazone analogues were designed,
synthesized, and evaluated for their inhibitory activity against cathepsins L and B.
Thiosemicarbazone inhibitors 3-benzoylbenzophenone thiosemicarbazone 1, 1,3-bis(4-
fluorobenzoyl)benzene thiosemicarbazone 8, and 1,3-bis(2-fluorobenzoyl)-5-bromobenzene
thiosemicarbazone 32 displayed the greatest potency against cathepsin L with low IC50 values of
9
.85 nM, 14.4 nM, and 8.12 nM, respectively. The benzoylbenzophenone thiosemicarbazone
analogues evaluated were selective in their inhibition of cathepsin L compared to cathepsin B.
Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast
cancer cells by 70% at 10 µM. Thiosemicarbazone analogue 8 significantly inhibited the
invasive potential of PC-3ML prostate cancer cells by 92% at 5 µM. The most active cathepsin L
inhibitors from this benzoylbenzophenone thiosemicarbazone series (1, 8, and 32) displayed low
cytotoxicity toward normal primary cells [in this case human umbilical vein endothelial cells
(
HUVECs)]. In an initial in vivo study, 3-benzoylbenzophenone thiosemicarbazone (1) was well-
tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed
efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo
tolerability are desirable characteristics for anti-metastatic agents functioning through an
inhibition of cathepsin L. Active members of this structurally diverse group of
benzoylbenzophenone thiosemicarbazone cathepsin L inhibitors show promise as potential anti-
metastatic, pre-clinical drug candidates.
2

3

Keywords
Small-molecule synthesis, thiosemicarbazone warhead, cathepsin L inhibitors, inhibition of
cancer cell invasion and migration, anti-metastatic agents
4

1
. Introduction
In various human cancers cathepsins L, B, K, H, S, and X display increased activity and
1
-5
play significant mechanistic roles in the invasion and migration of malignant cells. Cysteine
protease cathepsins degrade proteolytic targets predominately within lysosomes under normal
1
-5
physiological conditions. In cancer, cathepsins promote metastasis through phagocytosis as
6
well as extracellulary through multiple pathways including direct proteolysis of E-cadherin and
7
-10
8-10
9-15
components in the extracellular matrix (ECM) such as fibronectin,
laminin,
and indirectly through the activation of other proteases resulting in the degradation of the ECM
Figure 1).
collagen,
1
6
(
Figure 1. Overview of cysteine cathepsins in invasion and metastasis. Compared to normal
physiological conditions, certain cathepsins are upregulated in the tumor microenvironment.
Additionally, changes occur within the tumor microenvironment such as downregulation of
endogenous inhibitors including the cystatins, extracellular acidification increasing the
proteolytic capability of cathepsins, and extracellular secretion of cathepsins. Degradation of the
extracellular matrix through the direct proteolysis of matrix and cell-adhesion proteins such as
laminin, fibronectin, collagen, E-cadherin facilitates tumor cell invasion from the primary tumor
siteand ultimately migration to a secondary tumor site. Small-molecule cathepsin inhibitors have
the potential to inhibit the invasive nature of malignant cells and may provide increased
1
7
effectiveness in combination with known chemotherapeutics.
5

Elevated levels of cathepsin L, a ubiquitous endopeptidase, and cathepsin B, an
endopeptidase as well as a carboxypeptidase, are associated with poor prognosis in breast cancer,
1
8, 19
colorectal cancer, lung cancer, brain tumors, melanoma, and head and neck cancer.
Cathepsin K, an endopeptidase involved in bone resorption, shows increased activity in breast
2
0
cancer, cervical cancer, and lung cancer. The cathepsin K inhibitor Odanacatib (MK-0822)
displayed promise in a recent clinical trial by reducing bone resorption in individuals with
2
1-22
metastatic bone disease.
In addition to metastasis, cathepsins are involved in other pathological processes. Drug
candidates currently in the pipeline of pharmaceutical companies include the cathepsin K
2
1-24
25
inhibitors Odanacatib (Merck)
and MIV-711 (Medivir) for the treatment of osteoporosis,
2
6-27
the cathepsin B inhibitor VBY-376 (Virobay)
and the pan cysteine protease inhibitor VBY-
for the treatment of liver fibrosis, the cathepsin C inhibitor GSK2793660
2
8-29
8
25
3
0-31
(
GlaxoSmithKline) for the treatment of bronchiectasis,
and the cathepsin S inhibitor MIV-
3
2
2
47 (Medivir) for the treatment of neuropathic pain. Although selective inhibitors of cathepsin
L have not yet reached clinical trials, several have been described in the literature (Figure 2).
6

Figure 2. Representative potent inhibitors of cathepsin L utilizing various warheads. Notable
3
3
selective inhibitors of cathepsin L include the natural product gallinamide A I, the
3
4
35
36
epoxysuccinamide inhibitor Clik 148 II, the oxocarbazate III, the vinyl sulfonate IV, the
3
7
38
dipeptidyl nitrile V, and the azepanone VI. Potent but non-selective inhibitors of cathepsin L
3
9
40
include the nonpeptidic cyanamide VII, the azadipeptide nitrile VIII, and the diketone VBY-
2
8-29
8
25 IX.
Figure 3. Activity of selected benzophenone thiosemicarbazone analogues against cathepsin L
7

Historically, molecules incorporating the thiosemicarbazone functionality have been used
4
1-43
44
as antiviral and antibacterial agents in the treatment of smallpox
and tuberculosis. In
incorporation of the thiosemicarbazone moiety as
an electrophilic warhead has proved useful in the identification of potent small-molecule
4
5-51, 52
addition to the inhibition of cathepsins,
5
3-57
inhibitors of cysteine proteases found in Trypanosoma cruzi and Trypanosoma brucei,
5
7-59
60
61
Plasmodium falciparum
of thiosemicarbazone inhibitors in our group
benzophenone, thiochromanone, thiochromanone sulfone, and dihydroquinoline scaffolds
,
Leishmania Mexicana, and Eimeria tenella. In the investigation
4
5-50
several cathepsin L inhibitors based on the
4
6-48
including (3-bromo-3'-hydroxybenzophenone) thiosemicarbazone X,
(3-bromo-2'-
4
5
fluorobenzophenone) thiosemicarbazone XI, and 6-nitrothio-4-chromanone thiosemicarbazone
4
9
XVI emerged as promising lead compounds (Figure 3).
8

2
2
. Results and discussion
.1 Design and synthetic chemistry
Scheme 1. Synthesis of benzoylbenzophenone thiosemicarbazone analogues. Reagents and
conditions: (a) Thiosemicarbazide (TSC), TsOH, THF, reflux; (b) TSC, TsOH, MeOH, reflux;
o
(
c) EtOH, NaBH
4
, 0 C.
Functional group considerations based on our initial benzophenone thiosemicarbazone
inhibitors included the incorporation of a benzoyl moiety which, in this study, led to the
discovery that appropriately functionalized benzoylbenzophenone thiosemicarbazone analogues
demonstrated potent inhibition of cathepsin L. In addition to incorporation of a benzoyl group,
structural motifs previously identified in potent benzophenone thiosemicarbazone inhibitors were
integrated into the design of this new series of benzoylbenzophenone thiosemicarbazone
5
1
45-46
analogues. Previously,
incorporation of the 3-bromo functionality in benzophenone
thiosemicarbazone analogues proved critical in providing analogues which displayed potent
inhibitory activity against cathepsin L as demonstrated by the large differences in IC50 values
found for X and XI compared to the non-brominated analogues XIII and XIV, respectively
9

(
Figure 3). Additionally, the 4-bromo analogue XII displayed reduced activity towards cathepsin
L compared to the 3-bromo analogue XI further emphasizing the importance of the 3-bromo
4
5-46
functionality. Drawing on previous SAR studies based on the benzophenone scaffold,
extension of key structural components of the potent cathepsin L benzophenone
thiosemicarbazone inhibitors X and XI led to the design of benzoylbenzophenone
thiosemicarbazone inhibitors 31 and 32 which incorporated the 3-bromo functionality in the
central aromatic ring. In order to avoid potential complications that could arise due to the
difficulty of separating thiosemicarbazone regioisomers, only symmetrical diketones were
utilized in the synthesis of the new target compounds. Various synthetic methodologies
(
Schemes 1-4) were employed to assemble each benzoylbenzophenone molecular scaffold.
Benzoylbenzophenone thiosemicarbazones 1, 2 and benzoylbenzhydrol thiosemicarbazone 4
were prepared from commercially available diketones as illustrated in Scheme 1.
Scheme 2. Synthesis of benzoylbenzophenone thiosemicarbazone analogues utilizing Friedel-
Crafts acylation. Reagents and conditions: (a) AlCl
3
(neat), reflux; (b) AlCl
3
, CH
2
Cl
2
, reflux; (c)
, CH Cl
TSC, TsOH, THF, microwave irradiation; (d) TSC, TsOH, THF, reflux; (e) BF
3
·SMe
2
2
2
,
o
rt; (f) isopropyl bromide, K
2
CO , DMF, 90 C.
3
10

Utilization of Friedel-Crafts acylation chemistry facilitated the synthesis of para
substituted benzoylbenzophenone analogues as depicted in Scheme 2. Reaction of isophthaloyl
dichloride and the appropriately substituted benzene ring with aluminum chloride afforded para
substituted 1,3-dibenzoylbenzene analogues 5-7 which underwent condensation with
thiosemicarbazide to yield target thiosemicarbazone analogues 8-11. Demethylation of 1,3-bis(4-
methoxybenzoyl)benzene 7 followed by condensation with thiosemicarbazide afforded
thiosemicarbazone 13 and reaction of diketone 12 with isopropyl bromide followed by
condensation with thiosemicarbazide afforded the p-isopropoxy derivative 14.
Scheme 3. Synthesis of benzoylbenzophenone thiosemicarbazone analogues utilizing
o
isophthaloyl chloride. Reagents and conditions: (a) Me(OMe)NH ·HCl, NEt
3
, CH
2
Cl
2
, 0 C -rt;
o
o
(
b) n-BuLi, THF, -78 C; (c) THF, -78 C; (d) TSC, TsOH, MeOH, reflux; (e) TSC, TsOH, THF,
o
microwave irradiation, 90 C; (f) TBAF, THF, rt.
In order to incorporate meta substituents in the outermost rings of the
benzoylbenzophenone scaffold (Scheme 3), isophthaloyl dichloride starting materials were used
as precursors to Weinreb amides 16 and 17. Diketones 18 and 19 were synthesized from an
intermediate organolithium reagent and Weinreb amides 16 and 17 (separately). Condensation of
11

the resulting meta substituted 1,3-dibenzoylbenzene analogues with thiosemicarbazide and
removal of any protecting groups afforded benzoylbenzophenone thiosemicarbazone analogues
2
0 and 22.
Scheme 4. Synthesis of benzoylbenzophenone thiosemicarbazone analogues utilizing 1,3,5-
o
tribromobenzene. Reagents and conditions: (a) Me(OMe)NH ·HCl, NEt
3
, CH
2
Cl
2
, 0 C -rt; (b)
o
o
o
TBSCl, DMF, imidazole, 0 C -rt; (c) (i) t-BuLi, ether, -78 C; (ii) 23, 24, or 25 in ether, -78 C;
o
o
(
d) PCC, celite, CH
2
Cl
2
, 0 C -rt; (e) TSC, TsOH, THF, microwave irradiation, 90 C; (f) TSC,
, THF, reflux (h) TBAF, THF, rt.
TsOH, THF, reflux; (g) TSC, Ti(OiPr)
4
4
5-48
In our previous studies, benzophenone thiosemicarbazone analogues
containing a
meta-bromo substituent were among those with the highest inhibitory activity against cathepsin
L. Incorporation of meta-bromo substituents on the central aromatic ring in the
benzoylbenzophenone scaffold was achieved by utilizing 1,3,5-tribromobenzene as a starting
material. Halogen-metal exchange of 1,3,5-tribromobenzene with t-BuLi followed by the
addition of two equivalents of either aldehyde 23 or Weinreb amides 24 and 25 (separately)
allowed for the incorporation of a meta-bromo substituent on the central ring of diketone
analogues 27-29 (Scheme 4). Subsequent condensation of the diketones 27-29 with
12

thiosemicarbazide yielded the benzoylbenzophenone thiosemicarbazone analogues 30, 32-33,
and deprotection of 30 afforded the m-hydroxy substituted analogue 31.
Figure 4. Isomerization of thiosemicarbazone analogue 33 in DMSO-d
6
. The red ellipses
1
indicate regions in the H NMR spectra where additional peaks emerge due to the presence of the
1
1
other geometrical isomer. (a) H NMR of compound 33 after 0 hours in DMSO-d6. (b) H NMR
1
of compound 33 after 24 hours in DMSO-d6. (c) H NMR of compound 33 after 16 days in
DMSO-d6.
Moderate to low yields were observed for the condensation reaction to form the mono
thiosemicarbazone analogues due to the competing formation of the bis-thiosemicarbazone
products. For some of the desired mono-thiosemicarbazone products a lower equivalency of
thiosemicarbazide proved effective at minimizing formation of the bis derivative and
maximizing yield, but not in all cases. With the exception of 1,3-bis(2-fluorobenzoyl)-5-
bromobenzene thiosemicarbazone 32, which was isolated as the Z isomer and did not isomerize
after standing in acetone for one week, the final products exist as an inseparable mixture of E/Z
1
isomers in solution (As evidenced by H NMR). Imines including thiosemicarbazones are well-
6
2-63
64-65
known for their propensity to isomerize in solution under catalyzed
and non-catalyzed
66
conditions or from heating while in the solid state. As an example, benzoylbenzophenone
thiosemicarbazone 33, after purification and drying, was isolated as a single isomer; however,
13

the compound slowly isomerized in solution (Figure 4). After 16 days of standing in DMSO at
room temperature, thiosemicarbazone 33 isomerized to a 1:1 mixture of E/Z isomers.
2
.2 Cathepsin Inhibition Studies
Table 1. Inhibitory Activity of Benzoylbenzophenone Thiosemicarbazone Analogues
a
IC50 Values (nM)
Cmpd
R
1
R
2
R
3
R
4
X
C=O
Cat L
9.9
Cat B
1
2
4
8
9
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
Bz
H
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
C=O
56.0
H
H
CH(OH)
C=O
23.8
F
H
H
14.4
Br
H
H
C=O
1522
>10000
5117
340
1
1
1
1
2
2
3
3
3
0
1
3
4
0
2
1
2
3
Br
H
H
C=NNHC(S)NH
2
OCH
3
H
H
C=O
OH
H
H
C=O
OCH(CH
3
)
2
H
H
C=O
>10000
654
H
H
H
H
H
CH
3
H
C=O
b
Br
OH
Br
Br
Br
C=O
~10000
71.6
OH
H
C=O
C=O
8.1
Br
H
C=O
10347
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 data.
b
Compound 22 inhibited cathepsin L activity by 56.9% at 10000 nM.
14

The thiosemicarbazone derivative of commercially available 1,3-dibenzoylbenzene (3-
benzoylbenzophenone thiosemicarbazone 1) displayed pronounced inhibitory activity against
cathepsin L with an IC50 value of 9.85 nM (Table 1). Interestingly, the analogous benzophenone
4
6
thiosemicarbazone XV (Figure 3) was inactive (IC50 >10000 nM) against cathepsin L. Since
thiosemicarbazone 1 displayed pronounced activity against cathepsin L, several analogues were
synthesized including compounds which incorporated previously reported molecular scaffolds
(
Figure 3) known to be effective in terms of providing compounds with strong inhibitory activity
4
5-48
against cathepsin L.
In addition, certain structural modifications of the unsubstituted
analogue 1 such as the incorporation of a structurally demanding benzoyl substituent on the
central aromatic ring, exemplified by tribenzoylbenzophenone thiosemicarbazone analogue 2
(
IC50 = 56.0 nM), and the incorporation of a secondary alcohol in place of the carbonyl,
exemplified by 3-benzoylbenzhydrol thiosemicarbazone 4 (IC50 = 23.8 nM), were well tolerated.
Various para substituted analogues were evaluated against cathepsin L. The p-fluoro analogue 8
had comparable activity to the extremely potent unsubstituted analogue 1. The activity against
cathepsin L diminished with increasing steric hindrance in the para-substituted series (p-hydroxy
1
3, p-methoxy 11, p-isopropoxy 14). Thiosemicarbazone analogues 9, 22, and 33 each with p-
bromo or m-bromo substituents on the outermost rings of the benzoylbenzophenone molecular
template, although still active, showed a significant reduction in regard to inhibitory activity
against cathepsin L compared to the other compounds. Additionally, the p-bromo substituted bis-
thiosemicabazone analogue 10 showed no activity against cathepsin L at a concentration of
1
0,000 nM. Compared to the parent benzophenone thiosemicarbazone XI (Figure 3) with an
IC50 value of 30.5 nM, 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32
exhibited a greater than three fold increase in activity against cathepsin L with an IC50 value of
15

8
.12 nM. Incorporation of the benzophenone thiosemicarbazone X (Figure 3) molecular design
into the benzoylbenzophenone scaffold led to two thiosemicarbazone analogues; compound 31
with meta-hydroxy substituents on the outermost rings and compound 22 with meta-bromo
substituents on the outermost rings. Thiosemicarbazone 31 showed a nearly two fold increase in
activity against cathepsin L with an IC50 value of 71.6 nM compared to the parent benzophenone
thiosemicarbazone X (IC50 = 131.4 nM) while analogue 22 showed a significant decrease in
activity against cathepsin L with an IC50 value of ~10000 nM. Overall, increasing steric bulk on
the outermost rings likely resulted in benzoylbenzophenone analogues with decreased inhibitory
activity. Interestingly, all active benzoylbenzophenone thiosemicarbazone analogues were
selective against cathepsin L compared to cathepsin B and thiosemicarbazone analogues (31 and
3
2) of the parent benzophenone thiosemicarbazones X and XI (which do not have bulky groups
on the outermost rings) displayed increased activity against cathepsin L. These studies have
demonstrated that the benzoylbenzophenone scaffold coupled with the thiosemicarbazone
electrophilic moiety represents an effective molecular design leading to low nM inhibitors of
cathepsin L and additionally allows for a variety of structural modifications in the core scaffold
while maintaining activity against cathepsin L.
16

Figure 5. Representative progress curves of cathepsin L (1 nM) activity using Z-FR-AMC (10
µM) as substrate and increasing concentrations of (A) inhibitor 1 and (B) inhibitor 32 (0–10
µM). Reactions were initiated by the addition of enzyme with no pre-incubation with
compounds. Release of the fluorescent product AMC was followed as a function of time.
Kinetic analysis of the closely related thiosemicarbazone analogue X (Figure 3) showed
4
7
that it was a time-dependent and slowly reversible inhibitor of cathepsin L. The reaction
progress curves for benzoylbenzophenone thiosemicarbazone analogues 1 and 32 (Figure 5)
were comparable to those previously observed for (3-bromo-3'-hydroxybenzophenone)
thiosemicarbazone X. The cathepsin L catalyzed reaction was carried out with N-
carbobenzyloxy-L-Phe-L-Arg-7-amino-4-methylcoumarin (Z-FR-AMC) as substrate in the
presence of varying concentrations (0–10 µM) of thiosemicarbazone analogues 1 and 32. The
progress curves (Figure 5) demonstrated that these compounds are not classical, readily
reversible inhibitors. The increase in inhibition as a function of time, as observed from a
reduction in the slopes of the curves over time compared to untreated control, indicated that both
compounds are time-dependent inhibitors of cathepsin L. Separate experiments demonstrated
17

that analogues 1 and 32 were slowly reversible inhibitors of cathepsin L (see Supplementary
data).
2
.3 Molecular modeling studies
Figure 6. Molecular docking of analogues 1 and 32 within the active site of cathepsin L. (A)
Analogue 1 and (B) analogue 32 are shown in ball and stick mode (C, gray; H, white; O, red; N,
blue; S, yellow; F, light blue; Br, brown). Cathepsin L is shown in ribbon mode with a
transparent molecular surface (Green) and enzyme active site amino acid residues are labeled
and shown in stick mode (C, gray; H, white; O, red; N, blue; S, yellow).
Thiosemicarbazone analogues 1 and 32 which displayed the most pronounced activity
against cathepsin L were selected for molecular docking studies. The co-crystal structure of
67
cathepsin L with a nitrile inhibitor (PDB: 2XU1) within the active site of cathepsin L was
chosen as a starting model for the docking studies which were performed with Discovery Studio
4
.1 (Accelrys). A number of different binding modes with similar interaction energies were
obtained from docking analogues 1 and 32. In each case, several of the top binding orientations
placed the thiocarbonyl carbon atom of the inhibitor in close proximity to the Cys25 thiolate in a
4
7
45
similar manner to that observed with benzophenone thiosemicarbazones X and XI. One of the
top binding orientations for each of benzoylbenzophenone thiosemicarbazone analogues 1 and
3
2 which positions the Cys25 thiolate within 5 Å of the thiocarbonyl carbon atom is illustrated in
18

Figure 6. For analogue 1, the benzophenone fragment of the inhibitor resides deep within the S2
pocket of cathepsin L which is considered an important subsite for binding among the papain
6
7-68
family of cysteine proteases.
the S3 subsite. A hydrogen bond between the carbonyl oxygen of the inhibitor and the backbone
NH of His163 and a hydrogen bond between the terminal NH of the thiosemicarbazone and the
The remaining outermost ring of analogue 1 is oriented towards
2
backbone carbonyl of Asp162 is observed for analogue 1. Molecular docking studies for
analogue 32 orient the aryl rings of the inhibitor within the active site of cathepsin L in a similar
fashion to analogue 1 with the benzophenone fragment occupying the S2 pocket and the
opposing ring oriented toward the S3 subsite. For comparison, molecular docking studies were
carried out with analogue 14, which was inactive against cathepsin L. Molecular modeling
indicated that analogue 14 did not bind in a manner that would facilitate formation of a covalent
bond. The benzophenone portion resides in the S2 pocket of cathepsin L in a similar fashion
observed for active cathepsin L inhibitors 1 and 32. However, the remaining outermost ring
resides in the S1 subsite instead of being oriented towards the S3 subsite and the
thiosemicarbazone moiety is oriented towards the solvent instead of residing in the S1 subsite. In
each of the top binding orientations, the thiocarbonyl carbon atom of analogue 14 was not in
close proximity to the Cys25 thiolate ion of cathepsin L. Active cathepsin L inhibitors 1 and 32
bind in such a manner to facilitate formation of a transient covalent bond between the enzyme
and inhibitor. This comparison emphasizes that formation of a transient covalent bond is
necessary for activity against cathepsin L in this series of compounds (see Supplementary data).
2
.4 Invasion and migration studies
19

A hallmark of invasive cancer cells is their ability to invade through the ECM. Cell
6
9
invasion through Matrigel as a model for the ECM of the highly metastatic prostate cancer cell
4
8,70
71-72
line PC-3ML
and the highly invasive breast cancer cell line MDA-MB-231
was inhibited
by selected benzoylbenzophenone thiosemicarbazone inhibitors. Additionally, selected
thiosemicarbazone analogues were evaluated for their ability to inhibit cell migration. To
separate anti-tumorigenic from anti-metastatic effects, non-cytotoxic doses of evaluated
thiosemicarbazones were used. The results showed that treatment with thiosemicarbazone
analogue 8 significantly impaired the invasive capacities of prostate PC-3ML cancer cells by
5
9% and 92% at 1 µM and 5µM, respectively (Figure 7). The unsubstituted analogue 1 inhibited
cell invasion of PC-3ML prostate cancer cells by 37% and 53% at 10 µM and 25 µM,
respectively.
Figure 7. Effect of cathepsin L inhibitors on tumor cell invasion. PC-3ML prostate cancer cell
invasion in the presence of cathepsin inhibitors compound 1 (A) or compound 8 (B). Data are
mean and standard error values of 3-5 independent experiments.
Thiosemicarbazone 32 displayed the greatest effect on inhibition of invasion of MDA-
MB-231 breast cancer cells with 70 % inhibition at 10 µM, slightly better than 60% inhibition
20

7
3
found for the irreversible pan cysteine protease inhibitor E64 which was used as a positive
control for direct comparison of the effectiveness of the inhibitors (Figure 8). A significant
change in inhibition of cell invasion was not observed for analogue 32 at 25 µM compared to 10
µM. However, the p-fluoro analogue 8 demonstrated an increase from 15% to 60% inhibition of
invasion of MDA-MB-231 cells at 10 µM compared to 25 µM. The unsubstituted analogue 1,
which inhibited cell invasion by 30% at 25 µM, did not impact invasion of MDA-MB-231 cells
as significantly as analogues 8 and 32.
Figure 8. Inhibition of invasion and migration of MDA-MB-231 breast cancer cells by
thiosemicarbazone analogues 1, 8, and 32. (a) Inhibition of MDA-MB-231 breast cancer cell
invasion through Matrigel at 10 µM concentration of inhibitor. (b) Inhibition of MDA-MB-231
breast cancer cell invasion through Matrigel at 25 µM concentration of inhibitor. (c) Inhibition of
MDA-MB-231 breast cancer cell migration at 10 µM concentration of inhibitor. (d) Inhibition of
MDA-MB-231 breast cancer cell migration at 25 µM concentration of inhibitor. All values are
normalized to 2% DMSO vehicle and are from a minimum of a triplicate of experiments (* p <
0
.05, ** p < 0.01, *** p < 0.001).
21

All three benzoylbenzophenone analogues when evaluated at 10 µM and 25 µM
concentrations, inhibited MDA-MB-231 cell migration; 3-benzoylbenzophenone
thiosemicarbazone 1 showed the greatest effect with 80% inhibition of migration at 25 µM
which represented a greater than 2-fold increase in inhibition compared with the positive control
E-64 (Figure 8). In addition, 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32
and 1,3-bis(4-fluorobenzoyl)benzene thiosemicarbazone 8 also inhibited migration greater than
5
0% at 25 µM.
2
.5 Growth Inhibition of Mammary Carcinoma
Figure 9. 3-Benzoylbenzophenone thiosemicarbazone (1) effects on the growth of C3H
mammary carcinomas implanted in the right rear foot of female CDF1 mice. Mice were given 5
daily intraperitoneal injections from the day of tumour implant (as indicated by the arrows).
Results show means + 1 S.E. from 5-8 mice/group. [Legend: ꢀ Control (10% Tween80)
ꢁ
Analogue 1 (20 mg/kg)]
22

To investigate the effect of 3-benzoylbenzophenone thiosemicarbazone (1) (20 mg/kg) on
tumor initiation, animals were observed on a daily basis and tumor volume determined, as
described in Figure 9, when tumors were measureable. From these data the endpoint of TGT500
3
(
time taken in days for tumors to reach 500 mm ) was determined. For control animals injected
with vehicle (10% Tween80) for 5 days from the time of tumor implant the mean TGT500 (+ 1
S.E.) was found to be 20.1 days (+ 0.6 days) and this was significantly (Student’s T-test;
significance level of p<0.05) increased to 22.1 days (+ 0.5 days) when mice were given 5 daily
treatments with 3-benzoylbenzophenone thiosemicarbazone (1). This initial in vivo study
demonstrated that analogue 1 was well tolerated in this mouse model and showed modest,
statistically relevant efficacy in tumor growth delay. Cathepsin L inhibitors, functioning as anti-
metastatic agents, are not anticipated to impart dramatic decreases in tumor growth when utilized
as single agents; however, it is anticipated that their full value will be realized in combination
1
7
therapy with standard chemotherapy and/or radiation.
2
.6 Cytotoxicity
Table 2. Evaluation of Cytotoxicity Against HUVECs
Compound
Doxorubicin Paclitaxel
0.0268 0.00148
1
8
31
32
Cytotoxicity GI50 (µM)
53.3
>126
13.3
>85.9
Benzoylbenzophenone thiosemicarbazone analogues 1, 8, 31, and 32 were evaluated for
cytotoxicity against normal endothelial cells (HUVECs). Compared to known
chemotherapeutics, doxorubicin and paclitaxel, none of the compounds displayed significant
cytotoxicity against HUVECs. Compounds 8 and 32 were the least toxic against HUVECs with
23

GI50 values greater than 126 µM and 85.9 µM, respectively. Low cytotoxicity to normal cells
HUVECs in this case) is a desirable feature since the development of cathepsin L inhibitors as
anti-metastatic agents would likely involve chronic dosing to achieve efficacy.
(
3
. Conclusions
Several benzoylbenzophenone thiosemicarbazone analogues were synthesized and
evaluated for their inhibitory activity against cathepsins L and B. Thiosemicarbazone inhibitors
1
, 8 and 32 displayed the highest activity against cathepsin L with low IC50 values of 9.85 nM,
1
4.4 nM and 8.12 nM, respectively. The benzoylbenzophenone thiosemicarbazone analogues
represent a class of potent inhibitors that can tolerate structural changes and maintain activity
against cathepsin L. The high versatility of the core molecular scaffold offers potential for
optimization of the benzoylbenzophenone thiosemicarbazone analogues as inhibitors of
cathepsin L. Both thiosemicarbazone analogues 1 and 32 demonstrated time-dependent
inhibition of cathepsin L. Molecular docking studies of analogues 1 and 32 revealed that the
benzophenone fragment of the benzoylbenzophenone thiosemicarbazone inhibitors is capable of
filling the lipophilic S2 pocket of cathepsin L thus placing the thiosemicarbazone moiety in close
proximity to the Cys25 thiolate. In contrast, molecular modeling of analogue 14 (inactive against
cathepsin L) indicated that it did not bind to the enzyme in a conformation that would promote
formation of a covalent bond. Kinetic studies combined with molecular docking studies indicate
that the formation of a covalent bond between the enzyme and the most potent of this subset of
inhibitors represents an important contribution to their efficacy. The most potent inhibitor of
cathepsin L from this series, 1,3-bis(2-fluorobenzoyl)-5-bromobenzene thiosemicarbazone 32
significantly inhibited the ability of MDA-MB-231 cells to invade through Matrigel by 70%.
24

Additionally, the p-fluoro analogue 8 inhibited cell invasion of PC-3ML cells by 92% at 5 µM.
-Benzoylbenzophenone thiosemicarbazone (1) delayed growth in vivo of recently implanted
3
tumors in a C3H mouse mammary carcinoma model. Cathepsin L inhibitors 1, 8, 31, and 32
demonstrated very low cytotoxicity toward HUVECs. The high potency against cathepsin L and
ability to inhibit the invasion of tumor cells in vitro coupled with the desirable property of low
cytotoxicity to normal cells positions these compounds as viable pre-clinical candidates for
further development.
4
4
4
. Experimental Section
.1 Chemistry
.1.1 General Synthetic Protocols.
All reactions were carried out under an inert atmosphere of nitrogen unless otherwise noted.
Anhydrous solvents were used for carrying out reactions. Unless otherwise noted all reagents and
solvents were purchased from commercial suppliers and used without further purification.
Hexanes used for column chromatography were distilled from 4 Å molecular sieves prior to use.
Anhydrous tetrahydrofuran and dichloromethane were purchased from commercial suppliers or
dried using a VAC (Vacuum Atmospheres Co.) solvent purification system. Reactions were
monitored by SiliaPlate™ silica gel thin layer chromatography (TLC) plates (250 µm, F-254, 60
Å). Manual flash chromatography and automated flash chromatography was carried out with
silica gel purchased from either Silicycle Inc (230-400 mesh) or Biotage (40-65 microns). Proton
1
13
19
(
H), Carbon ( C), and Fluorine ( F) NMR spectra were recorded using a Varian Inova 500
25

MHz NMR system, Bruker Avance III HD 600 MHz NMR system, or a Bruker Avance III HD
00 MHz NMR system. Chemical shifts are reported in ppm (δ), coupling constants (J) are
reported in hertz (Hz), and peak patterns are reported as broad (br), singlet (s), doublet (d), triplet
t), quartet (q) and multiplet (m). High resolution mass spectra (HRMS) were obtained in the
4
(
Baylor University Mass Spectrometry Core Facility on a Thermo Scientific LTQ Orbitrap
Discovery using electrospray ionization (ESI). Purity of final compounds was analyzed using an
Agilent Technologies 1200 series HPLC system with a Diode Array and Multiple Wavelength
Detector SL, equipped with a Zorbax reliance cartridge guard-column; Agilent Eclipse XDB-
o
C18 column (4.6 mm ID X 250 mm, 5 µm particle size, 80 Å pore size); T = 25 C; flow rate 1.0
mL/min; injection volume 20 µL; monitored at 254 nm, 300 nm and 320 nm. Two HPLC
methods were used in the purity analysis of final compounds: Method A: water:acetonitrile,
gradient 50:50 to 10:90 from 0-25 min and isocratic 10:90 from 25-30 min Method B:
water:acetonitrile, gradient 70:30 to 10:90 from 0-25 min and isocratic 10:90 from 25-30 min.
5
1
4
.1.2 3-Benzoylbenzophenone thiosemicarbazone (1). p-Toluenesulfonic acid monohydrate
0.026 g, 0.136 mmol) was added to a solution of 1,3-dibenzoylbenzene (2.00 g, 6.98 mmol) in
anhydrous methanol (20 mL). After stirring at reflux for 10 min, thiosemicarbazide (0.476 g,
.235 mmol) was added to the flask. After 26 h, methanol was removed under reduced pressure
(
5
and water (50 mL) was then added. The products were extracted with ethyl acetate (2 x 50 mL)
and the combined organic phases were washed with brine, dried over anhydrous sodium sulfate,
and the solvent was removed under reduced pressure. Purification using flash chromatography
(
silica gel, hexanes:ethyl acetate, gradient 90:10 to 52:48) afforded 3-benzoylbenzophenone
1
thiosemicarbazone (0.829 g, 5.24 mmol, 44% yield) as a light yellow solid. H NMR (500 MHz,
26

CDCl ): δ 8.71 (1H, s), 8.00-7.99 (1H, m), 7.85-7.83 (1H, m), 7.77-7.67 (3H, m), 7.60-7.29
3
1
3
(
10H, m), 6.61-6.57 (1H, m). C NMR (125 MHz, CDCl ): δ 195.97, 195.29, 179.04, 178.99,
3
1
1
1
3
49.85, 149.70, 139.13, 137.95, 136.99, 136.94, 136.70, 136.01, 132.97, 132.81, 132.28, 131.84,
31.47, 131.45, 131.34, 130.68, 130.56, 130.48, 130.11, 130.09, 130.05, 128.86, 128.62, 128.58,
+
+
28.39, 128.34, 127.76. HRMS (ESI) calculated for C21
H
17
N
3
OSH (M+H) 360.11651, found
60.11667. HPLC retention time (Method A): 7.50 min. (Obtained as a mixture of E/Z isomers)
5
1
4
.1.3 1,3,5-Trisbenzoylbenzene thiosemicarbazone (2). 1,3,5–Tribenzoylbenzene (2.34 g,
6
.00 mmol) (purified by flash chromatography before using) and p-toluenesulfonic acid
monohydrate (11.4 mg, 0.06 mmol) were dissolved in anhydrous THF (20 mL) and heated to
reflux. Thiosemicarbazide (546 mg, 6.00 mmol) was then added to the flask and the reaction
mixture was stirred at reflux for 35 h. The reaction mixture was concentrated under reduced
pressure and the products were extracted from water (100 mL) with ethyl acetate (2 x 50 mL).
The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, and
the solvent was removed under reduced pressure. Purification using flash chromatography (silica
gel, hexanes: dichloromethane: ethyl acetate, gradient 80:10:10 to 40:30:30) afforded 1,3,5-
1
trisbenzoylbenzene thiosemicarbazone (0.961 g, 2.07 mmol, 35% yield) as a yellow solid. H
NMR (500 MHz, Acetone-d
6
): δ 9.40 (0.2H, s), 8.72 (0.8H, s), 8.33 (.2H, t, J = 1.5 Hz), 8.28
1.6H, d, J = 1.5 Hz), 8.21 (0.8H, s), 8.13 (0.2H, s), 8.07 (0.8H, t, J = 1.5 Hz), 8.05 (0.4H, d, J =
.5 Hz), 7.96-7.95 (0.8H, m), 7.85-7.84 (3.2H, m), 7.78 (0.8H, s), 7.73-7.52 (10.4 H, m), 7.43-
(
1
7
1
1
1
3
.37 (0.8H, m). C NMR (125 MHz, Acetone-d ): δ 194.47, 194.33, 180.08, 179.66, 147.62,
6
47.43, 139.25, 138.02, 137.94, 136.77, 136.74, 136.68, 133.68, 133.01, 132.99, 132.59, 131.83,
31.62, 131.37, 130.94, 130.47, 130.06, 130.05, 130.00, 129.99, 129.90, 128.67, 128.62, 128.53,
27

1
28.42, 127.85. X-Ray crystallographic data obtained for 1,3,5-trisbenzoylbenzene
thiosemicarbazone has been deposited in the Cambridge Crystallographic Data Centre and was
+
allocated the deposition number CDCC 1042567. HRMS (ESI) calculated for C28
H
21
O
2
3
N SH
+
(
(
[
M+H) 464.14272, found 464.14280. HPLC retention time (Method A): 10.53, 10.81 min.
Obtained as a mixture of E/Z isomers)
1
Note: The use of 0.8H and 0.2H for H NMR integral values relate to isomer ratios. This system
is use throughout.]
4
.1.4 3-benzoylbenzyhdrol (3).51,74 1,3-Dibenzoylbenzene (2.00 g, 6.98 mmol) was dissolved in
o
anhydrous ethanol (20 mL). The reaction mixture was cooled to 0 C followed by the addition of
sodium borohydride (0.090g, 2.094 mmol). The reaction mixture was stirred for 4 h and
quenched by the addition of a small amount of water. The reaction mixture was concentrated
under reduced pressure and the products were extracted from water with ethyl acetate (2 x 50
mL). The combined organic phases were washed with brine, dried over anhydrous sodium
sulfate, and the solvent was removed under reduced pressure. Purification using flash
chromatography (silica gel, hexanes: ethyl acetate, gradient 93:7 to 30:70) afforded 3-
1
benzoylbenzhydrol (0.601 g, 2.084 mmol, 30% yield). H NMR (500 MHz, Acetone-d
6
): δ 7.90
(
1H, t, J = 1.7 Hz), 7.77-7.74 (2H, m), 7.72-7.69 (1H, m), 7.67-7.62 (2H, m), 7.55-5.53 (2H, m),
7
.51-7.48 (1H, m), 7.46-7.44 (2H, m), 7.34-7.30 (2H, m), 7.25-7.21 (1H, m), 5.95 (1H, s), 5.04
1
3
(
1H, brs). C NMR (125 MHz, Acetone-d
6
): δ 196.47, 146.94, 146.03, 138.59, 139.40, 133.24,
1
31.36, 130.58, 129.24, 129.23, 129.11, 128.54, 127.96, 127.36, 75.71. HRMS (ESI) calculated
+
+
for C20
H
16
O H (M+H) 289.12231, found 289.12266.
2
28

51
4
.1.5 3-Benzoylbenzhydrol thiosemicarbazone (4). p-Toluenesulfonic acid monohydrate
(
0.007 g, 0.03 mmol) was added to a solution of 3-benzoylbenzyhdrol (0.396 g, 1.09 mmol) in
anhydrous tetrahydrofuran (10 mL). After stirring at reflux for 10 min, thiosemicarbazide (0.199
g, 2.19 mmol) was added to the reaction mixture. After 2 days, tetrahydrofuran was removed
under reduced pressure and water (50 mL) was added. The products were extracted with ethyl
acetate (2 x 20 mL) and the combined organic phases were washed with brine, dried over
anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Purification
using flash chromatography (silica gel, hexanes:ethyl acetate, gradient 88:12 to 0:100) afforded
1
3
-benzoylbenzhydrol thiosemicarbazone (0.228 g, 0.631 mmol, 57% yield) as a white solid. H
NMR (500 MHz, Acetone-d
6
): δ 8.51 (0.5H, brs), 8.50 (0.5H, brs), 8.08 (0.5H, brs), 8.00 (0.5H,
brs), 7.82 (0.5H, t, J = 1.7 Hz), 7.74-7.61 (4.5H m), 7.49-7.19 (10H, m), 5.97 (0.5H, d, J = 3.9
1
3
Hz), 5.82 (0.5H, d, J = 3.9 Hz), 5.10 (0.5H, d, J = 3.9 Hz), 4.87 (0.5H, d, J = 3.9 Hz). C NMR
125 MHz, Acetone-d ): δ 179.44, 179.41, 149.48, 149.42, 147.57, 145.86, 145.21, 144.96,
36.76, 136.61, 131.66, 131.35, 130.05, 129.81, 129.72, 129.70, 128.46, 128.29, 128.28, 128.19,
28.07, 127.96, 127.61, 127.07, 126.89, 126.55, 126.39, 126.35, 126.24, 125.55, 74.99, 74.48.
(
6
1
1
+
+
HRMS (ESI) calculated for C21
H
19
N OSNa (M+Na) 384.11410, found 384.11447. HPLC
3
retention time (Method B): 11.73, 12.32. (Obtained as a mixture of E/Z isomers)
4
.1.6 1,3-Bis(4-fluorobenzoyl)benzene (5).51,75 Aluminum trichloride (6.53 g, 49.5 mmol) was
added to a solution of isophthaloyl dichloride (5.00 g, 24.8 mmol) in dichloromethane (100 mL).
After heating at reflux for 30 min, fluorobenzene (3.41 g, 37.3 mmol) was added. After stirring
for 12 h, the reaction mixture was poured onto crushed ice, neutralized with 10% NaOH (100
mL), and extracted with dichloromethane (3 × 100 mL). The combined organic layers were
29