Functionalized benzophenone, thiophene, pyridine, and fluorene thiosemicarbazone derivatives as inhibitors of cathepsin L

Article abstract of DOI:10.1016/j.bmcl.2010.09.026

A series of thiosemicarbazone analogs based on the benzophenone, thiophene, pyridine, and fluorene molecular frameworks has been prepared by chemical synthesis and evaluated as small-molecule inhibitors of the cysteine proteases cathepsin L and cathepsin B. The two most potent inhibitors of cathepsin L in this series (IC₅₀ <135 nM) are brominated-benzophenone thiosemicarbazone analogs that are further functionalized with a phenolic moiety (2 and 6). In addition, a bromo-benzophenone thiosemicarbazone acetyl derivative (3) is also strongly inhibitory against cathepsin L (IC₅₀ = 150.8 nM). Bromine substitution in the thiophene series results in compounds that demonstrate only moderate inhibition of cathepsin L. The two most active analogs in the benzophenone thiosemicarbazone series are highly selective for their inhibition of cathepsin L versus cathepsin B.

Full text of DOI:10.1016/j.bmcl.2010.09.026

Bioorganic & Medicinal Chemistry Letters 20 (2010) 6610–6615
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Functionalized benzophenone, thiophene, pyridine, and fluorene
thiosemicarbazone derivatives as inhibitors of cathepsin L
G. D. Kishore Kumar a, Gustavo E. Chavarria a, Amanda K. Charlton-Sevcik a, Grace Kim Yoo a, Jiangli Song a,
Tracy E. Strecker a, Bronwyn G. Siim b, David J. Chaplin b, Mary Lynn Trawick a, Kevin G. Pinney a,
⇑
a Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, TX 76798-7348, USA
b OXiGENE Inc., 701 Gateway Blvd, Suite 210, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2010
Revised 3 September 2010
Accepted 7 September 2010
Available online 15 September 2010
A series of thiosemicarbazone analogs based on the benzophenone, thiophene, pyridine, and fluorene
molecular frameworks has been prepared by chemical synthesis and evaluated as small-molecule inhib-
itors of the cysteine proteases cathepsin L and cathepsin B. The two most potent inhibitors of cathepsin L
in this series (IC50 <135 nM) are brominated-benzophenone thiosemicarbazone analogs that are further
functionalized with a phenolic moiety (2 and 6). In addition, a bromo-benzophenone thiosemicarbazone
acetyl derivative (3) is also strongly inhibitory against cathepsin L (IC50 = 150.8 nM). Bromine substitu-
tion in the thiophene series results in compounds that demonstrate only moderate inhibition of cathepsin
L. The two most active analogs in the benzophenone thiosemicarbazone series are highly selective for
their inhibition of cathepsin L versus cathepsin B.
Keywords:
Cathepsin L
Inhibitors
Cancer metastasis
Ó 2010 Elsevier Ltd. All rights reserved.
Cathepsins L and B belong to the papain family of lysosomal
cysteine proteases including cathepsins C, F, H, K, O, S, V, W, and
X.1 Recent studies have shown that these two specific cysteine
proteases participate in tumor progression, hyperproliferation,
apoptosis, angiogenesis, and metastasis by malignant cells.2 They
have a broad tissue distribution and are overexpressed in several
types of human cancer including breast, prostate, lung, gastrointes-
tinal, and epidermal.3 Therefore, cysteine proteases, such as
cathepsins L and B, are of special interest as targets for the devel-
opment of novel chemotherapeutic agents.4
The thiosemicarbazone moiety has been utilized as a key func-
tional group in a variety of anticancer agents.5 Earlier studies have
reported that benzophenone thiosemicarbazone analogs have po-
tential application in the treatment of Chagas’ disease, sleeping
sickness, and malaria.6
More recently, we have designed and prepared by chemical
synthesis a variety of benzophenone thiosemicarbazone analogs
(e.g., I and II {IC50 = 30.5 and 46.5 nM, respectively}) that are potent
inhibitors of cathepsin L (Fig. 1).7 In addition other small molecules
including further benzophenone analogs,8 thiophene (III),8
oxadiazole (IV),9 pyrazole (V),10 oxocarbazate (VI),11 aldehyde
(VII),12 cyanopyrrolidine (VIII),13 epoxide (IX),14 and sulfonamide
(X)15 have been identified as inhibitors of cathepsin L (Fig. 1).
Presumably, the thiosemicarbazone moiety mimics the hydro-
lytic cleavage of peptide bonds that takes place in the active site
of cathepsin L (Fig. 2).16 Inspired by our previous success with
benzophenone-based inhibitors,7 we have extended the known
SAR to include various functionalized thiophene, pyridine,
fluorene, and additional benzophenone analogs bearing the thio-
semicarbazone moiety.
Synthesis of certain benzophenone thiosemicarbazone
derivatives utilized 3-hydroxybenzaldehyde as a starting material
(Scheme 1). The appropriate hydroxy protected benzaldehyde
was reacted with 3-bromophenylmagnesiumbromide to afford a
secondary alcohol that was oxidized to its corresponding ketone.
After formation of the thiosemicarbazone moiety, the silyl group
was deprotected using TBAF to afford analogs 2 and 6. Interest-
ingly, attempts to form the thiosemicarbazone group through a
condensation reaction with 3,30-dibromo-20-tert-butyldimethylsi-
lyloxy benzophenone were not successful. The desired analog 10
was prepared by deprotection of the TBS group prior to formation
of the thiosemicarbazone moiety (not shown in Scheme 1).
An alternative synthetic route, utilizing the appropriate
Weinreb amide, was employed for the synthesis of benzophenone
thiosemicarbazone analog 8 (Scheme 2).
In the non-symmetrical thiosemicarbazone analogs, E/Z double
bond isomers were detectable by HPLC, however they were
inseparable by chromatography. These ratios are reported in Table
1 although it is not possible to assign an individual HPLC peak to
either the E or Z isomer. In addition, for certain thiosemicarbazone
⇑
Corresponding author. Tel.: +1 254 710 4117; fax: +1 254 710 4272.
E-mail address: Kevin_Pinney@baylor.edu (K.G. Pinney).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.09.026

G. D. Kishore Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6610–6615
6611
Figure 1. Representative small-molecule inhibitors of cathepsin L.
S
R1
N
R1
N
R2
N
H
NH2
R2
H N
S
S
H
H
HN
N
HN
N
NH2
S
His - 159
His - 159
Cys - 25
Cys - 25
Figure 2. Proposed mechanism delineating the inhibition of cathepsin L with thiosemicarbazone analogs.
derivatives that resulted from the 1,3-dibromobenzene Grignard
chemistry, a small impurity (H vs Br)7 proved inseparable (Table 1).
Each of the synthesized thiosemicarbazone analogs was evalu-
ated in terms of its ability to inhibit cathepsin L (Table 1). For
comparison, these compounds were also evaluated for their
inhibitory activity against the widely expressed enzyme cathepsin
B. Three of the five benzophenone analogs (2, 6, and 8) that incor-
porate the phenolic functionality were active inhibitors of cathep-
sin L (IC50 <250 nM). This is potentially significant in terms of drug
design and development since the phenolic moiety provides a
molecular handle on which to incorporate prodrug moieties to
improve bioavailability. Importantly, a comparison of the cathep-
sin L activity between benzophenone thiosemicarbazone analogs
1 and 2 confirms the importance of a meta-bromo substituent in
the ‘A-ring’ in many of these compounds.7a This similar trend is
seen in a comparison of brominated analog 17 (IC50 = 1000 nM)
bearing a ‘B-ring’ pyridine functionality versus the non-bromi-
nated corresponding compound 16 (IC50 >10,000 nM). The
bromo-benzophenone acetyl derivative (3) is also strongly inhibi-
tory against cathepsin (IC50 = 150.8 nM). The two fluorene
L
analogs 24 and 25 are inactive as inhibitors of cathepsin L
(IC50 >10,000 nM). In this preliminary grouping of compounds,
only benzophenone analogs 3 and 11 showed any detectable activ-
ity against cathepsin B (IC50 = 1600 nM and 1424 nM, respectively).
These results confirm the importance of a benzophenone scaffold
in thiosemicarbazone inhibitors of cathepsin L reported in our pre-
vious study.7a The best inhibitors in our previous study7a all con-
tained a meta-bromo substituent in the A-ring and that is the
case with the current results. A number of additional SAR consid-
erations have emerged. The most potent inhibitor in our current
study (compound 6, IC50 = 126.1 nM) features
a meta-bromo,
para-hydroxy substitution pattern in the B-ring, thus extending
the SAR with functional group diversity at these positions while
maintaining strong inhibition against cathepsin L. The extension
of this work to now include a meta-hydroxy substituent in the B-
ring (compound 2) in place of a corresponding fluorine atom

6612
G. D. Kishore Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6610–6615
O
H
HO
X'
a
O
H
TBSO
O
OH
MgBr
Br
d
b
X'
OTBS
OTBS
c
X
X
X
X'
X
X'
S
N
NH2
S
N
NH2
NH
S
N
NH2
NH
NH
f
g
e
OAc
OTBS
OH
X
X'
X
X'
X' OAc
X
X'
X' OH
compd #
X
compd #
X
X' OTBS
compd #
X
2
6
5
7
Br H meta (C-3')
Br Br para (C-4')
3
Br H meta (C-3')
Br Br para (C-4')
Br H meta (C-3')
Scheme 1. Synthesis of various benzophenone-based thiosemicarbazone derivatives. Reagents and conditions: (a) t-butyldimethylsilylchloride (TBSCl), imidazole, DMF, rt,
6–12 h; (b) Mg, Et2O, reflux, 1–2 h; (c) Et2O, rt, 6–12 h; (d) pyridiniumchlorochromate (PCC), Celite, CH2Cl2, rt, 3–8 h; (e) NH2NHCSNH2, para-toluenesulfonic acid, MeOH,
reflux, 6–12 h; (f) tetrabutylammonium fluoride (TBAF), THF, rt, 3–4 h; (g) Ac2O, pyridine, CH2Cl2, rt, 4 h.
O
HO
OH
Br
a, b
O
TBSO
OMe
S
N
NH2
NH
N
Me
O
MgBr
Br
Br
OTBS
OH
c
e, f
d
Br
Br
Br
Br
Br
Br
8
Analog
Scheme 2. Synthesis of benzophenone-based thiosemicarbazone analog 8. Reagents and conditions: (a) t-butyldimethylsilylchloride (TBSCl), imidazole, DMF, rt, 18–24 h;
(b) 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT), N-methylmorpholine (NMM), HClꢀNH(OMe)Me, THF, rt, 24 h; (c) Mg, Et2O, reflux, 1–2 h; (d) Et2O, rt, 3–12 h;
(e) NH2NHCSNH2, para-toluenesulfonic acid, MeOH, reflux, 6–12 h; (f) tetrabutylammonium fluoride (TBAF), THF, rt, 1–3 h.
(compound 5 from our previous study)7a actually improves the
The most active inhibitor of cathepsin L in this series was
evaluated for its cytotoxicity (Table 2). Consistent with the pro-
posed mechanism of action (with cathepsin L), this compound
inhibitory activity towards cathepsin
250 nM, respectively).
L (IC50 = 131.4 nM vs

G. D. Kishore Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6610–6615
6613
Table 1
Inhibition of human cathepsins L and B for various functionalized benzophenone, thiophene, pyridine, and fluorene thiosemicarbazones
IC50 (nM)b
Cat B
S
N
NH2
NH
Cat L
Isomer ratio estimated
by HPLCa
H replaced by Br
(% determined by HPLC)
R1
R2
R1
R2
OH
1c
1:0.4
1:0.1
1:1
NAd
0.1
1.0
3.0
0.8
>10,000
>10,000
>10,000
1600
Br
Br
Br
Br
Br
OH
2
3
131.4
OAc
OBn
OTBS
150.8
4f
5f
NDe
>10,000
>10,000
>10,000
1:0.1
>10,000
6f
1:0.6
1:0.9
1:0.2
1:0.7
NDe
0.1
0.3
0.4
0.8
0.2
NAd
126.1
9493
>10,000
>10,000
>10,000
>10,000
1424
OH
Br
Br
Br
Br
Br
Br
Br
Br
7f
>10,000
OTBS
OH
8g
232.4
OTBS
9g
>10,000
>10,000
10f
11c
HO
Br
MeO
MeO
OH
1:0.7
3600
OMe
OMe
S
12c
13f
NDe
NAd
0.4
>10,000
>10,000
>10,000
Br
Br
S
1:0.2
369.7
Br
S
14f
15f
1:0.1
NDe
0.1
1.8
814.5
931.3
>10,000
>10,000
Br
Br
S
Br
(continued on next page)

6614
G. D. Kishore Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6610–6615
Table 1 (continued)
IC50 (nM)b
S
N
NH2
NH
Cat L
Cat B
Isomer ratio estimated
by HPLCa
H replaced by Br
(% determined by HPLC)
R1
R2
R1
R2
16c
NDe
NDe
NDe
NDe
NAd
4.0
1.5
0.5
>10,000
1000
>10,000
>10,000
>10,000
>10,000
N
N
N
Br
Br
Br
17f
18g
19g
>10,000
>10,000
Br
Br
N
Br
Br
20h
21g
22c
23c
NDe
1:0.5
NDe
NDe
0.3
>10,000
9650
>10,000
>10,000
>10,000
>10,000
1.4
NAd
NAd
>10,000
5410
24c
25c
NDe
NDe
NAd
NAd
>10,000
>10,000
>10,000
>10,000
Br
Br
a
b
c
E/Z isomer not assigned.
2% DMSO.
Synthesis begins with commercially available ketone.
NA = not applicable.
ND = not detected
Synthesis begins with aldehyde derivatives (see Scheme 1).
Synthesis begins with carboxylic acid derivatives (see Scheme 2).
Synthesis begins with acid chloride (see Ref. 7a).
d
e
f
g
h
was not particularly cytotoxic against these selected human cancer
cell lines.
In summary, this study has identified three new bromo-benzo-
phenone thiosemicarbazone analogs 2, 6, and 8 each bearing a
phenolic moiety that are potent inhibitors of cathepsin L. Future
studies will center on additional synthetic manipulations along
with further biochemical and biological evaluation of these impor-
tant new lead compounds and related analogs.
Table 2
Cytotoxicity against selected human cancer cell lines for analog 2
Cytotoxicity GI50
NCI-H460
(
l
M)
S
N
NH2
NH
DU-145
Acknowledgments
The authors are grateful to Oxigene Inc. (grants to K.G.P. and
M.L.T.), The Welch Foundation (Grant AA-1278 to K.G.P.), and
the Department of Chemistry and Biochemistry at Baylor Univer-
sity for generous financial support of this research, and the
National Science Foundation for funding the Varian 500 MHz
Br
OH
2
23.3
21.6

G. D. Kishore Kumar et al. / Bioorg. Med. Chem. Lett. 20 (2010) 6610–6615
6615
K. G. American Society for Biochemistry and Molecular Biology (ASBMB),
Experimental Biology Meeting, San Francisco, USA, 2006 (poster presentation).;
(c) Siles, R.; Chen, S.-E.; Zhou, M.; Pinney, K. G.; Trawick, M. L. Bioorg. Med.
Chem. Lett. 2006, 16, 4405; (d) Mallari, J. P.; Guiguemde, W. A.; Guy, R. K. Bioorg.
Med. Chem. Lett. 2009, 19, 3546.
NMR spectrometer (Award CHE-0420802). The authors thank Ms.
Ashleigh Locke and Ms. Erica N. Parker for valuable assistance with
portions of the chemical synthesis, Dr. Alejandro Ramirez (Mass
Spectrometry Core Facility, Baylor University) for HRMS analysis,
and Dr. James Karban and Dr. Michelle Nemec (Director) for use
of the shared Molecular Biosciences Center. The authors are grate-
ful to Dr. Michael P. Hay (Auckland Cancer Society Research
Centre) for valuable consultation regarding certain aspects of this
project.
7. (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.;
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G. D. K.; Lindsay, J. M.; Song, J.; Matthew, M.; Elizabeth, C.; Akash, D.; Grace, Y.;
Conner, E.; Arispe, W. M.; Chavarria, G. E.; Charlton-Sevcik, A. K.; Strecker, T. E.;
Chen, S.-E.; Trawick, M. L.; Pinney, K. G. 41st National Organic Symposium,
Colorado, Boulder, USA, 2009 (poster presentation).
8. Mallari, J. P.; Shelat, A.; Konsinki, A.; Caffrey, C. R.; Connelly, M.; Zhu, F.;
McKerrow, J. H.; Guy, R. K. Bioorg. Med. Chem. Lett. 2008, 18, 2883.
9. Myers, M. C.; Shah, P. P.; Diamond, S. L.; Huryn, D. M.; Smith, A. B., III Bioorg.
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Supplementary data
10. 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.;
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Supplementary data (detailed experimental syntheses and de-
tails regarding biological assays) associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.2010.09.026.
11. Myers, M. C.; Shah, P. P.; Beavers, M. P.; Napper, A. D.; Diamond, S. L.; Smith, A.
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