Synthesis and biochemical evaluation of thiochromanone thiosemicarbazone analogues as inhibitors of cathepsin L

Article abstract of DOI:10.1021/ml200299g

A series of 36 thiosemicarbazone analogues containing the thiochromanone molecular scaffold functionalized primarily at the C-6 position were prepared by chemical synthesis and evaluated as inhibitors of cathepsins L and B. The most promising inhibitors f

Full text of DOI:10.1021/ml200299g

Letter
pubs.acs.org/acsmedchemlett
Synthesis and Biochemical Evaluation of Thiochromanone
Thiosemicarbazone Analogues as Inhibitors of Cathepsin L
Jiangli Song,† Lindsay M. Jones,† G. D. Kishore Kumar,† Elizabeth S. Conner,† Liela Bayeh,†
Gustavo E. Chavarria,† Amanda K. Charlton-Sevcik,† Shen-En Chen,† David J. Chaplin,‡
Mary Lynn Trawick,† and Kevin G. Pinney*,†
†Department of Chemistry and Biochemistry, Baylor University, One Bear Place #97348, Waco, Texas, 76798-7348 United States
‡OXiGENE Inc., 701 Gateway Boulevard, Suite 210, South San Francisco, California, 94080, United States
S
* Supporting Information
ABSTRACT: A series of 36 thiosemicarbazone analogues containing the thiochromanone
molecular scaffold functionalized primarily at the C-6 position were prepared by chemical
synthesis and evaluated as inhibitors of cathepsins L and B. The most promising inhibitors
from this group are selective for cathepsin L and demonstrate IC50 values in the low nanomolar
range. In nearly all cases, the thiochromanone sulfide analogues show superior inhibition of
cathepsin L as compared to their corresponding thiochromanone sulfone derivatives. Without
exception, the compounds evaluated were inactive (IC50 > 10000 nM) against cathepsin B. The
most potent inhibitor (IC50 = 46 nM) of cathepsin L proved to be the 6,7-difluoro analogue 4. This small library of compounds
significantly expands the structure−activity relationship known for small molecule, nonpeptidic inhibitors of cathepsin L.
KEYWORDS: thiochromanone, thiosemicarbazone, cathepsin L, cathepsin B, inhibitor
here are five classes of proteases including matrix
metalloproteases (MMPs), cysteine proteases, serine
T
proteases, aspartic proteases, and threonine proteases, which
catalyze the hydrolysis of peptide bonds.1,2 Because of their
function in many disease states, including cancer metastasis and
cardiovascular disease, proteases have become well-investigated
therapeutic targets.3−6 Cysteine protease cathepsins, members
of the papain family, have recently been validated as an
important enzymatic class to target in cancer research.1,2,5 In
this family, there are 11 cathepsin enzymes known to date in
humans: B, C, F, H, K, L, O, S, V, W, and X.5,7−9 Odanacatib,
an inhibitor of cathepsin K developed by Merck, is currently in
phase III clinical trials for the treatment of osteoporosis.10
Cathepsins are found in the highest concentration in cellular
lysosomes, and during cancer progression, they are secreted at
an increased rate and degrade the extracellular matrix and
basement membrane, which aid in cancer metastasis.5 Small
molecule inhibitors of cathepsin L have been previously
identified (Figure 1), including azapenone (I),11 cyanamide
derivative (II),12 and a purine nitrile analogue (III),13 as well as
amino acid-based molecules including an oxocarbazate
analogue (IV)14 and an epoxide derivative (V).15
Figure 1. Representative inhibitors of cathepsin L.
cathepsin L. Several compounds, based primarily on the
benzophenone or thiochromanone thiosemicarbazone molec-
ular structure, were identified as lead compound inhibitors of
cathepsin L.20,22−24 These original findings provided the
foundational support for the current studies involving
functionalized thiochromanone thiosemicarbazone inhibitors
of cathepsin L. To the best of our knowledge, we are the first to
Although structurally similar, the active sites of the individual
cathepsins have small, but significant, differences in size and
shape.16 Taking advantage of these differences facilitates the
design of selective inhibitors.17,18 Previous research from our
group has focused on inhibitors of the cysteine protease,
cruzain, which has implications in the treatment of Chagas'
disease.19−21 As a preliminary assay to evaluate selectivity for
cruzain inhibition, a series of compounds was also screened
against the structurally similar human cysteine protease
Received: December 16, 2011
Accepted: April 18, 2012
Published: April 18, 2012
© 2012 American Chemical Society
450
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ACS Medicinal Chemistry Letters
Letter
report20,24 thiochromanone thiosemicarbazones and their
corresponding sulfone analogues as molecular scaffolds that
inhibit cathepsin L. A sampling of previously reported
inhibitors (Figure 2) containing the thiosemicarbazone moiety
includes VI,22 VII,23 VIII,22 and IX.25
methoxythiochromanone. The reduction of 6-nitrothiochroma-
none introduced the amine functionality in analogue 18, while
subsequent acylation provided the amide analogues 19 and 36.
While geometrical isomers (E and Z) are possible for
nonsymmetrical thiosemicarbazone analogues, it is interesting
to note that with the exception of two compounds (18 and 34),
evidence for such isomeric mixtures was not observable by
NMR or HPLC for the compounds in this study. Furthermore,
nuclear Overhauser effect spectroscopy (NOESY) studies
(carried out for compounds 4, 13, 16, 17, 27−29, 31, and
32) suggested the presence of only the E-isomer in each of
these compounds. In addition, a single-crystal X-ray diffraction
study of compound 27 indicated the E-isomer. Details
regarding the NOESY and X-ray studies are located in the
Supporting Information.
All of the thiochromanone thiosemicarbazone analogues
were evaluated for their ability to inhibit cathepsins L and B in
separate assays. In general, the sulfide series of analogues was
more active, in terms of cathepsin L inhibition, than their
corresponding sulfone derivatives with individual IC50 values
either much more active or equivalent. The most active
inhibitors of cathepsin L in this study were the 6,7-difluoro
analogue 4 and the 6-nitro analogue 17 (IC50 values of 46 and
68 nM, respectively). The lower IC50 values of compounds 5−
7, 15, and 25−27 suggested that substitution at the C-8
position is not productive. Incorporation of a bromine atom at
the C-6 position (compound 3) resulted in improved inhibition
of cathepsin L as compared to fluorine and chlorine atom
replacement (compounds 1 and 2). An increase in aliphatic
substituent size at the C-6 position (compare compounds 8/13,
9, 10) reflected a dramatic decrease in inhibition of cathepsin L.
Compounds 3, 4, 17, and 35 were strong inhibitors of
cathepsin L. It is interesting to note that incorporation of a
slightly electron-donating methyl group at C-6 (compound 8)
and a strongly electron-withdrawing trifluoromethyl group at
C-6 (compound 13) in each case led to excellent inhibitors of
cathepsin L. Incorporation of hydroxyl, amino, methoxy,
thiomethyl, and acetamide functionalities (compounds 16, 18,
11, 12, and 19) at C-6 all led to loss of cathepsin L inhibition.
The 6-bromo and 6-nitro substituted sulfones (compounds 23
and 35) demonstrated significant inhibition of cathepsin L
although less active than their sulfide counterparts (compounds
3 and 17). The C-6 trifluoromethyl sulfone (32) was
comparable in activity to its corresponding sulfide (13). All
of the compounds evaluated in this study were inactive against
cathepsin B (IC50 > 10000 nM). This reflects a selectivity ratio
(cathepsin L/cathepsin B) of <0.0046 for the most active
cathepsin L inhibitor (4). The inhibition of cathepsin L assay is
very sensitive to experimental parameters such as preincubation
time and substrate concentration.18 Therefore, it is difficult to
compare IC50 values, in an absolute sense, obtained for small
molecule inhibitors of cathepsin L between separate
laboratories under varying experimental conditions. With this
perspective in mind, it is encouraging to note that the most
potent thiochromanone thiosemicarbazone inhibitors of
cathepsin L described in this study (4 and 17) demonstrated
activity comparable to an exemplary benzophenone thiosemi-
carbarzone inhibitor (VII, Figure 2) assayed under comparable
conditions.
Figure 2. Representative thiosemicarbazone inhibitors of cathepsin L
(IC50 values for inhibition of cathepsin L, preincubation time, and
substrate concentration are indicated).
To incorporate structural diversity within the aryl portion of
the target compounds, as shown in Scheme 1, 3-bromopro-
Scheme 1. General Synthetic Route toward
Thiochromanone Thiosemicarbazone Analogues
pionic acid was reacted with various commercially available
thiophenols appropriately functionalized on the aromatic ring.
The resulting carboxylic acid intermediates underwent a ring-
closing reaction upon treatment with polyphosphoric acid
(PPA) to produce the functionalized thiochromanone ana-
logues. Condensation of the resultant ketone moiety with
thiosemicarbazide afforded the thiosemicarbazone derivatives in
good yields. The corresponding 1,1-dioxo analogues were
prepared in an analogous fashion by oxidation to obtain the
sulfone derivatives, prior to the condensation reaction.
Additional synthetic steps were necessary for the preparation
of analogues 3, 16, 18, 19, 23, 34, and 36 (Table 1 and
Supporting Information). Bromination of unsubstituted,
commercially available thiochromanone afforded 6-bromothio-
chromanone,19 which served as a starting material for the
synthesis of analogues 3 and 23. The preparation of the
hydroxy analogues 16 and 34 involved demethylation of 6-
In summary, from a focused small library of 36 analogues
with a diverse functionality, nine were found to be potent
inhibitors of cathepsin L (IC50 < 300 nM). The two most active
analogues were the 6,7-difluorothiochromanone thiosemicarba-
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ACS Medicinal Chemistry Letters
Letter
Table 1. Inhibitory Activity of Thiosemicarbazone Derivatives against Cathepsins L and B
a
a
IC50 (nM)
IC50 (nM)
Cat L
compd
R
X
Cat L
Cat B
compd
R
X
Cat B
1
2
6-F
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
741
228
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
6-NHAc
S
S
>10000
>10000
>10000
>10000
574
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
6-Cl
6-Br
fused 5,6-phenyl
6-F
3
152
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
SO2
4
6,7-F
46
6-Cl
5
6,8-F
1500
6-Br
6
6-F,8-Br
6-Br,8-F
6-CH3
6-CH2CH3
6-i-Pr
>10000
434
6,7-F
3650
7
6,8-F
>10000
>10000
1117
8
214
6-F,8-Br
6-Br,8-F
6-CH3
9
2,720
>10000
>10000
>10000
284
10
11
12
13
14
15
16
17
18
>10000
6521
6-OCH3
6-SCH3
6-CF3
6-CH2CH3
6-i-Pr
>10000
>10000
260
6-OCH3
6-CF3
6-OCF3
8-OCF3
6-OH
256
>10000
>10000
68
6-OCF3
6-OH
3960
>10000
112
6-NO2
6-NH2
6-NO2
6-NHAc
>10000
>10000
a
Each assay utilized 2% DMSO with a 5 min preincubation and 50 μM substrate (cat. L) or 60 μM substrate (cat. B).
zone 4 and the 6-nitro derivative 17 (IC50 = 46 and 68 nM,
respectively).
Baylor University) for mass spectroscopic analysis, Dr. Craig
Moehnke for assistance with NMR studies, and Dr. James
Karban and Dr. Michelle Nemec (Director) for use of the
shared Molecular Biosciences Center at Baylor University.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis and characterization, molecular modeling and X-ray
crystallography, and details regarding biological assays. This
material is available free of charge via the Internet at http://
pubs.acs.org.
REFERENCES
■
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AUTHOR INFORMATION
Corresponding Author
*Tel: 254-710-4117. Fax: 254-710-4272. E-mail: Kevin_
Pinney@baylor.edu.
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■
Funding
We express our appreciation to OXiGENE Inc. (grants to
K.G.P. and M.L.T.) for their financial support of this project
and to the National Science Foundation for funding both the
Varian 500 MHz NMR spectrometer (Grant No. CHE-
0420802) and the Bruker X8 APEX diffractometer (Grant
CHE-0321214).
Notes
The authors declare the following competing financial
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potential conflict of interest in regard to a paid consulting
relationship with Oxigene Inc.
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ACKNOWLEDGMENTS
■
We are grateful to Dr. Kevin K. Klausmeyer for his valuable
assistance with the X-ray crystallographic study. We also thank
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