Discovery of trypanocidal thiosemicarbazone inhibitors of rhodesain and TbcatB

Article abstract of DOI:10.1016/j.bmcl.2008.03.083

Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. The cysteine proteases of T. brucei have been shown to be crucial for parasite replication and represent an attractive point for therapeutic intervention. Herein we describe the synthesis of a series of thiosemicarbazones and their activity against the trypanosomal cathepsins TbcatB and rhodesain, as well as human cathepsins L and B. The activity of these compounds was determined against cultured T. brucei, and specificity was assessed with a panel of four mammalian cell lines.

Full text of DOI:10.1016/j.bmcl.2008.03.083

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2883–2885
Discovery of trypanocidal thiosemicarbazone inhibitors
of rhodesain and TbcatB
Jeremy P. Mallari,a,b Anang Shelat,b Aaron Kosinski,b Conor R. Caffrey,c
Michele Connelly,b Fangyi Zhu,b James H. McKerrowc and R. Kiplin Guya,b,
*
aGraduate Program in Chemistry and Chemical Biology, University of California, San Francisco, CA 94143-2280, USA
bDepartment of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
cSandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, CA 94143-2280, USA
Received 14 December 2007; revised 26 March 2008; accepted 31 March 2008
Available online 8 April 2008
Abstract—Human African trypanosomiasis (HAT) is caused by the protozoan parasite Trypanosoma brucei. The cysteine proteases
of T. brucei have been shown to be crucial for parasite replication and represent an attractive point for therapeutic intervention.
Herein we describe the synthesis of a series of thiosemicarbazones and their activity against the trypanosomal cathepsins TbcatB
and rhodesain, as well as human cathepsins L and B. The activity of these compounds was determined against cultured
T. brucei , and specificity was assessed with a panel of four mammalian cell lines.
Ó 2008 Elsevier Ltd. All rights reserved.
The protozoan parasite Trypanosoma brucei causes Hu-
man African trypanosomiasis (HAT), a major health
concern in sub-Saharan Africa with an estimated
50,000 cases and 60 million at risk of infection.1 The tox-
icity and impractical dosing regimens of the currently
available drugs require the development of new thera-
pies. The urgency of the situation is further underscored
by emerging clinical resistance to Melarsoprol, the
front-line therapy for late stage parasitemia.2–4
may be involved in nutrient aquisition, degradation of
host proteins, evasion of the host immune response, or
crossing of the blood brain barrier.8,11 Because T. brucei
expresses only two proteases of the papain-like protease
family, the biology of these two enzymes should be ame-
nable to study by specific small molecule inhibitors.
Our prior work has shown that thiosemicarbazones
have potent activity against rhodesain.12,13 However,
the relationships between these compounds’ in vitro
activity against rhodesain and TbcatB and their in vivo
activity in cultured T. brucei have not been assessed.
Here we report the synthesis of a third generation thio-
semicarbazone series and its activity against cultured
T. brucei proliferation and the parasite’s two cathepsins.
In addition, activity against human cathepsins B and L
was determined, and cytotoxicity evaluated in a panel of
four mammalian cell lines to determine a cellular thera-
peutic index.
One potential strategy for discovering new chemothera-
pies is to target T. brucei ’s cysteine proteases. Irrevers-
ible peptidyl cysteine protease inhibitors are potent
trypanocides5,6 and can arrest T. brucei infections in a
mouse model.7 The parasite’s major papain-like prote-
ase,8 known interchangeably as brucipain, trypanopain,
and rhodesain, was presumed until recently to be the
target of these inhibitors. However, recent studies indi-
cate that a second cysteine protease, TbcatB, may also
be a target for these inhibitors.9,10
Thiosemicarbazones were synthesized by the general
route (Fig. 1) previously described.13 Briefly, acid chlo-
ride 1 was reacted with the appropriate boronic acid
to yield the ketone intermediate 2. The crude reaction
mixture was filtered and concentrated in vacuo, and
the resulting solid was partially purified by silica chro-
matography. Acid catalyzed reaction with thiosemicar-
bazide afforded the target thiosemicarbazones 3a–m.
The biological functions of TbcatB and rhodesain are
poorly understood. It has been suggested that they
Keywords: Sleeping sickness; HAT; African trypanosomiasis; Cathep-
sins; TbCatB; Rhodesain; Thiosemicarbazone; Protease inhibitors.
*
Corresponding author. Tel.: +1 901 495 5714; fax: +1 901 495
5715; e-mail: kip.guy@stjude.org
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.083

2884
J. P. Mallari et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2883–2885
Figure 1. Reagents and conditions: (i) PdCl2(PPh3)2, K3PO4, toluene, 70 °C, 2–5 h; (ii) thiosemicarbazide, HOAc, H2O/EtOH, 80 °C, 72–96 h.
Purification was achieved by silica chromatography, and
the overall yield was 15–40%. Purity of target com-
pounds was confirmed by LCMS using both C4 and
C18 columns.
suggests that at least some of the compounds in this ser-
ies exert their effects at unknown targets. This is not
unexpected, as the thiosemicarbazone scaffold has re-
ported activity against a wide range of cell types and
molecular targets.12,14–18
Each inhibitor was tested for activity against the trypan-
osomal cathepsins TbcatB and rhodesain as well as
against T. brucei proliferation. In order to assess poten-
tial therapeutic utility, activity against human cathepsins
B and L was determined, and general human cytotoxic-
ity was evaluated in cultures of Raji (a lymphoblastoid
cell line derived from a Burkitt’s lymphoma), HEK
293 (a human embryonic kidney cell line), BJ (a human
fibroblast line), and HEP G2 (a human liver cell line de-
rived from a hepatoblastoma). Membrane permeability
was assessed in a parallel artificial membrane permeabil-
ity assay (PAMPA).
Significant specificity for the parasitic proteases was not
achieved in this compound series relative to the human
cathepsins. However, a high degree of specificity was ob-
served for the two cathepsin L-like enzymes (cathepsin L
and rhodesain) relative to the two cathepsin B-like en-
zymes (cathepsin B and TbcatB). These results are sim-
ilar to previous TbcatB studies with purine nitrile
inhibitors, in which TbcatB was generally less sensitive
to inhibition than cathepsin L.10 A number of inhibitors
displayed absolute specificity for rhodesain over TbcatB.
These compounds are potent inhibitors of rhodesain and
show little or no toxicity against the parasite. They are
also membrane permeable, making these compounds
attractive tools for studying rhodesain function in
T. brucei.
Previous research demonstrated that aryl substituents
are tolerated at the R and R1 positions by rhodesain.13
We further explored this observation and found that a
variety of aryl moieties were well tolerated at these posi-
tions (Table 1). Nearly all compounds displayed submi-
cromolar potency against rhodesain. Although several
compounds displayed submicromolar potency against
TbcatB, the protease was less sensitive to inhibition by
this compound series. Unlike rhodesain, TbcatB did
not tolerate phenylethyl substituted compounds.
General cytotoxicity of each inhibitor was evaluated by
EC50 determination in cultures of BJ, Raji, HEK 293,
and HEP G2. Of the four cell lines, Raji was the most
sensitive. A cellular therapeutic index for each cell line
was determined and defined as: (EC50 Raji)/(EC50
T. brucei ). Compounds in this series generally killed
the parasites with significant selectivity relative to mam-
malian cells, with several exhibiting index values up-
wards of 20-fold in various cell lines (Supporting
Information). These data indicate that the trypanocidal
effects of these inhibitors are not due to general cytotox-
icity, and it is notable that 3f displayed an index value of
over 20-fold in all four cell lines.
We hypothesized that thiosemicarbazones might act
through TbcatB, or through both rhodesain and
TbcatB, to kill the parasite. Regression analysis con-
ducted on the compound series detected only a weak po-
sitive association between rhodesain inhibition and
trypanocidal activity (R2 = 0.3). For TbcatB, no statisti-
cally significant relationship between inhibition and try-
panocidal
activity
was
observed.
Membrane
We have reported the synthesis and evaluation of a
series of thiosemicarbazones against two human and
two trypanosomal cathepsins. Each compound was as-
sayed for activity against T. brucei and for cytotoxic-
ity in a panel of four mammalian cell lines. This
inhibitor series was determined to have low overall
cytotoxicity, with several compounds killing the para-
site selectively relative to the mammalian cell lines
tested. All inhibitors from this series exhibited potent
activity against rhodesain, and a subset of these com-
pounds was active against TbcatB. Several compounds
were potent trypanocides. However, no strong correla-
tion was observed between trypanocidal activity and
potency against either protease. Although the mecha-
nism of action of these compounds remains unclear,
the low cytotoxicity and good membrane permeability
of this series suggests that thiosemicarbazones warrant
further examination as leads for the therapy of Hu-
man African trypanosomiasis.
permeability of the compound series was tested by
PAMPA, and it was found that the inhibitors generally
exhibited similar permeabilities (Supporting Informa-
tion). This suggests that differences in intracellular accu-
mulation are unlikely to explain the lack of correlation
between protease inhibition and trypanocidal activity.
It is clear that activity against the parasite cannot be ex-
plained by either rhodesain or TbcatB inhibition alone,
or simply by their acting in synergy. Although it is diffi-
cult to interpret the mechanism of action of these inhib-
itors, it is interesting to note that all reasonably active
compounds against TbcatB were also active against
T. brucei (3a–c). In contrast, at least one compound
highly active against rhodesain and inactive against
TbcatB was completely inactive against the parasite
(3h). The lack of correlation observed between trypano-
cidal activity and activity against either protease target

J. P. Mallari et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2883–2885
Table 1. Activity of thiosemicarbazones
2885
Compound
R
R1
IC50 versus
rhodesain
(lM)
IC50 versus
TbcatB (lM)
EC50 versus IC50 versus cat
T. brucei
IC50 versus cat Cellular
B (lM)
L. (lM)
therapeutic
index
(lM)
3a
3b
3c
3d
3e
3f
3,5-(CF3)2Ph-2-thiophene
0.007
0.008
2-Thiophene 0.011
0.001
0.15
0.21
0.45
6
0.01
7
1.5
7
1
0.0101
0.0009
0.0006
0.0003
0.0007
0.004
0.0006
0.001
0.003
0.004
0.08
0.30
0.21
0.30
1.8
0.05 >4
0.01 >17
4-Me Ph–
3,5-Cl2 Ph–
3,5-Cl2 Ph–
3-CF3 Ph–
0.001
0.001
0.003
0.004
0.008
0.03
0.06
1
0.2 0.0094
0.0036
2
0.02
0.2
0.1
0.9
0.4
0.5
0.6
1
>4
>9
4-Me Ph–
0.046
0.029
0.044
1.1
2.5
1.3
2
0.2 0.0110
0.3 0.081
0.8 0.0074
Ph–CH@CH– 3-CF3 Ph–
3,5-(CF3)2 Ph– 4-MePh–
7
15
1
1.7
3.3
>10
>19
>10
na
4
3g
3h
3i
3,5-(CF3)2 Ph– Ph–(CH2)2– 0.053
0.006 >25
0.009 >25
0.008 >25
1
0.017
0.044
3.0
4.5
4–Me Ph–
3,5-Cl2 Ph–
4–Me Ph–
Ph–(CH2)2–
Ph–(CH2)2–
Ph–(CH2)2–
3-F Ph–
Ph–(CH2)2– 0.057
0.089
>10
1.3
>10
>10
3.7
6
0.1 0.039
1.02
0.91
4.9
>19
na
3j
Ph–
Ph–
0.50
1.5
0.06
0.2
>25
>25
13
3k
3l
0.06
0.001
0.007
>25
6
na
3-Cl Ph–
3-F Ph–
0.042
0.39
0.006 >25
0.03 >25
0.7 0.021
1
1
4
>6.7
>4
3m
0.118
22
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Acknowledgments
8. Caffrey, C. R.; Hansell, E.; Lucas, K. D.; Brinen, L. S.;
Alvarez Hernandez, A.; Cheng, J.; Gwaltney, S. L., 2nd;
Roush, W. R.; Stierhof, Y. D.; Bogyo, M.; Steverding, D.;
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9. Mackey, Z. B.; O’Brien, T. C.; Greenbaum, D. C.;
Blank, R. B.; McKerrow, J. H. J. Biol. Chem. 2004,
279, 48426.
This work was supported by the American Lebanese
Syrian Associated Charities (ALSAC) and St. Jude Chil-
dren’s Research Hospital, NIH Grant AI35707, and the
Sandler Family Supporting Foundation.
Supplementary data
10. Mallari, J. P.; Shelat, A.; Obrien, T.; Caffrey, C.; Kosinski,
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Spectroscopic data, LCMS data, PAMPA data, and
complete cytotoxicity data for all listed compounds.
Details for regression analysis and cell based assays.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.03.083.
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