Discovery of potent thiosemicarbazone inhibitors of rhodesain and cruzain

Article abstract of DOI:10.1016/j.bmcl.2004.10.023

Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas' disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum. Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine proteases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing Chagas' disease and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T. brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.

Full text of DOI:10.1016/j.bmcl.2004.10.023

Bioorganic & Medicinal Chemistry Letters 15 (2005) 121–123
Discovery of potent thiosemicarbazone inhibitors of rhodesain
and cruzain
a
Naoaki Fujii, Jeremy P. Mallari, Elizabeth J. Hansell, Z. Mackey, Patricia Doyle,
a
c
c
c
c
d
d
c
Y. M. Zhou, Jiri Gut, Philip J. Rosenthal, James H. McKerrow and R. Kiplin Guy
a,b,c,
*
a
Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143, USA
b
Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94143, USA
Sandler Center for Basic Research in Parasitic Diseases, University of California, San Francisco, CA 94143, USA
c
d
Medicine, University of California, San Francisco, CA 94143, USA
Received 10 September 2004; revised 7 October 2004; accepted 7 October 2004
Available online 28 October 2004
Abstract—Herein we report the synthesis and evaluation of a series of thiosemicarbazones as potential inhibitors of cysteine pro-
teases relevant to parasitic diseases. Derivatives of thiosemicarbazone 1 were discovered to be potent inhibitors of cruzain and
rhodesain, crucial proteases in the life cycles of Trypanosoma cruzi and T. brucei rhodesiense, the organisms causing ChagasÕ disease
and sleeping sickness. However, the entire series had only modest potency against falcipain-2, an essential protease for Plasmodium
falciparum, the organism causing malaria. Among the active inhibitors, several potently inhibited proliferation of cultures of T.
brucei. However, only modest activity was observed in inhibition of proliferation of T. cruzi or P. falciparum.
Ó 2004 Elsevier Ltd. All rights reserved.
Unicellular protozoal parasites of humans cause several
of the major neglected diseases of the world including
malaria, ChagasÕ disease, and sleeping sickness. For
the latter two diseases, all existing drugs have inade-
quate efficacy and/or unacceptable toxicity profiles.
While useful drugs do exist for malaria, worldwide
resistance has emerged for many of the available thera-
peutics. Therefore, there is an urgent need for new clin-
ical candidates for these diseases.
teine proteases, which appear to be required for hemo-
,5
globin hydrolysis and possibly other processes.
4
ChagasÕ disease, the leading cause of heart disease in
Latin America, is transmitted to humans by fecal to
6
wound contact during feeding of the Ôkissing beetleÕ
host, as an infectious trypomastigote form of the proto-
zoan parasite Trypanosoma cruzi. Cruzain (also referred
to as cruzipain) is the major cysteine protease of T. cruzi.
The protease is expressed in all life cycle stages of the
parasite. It may function in nutrition, remodeling of
the mammalian cell, or evasion of host defense mecha-
nisms. Addition of a cruzain inhibitor to cultures of
mammalian cells exposed to trypomastigotes or to mam-
malian cells already infected with T. cruzi amastigotes
blocks replication and differentiation of the parasite,
Malaria is one of the most profound human health
problems, with several hundred million cases world-
1
wide. Most morbidity can be attributed to Plasmodium
falciparum. Unfortunately, cheap, effective drugs such as
chloroquine and Fansidar now suffer from worldwide
2
resistance problems. This problem is made worse by
slow anti-malarial drug development. One reason for
7
thus arresting the parasite life cycle. Therefore, cruzain
is essential for replication of the intracellular parasite.
3
the paucity of new drugs is the lack of validated targets.
Among targets of recent interest have been falcipain cys-
Sleeping sickness, which in its late stages induces a ter-
minal somnolent state, is transmitted to humans by
the bite of the tsetse fly as an infectious form of the pro-
tozoan parasite Trypanasoma brucei. There are two
dominant subspecies of T. brucei: gambiense and rhodes-
iense. Sleeping sickness is widespread in sub-Saharan
Africa. The disease is fatal if untreated and no agents
Keywords: Protease inhibitor; Thiosemicarbazone; Malaria; Chagas;
Sleeping sickness.
*
Corresponding author. Tel.: +1 415 5027051; fax: +1 415 5140689;
e-mail: rguy@cgl.ucsf.edu
0
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.023

122
N. Fujii et al. / Bioorg. Med. Chem. Lett. 15 (2005) 121–123
8
,9
Table 1. Chemical structures and yields of thiosemicarbazone cysteine
protease inhibitors 1a–j and 2
atives as promising lead compounds against cruzain.
In early structure–activity relationship (SAR) studies,
they explored thiosemicarbazone derivatives of aromatic
aldehydes, acetophenones, and propiophenones, finding
that 3,4-dichlorophenyl- or 3-trifluoromethylphenyl thio-
semicarbazones such as 1a were potent inhibitors of
cruzain.
a
Yield (%)
#
R
However, these studies did not investigate thio-
semicarbazones possessing larger alkyl groups than
ethyl at the adjoining alkyl position (R in 1, Table 1).
Therefore, we began optimizing 1a by chemical modifi-
cation of its ethyl side chain to obtain new SAR data
(Table 1). Here we report potent inhibitors having sig-
nificantly improved activity against both cruzain and
rhodesain relative to the prior best inhibitor, 1a.
Step (a)
—
Step (b)
—
1
1
1
a
b
c
40
22
59
36
1
1
1
d
e
f
42
12
13
35
88
27
To produce diverse substituents (R in 1, Table 1), we
needed a reliable protocol to synthesize ketones having
a 3,4-dichlorophenyl group. A straightforward method
to synthesize diverse ketones is the palladium-catalyzed
coupling of acid chlorides and organometallic reagents
such as organoboranes and organostannanes. Organo-
boronic acids are most useful reagents for chemical
library synthesis because of commercial availability
and lower toxicity of any residual starting materials.
However, couplings of boronic acids are generally car-
ried out under basic aqueous conditions, thus giving
hydrolytic decomposition of the acid chlorides. We
found that 3,4-dichlorobenzeneboronic acid 3 coupled
with diverse acid chlorides using potassium phosphate
1
1
1
1
g
h
i
20
45
6
19
22
48
49
j
33
1
0
monohydrate in nonaqueous conditions. However, a
decarbonylative coupling product was produced as a
significant side product along with the desired ketone.
We have optimized the reaction conditions and found
that 80°C is the appropriate temperature to give ketones
4. The ketones were converted to thiosemicarbazones
1b–j (Table 2) in the conventional manner (Fig. 1).
Because of the difficult separation of 4 from the decar-
bonylative coupling products and incomplete conver-
sion to thiosemicarbazones, overall chemical yields
were generally modest.
a
Isolated, not optimized. See Figure 1.
exist that are efficacious, nontoxic, and affordable in the
developing world. Rhodesain is a cysteine protease
expressed in T. brucei rhodesiense that regulates replica-
tion of the parasite. Inhibitors of rhodesain block the
parasite life cycle in infected mammalian cells.
During our continuous effort to find mechanism-based
small molecule inhibitors for these parasite proteases,
Du and co-workers discovered thiosemicarbazone deriv-
Compound series 1 was characterized for activity
against the cysteine proteases cruzain, falcipain-2, and
Table 2. Inhibition of cruzain, rhodesain, and falcipain enzymes and T. brucei and P. falciparum growth by thiosemicarbazone derivatives 1
Compounds
Enzymatic activity inhibition
Proliferation inhibition
Cruzain
Rhodesain
a
IC50 (nM )
Falcipain-2
a
IC50 (nM )
T. cruzi
T. brucei rhodesiense
a
ED50 (nM )
P. falciparum W2
a
ED50 (nM )
a
a
IC50 (nM )
ED50 (nM )
1
1
1
1
1
1
1
1
1
1
2
a
b
c
d
e
f
30
19
110
60
nt
nt
nt
nt
nd
620
nd
3000
nt
130
nt
>10,000
3000
30
3330
1100
38
nd
14,000
470
8400
1800
640
375
2600
nt
nt
nt
6000
9000
19,000
nd
nd
nd
nd
nd
nd
nd
nt
72
80
40
80
38
nt
g
h
i
nt
30
30
60
70
570
1500
69
nd
nd
j
40
310
90
230
nd
nt
nt
Untested compounds are designated ÔntÕ; Compounds with no detectable activity are designated ÔndÕ. Activities stronger than 1a are bolded.

N. Fujii et al. / Bioorg. Med. Chem. Lett. 15 (2005) 121–123
123
had inhibitory activities in the 10lM concentration
range against a drug resistant strain of P. falciparum.
Finally, in attempts to discern activity against T. cruzi
cultured in human macrophages, it was discovered
that the majority of the compounds were toxic to
macrophages at dosages higher than 10lM, before they
arrested parasite growth. However, one compound, 1b,
completely cured the infected macrophages at a dosage
of 3lM.
In this study, we intended to expand the SAR of thio-
semicarbazone derivatives for inhibition of cysteine
proteases of protozoans. The SAR presented in this re-
port finishes the preliminary definition of functional
groups from this scaffold that define activity against
these proteases. It will facilitate the design of lead opti-
mization libraries of thiosemicarbazone inhibitors hav-
ing potent parasiticidal activities. In particular, it
points out the value of fully exploring the benzophenone
series of thiosemicarbazones.
Figure 1. Synthesis of thiosemicarbazone 1b–j. Reagents and condi-
tions: (a) RCOCl, PdCl (PPh , K PO
-monohydrate, toluene, 80°C;
b) H NNHC(S)NH , EtOH, AcOH, H
2
3
)
2
3
4
(
2
2
2
O, 70°C, 3days.
8
rhodesain. Recombinantly expressed cruzain, falcipain-
4
11
2
, or rhodesain was incubated with inhibitors 1a–j
and 2 in sequential dilution followed by addition of a
fluorescent indicator of protease activity (e.g., Z-Phe-
Arg-AMC). The increase in fluorescence produced by
cleavage of the substrate allows determination of pro-
9
tease inhibition. IC s were determined in the linear
5
0
Acknowledgements
portion of a plot of inhibition versus log of inhibitor
concentration.
The Sandler Center for Basic Research in Parasitic Dis-
eases and the National Institutes of Health.
The results are summarized in Table 2. For inhibition of
cruzain, simple extension of the ethyl of 1a to n-butyl
gives two times improvement of activity (1b), however,
incorporating bulkier alkyl substituents having a branch
drastically reduces the potency (1c,d). Interestingly, a
compound with a small branched alkyl (cyclopropyl)
group having an aromatic ring (1h) possesses the same
potency as 1a. This led us to investigate compounds hav-
ing aromatic rings connected with several linkages to the
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2004.10.023.
3
,4-dichlorobenzoyl moiety. These compounds (direct
bond: 1g; one methylene: 1f; two methylenes: 1j; alkene:
e; ether: 1i) generally possess potency equivalent to 1a.
References and notes
1
1
. Breman, J. G.; Egan, A.; Keusch, G. T. Am. J. Trop. Med.
Hyg. 2001, 64, iv–vii.
Next, we tested these compounds against rhodesain. As
expected, all of the compounds except 1c,d gave potent
inhibition, which was stronger than that of 1a (Table
2. DÕAlessandro, U.; Buttiens, H. Trop. Med. Int. Health
2001, 6, 845–848.
3. Milhous, W. K. Med. Trop. 2001, 61, 48–50.
4
. Sijwali, P. S.; Rosenthal, P. J. Proc. Natl. Acad. Sci.
U.S.A. 2004, 101, 4384–4389.
2
). It is noteworthy that both n-butyl and bulkier aryl-
alkyls possess the same level of potency, while bulkier al-
kyl substituents drastically reduce the potency. These
inhibitors represent the most potent inhibitors discov-
ered against rhodesain to date.
5
. Sijwali, P. S.; Kato, K.; Seydel, K. B.; Gut, J.; Lehman, J.;
Klemba, M.; Goldberg, D. E.; Miller, L. H.; Rosenthal, P.
J. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 8721–8726.
. Libow, L. F.; Beltrani, V. P.; Silvers, D. N.; Grossman,
M. E. Cutis 1991, 48, 37–40.
6
7
Finally, the compounds were tested against falcipain-2.
All of the compounds were significantly less active inhib-
itors of this protease, with none having potencies less
than 100nM.
. Harth, G.; Andrews, N.; Mills, A. A.; Engel, J. C.; Smith,
R.; McKerrow, J. H. Mol. Biochem. Parasitol. 1993, 58,
17–24.
8. Du, X.; Guo, C.; Hansell, E.; Doyle, P. S.; Caffrey, C. R.;
Holler, T. P.; McKerrow, J. H.; Cohen, F. E. J. Med.
Chem. 2002, 45, 2695–2707.
A subset of the active compounds were also evaluated in
whole cell assays for inhibition of proliferation of T.
brucei rhodesiense, T. cruzi, and P. falciparum. As is
common with protease inhibitors, the overall activities
were lower than in the enzymatic assays, presumably
due to limitations in transport. Nevertheless, a number
of the compounds were highly potent against T. brucei.
Although most were inactive, several of the compounds
9
. Greenbaum, D. C.; Mackey, Z.; Hansell, E.; Doyle, P.;
Gut, J.; Caffrey, C. R.; Lehrman, J.; Rosenthal, P. J.;
McKerrow, J. H.; Chibale, K. J. Med. Chem. 2004, 47,
3
212–3219.
0. Urawa, Y.; Ogura, K. Tetrahedron Lett. 2003, 44, 271–
73.
1
2
11. Gotz, M. G.; Caffrey, C. R.; Hansell, E.; McKerrow, J. H.;
Powers, J. C. Bioorg. Med. Chem. 2004, 12, 5203–5211.