Synthesis and structure-activity relationships of parasiticidal thiosemicarbazone cysteine protease inhibitors against Plasmodium falciparum, Trypanosoma brucei, and Trypanosoma cruzi

Article abstract of DOI:10.1021/jm030549j

We have synthesized a library of thiosemicarbazones and screened them against three parasitic cysteine proteases, cruzain, falcipain-2, and rhodesain, and against the respective parasite sources of these three proteases, Trypanosoma cruzi, Plasmodium falciparum, and Trypanosoma brucei. The screens identified compounds that were effective against the enzymes and the parasites but also some compounds that were parasiticidal despite a lack of activity against the proteases. Several compounds were effective in killing all tested parasites. These promising lead compounds were tested for general toxicity in mice, and only one produced observable toxicity after 62 h. Our results suggest that thiosemicarbazones represent validated drug leads that kill several species of protozoan parasites through the inhibition of cysteine proteases as well as other novel targets.

Full text of DOI:10.1021/jm030549j

3212
J . Med. Chem. 2004, 47, 3212-3219
Syn th esis a n d Str u ctu r e-Activity Rela tion sh ip s of P a r a siticid a l
Th iosem ica r ba zon e Cystein e P r otea se In h ibitor s a ga in st P la sm od iu m
fa lcipa r u m , Tr ypa n osom a br u cei, a n d Tr ypa n osom a cr u zi
Doron C. Greenbaum,*,† Zachary Mackey,† Elizabeth Hansell,† Patricia Doyle,† J iri Gut,‡ Conor R. Caffrey,†
J ulia Lehrman,‡ Philip J . Rosenthal,‡ J ames H. McKerrow,† and Kelly Chibale*,§
Sandler Center for Basic Research in Parasitic Diseases, Department of Pathology, University of California,
San Francisco, California 94143, Department of Medicine, San Francisco General Hospital, San Francisco, California 94143,
and Department of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
Received October 27, 2003
We have synthesized a library of thiosemicarbazones and screened them against three parasitic
cysteine proteases, cruzain, falcipain-2, and rhodesain, and against the respective parasite
sources of these three proteases, Trypanosoma cruzi, Plasmodium falciparum, and Trypanosoma
brucei. The screens identified compounds that were effective against the enzymes and the
parasites but also some compounds that were parasiticidal despite a lack of activity against
the proteases. Several compounds were effective in killing all tested parasites. These promising
lead compounds were tested for general toxicity in mice, and only one produced observable
toxicity after 62 h. Our results suggest that thiosemicarbazones represent validated drug leads
that kill several species of protozoan parasites through the inhibition of cysteine proteases as
well as other novel targets.
In tr od u ction
substrates in their active site cleft through backbone
and side chain interactions that lie around several
defined pockets in the enzyme termed S1, S2, S3, S4
for those pockets that bind to the corresponding amino
acids that are N-terminal to the scissile amide bond
while the S1′, S2′, S3′, S4′ pockets bind to the amino
acids C-terminal to the scissile bond.
Thiosemicarbazones are a class of small molecules
that have been evaluated over the last 50 years as
antivirals1,2 and as anticancer therapeutics,3 as well as
for their parasiticidal action against Plasmodium fal-
ciparum4 and Trypanosoma cruzi,5,6 which are the
causative agents of malaria and Chagas’s disease,
respectively. Currently, a thiosemicarbazone, triapine,
is being evaluated in human phase II trials as an
antineoplastic therapeutic.7
In view of these SAR results, we synthesized a second
generation series of thiosemicarbazones. These com-
pounds were tested in vitro against cruzain as well as
the cysteine proteases falcipain-2 and rhodesain. In
addition, we screened all compounds, whether active
against the target enzyme or not, in culture against the
respective parasites, T.cruzi, P. falciparum, and T.
brucei. All three parasites are major public health
problems in tropical and subtropical countries, and for
which new chemotherapies are desperately needed.
In this synthetic design of the SAR library, attention
was paid to further explore substituents around the
aromatic ring as well as the previously unexplored N1
NH2-monosubstitution of the original thiosemicarbazone
scaffold. We reasoned that a diversity-oriented synthesis
using Suzuki- or copper-mediated coupling of a range
of commercially available boronic acids could be ef-
ficiently employed for optimization of interactions with
the S2 pocket. In addition, we expanded the SAR to
include possible prime side interactions at the S1′ or
S2′ positions by exploring N1 NH2-disubstitution. It is
noteworthy that thiosemicarbazones have only recently
been reported to inhibit falcipain-2 and rhodesain, and
these reported compounds are based on the isatin
scaffold. Their antiparasitic activities in cell culture
have not been determined.8
A series of thiosemicarbazones was recently shown
to inhibit a Trypanosoma cruzi-derived cysteine pro-
tease, cruzain.6 This study generated basic structure-
activity relationships (SAR) against cruzain around
three positions, using the scaffold shown in Figure 1.
The authors found that halides such as bromine and
chlorine were tolerated at the 3′- and/or 5′-position on
the aryl ring. In addition, changing the methyl sub-
stituent at the 2-position had little effect, and N1 NH2-
monosubstitution at position 3 was deleterious. Last,
the sulfur and methine bonds were critical to the
inhibition of cruzain. The authors concluded that the
probable site of attack by the active site cysteine thiol
on the thiosemicarbazone scaffold was the thiocarbonyl
group and accordingly suggested binding modes using
DOCK-based algorithms.6 This model placed the 3′-
substituent of the thiosemicarbazone into the main
specificity S2 pocket of cruzain. Proteases bind peptide
* Corresponding authors. D.C.G. current address: Department of
Infection and Immunity, Walter and Eliza Hall Institute of Medical
Research, 1G Royal Parade, Parkville, VIC 3050, Australia. Tel +61 3
9345 2479, fax +61 3 9347 0852, e-mail greenbaum@wehi.edu.au.
K.C.: Tel 27 21 650 2553, fax 27 21 689 7499, e-mail chibale@
science.uct.ac.za.
Ch em istr y
† University of CaliforniasSan Francisco.
Biphenyl thiosemicarbazones, 1a -c, were prepared
in high yield (80-90%) from biphenyl acetophenones
‡ San Francisco General Hospital.
§ University of Cape Town.
10.1021/jm030549j CCC: $27.50 © 2004 American Chemical Society
Published on Web 05/04/2004

Thiosemicarbazone Cysteine Protease Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3213
F igu r e 1. Summary of known thiosemicarbazone structure-activity relationships with cruzain.
Sch em e 1a
a
Reagents and conditions: (i) Pd(Ph3P)4, K2CO3(aq), PhMe, reflux, phenylboronic acid. (ii) thiosemicarbazide, MeOH, AcOH, reflux.
Sch em e 2a
a
Reagents and conditions: (i) Cu(OAc)2 (xs), pyridine, CH2Cl2, rt, phenylboronic acid. (ii) thiosemicarbazide, MeOH, AcOH, reflux.
Sch em e 3a
a
Reagents and conditions: (i) Thiosemicarbazide, MeOH, AcOH, reflux.
which were in turn prepared in good yield (60-70%) via
a Suzuki cross-coupling procedure from the correspond-
ing bromoacetophenones and phenylboronic acid (Scheme
1). The biarylamine and ether derivatives, 2a -e, were
also prepared in high yield (80-90%) via moderate
yielding (50-60%) N- and O-arylation, respectively
(Scheme 2). Treatment of the intermediate biphenyl,
biarylamine, and biaryl ether acetophenones with thio-
semicarbazide afforded the title compounds in high yield
(80-90%). Thiosemicarbazones 3a -h were prepared
with high yield (80-90%) from commercially available
starting materials by reaction with thiosemicarbazide
(Scheme 3). On the other hand, N,N-disubstituted
derivatives 4a -e were synthesized in good yield (70-
80%) by reacting thiosemicarbazone thioesters with
selected secondary amines (Scheme 4).
In theory, two geometrical isomers (E and Z) about
the imine (CdN) double bond are possible for the above
1
thiosemicarbazones. However, analysis of the H NMR
spectra of the target compound indicated one predomi-
nant isomer. This predominant isomer is presumed to
have the E-configuration for the aryl-based thiosemi-
carbazones and the Z-configuration for the 2-acetylpyr-
idine-based thiosemicarbazones.
Biologica l Resu lts
Cr u za in a n d T. cr u zi SAR. Previously, Du et al.6
showed that the best thiosemicarbazone inhibitor against

3214 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Greenbaum et al.
Sch em e 4a
a
Reagents and conditions: (i) NH2NH2 (xs), KOH i-PrOH. (ii) MeI. (iii) i-PrOH. (iv) i-PrOH.
Ch a r t 1. Structure-Activity Relationships against Cruzain and T. cruzi
cruzain contained a bromo substituent at the 3′-position
(depicted as compound 3d in Chart 1a, 2a, and 3a) or
3′,5′-bis(trifluoromethyl) substituents on the aryl ring.
Du et al.6 proposed a reversible covalent interaction
between thiosemicarbazones and cruzain in which the
aryl group lies in the S2 pocket of the enzyme. From
this we elaborated a series of biphenyl, biarylamine, and
biaryl ether derivatives with differing positions around
the aryl ring. Thus, the rationale for our choice of these
derivatives is based on both experimental and modeling
results.
Against recombinant cruzain, a number of compounds
with a substituent at the 3′- (1b, 2d , 3d , 3i, 4d , 4e, 4c)
and 4′- (1c, 2e) positions on the aromatic ring were
active in the mid-nanomolar to low micromolar range
(Chart 1a). The best compound, the parent molecule 3d ,
contained a bromine at the 3′-position. Thus, the 3′- and
4′-positions appear to be promising areas for further
expansion. Overall, there was no significant advantage
in cruzain inhibition between the biaryls (1b, 1c) and
aryl ethers (2d , 2e), while the arylamines (2b, 2c) were
ineffective. Replacing the terminal thioamide amino
group with disubstituted derivatives (4c, 4d , 4e) low-
ered potency by 2 orders of magnitude compared to the
parent compound (3d ).
Seven compounds (1b, 2b, 2c, 2d , 2e, 3d , 4d ) were
effective at protecting macrophage host cells against
T.cruzi (Chart 1). Two compounds (1b, 3d ) were effec-
tive against both cruzain and parasites, both of which
contain a 3′-substituent on the aromatic ring. Interest-
ingly, two compounds with 3′- and 4′-substituents (2b,
2c) did not inhibit cruzain in vitro but were successful
in killing parasites in culture (Charts 1a and 1b). This
suggests that the biarylamine substituents at the 3′-
and 4′-positions may be important for binding to both
cruzain as well as another protease or an unrelated
target. Finally, the differences in cell culture activity
between 2-, 3-, and 4-acetylpyridine thiosemicarbazones
3a , 3b, and 3c is noteworthy (Chart 1 and Supporting
Information Chart 1S). Compound 3a , acetylpyridine
thiosemicarbazone, is likely a tridentate metal chelator
in which the pyridine ring nitrogen is involved in
chelation and may act by binding endogenous iron and/
or copper.
Rh od esa in a n d T. br u cei SAR. Many more com-
pounds were effective against rhodesain as compared
to cruzain although the basic SAR was similar. This
may indicate that the S2 pocket of rhodesain is larger
than that of cruzain. Those compounds with substitu-

Thiosemicarbazone Cysteine Protease Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3215
Ch a r t 2. Structure-Activity Relationships against Rhodesain and T. brucei
Ch a r t 3. Structure-Activity Relationships against Falcipain and P. falciparum
ents at the 3′- (1b, 2b, 2d , 3d , 3i, 4c, 4d , 4e) and 4′-
(1c, 2c, 2e) position were effective in the mid-nanomolar
to low micromolar range (Charts 2a and 2b). This agrees
with the SAR data for cruzain. In contrast to the cruzain
SAR, the biarylamine inhibitors (2b, 2c) were effective
against rhodesain, which indicates that these com-
pounds can, in fact, bind to a cysteine protease. This
further indicates that substituents at these positions are
important for binding to cysteine proteases.
4b may be killing T. brucei via a protease-independent
mechanism. We have investigated the possibility of
metal chelation as a route by which some of these
inhibitors act on T. brucei. Inhibitors were assayed for
the ability to kill parasites in the presence or absence
of FeCl3. There was no significant effect (data not
shown), although these results are complicated by the
fact that the compounds can exchange precomplexed
iron for endogenous metals.
In screens against T. brucei in culture, there were
many inhibitors that were able to inhibit both rhodesain
and T. brucei (1b, 1c, 2b, 2c, 2d , 2e, 4c, 4d , 4e). This
indicates that inhibition of rhodesain may be the
primary action of these inhibitors. On the other hand,
the most potent compound in the present series against
T. brucei is a potential metal chelator (4b) with no
ability to inhibit the enzyme rhodesain. Thus, compound
F a lcip a in 2 a n d P . fa lcipa r u m SAR. The SAR
against falcipain-2 is striking in that most compounds
failed to inhibit the target enzyme. The exception (1c)
was a weak inhibitor with no efficacy against the
parasite. These data give some insight into the signifi-
cant differences in the active site topology between all
three closely related target proteases. On the basis of
the data for all three enzymes, rhodesain is by far the

3216 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Greenbaum et al.
F igu r e 2. Summary of (A) protease and (B) parasite structure-activity relationships. Bold lettering indicates significant differences
between parasite and enzyme SAR.
most promiscuous enzyme, while falcipain-2 is ex-
tremely restrictive.
In addition to the primary SAR, the results from our
unbiased screening approach uncovered four scaffolds
that represent more general parasiticidal compounds
capable of killing all parasites. Two of these are non-
metal chelators (2c, 4d ) and two are potential metal
chelators (3a , 4b). The mechanism of action of these
compounds appears to be complex. Compounds 2c and
4d can inhibit proteases, although 2c inhibits only
rhodesain, while 4d inhibits both rhodesain and cruzain.
Yet both compounds can kill all three organisms. This
highlights the fact that there must be other targets that
may or may not include cysteine proteases. On the other
hand, compounds 3a and 4b do not inhibit the target
cysteine proteases but are very effective at killing at
least two or all three tested parasites. Clearly these
inhibitors work through a different target and may
chelate endogenous metals.
Ultimately, a compound that can kill parasites is
necessary but not sufficient to represent a good drug
lead unless it also demonstrates low or no toxicity
among other criteria. Thus the most promising lead
compounds (1b, 2c, 3a , 3j, 4b, 4d ) were tested for
general toxicity in mice (Supporting Information Chart
4). Compounds 1b, 2c, 3j, 4b, and 4d generated no
toxicity in mice after 62 h while compound 3a was toxic.
In addition to having no obvious toxicity, the four best
general scaffolds (2c, 4d , 3a ,4b) are nonpeptidic and
achiral and utilize chemistries that are relatively inex-
pensive and amenable to chemical library generation.
For example, the synthesis of compound 2c utilizes
copper-mediated coupling chemistry using both com-
mercially and synthetically accessible boronic acids as
diversity elements. Thus, we are pursuing the genera-
tion of a second library based on this scaffold. These
results are encouraging, and we will conduct further
studies on their mechanism of action as well as efficacy
studies using these inhibitors in mouse models of
African sleeping sickness, Chagas’s disease, and ma-
laria.
However, with P. falciparum in culture, there were
five inhibitors that were able to kill or halt growth in
live P.falciparum cultures. The best inhibitors (3a , 3j,
4b) were potent having mid-nanomolar ED50s. While the
antimalarial activity of compound 3a has been previ-
ously reported,4 its protein target(s) in P. falciparum is
unknown. Once again, these inhibitors have the ability
to chelate a transition metal, and it is likely that this
mechanism of action is responsible for the activity of
these compounds.
Discu ssion
In this study we endeavored to expand the structure-
activity relationships of thiosemicarbazones against
parasitic cysteine proteases. To do this we took an
unbiased approach to screening these compounds against
both the target enzymes and the appropriate parasites.
Our screening strategy allowed us to analyze SAR
across protease targets as well as parasites. The pro-
tease SAR revealed that substituents at the 3′- or 4′-
positions on the aryl ring are most favored to bind the
protease targets and merit expansion (Figure 2a). The
parasite SAR corroborates these data, indicating that
most thiosemicarbazones with substituents at the 3′-
and 4′-positions are acting via a protease-dependent
mechanism. In contrast to the protease SAR, the
parasite SAR shows that the pyridine thiosemicarba-
zones, which do not inhibit the target cysteine proteases,
are excellent parasiticidal compounds (Figure 2b; 3a and
4b). Therefore, these types of compounds must be acting
either through inhibition of another cysteine protease
or by a protease-independent mechanism. Last, N1 NH2-
disubstitution as in compound 4d lowered potency
against target proteases but enhanced killing of T.
brucei and P. falciparum, especially when a protonat-
able terminal nitrogen is present as in compound 4d .
This may be due to increased uptake of the compound
across the cell membranes or enhanced binding to
another target protein.
The detailed mechanism of action of thiosemicarba-
zones has been elusive. The antimalarial 2-acetylpyri-
dine thiosemicarbazones (3a , 4b, and 3j) may be acting

Thiosemicarbazone Cysteine Protease Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3217
in a Fluoroskan Ascent spectrofluorometer (Labsystems). IC50
values were determined from plots of percents of activity over
the compound concentration using the data analysis program
Prism (GraphPad software).
as iron chelators in P. falciparum. Tridentate chelating
thiosemicarbazones have been shown to inhibit ribo-
nucleotide reductase, RR, an enzyme essential for DNA
synthesis.9,10 Tridentate chelating thiosemicarbazones
have also been proposed to act by inhibiting dihydro-
folate reductase.11,12 Last, there is evidence that copper
complexes of thiosemicarbazones produce significant
oxidative stress by binding endogenous reducing agents
such as glutathione.13 Our work suggests that the
mechanism of action of thiosemicarbazones is indeed
complex and may be mediated through inhibition of
multiple targets.
Finally, we hope that these results encourage the
screening of all compounds in a SAR series against both
the protein target as well as the relevant organism since
the combined data may reveal hidden target complexity.
If we had tested, in culture, only compounds in this
series that inhibited the target proteases, several very
potent parasiticidal compounds would have been over-
looked. Furthermore both types of data, against target
enzyme and parasite, help to optimize the next round
of library synthesis to optimize the properties necessary
for both inhibiting the target as well as killing the
organism.
T. cr u zi Cu ltu r e Assa y. Mammalian cells were cultured
in RPMI-1640 medium supplemented with 5-10% heat-
inactivated fetal calf serum (FCS) at 37 °C in 5% CO2. The Y
strain of T. cruzi was maintained by serial passage in bovine
embryo skeletal muscle (BESM) cells. Infectious trypomasti-
gotes are collected from culture supernatants. For drug assays,
J 774 macrophages were irradiated (5000 rad) and plated onto
six-well tissue culture plates 24 h prior to infection with about
106 trypomastigotes/well. Parasites were removed 2 h postin-
fection, and the medium was supplemented with the appropri-
ate inhibitor (10 µM). Inhibitor stocks (10 mM) in DMSO were
stored at -20 °C. J 774 monolayers treated with a blank
containing DMSO were used as a negative control. RPMI
medium with or without inhibitor was replaced every 48 h.
Cultures were maintained for up to 46 days and monitored
daily by contrast phase microscopy. T. cruzi completed the
intracellular cycle in 5-6 days in the untreated controls. The
comparative effectiveness of each inhibitor was estimated from
plots of the duration of the intracellular cycle of T. cruzi (days)
in treated vs untreated control wells.
T. br u cei Cu ltu r e Assa y. T. brucei rhodesiense were grown
to 106 cells/mL at 37 °C with 5% CO2 in complete HMI-9
medium containing 10% FBS, 10% Serum Plus, 1× penicillin/
streptomycin. To carry out drug screens, parasites were diluted
to 104 cells/mL in complete HMI-9 medium and aliquoted into
5 mL for growth in culture flasks or 100 mL for growth in 96-
well cultures plates. Each inhibitor was added to the appropri-
ate flask or well containing cultured parasites beginning at
the highest concentration. The inhibitors were then directly
diluted in the cultured parasites by serial dilutions until the
concentration of the inhibitors reached 1 nM. Parasites were
then incubated in the presence of each inhibitor for 48 h at 37
°C with 5% CO2 before monitoring viability. To assay for
viability after treatment with inhibitors, parasites were tested
for the production of ATP.17 To do this, 100 mL of parasites
from each flask were transferred to 96-well plates. An equal
volume of CellTiter-Glo (Promega) was added to each well of
the transferred parasites or parasites originally grown in 96-
well plates separately. The mixture was then shaken at room
temperature for 5 min before reading the plates using a
SpectraFluor Plus multidetection plate reader (Tecan). Alter-
natively, the treated parasites were counted by hemacytometer
48 h after incubating them with inhibitors.
P . fa lcipa r u m Cu ltu r e Assa y. W2-strain P. falciparum
parasites (1% parasitemia, 2% hematocrit) were cultured in
0.5 mL of medium in 48-well culture dishes.18 Appropriate
inhibitors from 10 mM stocks in DMSO were added to cultured
parasites to a final concentration of 20 µM. From 48-well
plates, 125 µL of culture was transferred to two 96-well plates
(duplicates). Serial dilutions (1:5) of inhibitors were made to
final concentrations of 10 µM, 2000 nM, 400 nM, 80 nM, 16
nM, and 3.2 nM. Cultures were maintained at 37 °C for 2 days.
The parasites were washed and fixed with 1% formaldehyde
in PBS. After 2 days, parasitemia was measured by flow
cytometry using the DNA stain YOYO-1 as a marker for cell
survival.
Mou se Toxicity Assa y. C3H female mice (mean weight,
18 g) (J ackson Laboratories) were injected daily via ip with
20 mg/kg weight or 5 mg/kg weight of selected compounds
(n ) 1 per treatment). Compounds were resuspended in 100
µL [70% DMSO (Sigma):30% ddH20] per dose and injected
twice daily for 48 h. Animals were monitored for signs of
toxicity, including behavior and feeding, and sacrificed 14 h
after the last treatment for necropsy. Major organs were
submitted for histological analysis.
Con clu sion
We identified several thiosemicarbazones that were
effective against parasite-derived cysteine proteases and
the appropriate parasites. Among the most effective
compounds, some were parasiticidal without activity
against the cognate protease. Four compounds were
effective in killing all tested parasites, and these
compounds showed no obvious toxicity in mice after 62
h. These results indicate that thiosemicarbazones rep-
resent promising drug leads capable of killing multiple
protozoal parasites through the inhibition of cysteine
proteases as well as other novel targets.
Exp er im en ta l P r oced u r es
P r otea se In h ibition a n d P a r a site Cell Cu ltu r e Assa ys.
Recombinant cruzain (from T. cruzi) and rhodesain (from T.
brucei rhodesiense) were recombinantly expressed as described
previously.14,15 Cruzain (2 nM) or rhodesain (3 nM) was
incubated with 0.5 to 10 µM inhibitor in 100 mM sodium
acetate, pH 5.5, containing 5 mM DTT (buffer A), for 5 min at
room temperature. Then buffer A containing Z-Phe-Arg-AMC
(Bachem, Km ) 1 µM) was added to enzyme inhibitor to give
20 µM substrate in 200 µL, and the increase in fluorescence
(excitation at 355 nM and emission at 460 nM) was followed
with an automated microtiter plate spectrofluorimeter (Mo-
lecular Devices, Flex station). Inhibitor stock solutions were
prepared at 20 mM in DMSO, and serial dilutions were made
in DMSO (0.7% DMSO in assay). Controls were performed
using enzyme alone and enzyme with DMSO. IC50 values were
determined graphically using inhibitor concentrations in the
linear portion of a plot of inhibition versus log [I] (seven
concentrations tested with at least two in the linear range).
IC50 values against recombinant Falcipain-2 were deter-
mined as described previously.16 Enzyme was incubated for
30 min at room temperature in 100 mM sodium acetate, pH
5.5, 10 mM DTT, with different concentrations of tested
inhibitors. Inhibitor solutions were prepared from stock in
DMSO (maximum concentration of DMSO in the assay was
1%).
Ch em istr y. Proton nuclear magnetic resonance (1H NMR)
spectra were obtained with a Varian Inova-400 MHZ spec-
trometer with Me4Si(TMS) as the internal reference. Coupling
constants (J ) are given in hertz. Elemental and mass spectra
analyses were performed at the Micro-Mass facility, University
After 30 min incubation, the substrate Z-Leu-Arg-AMC
(benzoxycarbonyl-Leu-Arg-7-amino-4-methylcoumarin) in the
same buffer was added to a final concentration of 25 µM.
Fluorescence was monitored for 15 min at room temperature

3218 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12
Greenbaum et al.
of California, Berkeley. Thin layer chromatography (TLC) was
carried out on aluminum-backed Merck silica gel 60 F254 using
the same solvent systems as those used in column chroma-
tography. Column chromatography was performed on silica gel
(70-230 mesh). Final products usually precipitated out and
were either rinsed with, and/or recrystallized from, methanol.
Yields for Suzuki cross-couplings and N- and O-arylation
reactions were in the range of 60-70% and 50-60%, respec-
tively, while yields for all thiosemicarbazone-forming reactions
were 80-90%.
Gen er a l P r oced u r e for t h e P r ep a r a t ion of Com -
p ou n d s. Suzuki Coupling (Scheme 1): 3 mmol of bromoac-
etophenone and 3 mmol of boronic acid were dissolved in a
mixture of toluene (30 mL) and ethanol (12 mL). Tetrakis-
triphenylphosphinepalladium(0) (100 mg) was added. After
addition of a saturated solution of potassium carbonate (30
mL), the mixture was heated under reflux for 5 h. Water was
added, and the mixture was extracted with dichloromethane.
Organic layers were combined and dried (MgSO4), and the
solvent was removed. Products were obtained by preparative
TLC.
8.32 (s, 1H), 10.25 (s, 1H); MS (EI) m/z 273.2 (MH+). Anal.
(C9H10BrN3S) C, H, N.
1-(3-Am in op h en yl)eth a n on e th iosem ica r ba zon e (3e):
δH (400 MHz; DMSO-d6) 2.22 (s, 3H), 5.04 (s, 2H), 6.60 (d, 1H,
J ) 4.4), 7.02 (d, 2H, J ) 4.4), 7.09 (s, 1H), 7.67 (s, 1H), 8.29
(s, 1H), and 10.18 (s, 1H); MS (EI) m/z 209.0 (MH+). Anal.
(C9H12N4S) C, H, N, S.
1-(2-Hyd r oxyp h en yl)eth a n on e th iosem ica r ba zon e (3f):
δH (400 MHz; DMSO-d6) 2.31 (s, 3H), 4.49 (s, 1H), 6.84 (s, 1H),
6.86 (t, 2H, J ) 8.4), 7.25 (t, 1H, J ) 7.6), 7.53 (s, 2H); MS
(EI) m/z 210.2 (MH+).
1-(3-Hydr oxyph en yl)eth an on e th iosem icar bazon e (3g):
δH (400 MHz; DMSO-d6) 2.25 (s, 3H), 6.79 (d, 1H, J ) 8.0),
7.19 (t, 1H, J ) 8.0), 7.24 (s, 1H), 7.33 (d, 1H, J ) 7.6), 7.78 (s,
1H), 8.27 (s, 1H) 9.43 (s, 1H), 10.21 (s, 1H); MS (EI) m/z. 210.2
(MH+). Anal. (C9H11N3OS) C, H, N, S.
1-(4-Hydr oxyph en yl)eth an on e th iosem icar bazon e (3h ):
δH (400 MHz; DMSO-d6) 2.23 (s, 3H), 6.75 (d, 2H, J ) 8.8),
7.76 (d, 2H, J ) 8.4), 7.80 (s, 1H), 8.16 (s, 1H), 9.72 (s, 1H),
and 10.06 (s, 1H); MS (EI) m/z. 210.2 (MH+). Anal. (C9H11N3-
OS) C, H, N, S.
Arylation (Scheme 2): A slurry of the substrate, phenylbo-
ronic acid (2-3 equiv), anhydrous Cu(OAc)2 (1-2 equiv), and
pyridine (2-3 equiv) in methylene chloride (10 mL/0.5 g of
substrate) was stirred at room temperature for 24-72 h.
Products were isolated by direct loading of the crude reaction
mixtures on silica gel.
Met h yl 3-[1-(3′-b r om op h en yl)et h ylid en e]h yd r a zin e-
ca r bod ith ioa te (4a ): δH (400 MHz; DMSO-d6) 2.38 (s, 3H),
2.52 (s, 3H), 7.42 (t, 1H, J ) 8), 7.65 (d, 1H, J ) 7.6), 7.85 (d,
1H,J ) 8.0), 8.02 (s, 1H), 12.53 (s, 1H); MS (EI) m/z 304.3
(MH+). Anal. (C10H11BrN2S2) C, H, N, S.
P ip er id in e-1-ca r both ioic a cid [1-(3-br om op h en yl)eth -
ylid en e] h yd r a zid e (4c): δH (400 MHz; DMSO-d6) 1.61 (s,
6H), 2.27 (s, 3H), 3.83 (s, 4H), 7.38 (t, 1H, J ) 7.6), 7.58 (d,
1H, J ) 7.6), 7.55 (d, 1H, J ) 7.6), 7.93 (s, 1H), 9.66 (s, 1H);
MS (EI) m/z 340.3 (MH+). Anal. (C14H18BrN3S) C, H, N.
4-Meth ylp ip er a zin e-1-ca r both ioic a cid [1-(3-br om o-
p h en yl)eth ylid en e] h yd r a zid e (4d ): δH (400 MHz; DMSO-
d6) 2.21 (s, 3H), 2.28 (s, 3H), 2.39 (s, 4H), 3.86 (s, 4H), 7.39 (t,
1H, J ) 7.2), 7.59 (d, 1H, J ) 6.8), 7.55 (d, 1H, J ) 7.6), 7.94
Thiosemicarbazone derivatives 1-4 were prepared essen-
tially as reported.6
1-Bip h en yl-2-yleth a n on e th iosem ica r ba zon e (1a ): 1H
NMR δH (400 MHz; DMSO-d6) 1.77 (s, 3H), 7.25 (s, 1H), 7.31
(d, 2H, J ) 7.6,), 7.36 (d, 1H, J ) 2.0), 7.39 (d, 2H, J ) 9.2),
7.43 (s, 1H), 7.45 (s, 1H), 7.55 (d, 1H, J ) 8.4), 8.12 (s, 1H),
10.13 (s, 1H), 11.95 (s, 1H); MS (EI) m/z 270.3 (MH+). Anal.
(C15H15N3S) C, H, N, S.
1
(s, 1H), 9.86 (s, 1H); MS (EI) m/z 355.5 (MH+). Anal. (C14H19
-
1-Bip h en yl-3-yleth a n on e th iosem ica r ba zon e (1b): H
NMR δH (400 MHz; DMSO-d6) 2.37 (s, 3H), 7.38 (t, 2H, J )
7.2), 7.48 (t, 2H, J ) 8.0), 7.67 (d, 1H, J ) 7.6), 7.74 (d, 2H,
J ) 7.2), 7.91 (d, 1H, J ) 8.0), 8.02 (s, 1H), 8.11 (s, 1H), 10.23
(s, 1H); MS (EI) m/z 270.3 (MH+). Anal. (C15H15N3S) C, H, N,
S.
1-Bip h en yl-4-yleth a n on e th iosem ica r ba zon e (1c): 1H
NMR δH (400 MHz; DMSO-d6) 2.34 (s, 3H), 7.39 (t, 1H, J )
7.2), 7.49 (t, 2H, J ) 8.0), 7.69 (dd, 4H, J ) 7.6), 7.99 (s, 1H),
8.03 (d, 2H, J ) 8.8), 8.30 (s, 1H), 10.25 (s, 1H); MS (EI) m/z
270.3 (MH+). Anal. (C15H15N3S) C, H, N, S.
1-(2-P h en ylam in oph en yl)eth an on e th iosem icar bazon e
(2a ): 1H NMR δH (400 MHz; CDCl3) 2.25 (s, 1H), 2.35 (s. 3H),
7.05 (m, 6H), 7.28 (m, 4H), 7.41 (d, 1H, J ) 7.2), and 8.73 (s,
1H); MS (EI) m/z 285.3 (MH+).
1-(3-P h en ylam in oph en yl)eth an on e th iosem icar bazon e
(2b): δH (400 MHz; CHCl3-d) 2.65 (s, 3H), 6.35 (broad s. 1H),
6.99 (t, 1H, J ) 7.2), 7.11 (t, 2H, J ) 8.4), 7.27 (m, 6H), 7.43
(s, 2H), 8.73 (s, 1H); MS (EI) m/z 285.3 (MH+). Anal. (C15H16N4S)
C, H, N.
1-(4-P h en ylam in oph en yl)eth an on e th iosem icar bazon e
(2c): δH (400 MHz; CHCl3-d) 2.27 (s, 3H), 6.25 (broad s. 2H),
7.04 (d, 2H, J ) 8.8), 7.15 (d, 1H, J ) 8.0), 7.26 (s, 2H), 7.32
(t, 2H, J ) 7.6), 7.63 (d, 2H, J ) 8.8), 8.62 (broad s, 1H); MS
(EI) m/z 285.3 (MH+).
1-(3-P h en oxyph en yl)eth an on e th iosem icar bazon e (2d):
δH (400 MHz; DMSO-d6) 2.28 (s, 3H), 6.99 (t, 2H, J ) 8.4),
7.13 (t, 1H, J ) 6.8), 7.39 (dd, 3H, J ) 7.2), 7.69 (s, 2H), 7.97
(s, 1H) 8.27 (s, 1H), 10.23 (s, 1H); MS (EI) m/z. 286.2 (MH+).
Anal. (C15H15N3OS) C, H, N, S.
1-(4-P h en oxyph en yl)eth an on e th iosem icar bazon e (2e):
δH (400 MHz; CHCl3-d) 2.28 (s, 3H), 6.98 (d, 2H, J ) 8.4), 7.02
(d, 2H, J ) 8.4), 7.14 (t, 1H, J ) 7.6), 7.34 (d, 2H, J ) 7.6),
7.37 (s, 1H), 7.67 (s, 2H), 8.82 (s, 1H); MS (EI) m/z 286.2 (MH+).
Anal. (C15H15N3OS) H, N, S.
3′-Br om oa cetop h en on e th iosem ica r ba zon e (3d ): δH
(400 MHz; DMSO-d6) 2.28 (s, 3H), 7.34 (t, 1H, J ) 8), 7.57 (d,
1H, J ) 8.4), 7.89 (d, 1H, J ) 7.6), 8.11 (s, 1H), 8.19 (s, 1H),
BrN4S) C, H, N.
3′-Br om oa cet op h en on e 4,4-d iet h yl-3-t h iosem ica r b a -
zon e (4e): δH (400 MHz; DMSO-d6) 1.20 (t, 6H), 2.28 (s, 3H),
3.73 (q, 4H), 7.39 (t, 1H, J ) 8.0), 7.59 (d, 1H, J ) 7.6), 7.76
(d, 1H, J ) 8.0), 7.95 (s, 1H), 9.45 (s, 1H); MS (EI) m/z 328.3
(MH+)
1-P yr id in -2-yleth a n on e th iosem ica r ba zon e (3a ): δH
(400 MHz; DMSO-d6) 2.39 (s, 3H), 7.38 (dt, 1H, J ) 1.2), 7.79
(dt, 1H, J ) 2.4), 8.13 (s, 1H), 8.42 (t, 2H, J ) 8.0), 8.57 (d,
1H, J ) 4.4), 10.31 (s, 1H); MS (EI) m/z 195.3 (MH+). Anal.
(C8H10N4S.0.5H2O) C, H, N, S.
1-P yr id in -3-yleth a n on e th iosem ica r ba zon e (3b): δH
(400 MHz; DMSO-d6) 2.28 (s, 3H), 7.35 (dd, 1H, J ) 4.4), 8.04
(s, 1H), 8.29 (d, 2H,J ) 6.8), 8.51 (d, 1H, J ) 3.6), 9.06 (s, 1H),
10.28 (s, 1H); MS (EI) m/z 0.195.3 (MH+). Anal. (C8H10N4S) C,
H, N, S.
1-P yr id in -4-yleth a n on e th iosem ica r ba zon e (3c): δH
(400 MHz; DMSO-d6) 2.30 (s, 3H), 7.90 (dd, 2H, J ) 2.0), 8.14
(s, 1H), 8.44 (s, 1H), 8.58 (dd, 2H, J ) 1.6), 10.42 (s, 1H); MS
(EI) m/z 195.3 (MH+). Anal. (C8H10N4S) C, H, N, S.
N′-(1-P yr id in -2-yleth ylid en e)h yd r a zin eca r bod ith ioic
a cid m eth yl ester (4b): δH (400 MHz; DMSO-d6) 2.26 (s, 3H),
2.50 (s, 3H), 7.44 (t, 1H, J ) 8.4), 7.67 (d, 1H, J ) 8.0), 7.90
(d, 1H,J ) 8.0), 8.65 (s, 1H); MS (EI) m/z 226.3 (MH+). Anal.
(C9H11N3S2) C, H, N, S.
1-(3-Acet ylp h en yl)et h a n on e t h iosem ica r b a zon e (3i):
δH (400 MHz; DMSO-d6) 2.35 (t, 6H), 7.40 (t, 1H, J ) 7.6),
7.97 (dd, 4H, J ) 1.6), 8.19 (s, 1H), 8.28(s, 2H), 10.22 (s, 2H);
MS (EI) m/z 309.4 (MH+). Anal. (C12H16N6S2‚H2O) H, N, S.
1-(6-Acetylp yr id in -2-yl)eth a n on e th iosem ica r ba zon e
(3j): δH (400 MHz; DMSO-d6) 2.44 (s, 6H), 7.78 (t, 1H, J )
7.6), 8.16 (broad s, 2H), 8.43 (d, 4H, J ) 8.4), 10.31 (s, 2H);
MS (EI) m/z 310.3 (MH+). Anal. (C11H15N7S2.1.5H2O) C, H.
1-(5-Acetyl-2,6-dim eth ylpyr idin -3-yl)eth an on e th iosem i-
ca r ba zon e(3k ): δH (400 MHz; DMSO-d6) 2.21 (s, 6H), 2.29
(t, 6H, J ) 6.8), 7.68 (d, 1H, J ) 2.8), 8.24 (broad s, 4H), 10.23
(s, 2H); MS (EI) m/z 338.1 (MH+). Anal. (C13H19N7S2) H, N, S.

Thiosemicarbazone Cysteine Protease Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 12 3219
carboxaldehyde thiosemicarbazone (3-AP) using a single intra-
venous dose schedule. Cancer Chemother. Pharmacol. 2002, 50,
223-229.
Ack n ow led gm en t. This research was supported by
the Sandler Family Support Foundation and NIH grant
AI35707. D.C.G. was also supported by a NIH Postdoc-
toral Training Fellowship (CA 09270-27). K.C. gratefully
acknowledges the award of a Sandler Sabbatical Fel-
lowship from the Sandler Centre For Basic Research
In Parasitic Diseases (University of CaliforniasSan
Francisco). The University of Cape Town and National
Research Foundation of South Africa are thanked for
approving leave of absence and additional support,
respectively (K.C.).
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Su p p or tin g In for m a tion Ava ila ble: Charts for com-
pounds with low efficacy against enzymes and organisms (1S,
2S, 3S). Chart showing toxicity of select compounds (4S).
Tables of elemental analyses and mass spectrometric and 1H
NMR data. This material is available free of charge via the
Internet at http://pubs.acs.org.
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