Synthesis and evaluation of isatins and thiosemicarbazone derivatives against cruzain, falcipain-2 and rhodesain

Article abstract of DOI:10.1016/S0960-894X(03)00756-X

While commercial isatins were practically inactive against the target proteases, thiosemicarbazone derivatives were found to be active. The most active compound from the series displayed an inhibitory IC₅₀ value of 1 μM against rhodesain. One thiosemicarbazone was found to be active against all three proteases with inhibitory IC₅₀ values of 10 μM or less. A combination of N-benzylation and appropriate substitution on the aromatic portion of the isatin scaffold was generally found to be beneficial especially against cruzain for ketone inhibitors.

Full text of DOI:10.1016/S0960-894X(03)00756-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3527–3530
Synthesis and Evaluation of Isatins and Thiosemicarbazone
Derivatives against Cruzain, Falcipain-2 and Rhodesain
Idan Chiyanzu,a Elizabeth Hansell,b Jiri Gut,c Philip J. Rosenthal,c James H. McKerrowb
and Kelly Chibalea,*
aDepartment of Chemistry, University of Cape Town, Rondebosch 7701, South Africa
bDepartment of Pathology, Tropical Diseases Research Unit, University of California San Francisco,
513 Parnassus Avenue, Box 0511, HSW 511, San Francisco, CA 94143, USA
cDepartment of Medicine, San Francisco General Hospital, University of California San Francisco, 1001 Potrero Avenue,
Box 0811, San Francisco, CA 94110, USA
Received 17 April 2003; accepted 9 July 2003
Abstract—While commercial isatins were practically inactive against the target proteases, thiosemicarbazone derivatives were found
to be active. The most active compound from the series displayed an inhibitory IC50 value of 1 mM against rhodesain. One thio-
semicarbazone was found to be active against all three proteases with inhibitory IC50 values of 10 mM or less. A combination of
N-benzylation and appropriate substitution on the aromatic portion of the isatin scaffold was generally found to be beneficial
especially against cruzain for ketone inhibitors.
# 2003 Elsevier Ltd. All rights reserved.
Isatin 1 is a natural product found in a number of plants
including those of the genus Isatis.1 It has also been
found as a metabolic derivative of adrenaline in
humans.2 Various derivatives of isatin are known to
possess a range of pharmacological properties including
antiprotozoal activities.3,4 Within the context of enzyme
inhibitors, isatins (also known as 2,3-dioxindoles) have
seen recent applications in the inhibition of cysteine and
serine proteases.5,6 Thus isatin is a biologically validated
starting point for the design and synthesis of chemical
libraries directed at these targets.7 Due to the privileged
nature of isatin, libraries designed and synthesized
around the basic structure of this scaffold should yield
medicinally active compounds with high hit rates at
significantly reduced library size compared to large
classical libraries obtained from combinatorial chem-
istry efforts based on non-privileged templates.
in trypanosomes (cruzain and rhodesain) and malaria
parasites (falcipain-2) respectively.8À11 It is noteworthy
that a series of highly potent reversible ketone inhibitors
of cruzain have previously been disclosed.12 This has
recently culminated in the first crystal structures of these
inhibitors being solved.13 We reasoned that the presence
of the ketone functionality adjacent to the aromatic ring
on the isatin scaffold provides a diversity point for
accessing the corresponding thiosemicarbazones, a class
of compounds recently identified as potent anti-
trypanosomal inhibitors of cruzain.14 Coupled with the
molecular simplicity, potentially cost effective synthesis
and non-peptidic nature, this makes isatins attractive
scaffolds for antiparasitic drug discovery. Cost-effective
synthesis should always be a consideration in deciding
on potential new antiparasitic agents given the
prevalence of major parasitic diseases in poor countries.
The aforementioned antiprotozoal and cysteine pro-
tease inhibitory activities of isatin derivatives prompted
us to investigate this class of compounds as potential
ketone inhibitors of parasitic cysteine proteases identified
The initial aim of our work on isatins is focused on
identifying the most promising commercially available
scaffold and ideal sites for introducing chemical diver-
sity before launching a full scale medicinal chemistry
programme. In this paper, we report on the evaluation
of isatin-based synthetic thiosemicarbazones 2 and N-
substituted isatins 3 against cruzain, falcipain-2 and
rhodesain (Fig. 1).
*Corresponding author. Tel.: +27-21-650-2553; fax: +27-21-689-
7499; e-mail: chibale@science.uct.ac.za
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00756-X

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I. Chiyanzu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3527–3530
Figure 1.
Scheme 1 depicts the straightforward and simple synth-
esis of target compounds from commercially available
isatins. The simplicity of the chemistry cannot be over-
emphasised. Reaction of commercially available isatins
with thiosemicarbazide in ethanol provided target thio-
semicarbazones 2. Isatins 3 were synthesized via alkyl-
ation, acylation and sulfonylation of the same
commercially available starting materials. Acetonitrile
was used as the solvent for all alkylation reactions while
tetrahydrofuran was used for all acylation and
sulfonylation reactions. The reactions mediated by
potassium fluoride supported on alumina provided
derivatives in generally high yields and purity as deter-
Scheme 1.
subject of a recent review.15 Its envisaged use in the
synthesis of N-alkylated thiosemicarbazones compli-
ments a recent literature report on the solid phase
synthesis of isatin b-thiosemicarbazone Mannich bases.
In this report a trityl resin-supported thiosemicarbazide
captures isatins from solution, permits N-alkylation and
is then easily removed from the solid support.16 Carba-
mates 3d and 3e were prepared from isatins using ben-
zylchloroformate in the presence of triethylamine and 4-
dimethylaminopyridine. All new compounds gave 1H
NMR, FAB-MS and microanalysis data consistent with
their structures.
1
mined by H NMR analysis before further purification
by recrystallisation to obtain analytically pure samples.
The results obtained with this support-bound base
augur well for the high throughput parallel solution
phase synthesis of libraries of these derivatives when
used in conjunction with scavenging protocols. Alkyl-
ation reactions of isatins using a support-bound base, 2-
tert-butylimino-2-diethylamino-1,3-dimethylperhydro-
1,3,2-diazaphosphorine on polystyrene (BEMP) in con-
junction with scavenging protocols have been reported.7
The major drawback of BEMP in these reactions is the
very high cost of this base. On the other hand KF-Al2O3
is relatively inexpensive and easy to prepare. The use of
this support-bound base in a variety of reactions is the
IC50 values against recombinant proteins (cruzain and
falcipain-2) were determined essentially as described
previously.14,17 IC50 values for rhodesain (recombinant,
cysteine protease from T. brucei rhodensiense, EC
3.4.22.X) were similarly determined at 3 nM. The results
are presented in Tables 1 and 2. Data for commercially
available isatins is included for comparison purposes.
From Table 1, it is immediately clear that commercially
available isatins were practically inactive while the
corresponding thiosemicarbazones showed some
activity (albeit modest in many cases). The most notable
Table 1. Inhibition of cruzain, falcipain-2 and rhodesain by commercial isatins and thiosemicarbazone derivatives 2
Compd
R1
R2
X
IC50 (mM)
Cruzain
Falcipain-2
Rhodesain
Isatin
5-Methylisatin
5-Fluoroisatin
5-Chloroisatin
5-Bromoisatin
5-Iodoisatin
5-Nitroisatin
5-(Trifluoromethoxy)isatin
5,7-Dimethylisatin
2a
2b
2c
2d
2e
2f
2g
2h
2i
H
Me
F
Cl
Br
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
H
Me
Me
O
O
O
O
O
O
O
O
> >10
> >10
> >10
> >10
> >10
> >10
> >10
> >10
> >10
8
NE
NE
NE
NE
NE
NE
42.6
NE
NE
38.8
43.9
38.6
32.8
29.6
28.7
4.4
> >10
ND
ND
ND
ND
ND
> >10
> >10
ND
3.5
I
NO2
CF3O
Me
H
Me
F
Cl
Br
I
O
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
N–NHC(S)NH2
20–50
30
21
15
15
6
7
1
17
20
9
30
16
10.5
NO2
Me
Cl
13.2
9.4
15
3
NE, no effect; ND, not determined.

I. Chiyanzu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3527–3530
3529
Table 2. Inhibition of cruzain, falcipain-2 and rhodesain by N-substituted isatins derivatives 3
Compd
R1
R2
R3
IC50 (mM)
Cruzain
Falcipain-2
Rhodesain
1-Methylisatin
1-Phenylisatin
3a
H
H
H
H
H
H
Me
Ph
> >10
> >10
> >10
NE
NE
NE
ND
ND
> >10
3b
3c
3d
3e
3f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
> >10
> >10
> >10
80
NE
NE
NE
NE
NE
21.9
9.2
15
100
H
30
Me
Me
Me
Me
Cl
Cl
Cl
Cl
I
> >10
> >10
> >10
> >10
> >10
> >10
> >10
> >10
> >10
> >10
> >10
2
3g
3h
3i
2.8
6
31.2
34.6
46.9
NE
ND
ND
3j
> >10
90
3k
3l
80
3m
3n
90
F
> >10
NE, no effect; ND, not determined.
thiosemicarbazone scaffold from this limited series is 2i
which showed activity against all targets with IC50
values of 10 mM or less. The most promising scaffolds in
the series for the homologous cruzain and rhodesain are
2a and 2f while, in addition to 2i, thiosemicarbazone 2g
provides a potential scaffold for future development of
falcipain-2 inhibitors.
Although the number of compounds synthesized is
small, from the data presented in Table 2, it is clear that
lack of substitution at the 5-position (R1 substituent) of
isatin did not generally improve activity. Results from
the commercially available 1-methylisatin and 1-phenyl-
isatin as well as from compounds 3a–3d confirm this. In
comparison to the data for N-unsubstituted commercially

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I. Chiyanzu et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3527–3530
available isatins (Table 1), it is evident that N-benzyla-
tion in combination with substitution at position 5 of
the isatin scaffold seems favorable. Cruzain was the
most sensitive protease to N-benzylation of the isatin
scaffold with rhodesain being the least sensitive.
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N-substituted derivatives is 3h which shows activity
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Acknowledgements
This material is based upon work supported by the
National Research Foundation of South Africa under
Grant number 2053362 (K.C.).
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