Privileged structure-guided synthesis of quinazoline derivatives as inhibitors of trypanothione reductase

Article abstract of DOI:10.1016/j.bmcl.2009.04.060

Novel quinazoline-type compounds were designed as inhibitors of the parasite specific enzyme trypanothione reductase (TR), and their biological activities were evaluated. Some of our compounds inhibited TR, showed selectivity for TR over human glutathione

Full text of DOI:10.1016/j.bmcl.2009.04.060

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3031–3035
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Privileged structure-guided synthesis of quinazoline derivatives as inhibitors
of trypanothione reductase
Andrea Cavalli a,b, Federica Lizzi a, Salvatore Bongarzone a, Reto Brun c,
R. Luise Krauth-Siegel d, Maria Laura Bolognesi a,
*
a Department of Pharmaceutical Sciences, Alma Mater Studiorum-Bologna University, Via Belmeloro 6, I-40126 Bologna, Italy
b Department of Drug Discovery and Development, Italian Institute of Technology (IIT), Via Morego 30, I-16163 Genova, Italy
c Swiss Tropical Institute, Socinstrasse 57, CH-4002 Basel, Switzerland
d Biochemie-Zentrum der Universität Heidelberg (BZH), Im Neuenheimer Feld 504, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 February 2009
Revised 6 April 2009
Accepted 6 April 2009
Available online 20 April 2009
Novel quinazoline-type compounds were designed as inhibitors of the parasite specific enzyme trypano-
thione reductase (TR), and their biological activities were evaluated. Some of our compounds inhibited
TR, showed selectivity for TR over human glutathione reductase, and inhibited parasite growth in vitro.
We propose that the quinazoline framework is a privileged structure that can be purposely modified to
design novel TR inhibitors. Furthermore, the use of privileged motifs might emerge as an innovative
approach to antiparasitic lead candidates.
Keywords:
Neglected tropical disease
Trypanosoma
Ó 2009 Elsevier Ltd. All rights reserved.
Leishmania
Leishmaniasis
Trypanosomiasis
Docking simulations
Neglected tropical diseases (NTD) do not have high visibility in
Western societies. Their name is also related to the fact that they
are often neglected when health agendas and budgets are set,
and at the level of research and development.1 This is despite the
one billion people worldwide suffering from NTD, and the inappro-
priateness of the currently available drugs, which have problems
with safety, administration, cost, and, increasingly, resistance.2
NTD, such as different forms of leishmaniasis and trypanosomiasis,
remain difficult to cure and often lead to fatalities. There are sev-
eral current medicinal chemistry strategies for tackling this prob-
lem. Among others, an exciting opportunity has emerged from
the genome-sequencing projects of these parasites.3 The knowl-
edge of the total genomes offers the possibility of identifying novel
drug targets that are vital to the pathogens but are absent in their
hosts, and that are open to selective inhibition.4,5
Trypanothione (T[SH]2; bis-(glutathionyl)-spermidine), is un-
ique to members of the order Kinetoplastida. T[SH]2 is the main
low molecular mass thiol in these parasitic protozoa and is respon-
sible for maintaining the intracellular reducing environment.
T[SH]2 is kept reduced by the essential flavoenzyme trypanothione
reductase (TR, E.C. 1.6.4.8).6 TR is an FAD-disulfide oxidoreductase.
The catalyzed NADPH-dependent reduction of trypanothione
disulfide (TS2) to T[SH]2 is homologous to the reduction of glutathi-
one disulfide (GSSG) by glutathione reductase (GR) in other organ-
isms, such as in cells of the mammalian host. Both enzymes have
three residues that are directly involved in catalysis, the redox ac-
tive dithiol/disulfide (Cys53-Cys58 in Trypanosoma cruzi TR) and a
His residue (His4610 in T. cruzi TR), which is provided by the second
subunit of the homodimer.7,8 Although TR and GR share 40% of all
residues, two major characteristics account for the selectivity of
inhibitors against the two enzymes. Because of the presence of
an extra protonated amino group in T[SH]2, TR possesses a nega-
tively charged disulfide substrate binding site whereas that of hu-
man GR carries an overall positive charge.8,9 Moreover, TR has a
larger active site to accommodate its bulkier substrate. This dis-
tinct profile, together with the availability of the coordinates of
several crystal structures, makes TR a validated target in the search
for antiparasitic drugs.10–17
The aim of the present study was to develop, by investigating
the active site topology of TR, novel quinazoline-based ligands as
selective TR inhibitors. We started from the consideration that a
scaffold protonated at physiological pH is a prerequisite for ensur-
ing TR/GR selectivity. However, antiparasitic molecules with posi-
tively charged groups, such as amidine or guanidine moieties, are
in general poorly orally available.18 Therefore, we searched for
new chemical entities with superior intrinsic drug-like properties.
Privileged structures with their inherent affinity for diverse
* Corresponding author. Tel.: +39 051 2099718; fax: +39 051 2099734.
E-mail address: marialaura.bolognesi@unibo.it (M.L. Bolognesi).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.04.060

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A. Cavalli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3031–3035
biological targets, represent an ideal source of core scaffolds for the
design of libraries targeted at various receptors.19 In addition, the
likely drug-like properties of privileged structures and substruc-
tures might produce more drug-like compound libraries and
leads.20 Properly substituted 2,4-diamino-quinazolines are privi-
leged sub-structures protonated at physiological pH.21 Numerous
quinazoline-based libraries have been synthesized, and have pro-
ven to be extremely powerful tools to the rapid discovery and opti-
mization of ligands for a wide variety of targets,22 also in the field
of antiparasitic drugs.23 On these bases, a series of 2-piperazin-1-
yl-quinazolin-4-ylamine derivatives (1–7) was, for the first time,
designed and tested against TR. Docking of the quinazoline core
1-(4-(4-amino-6,7-bis(2-(dimethylamino)ethoxy)quinazolin-2-
yl)piperazin-1-yl)ethanone showed that such a scaffold was able to
interact with the TR active site. Then, all compounds were de-
signed in such a way as bearing structural features of typical TR
inhibitors, namely the overall positive charge together with ex-
tended hydrophobic parts.24 Different moieties, such as methoxy
NH2
2,4-diamino-quinazoline
core fragment protonated
at physiological pH
O
R1
R1
N
O
N
H
N
N
R2
R1 CH3
R2
R1 CH2CH2N(CH3)2
O
O
O
O
NH
NH
O
N
H
O
O
1
2
O
7
6
N
O
H
O
O
4
3
5
Figure 1. Design strategy for compounds 1–7.
Figure 2. (A) Binding mode of a statistically populated cluster of 1-(4-(4-amino-6,7-dimethoxyquinazolin-2-yl)piperazin-1-yl)ethanone (carbon atoms cyan) at the TR
binding site. The key amino acids responsible for the binding are explicitly reported. (B) Binding mode of 6 at the TR binding site. The electrostatic potential of TR (C) and GR
(D), calculated by the APBS plug-in of Pymol, is shown: negative and positive electrostatic potential is red and blue, respectively. The electrostatic potential helps explaining
the rather good TR/GR selectivity of 6.

A. Cavalli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3031–3035
3033
and dimethylaminoethoxy groups in R1, and indole, quinone, and
tetrahydroquinoline moieties in R2, were attached to the quinazo-
line core to provide additional interactions with the target (Fig. 1).
Figure 2A shows the suggested binding mode of the starting
fragment 1-(4-(4-amino-6,7-bis(2-(dimethylamino)ethoxy)qui-
nazolin-2-yl)piperazin-1-yl)ethanone. An electrostatic interaction
could be identified between the protonated N1 nitrogen of the qui-
nazoline moiety and Glu4660. The substituents on position R1 were
required to capture additional interactions with Glu19 and Trp22
that could confer selectivity versus the GR enzyme. Then, struc-
tural modifications on position R2 could be envisaged to capture
further interactions with Lys62. The binding mode of 6, which
can be considered the end point of the above described design
ther docking simulations were carried out, being however the
binding mode of quinone containing compounds not univocally
determined. Indeed, such molecules could bind to TR enzyme at
different sites, thus accounting for their non-competitive or mixed
inhibition mechanisms. This has been already reported in the liter-
ature for similar quinone derivatives.24
The novel substituted 2-piperazin-1-yl-quinazolin-4-ylamines
1–6 were synthesized using the readily accessible methodologies
developed for this class of compounds, whereas the synthesis of
725 was previously reported.25,26 The only new chemistry con-
cerned the insertion of the dimethylaminoethyl chain in positions
6 and 7 of the quinazoline ring of 8,27 to afford 9: the nucleophilic
substitution strictly required the use of cesium carbonate as a base
(Scheme 1, complete synthetic details and spectroscopic analysis
are available online in Supplementary data). Afterwards, the proto-
col proceeded by allowing 9 to react with excess piperazine. Then,
intermediates 10 and 11 were reacted with quinone, indole, and
tetrahydroisoquinoline derivatives, affording directly, or after a
deprotection step, the final compounds 1–7 (Scheme 2).
strategy, is reported in Figure 2B. Indeed, a cation-p interaction
was detected between the indole ring of 6 and Lys62, as well an
electrostatic interaction between the protonated nitrogen of the
2-dimethylaminoethoxy chain and both Glu19 and Trp22. A fur-
ther interaction was observed between the chain and the OH group
of Tyr111. In Figure 2C and D, the electrostatic surfaces of TR and
GR, respectively, are depicted. While TR bears a negative electro-
static potential, GR has positive values for the potential at the same
region of the active site. This electrostatic feature could account for
the TR/GR selectivity of 6 and parent molecules of this series. Fur-
The assays contained NADPH, TR, and 100 lM of each inhibitor
and TS2 which corresponds to a substrate concentration of 5 ꢀ Km.
All of the quinazolines proved to be inhibitors of TR, except 1 and 2.
The inhibition ranged from 23% to 76%, with the naphthoquinone
N
NH2
N
N
NH2
N
NH2
N
O
O
O
O
O
O
b
a
H
H
N
N
N
Cl
N
Cl
N
NH
N
8
9
10
Scheme 1. Reagents and conditions: (a) CsCO3, (2-chloroethyl)-dimethylamine, N2, DMF, reflux, 2 h, 10%; (b) piperazine, isoamylic alcohol, 160 °C, 30 h, 34%.
NH2
O
X
X
N
O
N
N
O
O
N
5: X = CH3
7: X = CH2CH2NMe2
NH2
N
NH2
N
O
O
O
O
O
X
X
O
O
O
c
O
b
N
N
N
N
O
N
O
NH2
O
N
a
4
O
N
HO
O
O
O
X
X
N
H
O
3: X = CH3
6: X = CH2CH2NMe2
O
N
N
N
H
N
11: X = CH3
10: X = CH2CH2NMe2
H
O
O
OH
NCbz
OH
NCbz
a
NH2
a
NH2
O
O
N
O
N
R
N
N
N
R
N
O
N
N
N
N
O
12: R = Cbz
1: R = H
13: R = Cbz
2: R = H
d
d
O
Scheme 2. Reagents and conditions: (a) EDCI, HOBt, DMF, rt, overnight, 24–61%; (b) CHCl3, reflux, 96 h, 4%; (c) EtOH, reflux, 96 h, 47%; (d) MeOH, 10 % Pd/C, 90–95%.

3034
A. Cavalli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3031–3035
derivatives 5 and 7 being the most effective inhibitors (Table 1).
From these results, we conclude that the 2,4-diaminoquinazoline
scaffold is a good motif for TR recognition. However, the presence
of a suitable capping fragment is fundamental for activity. In fact,
the tetrahydroisoquinolines 1 and 2 did not inhibit the enzyme,
whereas quinones-bearing molecules 4, 5 and 7, by virtue of the
known quinone ability to interact with TR, display a more pro-
nounced inhibition.28 For 4, 5, and 7 the inhibitor constants were
determined. The naphthoquinone derivatives 5 and 7 inhibited
The anti-parasitic potential of all compounds was analyzed in
light of the WHO/TDR criteria, as previously described.31 Table 2
summarizes the activity of compounds 1–7 against Trypanosoma
brucei rhodesiense, T. cruzi, Leishmania donovani, and Plasmodium
falciparum, as well as their cytotoxicity against L6 cells. The results
show good correlation between enzyme inhibition and trypanoci-
dal activity for compound 5, which is a TR inhibitor and the most
active against the different trypanosomatids. On the other hand,
compounds 1 and 2, inactive at 100 lM against TR, also interfered
TR with Ki-values of about 10
l
M, not dissimilar to that of 4,40-
with parasite growth, confirming the potential of the quinazoline
scaffold for antiparasitic activity. As a drawback, some derivatives
showed cytotoxicity towards L-6 myoblast cells. However, against
the extracellular African trypanosomes compounds 1–5 were
7–27-fold more active than against the mammalian control cells.
The mammalian cytotoxicity proved to be highest for the naphtho-
quinone-containing quinazolines 5 and 7. It can, therefore, be ar-
gued that toxicity can be tuned down by properly modifying or
replacing the naphthoquinone moieties. It is encouraging that 11
itself was not found to modify cell proliferation.25 It is also impor-
tant to emphasize that all the designed compounds are active in
the context of the cell environment and therefore are able to pen-
etrate cells and reach the intracellular target.32 However, we can
not exclude that other proteins are (also) modulated by our privi-
leged quinazoline compounds. In this respect, we were pleased to
note that all seven derivatives exhibited remarkable activity
against P. falciparum, with IC50-values in the micromolar and sub-
micromolar range, although P. falciparum does not have the trypa-
nothione metabolism.
bis(4-benzyloxy-3-methoxybenzimidoylamino)di-cyclohexylme-
thane (14, see SI Fig. 2), used as positive control.24 The degree of
inhibition is comparable to that exerted by other known TR inhib-
itors such as mepacrine (Ki 19
5 l
M)29 and clomipramine (Ki
6.5 0.6
l
M).30 These results suggest that 5 and 7 are a good start-
ing point for a medicinal chemistry program aimed at identifying
derivatives with higher TR inhibitory potency. Inhibition of TR
was not purely competitive, confirming that quinone-bearing com-
pounds might bind to multiple sites.
Compounds 5 and 7 were also tested against human GR. At
10
pound 5 did not show any inhibition of GR in the presence of
100 M GSSG. 125 or 250 M of compound 7 resulted in about
10 and 20% inhibition, respectively. Taking into account that
100
lM, the highest concentration soluble in the assay buffer, com-
l
l
l
M GSSG corresponds to only 1.5 ꢀ Km, inhibition of the host
GR is negligible when compared to that of the parasite TR. Thus,
both inhibitors displayed selectivity for the parasite enzyme, con-
firming the design rationale.
The quinazoline ring is a heterocyclic motif that meets the
requirements of a privileged structure that is, according to Evans’
original definition, ‘a single molecular framework able to provide
ligands for diverse receptors’.33 The inherent promiscuity of privi-
leged scaffolds might also be a drawback, as selectivity might be-
come an issue. However, several reports confirm that small
modifications in and around the core cause difference in activity,
giving raise to the desired selectivity.34 Compounds based on this
motif might have favourable pharmacokinetic features, a high de-
gree of drug-likeness.35 In antiparasitic drug discovery, where eco-
nomic reality raises the need for inexpensive chemotherapeutics,
the importance of using pre-validated chemical scaffolds might ac-
quire relevance. Cost of goods is a crucial issue because the drugs
will have to be cheap to produce and distribute. The drugs should
also be orally available since other routes of administration are
problematic in rural settings.18 In this scenario, this small library
holds promise for the further exploration of quinazoline substruc-
ture for interaction with TR, with the potential to generate,
through iterative medicinal chemistry and DMPK (Drug Metabo-
lism and Pharmacokinetics) network activities, preliminary SAR
and in vivo activities. All these considerations, especially those re-
lated to economic issues, indicate that the development of quinaz-
oline- or other privileged structure-based antiparasitic compounds
might have potential for the research and development of new
drugs for NTD.
Table 1
Inhibition of T. cruzi TR by quinazoline derivatives
No. [Inhibitor]
M)
% inhibition of TR at
100 50
Ki
(lM)
Inhibition type
(l
lM
lM
(TS2)
(TS2)
1
2
3
4
5
100
100
100
100
25
0
0
0
0
nda
nd
nd
32
23
75
57
13
69
53
4
2
Non competitive
Mixed-type, Ki0
7.5
22
6 lM
6
7
100
40
33
76
97
44
79
94
nd
11
2
3
Non competitive
Mixed-type, Ki0 16
lM
14
100
a
Not determined.
Table 2
Antiparasitic activity of compounds 1–7 in whole cell assays. IC50-values (
l
M) for
amastigote forms of T. cruzi in L6 cells, bloodstream trypomastigote forms of T. brucei
rhodesiense, axenic amastigote form of Leishmania donovani, and the intra-erythro-
cytic form of Plasmodium falciparum are reported. Activity for mammalian L6 cells
(cytotoxicity) is also shown. 15, 16, 17, 18 and 19 are the reference compounds
benznidazole, melarsoprol, miltefosine, chloroquine, and podophyllotoxin,
respectively
Compd
T. cruzi
T. b. rhodesiense
L. donovani
P. falciparum
L6
Acknowledgements
1
2
3
4
5
6
7
15
16
17
18
19
14.3
27.6
10.2
35.6
3.68
>50
1.81
5.37
2.21
1.98
0.12
21.8
2.54
>67
>67
42.2
45.7
1.78
>54
67.1
2.10
1.24
1.35
3.01
0.53
2.19
0.19
14.2
37.2
19.6
52.5
2.76
>161
5.25
This work was supported by the University of Bologna (MLB)
and the Deutsche Forschungsgemeinschaft (SFB 544, project B3
to LKS). Edith Röckel and Natalie Dirdjaja are acknowledged for
excellent technical assistance.
10.5
1.48
Supplementary data
0.01
0.34
0.22
Supplementary data (the detailed synthetic and biological pro-
cedures, as well as full spectral characterization of the new com-
0.012

A. Cavalli et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3031–3035
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