5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors of Trypanosoma brucei bloodstream forms

Article abstract of DOI:10.1016/j.bmc.2016.06.023

Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and synthesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanosomal activity in double digit nanomolar concentrations and a selectivity index of three orders of magnitude versus murine macrophage cells.

Full text of DOI:10.1016/j.bmc.2016.06.023

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-Substituted 3-chlorokenpaullone derivatives are potent inhibitors
of Trypanosoma brucei bloodstream forms
Oliver C. F. Orban a,y, Ricarda S. Korn a,y, Diego Benítez b, Andrea Medeiros b,c, Lutz Preu a, Nadège Loaëc d,
Laurent Meijer d, Oliver Koch e, Marcelo A. Comini b, , Conrad Kunick a,f,
⇑
⇑
a Technische Universität Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstraße 55, D-38106 Braunschweig, Germany
b Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, CP 11400 Montevideo, Uruguay
c Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, Montevideo, Uruguay
d ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, France
e Faculty of Chemistry and Chemical Biology, TU Dortmund University, Otto-Hahn-Straße 6, 44227 Dortmund, Germany
f Technische Universität Braunschweig, Center of Pharmaceutical Engineering (PVZ), Franz-Liszt-Straße 35A, D-38106 Braunschweig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanothione synthetase is an essential enzyme for kinetoplastid parasites which cause highly disabling
and fatal diseases in humans and animals. Inspired by the observation that N(5)-substituted paullones
inhibit the trypanothione synthetase from the related parasite Leishmania infantum, we designed and syn-
thesized a series of new derivatives. Although none of the new compounds displayed strong inhibition of
Trypanosoma brucei trypanothione synthetase, several of them caused a remarkable growth inhibition of
cultivated Trypanosoma brucei bloodstream forms. The most potent congener 3a showed antitrypanoso-
mal activity in double digit nanomolar concentrations and a selectivity index of three orders of
magnitude versus murine macrophage cells.
Received 11 April 2016
Revised 9 June 2016
Accepted 11 June 2016
Available online xxxx
Keywords:
Leishmaniasis
Neglected tropical diseases
Paullone
Ó 2016 Elsevier Ltd. All rights reserved.
Trypanosomiasis
Trypanothione synthetase
Trypanosoma brucei
1. Introduction
asites which are injected by tsetse flies during a blood meal. With-
out proper treatment, the infection is fatal in its final stage. Chagas
Of the seventeen ‘neglected tropical diseases’ designated by the
World Health Organization, three groups of infectious diseases
(Human African trypanosomiasis, Chagas disease, and Leishmania-
sis) are caused by hemoflagellated parasites belonging to the fam-
ily Trypanosomatidae.1 The Human African Trypanosomiasis (HAT,
also called sleeping sickness) is caused by Trypanosoma brucei par-
disease is a life-threatening infection caused by Trypanosoma cruzi
which is transmitted through contact with biting triatomine bugs.
The different forms of leishmaniasis are caused by parasites of var-
ious Leishmania species which use biting female sandflies as vec-
tors. The most dangerous form is visceral leishmaniasis (VL). It
affects internal organs, a deadly situation if patients do not receive
a proper medication.1 While all these infections occur in different
forms and stages, they have in common that mainly the poverty-
stricken population in tropical and subtropical areas of the third
world is affected and that they may lead to serious impairment
of body functions or a fatal outcome if left untreated. The number
of drugs available for a curative treatment of these infections is
rather limited, with most of them suffering from serious shortcom-
ings such as high costs, long treatment periods, complicated appli-
cation procedures, need for hospitalization, or severe side effects.
Moreover, in some areas parasites have developed resistance
against drugs that have been successfully used for treatment over
decades, the most prominent example being the resistance of
Leishmania parasites against pentavalent antimonials in the
Abbreviations: ACN, acetonitrile; CDK, cyclin-dependent kinase; CLK, cdc2-like
kinase; CK, casein kinase; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s
modified Eagle’s medium; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfox-
ide; DYRK1A, dual specificity tyrosine phosphorylation-regulated protein kinase
1A; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGTA, ethylene
glycol-bis(b-aminoethyl ether)-N,N,N0,N0-tetraacetic acid; FCS, fetal calf serum; FSC,
forward scatter; GSK-3, glycogen synthase kinase-3; HEPES, 4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid; PyBOP, benzotriazol-1-yl-oxytripyrrolidinophos-
phonium-hexafluorophosphate; SSC, side scatter; SD, standard deviation; THF,
tetrahydrofuran; TryS, trypanothione synthetase.
⇑
Corresponding authors. Tel.: +598 25220910 (M.A.C.), +49 5313912751 (C.K.).
E-mail addresses: mcomini@pasteur.edu.uy (M.A. Comini), c.kunick@
tu-braunschweig.de (C. Kunick).
y
Co-first authors.
http://dx.doi.org/10.1016/j.bmc.2016.06.023
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Orban, O. C. F.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.023

2
O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
province Bihar in India.2,3 Despite the fact that leishmaniasis and
trypanosomiasis account for 2 million new cases of infection per
year, the development of new drugs is unlikely to be profitable,
since most affected patients are unable to afford expensive new
medicines. Nevertheless, to overcome problems with emerging
resistances and safety issues, the development of novel efficacious
and safer drugs against Trypanosoma and Leishmania infections is
urgently needed.4,5 In order to avoid cross-resistance with estab-
lished medications, the novel drugs should represent new chemo-
types and target novel and indispensable parasite’s biomolecules.
In this respect, the thiol-redox metabolism of trypanosomatids
offers unique opportunities for a selective and efficacious impair-
ment of pathogen’s viability. In Trypanosomatidae parasites, the
dithiol trypanothione (bis-glutathionylspermidine) substitutes
glutathione as major redox co-substrate that supports the intracel-
lular redox homeostasis providing protection against oxidative
stress and reducing equivalents for the biosynthesis of nucleic
acids. The enzyme trypanothione synthetase (TryS) synthesizes
trypanothione by ATP-dependent conjugation of spermidine with
two molecules of glutathione (Fig. 1).6–12
H
N
HN
N
CH3
HN
N
Aryl
H
N
O
4
O
5
O
O
6
3
O
7
Cl
2
1
8
HN
12
R
HN
HN
9
11
CF3
Br
10
1a (R = Br)
1b
2
3
(R = NO2)
Chart 1. Paullones mentioned in the text: kenpaullone (1a), alsterpaullone (1b),
FS-554 (2), and title compounds 3.
chain at the N(5)-position (e.g., 2) inhibited both recombinant
L. infantum TryS (LiTryS) and the in vitro growth of L. infantum
promastigotes (insect stage).19,20 The most potent derivative
(IC50 0.15 lM and EC50 12 lM), containing halogen substituents
in position 3 (chloro) and 9 (bromo), produced a significant deple-
tion of trypanothione in L. infantum, which confirmed its on-target
effect.19,20 We here report an approach to develop new paullones
with potent growth inhibitory activity against the infective form
of T. brucei. The new compounds were also screened for their
anti-TryS activity against the enzyme from three major pathogenic
species of trypanosomatids: T. brucei, T. cruzi and L. infantum.
Details of the chemical mechanisms catalyzed by TryS have
been reported.13–15 TryS has been suggested as chemotherapeutic
target against trypanosomatid parasites based on its demonstrated
indispensability for T. brucei16–18 and L. infantum19 and the com-
plete lack of homologue sequences in the human host. Recently,
N(5)-substituted paullones were identified as inhibitors of
TryS.19,20
Paullones (Chart 1) are a group of 7,12-dihydroindolo[3,2-d][1]benz-
azepin-6(5H)-ones which besides other biological properties21–23
display inhibitory activities against mammalian protein kinases
of the CMGC superfamily, e.g., cyclin-dependent kinases
(CDKs)24–26 and glycogen synthase kinase-3 (GSK-3).27,28
Kenpaullone (1a),29 the prototype of the series, is used as a stan-
dard GSK-3 inhibitor which displays selectivity versus a wide array
of protein kinases (e.g., ERK1, DYRK1A, VEGF-R2, PLK1, INS-R).30,31
Of note, paullones bind to the ATP binding site of kinases via the
nitrogen atom in 5-position which acts as hydrogen bond donor.32
In consequence, N(5)-substituted paullones lose the kinase inhibi-
tory activity.33
2. Drug design and synthesis
The N(5)-substituted paullone FS-554 (2) inhibited very selec-
tively recombinant LiTryS but only moderately the trypanosomal
TryS. Here we employed a structure-based rational approach
aimed to design novel paullones with increased TryS inhibitory
activity and a concomitant selective anti-parasitic activity. A model
of Leishmania major TryS (LmTryS) with mechanistically important
regions involved in substrate (ATP) binding was previously gener-
ated.13 Analysis of the binding mode of ATP to LmTryS revealed
that it is located in a large and solvent accessible pocket, anchored
by a double hydrogen bond motif between the aminopyrimidine
substructure and the backbone amide groups of Gln583 and
Phe585 of the protein. This hydrogen bonding pattern to an
extended part of the protein chain shows a high similarity to the
Moreover, previous studies involving paullones as anti-TryS
scaffolds revealed that substitution of 4-azapaullones in position
9 or 11 with alkyl or aryl groups is detrimental for target inhibi-
tion34, whereas paullones harboring a methyl ethylendiamine side
SH
O
H
H
N
H2N
CO2H
NH2
N
2
+
H2N
N
H
CO2H
GlutathioneO
Spermidine
2 ATP
2 ADP
Trypanothione
Synthetase (TryS)
SH
O
O
O
CO2H
NH2
H
N
H
N
H
N
H
H2N
N
N
H
N
N
H
H
CO2H
O
O
O
HS
Trypanothione
Figure 1. Synthesis of trypanothione from spermidine and glutathione by trypanothione synthetase (TryS). The reaction is a two-step process, of which both steps are
reversible by the action of an amidase activity located in the N-terminal domain of TryS. ATP is necessary to activate glutathione by phosphorylation of the glycine
carboxylate function. Modified from Ref. 13.
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O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
interaction of the well-known hinge region of protein kinases to
both ATP and ATP-competitive kinase inhibitors.35
hydrogen bonding interaction to the backbone carbonyl group of
Phe586 that is slightly moved upwards in contrast to the
corresponding kinase hinge region which points into the ATP
binding site. Interestingly, the methylaminoethyl part is placed
near a hydrophobic region of Leu585. Because the N(5) side chain
is needed for selective TryS inhibition on the one hand and an
extension of the hydrophobic part in this area can lead to a gain
of affinity on the other hand, we designed the series 3 by replacing
the methylaminoethyl chain of 2 by benzyl or heteroarylmethyl
moieties to increase the hydrophobic interaction potential
between ligand and protein.
The N(5)-substituted paullones 3a–l were prepared from
3-chlorokenpaullone 8 as central building block (Scheme 1).
Although innovative new paullone syntheses catalyzed by transi-
tion metals have been reported lately,36–39 we prepared 8 by a tra-
ditional acid-induced Fischer indolization procedure40 for the sake
of simplicity.41 In brief, the synthesis of 8 started by the reaction of
methyl 4-chloroanthranilate 4 with methyl succinylcloride. A sub-
sequent Dieckmann reaction of the resulting amide 5 yielded the
Figure 2 shows a comparison between a TryS model containing
FS-554 (2) and a crystal structure of the mammalian kinase GSK-3
in complex with alsterpaullone (1b). LmTryS and GSK-3 show a
high structural and sequence similarity within the ATP binding site
(see Fig. 2b). Based on this similarity FS-554 (2) was modeled into
the LmTryS structure using the binding mode of alsterpaullone (1b)
to GSK-3 as template. The resulting model can be used to find a
possible explanation of the different activities of paullone scaf-
folds. FS-554 does not show any kinase inhibitory activity since
the characteristic hydrogen bond pattern to the hinge region of
the kinase found with N(5)-unsubstituted paullones like alster-
paullone is hampered by the N(5) side chain in FS-554. In the
GSK-3 structure, this side chain leads to a steric clash of FS-554
with Tyr134 of the hinge region (see Fig. 2b). In contrast, the
LmTryS structure shows a cleft at the corresponding TryS ‘hinge
region’, where the N(5) side chain of FS-554 can be accommodated.
Moreover, in TryS the side chain amide of FS-554 can form a
Figure 2. Binding mode comparison between GSK-3 (lime green surface) in complex with alsterpaullone (1b, green) and LmTryS (light blue surface) with modeled FS-554
(2, blue). (a) LmTryS surface with FS-554 and alsterpaullone. (b) Ribbon representation and important amino acid side chains with high similarity. (c) GSK-3 surface with
alsterpaullone and FS-554.
O
CO2CH3
H
N
O
H
N
O
Cl
NH2
Cl
Cl
NH
Cl
i
ii
iii
iv
CO2CH3
CO2CH3
CO2CH3
HO
O
4
5
6
7
HN
Aryl
CO2-t-Bu
CO2H
O
H
N
O
O
O
O
Cl
N
Cl
N
HN
9
N
Cl
Cl
v
vi
vii
HN
HN
HN
Br
Br
Br
Br
8
10
3a-l
Scheme 1. Synthesis of N(5)-substituted 3-chlorokenpaullones 3a–l. For aryl substituents refer to Table 1. Reagents and conditions: (i) methyl succinyl chloride, pyridine,
toluene, 0 ? 80 °C, 2 h, 89%; (ii) KOtBu, DMF, toluene, 0 ? 80 °C, 3.5 h, 65%; (iii) DMSO/H2O 9:1, 150 °C, 6 h, 53%; (iv) 1. 4-bromophenylhydrazine hydrochloride, AcOH,
NaOAc, 70 °C, 1 h 2. AcOH, H2SO4, 70 °C, 1 h, 41%; (v) 1. KOtBu, THF, rt, 1 h 2. tert-butyl 2-bromoacetate, THF, rt, 22 h, 62%; (vi) TFA, CH2Cl2, rt, 22 h, quantitative conversion;
(vii) DMF, PyBOP, DIPEA, arylmethylamines, 0 °C ? rt, 20 h, 10–53%.
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4
O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
enolized cyclic b-oxocarboxylic acid ester 6 which was then
dealkoxycarbonylated by heating in wet DMSO under neutral
conditions. The so-prepared cyclic ketone 7 was reacted with
4-bromophenylhydrazine, yielding the 3-chlorokenpaullone 8. A
selective alkylation of 8 at N(5) was accomplished by deprotona-
tion with potassium tert-butoxide in THF and subsequent reaction
with tert-butyl 2-bromoacetate. Subsequently, the resulting ester 9
was deprotected by means of trifluoroacetic acid yielding
2-(3-chlorokenpaullone-5-yl)acetic acid (10). Eventually, the
desired title compounds 3a–l were prepared by PyBOP-promoted
reaction of 10 with appropriate arylmethylamines in the presence
of diisopropylethylamine (DIPEA).
the 4-pyridyl compound 3j (IC50 1.2
revealed that an H-bond donor group (OH) at para position of the
phenyl ring (3h, IC50 10 M) contributes to the inhibitory activity
lM). SAR analysis further
l
since 4-unsubstituted derivatives or bearing H-bond acceptors
were less active (Table 1). The most potent LiTryS inhibitor was
the aminoalkyl derivative 2 (FS-554). It harbors at N(5) position
an ethylendiamine moiety with two nitrogen atoms that also
may potentially be engaged in H-bond formation with protein resi-
dues from the active site and might explain the higher activity of
this derivative. On the other hand, an additional explanation for
the lower activity displayed by the new paullone derivatives 3
compared to FS-554 may lie on the fact that the aryl rings of 3 pre-
sent a more rigid conformation than an alkyl chain and, thus, offer
a reduced repertoire of conformers suited for proper accommoda-
tion into the active site pocket of the enzyme.
3. Biological evaluation and discussion
Although the rationale behind our design strategy for series 3
was not corroborated by the inhibition results for LiTryS, our
results support the occurrence of important structural differences
at the ATP-binding site of these enzymes.7,14,19 Noteworthy, TryS
belongs to the ATP-grasp family of proteins, which undergo impor-
tant conformational changes in regions and structural elements
shaping their ATP-binding site upon substrate binding. Based on
this and given the high degree of sequence conservation for the
ATP-binding region among trypanosomatid’s TryS, it is tempting
to speculate that secondary residues or elements in the active site
of these enzymes play an important role in modulating the
interaction with ligands. In a more general context, these data
add value to a recent biochemical study with TryS from trypanoso-
matids performed by our groups, which proposed the existence of
species-specific differences between TryS to explain the selective
interaction of the enzymes with different ligands (substrates,
inhibitors).20
While the tests on isolated TryS enzymes produced disappoint-
ing results, several compounds of series 3 exhibited potent antipar-
asitic activity on cultivated T. brucei bloodstream forms with single
digit or submicromolar EC50 values. This biological activity was
clearly related to the structure of the congeners, since the three
compounds 3j–l with a pyridylmethyl moiety as part of the side
chain in 5-position were less potent compared to the derivatives
with a benzyl structure (3a–3i), except for compound 3c as dis-
cussed below. Among this subgroup the parent compound 3a,
which is unsubstituted at the phenyl ring, was tenfold more active
against the parasites compared to 4- or 3-mono- or di-substituted
The new 5-substituted compounds 3a–l were evaluated for
inhibitory activity on the TryS of three trypanosomatid parasite
species: T. brucei, T. cruzi, and L. infantum. All compounds of series
3 and FS-554 (2), which was used as a standard, were tested for
15 min at an initial concentration of 30 lM using an end point
assay based on the determination of inorganic phosphate released
from ATP during TryS catalysis. If more than 50% inhibition was
observed, IC50 values were determined. The results (Table 1) show
that most compounds from the new series 3 exhibit only a modest
improvement of inhibitory activity over 2 towards T. brucei TryS
(e.g., 40–60% enzyme inhibition for 3a–h at 30
lM versus 30% TryS
inhibition for FS-554 at 30 M). In general, in the N(5) side chain
l
terminal phenyl rings were preferred over pyridyl moieties (25–
35% TryS inhibition for 3j–l). Interestingly, with the exception of
3a, 3j, and 3l, several of the new 5-substituted paullones of series
3 inhibited TryS of T. cruzi with IC50 values near 10
lM and in this
regard were superior to FS-554 (IC50 ꢀ 30 M). At variance with
l
the T. brucei TryS, the enzyme from T. cruzi displayed a higher
promiscuity for the terminal aryl substituent of the N(5) side chain.
Overall, the results obtained for the trypanosomal TryS indicate
that the inclusion of a N(5) side chain with a terminal aryl group
positively affects inhibition, albeit to different extent depending
on the TryS species.
Strikingly, the situation was different for TryS of L. infantum.
Exhibiting an IC50 value of 0.35 lM, FS-554 (2) was in this case
clearly more potent compared to all new N(5)-substituted
paullones 3 reported in this study. The only congener with a
comparable, albeit lower, activity against L. infantum TryS was
Table 1
Biological activities and physicochemical properties of N(5)-substituted paullones 3
e
Aryl
TbTryS activitya
[%]
TcTryS activity
LiTryS activity
Viability of T. b. brucei
Selectivity
indexc
Predicted
S0,exp
LogPd
[lmol/L]
a
a
b
[%] or IC50
[lM]
[%] or IC50
50.0 ( 4.0)%
109.1 ( 8.3)%
[
l
M]
[%] or EC50
[lM]
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
Ph
45.2 ( 4.1)
50.1 ( 7.9)
55.3 ( 8.2)
55.9 ( 6.9)
42.0 ( 4.2)
53.7 ( 3.6)
53.5 ( 3.0)
58.0 ( 7.2)
63.5 ( 7.2)
65.2 ( 3.7)
64.8 ( 4.5)
75.8 ( 4.5)
70.0 ( 4.9)
72.8 ( 5.0)%
0.04 0.01
l
M
1410
>62
ND
ꢀ91
ꢀ12
>167
22
ꢀ238
>18
ND
4.23
4.79
4.79
4.79
5.34
4.72
4.10
3.84
5.15
2.89
2.89
3.32
1.65
<0.49
ND
4-Cl-Ph
2-Cl-Ph
3-Cl-Ph
3,4-Cl2-Ph
4-Me-Ph
4-MeO-Ph
4-HO-Ph
4-CF3-Ph
4-Pyridyl
3-Pyridyl
2-Pyridyl
n.a.
11 ( 2)
11 ( 3)
11 ( 2)
lM
lM
lM
0.8 ( 0.2)
53.2 ( 4.0)%
1.1 ( 0.2)
0.84 ( 0.2)
0.3 ( 0.1)
1 ( 0.3)
0.84 ( 0.2)
1.8 ( 0.3)
lM
23 ( 4)
17 ( 3)
19 ( 3)
lM
lM
lM
ND
ND
ND
lM
9 ( 1)
9 ( 2)
l
l
M
M
lM
48.6 ( 8.2)%
lM
<0.48
ND
11 ( 4)
lM
24 ( 5)
10 ( 3)
l
l
M
M
lM
9 ( 3)
9 ( 2)
l
l
M
M
lM
<4.8
ND
ND
ND
ND
55.4 ( 3.7)%
1.2 ( 0.5)
11 ( 3)
95.5 ( 3.8)%
0.35 ( 0.5)
lM
58.1 ( 5.2)%
9 ( 3)
l
M
M
49.1 ( 0.7)%
59.4 ( 2.4)%
33.8 ( 2.2)%
l
M
l
ND
ND
ꢀ1
54.8 ( 9.9)%
44.5 ( 3.8)%
FS-554 (2)
lM
8.3 ( 0.8)
lM
ND
n.a. = not applicable.
ND = not determined.
a
Residual TryS activity ( 2 SD) [%] at 30
Viability of infective T. b. brucei ( SD) [%] at 5 M, or EC50 ( SD) [lM].
Ratio EC50 murine macrophages/EC50 T. b. brucei.
Calculated with ChemBioDraw Ultra, version 13.02.3021.
l
M, or IC50 ( 2 SD) [
lM].
b
c
l
d
e
Solubility determined by shake-flask-method, pH = 7.4; 25 °C; 48 h incubation time.
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O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
derivatives (3b, 3d–3i). The effect was even more dramatic on
2-substitution, as the ortho chloro-substituted derivative 3c was
explicitly inferior to the para and meta isomers 3b and 3d
enzymes from different pathogens during screening campaigns
aimed at detecting broad-range TryS inhibitors.
Importantly, several compounds showed potent antiparasitic
activity and selectivity against the infective form of African
trypanosomes. The wide difference between enzyme inhibitory
concentration and antiparasitic activity casts doubt on TryS as
responsible target. Because of its remarkable potent and selective
antitrypanosomal activity together with its lack of inhibitory activ-
ity towards host kinases, structure 3a (EC50 40 nM and SI > 1000)
can be rated as a promising new hit against African trypanosomiasis
identified from this study. Thus, compound 3a could serve as start-
ing point for the development of derivatives with improved phar-
macological properties. The identification of the responsible
intracellular target underlying the antitrypanosomal activity of 3a
as well as its biological activity towards other pathogenic species
of trypanosomatids will be of utmost importance for a rational drug
development campaign in the future and therefore will be the sub-
ject of further studies, together with the optimization of physico-
chemical properties, namely the solubility in aqueous media.
(EC50 ꢀ 1
lM). Compounds with single digit micromolar or submi-
cromolar antitrypanosomal activity were also tested on murine
macrophages. Three compounds (3a, 3f and 3h) showed a signifi-
cant selectivity profile (SI P 100) for the parasite cells. Although
the 5-substitution at the paullone heterocyclic parent scaffold in
3a–l should, on a structural basis, prevent any inhibition of protein
kinases, the compound showing the most potent antitrypanosomal
activity (3a) was evaluated on mammalian kinases that are typi-
cally inhibited by paullones (CDK1/cyclin B, CDK2/cyclin A,
CDK5/p25, CDK9/cyclin T, GSK-3) and related kinases (CK1, CLK1,
DYRK1A). 3a failed to inhibit all protein kinases in the panel at
the highest tested concentration (10
tivity window of at least more than two orders of magnitude
between the antitrypanosomal activity (EC50 0.04 M) and a
lM), displaying a wide selec-
l
potential inhibition of host’s kinases. Since FS-554 (2), the com-
pound which was taken as the starting point of the study, showed
only a modest antitrypanosomal activity and selectivity in our test
model (EC50 8.3
3h) of the most active and selective derivatives identified here
(EC50 6 1 M and SI P 60) as substantial progress in our efforts
l
M and SI ꢀ 1), we rate 3a and four (3b, 3d, 3f and
5. Experimental section
5.1. Chemistry
l
to develop antitrypanosomal paullones. However, considering the
wide difference between antitrypanosomal and TryS inhibitory
activity, it is very improbable that TryS is the molecular target
underlying the antiparasitic activity of 3a and its congeners. Fur-
ther studies are warranted to identify this target, e.g., by immobi-
lizing compounds of series 3 on solid matrices and performing pull
down assays with parasite cell homogenates.
Although some of the structures presented here exhibit potent
antitrypanosomal activity and reasonable selectivity (e.g., 3a, 3f,
3h), these are not looking very drug-like from a medicinal chemists
point of view, displaying both high lipophilicity values and molec-
ular masses. This impression is underlined by the very low solubil-
ity of the three mentioned compounds in aqueous phosphate
buffer (Table 1). The optimization of the physicochemical parame-
ters within this new antitrypanosomal compound family will
therefore be a priority in further optimization studies.
5.1.1. General
Melting points (mp) were determined on an electric variable
heater (Barnstead Electrothermal IA 9100) in open glass capillaries
and are not corrected. IR-spectra were recorded as KBr pellets on a
Thermo Nicolet FT-IR 200. 1H NMR spectra and 13C NMR spectra
were recorded on the following instruments: Bruker Avance
DRX-400 and Bruker Avance II-600; solvent DMSO-d6 if not stated
otherwise; internal standard tetramethylsilane; signals in ppm
(d scale). Elemental analyses were determined on a CE Instruments
FlashEAÒ 1112 Elemental Analyser (Thermo Quest). Mass spectra
were recorded on a double-focused sector field mass spectrometer
Finnigan-MAT 90 or
a
MAT95XL (Thermofinnigan MAT,
Department of Mass Spectrometry of the Chemical Institutes of
the Technische Universität Braunschweig). Accurate measurements
where conducted according to the peakmatch method using
perfluorokerosene (PFK) as an internal mass reference; (EI)-MS:
ionization energy 70 eV. TLC: PolygramÒ Sil G/UV254, Macherey-
Nagel, 40 ꢁ 80 mm, visualization by UV-illumination (254 nm).
Purity was determined by HPLC using the following devices and set-
tings: Merck Hitachi LaChrom Elite system (DAD detector: L-2450
(isocrat.), UV detector: L-2400 (gradient); pump: L-2130; autosam-
pler: L-2200; column: Merck LiChroCART 125-4, LiChrospher 100
4. Conclusion
With a view to develop drugs against the unicellular parasite
T. brucei and based on the observation that paullone FS-554 (2) is
a potent or modest to weak inhibitor of TryS from the parasites L.
infantum or T. cruzi and T. brucei, respectively, we have designed
and synthesized 3-chlorokenpaullone derivatives with aryl instead
of alkyl substitutions as potentially improved inhibitors of try-
panosomatids TryS. This goal has, to some extent, been fulfilled
for TcTryS, with some of the novel paullones presenting a 3-fold
higher activity than the lead structure scaffold. Although to an even
RP-18 (5 lm); isocratic eluent: acetonitrile/water mixture;
gradient elution (Method A): concentration acetonitrile 0–2 min:
10%; 2–12 min: 10% ? 90% (linear) 12–20 min: 90%; gradient elu-
tion (Method B): concentration acetonitrile: 0–13 min: 10% ? 90%
(linear), 13–20 min: 90%.; elution rate: 1.000 mL/min; detection
wavelength: 254 nm and 280 nm (isocrat.), 254 nm (gradient);
overall run time: 15 min (isocrat.); tM = dead time related to DMSO;
tR = total retention time. All compounds employed in biological
tests presented a purity >95%. Wavelength maxima were extracted
from spectra generated by the DAD detector during HPLC analyses.
Preparation of buffer for HPLC: To a mixture of trimethylamine
(20.0 mL), sodium hydroxide (242 mg) and water (980 mL), H2SO4
was added until the pH reached 2.7. THF was pre-dried over
potassium hydroxide and subsequently distilled from CaH2. CH2Cl2
was dried by distillation from CaH2. 4-Hydroxybenzylaminehydro-
bromide was prepared by refluxing 4-methoxybenzylamine with
5 equiv of 46% aqueous hydrobromic acid overnight, cooling to
0 °C and collecting the filtrate.42 3-Chlorokenpaullone 8 was
synthesized according to a published method.41 Other starting
minor level, most new derivatives also displayed
a slightly
improved inhibition of TbTryS. In contrast, none of the new com-
pounds could surpass the strong inhibition exerted by FS-554 (2)
against TryS from L. infantum. Compared to the formerly investi-
gated 9- and 11-substituted paullones the new series presented
here represent an important advance in the generation of paullones
with wider species-specificity for TryS. Nevertheless, the SAR anal-
ysis derived from testing paullones against TryS from three major
trypanosomatid species provides strong evidence for the existence
of peculiar demands for steric occupancy and polar interactions in
the ATP-binding pocket of these enzymes. In this respect, future
synthetic strategies of paullone analogues will address this subject
by including novel alkyl substitutions at N(5) that may satisfy these
demands. Not least, our data re-inforce the importance of testing
Please cite this article in press as: Orban, O. C. F.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.023

6
O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
materials were purchased from commercial suppliers (Acros
Organics, Geel, Belgium; Sigma Aldrich, Steinheim, Germany) and
were used without further purification.
[4,5-b]indol-5-yl)acetic acid (10) in DMF under nitrogen atmo-
sphere. The solution was stirred at 0 °C for 20 min. The appropriate
primary amine was added dropwise. The mixture was covered
with a layer of argon and stirred for 20 h at room temperature.
After addition of ethyl acetate (100 mL) the mixture was filtered
and the collected precipitate was set aside. The organic solution
was washed successively with 0.1 M aqueous hydrochloric acid
(100 mL), 0.1 M aqueous sodium hydroxide solution (100 mL)
and water (100 mL), then dried with Na2SO4 and evaporated. The
residue was combined with the precipitate collected by filtration
and then crystallized from the indicated solvent.
5.1.2. tert-Butyl 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-tetrahydro-
benzo[2,3]azepino[4,5-b]indol-5-yl)acetate (9)
9-Bromo-3-chloro-7,12-dihydroindolo[3,2-d][1]benzazepin-6
(5H)-one (8, 610 mg, 1.68 mmol) was dissolved in dry THF (25 mL)
under nitrogen. Potassium tert-butoxide (224 mg, 1.99 mmol) was
added and the mixture stirred for 1 h at room temperature under
nitrogen. After addition of tert-butyl 2-bromoacetate (390 mg,
1.99 mmol), dissolved in dry THF (50 mL), stirring was continued
overnight at room temperature. Subsequently, the mixture was
evaporated and the residue was dissolved in CH2Cl2 (100 mL).
The organic layer was washed with water (100 mL) and evapo-
rated. The residue was crystallized from ethanol to yield a yellow
powder (446 mg; 56%); mp: 278–281 °C; IR (KBr): 3333 cmꢂ1
(NH), 1736 cmꢂ1 (C@O), 1653 cmꢂ1 (C@O); 1H NMR (400 MHz,
5.1.4.1. N-Benzyl-2-(9-bromo-3-chloro-6-oxo-5,6,7,12-tetrahy-
drobenzo[2,3]azepino[4,5-b]indole-5-yl)acetamide (3a). Synthe-
sized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo
[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10, 202 mg, 481
DMF (1.0 mL), DIPEA (370 L, 2.12 mmol), PyBOP (283 mg,
544 mol) and benzylamine (65.4 L, 601 mol). The material
lmol),
l
l
l
l
d6-DMSO):
d
(ppm) = 12.00 (s, 1H, indole-NH), 7.95 (d, 1H,
was crystallized from tetrahydrofurane/toluene (1:1) to yield a col-
orless solid (128 mg, 53%); dec. starting at 337 °C; IR (KBr):
3316 cmꢂ1 (NH), 1663 cmꢂ1 (C@O); 1H NMR (400 MHz, DMSO-d6)
d (ppm) = 11.99 (s, 1H, indole-NH), 8.66 (t, 1H, J = 6.0 Hz, NH),
7.97 (d, J = 2.0 Hz, 1H, ArH), 7.71 (m, 2H, ArH), 7.49 (dd, J = 8.4,
2.1 Hz, 1H, ArH), 7.44–7.40 (m, 1H, ArH), 7.36–7.21 (m, 6H, ArH),
4.35 (m, 4H, AN(5)-CH2 and ACH2), 4.01 and 3.11 (bs, 2H, aze-
pine-CH2); 13C NMR (100.6 MHz, DMSO-d6): d (ppm) = 31.2, 42.1,
53.5 (CH2); 113.6, 120.7, 124.1, 124.9, 125.1, 126.7, 127.0, (2C),
128.2 (2C), 128.6 (CH); 109.4, 111.8, 124.0, 127.8, 132.4, 133.0,
136.0, 139.2, 141.0, 168.3, 170.2 (C); CHN: calcd C 59.02, H 3.76;
N 8.28, found C 58.86, H3.74, N 8.75; C25H19BrClN3O2 (508.79);
MS (EI): m/z (%): 509 [M]+ꢀ (23), 402 [Mꢂ107(AC7H9N)]+ (100);
HRMS (EI): m/z [M]+ꢀ calcd 507.03437, found 507.03409; HPLC (iso-
crat.): 96.9% at 254 nm, 96.2% at 280 nm, tR = 3.93 min, tM (DMSO) =
1.07 min (ACN/H2O = 60:40); kmax: 237 nm, 318 nm, 389 nm.
J = 1.9 Hz, ArH.), 7.74 (d, 1H, J = 8.4 Hz, ArH), 7.59 (d, 1H,
J = 2.1 Hz, ArH.), 7.50 (dd, 1H, J = 8.4, 2.1 Hz, ArH.), 7.42 (d, 1H,
J = 8.6 Hz, ArH), 7.30 (dd, 1H, J = 8.6, 1.9 Hz, ArH), 4.42 (s, 2H, N
(5)-CH2), 3.97 and 3.09 (bs, 2H, azepine-CH2), 1.25 (s, 9H, tert-
butyl); 13C NMR (100.6 MHz, d6-DMSO): d (ppm) = 27.4 (3C, tert-
butyl); 30.9 (azepine-CH2), 52.7 (N(5)-CH2); 113.6, 120.7, 123.9,
124.9, 125.3, 128.7 (CH); 80.9, 109.2, 111.8, 124.3, 127.8, 132.5,
133.0, 136.0, 140.3, 168.1, 170.2 (C); CHN: calcd C 55.54, H 4.24,
N 5.89, found C 55.93, H 4.33, N 5.59; C22H20BrClN2O3 (475.76);
MS (EI): m/z (%) = 476 ([M]+ꢀ, 74), 420 ([Mꢂ56(AC4H8)]+, 100),
361 ([Mꢂ115(AC6H11O2)]+, 79); HPLC (isocrat.): 99.0% at 254 nm,
99.1% at 280 nm, tR = 3.96 min, tM (DMSO) = 1.07 min (ACN/H2O
50:50); kmax: 233 nm, 319 nm, 385 nm; HPLC (gradient): 95.2% at
254 nm, tR = 13.40 min, tM (DMSO) = 1.07 min (time: ACN/H2O;
0 min: 10/90; 10 min: 70/30; 10.5 min: 90/10; 22 min: 90/10).
5.1.3. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10)
5.1.4.2. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indole-5-yl)-N-(4-chlorobenzyl)acetamide
Trifluoroacetic acid (2 mL) was added to a suspension of tert-
butyl 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo[2,3]
azepino[4,5-b]indol-5-yl)acetate (9, 296 mg, 0.622 mmol) in dry
CH2Cl2 (30 mL) under nitrogen. After stirring for 20 h at room tem-
perature, the mixture was evaporated. Diethyl ether (3 mL) was
added to the oily residue and the mixture was refluxed for 1 h.
The resulting precipitate was filtered off, washed with diethyl
ether and dried to yield a colorless powder (132 mg, 51%); mp:
207–208 °C (dec); IR (KBr): 3325 cmꢂ1 (NH), 1655 cmꢂ1 (C@O);
1H NMR (400 MHz, d6-DMSO): d (ppm) = 12.83 (bs, 1H, AOH),
12.01 (s, 1H, indole-NH), 7.96 (d, 1H, J = 1.9 Hz, ArH), 7.73 (d, 1H,
J = 8.4 Hz, ArH), 7.58 (d, 1H, J = 2.0 Hz, ArH), 7.50 (dd, 1H, J = 8.4,
2.1 Hz, ArH), 7.42 (d, 1H, J = 8.6 Hz, ArH), 7.30 (dd, 1H, J = 8.6,
1.9 Hz, ArH), 4.39 (s, 2H, N(5)-CH2), 3.99 and 3.11 (bs, 2H, aze-
pine-CH2); 13C NMR (100.6 MHz, d6-DMSO): d (ppm) = 31.0 (aze-
pine-CH2), 52.3 (AN(5)-CH2); 113.6, 120.7, 123.7, 124.9, 125.3,
128.7 (CH2); 109.2, 111.9, 124.1, 127.8, 132.6, 133.0, 136.0,
(3b).
12-tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic
(10, 293 mg, 698 mol), DMF (1.4 mL), DIPEA (511
2.94 mmol), PyBOP (443 mg, 851 mol) and 4-chlorobenzy-
lamine (100 L, 827 mol). The material was crystallized twice
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,
acid
L,
l
l
l
l
l
from methanol to yield a colorless solid (114 mg, 30%); dec. start-
ing at 305 °C; IR (KBr): 3316 cmꢂ1 (NH), 1639 cmꢂ1 (C@O),
1657 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-d6): d (ppm) =
11.99 (s, 1H, indole-NH), 8.69 (t, J = 6.0 Hz, 1H, ANH), 7.97 (d,
J = 1.9 Hz, 1H, ArH), 7.72 (d, J = 8.4 Hz, 1H, ArH), 7.67 (d,
J = 2.1 Hz, 1H, ArH), 7.49 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 7.42 (d,
J = 8.6 Hz, 1H, ArH), 7.39–7.35 (m, 2H, ArH), 7.30 (dd, J = 8.6,
2.0 Hz, 1H, ArH), 7.29–7.26 (m, 2H, ArH), 4.31 (bs, 4H, AN(5)-
CH2 and ACH2), 3.99 and 3.12 (bs, 2H, azepine-CH2); 13C NMR
(151 MHz, DMSO-d6): d (ppm) = 31.1, 41.4, 53.5 (CH2); 113.6,
120.7, 124.0, 124.8, 125.1, 128.1 (2C), 128.5, 128.8 (2C) (CH);
109.3, 111.8, 123.9, 127.7, 128.8, 131.2, 132.4, 135.9, 138.3,
140.9, 168.4, 170.2 (C); CHN: calcd C 55.27, H 3.34, N 7.74, found
C 55.28, H 3.39, N 7.47; C25H18BrCl2N3O2 (543.24); MS (EI): m/z
(%): 542.9 [M]+ꢀ (24), 401.9 [Mꢂ141(AC7H7ClN)]+ (100); HPLC
(isocrat.): 99.2% at 254 nm, 99.5% at 280 nm, tR = 5.40 min,
tM (DMSO) = 1.06 min (ACN/H2O = 60:40); kmax: 234 nm, 318 nm;
HPLC (gradient method A): 98.5% at 254 nm, tR = 13.14 min.
140.6, 170.3, 170.7 (C);
C18H12BrClN2O3 (419.66); MS (EI):
m/z (%) = 420 ([M]+ꢀ, 100), 391 ([Mꢂ29(ACHO)]+, 40), 361
([Mꢂ59(AC2H3O)]+, 61); HRMS (EI): m/z [M]+ꢀ calcd 417.97143,
found 417.97144; HPLC (isocrat.): 98.2% at 254 nm, 98.5% at
280 nm, tR = 3.99 min, tM (DMSO) = 1.07 min (ACN/H2O = 50:50);
kmax: 234 nm, 318 nm, 398 nm.
5.1.4. General procedure for the preparation of N-substituted 2-
(9-bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo[2,3]azepino-
[4,5-b]indole-5-yl) acetamides 3a–3l
PyBOP and DIPEA were added to an ice-cooled solution of
2-(9-bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo[2,3]azepino
5.1.4.3. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indole-5-yl)-N-(2-chlorobenzyl)acetamide
(3c).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
293 mg, 698 mol), DMF (1.4 mL), DIPEA (511 L, 2.94 mmol),
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
Please cite this article in press as: Orban, O. C. F.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.023

O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
PyBOP (441 mg, 847
l
mol) and 2-chlorobenzylamine (100
l
L,
402.0 [Mꢂ174.9(AC7H6Cl2N)]+ (100); HPLC (isocrat.) 99.5% at
254 nm, 99.6% at 280 nm, tR = 7.5 min, tM(DMSO) = 1.06 min
(ACN/H2O = 60:40); kmax: 228 nm, 319 nm; HPLC (gradient method
A): 99.7% at 254 nm, tR = 13.6 min.
827 mol). The material was crystallized from methanol to yield
l
a colorless solid (159 mg, 42%); dec. starting at 345 °C; IR (KBr):
3422 cmꢂ1 (NH), 3312 cmꢂ1 (NH), 3262 cmꢂ1 (NH), 1654 cmꢂ1
(C@O); 1H NMR (600 MHz, DMSO-d6): d (ppm) = 11.99 (s, 1H,
indole-NH), 8.70 (t, J = 5.9 Hz, 1H, ANH), 7.97 (d, J = 1.9 Hz, 1H,
ArH), 7.72 (d, J = 8.4 Hz, 1H, ArH), 7.69 (d, J = 2.1 Hz, 1H, ArH),
7.49 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 7.47–7.43 (m, 1H, ArH), 7.42 (d,
J = 8.6 Hz, 1H, ArH), 7.40–7.36 (m, 1H, ArH), 7.35–7.28
(m, 3H, ArH), 4.38 (bs, 4H, AN(5)-CH2 and ACH2), 4.01 and 3.12
(bs, 2H, azepine-CH2); 13C NMR (151 MHz, DMSO-d6): d (ppm)
= 31.2, 40.1, 53.5 (CH2); 113.6, 120.8, 124.0, 124.9, 125.2, 127.1,
128.6 (3C), 129.1 (CH); 109.4, 111.9, 124.1, 127.8, 131.9,
132.5, 133.0, 136.0, 136.1, 141.0, 168.6, 170.33 (C); CHN:
calcd C 55.27, H 3.34, N 7.74, found C 55.13, H 3.42, N 7.48;
5.1.4.6. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(4-methylbenzyl)acetamide
(3f).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
296 mg, 705 mol), DMF (1.4 mL), DIPEA (511 L, 2.94 mmol),
PyBOP (440 mg, 846 mol) and 4-methylbenzylamine (100 L,
786 mol). Crystallization from methanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
l
l
l
(120 mg, 33%); dec. starting at 326 °C; IR (KBr): 3317 cmꢂ1 (NH),
1653 cmꢂ1 (C@O), 1640 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-
d6): d (ppm) = 11.99 (s, 1H, indole-NH), 8.60 (t, J = 6.0 Hz, 1H,
ANH), 7.97 (d, J = 1.9 Hz, 1H, ArH), 7.72 (d, J = 8.4 Hz, 1H, ArH),
7.68 (d, J = 2.1 Hz, 1H, ArH), 7.49 (dd, J = 8.4, 2.1 Hz, 1H, ArH),
7.42 (d, J = 8.6 Hz, 1H, ArH), 7.30 (dd, J = 8.6, 1.9 Hz, 1H, ArH),
7.13 (q, J = 8.0 Hz, 4H, ArH), 4.28 (bs, 4H, AN(5)-CH2 and ACH2),
3.99 and 3.11 (bs, 2H, azepine-CH2), 2.28 (s, 3H, ACH3); 13C NMR
C
25H18BrCl2N3O2 (543.24); MS (EI): m/z (%): 543.0 [M]+ꢀ (14),
401.9 [Mꢂ141(AC7H7ClN)]+ (100); HPLC (isocrat.): 98.4% at
254 nm, 98.5% at 280 nm, tR = 5.47 min, tM (DMSO) = 1.06 min
(ACN/H2O = 60:40); kmax
(gradient method A): 98.3% at 254 nm, tR = 13.01 min.
:
233 nm, 231 nm, 318 nm; HPLC
(151 MHz, DMSO-d6):
d (ppm) = 20.7 (CH3); 31.2, 41.9, 53.5
5.1.4.4. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indole-5-yl)-N-(3-chlorobenzyl)acetamide
(CH2); 113.6, 120.8, 124.1, 124.9, 125.2, 127.0 (2C), 128.6, 128.8
(2C) (CH); 109.4, 111.9, 124.0, 127.8, 132.5, 133.0, 135.8, 136.0,
136.2, 141.1, 168.2, 170.2 (C); CHN: calcd C 59.73, H 4.05, N 8.04,
found C 59.70, H 4.03, N 7.81; C26H21BrClN3O2 (522.83); MS (EI):
m/z (%): 523.0 [M]+ꢀ (24), 402.0 [Mꢂ121(AC8H11N)]+ (100); HPLC
(isocrat.): 99.2% at 254 nm, 99.2% at 280 nm, tR = 5.37 min, tM
(3d).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
294 mg, 700 mol), DMF (1.4 mL), DIPEA (511 L, 2.94 mmol),
PyBOP (442 mg, 849 mol) and 3-chlorobenzylamine (100 L,
827 mol). Crystallized from methanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
l
l
l
(DMSO) = 1.06 min (ACN/H2O = 60:40); kmax: 236 nm, 318 nm,
(95 mg, 25%); dec. starting at 342 °C; IR (KBr): 3308 cmꢂ1 (NH),
1639 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-d6): d (ppm) = 11.99
(s, 1H, indole-NH), 8.73 (t, J = 6.0 Hz, 1H, NH), 7.98 (d, J = 1.9 Hz,
1H, ArH), 7.72 (d, J = 8.4 Hz, 1H, ArH), 7.67 (d, J = 2.1 Hz, 1H,
ArH), 7.49 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 7.42 (d, J = 8.6 Hz, 1H,
ArH), 7.38–7.28 (m, 4H, ArH), 7.26–7.21 (m, 1H, ArH), 4.35 (bs,
4H, AN(5)-CH2 and ACH2), 3.99 and 3.13 (bs, 2H, azepine-CH2);
13C NMR (151 MHz, DMSO-d6): d (ppm) = 31.2, 41.6, 53.6 (CH2);
113.6, 120.8, 124.0, 124.1, 124.9, 125.2, 126.7, 126.9, 128.6, 130.1
(CH); 109.4, 111.9, 125.7, 127.8, 132.4, 133.0, 133.0, 136.0, 141.1,
141.9, 170.3, 168.6 (C); CHN: calcd C 55.27, H 3.34, N 7.74, found
C 54.88, H 3.38, N 7.56; C25H18BrCl2N3O2 (543.24); MS (EI): m/z
(%): 542.9 [M]+ꢀ (24), 401.9 [Mꢂ141(AC7H7ClN)]+ (100); HPLC (iso-
crat.): 95.9% at 254 nm, 96.3% at 280 nm, tR = 5.40 min, tM(DMSO) =
1.06 min (ACN/H2O = 60:40); kmax: 232 nm, 319 nm, 383 nm; HPLC
(gradient method B): 96.7% at 254 nm, tR = 13.38 min.
391 nm; HPLC (gradient method B): 99.1% at 254 nm,
tR = 13.21 min.
5.1.4.7. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(4-methoxybenzyl)acetamide
(3g).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
217 mg, 517 llmol), DMF (1.0 mL), DIPEA (370 L, 2.12 mmol),
PyBOP (318 mg, 611 llmol) and 4-methoxylbenzylamine (83 L,
635 mol). Crystallization from methanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
l
(82 mg, 29%); dec. starting at 313 °C; IR (KBr): 3314 cmꢂ1 (NH),
1654 cmꢂ1 (C@O), 1640 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-
d6): d (ppm) = 12.00 (s, 1H, indole-NH), 8.59 (t, J = 5.9 Hz, 1H,
ANH), 7.98 (d, J = 1.9 Hz, 1H, ArH), 7.73 (d, J = 8.4 Hz, 1H, ArH),
7.69 (d, J = 2.1 Hz, 1H, ArH), 7.50 (dd, J = 8.4, 2.1 Hz, 1H, ArH),
7.43 (d, J = 8.6 Hz, 1H, ArH), 7.31 (dd, J = 8.6, 1.9 Hz, 1H, ArH),
7.23–7.16 (m, 2H, ArH), 6.94–6.85 (m, 2H, ArH), 4.26 (bs, 4H, AN
(5)-CH2 and ACH2), 4.00 and 3.13 (bs, 2H, azepine-CH2), 3.75 (s,
5.1.4.5. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(3,4-dichlorobenzyl)acetamide
3H, AOCH3); 13C NMR (151 MHz, DMSO-d6):
d (ppm) = 54.8
(3e).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
295 mg, 703 mol), DMF (1.4 mL), DIPEA (511 L, 2.94 mmol),
PyBOP (447 mg, 859 mol) and 3,4-dichlorobenzylamine (100 L,
858 mol). Crystallization from ethanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
(CH3); 30.9, 41.4, 53.3 (CH2); 113.4 (2C), 120.5, 123.9, 124.7,
124.9, 128.2 (2C), 128.4 (CH); 109.4, 111.9, 124.0, 127.8, 131.1,
132.5, 133.0, 136.0, 141.1, 158.2, 168.2, 170.2 (C); CHN:
calcd C 57.96, H 3.93, N 7.80, found C 57.76, H 3.74, N 7.45;
l
l
l
l
l
C
26H21BrClN3O3 (538.83); MS (EI): m/z (%): 539.0 [M]+ꢀ (24),
(81 mg, 20%); mp: 325 °C; IR (KBr): 3312 cmꢂ1 (NH), 1638 cmꢂ1
(C@O), 1654 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-d6): d (ppm)
= 12.00 (s, 1H, indole-NH), 8.76 (t, J = 6.0 Hz, 1H, ANH), 7.98 (d,
J = 1.9 Hz, 1H, ArH), 7.73 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d,
J = 2.1 Hz, 1H, ArH), 7.59 (d, J = 8.3 Hz, 1H, ArH), 7.54 (d,
J = 2.0 Hz, 1H, ArH), 7.50 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 7.43 (d,
J = 8.4 Hz, 1H, ArH), 7.31 (dd, J = 8.6, 1.9 Hz, 1H, ArH), 7.27 (dd,
J = 8.3, 2.0 Hz, 1H, ArH), 4.34 (bs, 4H, AN(5)-CH2 and ACH2), 4.00
and 3.14 (bs, 2H, azepine-CH2); 13C NMR (151 MHz, DMSO-d6): d
(ppm) = 31.2, 41.1, 53.7 (CH2); 113.7, 120.8, 124.1, 124.9, 125.2,
127.4, 128.6, 129.0, 130.4 (CH); 109.4, 111.9, 124.0, 127.8, 129.3,
131.0, 132.5, 133.0, 136.0, 140.6, 141.0, 168.7, 170.4 (C); CHN:
calcd C 51.98, H 2.97, N 7.27, found C 52.13, H 2.79, N 7.07;
402.0 [Mꢂ137(AC8H11NO)]+ (100); HPLC (isocrat.): 99.1% at
254 nm, 99.4% at 280 nm, tR = 5.37 min, tM (DMSO) = 1.06 min
(ACN/H2O = 60:40); kmax
:
235 nm, 318 nm, 389 nm; HPLC
(gradient method B): 99.6% at 254 nm, tR = 12.45 min.
5.1.4.8. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(4-hydroxybenzyl)acetamide
(3h).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
295 mg, 703 mol), DMF (1.4 mL), DIPEA (1.02 mL, 5.88 mmol),
PyBOP (449 mg, 862 mol) and 4-hydroxylbenzylaminehydrobro-
mide (178 mg, 873 mol). Crystallization from ethanol yielded a
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
l
colorless solid (146 mg, 40%); dec. starting at 280 °C; IR (KBr):
C
25H17BrCl3N3O2 (577.68); MS (EI): m/z (%): 576.9 [M]+ꢀ (24),
3312 cmꢂ1 (NH), 1638 cmꢂ1 (C@O), 1654 cmꢂ1 (C@O); 1H NMR
Please cite this article in press as: Orban, O. C. F.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.023

8
O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
(600 MHz, DMSO-d6): d (ppm) = 12.00 (s, 1H, indole-NH), 9.31 (s,
1H, AOH), 8.54 (t, J = 5.9 Hz, 1H, ANH), 7.98 (d, J = 1.9 Hz, 1H,
ArH), 7.73 (d, J = 8.4 Hz, 1H, ArH), 7.68 (d, J = 2.1 Hz, 1H, ArH),
7.49 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 7.43 (d, J = 8.6 Hz, 1H, ArH),
7.10–7.05 (m, 2H, ArH), 7.31 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 6.79–
6.65 (m, 2H, ArH), 4.22 (s, 4H, AN(5)-CH2 and ACH2), 4.01 and
3.13 (s, 2H, azepine-CH2); 13C NMR (151 MHz, DMSO-d6):
d (ppm) = 31.2, 41.7, 53.5 (CH2); 113.7, 115.0 (2C), 120.8, 124.1,
124.9, 125.2, 128.4 (2C), 128.6 (CH); 109.4, 111.9, 124.0, 127.8,
129.3, 132.5, 133.0, 136.0, 141.1, 156.3, 168.1, 170.2 (C); CHN:
calcd C 57.22, H 3.65, N 8.01, found C 56.88, H 3.85, N 7.74;
5.1.4.11. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(pyridin-3-ylmethyl)acetamide
(3k).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
218 mg, 519 mol), DMF (1.0 mL), DIPEA (370 L, 2.12 mmol),
PyBOP (340 mg, 653 mol), and 3-pyridinemethanamine (61 L,
599 mol). Crystallization from methanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
l
l
l
(38 mg, 14%); dec. starting at 308 °C; IR (KBr): 3348 cmꢂ1 (NH),
1698 cmꢂ1 (C@O), 1645 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-
d6): d (ppm) = 12.00 (s, 1H, indole-NH), 8.79 (t, J = 6.0 Hz, 1H,
ANH), 8.54–8.52 (m, 1H, ArH), 7.98 (d, J = 1.9 Hz, 1H, ArH), 7.78
(dd, J = 7.7, 1.8 Hz, 1H, ArH), 7.76–7.72 (m, 2H, ArH), 7.51 (dd,
J = 8.4, 2.1 Hz, 1H, ArH), 7.43 (d, J = 8.6 Hz, 1H, ArH), 7.36–7.23
(m, 3H, ArH), 4.34 (bs, 4H, AN(5)-CH2 and ACH2), 4.01 and 3.15
(bs, 2H, azepine-CH2); 13C NMR (151 MHz, DMSO-d6): d (ppm)
= 31.0, 43.9, 53.5 (CH2); 113.4, 120.5, 120.6, 121.8, 123.9, 124.7,
125.0, 128.4, 136.4, 148.6 (CH); 109.4, 111.9, 124.0, 127.8,
132.5, 133.0, 136.0, 141.1, 158.4, 168.6, 170.3 (C); CHN: calcd C
C
25H19BrClN3O3 (524.80); MS (EI): m/z (%): 525.0 [M]+ꢀ (21),
402.0 [Mꢂ123(AC7H9NO)]+ (100); HPLC (isocrat.): 98.3% at
254 nm, 99.0% at 280 nm, tR = 4.50 min, tM (DMSO) = 1.06 min
(ACN/H2O = 50:50); kmax
:
228 nm, 319 nm, 390 nm; HPLC
(gradient method A): 95.5% at 254 nm, tR = 11.3 min.
5.1.4.9. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(4-(trifluoromethyl)benzyl)ace-
56.55, H 3.56, N 10.99, found C 56.47, H 3.80, N 10.65;
tamide (3i).
6,7,12-tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic
(210 mg, 500 mol), DMF (1.0 mL), DIPEA (370 L, 2.12 mmol),
PyBOP (316 mg, 607 mol) and 4-trifluoromethylbenzylamine
(88 L, 615 mol). Crystallization from ethanol yielded a colorless
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,
C
24H18BrClN4O2 (509.79); MS (EI): m/z (%): 510.0 [M]+ꢀ (49), 402.0
acid
[Mꢂ108.0(AC6H8N2)]+ (100); HPLC (isocrat.): 99.6% at 254 nm,
99.6% at 280 nm, tR = 3.40 min, tM(DMSO) = 1.06 min (ACN/Buffer =
40:60); kmax: 232 nm, 319 nm.
l
l
l
l
l
solid (43 mg, 15%); dec. starting at 321 °C; IR (KBr): 3312 cmꢂ1
(NH), 1639 cmꢂ1 (C@O), 1659 cmꢂ1 (C@O); 1H NMR (600 MHz,
DMSO-d6): d (ppm) = 12.01 (s, 1H, indole-NH), 8.79 (t, J = 6.0 Hz,
1H, ANH), 7.98 (d, J = 2.0 Hz, 1H, ArH), 7.73 (d, J = 8.4 Hz, 1H,
ArH), 7.71–7.66 (m, 3H, ArH), 7.51–7.49 (m, 3H, ArH), 7.45–7.42
(m, 1H, ArH), 7.31 (dd, J = 8.6, 2.0 Hz, 1H, ArH), 4.43 (bs, 4H, AN
(5)-CH2 and ACH2), 4.01 and 3.14 (bs, 2H, azepine-CH2); 13C NMR
(151 MHz, DMSO-d6): d (ppm) = 31.2, 41.8, 53.6 (CH2); 113.4,
120.5, 123.9, 124.7, 124.9 (2C, q, J = 3.8 Hz), 125.0, 127.5 (2C),
128.4 (CH); 109.4, 111.9, 124.0, 124.4 (q, J = 271.8 Hz), 127.5 (q,
J = 31.6 Hz), 132.5 133.0, 136.0, 141.0, 144.3, 168.6, 170.3 (C);
CHN: calcd C 54.14, H 3.15, N 7.29, found C 53.97, H 3.01, N 6.95;
5.1.4.12. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(pyridin-2-ylmethyl)acetamide
(3l).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic acid (10,
218 mg, 519 mol), DMF (1.0 mL), DIPEA (370 L, 2.12 mmol),
PyBOP (284 mg, 546 mol), and 2-pyridinemethanamine (61 L,
623 mol). Crystallization from methanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
l
l
l
l
l
(26 mg, 10%); dec. starting at 313 °C; IR (KBr): 3300 cmꢂ1 (NH),
1643 cmꢂ1 (C@O); 1H NMR (600 MHz, DMSO-d6): d (ppm) = 12.01
(s, 1H, indole-NH), 8.79 (t, J = 6.1 Hz,1H, ANH), 8.60–8.39 (m, 2H,
ArH), 7.99 (d, J = 1.9 Hz, 1H, ArH), 7.74 (d, J = 8.4 Hz, 1H, ArH),
7.70 (d, J = 2.1 Hz, 1H, ArH), 7.51 (dd, J = 8.4, 2.1 Hz, 1H, ArH),
7.43 (d, J = 8.5 Hz, 1H, ArH), 7.31 (dd, J = 8.6, 2.0 Hz, 1H, ArH),
7.29–7.26 (m, 2H, ArH), 4.37 (bs, 4H, AN(5)-CH2 and ACH2), 4.02
and 3.14 (bs, 2H, azepine-CH2); 13C NMR (151 MHz, DMSO-d6): d
(ppm) = 30.9, 40.9, 53.4 (CH2); 113.4, 120.5, 121.7 (2C), 123.91,
124.7, 125.0, 128.4, 149.2 (2C) (CH); 109.4, 111.9, 124.1, 127.8,
132.5, 133.0, 136.0, 141.0, 148.4, 168.8, 170.4 (C); CHN: calcd C
C
26H18BrClF3N3O2 (576.80); MS (EI): m/z (%): 577.0 [M]+ꢀ (28),
402.0 [Mꢂ175(AC8H8F3N)]+ (100); HPLC (isocrat.): 98.5% at
254 nm, 98.8% at 280 nm, tR = 6.51 min, tM (DMSO) = 1.06 min
(ACN/H2O = 60:40); kmax: 228 nm, 319 nm, 390 nm; HPLC (gradient
method B): 98.0% at 254 nm, tR = 13.2 min.
5.1.4.10. 2-(9-Bromo-3-chloro-6-oxo-5,6,7,12-tetrahydrobenzo-
[2,3]azepino[4,5-b]indol-5-yl)-N-(pyridin-4-ylmethyl)acetamide
56.55, H 3.56, N 10.99, found C 55.95, H 3.42, N 10.43;
C
24H18BrClN4O2 (509.79); MS (EI): m/z (%): 510.0 [M]+ꢀ (13), 402.0
(3j).
tetrahydrobenzo[2,3]azepino[4,5-b]indol-5-yl)acetic
211 mg, 503 mol), DMF (1.0 mL), DIPEA (370
PyBOP (280 mg, 538
601 mol). Crystallization from methanol yielded a colorless solid
Synthesized from 2-(9-bromo-3-chloro-6-oxo-5,6,7,12-
[Mꢂ108.0(AC6H8N2)]+ (100); HRMS (EI): m/z [M]+ꢀ calcd
508.02962, found 508.03027; HPLC (isocrat.): 99.5% at 254 nm,
99.9% at 280 nm, tR = 3.75 min, tM (DMSO) = 1.06 min (ACN/
Buffer = 50:50); kmax: 232 nm, 319 nm, 389 nm.
acid (10,
l
lL, 2.12 mmol),
l
mol) and 4-pyridinemethanamine (61 lL,
l
(83 mg, 33%); dec. starting at 301 °C; IR (KBr): 3401 cmꢂ1 (NH),
3231 cmꢂ1 (NH), 1654 cmꢂ1 (C@O), 1687 cmꢂ1 (C@O); 1H NMR
(600 MHz, DMSO-d6): d (ppm) = 12.00 (s, 1H, indole-NH), 8.78 (t,
J = 6.0 Hz, 1H, ANH), 8.55–8.45 (m, 2H, ArH), 7.98 (d, J = 1.9 Hz,
1H, ArH), 7.73 (d, J = 8.4 Hz, 1H, ArH), 7.69 (d, J = 2.1 Hz, 1H, ArH),
7.50 (dd, J = 8.4, 2.1 Hz, 1H, ArH), 7.42 (d, J = 8.6 Hz, 1H, ArH),
7.30 (dd, J = 8.6, 1.9 Hz, 1H, ArH), 7.28–7.25 (m, 2H, ArH), 4.36
(bs, 4H, AN(5)-CH2 and ACH2), 4.01 and 3.13 (bs, 2H, azepine-
CH2); 13C NMR (151 MHz, DMSO-d6): d (ppm) = 31.2, 41.2, 53.6
(CH2); 113.6, 120.8, 121.9 (2C), 124.2, 124.9, 125.2, 128.6, 149.4
(2C) (CH); 148.4, 109.4, 111.9, 124.0, 127.8, 132.5, 133.0, 136.0,
141.0, 168.8, 170.4 (C); CHN: calcd C 56.55, H 3.56, N 10.99, found
C 56.31, H 3.54, N 10.60; C24H18BrClN4O2 (509.79); MS (EI): m/z
(%): 510.0 [M]+ꢀ (24), 402.0 [Mꢂ108.0(AC6H8N2)]+ (100); HPLC
(isocrat.): 97.7% at 254 nm, 98.2% at 280 nm, tR = 4.79 min,
tM (DMSO) = 1.06 min (ACN/Buffer = 40:60); kmax: 232 nm, 319 nm.
5.2. Biochemical and biological assays
5.2.1. Expression, purification and activity assay of
trypanothione synthetase (TryS)
5.2.1.1. Expression and purification of recombinant
TryS.
The recombinant expression and purification of His-
tagged TryS from T. brucei, T. cruzi and L. infantum was conducted
as essentially described in Maiwald et al.34, and Sousa et al.19
The expression vectors for TbTryS, LiTryS and TcTryS were kindly
provided by Dr. Alan Fairlamb (Dundee University, UK), Dr. Ana
Tomás (IBMC, Portugal) and Dr. Sergio Guerrero (Universidad
Nacional del Litoral, Argentina). All recombinant proteins were
expressed using Escherichia coli strain BL21(DE3) as host and Terri-
fic Broth or auto-induction medium devoid of trace elements. For
the first growth medium, 0.5 mM isopropyl 1-thio-b-
ranoside were added to induce recombinant protein expression
D-galactopy-
Please cite this article in press as: Orban, O. C. F.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.023

O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
during 5 h at 25 °C. In the autoinduction medium, cells were grown
at 37 °C for 5 h and then at 25 °C for further 16–18 h. After physical
(sonication) and enzymatic (lysozyme) cell lysis, the clarified
supernatant was applied to a Ni-NTA resin and the recombinant
TryS further purified to homogeneity by gel filtration and/or ion-
exchange chromatography at room temperature and using an
Äkta-FPLC device (GE Healthcare). Protein purity (for all
TryS P 95%) was assessed after each purification step by SDS–
PAGE 12% gel under reducing conditions. Protein concentration
was determined by the Bicinchoninic Acid assay with bovine
serum albumin as standard and enzyme activity was determined
by the end-point assay detailed below (see ‘TryS activity assay’).
The purified proteins were stored at ꢂ20 °C in reaction buffer
added of 40% (v/v) glycerol (Carlo Erba Reagents SA) without
significant loss of activity for at least 6 months.
costarÒ 3599) at a cell density of 5 ꢁ 105 cells/mL and treated with
2 lL compound or DMSO (proliferation control). After 24 h, 100 lL
from each well was transferred to individual 1.1 mL tubes (T100,
BiotubeTM System), diluted with 200
L sterile phosphate buffered
saline, pH 7.0, with 1% (w/v) glucose and added of 2 L propidium
iodide (exclusion dye) at 200 g/mL, homogenized by quick vor-
texing and analyzed using a CyAnTM ADP (DakoCytomation) flow
cytometer. Flow cytometry analysis was performed with
l
l
l
a
488 nm solid-state laser as the excitation light source. The positive
events (cells) were gated by forward-scatter (FSC) and side-scatter
(SSC) parameters whereas the fluorescence of propidium iodide
was collected at kem = 613/30 nm to distinguish dead cells. The sig-
nals were detected with logarithmic amplifiers. All measurements
were made at constant flow and acquisition time (60 s per sample).
The data were analyzed using the Summit (Dako) and Flow-Jo
(Tree star Inc.) software. A positive control included Nifurtimox
5.2.1.2. TryS activity assay.
The end-point assay is based on
(LampitÒ from Bayer) tested at its EC50 (15
were tested by triplicate.
lM). All conditions
the determination of inorganic phosphate released from ATP dur-
ing TryS catalysis, by its complexation (phosphomolybdate) to
the cationic triphenylmethane dye, malachite green (BIOMOL
GREENTM EnzoÒ Life Sciences). The green complex presents an
absorption maximum at 650 nm. For all TryS, ATP was used at
The relative percentage of viable parasites at 5 or 30
expressed as follow:
lM is
%viability ¼ ½ðnumber of parasites for compound X at concentration YÞ=
ðnumber of parasites in the proliferation controlÞꢃꢁ100:
150 lM because of high background signal at higher concentra-
tions and spermidine fixed at 2 mM, a concentration that resem-
The EC50 values were obtained from dose response curves fitted
to a sigmoidal Boltzmann equation (errors calculated using errors’
propagation) or extrapolated from non-linear fitting plots. The
bles the intracellular levels calculated from data reported in the
literature for different trypanosomatids43–45
. Glutathione was
adjusted to 0.05, 0.57 and 0.25 mM for TbTryS, TcTryS and LiTryS,
respectively, concentrations that avoid substrate inhibition or cor-
respond with reported intracellular contents.46
A master mix (MM) solution containing all the substrates at
1.25-fold their end concentration in assay was prepared in reaction
buffer (5 mM DTT, 10 mM MgSO4, 0.5 mM EDTA, 100 mM HEPES pH
7.4, 9 mM NaCl, 10% v/v DMSO) and kept on ice until use. Microtiter
error is expressed as 2 S.D., estimated as 2rnꢂ1
.
5.2.3. Cytotoxicity in murine macrophages
The J774 mouse macrophage cell line was cultivated in a
humidified 5% CO2/95% air atmosphere at 37 °C in DMEM medium
supplemented with 10% (v/v) FCS, 10 U/mL penicillin and 10 lg/mL
streptomycin. The EC50 were determined from dose–response
assays at seven experimental concentrations (from 200 to
plate wells were loaded with 5
lL of test compound, DMSO (reac-
tion control) or TryS-specific inhibitor (inhibition control) and
0.001
l
M) of compounds tested in triplicate and essentially as
described in Demoro et al.48, except that 200
l
L of a cell suspen-
40
l
L of MM. The reactions were then started by adding 5
l
L of TryS
mol minꢂ1 mLꢂ1 for TcTryS, LiTryS and TbTryS,
L BIOMOL
(1, 2 and 3 ꢁ 10ꢂ5
l
sion at 6 ꢁ 104 cells/ mL was added per well in a 96-well culture
plate. The cytotoxicity was evaluated with a colorimetric assay
(WST-1 reagent) that measures the absorbance at 450 nm of the
formazan dye produced by metabolically active cells. The photo-
metric measurements were performed with an EL 800 microplate
reader (Biotek). The following equation was used to estimate the
cytotoxicity: Cytotoxicity (%) = (experimental value ꢂ DMSO con-
trol)/(growth control ꢂ DMSO control) ꢁ 100. The data were plot-
ted as percentage cytotoxicity versus compound concentration.
EC50 values were obtained as described above for anti-T. brucei
activity.
respectively) and stopped after 15 min with 200
l
GREENTM reagent. Blanks were prepared for each condition by add-
ing 5 L of reaction buffer with 150 mM NaCl instead of enzyme.
The TryS activity is calculated as follow: % TryS activity =
{[(A650 CiB)/(A650 E ꢂ A650 EB)] ꢁ 100}, where
Ci ꢂ A650
l
nm
nm
nm
nm
A650 nm Ci is the mean absorbance at 650 nm for the reaction test
with compound i, A650 is the mean absorbance at 650 nm
nm CiB
for the blank control with compound i, A650
is the mean
nm
E
absorbance at A650
for the reaction control with DMSO and
nm
A650 nm EB is the mean absorbance at A650 nm for the blank control
with DMSO. The assays yielded an intra-assay coefficient variation
(CV = rnꢂ1E/E) 6 2.5%, a signal to background coefficient (E/EB) of
ꢀ3.5 and a Z0 factor P0.85.
5.2.4. Assay for inhibition of mammalian protein kinases
5.2.4.1. Protein kinase assay buffers.
1 mM EGTA, 1 mM DTT, 25 mM Tris–HCl pH 7.5, 50
Buffer B: 50 mM MgCl2, 90 mM NaCl, 30 mM Tris–HCl pH 7.4.
Buffer A: 10 mM MgCl2,
lg heparin/mL.
5.2.2. Proliferation assay for infective T. brucei brucei
The biological activity of the compounds was evaluated in the
infective form of T. brucei brucei strain 427, cell line 449 (encoding
one copy of the tet-represor protein: Pleo)47 transfected with a
construct encoding a redox biosensor (Sardi, Comini unpublished).
The cultivation of the parasites and the viability assay were per-
formed as previously described by Maiwald et al.34 Stock solutions
at 3–24 mM in DMSO were prepared for each compound according
to their solubility. All compounds were subjected to a primary
5.2.4.2. Kinase preparations and assays.
each enzyme were assayed in buffer A or B, with their correspond-
ing substrates, in the presence of 15 M ATP in a final volume of
30 L. After 30 min incubation at 30 °C, the reaction was stopped
Kinase activities for
l
l
by harvesting, using a FilterMate harvester (Packard), onto P81
phosphocellulose papers (GE Healthcare) which were washed in
1% phosphoric acid. Scintillation fluid was added and the radioac-
tivity measured in a Packard counter. Blank values were subtracted
and activities calculated as pmoles of phosphate incorporated dur-
ing the 30 min incubation. The activities were expressed in % of the
maximal activity, i.e., in the absence of inhibitors. Controls were
performed with appropriate dilutions of DMSO.
screening at 5 and 30
lM. For compounds showing more than
90% parasite growth inhibition at 5
lM, EC50 values were deter-
mined. To determine EC50, twofold serial dilutions in DMSO were
prepared from the compound’s stock solution. Two hundred micro-
liter of trypanosomes in mid-exponential phase (ꢀ2 million cells/
mL) were seeded in a 96-well culture plate (Corning Incorporated,
Please cite this article in press as: Orban, O. C. F.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.06.023

10
O. C. F. Orban et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin A
(human, recombinant, from A. Echalier, University of Leicester),
Therefore the lowest concentrations used for the calibration are
indicated as upper limit of solubility in Table 1.
CDK5/p25 (human, recombinant, expressed in E. coli) were assayed
in Buffer
BSA/mL, except for CDK2) with 25
T (human, recombinant, expressed in insect cells) was assayed as
A
(supplemented extemporaneously with 0.15 mg
Acknowledgements
lg of histone H1. CDK9/cyclin
This project was funded by the German Federal Ministry of Edu-
cation and Research (BMBF, program KMU-innovativ 5, grant pro-
ject code 0315814; to R. S. K., O. K., and C. K.) and supported by the
COST action CM1307 (to O. C. F. O. and C. K.). The support of FOCEM
(MERCOSUR Structural Convergence Fund, COF 03/11), Institut
Pasteur ACIP call 2015 (project ACIP 17-2015) and ANII – Uruguay
(POS_NAC_2013_1_114477) is acknowledged by M.A.C., D.B. and A.
M., L.M. was supported by a grant from the ‘Agence Nationale de la
Recherche’ (ANR), Transleish. Molecular graphics were performed
with the UCSF Chimera package. Chimera is developed by the
Resource for Biocomputing, Visualization, and Informatics at the
University of California, San Francisco (supported by NIGMS
9P41GM103311). All funding sources were not involved in study
design, collection, analysis and interpretation of data; in the writ-
ing of the paper; and in the decision to submit the article for
publication.
described for CDK1/cyclin B, but using 8.07
lg of CDK7/9-Tide
(YSPTSPSYSPTSPSYSPTSPSKKKK).49
DYRK1A (human, recombinant, expressed in E. coli as a GST
fusion protein), and CLK1 (mouse, recombinant, expressed in
E. coli as a GST fusion protein) were assayed as described for
CDK1/cyclin B with 1
lg of RS peptide (GRSRSRSRSRSR) as a
substrate.50
GSK-3a/b (porcine brain, native, affinity purified on axin
1-sepharose beads51 was assayed as described for CDK1/cyclin B,
but using a GSK-3 specific substrate (GS-1: YRRAAVPPSPSLSRHS
SPHQpSEDEEE where pS stands for phosphorylated serine).
Casein kinase 1 (CK1d/
e) (porcine brain, native, purified by
affinity chromatography on axin 2-sepharose beads52) was assayed
with 0.67
substrate.
lg of CKS peptide (RRKHAAIGpSAYSITA), a CK1 specific
5.3. In-silico analysis
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