9- and 11-substituted 4-azapaullones are potent and selective inhibitors of African trypanosoma

Article abstract of DOI:10.1016/j.ejmech.2014.06.020

Trypanosomes from the "brucei" complex are pathogenic parasites endemic in sub-Saharan Africa and causative agents of severe diseases in humans and livestock. In order to identify new antitrypanosomal chemotypes against African trypanosomes, 4-azapaullone

Full text of DOI:10.1016/j.ejmech.2014.06.020

European Journal of Medicinal Chemistry 83 (2014) 274e283
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
9- and 11-substituted 4-azapaullones are potent and selective
inhibitors of African trypanosoma
,
,
Franziska Maiwald a 1, Diego Benítez b 1, Diego Charquero b, Mahin Abad Dar c,
c
a
d
c
e
€
ꢀ
€
Hanna Erdmann , Lutz Preu , Oliver Koch , Christoph Holscher , Nadege Loaec ,
Laurent Meijer e, Marcelo A. Comini b *, Conrad Kunick a
,
, *
a
€
Technische Universitat Braunschweig, Institut für Medizinische und Pharmazeutische Chemie, Beethovenstra
ße 55, D-38106 Braunschweig, Germany
b Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Mataojo 2020, CP 11400 Montevideo, Uruguay
c Forschungszentrum Borstel, Forschungsgruppe Infektionsimmunologie, Parkallee 22, D-23845 Borstel, Germany
d
€
€
Technische Universitat Dortmund, Department of Chemistry and Chemical Biology, Technische Universitat Dortmund, Otto-Hahn-Stra
ße 6,
44227 Dortmund, Germany
e ManRos Therapeutics, Perharidy Research Center, 29680 Roscoff, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanosomes from the “brucei” complex are pathogenic parasites endemic in sub-Saharan Africa and
causative agents of severe diseases in humans and livestock. In order to identify new antitrypanosomal
chemotypes against African trypanosomes, 4-azapaullones carrying a,b-unsaturated carbonyl chains in
9- or 11-position were synthesized employing a procedure with a Heck reaction as key step. Among the
so prepared compounds, 5a and 5e proved to be potent antiparasitic agents with antitrypanosomal
activity in the submicromolar range.
Received 31 March 2014
Received in revised form
4 June 2014
Accepted 10 June 2014
Available online 11 June 2014
© 2014 Published by Elsevier Masson SAS.
Keywords:
Chalcones
Cinnamides
Nagana
Paullones
Trypanosoma brucei brucei
Human trypanosomiasis
1. Introduction
Interestingly, the proposal by MacLeod et al. that human pathogens
from the “brucei” clade are host-range variants of the local T. b.
Protozoan parasites from the genus Trypanosoma are respon-
sible for zoonotic diseases affecting human and livestock. In Africa,
Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense
cause human sleeping sickness whereas Trypanosoma brucei brucei,
Trypanosoma vivax and Trypanosoma congolense produce Nagana in
cattle. These unicellular pathogens are transmitted by the bites of
tsetse flies abundant in sub-Saharan Africa where more than 70
million people and 40 million domestic animals are at risk of
trypanosomiasis [1].
brucei populations [2] has recently been supported by a thorough
genetic survey of field isolates that demonstrate inter-subspecies
genetic exchange [3,4] and the identification of single genes as
determiner of human infectivity [5,6]. These findings also raise
alarms about the genetic diversity available to T. brucei for coun-
teracting natural or chemical (drugs) selection [3].
Among the animals affected by the disease are cattle, horses,
sheep and goats that after severe infection show anemia, body
weight loss, changes in food intake, which increases the suscepti-
bility to secondary infections and decreases productivity [7]. Since
the domestic animals are a major source of food and financial
earnings of the rural population, Nagana accounts also for human
malnutrition and impairment of social development. Affecting
almost one third of the cattle population, Nagana is economically
the most important African livestock disease [8].
The infection in humans has two major clinical presentations,
acute (haemolymphatic form) and chronic (neurological form), that
despite being highly disabling are fatal if left untreated.
* Corresponding authors.
The development of effective immunoprophylaxis against Afri-
can trypanosomes remains unattainable due to sophisticated and
successful evasion mechanism employed by the pathogens [9].
E-mail addresses: mcomini@pasteur.edu.uy (M.A. Comini), c.kunick@tu-
braunschweig.de, c.kunick@tu-bs.de (C. Kunick).
1
These authors contributed equally.
http://dx.doi.org/10.1016/j.ejmech.2014.06.020
0223-5234/© 2014 Published by Elsevier Masson SAS.

F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
275
Chart 1. Paullones mentioned in the text.
Since more than six decades these diseases are treated using a
limited number of drugs that suffer from poor safety and efficacy,
and require an administration regime difficult to apply in humans.
For instance, pentamidine and suramin are recommended for the
treatment of the first stage of human African trypanosomiasis,
whereas melarsoprol, efluornithine and, more recently, a combi-
nation of efluornithine/nifurtimox are drugs of choice when the
parasites invade the central nervous system [10]. The lack of
effectiveness of efluornithine and nifurtimox against T. b. rhode-
siense and the increasing number of cases reporting drug resistance
[11e14] exemplify how limited is the current chemotherapy.
Nevertheless, an agreement between WHO and pharmaceutical
companies (Sanofi and Bayer AG) make these drugs available to
endemic countries free of charge [1].
Nagana is treated with: the phenanthridinium ethidium chlo-
ride, the diamidine diminazene aceturate (DA), and iso-
methamidium chloride (ISM) [8,15]. Because of its mutagenic
properties the use of ethidium for prophylaxis or therapy of
infected animals is no longer recommended [16] and its effective-
ness is furthermore hampered by widespread resistance of the
parasites [17]. DA and ISM are cheap and easy to obtain, their pri-
vate use is often not controlled by trained veterinary personnel.
Thus, inadequate application regimes might have contributed to
the numerous cases of resistance reported from 17 African coun-
tries between Mali and Mozambique [16,18].
Unfortunately, the market volume for human and veterinary
drugs against African trypanosomiasis is not attractive for inter-
national pharmacy companies [16]. However, the pharmacological
weakness of the drugs currently available, the lack of back-up
substitutes and the emergence of resistance urge on the develop-
ment of new chemotherapeutics against African trypanosomes. To
avoid the risk of cross-resistance with established drugs, new
antitrypanosomal candidates should be unrelated to existing drugs
by means of structure and potential mechanism of action. The study
presented here was directed to the design of new antitrypanosomal
compounds against African trypanosomes.
In preceding papers we reported upon the antileishmanial and
antitrypanosomal activity of paullone derivatives substituted at the
2-position with side chains containing
a,b-unsaturated carbonyl
elements, e.g. the paulloneechalcone hybrid 1 [19,20]. However,
these compounds showed some signs of toxicity when tested on
human cell lines. Since the structures are based on the heterocyclic
scaffold paullone (7,12-dihydroindolo[3,2-d][1]benzazepin-6(5H)-
one, 2a), which represents a well established class of mammalian
protein kinase inhibitors [21e25], toxic effects of antitrypanosomal
paullones may stem from undesired inhibition of host kinases. It
has been shown that insertion of a nitrogen atom in position 4 of
the paullone scaffold abrogates the kinase inhibitory potency in
paullone congeners [26]. For example, 4-azakenpaullone 3 is more
than two orders of magnitude less active as a GSK-3 inhibitor
compared to its 4-carbaanalogue kenpaullone 2b (Chart 1) [27]. We
therefore designed new 4-azapaullones as potential anti-
trypanosomal compounds. To introduce an additional aspect of
novelty, the side chains were attached to the 9-position (derivatives
4) or to the 11-position (derivatives 5), respectively.
2. Chemistry
For the preparation of the desired compounds 4 and 5, an
improved synthesis for the pivotal building block 9 was developed
(Scheme 1) [27]. First, commercially available 2-aminopyridine-3-
carboxylic acid (6) was esterified by means of iodoethane. The
resulting ethyl ester 7 was acylated by ethyl succinyl chloride. The
raw resulting amide was treated with potassium tert-butylate in a
toluene/DMF mixture to yield the benzazepine derivative 8 by
means of
a Dieckmann condensation reaction. A dealk-oxy-
carbonylation of 8 in wet DMSO under neutral conditions furnished
the cyclic ketone 9 which was reacted at 40 ꢀC with either 4-iodo-
or 2-iodophenylhydrazine in the presence of sulfuric acid, respec-
tively, yielding the phenylhydrazones 10a and 10b. After purifica-
tion, these phenylhydrazones were heated to 70 ꢀC in the presence
of sulfuric acid to produce the 4-azapaullones 11a and 11b by

276
F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
Scheme 1. Synthesis of 9- or 11-iodo-4-azapaullones. Reagents and conditions: (i) ICH2CH3, K2CO3, DMF, rt, 1 h, 81%; (ii) ethyl succinyl chloride, pyridine, THF, rt; (iii) KO-tert-Bu,
toluene, DMF, N2, 0 ꢀC / rt, 33% from 7; (iv) DMSO, H2O, N2, 150 ꢀC, 5e7 h, 87%; (v) 2- or 4-iodophenylhydrazine, AcOH, H2SO4, rt / 40 ꢀC, 67% and 89%, respectively; (vi) AcOH,
H2SO4, 70 ꢀC, 63% and 51%, respectively.
Fischer indole ring closure reactions. Starting from the iodoarenes
11a or 11b, respectively, the preparation of the title compounds was
accomplished by Heck reactions as exemplified in Scheme 2. For the
synthesis of the paulloneechalcone hybrid structure 5a, 11b was
reacted with N,N-dimethyl-3-oxo-3-phenylpropan-1-aminium
chloride (12) as precursor of phenyl vinyl ketone in the presence
of palladium acetate [28]. The product 5a resulted in (E)-configu-
ration as deduced from the coupling constant of the vinylic protons
in the 1H NMR spectrum. The low yield of this compound was
caused by a cumbersome purification procedure during which most
of the product was lost. A classical Heck reaction of 11b with N-
phenylacrylamide (13) yielded the paulloneecinnamide hybrid
structure 5e that also displayed (E)-configuration. The other test
compounds 4 and 5 were synthesized by similar procedures.
the related pathogenic subspecies. The screening tests were per-
formed at compound concentrations of 5 and 30 M and cell
viability was assessed by flow cytometry after 24 h incubation. For
those compounds that at 5 M reduced more than 90% of the cell
m
m
population, EC50 values were determined. The results displayed in
Table 1 show that indeed most of the new 4-azapaullones 4 and 5
are potent inhibitors of T. b. brucei parasites. In the series 4
substituted at the 9-position, the secondary cinnamides 4f and 4g
showed higher antiparasitic activity than the tertiary amide 4e or
the chalcone-4-azapaullone hybrids 4ae4d. This observation in-
dicates that both an H-bond donor motif and an aromatic ring
appear advantageous at the end of the side chain in the derivatives
4. The results with the congeners 5 were more heterogeneous.
While the chalcone-4-azapaullone hybrid 5a showed a stronger
antiparasitic activity compared to its 9-substituted analogue 4a, p-
substitution with a methoxy (5b) or a chloro substituent (5c) at the
terminal phenyl ring led to derivatives that were significantly less
potent compared to the analogues 4b and 4c, respectively. With an
EC50 of 120 nM, the N-phenylcinnamide 5e turned out to be the
most potent antitrypanosomal compound in the series. The com-
parison with the modest activity displayed by the iodo-substituted
3. Biological evaluation and discussion
All new 4-azapaullones 4 and 5 were tested for growth inhibi-
tion of infective T. b. brucei cultivated in vitro. T. b. brucei appears as
a suitable laboratory model for in vitro screening tests because it is
not pathogenic for humans but expresses the full repertoire of
genes controlling metabolic and other major cellular processes as
4-azapaullones 11a and 11b demonstrates that the a,b-unsaturated
Scheme 2. Synthesis of azapaullones 5a and 5e substituted in 11-position. The synthesis of azapaullones 4 substituted in 9-position was performed by similar methods. Reagents
and conditions: (i) TEA, Pd(OAc)2, DMF, 140 ꢀC, 1 h, 6%; (ii) TEA, Pd(OAc)2, DMF, microwaves, 100 ꢀC, 40 min, 80%.

F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
277
Table 1
Structures, antitrypanosomal Activity, trypanothione synthetase inhibition and toxicity of 4-Azapaullones
R
Viabilitya of bloodstream
Residual activityc of TbTryS [%] 2SD @ 30
m
M; n
Toxd [%] SD; n
T. b. brucei @ conc. [%] 2SD;
n or EC50 T.b.b. [m
M]b
4a
4b
4c
4d
4e
4f
4g
5a
5b
5c
5d
5e
5f
Ph-
4-MeO-Ph-
4-Cl-Ph-
Furan-2-yl-
Diethylamino-
Phenylamino-
Benzylamino-
Ph-
54.0 3.1 @ 5
44.5 2.7 @ 5
38.4 2.7 @ 5
m
m
m
M; 2
M; 2
M; 2
61.9 6.4; 4
77.6 5.4; 4
79.8 5.7; 4
90.2 3.8; 4
91.5 5.2; 4
68.1 2.4; 4
67.5 4.2; 6
91.1 7.2; 3
80.4 3.1; 3
73.2 7.8; 4
102.0 8.3; 4
94.6 2.7; 4
81.8 5.5; 4
96.7 6.1; 4
111.7 8.1; 3
7.6 3.1 @ 26
20.3 12.7 @ 12
4.73 1.8 @ 48
17.6 6.9 @ 54
m
M; 3
M; 3
m
m
m
M; 3
M; 3
21.9 0.001 @ 30
22.1 0.6 @ 30 M; 2
EC50 ~ 2.5
12.1 4.4 @ 5
EC50 ¼ 0.18 0.02
79.9 10.5 @ 30
74.6 7.3 @ 5 M; 2
81.6 3.9 @ 30 M; 2
m
M; 3
m
<0 @ 59
<0 @ 58
<0 @ 29
mM; 3
mM; 4
mM; 4
m
M
mM; 2
mM
28.3 7.9 @ 26
m
M; 4
4-MeO-Ph-
4-Cl-Ph-
m
M; 2
16.5 11.7 @ 48
9.2 6.6 @ 12 M; 4
<0 @ 56 M; 4
mM; 4
m
m
Diethylamino-
Phenylamino-
Benzylamino-
R1 ¼ I, R2 ¼ H
R1 ¼ H, R2 ¼ I
m
m
EC50 ¼ 0.12 0.04
m
m
M
M
74.7 4.4 @ 60
4.16 0.7 @ 24
12.3 5.7 @ 10
m
m
m
M; 4
M; 2
M; 4
EC50 ¼ 0.45 0.12
11a
11b
72.1 7.6@ 30
m
M; 2
M; 2
61.3 18.5 @ 30
m
4.4 4.1 @ 5 mM; 3
a
Compounds were simultaneously tested @ 5 and 30 mM. Parasite viability was assessed by flow cytometry after 24 h incubation with test compounds. Nifurtimox 15 mM
was used as 50% growth inhibition control drug.
b
For the compounds reducing parasite viability for more than 90% @ 5 mM, EC50 values were determined. The EC50 is the compound concentration that inhibits parasite
growth to 50% compared to untreated controls.
c
The activity of recombinant T. b. brucei trypanothione synthetase (TbTryS) was evaluated after incubation for 15 min without (control) or with test compounds by
measuring the phosphate released from ATP with Biomol Green™ reagent. The values are expressed as % residual activity with respect to the control.
d
Toxicity for mammalian cells measured with mouse macrophages using the xCELLigence system. Shown is the toxicity [%] at the highest concentration [
depending on the solubility of the test compounds.
mM] that was used
carbonyl motif plays an important role for antitrypanosomal ac-
tivity in 4 and 5. All compounds were also evaluated for non-
specific toxicity using a xCELLigence assay with mouse macro-
phages as mammalian cell model. At the highest tested concen-
tration, which depended on individual compound solubility, none
of the derivatives (with the exception of 5e) provoked a reduction
in viability of more than 50%. Hence, EC50 values for these com-
pounds were not calculated and compounds were rated to be of
limited toxicity, at least in this test system. For instance, by
comparing the toxicity parameters for 5a it can be estimated that it
presents selective inhibition towards parasites that is at least two-
orders of magnitude higher than against macrophages. For an
assessment of potential off-targets of the substituted 4-
azapaullones, the derivatives showing highest antitrypanosomal
activity (5a and 5e) were tested on mammalian kinases that are
typically inhibited by paullones. In the test panel also related ki-
nases from the CMGC family were included (Table 2). Both 5a and
5e failed to inhibit all and most, respectively, protein kinases at the
Up to now, the biochemical mechanism underlying the anti-
trypanosomal activity of 4 and 5 remains speculative. Recently it
was reported that N5-substituted paullones inhibit the trypano-
thione synthetase (TryS) by competing with ATP-binding [29]. Be-
ing essential for maintaining the cellular redox status in
Kinetoplastidae and absent in mammalian species, TryS was sug-
gested as a druggable target for treatment of trypanosomiasis
[30e32]. We therefore constructed a homology model of T. b. brucei
TryS based on the published structure of the Leishmania major TryS
(pdb file 2vpm) and the T. b. brucei TryS sequence (Q82MC2 from
the Uniprot data base) (data not shown). All attempts to generate
meaningful ligandeprotein complexes by docking compounds 4
and 5 into the ATP binding site of the T. b. brucei TryS 3D-model
failed. TryS have been shown to contain highly dynamic structural
elements involved in substrate binding [33e35]. Thus, a structural
Table 2
highest tested concentration (10
mM) with the only exception that
Inhibition of Mammalian Kinases of the CMGC Family by 4-Azapaullones 5a and 5e
compound 5e exhibited an IC50 of 0.49
m
M against the casein kinase
[IC50
,
m
M].
1 (CK1). Overall, this result corroborates our assumption that
introduction of the nitrogen atom in 4-position of the paullone
basic structure leads to loss of undesired inhibitory activity on a
wide diversity of mammalian kinases.
CDK1 CDK2/A CDK5 CDK9/T CK1
CLK1 DYRK1A GSK-3
5a >10
5e >10
>10
>10
>10
>10
>10
>10
>10
0.49 >10
>10
>10
>10
>10
>10

278
F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
model of TryS might not provide reliable information about
conformational changes shaping the sites of ligand binding.
Therefore, we decided to test all derivatives 4 and 5 for in vitro
inhibition of recombinant T. b. brucei TryS (Table 1). Although some
congeners (4aec, 4feg, 5c) showed a modest inhibition, the dif-
ferences observed in potency against enzyme (e.g. 20e30% activity
devices and settings [38]: Elite LaChrom (Merck/Hitachi), Pump L-
2130, autosampler L-2200, Diode Array Detector L-2450, organizer
box L-2000; column: Merck LiChroCART 125-4, LiChrosphere 100,
RP 18, 5
mm; flow rate 1.000 mL/min, isocratic, volume of injection:
10 L; detection (DAD) at 254 and 280 nm; AUC-%-method.; time of
m
detection 15 min, net retention time (tN), dead time (tm) related to
DMSO. All compounds employed in biological tests were used in
>90% purity. Microwave accelerated reactions were carried out in
sealed 10 mL microwave glass tubes using a mono mode microwave
device (CEM discover, Kamp-Lintfort, Germany). For reactions in a
parallel synthesis reactor a carousel 12 place reaction station was
used (Radley Discovery Technologies, Shire Hill, Saffron Walden,
Essex, UK). Mannich bases [39] and N-substituted acrylamides
[40,41] employed as reagents in Heck reactions were synthesized
according to published standard methods. Other starting materials
were purchased from commercial suppliers (Acros Organics, Geel,
Belgium), Sigma Aldrich (St. Louis, MO, USA), Merck KGaA (Darm-
stadt, Germany) and were used without further purification. Syn-
thesis procedures and characterization of 2-iodophenylhydrazine
as well as test compounds 4aeg, 5bed and 5f are described in the
supporting information.
inhibition at 30
m
M) and parasite growth (EC50 ꢁ 5 uM) suggests
that other targets than TryS are responsible for the anti-
trypanosomal activity of the title compounds.
It may be speculated that the Michael acceptor motif present in
4 and 5 could react with vital thiols in the parasite cells. This mode
of action has recently been proven for the antitrypanosomal
sesquiterpene lactone cynaropicrin which leads to depletion of
trypanothione and glutathione in the parasite by formation of co-
valent adducts [36]. On the other hand, based on the inhibition of
mammalian CK1 by 5e and the finding that the isoform 2 of the
homologue protein from Trypanosoma brucei has been shown to be
indispensable for the infective form of the parasite [37] it is
tempting to speculate that this compound exerts its trypanosomal
toxicity by inhibiting this molecular target. Future studies will
address the mechanism of action of the most potent paullones
described in this work.
4. Conclusion
5.1.2. 2-Aminopyridine-3-carboxylic acid ethyl ester (7)
To a solution of 2-aminopyridine-3-carboxylic acid (6, 2.00 g,
14.0 mmol) in DMF (56 mL) was added potassium carbonate
(2.50 g, 28.0 mmol). After stirring the suspension for 30 min at
room temperature, a solution of iodoethane (2.20 g, 14.0 mmol,
1.10 mL) in DMF (14 mL) was added dropwise. After stirring for 1 h
the mixture was poured on ice water (300 mL). The precipitated
crystals were collected by filtration. The filtrate was extracted with
ethyl acetate (4 ꢂ 75 mL). The combined ethyl acetate layers were
dried (Na2SO4) and evaporated. To the remaining residue diethyl
ether (100 mL) was added and the resulting solution was washed
with aqueous 30% lithium chloride solution (4 ꢂ 25 mL). After
drying with Na2SO4, the organic solvent was evaporated. The res-
idue was combined with the precipitate that was collected earlier
and the material was crystallized from ethanol to yield 1.90 g
(80.8%) colorless crystals. mp: 93e94 ꢀC (Lit.: 94e96 ꢀC [42]); IR
The design and synthesis of 4-azapaullones carrying
a,b-un-
saturated carbonyl chains in 9- or 11-position led to compounds 4
and 5 showing inhibitory activity towards the infective stage of T. b.
brucei. The most potent compounds 5a and 5e inhibited the para-
sites in submicromolar concentrations. A low degree of toxicity for
these compounds can be assumed since mammalian cells (mouse
macrophages) were only slightly affected in significant higher
concentrations on the one hand and a selection of mammalian ki-
nases was not inhibited by 5a and 5e, respectively, on the other
hand. The exception was compound 5e that has been identified
here as a selective inhibitor of mammalian CK1. The mode of action
of these antitrypanosomal agents is still unknown. The trypano-
thione synthetase of T. b. brucei was eliminated as possible intra-
cellular target by docking experiments and by inhibition tests with
recombinant enzyme. Since 5a and 5e offer many options for mo-
lecular variations they appear as appropriate starting points for a
further development of drugs against African trypanosomes.
(KBr): 3428 (NH2), 1695 (C]O), 1625 (NH2), 1245, 770 cmꢃ1
;
1H
3
NMR (600 MHz, CDCl3):
d
¼ 8.22 (dd, 1H, JH,H ¼ 4.8 Hz,
4JH,H ¼ 1.9 Hz), 8.15 (dd, 1H, 3JH,H ¼ 7.8 Hz, 4JH,H ¼ 1.9 Hz), 6.63 (dd,
1H, 3JH,H ¼ 7.8 Hz, 3JH,H ¼ 4.8 Hz), 4.35 (q, 2H, 3JH,H ¼ 7.1 Hz), 1.39 (t,
3
5. Experimental protocols
3H, JH,H ¼ 7.1 Hz); 13C NMR (150.9 MHz, CDCl3):
d
¼ 167.0, 159.4,
106.5 (quat C); 153.5, 140.1, 112.7 (tert C); 60.9 (sec C); 14.3 (prim.
C); C8H10N2O2 (166.18); calcd. C 57.82, H 6.07, N 16.86; found C
57.68, H 6.22, N 17.20.
5.1. Synthetic chemistry
5.1.1. General
Melting points (mp) were determined on an electric variable
heater (Barnstead Electrothermal IA 9100) and were not corrected.
IR-spectra were recorded as KBr discs 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
5.1.3. 2-[(4-Ethoxy-1,4-dioxobutyl)amino]pyridine-3-carboxylic
acid ethyl ester
To a solution of 2-aminopyridine-3-carboxylic acid ethyl ester
(7, 4.00 g, 24.00 mmol) in THF (80 mL) were successively dropwise
added pyridine (2.00 g, 24.8 mmol, 2.00 mL) and succinic acid ethyl
ester chloride (7.20 g, 44.0 mmol, 6.00 mL). After stirring the
mixture for 2 h at room temperature, water (160 mL) was added
and the resulting mixture was extracted with ethyl acetate
(3 ꢂ 120 mL). The combined organic layers were dried (Na2SO4) and
evaporated. The remaining viscous oil (4.96 g) was used for the
following synthesis step without further purification. 1H NMR
tetramethylsilane; signals in ppm (
d scale). Elemental analyses
were determined on a CE Instruments FlashEA® 1112 Elemental
Analyser (Thermo Quest). Mass spectra were recorded on a double-
focused sector field mass spectrometer Finnigan-MAT 90. Accurate
measurements where conducted according to the peakmatch
method using perfluorokerosene (PFK) as an internal mass refer-
ence; (EI)-MS: ionization energy 70 eV. TLC: Polygram® Sil G/
UV254, MachereyeNagel, 40 ꢂ 80 mm, visualization by UV-
illumination (254 nm). Column chromatography: silica gel 60
(Merck), column width: 2 cm, column height 10 cm unless stated
otherwise. Purity was determined by HPLC using the following
(600 MHz, CDCl3):
d
¼ 10.94 (s, 1H), 8.57 (d, 1H, 3JH,H ¼ 4.4 Hz), 8.33
(d,1H, 3JH,H ¼ 7.8 Hz), 7.09 (dd,1H, 3JH,H ¼ 7.4 Hz, 3JH,H ¼ 5.0 Hz), 4.40
3
3
(q, 2H, JH,H ¼ 7.1 Hz), 4.16 (q, 2H, JH,H ¼ 6.9 Hz), 2.98 (t, 2H,
3JH,H ¼ 6.8 Hz), 2.75 (t, 2H, 3JH,H ¼ 6.8 Hz), 1.41 (t, 3H, 3JH,H ¼ 7.2 Hz),
3
1.26 (t, 3H, JH,H ¼ 7.1 Hz).

F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
279
5.1.4. 5-Hydroxy-8-oxo-8,9-dihydro-7H-pyrido[2,3-b]azepine-6-
carboxylic acid ethyl ester (8)
The raw 2-[(4-ethoxy-1,4-dioxobutyl)amino]pyridine-3-
5.1.7. 11-Iodo-7,12-dihydropyrido[20,30:2,3]azepino[4,5-b]indol-
6(5H)-one (11b)
To a mixture of 5-[2-(2-iodophenyl)hydrazono]-6,7-dihydro-
5H-pyrido[2,3-b]azepin-8(9H)-one (10b, 230 mg, 0.580 mmol) and
glacial acetic acid (10 mL) was added concentrated sulfuric acid
(0.7 mL). After stirring for 1 h at 70 ꢀC under nitrogen the mixture
was cooled to room temperature. A 5% aqueous solution of sodium
acetate (40 mL) was added. A precipitate formed which was filtered
off, washed with water and crystallized from ethanol to yield a
colorless solid (65.0 mg, 51.2%); dec. starting at 330 ꢀC; IR (KBr):
carboxylic acid ethyl ester (4.96 g) prepared as described above
was dissolved in a mixture of toluene (53 mL) und DMF (8 mL). This
solution was added dropwise to a precooled solution of potassium
tert-butoxide (10.0 g, 89.0 mmol) in toluene (27 mL) at 0 ꢀC under
nitrogen. The mixture was allowed to warm up to room tempera-
ture and was stirred for further 4 h under nitrogen. After addition of
glacial acetic acid (5.3 mL) and water (53 mL) a precipitate formed
which was filtered and washed with petrol ether. The material was
crystallized from ethanol to yield a colorless solid (1.90 g, 33.4%
over two steps from 7); mp: 207e208 ꢀC; IR (KBr): 3204 (NH), 1690
3421, 3192 (NH), 1667 (C]O) cmꢃ1; 1H NMR (600 MHz, DMSO-d6):
3
d
¼ 11.40 (s, 1H), 10.36 (s, 1H), 8.45 (dd, 1H, JH,H ¼ 4.7 Hz,
4JH,H ¼ 1.8 Hz), 8.35 (dd,1H, 3JH,H ¼ 7.8 Hz, 4JH,H ¼ 1.8 Hz), 7.75 (d,1H,
(C]O), 1403, 1193, 764 cmꢃ1
;
1H NMR (600 MHz, DMSO-d6):
3JH,H ¼ 7.7 Hz), 7.62 (dd, 1H, 3JH,H ¼ 7.4 Hz, 5JH,H ¼ 0.9 Hz), 7.34 (dd,
3
3
3
3
d
¼ 12.47 (s, 1H), 10.74 (s, 2H), 8.58 (dd, 1H, JH,H ¼ 4.7 Hz,
1H, JH,H ¼ 7.8 Hz, JH,H ¼ 4.7 Hz), 6.92 (dd, 1H, JH,H ¼ 7.9 Hz,
4JH,H ¼ 1.9 Hz), 8.23 (dd, 1H, 3JH,H ¼ 7.9 Hz, 4JH,H ¼ 1.9 Hz), 7.34 (dd,
1H, 3JH,H ¼ 7.9 Hz, 3JH,H ¼ 4.7 Hz), 4.31 (q, 2H, 3JH,H ¼ 7.1 Hz), 3.03 (s,
3JH,H ¼ 7.5 Hz), 3.59 (s, 2H); 13C NMR (100.6 MHz, DMSO-d6):
d
¼ 171.5, 147.6, 139.8, 131.5, 126.9, 117.3, 109.6, 76.8 (quat C); 147.7,
1H), 1.31 (t, 3H, JH,H ¼ 7.1 Hz); 13C NMR (150.9 MHz, DMSO-d6):
137.2, 131.9, 121.3, 118.9, 118.3 (tert C); 31.9 (sec C); C15H10IN3O
3
ꢄ
d
¼ 171.7, 170.2, 164.3, 149.2, 119.8, 96.1 (quat C); 151.5, 137.7, 119.3
(375.16); MS (EI): m/z ¼ 375 ([M]þ , 100), 346 ([M-CHO]þ, 51), 219
ꢄ
(tert C); 61.5, 30.9 (sec C); 14.0 (prim. C); C12H12N2O4 (248.23);
calcd. C 58.06, H 4.87, N 11.29; found C 57.99, H 4.77, N 11.30.
(25); HRMS (EI): m/z ([M]þ ) calcd. 374.98631, found 374.98555;
HPLC: 98.1% at 254 nm, 97.8% at 280 nm, tN ¼ 6.7 min, tM ¼ 1.09 min
(ACN/H2O; 40:60).
5.1.5. 6,7,8,9-Tetrahydro-5H-pyrido[2,3-b]azepine-5,8-dione (9)
A mixture of 5-hydroxy-8-oxo-8,9-dihydro-7H-pyrido[2,3-b]
azepine-6-carboxylic acid ester (8, 678 mg, 2.70 mmol), DMSO
(48.6 mL) and water (5.4 mL) was heated for 5e7 h to 150 ꢀC under
nitrogen. After cooling to room temperature, water (54 mL) was
added. Upon standing at room temperature a precipitate formed
which was filtrated and crystallized from ethanol to yield a beige
solid (412 mg, 86.6%); mp: 218e221 ꢀC; IR (KBr): 3433 (NH), 2976
(CH aliphat.), 2917 (CH aliphat.), 1674 (C]O), 1592 (CONH),
5.1.8. (E)-11-(3-Oxo-3-phenylprop-1-en-1-yl)-7,12-dihydropyrido
[20,30:2,3]azepino-[4,5-b]indol-6(5H)-on (5a)
11-Iodo-7,12-dihydropyrido[20,30:2,3]azepino[4,5-b]indol-
6(5H)-one (11b, 200 mg, 0.530 mmol), N,N-dimethyl-3-oxo-3-
phenylpropan-1-aminium chloride (12, 126 mg, 0.590 mmol),
triethylamine (1 mL), Pd(OAc)2 (7 mg) and DMF (5 mL) were stirred
for 1 h at 140 ꢀC in a carousel parallel synthesis reactor station. After
filtration of the reaction mixture through a charcoal frit ethyl ace-
tate (220 mL) was added. A precipitate formed, which was suc-
cessively filtered off and washed with n-hexane (320 mL). In the
combined washing solutions a precipitate formed, which was
filtered off and crystallized from ethanol to yield a brown solid
(12 mg, 6.0%) dec. starting at 319 ꢀC; IR (KBr): 3246 (NH), 2909 (CH
767 cmꢃ1; 1H NMR (600 MHz, DMSO-d6):
d
¼ 10.40 (s, 1H), 8.56 (dd,
3
4
3
1H, JH,H ¼ 4.6 Hz, JH,H ¼ 1.9 Hz), 8.26 (dd, 1H, JH,H ¼ 7.8 Hz,
4JH,H ¼ 1.9 Hz), 7.26 (dd, 1H, 3JH,H ¼ 7.8 Hz, 3JH,H ¼ 4.6 Hz), 3.08e2.85
(m, 2H), 2.85e2.63 (m, 2H); 13C NMR (150.9 MHz, DMSO-d6):
d
¼ 197.2, 172.8, 150.78, 120.9 (quat. C); 153.1, 139.8, 119.2 (tert C);
aliphat.), 1672 (C]O), 1645 (C]O), 1326, 1218 cmꢃ1 1H NMR
;
37.1, 29.3 (sec C); C9H8N2O2 (176.17); calcd. C 61.36, H 4.58, N 15.90;
found C 60.70, H 4.43, N 15.87.
(600 MHz, DMSO-d6):
d
¼ 12.09 (s, 1H), 10.41 (s, 1H), 8.58 (d, 1H,
3JH,H ¼ 15.4 Hz), 8.46 (dd, 1H, 3JH,H ¼ 4.7 Hz, 4JH,H ¼ 1.8 Hz), 8.37 (dd,
3
4
1H, JH,H ¼ 7.8 Hz, JH,H ¼ 1.8 Hz), 8.25e8.20 (m, 2H), 8.08 (d, 1H,
3JH,H ¼ 15.3 Hz), 8.04e7.98 (m, 1H), 7.86 (d, 1H, JH,H ¼ 7.8 Hz),
3
3
3
5.1.6. 5-[2-(2-Iodophenyl)hydrazono]-6,7-dihydro-5H-pyrido[2,3-
b]azepine-8(9H)-one (10b)
7.72e7.67 (m, 1H), 7.60 (dd, 2H, JH,H ¼ 8.1 Hz, JH,H ¼ 7.2 Hz), 7.38
(dd, 1H, 3JH,H ¼ 7.8 Hz, 3JH,H ¼ 4.7 Hz), 7.22 (t, 1H, 3JH,H ¼ 7.7 Hz), 3.66
6,7,8,9-Tetrahydro-5H-pyrido[2,3-b]azepine-5,8-dione
(9,
(s, 2H); 13C NMR (100.6 MHz, DMSO-d6):
d
¼ 188.9, 171.3, 147.6,
200 mg, 1.13 mmol), 2-iodophenylhydrazin hydrochloride (460 mg,
1.70 mmol) and sodium acetate (139 mg,1.70 mmol) were stirred in
glacial acetic acid (10 mL) under nitrogen. After stirring for 5 h,
concentrated sulfuric acid (0.4 mL) was added and stirring at room
temperature was continued for 2 h. Subsequently the mixture was
stirred for 2 h at 40 ꢀC. The reaction was terminated by addition of
5% aqueous sodium acetate solution (40 mL). The resulting pre-
cipitate was filtered, washed with water and crystallized from
ethanol to yield a colorless solid (295 mg, 66.6%); mp: 253e254 ꢀC;
137.7, 137.2, 131.3, 127.5, 188.8, 117.4, 108.4 (quat C); 147.7, 139.3,
136.3, 133.0, 128.7 (2C), 128.4 (2C), 121.5, 121.0, 120.9, 119.7, 119.0
(tert C); 31.7 (sec C); C24H17N3O2 (379.41); MS (EI): m/z ¼ 379
ꢄ
([M]þ , 100), 362 (55), 302 ([C18H12N3O2]þ, 39), 274 ([C17H12N3O]þ,
ꢄ
70), 105 (59); HRMS (EI): m/z ([M]þ ) calcd. 379.13153, found
379.13143; HPLC: 91.1% at 254 nm, 95.6% at 280 nm, tN ¼ 5.6 min,
tM ¼ 1.09 min (ACN/H2O; 50:50).
5.1.9. (E)-3-(6-oxo-5,6,7,12-tetrahydropyrido[20,30:2,3]azepino[4,5-
b]indol-11-yl)-N-phenylacrylamide (5e)
IR (KBr): 3433, 3048 (NH), 2893, 1688 (C]O), 1589, 1452, 741 cmꢃ1
;
1H NMR (600 MHz, DMSO-d6):
d
¼ 10.12 (s, 1H), 8.39 (dd, 1H,
A mixture of 11-iodo-7,12-dihydropyrido[20,30:2,3]azepino[4,5-
b]indol-6(5H)-one (11b, 200 mg, 0.530 mmol), N-phenylacrylamide
(13, 87 mg, 0.59 mmol), triethylamine (101 mg, 1.00 mmol,
0.14 mL), Pd(OAc)2 (7 mg, 0.03 mmol), and DMF (3 mL) were heated
for 40 min to 100 ꢀC (power: 100 W; pressure 150 PSI) in a mi-
crowave device in a sealed microwave reaction vessel. After filtra-
tion of the mixture through a charcoal frit, ethyl acetate (220 mL)
was added to the filtrate. A solid precipitated, which was filtered off
and washed with n-hexane (320 mL). Further material precipitated
from the washing liquid, which was collected by filtration and
combined with the material collected previously. Crystallization of
4
3
3JH,H ¼ 4.7 Hz, JH,H ¼ 1.9 Hz), 8.11 (dd, 1H, JH,H ¼ 7.7 Hz,
4JH,H ¼ 1.8 Hz), 7.94 (s, 1H), 7.75 (dd, 1H, JH,H ¼ 7.8 Hz,
3
4JH,H ¼ 1.4 Hz), 7.43 (dd, 1H, 3JH,H ¼ 8.2 Hz, 4JH,H ¼ 1.5 Hz), 7.36e7.28
3
3
(m, 1H), 7.23 (dd, 1H, JH,H ¼ 7.7 Hz, JH,H ¼ 4.7 Hz), 6.70 (ddd, 1H,
3
4
3JH,H ¼ 7.8 Hz, JH,H ¼ 7.2 Hz, JH,H ¼ 1.6 Hz), 3.10 (dd, 2H,
3JH,H ¼ 7.4 Hz, JH,H ¼ 5.6 Hz), 2.68e2.61 (m, 2H); 13C NMR
3
(100.6 MHz, DMSO-d6):
d
¼ 172.4, 148.9, 145.2, 144.7, 125.2, 84.1
(quat C); 148.5, 138.6, 138.4, 129.2, 122.3, 119.9, 114.7 (tert C); 30.9,
28.0 (sec C); C15H13IN4O (392.19); HPLC: 99.3% at 254 nm, 99.5% at
280 nm, tN ¼ 13.1 min, tM ¼ 1.09 min (ACN/H2O; 40:60).

280
F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
the combined materials from ethanol yielded a gray-brown solid
(168 mg, 80.4%); decomposition starting at 289 ꢀC; IR (KBr): 3435
(NH), 2908 (CH aliphat.), 1652 (C]O), 1624 (C]O), 1442,
with reaction buffer. TryS recovered from the PD-10 column was
applied to the MonoQ column pre-equilibrated with reaction
buffer. The column was washed with reaction buffer until absor-
bance 280 nm approaches the baseline and bound proteins eluted
with a linear gradient from 0 to 500 mM NaCl in reaction buffer. The
last two chromatographies were performed at room temperature
1359 cmꢃ1; 1H NMR (400 MHz, DMSO-d6):
d
¼ 12.03 (s, 1H), 10.40
3
4
(s, 1H), 10.25 (s, 1H), 8.46 (dd, 1H, JH,H ¼ 4.7 Hz, JH,H ¼ 1.8 Hz),
3
8.42e8.32 (m, 2H), 7.83e7.72 (m, 3H), 7.58 (d, 1H, JH,H ¼ 7.4 Hz),
7.44e7.29 (m, 3H), 7.21 (t, 1H, 3JH,H ¼ 7.7 Hz), 7.13e7.04 (m, 1H), 6.99
using an Akta-FPLC device (GE Healthcare).
€
(d, 1H, JH,H ¼ 15.5 Hz), 3.65 (s, 2H); 13C NMR (100.6 MHz, DMSO-
Protein purity was assessed after each purification step by SDS-
PAGE 12% gel under reducing conditions. Protein concentration was
determined by the Bicinconinic Acid assay with bovine serum al-
bumin as standard and enzyme activity was determined by the
end-point assay detailed below (see “TryS activity assay”). The
protocol described above yielded ~1e1.5 mg recombinant protein
per liter of culture medium with a purity ꢅ95%. The protein was
stored at e 20 ꢀC in reaction buffer added of 40% (v/v) glycerol
(Carlo Erba Reagents SA) without significant loss of activity for at
least 6 months.
3
d6):
d
¼ 171.4, 163.8,147.7,139.4,136.7,131.3, 127.5, 119.1, 117.5,108.4
(quat C); 147.6, 136.3, 135.6, 128.8 (2C), 123.3, 122.3, 119.9, 119.8,
119.4, 119.2 (2C), 119.1 (tert C); 31.8 (sec C); C24H18N4O2 (394.43);
ꢄ
MS (EI): m/z ¼ 394 ([M]þ , 52), 302 ([C18H12N3O2]þ, 100), 274
ꢄ
([C17H12N3O]þ, 99), 259 (56); HRMS (EI): m/z ([M]þ
) calcd.
394.14243, found 394.14233; HPLC: 98.1% at 254 nm, 98.9% at
280 nm, tN ¼ 8.5 min, tM ¼ 1.09 min (ACN/H2O; 40:60).
5.2. Biological assays
5.2.1. Expression, purification and activity assay of T. b. brucei
trypanothione synthetase (TbTryS)
5.2.1.2. TryS activity assay. The end-point assay is based on the
determination of inorganic phosphate released from ATP during
TryS catalysis, by its complexation (phosphomolybdate) to the
cationic triphenylmethane dye, malachite green (BIOMOL GREEN™
Enzo® Life Sciencies). The green complex presents an absorption
maximum at 650 nm. The reaction buffer consisted of 100 mM
HEPES-K, 0.5 mM EDTA, 10 mM MgSO4 and 5 mM DTT, pH 7.4.
Master Mix (MM) solutions containing substrates at concentrations
adjusted according to their intracellular levels in T. brucei [44,45]
and the kinetic parameters reported in the literature for TbTryS
[30,43] were prepared. Thus, spermidine concentration was fixed to
5.2.1.1. Expression and purification of recombinant TbTryS.
TbTryS was produced in recombinant form with an N-terminal His-
tag using Escherichia coli strain BL21(DE3) transformed with the
construct pET-15b TbTryS [43] (a kind gift of Dr. Alan Fairlamb,
Dundee University, Scotland; Gen Bank™ accession number
CAC87573.1). Starter cultures of freshly transformed cells were
prepared in LB medium containing 100
mg/mL ampicillin and
grown overnight under aerobic conditions at 37 ꢀC and 180 rpm.
Five mL of the overnight culture was inoculated into a 2 L Erlen-
meyer flask containing 500 mL of Terrific Broth medium supple-
2 mM and glutathione concentration was adjusted to 50
interference with the assay, ATP concentration was adjusted to
150 M.
mM. Due to
mented with 10 g/L glucose and 100
mg/mL ampicillin. Culture
growth was initiated by incubation at 37 ꢀC and 180 rpm and
extended until an optical density at 600 nm between 0.8 and 1.0
was achieved. The flask was then chilled at 4 ꢀC for 15 min. After
m
Assays were conducted at room temperature, using 96-well
plates (Corning Incorporated, costar® 3590) and a total reaction
addition of 0.5 mM isopropyl 1-thio-b-D-galactopyranoside bac-
volume of 50
DMSO (J.T. Baker) and 5
~0.070 mol/min mg). A reaction control was prepared by adding
L DMSO. An inhibitory control was performed for the enzyme by
adding 5
L of a N5-substituted paullone at its respective IC50
concentration of 30 M [46]. Blanks lacking enzyme were prepared
for each tested compound and controls. Compounds, controls and
blanks were tested in quadruplicates.
The reaction was initiated by addition of enzyme (or reaction
buffer, in the case of blanks) and stopped after 15 min by adding
m
L: 40
m
L of MM, 5
mL of test compound dissolved in
terial growth was resumed at 25 ꢀC and 180 rpm for further 5 h.
Cells were harvested by centrifugation at 4000ꢂ g for 15 min at 4 ꢀC
(centrifuge Sorvall RC-6, Thermo Fisher Scientific) and resuspended
in 50 mM NaH2PO4, pH 7.2, 300 mM NaCl (buffer A) and 10 mM
imidazol at a ratio of 1 g wet weight pellet per 5 mL buffer. Cell lysis
was achieved by gently shaking with lysozyme (30 mg % w/v) for
1 h at 4 ꢀC followed by sonication on ice (4 pulses during 30 s
interspersed with pauses of 60 s) at an amplitude of 40% using a
Digital Sonifier 450 (Branson). The lysate was centrifuged twice at
20,000ꢂ g for 20 min at 4 ꢀC (centrifuge Sorvall RC-6, Thermo
Fisher Scientific) to remove debris and the supernatant containing
mL enzyme solution (TbTryS: 230 nM,
m
5
m
m
m
200
mL BIOMOL GREEN™ reagent. The colorimetric signal was
allowed to develop for 20 min and then absorbance at 650 nm was
measured with a MultiScan EX plate reader (Thermo SCIENTIFIC).
The results are expressed as residual activity (%) and are calculated
as follow:
soluble protein was cleared by filtration (0.7 mM filter) and then
loaded onto a 1 mL HisTrap Fast Flow column (GE Healthcare) pre-
equilibrated in buffer A. The column was washed with buffer A
added with 25 mM imidazol and the recombinant protein eluted in
an isocratic fashion with 10 mL of buffer B (buffer A with 500 mM
imidazol). This chromatography was performed at 4 ꢀC and at a
flow rate of 1 mL/min using a peristaltic pump (TRIS Teledyne
ISCO). Fractions containing homogenous and active protein (tested
by a kinetic assay in saturating conditions [33]) were pooled and
concentrated with a 30 kDa cut-off Amicon filter (Millipore). Re-
combinant TryS was subjected to a second purification step by size
exclusion chromatography (SEC) in a Superdex™ 200 10/300 GL
(GE healthcare) run on reaction buffer (see below “TryS activity
assay”) added of 150 mM NaCl. Elution fractions displaying ho-
mogenous and active enzyme were pooled (see below). The final
purification step consisted of an anion exchange chromatography
using a MonoQ HR 5/5 column (GE Healthcare). Prior to injection
into the MonoQ column, the protein samples were desalted on a
PD-10 column (Sephadex G25 matrix; GE Healthcare) equilibrated
½ðA650 nmcorrcompound=A650 nmcorrreactioncontrolÞꢂ100ꢆ;
where
A650 nm corr compound ¼ A650 nm compound
ꢃ A650 nm compound blank;
A650 nmcorrreactioncontrol¼A650 nmreactioncontrol
ꢃA650 nmreactioncontrolblank;
A650 nm corr compound is the corrected absorbance measured
for a given compound, and A650 nm corr reaction control is the
corrected absorbance measured for the reaction control. The error
is expressed as 2 S.D., estimated as 2snꢃ1
.

F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
281
5.2.2. Proliferation assay for infective T. b. brucei
After 30 min incubation at 30 ꢀC, the reaction was stopped by
harvesting, using a FilterMate harvester (Packard), onto P81 phos-
phocellulose papers (GE Healthcare) which were washed in 1%
phosphoric acid. Scintillation fluid was added and the radioactivity
measured in a Packard counter. Blank values were subtracted and
activities calculated as pmoles of phosphate incorporated during
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.
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).
These parasites were cultivated aerobically in HMI-9 medium [48]
supplemented with 10% (v/v) fetal bovine serum (FBS) (PAA), 10 U/
mL penicillin (Gibco®), 10
m
m
g/mL streptomycin (Gibco®), 0.2
g/mL hygromicyn (Invitrogen), inside a
mg/mL
phleomycin (Gibco®) and 5
humidified incubator (Thermo SCIENTIFIC) with controlled tem-
CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin E
(human, recombinant, from A. Echalier), CDK5/p25 (human, recom-
binant, expressed in E. coli) were assayed in Buffer A (supplemented
extemporaneously with 0.15 mg BSA/mL, except for CDK2) with
perature (37 ꢀC) and CO2 (5%).
Stock solutions at 3e24 mM in DMSO were prepared for each
compound according to their solubility. All compounds were sub-
jected to a primary screening at 5 and 30
For compounds showing more than 90% parasite growth inhi-
bition at 5 M, EC50 values were determined. To determine EC50
m
M.
25
in insect cells) was assayed as described for CDK1/cyclin B, but using
8.07 g of CDK7/9-Tide (YSPTSPSYSPTSPSYSPTSPSKKKK) [49].
mg of histone H1. CDK9/cyclin T (human, recombinant, expressed
m
,
m
two-fold serial dilutions in DMSO were prepared from the com-
pound's stock solution.
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
Trypanosomes were grown to mid-exponential phase (~2 million
cells/mL), counted with a Neubauer chamber (Precicolor HBG, Ger-
many) and harvested by centrifugation at 2000 g for 10 min at room
temperature. The cell pellet was resuspended at a density of
B with 1
mg of RS peptide (GRSRSRSRSRSR) as a substrate [50].
GSK-3a/b (porcine brain, native, affinity purified on axin 1-
sepharose beads [51]) was assayed as described for CDK1/cyclin
B, but using GSK-3 specific substrate (GS-1:
5 ꢂ105 cells/mL in fresh culture medium. Two
m
L from the compound
a
solutions or DMSO (proliferation control) was added onto each well of
YRRAAVPPSPSLSRHSSPHQpSEDEEE where pS stands for phos-
phorylated serine).
a 96-well culture plate (Corning Incorporated, costar® 3599). Imme-
diately after, 200
and the culture plate was incubated at 37 ꢀC with 5% CO2. After 24 h,
100 L from each well was transferred to individual 1.1 mL tubes
(T100, Biotube™ System), diluted with 200 L sterile phosphate
buffered saline, pH 7.0, with 1% (w/v) glucose and added of 2
m
L of the cell suspension was plated into each well
Casein kinase 1 (CK1
finity chromatography on axin 2-sepharose beads [52]) was
assayed with 0.67 mg of CKS peptide (RRKHAAIGpSAYSITA), a CK1
specific substrate.
d/ε) (porcine brain, native, purified by af-
m
m
mL
propidium iodide (exclusion dye) at 200 mg/mL, homogenized by
quick vortexing and analyzed using a CyAn™ ADP (DakoCytomation)
flow cytometer. Flow cytometry analysis was performed with 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 lem ¼ 613/30 nm to distinguish dead cells. The signals
were detected with logarithmic amplifiers. All measurements were
madeatconstantflowand 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 (Lampit® from Bayer)
5.2.4. Toxicity assay on murine macrophages by xCELLingence
device
Toxicity assays were performed with the xCELLigence System
(Roche Applied Systems) using mouse bone marrow-derived mac-
rophages generated as described previously [53]. After seeding
7.5 ꢂ 104 macrophages in 75
m
L medium (Dulbecco's Modified Eagle
Medium (Gibco), supplemented with 2 mM
L-glutamine, 10% heat-
inactivated fetal calf serum, penicillin and streptomycin) to each
wellofanE-plateVIEW96(RocheApplied Science)for24 h, cells were
treated with 125 mL medium containing the different test compounds
at the highest concentration depending on the respective solubility.
Impedance measurements were performed on the Real-Time Cell
Analyzer SP instrument (Roche Applied Systems) every 30 min. All
experiments were run for 24 h. Cell indices obtained were analyzed
tested at its EC50 (15 mM). All conditions were tested by triplicate.
The relative percentage of viable parasites at 5 or 30 uM is
expressed as follow:
% Viability ¼ ½ðnumber of parasites for compound X at concentration YÞ=ðnumber of parasites in the proliferation controlÞꢆ ꢂ 100:
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 er-
using the Real-Time Cell Analyzer Software 1.2 (Roche Applied Sci-
ence). The percentage of toxicity was calculated as follows:
ror is expressed as 2 S.D., estimated as 2snꢃ1
.
%Toxicity¼100ꢃðcellindextestcompound=cellindexmediumÞ
ꢂ100
5.2.3. Assay for inhibition of mammalian protein kinases
5.2.3.1. Protein kinase assay buffers. Buffer A: 10 mM MgCl2, 1 mM
EGTA, 1 mM DTT, 25 mM TriseHCl pH 7.5, 50 mg heparin/mL.
5.3. In-silico analysis
Buffer B: 50 mM MgCl2, 90 mM NaCl, 30 mM TriseHCl pH 7.4.
5.3.1. Homology modeling
5.2.3.2. Kinase preparations and assays. Kinase activities for each
enzyme were assayed in buffer A or B, with their corresponding
substrates, in the presence of 15 mM ATP in a final volume of 30 mL.
Homology Modeling was performed using MOE [54] and the ho-
mology modeling module. The available X-ray structure for L. major
TryS (pdb 2vpm) was used as template structure and the T. b. brucei

282
F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
[16] V. Delespaux, D. Geysen, P. Van den Bossche, S. Geerts, Molecular tools for the
rapid detection of drug resistance in animal trypanosomes, Trends Parasitol.
24 (2008) 236e242.
[17] Diseases, treatment, therapy, in: H. Mehlhorn (Ed.), Enzyclopedic Reference of
Parasitology, 2 ed., Springer-Verlag, Berlin, 2001, pp. 178e180.
[18] E.O. Mungube, H.S. Vitouley, E. Allegye-Cudjoe, O. Diall, Z. Boucoum, B. Diarra,
Y. Sanogo, T. Randolph, B. Bauer, K.H. Zessin, P.H. Clausen, Detection of mul-
tiple drug-resistant Trypanosoma congolense populations in village cattle of
south-east Mali, Parasit. Vectors 5 (2012) 155.
TryS sequence from UniProt data base with the reference number:
Q82MC2 was used as modeling sequence. For model creation stan-
dard settings together with the Amber99 force field were used.
5.3.2. Molecular docking
For docking GOLD 5.2 was used with standard settings and
ChemPLP for pose creation and scoring [55,56]. For each molecule,
50 poses were created.
[19] C. Reichwald, O. Shimony, U. Dunkel, N. Sacerdoti-Sierra, C.L. Jaffe, C. Kunick,
2-(3-Aryl-3-oxopropen-1-yl)-9-tert-butyl-paullones:
chemotype, J. Med. Chem. 51 (2008) 659e665.
a new antileishmanial
[20] J. Ryczak, M. Papini, A. Lader, A. Nasereddin, D. Kopelyanskiy, L. Preu, C.L. Jaffe,
C. Kunick, 2-Arylpaullones are selective antitrypanosomal agents, Eur. J. Med.
Chem. 64 (2013) 396e400.
[21] C.F. McGrath, N. Pattabiraman, G.E. Kellogg, T. Lemcke, C. Kunick,
E.A. Sausville, D.W. Zaharevitz, R. Gussio, Homology model of the CDK1/cyclin
B complex, J. Biomol. Struct. Dyn. 22 (2005) 493e502.
[22] T. Pies, K.-J. Schaper, M. Leost, D.W. Zaharevitz, R. Gussio, L. Meijer, C. Kunick,
CDK1-inhibitory activity of paullones depends on electronic properties of 9-
substituents, Arch. Pharm. Pharm. Med. Chem. 337 (2004) 486e492.
[23] N. Tolle, C. Kunick, Paullones as inhibitors of protein kinases, Curr. Top. Med.
Chem. 11 (2011) 1320e1332.
[24] K. Wieking, M. Knockaert, M. Leost, D.W. Zaharevitz, L. Meijer, C. Kunick,
Synthesis of paullones with aminoalkyl side chains, Arch. Pharm. Pharm. Med.
Chem. 335 (2002) 311e317.
[25] X. Xie, T. Lemcke, R. Gussio, D.W. Zaharevitz, M. Leost, L. Meijer, C. Kunick,
Epoxide-containing side chains enhance antiproliferative activity of paul-
lones, Eur. J. Med. Chem. 40 (2005) 655e661.
[26] C. Kunick, K. Lauenroth, K. Wieking, X. Xie, C. Schultz, R. Gussio, D. Zaharevitz,
M. Leost, L. Meijer, A. Weber, F.S. Jorgensen, T. Lemcke, Evaluation and
comparison of 3D-QSAR CoMSIA models for CDK1, CDK5, and GSK-3 inhibi-
tion by paullones, J. Med. Chem. 47 (2004) 22e36.
[27] C. Kunick, K. Lauenroth, M. Leost, L. Meijer, T. Lemcke, 1-Azakenpaullone is a
selective inhibitor of glycogen synthase kinase-3, Bioorg. Med. Chem. Lett. 14
(2004) 413e416.
Acknowledgments
This project was funded by the German “Bundesministerium für
Bildung und Forschung” (KMU-innovativ 5, Forderkennzeichen
€
0315814; to F. M., M. A. D., O. K., C. H., and C. K.). The support of
FOCEM (MERCOSUR Structural Convergence Fund, COF 03/11) and
ANII (grant Innova Uruguay, agreement DCI-ALA/2007/19.040 be-
tween Uruguay and the European Commission; INI_X_2011_1_4077
and POS_NAC_2013_1_114477) is acknowledged by M. A. C., D. B. and
D. C.. L. M. was supported by a grant from the ‘Agence Nationale de la
Recherche’ (ANR- 11-RPIB-0016 grant, Transleish).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.06.020.
References
[28] C. Reichwald, O. Shimony, N. Sacerdoti-Sierra, C.L. Jaffe, C. Kunick, A new Heck
reaction modification using ketone Mannich bases as enone precursors:
parallel synthesis of anti-leishmanial chalcones, Bioorg. Med. Chem. Lett. 18
(2008) 1985e1989.
[1] W.H.O, Trypanosomiasis, Human African (Sleeping Sickness), Factsheet 259,
2013. http://www.who.int/mediacentre/factsheets/fs259/en/.
[2] A. MacLeod, A. Tait, C.M.R. Turner, The population genetics of Trypanosoma
brucei and the origin of human infectivity, Phil. Trans. R. Soc. Lond. B B 356
(2001) 1035e1044.
[3] O. Balmer, J.S. Beadell, W. Gibson, A. Caccone, Phylogeography and taxonomy
of Trypanosoma brucei, PLoS Negl. Trop. Dis. 5 (2011) e961.
[4] I. Goodhead, P. Capewell, J.W. Bailey, T. Beament, M. Chance, S. Kay,
S. Forrester, A. MacLeod, M. Taylor, H. Noyes, N. Hall, Whole-genome
sequencing of Trypanosoma brucei reveals introgression between subspecies
that is associated with virulence, mBio 4 (2013) e00197-13.
[5] R.E. Symula, J.S. Beadell, M. Sistrom, K. Agbebakun, O. Balmer, W. Gibson,
S. Aksoy, A. Caccone, Trypanosoma brucei gambiense group 1 is distinguished
by a unique amino acid substitution in the HpHb receptor implicated in hu-
man serum resistance, PLoS Negl. Trop. Dis. 6 (2012) e1728.
[6] H.V. Xong, L. Vanhamme, M. Chamekh, C.E. Chimfwembe, J. Van Den Abbeele,
A. Pays, N. Van Meirvenne, R. Hamers, P. De Baetselier, E. Pays, A VSG
expression site-associated gene confers resistance to human serum in Try-
panosoma rhodesiense, Cell 95 (1998) 839e846.
[7] P.H. Holmes, E. Katunguka-Rwakishaya, J.J. Bennison, G.J. Wassink, J.J. Parkins,
Impact of nutrition on the pathophysiology of bovine trypanosomiasis, Para-
sitology 120 (Suppl) (2000) S73eS85.
[8] R.C. Ezeokonkwo, I.O. Ezeh, C.N. Iheagwam, W.E. Agu, R.I.S. Agbede, Efficacy of
Kelamidium in the prevention and treatment of Trypanosoma brucei brucei
infection in albino rats, Comp. Clin. Pathol. 22 (2013) 219e226.
ꢁ
[29] L. Flohe, The trypanothione system and the opportunities it offers to create
drugs for the neglected kinetoplast diseases, Biotechnol. Adv. 30 (2012)
294e301.
[30] L.S. Torrie, S. Wyllie, D. Spinks, S.L. Oza, S. Thompson, J.R. Harrison, I.H. Gilbert,
P.G. Wyatt, A.H. Fairlamb, J.A. Frearson, Chemical validation of trypanothione
synthetase: a potential drug target for human trypanosomiasis, J. Biol. Chem.
284 (2009) 36137e36145.
ꢁ
[31] M.A. Comini, S.A. Guerrero, S. Haile, U. Menge, H. Lünsdorf, L. Flohe, Validation
of Trypanosoma brucei trypanothione synthetase as drug target, Free Radic.
Biol. Med. 36 (2004) 1289e1302.
ꢁ
[32] L. Flohe, The trypanothione system and its implications in the therapy of
trypanosomatid diseases, Int. J. Med. Microbiol. 302 (2012) 216e220.
ꢁ
[33] M.A. Comini, U. Menge, J. Wissing, L. Flohe, Trypanothione synthesis in cri-
thidia revisited, J. Biol. Chem. 280 (2005) 6850e6860.
[34] P.K. Fyfe, S.L. Oza, A.H. Fairlamb, W.N. Hunter, Leishmania trypanothione
synthetase-amidase structure reveals a basis for regulation of conflicting
synthetic and hydrolytic activities, J. Biol. Chem. 283 (2008)
17672e17680.
€
ꢁ
[35] O. Koch, D. Cappel, M. Nocker, T. Jager, L. Flohe, C. Sotriffer, P.M. Selzer, Mo-
lecular dynamics reveal binding mode of glutathionylspermidine by trypa-
nothione synthetase, PLoS One 8 (2013) e56788.
[36] S. Zimmermann, M. Oufir, A. Leroux, R. Krauth-Siegel, K. Becker, M. Kaiser,
R. Brun, M. Hamburger, M. Adams, Cynaropicrin targets the trypanothione
redox system in Trypanosoma brucei, Bioorg. Med. Chem. 21 (2013)
7202e7209.
[9] C. Gadelha, J.M. Holden, H.C. Allison, M.C. Field, Specializations in a successful
parasite: what makes the bloodstream-form African trypanosome so deadly?
Mol. Biochem. Parasitol. 179 (2011) 51e58.
[37] M.D. Urbaniak, Casein kinase 1 isoform 2 is essential for bloodstream form
Trypanosoma brucei, Mol. Biochem. Parasitol. 166 (2009) 183e185.
[38] A.-M. Egert-Schmidt, J. Dreher, U. Dunkel, S. Kohfeld, L. Preu, H. Weber,
[10] A.H. Fairlamb, Chemotherapy of human African trypanosomiasis: current and
future prospects, Trends Parasitol. 19 (2003) 488e494.
[11] F.E. Graf, P. Ludin, T. Wenzler, M. Kaiser, R. Brun, P.P. Pyana, P. Büscher, H.P. de
€
J.E. Ehlert, B. Mutschler, F. Totzke, C. Schachtele, M.H.G. Kubbutat, K. Baumann,
€
Koning, D. Horn, P. Maser, Aquaporin 2 mutations in Trypanosoma brucei
C. Kunick, Identification of 2-anilino-9-methoxy-5,7-dihydro-6H-pyrimido
[5,4-d][1]benz:azepin-6-ones as dual PLK1/VEGF-R2 kinase inhibitor che-
motypes by structure-based lead generation, J. Med. Chem. 53 (2010)
2433e2442.
gambiense field isolates correlate with decreased susceptibility to pentami-
dine and melarsoprol, PLoS Negl. Trop. Dis. 7 (2013) e2475.
[12] M.L. Stewart, R.J. Burchmore, C. Clucas, C. Hertz-Fowler, K. Brooks, A. Tait,
A. Macleod, C.M. Turner, H.P. De Koning, P.E. Wong, M.P. Barrett, Multiple
genetic mechanisms lead to loss of functional TbAT1 expression in drug-
resistant trypanosomes, Eukaryot. Cell. 9 (2010) 336e343.
[39] F.F. Blicke, The Mannich reaction, Org. React. 1 (1942) 303e341.
€
[40] J. Eriksson, O. Åberg, B. Långstrom, Synthesis of [11C]/[13C]acrylamides by
palladium-mediated carbonylation, Eur. J. Org. Chem. 2007 (2007)
455e461.
[41] N. Kuhnert, A. Le-Gresley, Synthesis and capsule formation of upper rim
[13] A.C. Likeufack, R. Brun, A. Fomena, P. Truc, Comparison of the in vitro drug
sensitivity of Trypanosoma brucei gambiense strains from West and Central
Africa isolated in the periods 1960e1995 and 1999e2004, Acta Trop. 100
(2006) 11e16.
substituted tetra-acrylamido calix[4]arenes, Org. Biomol. Chem.
3 (2005)
2175e2182.
€
[14] N. Maina, K.J. Maina, P. Maser, R. Brun, Genotypic and phenotypic charac-
[42] H.H. Fox, Synthetic tuberculostats. II. Amino- and hydroxypyridine carboxylic
acid derivatives, J. Org. Chem. 17 (1952) 547e554.
[43] S.L. Oza, M.R. Ariyanayagam, N. Aitcheson, A.H. Fairlamb, Properties of try-
panothione synthetase from Trypanosoma brucei, Mol. Biochem. Parasitol. 131
(2003) 25e33.
terization of Trypanosoma brucei gambiense isolates from Ibba, South Sudan,
an area of high melarsoprol treatment failure rate, Acta Trop. 104 (2007)
84e90.
[15] L.D.B. Kinabo, Pharmacology of existing drugs for animal trypanosomiasis,
Acta Trop. 54 (1993) 169e183.

F. Maiwald et al. / European Journal of Medicinal Chemistry 83 (2014) 274e283
283
[44] R.L. Krauth-Siegel, M. Comini, Redox control in trypanosomatids, parasitic
protozoa with trypanothione-based thiol metabolism, Biochim. Biophys. Acta
1780 (2008) 1236e1248.
structures and neuroprotective properties of leucettines, a family of protein
kinase inhibitors derived from the marine sponge alkaloid leucettamine B,
J. Med. Chem. 55 (2012) 9312e9330.
[45] M.C. Taylor, H. Kaur, B. Blessington, J.M. Kelly, S.R. Wilkinson, Validation of
spermidine synthase as a drug target in African Trypanosomes, Biochem. J.
409 (2008) 563e569.
[51] A. Primot, B. Baratte, M. Gompel, A. Borgne, S. Liabeuf, J.L. Romette,
F. Costantini, L. Meijer, Purification of GSK-3 by affinity chromatography on
immobilised axin, Protein Expr. Purif. 20 (2000) 394e404.
ꢁ
[46] L. Flohe, In search of trypanocidal drugs, in: P.M. Selzer (Ed.), Drug Discovery
[52] J. Reinhardt, Y. Ferandin, L. Meijer, Purification of CK1 by affinity chroma-
tography on immobilised axin, Protein Expr. Purif. 54 (2007) 101e109.
in Infectious Diseases: From Molecular Targets to Drug Candidates, Wiley-
VCH, Weinheim, 2009, pp. 211e226.
[47] S. Biebinger, L.E. Wirtz, P. Lorenz, C. Clayton, Vectors for inducible expression
of toxic gene products in bloodstream and procyclic Trypanosoma brucei, Mol.
Biochem. Parasitol. 85 (1997) 99e112.
[48] H. Hirumi, K. Hirumi, Continuous cultivation of Trypanosoma brucei blood
stream forms in a medium containing a low concentration of serum protein
without feeder cell layers, J. Parasitol. 75 (1989) 985e989.
€
€
[53] C. Holscher, G. Kohler, U. Müller, H. Mossmann, G.A. Schaub, F. Brombacher,
Defective nitric oxide effector functions lead to extreme susceptibility of
Trypanosoma cruzi-infected mice deficient in gamma interferon receptor or
inducible nitric oxide synthase, Infect. Immun. 66 (1998) 1208e1215.
[54] Molecular Operating Environment (MOE), 2013.08; Chemical Computing
Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC, Canada, H3A
2R7, 2013.
[49] K. Bettayeb, N. Oumata, A. Echalier, Y. Ferandin, J.A. Endicott, H. Galons,
L. Meijer, CR8, a potent and selective, roscovitine-derived inhibitor of cyclin-
dependent kinases, Oncogene 27 (2008) 5797e5807.
[55] G. Jones, P. Willett, R.C. Glen, A.R. Leach, R. Taylor, Development and valida-
tion of a genetic algorithm for flexible docking, J. Mol. Biol. 267 (1997)
727e748.
[50] T. Tahtouh, J.M. Elkins, P. Filippakopoulos, M. Soundararajan, G. Burgy,
E. Durieu, C. Cochet, R.S. Schmid, D.C. Lo, F. Delhommel, A.E. Oberholzer,
L.H. Pearl, F. Carreaux, J.P. Bazureau, S. Knapp, L. Meijer, Selectivity, cocrystal
[56] J.W. Liebeschuetz, J.C. Cole, O. Korb, Pose prediction and virtual screening
performance of GOLD scoring functions in a standardized test, J. Comput.
Aided Mol. Des. 26 (2012) 737e748.