Bistacrines as potential antitrypanosomal agents

Article abstract of DOI:10.1016/j.bmc.2017.06.051

Human African Trypanosomiasis (HAT) is caused by two subspecies of the genus Trypanosoma, namely Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. The disease is fatal if left untreated and therapy is limited due to only five non-adequate drugs currently available. In preliminary studies, dimeric tacrine derivatives were found to inhibit parasite growth with IC₅₀-values in the nanomolar concentration range. This prompted the synthesis of a small, but smart library of monomeric and dimeric tacrine-type compounds and their evaluation of antiprotozoal activity. Rhodesain, a lysosomal cathepsin-L like cysteine protease of T. brucei rhodesiense is essential for parasite survival and likely target of the tacrine derivatives. In addition, the inhibition of trypanothione reductase by bistacrines was found. This flavoprotein oxidoreductase is the main defense against oxidative stress in the thiol redox system unique for protozoa.

Full text of DOI:10.1016/j.bmc.2017.06.051

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Bistacrines as potential antitrypanosomal agents
Ines Schmidt a, Sarah Göllner e, Antje Fuß b, August Stich b, Anna Kucharski a, Tanja Schirmeister c,
Elena Katzowitsch d, Heike Bruhn d, Alexandra Miliu e, R. Luise Krauth-Siegel e, Ulrike Holzgrabe a,
⇑
a Institute for Pharmacy and Food Chemistry, Julius-Maximilians-University of Würzburg, Am Hubland, 97074 Würzburg, Germany
b Medical Mission Institute, Hermann-Schell-Strasse 7, 97074 Würzburg, Germany
c Institute for Pharmacy and Biochemistry, Johannes-Gutenberg-University of Mainz, Staudinger Weg 5, 55128 Mainz, Germany
d Institute for Molecular Infection Biology, Julius-Maximilians-University of Würzburg, Josef-Schneider-Strasse 2, 97080 Würzburg, Germany
e Biochemistry Center (BZH), Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Human African Trypanosomiasis (HAT) is caused by two subspecies of the genus Trypanosoma, namely
Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. The disease is fatal if left untreated
and therapy is limited due to only five non-adequate drugs currently available. In preliminary studies,
dimeric tacrine derivatives were found to inhibit parasite growth with IC50-values in the nanomolar con-
centration range. This prompted the synthesis of a small, but smart library of monomeric and dimeric
tacrine-type compounds and their evaluation of antiprotozoal activity. Rhodesain, a lysosomal cathep-
sin-L like cysteine protease of T. brucei rhodesiense is essential for parasite survival and likely target of
the tacrine derivatives. In addition, the inhibition of trypanothione reductase by bistacrines was found.
This flavoprotein oxidoreductase is the main defense against oxidative stress in the thiol redox system
unique for protozoa.
Received 15 February 2017
Revised 28 June 2017
Accepted 30 June 2017
Available online xxxx
Keywords:
Bistacrine
Antitrypanosomal drugs
Rhodesain
Trypanothione reductase
Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction
Preliminary studies showed the antitrypanosomal activity of
tacrine (1,2,3,4-tetrahydroacridin-9-amine, THA, see Fig. 1) in the
Human African Trypanosomiasis (HAT), a parasitic disease
caused by protozoa of the genus Trypanosoma, imperils more than
60 million people living in 36 sub-Saharan countries. The patho-
gens are transmitted by the bite of the tsetse fly.1 The disease
occurs in two manifestations depending on the species of the
pathogen. Trypanosoma brucei rhodesiense initiates an acute infec-
tion and is primarily found in eastern and southern Africa, whereas
Trypanosoma brucei gambiense causes the protracted form mainly
in central and western Africa. HAT, also called sleeping sickness,
is fatal if left untreated. Treatment options are very limited to only
five drugs currently available. However, increasing resistance and
marked host cytotoxicity are related to these drugs. Pentamidine
and suramine are predominantly used for treatment of the
hemolymphatic stage of the disease.2 During the second stage of
the disease, after the parasites having crossed the blood brain bar-
rier, only melarsoprol, eflornithine, and the recently introduced
combination therapy of nifurtimox and eflornithine (NECT) proved
impact. This medication is expensive, painful, and associated with
severe side effects. Therefore, the development of new and afford-
able drugs is urgently needed.3
micromolar concentration range (IC50 (cell line TC221)
= 14.6 mM). Linking of two tacrine moieties with a hexamethylene
chain led to a significant increase of the activity being in the sub-
micromolar concentration range. This bistacrine was originally
synthesized because of its activity against Plasmodium falciparum.4
These results prompted us to synthesize monomeric and
dimeric tacrine congeners and to evaluate their antitrypanosomal
activity and putative mode of action. Since a couple of tacrine-type
compounds has already shown inhibitory activity towards falci-
pain,4 which is closely related to rhodesain,5 a cysteine protease
being a cathepsin-L like enzyme of the papain family expressed
by bloodstream form of Trypanosoma brucei brucei (Tbb), the new
library of compounds was tested for the inhibitory activity towards
rhodesain. Of note, rhodesain is essential for the development and
growth of parasites and facilitates the crossing of the blood brain
barrier to enter the central nervous system.6
Additionally, for representative compounds the inhibition of try-
panothione reductase (TryR) was tested, because a couple of similar
tricyclic systems, such as phenothiazines, xanthenes, bis-acridines,
and 4-aminoquinoline-type compounds7 showed inhibition of the
NADPH-dependent flavoenzyme.8–10 The TryR is responsible for
keeping the intracellular reducing environment for defence against
oxidative stress and thus constitutes a promising target.11
⇑
Corresponding author.
E-mail address: ulrike.holzgrabe@uni-wuerzburg.de (U. Holzgrabe).
http://dx.doi.org/10.1016/j.bmc.2017.06.051
0968-0896/Ó 2017 Elsevier Ltd. All rights reserved.
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I. Schmidt et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
using the alamarBlueÒ-based assay.13 The data are summarized
in Tables 1 and 2. Of note, the oxotacrine dimers 5 were not
screened because of their very low aqueous solubility, and the
hydroxytacrine dimers 6 were not tested because they are prone
to dehydratisation which results in compounds 4.
The comparison of the series 1a–1d, 1f–1i, and 1j–1m with
either hydrogen, methyl, ethyl or propyl substituents in position
2 indicates a stepwise increase of activity with enlargement of this
residue. Though cytotoxicity increases with an additional chlorine
substituent in the aromatic moiety, this does not affect activity.
Thus, 1d shows a good activity with acceptable toxicity resulting
in a selectivity index of 16.5. An additional nitro group in position
7 (1n-p) is also advantageous in leading to a slight improvement in
both antitrypanosomal activity and cytotoxicity.
Next, the influence of the linker length between two tacrine-
type molecules was studied for the compounds 2a-2i. The dimeri-
sation shifts both antitrypanosomal activity and cytotoxicity to
lower IC50 values resulting in almost constant selectivity indices.
The compounds 2h and 2i with nine- and ten-membered linkers
exhibit the best antitrypanosomal activities (IC50 = 130 nM and
120 nM) and selectivity index (SI) values (i.e., 13.8 and 15.0,
respectively).
Fig. 1. Monomeric tacrine derivatives, R1 and R2 are outlined in Table 1.
2. Results and discussion
2.1. Synthesis pathways
Monomeric tacrine derivatives 1 are easily available by conden-
sation and cyclization of correspondingly substituted 2-aminoben-
zonitriles and cyclohexanones with catalytic amounts of p-toluene
sulfonic acid in toluene as solvent. Some members of the library
(1a, 1d, 1e, 1f, 1j, 1m, and 1n) have already been reported in a pre-
vious paper.3
For the synthesis of the dimeric tacrine-type compounds 2 and
3, 9-chloro-1,2,3,4-tetrahydroacridine derivatives were produced
by condensation of correspondingly substituted 2-aminobenzoni-
triles and cyclohexanones using an excess of POCl3. Two of the
Interestingly, the different substitution pattern of compound 3
has as a minor influence on the inhibition of parasite growth which
holds also true for the cytotoxicity, with 3d and 3g being the most
active antitrypanosomal derivatives (Fig. 2).
intermediates were linked by conversion with 1,x-dibromoalkanes
to give the compounds 2 and 3. 2a–2i, and 3a, 3b, 3c, 3g, and 3f
were already reported.4
The synthesis of dimeric N1,N9-bis(3,4-dihydroacridin-9-yl)
alkanes 4 started off with the condensation of 2-aminobenzonitrile
and 1,3-cyclohexanedione to give the 1-oxotacrine 1q, which was
dimerized by means of the corresponding dibromoalkane in pres-
ence of KOH and tetrabutylammonium iodide. The oxotacrine
dimers 5a-e were reduced with sodium borohydride to give the
corresponding alcohols 6a-e whose hydroxyl groups were elimi-
nated by usage of HCl in isopropanol to achieve the compounds
4a-e (see Scheme 1).
Of note, the compounds 4a–4e having an additional double
bond in the saturated region of the tacrine moiety (Fig. 3) showed
a very good activity against Trypanosoma. As can be seen in Table 3,
especially compounds 4b, 4c, and 4d with a linker length of seven,
eight, and nine methylene units, respectively, are active in the two-
digit nanomolar range of concentration. Although being toxic to
macrophages in low micromolar concentrations, these derivatives
displayed the best SIs in the entire series of bistacrines. The most
active compound 4b shows a SI of about 150, which makes it an
interesting lead compound. All compounds 4 are stable throughout
the assays.
2.2. Biology
2.2.1. In vitro activity against Trypanosoma brucei brucei
Monomeric and dimeric tacrine derivatives were tested against
Trypanosoma brucei brucei, cell line TC221, using the cell prolifera-
tion alamarBlueÒ assay. Living cells are able to reduce the redox
indicator resazurine to resorufine, resulting in a color change,
whereas dead cell reduce this reaction. IC50 values were deter-
mined according to the method published by Räz et al.12 Cytotox-
icity was measured against murine macrophages, cell line J774.1
2.2.2. Inhibition of rhodesain
The cysteine protease rhodesain is essential as a digestive
enzyme14 and enables African trypanosomes to cross the blood
brain barrier.15 Dimeric tacrine derivatives 2a–2i and 3a–3g were
evaluated in vitro for their ability to inhibit rhodesain using a fixed
concentration of 20 mM (see Table 4). In this assay, an equivalent
volume of DMSO was used as negative control. Since fluorogenic
Scheme 1.
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I. Schmidt et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
3
Table 1
Antitrypanosomal activities, cytotoxicities, and selectivity indices of monomeric tacrine derivatives; all compounds were tested in duplicate; values represent the mean of the
two measurements. Selectivity indices were calculated by IC50 (J774.1)/IC50 (Tbb).
Compound
T. brucei brucei IC50 [mM]
Cytotoxicity J774.1
SI
R1
R2
48 h
72 h
IC50 [mM]
1a
1b
1c
1d
1e
1f
1 g
1 h
1i
1j
1k
1l
1m
1n
1o
H
H
H
H
H
14.6 7.8
15.8 1.1
12.3 0.88
2.6 0.74
14.5 1.6
12.6 0.59
6.0 1.2
2.8 0.02
2.6 0.13
12.0 1.0
3.9 0.71
2.7 0.15
2.7 0.53
8.6 0.23
3.2 0.14
6.1 3.5
18.4 1.2
18.1 0.14
17.2 0.85
3.5 0.46
17.6 0.42
17.1 0.48
13.4 0.39
3.5 0.07
3.4 0.12
16.5 0.50
10.3 4.8
3.7 0.42
3.4 0.11
15.1 0.06
4.8 0.48
3.7 0.58
18.0 0.89
>100
44.9
43.6
43.0
45.0
41.0
41.2
24.4
8.4
>6.8
2.8
3.5
16.5
3.1
3.3
6.9
8.7
3.2
3.6
11.2
3.1
3.1
5.2
14.5
7.1
2-Me
2-Et
2-Pr
3-Me
H
2-Me
2-Et
2-Pr
H
2-Me
2-Et
2-Pr
H
2-Me
3-Me
H
H
6-Cl
6-Cl
6-Cl
6-Cl
7-Cl
7-Cl
7-Cl
7-Cl
7-NO2
7-NO2
7-NO2
H
43.2
43.8
8.3
8.4
44.6
46.4
43.3
>100
38.6
1p
1q*
14.6 1.38
5.3 nM
>6.8
7283
Pentamidine
*
Oxotacrine.
Table 2
Antitrypanosomal activities, cytotoxicities and selectivity indices of dimeric tacrine derivatives; all compounds were tested in duplicate; values represent the mean of the two
measurements. SIs were calculated by IC50 (J774.1)/IC50 (Tbb).
Compound
Linker (methylene units)
T. brucei brucei IC50 [mM]
Cytotoxicity J774.1 IC50 [mM]
Selectivity index
48 h
72 h
2a
2b
2c
2d
2e
2f
2g
2h
2i
3a
3b
3c
3d
3e
3f
2
3
4
5
6
7
8
9
10
6
6
6
9
9
9
9
3.6 0.53
3.3 0.15
0.75 0.01
5.2 0.42
5.1 1.1
45.2
42.6
8.3
1.8
3.0
1.9
1.9
1.8
1.8
2.8
1.7
25.0
1.8
8.6
1.7
1.7
12.6
12.9
11.1
0.35
11.5
11.9
4.8
13.8
15.0
4.4
5.6 0.30
2.1 0.36
6.4 2.5
0.26 0.09
0.16 0.05
0.40 0.02
0.13 0.02
0.12 0.01
0.64 0.04
0.64 0.01
0.62 0.05
0.16 0.01
0.43 0.38
0.63 0.08
0.12 0.01
0.33 0.13
0.59 0.01
0.66 0.05
0.16 0.01
0.15 0.01
0.71 0.04
0.72 0.03
0.68 0.03
0.21 0.07
0.42 0.21
0.71 0.01
0.14 0.01
2.7
38.5
11.3
20.0
2.8
3g
10.6
inhibition, respectively. Compounds which showed an inhibition
superior than >50% at 20 mM concentration were selected for deter-
mination of their IC50-values by varying the inhibitor concentra-
tion between
0 and 100 mM. Progress curves which were
recorded over a period of 10 min confirmed a time-independent
inhibition for all tested compounds.
Compounds 2g, 2h, 3c, 3f, and 3g seem to be reasonable inhibi-
tors, all of them showing IC50-values below 30 mM whereby 3a and
3c have values being in the one-digit micromolar range. This sup-
ports rhodesain as a possible target of dimeric bistacrines. Of note,
these compounds show similar activities against falcipain-2,4 an
enzyme related to rhodesain expressed by the malarial parasite
Plasmodium falciparum. However, the IC50-values (rhodesain) are
some 10-times higher than the antitrypanosomal activity towards
Tbb, indicating that the compounds may be enriched in the parasite
or hit an additional target. Thus, no other compounds of the library
were tested.
Fig. 2. Chemical structures of compounds 2a–2i and 3a–3g.
Fig. 3. Chemical structures of compounds 4a–4e.
2.2.3. Inhibition of trypanothione reductase
7-amino-4-methylcoumarin (AMC) is released from the substrate
Cbz-Phe-Arg-AMC by the enzyme, fluorescence of the reaction
can be monitored and allows conclusions of enzyme activity and
The antitrypanosomal activity of tricyclic, hydrophobic com-
pounds such as phenothiazines, xanthenes,8 acridines,9,7 and het-
erodimeric 4-aminoquinoline-type compounds7 is well known
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I. Schmidt et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 3
Antitrypanosomal activities, cytotoxicities, and selectivity indices of compounds 4a–4e; all compounds were tested in duplicates. Values represent the mean of the two
measurements. SIs were calculated by IC50 (J774.1)/IC50 (Tbb).
Compound
Linker (methylene units)
T. brucei brucei IC50 [mM]
Cytotoxicity J774.1 IC50 [mM]
SI
48 h
72 h
4a
4b
4c
4d
4e
6
7
8
9
0.51 0.09
0.01 0.01
0.08 0.01
0.09 0.03
0.29 0.37
0.70 0.01
0.01 0.01
0.25 0.18
0.25 0.18
0.88 0.23
1.8
1.5
1.7
1.6
9.7
3.5
152
21.3
16.7
33.4
10
Table 4
Inhibition of rhodesain by compounds 2d–2h, 3a–3g; all compounds were tested in duplicate. Values represent the mean of the two measurements with a deviation less than
10%; nd = not determined.*
Compound
R1
R2
Linker (methylene units)
% Inhibition (at 20 mM)
IC50 [mM]
2d
2e
2f
H
H
H
H
H
H
H
H
H
H
5
6
7
8
9
6
6
6
9
9
9
9
9
nd
nd
nd
25
44
87
82
61
64
82
68
26
89
82
2g
2h
3a
3b
3c
3d
3e
3f
16.9
19.4
7.4
26.6
5.8
26.6
nd
13.3
18.1
H
6-Cl
6-Cl
7-NO2
H
6-Cl
6-Cl
7-OMe
3-(CH3)2
3-Me
2-Me
2-OBu
2-OEt
H
3g
*
Reference inhibitor K11777: k2nd = 555,000 [1/(MÁs)]30; k2nd (measured here) = 500,000 [1/(MÁs)].
Fig. 4. Lineweaver-Burk-Plots for compound 2h inhibition ofTcTryR (A) and TbTryR (B); 0 mM = control DMSO.
and attributed to the large, hydrophobic active site of TryR. This
essential enzyme is unique to trypanosomatid parasites and repre-
sents the main weapon against oxidative stress, rendering it an
attractive drug target. Hence, a representative selection of com-
pounds was tested as putative inhibitors of the reductase.
First, compounds 2e–2h showing the lowest IC50 values in vitro,
were studied at two fixed concentrations each (2e–2g: 2 mM and
5 mM, 2h: 1 mM and 3 mM, respectively) and two concentrations
of trypanothione disulfide (TS2). 40 and 100 mM TS2 correspond
to about twice and five-times the Km-value for this substrate
(18 mM in the case of Trypanosoma cruzi (Tc) TryR.16 A decrease of
the substrate concentration resulted in an increase of inhibition
suggesting a competitive type of inhibition. In the presence of
40 mM substrate, 2 mM 2e, 2f, and 2g resulted in 28, 36, and 46%
inhibition of TcTryR, respectively. In comparison, 1 mM 2h yielded
51% inhibition (for details, see Table S1).
Third, since compound 4b had the highest antitrypanosomal
activity and the highest selectivity index, the inhibitory activity
of this compound was tested towards TbTR. At a concentration of
5 mM and a substrate concentration of 100 and 40 mM TS2, 4b
resulted in 53 and 47% inhibition (see Fig. 5). 4b is a mixed-type
Thus, compound 2h was subjected to a detailed kinetic analysis
using TryR species from both Tb and Tc. Lineweaver-Burk-Plots
confirmed the competitive type of inhibition (Fig. 4). The Ki-values
were calculated from the direct plots giving a value of 0.4 mM for
both TcTryR and TbTryR.
Fig. 5. Lineweaver-Burk-Plot for compound 4b and TbTryR.
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I. Schmidt et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
inhibitor with Ki = 1.2 mM and Ki’ = 12 mM, indicating a higher affin-
ity for the free enzyme than for the enzyme-substrate complex.
Comparing the inhibitory potency of 5 mM 2f and 4b, both hav-
ing a heptane linker, in the presence of 100 and 40 mM of TS2
revealed a stronger inhibition for 2f (53 and 66%) and a slightly
greater difference. This might be due to the different mode of inhi-
bition, i.e. competitive versus mixed type.
Since TryR and human glutathione reductase (hGR) share almost
40% of the residues and thus have a similar structure and mecha-
nism of action,17 compound 2h was tested for its inhibitory activity
towards hGR as well. At 100 mM GSSG, 2 and 50 mM 2h only weakly
inhibited the host enzyme by 2% and 19%, respectively. Thus, the
compounds are likely to be selective for the protozoal TyrR.
substrate stock solution (400 mM in DMSO) were added and the
increase of the fluorescence was observed over 5 min. The enzyme
activity was calculated from the slopes of the curves obtained (DF
vs. time). Compounds having an inhibitory effect greater than 50%
at 20 mM were chosen for detailed assays. Therefore, 185 ml buffer,
5 ml enzyme solution, 5 ml inhibitor solution, and 5 ml substrate
solution were mixed and the fluorescence intensity was recorded
over 10 min. Inhibitor concentrations were in the range of 0–
100 mM in case of compounds 3b, 3d, and 3g; 0–50 mM in case of
compounds 2h; 0–40 mM in case of compounds 2g, 3a, and 3f;
and 0–20 mM in case of compounds 3c. IC50-values were obtained
by nonlinear regression analysis using OriginÒ Pro 2015 software.
4.2.2.2. Trypanothione reductase assay. Trypanothione disulfide
(TS2) was synthesized enzymatically.28 TryR activity was measured
in a total volume of 1 ml of 40 mM of HEPES buffer, 1 mM EDTA, pH
7.5, containing 100 mM NADPH and the enzyme. The reaction was
started by adding TS2; the absorbance decrease at 340 nm was fol-
lowed at 25 °C using a Jasco V-650 spectrophotometer. The con-
3. Conclusion
Substitution variations of both ends of the bistacrines and the
length of the linker led to compounds of inhibitory activities
against Trypanosoma brucei brucei covering three orders of magni-
tude which allow to establish pronounced structure-activity rela-
tionships (SARs). SARs point to a specific interaction with a
defined target. Whereas some compounds showed inhibition of
rhodesain in micromolar range of concentration, the inhibition of
TryR was partially found in submicromolar concentrations, which
is in line with the in vitro activity. Interestingly, some compounds
are competitive (series 2) and some are mixed-mode inhibitors.
This may suggest TryR being a more important target of these com-
pounds compared to rhodesain. Of note, preliminary investigations
showed that the bistacrine compounds show antileishmanial activ-
ity and inhibit the corresponding TryR of Leishmania infantum (data
not shown).
However, the compounds were found to have a high lipophilic-
ity4 which might affect the toxicity adversely.18 It might be specu-
lated that a decrease of logP – e.g. by introducing carbonyl groups
or polyoxyethylene moieties into the linker – may lead to reduced
promiscuity and thus, to reduced toxicity. Anyway, the bistacrine
moiety and especially compound 4b is a promising lead structure
for further development of an antitrypanosomal drug.
sumption of NADPH (e
= 6.2 mMÀ1 cmÀ1) was used to calculate
the volume activity of the enzyme. The amount of TryR in the assay
was chosen to obtain an absorbance decrease of À0.02 to
À0.08 minÀ1 (corresponding to 10 mU per 1 mL assay solution,
i.e. 2 nM).
Millimolar stock solutions of the inhibitors were prepared in
DMSO. Each assay contained a total volume of 50 ml of DMSO, either
of inhibitor solution or DMSO as control. The preliminary screening
was performed with inhibitor concentrations from 1 to 5 mM and a
fixed concentration of either 40 or 100 mM TS2. For the detailed
kinetic analyses, the TS2 concentration was varied from 20 to
200 mM. The inhibitor constants were calculated by means of Graph-
pad Prism 7 (GraphPad Software, Inc.La Jolla, CA, USA).
4.2.2.3. Glutathione reductase assay. The assay was performed
according to 29. In brief, the activity of hGR was measured in
47 mM potassium phosphate, 200 mM KCl, 1 mM EDTA, pH 6.9.
In analogy to the TR assay, the assays contained in a total volume
of 1 ml buffer 100 mM NADPH and 6–30 mU of hGR (i.e. 1–2 nM),
and the activity was measured at 25 °C. The reaction was started
by addition of glutathione disulfide (GSSG) and the decrease of
absorbance was followed at 340 nm.
4. Experimental
4.1. Chemistry
Acknowledgements
Tacrine (1a)19 and compounds 1d,4 1e,20 1f,4,21 1j,22 1m,4 1n,22
2a–i,4,23–25 3a–c,4 3f,4 and 3g4 were prepared as previously
described. The new compounds were synthesized accordingly.
Details and the synthetic pathway for compounds 4a–4e can be
found in the Supporting information.
This work was funded by the SFB 630 of the Deutsche
Forschungsgemeinschaft. Thanks are due to Anja Hasenkopf,
Würzburg, for the re-synthesis of the compounds 4, and Alejandro
Leroux and Sarah Göllner, Heidelberg, who contributed to the TR
kinetics.
4.2. Biological assays
A. Supplementary data
4.2.1. Trypanosoma and macrophage assay
AlamarBlueÒ assays for investigating the activity against T. bru-
cei brucei laboratory strain TC221 and J774.1 macrophages were
conducted as previously reported.12,26
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2017.06.051.
References
4.2.2. Enzyme assays
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