Inhibitory effect of phenothiazine- and phenoxazine-derived chloroacetamides on Leishmania major growth and Trypanosoma brucei trypanothione reductase

Article abstract of DOI:10.1016/j.ejmech.2015.11.023

A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania major (L. major) promastigotes. Several analogs were remarkably potent inhibitors, with antileishmanial activities being comparable or superior to those of the reference antiprotozoal drugs. Furthermore, we explored the structure-activity relationships of N-10 haloacetamides that influence the potency of such analogs toward inhibition of L. major promastigote growth in vitro. With respect to the mechanism of action, selected compounds were evaluated for time-dependent inactivation of Trypanosoma brucei trypanothione reductase. Our results are indicative of a covalent interaction which could account for potent antiprotozoal activities.

Full text of DOI:10.1016/j.ejmech.2015.11.023

Accepted Manuscript
Inhibitory Effect of Phenothiazine- and Phenoxazine-Derived Chloroacetamides on
Leishmania major Growth and Trypanosoma brucei Trypanothione Reductase
Ana Marcu, Uta Schurigt, Klaus Müller, Heidrun Moll, R.Luise Krauth-Siegel, Helge
Prinz
PII:
S0223-5234(15)30359-7
10.1016/j.ejmech.2015.11.023
EJMECH 8213
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 27 May 2015
Revised Date: 23 September 2015
Accepted Date: 16 November 2015
Please cite this article as: A. Marcu, U. Schurigt, K. Müller, H. Moll, R.L. Krauth-Siegel, H. Prinz,
Inhibitory Effect of Phenothiazine- and Phenoxazine-Derived Chloroacetamides on Leishmania major
Growth and Trypanosoma brucei Trypanothione Reductase, European Journal of Medicinal Chemistry
(2015), doi: 10.1016/j.ejmech.2015.11.023.
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ACCEPTED MANUSCRIPT
Inhibitory Effect of Phenothiazine- and Phenoxazine-Derived
Chloroacetamides on Leishmania major Growth and Trypanosoma brucei
Trypanothione Reductase
#
#
‡
#
ƒ
Ana Marcu Uta Schurigt* , Klaus Müller , Heidrun Moll , R. Luise Krauth-Siegel , Helge
,
,‡
Prinz*
*
To whom correspondence should be addressed. Phone: +49 251-8332195.
Fax: +49 251-8332144. E-mail: prinzh@uni-muenster.de.
‡
Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster, Corrensstraße
4
8, D-48149 Muenster, Germany
#
Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2,
D-97080 Wuerzburg, Germany
ƒ
Biochemistry Center of Heidelberg University (BZH), Im Neuenheimer Feld 504,
D-69120
Heidelberg

1
ACCEPTED MANUSCRIPT
Inhibitory Effect of Phenothiazine- and Phenoxazine-Derived
Chloroacetamides on Leishmania major Growth and
Trypanosoma brucei Trypanothione Reductase
#
#
‡
#
ƒ
Ana Marcu Uta Schurigt
*
, Klaus Müller , Heidrun Moll , R. Luise Krauth-Siegel , Helge
,
,‡
Prinz
*
‡
Institute of Pharmaceutical and Medicinal Chemistry, University of Muenster,
Corrensstraße 48, D-48149 Muenster, Germany
#
Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2,
D-97080 Wuerzburg, Germany
ƒ
Biochemistry Center of Heidelberg University (BZH), Im Neuenheimer Feld 504,
D-69120
Heidelberg
*
To whom correspondence should be addressed. Tel: +49 251-8332195.
Fax: +49 251-8332144. E-mail: prinzh@uni-muenster.de
or Tel: +49 931-3184671. E-mail: uta.schurigt@uni-wuerzburg.de
Abbreviations
L. major: Leishmania major, MDR: multiple drug resistance, ND: not determined, Px: nonselenium glutathione
peroxidase type tryparedoxin peroxidase, SAR: structure–activity relationship, SRB: sulforhodamine B, TBAB:
tetra-n-butylammonium bromide, TR: trypanthione reductase, Tpx: tryparedoxin, T(SH) : trypanothione
2
[
bis(glutathionyl)spermidine], TS : trypanothione [bis(glutathionyl)spermidine] disulfide, NADPH: nicotinamide
2
adenine dinucleotide phosphate, T. brucei: Trypanosoma brucei
Key words
Phenoxazines, phenothiazines, antileishmanial activity, chloroacetamides, trypanothione reductase

2
ACCEPTED MANUSCRIPT
Abstract
A number of phenothiazine-, phenoxazine- and related tricyclics-derived chloroacetamides
were synthesized and evaluated in vitro for antiprotozoal activities against Leishmania major
(L. major) promastigotes. Several analogs were remarkably potent inhibitors, with
antileishmanial activities being comparable or superior to those of the reference antiprotozoal
drugs. Furthermore, we explored the structure–activity relationships of N-10 haloacetamides
that influence the potency of such analogs toward inhibition of L. major promastigote growth
in vitro. With respect to the mechanism of action, selected compounds were evaluated for
time-dependent inactivation of T. brucei trypanothione reductase. Our results are indicative of
a covalent interaction which could account for potent antiprotozoal activities.

3
ACCEPTED MANUSCRIPT
1
.
Introduction
Protozoan parasites of the order Kinetoplastida are causative to Chagas disease (Trypanosoma
cruzi), sleeping sickness (Trypanosoma brucei subspp.) and the different manifestations of
leishmaniasis (Leishmania spp.). Although the latter can be treated, limited therapeutic
options including pentavalent antimonials, miltefosine or amphotericin B need a long-term
parenteral administration and show severe side effects and toxicity. Additionally, these drugs
are expensive and require adequate medical care which is not readily available in the most
affected regions of India or Africa. Clearly more efficient and affordable antileishmanial
therapeutics are needed. In our search strategy for novel, potent antileishmanial compounds
we extended our studies to tricyclic heterocycles, which are attractive scaffolds in medicinal
chemistry and part of numerous important therapeutic agents (Chart 1).[1]
Phenothiazine (1, Chart 1), phenoxazine (2) and related structures are building blocks for a
large number of drugs that have shown diverse biological activities including antipsychotic,
anticancer, antihelminthic and other pharmacological properties.[2]
Chart 1 should be inserted here
Chlorpromazine (3), prepared in 1950, is the prototypical phenothiazine antipsychotic drug
and perhaps the best known of the phenothiazine drugs for treatment of neurological
disorders. Many other 10-substituted phenothiazines and related structures such as
clomipramine (4) are also well-known neuroleptic and antidepressant drugs. The
phenothiazine dye methylene blue (5) is employed as a fast-acting antidote for the treatment
of methemoglobinemia[3]. The antileishmanial activity of chlorpromazine against both
promastigotes and amastigotes of L. donovani has been demonstrated by Pearson et al.[4] A
trypanocidal effect of neuroleptic phenothiazines such as chlorpromazine and thioridazine on
Trypanosoma brucei was described by Seebeck et al., most likely due to an interaction of the

4
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drugs with pellicular microtubules.[5] Recently, Fauro et al. found clomipramine to be a good
candidate for Chagas disease in the chronic cardiac form of the infection.[6] Notably, no
single component of the molecular structure of the tricyclic neuroleptics and antidepressants
appears to be critical for activity.[7]
Moreover, antifungal activities of α-chloro-N-acetyl derivatives of phenoxazine, carbazole
and diphenylamine (13a, 17 and 18, see Scheme 1) have been described by Sarmiento et al.[8]
We were able to identify a panel of leishmanicidal phenothiazine-, phenoxazine- and related
tricyclics-derived chloroacetamides. Parent compounds were originally obtained during
synthetic studies on phenothiazine– and phenoxazine–based tubulin inhibitors,[9] when
subjecting selected analogs to a L. major promastigote screening. L. major may cause
cutaneous leishmaniasis with symptoms ranging from skin sores, cutaneous and muco-
cutaneous lesions, leading to the formation of disfiguring scars after wound healing. Though
the leishmanicidal activities of phenothiazines and related compounds have been intensively
investigated,[10-12] structure–activity relationships of their central nitrogen N-10
haloacetamides have never, to our knowledge, been reported systematically.
Some of the compounds described herein were remarkably potent inhibitors, with
antileishmanial activities of the most active compounds being comparable or superior to those
of the reference compounds, such as miltefosine, paromomycine and pentamidine. To get an
insight in the specifity, the cytotoxicity of selected compounds was assayed against human
leukemia K562 cells. To further elucidate the mechanism of action, time-dependent
inactivation of T. brucei TR by chloroacetamides was investigated.
2
2
.
Results and discussion
Chemistry
.1
Synthetic precursors (Chart 2) were available from commercial sources or prepared according
to literature protocols.[13]

5
ACCEPTED MANUSCRIPT
Chart 2 should be inserted here
The synthesis of haloacetamides was accomplished by chloroacylation of the respective
tricyclics and related structures with appropriately substituted acyl chlorides under reflux in
toluene (Scheme 1). Compound 20 was prepared by alkylation with 1-bromo-2-chloroethane
using catalytic tetrabutylammonium bromide (TBAB).
Scheme 1 should be inserted here.
2
2
.2
Biological evaluation
.2.1 Growth inhibition of L. major promastigotes
Although the Leishmania parasite exists in the human host in its intra-macrophage amastigote
form, the extracellular insect-infective promastigote form of Leishmania is addressed in many
drug screening assays against Leishmania, which can be performed readily, thus being more
suitable for automation. Detailed aspects of promastigote versus amastigote screening have
recently been excellently reviewed by De Muylder et al.[14]
Table 1 displays the inhibition data of the novel compounds against promastigotes of L.
®
major, which were assesssed by colorimetric AlamarBlue assay.[15] These data explore the
effect of altering not only the nature of the tricyclic nucleus but also substitutions on the
nitrogen atom. The most potent promastigote inhibitors within the phenothiazine- and
phenoxazine series displayed IC values in the range of 10 µM and include a tricyclic 6-6-6
5
0
(12a, 12b, 13b, 13f, 13i) or 6-7-6 scaffold (14) and – with the exception of compound 13l –
the N-chloroacetamido moiety.
The comparison of the IC values obtained for 13a bearing an acetamide moiety with those
50
obtained for chloracetamides 12a, 12b, 13b, 13f and 13i clearly suggests the importance of

6
ACCEPTED MANUSCRIPT
the intact haloacetamide for potent promastigote inhibition. The mere presence of an amide
functional group is apparently not sufficient to impart antileishmanial activity, as seen with
1
3a (L. major IC > 100 µM, Table 1), and did not induce antiproliferative potency. Other
50
evidence that the chloroacetamido provides an important partial structure comes from the
synthetic precursor 2-chloro-10H-phenoxazine (2a), which does not show substantial activity.
In particular the replacement of the carbonyl functional group (20, Scheme 1, Table 1) by a
methylene spacer or substitution of the terminal chloro atom by a bromo- or a cyano group
(
13g, 13h, Table 1) caused a dramatic decrease in the IC value, again pointing to the
50
particular importance of the α-chloroacetamido as a main determinant for inhibiton of
parasite proliferation. In this connection it is interesting to note that chain elongation by
inserting methylene spacers (13c–13e) between the amide and the terminal chloro did not
cause an activity enhancement, but even resulted in a reduced antipromastigote potency.
Thus, the spatial disposition of the terminal chloro atom and the heterocycle is an influential
factor in determining antileishmanial activity. Ring-opened variants 18 and 19, both bearing
the chloroacetamide, proved considerably less effective (18, IC50 40.30 µM and 19, IC50
4
2.60 µM vs. 12a, IC 9.30 µM and 13b, IC 7.60 µM) compared to the most active analogs,
50 50
indicating that rigidifying the diphenylamine or the diphenyl ether is important for target
binding. This is supported by similar observations made by Richardson et al. with
diphenylmethane analogs.[16]
On the one hand, efficiency against promastigotes could be attained by 7-chloro-5,11-
dihydrodibenzo[b,e][1,4]oxazepine–derived 14, leading to one of the most active compounds
of the series (IC 7.20 µM). This finding indicates that compared with 13b the ring-enlarged
5
0
tricyclic 6-7-6 oxazepine is also a favored scaffold. On the other hand, 5H-
dibenzo[b,f]azepines 15 and 16 are only moderate inhibitors of promastigote growth (15, IC50
4
3.30 µM and 16, IC50 48.10 µM). Interestingly, decreasing the ring size to obtain the

7
ACCEPTED MANUSCRIPT
carbazole 17 completely abolished activity against L. major. In general, introduction of
different substituents into the phenothiazine- or phenoxazine, such as chloro (12b), dichloro
(13f), trifluoromethyl (13i) or cyano (not shown) had no significant impact on the
antiparasitic activities. Based on the broad range of potencies, inhibition of L. major
promastigote growth by the chloroacetamides cannot be considered unselective. Fig. 1
summarizes the SAR on promastigote inhibition.
Fig. 1 should be inserted here
2
.2.2 Growth inhibition of K562 cells
Additionally, we preliminarily evaluated the putative cytotoxicity of the compounds towards
K562 cells (human chronic myelogenous leukemia, DSMZ ACC-10).[17] Cell proliferation
was determined directly by counting the cells with a hemocytometer after 48 h of treatment.
Table 1 summarizes the data for inhibition of K562 cell growth. The most active
chloroacetamides displayed IC50 values in the range ≤ 5 µM, indicating strong cytotoxic
potential. The chloro acetamides such as 15, 16 and especially 17, which were not or only
weakly active against promastigotes showed antiproliferative potencies in the range of 2-5
µM. Obviously, the chloroacetamide is crucial for the cytotoxicity against K562 cells with
cytotoxic potencies being more or less similar. These finding suggests a nonselective growth
inhibition in the K562 cell-based assay and is in contrast with the results from the
promastigote assay, where the analogs may act specifically toward a target-protein with the
nature of the tricyclic ring playing a critical role for target interaction.
Table 1 should be inserted here
2
.2.3 Studies on the mechanism of antiprotozoal action

8
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Earlier, molecular modeling approaches as well as kinetic analyses and crystallographic
analysis revealed phenothiazine (1), imipramine and related tricyclic antidepressants are
competitive inhibitors of TR.[10-12]
The flavoenzyme trypanothione reductase (TR), which does not occur in the mammalian host,
is a prominent target for antitrypanosomal and -leishmanial drug development
approaches.[18] TR catalyzes the NADPH–dependent reduction of trypanothione
[
bis(glutathionyl)spermidine] disulfide TS2 to trypanothione [T(SH) ], the main low
2
molecular mass thiol in trypanosomatids (Equation 1).[19]
Equation 1 should be inserted here
The tricyclic structures are proposed to bind to TR with the tricyclic ring lodged against the
hydrophobic wall of TR active site formed by Trp21 and Met 113 and the aminopropyl side
chain extending towards Glu466´and Glu467` residues.[10, 11] By inhibiting TR, the parasite
should become susceptible to oxidative stress induced by drugs or the host defence system
(Fig. 1). Notably, N-substituted 12a (Scheme 1), has been noted previously as a moderate
inhibitor of T. cruzi TR.[11]
We suggest that an irreversible inhibition by alkylation of biologically important nucleophiles
such as susceptible SH- or NH-groups on the active site of distinct proteins could possibly be
involved in the modes of action of the chloroacetamides against the L. major promastigotes,
as described for other compounds.[20, 21] Several drugs, such as the 2-chloroethyl
nitrosourea carmustine[22], and quinacrine analogs[23] are known to inhibit trypanothione
reductase irreversibly. Basically, the reactive α-haloacetamide moiety itself is prone to form
covalent bonds by attacking nucleophiles with concomitant displacement of the halogen.[24]
However, since numerous enzymes contain SH-groups vital to the activity of the enzyme, it

9
ACCEPTED MANUSCRIPT
would seem obvious that a large variety of enzymes might be nonselectively inhibited by
chloroacetamides.
Trypanosoma and Leishmania are protozoan parasites which have a unique trypanothione-
based thiol redox metabolism (Fig. 2). In order to get more insight into the mechanism of
action of the chloroacetamides, we performed additional studies using recombinant T. brucei
TR. TR is common to all parasites of the Trypanosomatidae family and it has been
documented, that kinetic and physical properties of T. brucei TR are consistent with
trypanothione reductases from other trypanosomatids such as T. cruzi and L. donovani.[25]
None of the compounds proved to be a reversible inhibitor of the enzyme (data not shown).
Thus, in the next step, selected derivatives were studied for their ability to interfere with the
unique peroxidase cascade of the parasites. All enzymes establishing the detoxification
system, namely TR, tryparedoxin (Tpx) and the tryparedoxin peroxidases have been shown to
be essential for T. brucei.
Fig. 2 should be inserted here
In a first set of experiments, to identify a distinct target protein, selected compounds were
characterized in a time-dependent peroxidase. For this purpose, a reaction mixture composed
of NADPH, T. brucei TR, Tpx, Px, T(SH) and inhibitor was incubated for 10 min and then
2
the reaction started by adding H O . Compounds 12a and 14 showed remarkable time-
2
2
dependent inhibition (data not shown).
In the next set of experiments, selected compounds were evaluated for their activity toward T.
brucei TR. TR was preincubated with the compounds in the presence or absence of NADPH.
After different times, an aliquot of the reaction mixture was subjected to a TR standard assay,
starting the reaction by adding TS2.

1
0
ACCEPTED MANUSCRIPT
Fig. 3 should be inserted here
Our cuvette-based assays showed a time-dependent inactivation of TR for the most active
compounds of the Leishmania assay. It has to be pointed out that preincubation mixtures that
lacked either NADPH or the potential inhibitor showed no decrease of enzyme activities with
time. Moreover, when the assay was started directly, no inactivation became visible (data not
shown).
Examination of time-dependent TR inhibition revealed that all tested analogs (Fig. 2) with
potent antileishmanial activities were also active as T. brucei TR inhibitors.
3
.
Conclusion
We have shown that phenothiazine-, phenoxazine- and related tricyclics-derived
haloacetamides are highly potent inhibitors of the promastigote form of L. major. Structural
comparisons of the compounds hints at a selective mechanism of action. Those analogs with
the highest antiprotozoal activities in the promastigote assay displayed strong time-dependent
inhibition of recombinant TR. The most active analogs described herein showed two essential
moieties. One is the tricyclic (6-6-6 or 6-7-6) scaffold which possibly interacts with
hydrophobic regions of the target enzyme active site, in a way that has been suggested for
several tricyclic, mainly competitive TR inhibitors.[10-12] The second is the chloroacetamido
moiety. Since alkylation of biologically important nucleophiles such as susceptible SH- or
NH-groups of active sites could contribute to antileishmanial and antitrypanosomal potencies,
we investigated the time-dependent effects of the chloroacetamides on inhibition of T. brucei
TR. Preincubation of TR with selected compounds in the presence – but not in the absence -
of NADPH resulted in a time-dependent inactivation of TR, pointing to a covalent
modification of Cys52, part of the redox active dithiol/disulfide. Our findings strongly suggest
that TR is a target of the chloroacetamides. This is strongly supported by literature findings

1
1
ACCEPTED MANUSCRIPT
for structurally related TR-inhibiting tricyclic systems.[10-12, 23] However, it cannot yet be
completely ruled out that putative drug targets of the peroxidase cascade such as Tpx are also
affected by our inhibitors.[24]
Taken together, our results are indicative of a covalent mechanism of action of the
haloacetamides and the effects of the compounds toward the peroxidase cascade. To our
knowledge this is the first report on covalent inhibitors derived from phenothiazines or
phenoxazines. Scope of future experiments will be expanded to the selectivity for TR over
human glutathione reductase, and the correlation of TR inhibition with antiparasitic growth-
inhibitory activity. Activities against L. major amastigotes have yet to be determined.
4
4
4
.
Experimental
Chemistry
.1
.1.1 General
Melting points were determined with a Kofler melting point apparatus and are uncorrected.
1
13
Spectra were obtained as follows: H NMR (400 MHz), C NMR (100 MHz) spectra were
recorded with a Varian Gemini 2000 or Varian Mercury 400 plus (400 MHz) spectrometers,
respectively. NMR signals were referenced to TMS (δ = 0 ppm) or solvent signals and
recalculated relative to TMS. Fourier-transform IR spectra were recorded on a Bio-Rad
laboratories Typ FTS 135 spectrometer and analysis was performed with WIN-IR Foundation
software. Mass spectra were obtained on Finnigan GCQ and LCQ apparatuses applying
electron beam ionization (EI) and electrospray ionization (ESI). Atmospheric pressure
chemical ionization (APCI) method was performed with a microTOF-QII apparatus (Bruker).
The purity of all target compounds was determined either by elemental analyses or by
reversed phase HPLC at 254 nm. Compound purity for all target compounds is ≥ 95%.
Elemental analyses were performed at the Münster microanalysis laboratory, using a Vario
EL elemental analyzer from Elementar Analysensysteme GmbH Hanau, and all values were

1
2
ACCEPTED MANUSCRIPT
within ± 0.4% of the calculated composition. The HPLC system applied a C18 phase
(Nucleosil, 3 ꢀm, 3.0 × 125 mm, CS-Chromatographie Service GmbH, Langerwehe,
Germany) eluting the compounds with an acetonitrile/H O gradient at a flow rate of 0.40
2
mL/min. All organic solvents were appropriately dried or purified prior to use. Acid chlorides
were obtained from commercial sources or prepared to literature protocols. Purification by
chromatography refers to column chromatography on silica gel (Macherey-Nagel, 70-230
mesh). In most cases, the concentrated pure fractions obtained by chromatography using the
indicated eluants were treated with a small amount of hexane to induce precipitation. All new
1
compounds displayed H NMR and MS spectra consistent with the assigned structure. Yields
have not been optimized. Analytical TLC was done on Merck silica 60 F254 alumina coated
plates (E. Merck, Darmstadt).
2
-Chloro-10H-phenoxazine (2a).[13]
The title compound was prepared according to the literature protocol.
-Chloro-1-(10H-phenothiazin-10-yl)ethan-1-one (12a).[8]
See SI for details.
-Chloro-1-(2-chloro-10H-phenothiazin-10-yl)ethan-1-one (12b).[11]
See SI for details.
-(2-Chloro-10H-phenoxazin-10-yl)ethan-1-one (13a). [9]
The title compound was prepared as recently described.[9]
2
2
1
2
-Chloro-1-(2-chloro-10H-phenoxazin-10-yl)ethan-1-one (13b).[26]
The title compound was prepared from 2a (2.12 g, 9.74 mmol) and chloroacetyl chloride
(1.22 g, 10.8 mmol) in toluene (60 mL) in a similar manner according to a literature
protocol.[26] Purification of the residue by chromatography (CH Cl ) afforded 13b as white
2
2
1
crystals (2.21 g, 94 %). mp 97 °C (Lit. [26] 96 °C); H NMR (400 MHz, Chloroform-d) δ
.71 – 7.67 (m, 1H), 7.50 (d, J = 7.9 Hz, 1H), 7.31 – 7.25 (m, 1H), 7.24 – 7.14 (m, 3H), 7.08
dd, J = 8.3, 0.6 Hz, 1H), 4.32 (s, 2H).
7
(

1
3
ACCEPTED MANUSCRIPT
3
-Chloro-1-(10H-phenoxazin-10-yl)propan-1-one (13c).[27]
See SI for details.
-Chloro-1-(2-chloro-10H-phenoxazin-10-yl)propan-1-one (13d).
See SI for details.
-Chloro-1-(2-chloro-10H-phenoxazin-10-yl)butan-1-one (13e).
See SI for details.
-Chloro-1-(2,8-dichloro-10H-phenoxazin-10-yl)ethan-1-one (13f).
See SI for details.
-Bromo-1-(2-chloro-10H-phenoxazin-10-yl)ethan-1-one (13g).
See SI for details.
-Oxo-3-(10H-phenoxazin-10-yl)propanenitrile (13h).
See SI for details.
-Chloro-1-(2-(trifluoromethyl)-10H-phenoxazin-10-yl)ethan-1-one (13i).[28]
See SI for details.
-Chloro-1-(10H-phenoxazin-10-yl)butan-1-one (13j).
See SI for details.
,3-Dichloro-1-(10H-phenoxazin-10-yl)propan-1-one (13k).
See SI for details.
,3-Dichloro-1-(10H-phenoxazin-10-yl)propan-1-one (13l).
See SI for details.
-Chloro-1-(7-chlorodibenzo[b,e][1,4]oxazepin-5(11H)-yl)ethan-1-one (14).
3
4
2
2
3
2
3
3
2
2
In a typical procedure, to a solution of 6 (0.59 g, 3 mmol) in toluene (30 mL), chloroacetyl
chloride (0.34 g, 3 mmol) was added. The mixture was stirred at reflux for 4 h. After that, the
solvent was evaporated in vacuo. Purification of the residue by chromatography (CH Cl )
2
2
-1 1
afforded 14 as white crystals (0.54 g, 58%). mp 142-143 °C; FTIR 1685 cm ; H NMR (400
MHz, Chloroform-d) δ 7.58 – 7.28 (m, 5H), 7.13 (s, 1H), 6.81 (s, 1H), 5.70 (d, J = 12.5 Hz,

1
4
ACCEPTED MANUSCRIPT
+
1
3
2
H), 4.87 (d, J = 12.5 Hz, 1H), 4.40 – 3.83 (m, 2H); MS (APCI) calcd for C H Cl NO [M]
15 11 2 2
07.02; found 308.03; purity (HPLC): 99.66%.
-Chloro-1-(10,11-dihydro-5H-dibenzo[b,f]azepin-5-yl)ethan-1-one (15).
See SI for details.
-Chloro-1-(5H-dibenzo[b,f]azepin-5-yl)ethan-1-one (16).
See SI for details.
-(9H-Carbazol-9-yl)-2-chloroethan-1-one (17).[8]
See SI for details.
-Chloro-N-(3-chlorophenyl)-N-phenylacetamide (18).[8]
See SI for details.
-Chloro-N-(5-chloro-2-phenoxyphenyl)acetamide (19).
See SI for details.
-Chloro-10-(2-chloroethyl)-10H-phenoxazine (20). To a solution of 2-chloro-10H-
phenoxazine (2a, 2.0 g, 9.22 mmol) in CH Cl (20 mL) were added KOH (6N, 30 mL), tetra-
2
1
2
2
2
2
2
n-butylammonium bromide (TBAB, 1.5 g, 4.65 mmol) and 1-bromo-2-chloroethane (3.17 g,
2.34 mmol, 1.84 mL). The reaction mixture was stirred at room temperature until the
2
reaction was completed, then treated with water (50 mL) and subsequently extracted with
CH Cl (2 × 25 mL). The combined organic phases were washed with water, dried over
2
2
Na SO , and concentrated in vacuo. Purification by chromatography (CH Cl /hexane 1/1 )
2
4
2
2
-
1 1
afforded 20 as a fine white powder (0.14 g, 20%): mp 84-85 °C; FTIR 1458, 1423 cm ; H
NMR (CDCl ) δ 6.67-6.63 (m, 1H), 6.56-6.52 (m, 1H), 6.48-6.44 (m, 2H), 6.37 (d, 1H, J =
3
8
.22 Hz), 6.34 (dd, 1H, J = 6.65 Hz, J = 1.18 Hz), 6.28 (d, 1H, J = 2.35 Hz), 3.65 (t, 2H, J =
.43 Hz), 3.45 (t, 2H, J = 7.04 Hz); MS m/z 353 (<1), 277 (100); Purity (HPLC), 96.67%.
7
4
.2
Biological evaluation
Materials and methods for the biological assays have been described before by the authors.

1
5
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AlamarBlue assay for investigation of antileishmanial activities against L. major
promastigotes was conducted as previously reported.[15]
4
.2.1 Assay of Cell Growth.
5
K562 cells were plated at 2 × 10 cells/mL in 24 well dishes (Costar, Cambridge, MA).
Untreated control wells were assigned a value of 100%. Test compounds were made soluble
in DMSO/methanol 1:1 (stock solution 3 mg/mL), and control wells received equal volumes
(0.5%) of vehicle alone. We demonstrated, that 0.5 % DMSO (final DMSO concentration in
all samples including the controls) did not affect K562 cell growth. To each well 5 µL of
compound were added and the final volume in the well was 500 µL. Cell numbers were
counted with a Neubauer counting chamber (improved, double grid) after 48-h compound
exposure. Each assay was prepared in triplicate, and the experiments were carried out three
TM
times. IC values were obtained by nonlinear regression (GraphPad Prism ) and represent
5
0
the concentration at which cell growth was inhibited by 50%. The adjusted cell number was
calculated as a percentage of the control, which was the number of cells in wells without the
test compound.
4
.2.2 Time-dependent TR inactivation assay.
In a total volume of 200 µL TR assay buffer, 400 µM NADPH, 1 µM T. brucei TR[18], and
either 25 (2a), 50 (13b) or 100 µM (12a, 13a, 14) compound or DMSO (5%) were
preincubated at 25 °C. After 10 min, 30 min, 60 min, 120 min and 180 min, respectively, a 5
µL aliquot was subjected to a 1 mL TR standard assay,[22] containing 100 µM NADPH and
the reaction was started by addition of 100 µM (5 µL) trypanothione disulfide (TS ). The
2
absorption decrease at 340 nm was monitored at 25 °C. The volume activity (U/mL) was
plotted against the pre-incubation time.
ACKNOWLEDGMENTS

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6
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We thank Martina Schultheis and Angelika Zinner for excellent technical assistance. Thanks
are due to the Deutsche Forschungsgemeinschaft (SFB 630) for financial support.

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Table 1.
EC values of selected compounds towards L. major in comparison to
50
reference compounds as well as mammalian cells
1
2
3
a)
b)
X
Y
n
R
R
R
L. major
K562
cmpd
IC [µM] EC [µM]
5
0
50
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
69.20
9.30
2a
2b
3a
3b
3c
3d
3e
3f
3g
3h
3i
S
Cl
1
1
1
1
2
1
2
1
1
1
1
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
5.00
S
Cl
Cl
Cl
Cl
H
10.4
3.20
> 30
3.00
15.23
10.22
> 90
2.03
6.15
23
O
O
O
O
O
O
O
O
O
O
O
O
CH
H
> 100
7.60
Cl
Cl
39.10
15.70
> 100
9.50
CH
CH
Cl
Cl
Cl
Cl
Cl
Cl
H
2
CH
Cl
2
2
Cl
H
H
H
H
H
H
Br
CN
Cl
> 100
> 100
9.60
CF
H
ND
3
3j
3k
3l
CH
3
> 100
82
> 30
> 30
ND
Cl
H
CH
Cl
Cl
H
9.60
2
4
2
-O
H
7.20
3.30

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15
16
17
18
CH
2
-CH
2
Cl
H
H
43.30
48.10
> 100
40.30
4.80
6.20
6.44
ND
CH=CH Cl
H
H
*
*
*
4
2.60
100
ND
1
2
9
0
*
>
ND
amphotericine B
miltefosine
0.33
36
ND
ND
ND
ND
pentamidine
isethionate
paromomycine
84
> 100
a
The drug concentrations resulting in 50% cell growth (EC ). Cell viability was determined after
5
0
®
b
4
8 h by the AlamarBlue assay. EC K562, concentration of drug required for 50% inhibition of
50
cell growth. Cells were treated with drugs for two days. EC values are the means of at least three
5
0
*
independent determinations (SD < 10%) . See Scheme 1 for chemical structure.

1
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Figure Legends
Chart 1
Chart 2
Scheme 1
Phenothiazine (1), phenoxazine (2) and related drugs 3, 4 and 5.
Synthetic precursors
1
3
R -R are defined in Table 1. Reagents and conditions: (a) haloacyl chloride,
toluene, 90 °C, N ; b) TBAB, CH Cl , 6N KOH, 1-bromo-2-chloroethane, room temperature;
2
2
2
Equation 1
Fig. 1
Fig. 2
Fig. 3
SAR summary
Detoxification of hydroperoxides (ROOH) by the trypanothione cascade.
Time-dependent inactivation of T. brucei trypanothione reductase. The enzyme
was incubated with the compounds in the presence or absence of NADPH. After different
times, an aliquot of the reaction mixture was subjected to a standard assay as outlined in
section 4.2.2. . Compounds 2a (A) and 13a (B) represent negative controls, which show no
inhibition over time. On the other hand compounds 13b (C), 12a (D) and 14 (E) show a
strong time-dependent inhibition, suggesting a covalent mechanism of action. The EC refers
5
0
to the L. major promastigote assay.

2
0
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2
1
ACCEPTED MANUSCRIPT

2
2
ACCEPTED MANUSCRIPT

2
3
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Equation 1

2
4
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ring contraction
NOT PERMITTED
X = -S- or -CH -O- RETAIN
2
Carbazole
INACTIVE
activity;
X = CH -CH , CH=CH
2
2
DIMINISH
activity
ring expansion
-7-6
TOLERATED
6
X
R = Cl, CN
TOLERATED
R
N
O
ring opening
REDUCES activity
CHLOROacetamide
CRITICAL
for activity
N-formyl, N-acetyl
ABOLISH activity
n
Y
carbonyl
REQUIRED
C=O vs CH2
ABOLISHES
activity
Y = Cl
spacers
DIMINISH
activity
REQUIRED
Y = H, Br, CN
INACTIVE
gem Cl INACTIVE
-
CH vs -CHCH3
2
INACTIVE
-
CH vs CHCH Cl
2 2
RETAINS activity
Fig. 1 SAR summary

2
5
ACCEPTED MANUSCRIPT
Fig. 2
NADPH
NADP
TRox
T(SH)2
Tpxox
Pxred
Pxox
ROOH
TRred
TS2
Tpxred
2
ROH + H O

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Fig. 3
A
2a
IC50 = 69.2 µM
3
2
2
1
1
0
0
,0
,5
,0
,5
,0
,5
,0
0
20 40 60 80 100 120 140 160 180 200
Time (min)
B
13a
IC50 > 100 µM
C
13b
IC50 = 7.6 µM
3
,0
,5
,0
,5
,0
,5
,0
3,0
2,5
2,0
1,5
1,0
0,5
0,0
2
2
1
1
0
0
0
20 40 60 80 100 120 140 160 180 200
0
20 40 60 80 100 120 140 160 180 200
Time (min)
Time (min)
D
12a
IC50 = 9.3 µM
E
14
IC50 = 7.2 µM
3
2
2
1
1
0
0
,0
,5
3,0
2,5
2,0
1,5
1,0
0,5
0,0
,0
,5
,0
,5
,0
0
20 40 60 80 100 120 140 160 180 200
0
20 40 60 80 100 120 140 160 180 200
Time (min)
Time (min)

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