Novel distamycin analogues that block the cell cycle of African trypanosomes with high selectivity and potency

Article abstract of DOI:10.1016/j.ejmech.2020.112043

Polyamides-based compounds related to the Streptomycetal distamycin and netropsin are potent cytostatic molecules that bind to AT-rich regions of the minor groove of the DNA, hence interfering with DNA replication and transcription. Recently, derivatives belonging to this scaffold have been reported to halt the proliferation of deadly African trypanosomes by different and unrelated mechanisms. Here we describe the synthesis and preliminary characterization of the anti-trypanosomal mode of action of new potent and selective distamycin analogues. Two tri-heterocyclic derivatives containing a central N-methyl pyrrole ring (16 and 17) displayed high activity (EC₅₀ < 20 nM) and selectivity (selectivity index >5000 with respect to mammalian macrophages) against the infective form of T. brucei. Both compounds caused cell cycle arrest by blocking the replication of the mitochondrial DNA but without affecting its integrity. This mode of action clearly differs from that reported for classical minor groove binder (MGB) drugs, which induce the degradation of the mitochondrial DNA. In line with this, in vitro assays suggest that 16 and 17 have a comparatively lower affinity for different template DNAs than the MGB drug diminazene. Therapeutic efficacy studies and stability assays suggest that the pharmacological properties of the hits should be optimized. The compounds can be rated as excellent scaffolds for the design of highly potent and selective anti-T. brucei agents.

Full text of DOI:10.1016/j.ejmech.2020.112043

Journal Pre-proof
Novel distamycin analogues that block the cell cycle of African trypanosomes with
high selectivity and potency
Jaime Franco, Laura Scarone, Marcelo A. Comini
PII:
S0223-5234(20)30010-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112043
EJMECH 112043
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 November 2019
Revised Date: 3 December 2019
Accepted Date: 6 January 2020
Please cite this article as: J. Franco, L. Scarone, M.A. Comini, Novel distamycin analogues that block
the cell cycle of African trypanosomes with high selectivity and potency, European Journal of Medicinal
Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2020.112043.
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© 2020 Published by Elsevier Masson SAS.

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Novel distamycin analogues that block the cell cycle of
African trypanosomes with high selectivity and potency
Jaime Francoa,b, Laura Scarone*a and Marcelo A. Comini*b
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Laboratorio de Química Farmacéutica, Departamento de Química Orgánica, Facultad de
Química, Universidad de la República, Montevideo, Uruguay.
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Group Redox Biology of Trypanosomes, Institut Pasteur de Montevideo, Montevideo,
Uruguay.
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* Corresponding authors:
(1) Laura Scarone
Laboratorio de Química Farmacéutica, Departamento de Química Orgánica
Facultad de Química, Universidad de la República
Gral. Flores 2124
Montevideo, CP 11800
Uruguay
E-mail: laurito@fq.edu.uy; Fax: +598 29241906; Phone: +598 29290290
(2) Marcelo Comini
Group Redox Biology of Trypanosomes
Institut Pasteur de Montevideo
Mataojo 2020
Montevideo, CP 11400
Uruguay
E-mail: mcomini@pasteur.edu.uy; Fax: +598 2522 4185; Phone: +598 2522 0910 ext. 164
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Abstract
Polyamides-based compounds related to the Streptomycetal distamycin and netropsin are
potent cytostatic molecules that bind to AT-rich regions of the minor groove of the DNA,
hence interfering with DNA replication and transcription. Recently, derivatives belonging to
this scaffold have been reported to halt the proliferation of deadly African trypanosomes by
different and unrelated mechanisms. Here we describe the synthesis and preliminary
characterization of the anti-trypanosomal mode of action of new potent and selective
distamycin analogues. Two tri-heterocyclic derivatives containing a central N-methyl pyrrole
ring (16 and 17) displayed high activity (EC50 <20 nM) and selectivity (selectivity index
>5000 with respect to mammalian macrophages) against the infective form of T. brucei. Both
compounds caused cell cycle arrest by blocking the replication of the mitochondrial DNA but
without affecting its integrity. This mode of action clearly differs from that reported for
classical minor groove binder (MGB) drugs, which induce the degradation of the
mitochondrial DNA. In line with this, in vitro assays suggest that 16 and 17 have a
comparatively lower affinity for different template DNAs than the MGB drug diminazene.
Therapeutic efficacy studies and stability assays suggest that the pharmacological properties
of the hits should be optimized. The compounds can be rated as excellent scaffolds for the
design of highly potent and selective anti-T. brucei agents.
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Keywords:
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mitochondrial DNA; cytostatic; Trypanosoma brucei; minor groove binder; distamycin
analogues; polyamides.
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1. Introduction
African trypanosomiasis encompasses a group of zoonotic diseases caused by hemoflagellate
parasites from different (sub)species of the Genus Trypanosoma that are endemic of the sub-
Saharan region of Africa. The subspecies that causes Human African Trypanosomiasis
(HAT), also known as African sleeping sickness, are Trypanosoma brucei gambiense and
Trypanosoma brucei rhodesiense. Although control programs implemented by WHO have
contributed to reduce the number of cases (< 10000 cases/year), 65 million people are at risk
of contracting a disease that is fatal without therapeutic intervention [1,2].
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T. b. brucei, T. congolense and T. vivax are responsible for Animal African Trypanosomiasis
(AAT) [3]. Current estimations indicate that more than 3 million stock animals are affected
by AAT, which accounts for large economic losses (several billions U$S/year) due to
impaired productivity [4].
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The current chemotherapy for HAT relies on old fashioned drugs (i.e. suramin, pentamidine,
melarsoprol, eflornithine and nifurtimox) characterized by their limited, stage- and parasite
subspecies-specific efficacy. Most of these drugs were developed based on empiric findings,
are toxic and with undesirable administration routes [1,2]. In addition, the indiscriminate use
of drugs to control AAT (e.g. isometamidium) has been linked to the emergence of cross-
resistance with the human frontline drug pentamidine [5].
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After 10 years of continued and joint efforts between academy, non-profit initiatives and
pharma companies, the drug fexinidazole has been “rediscovered” and approved for the oral
treatment of both the systemic and neurological stages of HAT [6]. Following a similar non-
profit, R&D model, an acoziborole (SCYX-7158) has been selected as a promising pre-
clinical candidate against HAT that is currently undergoing clinical studies [7]. To our
knowledge, there are no more compounds in the clinical pipeline for this neglected disease.
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Organic chemistry have been often inspired by the discovery of biological activities present
in natural molecules. A good example is distamycin and netropsin, both being polyamides
produced by the bacteria Streptomyces netropsin and harboring antifungal activity.
Compounds of this family can bind into the minor groove of the DNA (hence named minor
groove binder: MGB), especially in AT-rich regions, and by doing so interfere with DNA
replication and transcription [8-11]. Several analogues have been synthesized and displayed
potent anti-proliferative activity against a variety of biological models such as tumoral cells,
viruses, fungi, bacteria and protozoan [12-18].
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In the search for new MGB-like compounds acting against infectious organisms, Lang and
col. reported a series of heterocycles linked by amide bonds some of which resulted active
against T. b. brucei, in particular a molecule lacking a typical basic and flexible terminal
group found in MGB [15]. Two years later, the same research group published on the anti-
trypanosomal activity of a series of 32 structurally diverse MGB consisting of heterocycles
linked by amide bonds [12]. Several of them presented a remarkable anti-proliferative
activity (≤ 40 nM) and selectivity (>500) towards bloodstream T. b. brucei. A strongly
fluorescent compound of this series displayed a granular distribution in the parasite cytosol
with predominant signal accumulated in the nucleus and mitochondrial DNA (kinetoplast), as
expected for compounds that interact with nucleic acids.
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Recently, we have prepared and characterized a series of bi and tri-thiazoles resembling the
distamycin scaffold 1 (Figure 1) [19]. The tri-thiazoles were more active and selective
against infective T. b. brucei than the bi-thiazoles. Notably, the mode of action of the most
potent (EC50 310 nM) and selective (selectivity index = 16 vs. murine macrophages) hit,
namely compound 1, involved the loss of lysosome integrity but no effect on cell cycle.
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Despite its potency, the low selectivity of 1 precluded advancing this compound to the next
steps of the drug development pipeline. In order to obtain derivatives with a better biological
profile and taking into account previous results with structurally related compounds, we
hypothesized that inclusion of an N-methyl pyrrole as the central ring (New series shown in
Figure 1) may confer a higher selectivity. To our surprise, this slight structural change
yielded compounds far more potent (>26-fold) and selective (>340-fold), respectively, than 1.
Interestingly, the new hits inhibited parasite proliferation by blocking the replication of the
kinetoplast. Despite the negative results obtained for one of the hits in therapeutic efficacy
studies conducted on a mouse model of African trypanosomiasis, the new scaffolds appear as
top candidates for optimization of their pharmacological properties.
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2. Results and discussion
2.1. Chemistry and organic synthesis
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In order to obtain a molecule with a N-methyl pyrrole as a central heterocyclic, we
synthesized the trifluoroketone 2 [20,21] from the commercially available N-methyl pyrrole,
which was nitrated in classical conditions to obtain the 2,4-disubstituted N-methyl pyrrole 3.
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Then, compound 3 was treated with NaH in aqueous medium to substitute the trifluoromethyl
by an hydroxyl group [21] that led to the central building block 4 in very good yields.
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We employed Hantzsch and Barton methodologies [22-24] to synthesize thiazoles 5 and 6,
that were derivatized to obtain two series of thiazoles containing esters and carboxylic acids
with different protective groups on their 2-amino position 7-12. (Scheme 1)
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The N-methyl pyrrole 4 was coupled with the 2-aminothiazoles 5 and 6 in the presence of the
iminium salt HBTU and DIPEA in CH2Cl2 at room temperature for 24 h obtaining 13 in 87 %
yield and 14 in 62 % yield, respectively (Scheme 2). The nucleophilicity of the 2-amino
thiazole has been reported as low due to the iminium tautomerism, in fact several reactions
pointed that the endo nitrogen can be the nucleophile instead of the exo amino group [25,26].
This can explain the lower yield of 14 in comparison to 13, whose isopropyl group likely
increases the electron density and makes the 2-amino group more reactive towards the
carboxylic group. As part of our studies, the HBTU activated acid of the N-methyl pyrrole
was isolated, re-dissolved in dry THF and refluxed in the presence of compound 6 without
increasing yields.
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Next, the third heterocyclic ring (9, 10 or 12) was coupled to 13 or 14 through the nitrogen at
position 4 of the N-methyl pyrrole. Therefore a reduction of the nitro group was performed in
the presence of H2, Pd/C for 3 h resulting on a free amine on the position 4. Due to the
reported instability of the pyrrole amine [27], the reaction crude was used without further
purification. The crude was filtered through celite, concentrated in vacuo and re-dissolved in
dry CH2Cl2 for coupling with carboxylic acids 9, 10 and 12 in the presence of HBTU for 24 h
(Scheme 2). Despite of being a two-step reaction, the tri heterocycles (15 from 12 and 13, 16
from 13 and 9, 17 from 14 and 10) were obtained in fair yields (e.g. 30 to 40 %). These three
heterocycles were hydrolyzed in the presence of aqueous LiOH, leading to compounds 18-20
in excellent yields (Scheme 2).
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2.2. Biological characterization
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Compounds 13-20 were preliminary screened at a concentration of 5 µM against bloodstream
T. b. brucei (Table 1). For compounds causing ≥ 75% parasite death at 5 µM, concentration-
response assays against the pathogen and murine macrophages were performed to estimate
EC50 and selectivity values.
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As previously observed for the first series of compounds [19], two major features correlated
positively with the biological activity of the second series: the number of heterocycles and the
state of the terminal amine and carboxylic acid derivatives. Regarding the first factor, at 5 µM
the bis-heterocycle (13 and 14) proved innocuous to trypanosomes while the tri-heterocyclic
compounds (15-17 and 19-20), with the exception of 18, exhibited a remarkable anti-parasitic
activity (cell viability <25% at 5 µM). Protection of the carboxylic acid rendered the tri-
heterocyclic compounds 15-17 (EC50 827 to 7 nM) significantly more active than their
corresponding un-protected analogues 18-20 (EC50 ≥ 3327 nM). For both compound classes,
the chemical nature of the group blocking the terminal amine (R3) and the R2 substituent
affected the biological potency. For position R3, the replacement of the acetyl group by a t-
butyl carbamate increased in more than 60 times the potency of 16 (EC50 12 nM) with respect
to 15 (EC50 827 nM), and at least twice for 19 (48 % viability at 5 µM) with respect to 18
(113 % viability at 5 µM). In contrast, the addition of an isopropyl group at 5 position of the
thiazoles proved slightly detrimental for the anti-trypanosomal activity as evidenced by the
lower activity of 16 (EC50 12 nM) vs. 17 (EC50 7 nM), and for 19 (48 % viability at 5 µM) vs.
20 (EC50 3 µM). This suggests that addition of two isopropyl groups to the tri-heterocycles
may interfere with the recognition of the molecular target. Interestingly, the replacement of
the central azole ring of 1 (EC50 310 nM) [19] by an N-methyl pyrrole increased significantly
(26- to 44-fold) the anti-trypanosomal activity of the tri-heterocycles bearing a carbamated
terminal amine and either propylated (16) or non-propylated (17) thiazoles.
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Worth noting, 18 was previously synthesized and described as a potent growth inhibitor of
bloodstream T. b. brucei (strain 427) with a minimum inhibitory concentration (defined as the
concentration of compound that inhibited more than 20% parasite growth compared to non-
treated control) of 64 nM [15]. However, under our experimental conditions (see section
4.6.1 ), the compound lacked activity against the same strain and stage of T. b. brucei. In
order to determine whether the different assay conditions were responsible for this conflicting
data, 18 was assayed under conditions (initial density of 1.2×104 parasites/mL and 48 h
treatment) resembling those reported in [15] (i.e. 2×104 parasites/mL and 48 h treatment) and
including nifurtimox and 1 as positive controls. Under this less stringent assay condition and
tested up to 60 µM, 18 resulted fully inactive whereas nifurtimox and 1 increased their EC50
by 20- and 5-fold (EC50 = 0.75 and 0.057 µM, respectively) when compared to the values
obtained in our standard assay (5×105 parasites/mL and 24 h treatment, Table 1). Our finding
is further supported by the fact that heterocycles with esterified azoles (15-17) are more
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potent than the corresponding carboxylic acid analogues (18-20, Table 1). Thus, the data
previously reported for 18 [15] remains intriguing.
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Among the compounds displaying the most potent anti-trypanosomal activity (15-19 and 20),
16 presented an EC50 ~40 µM against murine macrophages, whereas the other ones lacked
toxicity at a concentration of 120 µM [note that higher concentrations could not be tested due
to low compound´ solubility at 1% (v/v) DMSO]. 16 and 17 displayed the highest selectivity
indexes (SI): > 10169 and 5560, respectively. Similar to the effect observed against T. brucei
and compared to the non-propylated analog 17 (EC50 for macrophages > 120 µM),
propylation of the thiazoles of 16 (EC50 for macrophages ~39.5 µM) reduced by at least 3-
fold macrophage toxicity. The replacement of the central thiazole ring by an N-methyl
pyrrole impacted also positively in the toxicity against host cells, reducing it by 8-fold (i.e.
EC50 against macrophages for 1 vs. 17 is 5 µM vs. ~40 µM, respectively).
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In summary, the incorporation of a central N-methyl pyrrole to bis-thiazoles yielded
derivatives significantly less cytotoxic and with higher anti-trypanosomal activity than the
original tri-thiazoles analogs.
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2.3. Mechanism of action
2.3.1. Cell membrane integrity
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The remarkable improvement in the biological activity of several compounds from the new
series with respect to 1 [19] raised the question whether this stem from an enhanced
compound uptake, affinity for the molecular target and/or from a change in the mechanism of
action (i.e. molecular target). To address this point, 16 and 17 were subjected to a thorough
characterization of their trypanosomal killing mechanism.
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Compound 1 has been shown to kill trypanosomes by disrupting the integrity of the
lysosomal membrane, which is followed by release of toxic proteases, iron, generation of
oxidative stress and permeabilization of plasma membrane [19]. Thus, we first investigated
whether 16 and 17 would induce a similar phenotype in infective T. brucei. Both compounds
added at their corresponding EC50 did not alter the integrity of the plasma membrane after a 4
h incubation, as observed by the lack of incorporation of the exclusion dye propidium iodide
by the parasites (PI; Figure 2A). In contrast, a very low concentration of the detergent Triton
X-100 (0.001 % v/v) damaged the membrane of 90% of the cells, an effect comparable to that
exerted by 1 added at its EC50 and assayed under identical conditions [19]. Next, the capacity
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of the compounds to induce oxidative stress at intracellular level was studied using a redox-
reporter cell line of bloodstream T. b. brucei. The redox biosensor responds rapidly and
specifically to changes in the ratio of reduced/oxidized glutathione and trypanothione (bis-
glutathionylspermidine) [19,28,29], which are the major low molecular weight thiols in
trypanosomes [30]. The level of oxidation of the redox biosensor was not significantly
different for parasites treated with the vehicle or exposed to 16 or 17 at 1X or 5X their EC50
for 4 h (Figure 2B). This contrast with the behavior of 1, which at concentrations ≤ EC50
induced intracellular oxidation [19], and strongly suggests that 16 and 17 possess a different
mode of action.
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2.3.2. Cell cycle analysis
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As discussed above, several distamycin and netropsin analogs were described as MGB that,
similarly to the natural products, bind to AT-rich regions of nucleic acids and thereby inhibit
cell cycle progression [8]. On this basis and given the potent parasitostatic effect exerted by
the new compounds, a potential interference with parasite cell cycle was studied. Before
treatment with the compound, cell cycle synchronization was achieved by incubating the
parasites with hydroxyurea for 6 h, which corresponds to the doubling time for non-treated
infective T. b. brucei.
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For this assay, we choose 17 as model compound of the new series and DAC, a well-known
MGB, as control drug. After 24 h incubation, the distribution of parasites within the major
phases of the cell cycle was estimated by measuring the DNA-content by flow cytometry
(Fig. 3). For the DMSO-treated culture, cells were distributed in the following
subpopulations: 49% in the growth phase (G0/G1), 31% in the synthesis phase (S) and 20% in
the division/mitosis phase (G2/M). Tested at its EC50 (7 nM), 17 produced a minor but
statistically significant perturbation of the parasite´s cell cycle (i.e. G0/G1 phase: 44% cells,
S-phase: 34% cells, and G2/M-phase: 22%). Interestingly, doubling the concentration of 17
led to a more remarkable change with 94% of the trypanosomes arrested at the G2/M phase
and the remaining 6% at the G0/G1 phase. An almost identical effect was caused by DAC,
which added at its EC50 (41 nM) blocked cell cycle at the division/mitosis (G2/M-phase: 93%
cells and G0/G1-phase: 7% cells).
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2.3.3. DNA-content analysis by fluorescence microscopy
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Kinetoplastid organisms are characterized for having a densely packed mitochondrial DNA
(kinetoplast, K or kDNA) rich in AT-regions, and whose replication precedes that of the
nucleus (N) and is perfectly synchronized with the cell cycle [31-33]. For instance, cells in
the G1 or S phase of the cell cycle possess a single kinetoplast and a single nucleus (1K+1N).
As the cell cycle progresses, the kinetoplast replicates and divides before the nucleus to
produce a 2K+1N cell. Cells then enter mitosis, the outcome of which is a 2K+2N cell that,
upon cytokinesis, divides into two 1K+1N siblings. To get a deeper insight into how 16 and
17 may interfere with this process, the nuclear and mitochondrial DNA organization was
analyzed by epifluorescence microscopy (Figure 4). For DMSO-treated parasites, 80% of the
cell population displayed 1K+1N (Figure 4A, B), which fully agrees with the results
obtained for T. brucei grown under standard conditions [34] and confirms that the vehicle
does not affect DNA replication. DAC treatment led to a major decrease in the 1K+1N cell
population (44%, Figure 4A) and to the appearance of a large percentage of dyskinetoplastic
trypanosomes (0K+1N: 39%, Figure 4C). Such phenotype has been reported for
trypanosomes treated with different DNA-binding drugs, DAC among them, and ascribed to
the linearization and protease-mediated degradation of the minicircle DNA, probably due to
the inhibition of a mitochondrial type II topoisomerase [35-37]. For trypanosomes treated
with 16 and 17, the major population was also represented by 1K+1N cells (48% and 46%,
respectively; Figure 4A). However, at variance with DAC, the second largest population
(38% and 26% for 16 and 17, respectively) corresponded to cells bearing a single kinetoplast
and a single, but with a septum-like or partitioned, nucleus, a phenotype that we termed:
1K+1N* (Figure 4A, D). Thus, the distamycin analogues 16 and 17 appear to exert their
trypanostatic effect by inhibiting the kDNA replication but without altering its structural
integrity, as DAC and other MGBs do [35-37].
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2.3.4. DNA-binding competition assays
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The kDNA consists of concatenated maxi and minicircles encoding for rRNA and
mitochondrial proteins, and for small guide RNAs, respectively [37, 38]. The minicircles are
required for the editing of mRNA, are rich in A-T bases and, therefore, preferred targets of
MGB [36-38]. As shown above, 16 and 17 interfere with the replication of the kDNA but do
not lead to its degradation as MGBs do (Fig. 4). This suggests that, if any, the interaction of
the distamycin analogues with the target DNA differs from that of the MGB. To test this,
fluorescence competition assays were performed incubating a DNA-vector lacking (pUC18, 5
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nM) or harboring a T. brucei minicircle sequence [38] (pUC18-kDNA, 5 nM) with an excess
DAC, 16 or 17 (20 µM) for 30 or 120 min, followed by DNA-staining with DAPI (4',6-
diamidino-2-phenylindole, 0.5 µM), a well-known fluorescent MGB with high affinity for A-
T rich DNA [39]. DNA incubated with the vehicle DMSO and stained with DAPI was used
as fluorescence normalization control.
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The low fluorescence intensity of the DAC-treated samples (25-22 %) indicates that DAPI
could not displace DAC-binding to either the plasmid and the minicircle DNA, even if
incubation was extended from 30 min (Fig. 5A) to 120 min (Fig. 5B). This is in agreement
with previous reports for MGB competitors [40,41] and confirms that DAC occupies DAPI
sites in the DNA. At time point 30 min, 17 displayed certain preference for competing with
DAPI-binding to the mock vector instead of the DNA containing the minicircle sequence
(Fig. 5A). At this time point, 16 increased (15-28%) DAPI signal from both DNA samples
when compared to the vehicle control. Interestingly, extending the incubation to 120 min, 16
and 17 diminished by 30% and 48%, respectively, the fluorescence of the DAPI-
pUC18kDNA complex but not that of the mock DNA (Fig. 5B). This suggests that the
interaction of 16 and 17 with the kDNA is a dynamic process.
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Overall, the results supports that the interaction of 16 or 17 with the target DNA clearly
differs from the MGB drug DAC. At shorter time points, 16 and 17 appear to have an overall
relaxing effect on the DNA that favors dye incorporation to MG regions, whereas at longer
incubation times both compounds are able to partially compete and/or allosterically affect
dye-binding to DNA. Further experiments should be performed to confirm this preliminary
finding.
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2.3.5. Therapeutic efficacy test
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Supported by the impressive performance of the compounds in vitro, a preliminary
therapeutic efficacy study on a mouse model of African trypanosomiasis was conducted with
17. Because the compound displayed a trypanostatic effect in vitro, the in vivo treatment was
extended for one-week. Balb/cJ mice susceptible to T. b. brucei infection were infected with
the parasite strain AnTaT 1.1, a pleomorphic cell line able to reproduce the natural infection
[42]. Infected mice were divided in 4 groups (n = 6/group) that at day 4th post-infection were
treated with 17 given daily for 7 days at 1000X (8.36 mg/Kg) or 250X (2.09 mg/Kg) the EC50
determined in vitro, DMSO (3.3 mg/Kg × day/7 days) or a single dose of DAC (40 mg/Kg).
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Parasitemia and animal status was monitored on a regular basis. For mice treated with vehicle
(DMSO), parasitemia showed the typical behavior with a peak (mean value = ~100 million
parasites/mL blood) at day 4th followed by a 2 to 3 orders of magnitude decrease between day
7-11 that can be associated to a temporary control of parasite proliferation by the host innate
immune response [43]. From day 11th on, parasitemia raised steadily until the end of the
assay (day 21st, Fig. 6). For mice treated with 17, and independently of the dose administered,
the parasitemia´ trend and absolute values were almost identical to those observed for the
DMSO-treated group. This clearly indicates that 17, even if given at 250-1000X its in vitro
EC50, was unable to control T. b. brucei proliferation in vivo. In contrast, from day 7
onwards, a single dose of the control drug DAC was sufficient to reduce parasites from blood
at levels undetectable by our counting method (detection limit ≥ 2.5×104 parasites/mL). Due
to the lack of therapeutic efficacy of 17 and the high parasitemia levels, the experiment was
finished at day 21st. For this time-window, and despite early (day 4th) deceases, each
occurring in the DMSO and the 250X group, no differences in animal survival rate were
observed among groups (Log-rank test, not shown).
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The lack of correlation between in vitro vs. in vivo potency of 17 may be ascribed to different
factors that likely have affected the bioavailability of the compound´s active form. Among
them, metabolization by host´s hydrolases or oxidases may have converted 17 into a non- or
significantly less-active species. In this respect, it is important to note that the tri-
heterocyclics prepared in this work harbor two amide bonds, which may be susceptible to
cleavage by liver esterases and amidases [44].
337
338
339
340
341
342
343
344
345
346
347
Among all these factors, we investigated in vitro a potential inactivation of 17 by serum
components given that, although minor, a fraction of liver esterases is present in serum
[45,46]. The compound (100 µM) was incubated for 24 h at 37 ºC with fresh or heat-
inactivated (54°C for 45 min) fetal bovine serum and further added at different concentrations
to T. brucei cultures. As shown in Table 2, in the absence of compound, either fresh or heat-
inactivated serum did not impair parasite viability. At 10 nM, value close to compound´s
EC50, the anti-trypanosomal activity of 17 was fully abrogated or reduced by 30% when the
compound was pre-incubated with heat-inactivated or fresh serum, respectively (p <0.01).
Both serum samples were still capable to lower the activity of a 100-fold excess (1 µM) 17.
This result shows that serum component(s) may contribute to reduce the fraction of free or
active compound.
348
11

349
3. Concluding remarks
350
351
352
353
354
355
356
357
358
359
A new series of distamycin analogues that are structure- and chemically-related to a scaffold
from which several derivatives were identified as potent inhibitors of T. brucei proliferation
[5-8] have been synthesized in good yields using classic organic chemistry reactions. Two tri-
heterocyclic derivatives (16 and 17) displayed low nM potency against bloodstream African
trypanosomes and high selectivity. In agreement with previous conclusions [9,19], our SAR
analysis revealed that increased compound lipophilicity, due to the addition of a third
heterocycle and blocking of the terminal amine and carboxylic acid with a carbamate and an
ester group, respectively, are important determinants of compound activity. In fact, 16 (LogP
= 3.7) and 17 (LogP = 2.4) have similar EC50 towards trypanosomes despite the first has two
isopropyl groups and an increased LogP [47].
360
361
362
363
364
365
366
367
368
369
370
Our data highlights how minor changes in the structure of the distamycin scaffold may have a
significant impact in the mode of action of related derivatives. For instance, the tri-thiazol 1
commits parasites to death by disrupting the integrity of the lysosome [19]. Here we show
that replacement of the former central thiazol ring of 1 by an N-methyl pyrrole proved key in
conferring the new analogues with a potent trypanostatic activity that involves the inhibition
of DNA replication. In contrast to MGB drugs [35-37], 16 and 17 did not affect the structure
of the kinetoplast but induced a partitioning of the nuclear DNA, which supports the blockade
of DNA replication at a specific step. Interestingly, also in vitro 16 and 17 displayed low
affinity for different template DNAs. Compared to the MGB-drug DAC, the compounds
added in a 40-fold excess with respect to DAPI, only to a minor extent displaced dye-binding
to a bacterial plasmid and T. brucei mitochondrial minicircle DNA.
371
372
373
374
375
376
Thus, the mode of action of 16 and 17 against trypanosomes clearly differ from that described
for classical MGBs. The molecular targets of these compounds remain unknown. However,
given the selectivity of 16 and 17, the uniqueness of the trypanosomal mitochondrial DNA
sequences and that kDNA replication is a major cell cycle checkpoint in these parasites, it is
tempting to propose that components of the mitochondrial DNA replication machinery might
be their potential molecular targets.
377
378
379
380
Despite 17 showed no activity in a mouse infection model of African trypanosomiasis,
probably as consequence of its efficient and rapid inactivation by host’s detoxication systems,
the compounds characterized in this work represent excellent starting points for designing
highly potent and selective anti-T. brucei agents.
12

381
382
383
4. Experimental
4.1. Chemical methods
1
13
384
385
386
387
388
IR spectra were recorded on a Shimadzu FTIR 8101A spectrophotometer. H NMR and C
NMR spectra were recorded on a Bruker Avance DPX- 400 (see Supplementary Material).
Chemical shifts are related to TMS as an internal standard. High resolution mass spectra
(HRMS) were obtained on a MicroQ-TOF (ESI,Bruker Daltonics), low resolution mass
spectra were obtained using a GCMS Shimadzu QP 1100-EX
389
390
391
392
393
394
395
Melting points were measured using a Fisher-Johns Melting Point Apparatus. Flash column
chromatography was carried out with Silica gel 60 (J.T. Baker, 40 µm average particle
diameter). All reactions and chromatographic separations were monitored by TLC, conducted
on 0.25 mm Silica gel plastic sheets (Macherey/Nagel, Polygram SIL G/UV 254). TLC plates
were analyzed under 254 nm UV light, iodine vapor, p-hydroxybenzaldehyde spray or
ninhydrine spray. Yields are reported for chromatographically and spectroscopically (1H and
13C NMR) pure compounds.
396
397
All solvents were purified according to literature procedures [48]. All reactions were carried
out in dry, freshly distilled solvents under anhydrous conditions unless otherwise stated.
398
4.2. General procedure for ester hydrolysis
399
400
401
402
403
An aqueous solution of LiOH or KOH [10% (w/v)] is added to a solution of the ester
dissolved in THF or MeOH on a 1:1 ratio, the reaction is stirred at room temperature until
TLC confirmed the disappearance of the ester. HCl [5% (v/v)] is added up to pH 2, the
solution is extracted with EtOAc, dried over Na2SO4 and concentrated in vacuo to afford the
corresponding acid.
404
405
406
407
408
409
410
4.3. General procedure for amide bond synthesis
HBTU (1.2 eq.), DIPEA (2 eq.) and 4-DMAP (0.2 eq.) are added under a N2 atmosphere to a
solution containing the amine (1.2 eq.) and the acid (1.0 eq.) on dry CH2Cl2 DCM at 0 °C.
The mixture is stirred for 24-72 h at room temperature. AcOEt is added to the crude and the
solution is washed with HCl [5% (v/v)] and then NaHCO3 (sat), the organic layer is dried
over Na2SO4, filtered and evaporated in vacuo. The crude is purified by flash
chromatography using the corresponding eluent to give the amide.
411
4.4. General procedure for nitro reduction
13

412
413
414
415
The nitro compound is dissolved in EtOH and Pd/C (10 %) is added to the mixture, the
reaction is carried at room temperature for 3 h under an H2 atmosphere (3 atm). The crude is
filtered through celite, concentrated in vacuo, redissolved in dry DCM and used without
purification.
416
417
418
419
420
421
422
423
4.5. Building blocks
4.5.1. 2,2,2-trifluoro-1-(1-methyl-1H-pyrrol-2-yl)ethan-1-one (2)
N-methyl pyrrole (5.4 g, 67 mmol) is dissolved in 18 mL of dry Et2O under a N2 atmosphere,
the reaction is then cooled to 0 ºC on an ice bath. TFAA (9.4 mL, 67 mmol) is added slowly
and the reaction is stirred for 6 h at 0 ºC. The reaction is washed with NaHCO3 [10% (w/v); 3
x 40 mL] and brine (2 x 10 mL). The organic layer is dried over Na2SO4, filtered and
1
concentrated in vacuo. Yellow oil. Y = 98 %. H-NMR (CDCl3, 400 MHz): δ 3.98 (s, 3H),
6.26 (dd, J = 4.6 , 2.3 Hz, 1H), 7.04 (m, 1H), 7.22 (dd, J = 4.6, 2.3 Hz, 1H).
424
425
426
427
428
429
430
4.5.2. 2,2,2-trifluoro-1-(1-methyl-4-nitro-1H-pyrrol-2-yl)ethan-1-one (3)
Once compound 2 (11 g, 67 mmol) is dissolved on acetic anhydride, HNO3 is added dropwise
(5.6 mL, 0.134 mmol) keeping the reaction at - 5 ºC. After addition, the reaction is stirred for
3 h at 10 ºC. CHCl3 is added and the mixture is extracted exhaustively with NaHCO3 [10%
(w/v)], then the organic layer is dried over Na2SO4, filtered and concentrated in vacuo.
1
Orange oil. R = 80 %. H -NMR (CDCl3, 400 MHz): δ 4.06 (s, 3H), 7.69 (s, 1H), 7.82 (s,
1H).
431
4.5.3. 1-methyl-4-nitro-1H-pyrrole-2-carboxylic acid (4)
432
433
434
435
436
437
Compound 3 (1 g, 4.5 mmol) dissolved in 20 mL of DMF:H2O (9:1) and added dropwise to a
solution containing NaH (0.43 g, 18 mmol) in 5 mL of dry DMF and then heated at 60 ºC for
3 h. HCl [5% (v/v)] is then added to the solution and the pH is adjusted at a value of 2, the
1
aqueous layer is extracted with Et2O. White solid. R = 86 %. M.p.: 204-205 °C [49]. H-
NMR (CDCl3, 400 MHz): δ 4.19 (s, 3H), 7.26 (d, J =2.0 Hz, 1H,), 8.23 (d, J =2.0 Hz, 1H),
13.16 (s, 1H). 13C-NMR (CDCl3, 100 MHz): δ 37.9, 111.8, 124.3, 129.6, 134.5, 161.5.
438
4.5.4. Ethyl 2-aminothiazole-4-carboxylate (5)
439
440
441
442
Compound 5 is prepared according to the literature [15]. White solid. Y = 80 %. M.p.: 171-
174 °C [50]. 1H-NMR (400 MHz, (CD6)2CO): δ 1.31 (t, J = 7.2 Hz, 3H), 4.25 (q, J = 7.2 Hz,
13
2H), 6.82 (s, 2H); 7.44 (s, 1H). C-NMR (100 MHZ, (CD6)2CO): 14.6, 60.9, 117.5, 144.1,
161.9, 169.2.
14

443
4.5.5. Methyl 2-amino-5-isopropylthiazole-4-carboxylate (6)
1
444
445
446
Compound 6 is prepared according to the literature [18,19]. Yellow oil. Y = 50 %. H-NMR
(400 MHz, CDCl3): δ 1.27 (d, J = 7.0 Hz, 6H); 3.88 (s, 3H); 4.07 (m, 1H), 5.18 (s, 2H). 13C-
NMR (100 MHz, CDCl3): δ 24.8, 27.6, 51.9, 134.6, 150.6, 162.7, 163.2.
447
4.5.6. Methyl 2-((tert-butoxycarbonyl)amino)-5-isopropylthiazole-4-carboxylate (7)
448
449
450
451
452
453
454
455
456
Compound 6 2.5 g (12.5 mmol) is dissolved in 40 mL of dry CH2Cl2 under a N2 atmosphere.
TEA (5.3,37 mmol), Boc2O (8.175 g, 37 mmol) and 4-DMAP (cat) are added to the mixture.
The reaction is refluxed for 6 h, and then 200 mL of EtOAc are added, the reaction is washed
with HCl [5% (v/v); 3 x 15 mL] and H2O (50 mL). The organic layer is dried over, filtered
and concentrated in vacuo. The crude is purified by flash chromatography using
EtOAc:Hexanes (1:4) as eluent. White solid. Rf = 0.4. Y = quantitative. M.p.: 140-141 °C.
1H-NMR (CDCl3, 400 MHz): δ 1.34 (d, J = 7.9 Hz, 6H), 1.50 (s, 9H), 3.90 (s, 3H), 4.16 (m,
1H). 13C NMR (CDCl3, 100 MHz): δ 24.9, 27.7, 28.0, 52.0, 84.7, 135.6, 149.7, 154.3, 159.1,
162.6.
457
4.5.7. Ethyl 2-((tert-butoxycarbonyl)amino)thiazole-4-carboxylate (8)
458
459
460
461
462
463
464
465
466
Compound 5 (0.439 g, 2.5 mmol) is dissolved in 10 mL in DCM:THF (1:1) under a N2
atmosphere. (Boc)2O (0.577 g, 2.65 mmol), TEA (0.744 g, 7.36 mmol) and 4-DMAP (cat)
were added and the reaction was refluxed for 72 h, then the solvent was removed in vacuo
and redissolved in EtOAc, washed with HCl [5% (v/v); 3 x 15 mL], dried over Na2SO4,
filtered and concentrated in vacuo. The residue was purified using flash chromatography
using EtOAc:Hexanes (3:7) as eluent. White solid. Rf = 0.38. R = 74 %. M.p.: 105-106 °C.
1H-NMR (400 MHz, (Acetone-d6): δ 1.33 (t, J = 7.2 Hz, 3H), 1.54 (s, 9H), 4.30 (q, J = 7.2
13
Hz, 2H), 7.92 (s, 1H), 10.37 (s, 1H). C-NMR (100 MHz, (Acetone-d6): δ 14.6, 28.2, 61.1,
82.2, 122.4, 142.8, 153.1, 160.4, 161.
467
4.5.8. 2-((tert-butoxycarbonyl)amino)-5-isopropylthiazole-4-carboxylic acid (9)
Compound 9 was obtained from 7 using the ester hydrolysis general procedure. White solid.
468
469
470
471
1
Y = 100 %. M.p.: 132-133 °C. H-NMR (DMSO-d6, 400 MHz) δ 1.25 (d, J = 7.2 Hz, 6H),
13
1.46 (s, 9H), 3.99 (m, 1H), 11.54 (s, 1H), 12.62 (bp, 1H). C-NMR (DMSO-d6, 100 MHz)
25.1, 27.2, 28.3, 81.8, 135.5, 150.0, 153.6, 155.4, 163.9.
472
4.5.9. 2-((tert-butoxycarbonyl)amino)thiazole-4-carboxylic acid (10)
15

473
474
475
476
Compound 10 was obtained from 8 using the ester hydrolysis general procedure. White solid.
1
Y= 100 %. M.p.: 112-113 °C. H-NMR (400 MHz, DMSO-d6): δ 1.48 (s, 9H), 7.92 (s, 1H),
11.71 (s, 1H), 12.73 (s, 1H).13C-NMR (100 MHz, DMSO-d6): δ 27.9, 81.6, 121.9, 142.4,
153.1, 159.6, 162.4.
477
4.5.10. Methyl 2-acetamido-5-isopropylthiazole-4-carboxylate (11)
478
479
480
481
482
483
484
485
486
Compound 9 (118 mg, 0.588 mmol) is dissolved under a N2 atmosphere in dry CH2Cl2 (4
mL) on an ice bath. Pyridine and Ac2O in excess and 4-DMAP (cat) are added to the mixture.
The reaction is then refluxed for 6 h and then EtOAc is added, the reaction is washed with
HCl [5% (v/v); 2 x 10 mL], and H2O (15 mL). The organic layer is dried over Na2SO4,
filtered and concentrated in vacuo. The crude is purified in silica flash chromatography using
1
EtOAc:Hexanes (4:1) as eluent. White solid. Rf = 0.48. Y = 83 %. M.p.: 97 ºC. H-NMR
(CDCl3, 400 MHz): δ 1.34 (d, J = 6.9 Hz, 6H), 2.26 (s, 3H), 3.90 (s, 3H), 4.07 (m, 1H), 11.65
13
(s, 1H). C-NMR (CDCl3, 100 MHz): δ 22.9, 24.7, 27.6, 51.9, 132.6, 151.9, 155.9, 162.3,
168.8. IE-MS (70 eV): 242 (M+, 35), 140 (C6H8N2S•, 100).
487
4.5.11. 2-acetamido-5-isopropylthiazole-4-carboxylic acid (12)
488
489
490
491
492
Compound 12 is obtained from 11 using the ester hydrolysis general procedure. White solid.
Y= 100 %. M.p: 104 ºC decomp .1H-NMR (DMSO-d6, 400 MHz) δ 1.24 (d, J = 6.8 Hz, 6H),
13
2.09 (s, 3H), 3.99 (dt, J = 6.8 Hz, 1H), 12.25 (s, 1H), 12.74 (bp, 1H). C NMR (DMSO-d6,
100 MHz): δ 22.8, 25.2, 27.3, 135.1, 150.1, 153.8, 164.1, 169.1. IE-MS (70 eV): 228 (M+,
20), 168 (C7H8N2OS•, 56), 140 (C6H8N2S•, 100).
493
4.5.12. Ethyl 2-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)thiazole-4-carboxylate (13)
494
495
496
497
498
499
Compound 13 was obtained from 4 and 5 using the general procedure for amide bond
formation. The crude is purified using EtOAc:Hexanes (3:2) as eluent. White solid. Rf = 0.41.
1
R = 62 %. M.p.: 215-217 °C. H-NMR (DMSO-d6, 400 MHz): δ 1.30 (t, J = 6.9 Hz, 3H),
3.99 (s, 3H), 4.29 (q, J = 6.9 Hz, 2H), 8.02 (d, J = 1.9 Hz, 1H), 8.11 (s, 1H), 8.30 (d, J = 1.9
Hz, 1H), 12.97 (s, 1H). 13C-NMR (CDCl3, 100 MHz): δ 14.7, 38.5, 61.1, 111.1, 123.6, 124.1,
130.4, 134.6, 141.5, 158.5, 158.8, 161.5.
500
501
4.5.13.
Methyl
5-isopropyl-2-(1-methyl-4-nitro-1H-pyrrole-2-carboxamido)thiazole-4-
carboxylate (14)
502
503
Compound 14 was obtained from 4 and 6 using the general procedure for amide bond
formation, the crude was purified using EtOAc:CHCl3 (1:4) as eluent. White solid. Rf = 0.51.
16

1
504
505
506
507
R = 87 %. M.p.: 118-120 °C. H-NMR (CDCl3, 400 MHz): δ 1.38 (d, J = 6.6 Hz, 6H), 3.72
13
(s, 3H), 4.10 (m, 4H), 7.11 (d, J = 1.9 Hz, 1H), 7.65 (d, J = 1.9 Hz, 1H), 11.13 (s, 1H). C-
NMR (CDCl3, 100 MHz): δ 24.8, 27.8, 38.2, 51.8, 109.6, 124.0, 127.9, 133.7, 135.2, 153.6,
154.6, 158.3, 162.2.
508
509
4.5.14. Methyl 2-(4-(2-acetamido-5-isopropylthiazole-4-carboxamido)-1-methyl-1H-pyrrole-
2-carboxamido)-5-isopropylthiazole-4-carboxylate (15) [11]
510
511
512
513
514
515
516
Compound 13 was reduced using the general procedure for nitro group reduction, and then
coupled with 12 using the general procedure for amide bond formation. The reaction is
purified using EtOAc as eluent. White solid. Rf = 0.4. Y = 30 %. M.p.: 170-171 °C. 1H-NMR
(CDCl3, 400 MHz): δ 0.99 (d, J = 7.1 Hz, 6H), 1.38 (d, J = 7.1 Hz, 6H), 1.85 (s, 3H), 3.91
(s, 3H), 4.04 (s, 3H), 4.14 (dt, J= 7.1 Hz, 1H), 4.96 (m, 1H), 6.56 (d, J = 1.48 Hz, 1H), 6.71
(d, J = 1.48 Hz, 1H), 9.52 (s, 1H). 13C-NMR (CDCl3, 100 MHz): δ 20.8, 23.2, 24.8, 27.7,
37.4, 44.8, 52.1, 114.3, 122.0, 122.4, 128.7, 133.5, 153.1, 153.8, 158.1, 162.7, 171.1.
517
518
4.5.15. Methyl 2-(4-(2-((tert-butoxycarbonyl)amino)-5-isopropylthiazole-4-carboxamido)-1-
methyl-1H-pyrrole-2-carboxamido)-5-isopropylthiazole-4-carboxylate (16)
519
520
521
522
523
524
525
526
527
Compound 14 was reduced using the general procedure for nitro group reduction, and then
coupled with 9 using the general procedure for amide bond formation. The reaction is
purified using EtOAc:Hexanes (6:4) as eluent. White solid. M.p.: 263 ºC decomp. Y = 40 %.
1H-NMR (CDCl3, 400 MHz): δ 1.34 (m, 12H), 1.56 (s, 9H), 3.90 (s, 3H), 3.97 (s, 3H), 4.12
(dt, J = 6.8 Hz, 1H), 4.37 ( dt, J = 6.8 Hz, 1H), 6.63 (s, 1H), 7.62 (s, 1H), 8.40 (s, 1H), 9.02
(s, 1H), 9.56 (s, 1H). 13C-NMR (DMSO-d6, 100 MHz): δ 24.8, 24.9, 27.1, 27.6, 28.2, 37.1,
52.0, 83.1, 104.7, 120.3, 122.1, 122.2, 133.4, 135.3, 148.9, 152.2, 152.8, 154.1, 154.6, 158.3,
159.8, 162.8. EI-MS (20 eV): 590 (M+, 0.1), 490 (C21H26N6O4S2, 100), 322 (C14H18N4O3S,
43), 291 (C13H15N4O2S•, 80). IR KBr ν (cm-1): 3351, 3124, 2915, 1765, 1491, 1101.
528
529
4.5.16. Ethyl 2-(4-(2-((tert-butoxycarbonyl)amino)thiazole-4-carboxamido)-1-methyl-1H-
pyrrole-2-carboxamido)thiazole-4-carboxylate (17) [51]
530
531
532
533
534
Compound 17 was reduced using the general procedure for nitro group reduction, and then
coupled with 6 using the general procedure for amide bond formation. The reaction is
purified using CHCl3:EtOAc (1:2). Rf = 0.51. White solid. R = 35 %. M.p.: 142 ºC. 1H-NMR
(CDCl3, 400 MHz): δ 1.34 (t, J = 7.1 Hz, 3H), 1.55 (s, 9H), 3.95 (s, 3H), 4.35 (q, J = 7.1 Hz,
1H), 6.88 (s, 2H), 7.60 (d, 1H, J = 1.97 Hz), 7.79 (s, 1H), 7.80 (s, 1H), 8.72 (s, 1H), 8.90 (s,
17

535
536
1H), 9.78 (s, 1H). 13C-NMR (CDCl3, 100 MHz): δ 14.2, 28.1, 37.2, 61.4, 104.6, 117.9,
120.0, 120.3, 121.9, 122.3, 141.5, 158.1, 158.3, 158.4, 159.4, 161.1, 161.5.
537
538
4.5.17.
2-(4-(2-acetamido-5-isopropylthiazole-4-carboxamido)-1-methyl-1H-pyrrole-2-
carboxamido)-5-isopropylthiazole-4-carboxylic acid (18)
539
540
541
542
543
544
545
546
547
Compound 18 was obtained from 15 using the general procedure for ester hydrolysis. White
solid. Y = 100 %. M.p.: 122 ºC decomp. 1H-NMR (CDCl3, 400 MHz): δ 1.04 (d, J = 7.1 Hz,
6H), 1.40 (d, J = 7.1 Hz, 6H), 1.92 (s, 3H), 4.10 (s, 3H), 4.25 (dt, J = 7.1 Hz, 1H), 4.98 (dt, J
= 7.1 Hz, 1H), 6.74 (d, J = 1.4 Hz, 1H), 7.39 (d, J = 1.4 Hz, 1H), 12.52 (s, 1H), 15.32 (s, 1H).
13C-NMR (CDCl3, 100 MHz): δ 20.9, 23.4, 24.4, 27.7, 37.9, 44.8, 116.2, 122.0, 122.4, 128.8,
1
132.6, 153.1, 156.6, 159.1 166.3, 170.9. H-NMR (DMSO-d6, 400 MHz): δ 1.29 (m, 12H),
2.13 (s, 3H), 3.90 (s, 3H), 4.05 (m, 1H), 4.16 (m, 1H), 7.35 (d, J = 1.4 Hz, 1H), 7.56 (d, J =
1.4 Hz, 1H), 9.61 (s, 1H), 12.15 (s, 1H), 12.45 (bp, 2H). EI-MS (70 eV); 518 (M+,5), 474
•
(C20H22N6O4S2 ,15), 333 (C15H17N4O3S•, 42), 211 (C9H11N2O2S•, 100).
548
549
4.5.18. 2-(4-(2-((tert-butoxycarbonyl)amino)-5-isopropylthiazole-4-carboxamido)-1-methyl-
1H-pyrrole-2-carboxamido)-5-isopropylthiazole-4-carboxylic acid (19)
550
551
552
553
554
555
556
557
Compound 19 was obtained from 16 using the general procedure for ester hydrolysis. White
solid. Y = 100 %. M.p.: 254-256 ºC. 1H-NMR (DMSO-d6, 400 MHz): δ 1.27 (m, 12H), 1.48
(s, 9H), 3.88 (s, 3H), 4.02 (dt, J = 7.1 Hz, 1H), 4.15 (dt, J = 7.1 Hz, 1H), 7.32 (s, 1H), 7.55 (s,
1H), 9.49 (s, 1H), 11.49 (s, 1H), 12.31 (s, 1H), 12.71 (s, 1H). 13C-NMR (DMSO-d6, 100
MHz): δ 25.1, 25.2, 26.8, 27.2, 28.3, 37.1, 81.9, 107.5, 120.7, 121.8, 122.6, 136.6, 146.1,
150.0, 154.3, 154.9, 155.7, 159.6, 164.1. EI-MS (20 eV): 476 (C20H24N6O4S2, 14), 432
(C19H24N6O2S2, 62) 291 (C13H15N4O2S•, 100), 264 (C12H16N4OS, 45). IR KBr ν (cm-1):
2935,2831, 1677, 1607, 1451,946.
558
559
4.5.19. 2-(4-(2-((tert-butoxycarbonyl)amino)thiazole-4-carboxamido)-1-methyl-1H-pyrrole-
2-carboxamido)thiazole-4-carboxylic acid (20)
560
561
562
563
564
Compound 20 was obtained from 17 using the general procedure for ester hydrolysis. Pale
yellow solid. Y = 100 %. M.p.: 198 ºC. 1H-NMR (DMSO-d6, 400 MHz): δ 1.49 (s, 9H), 3.90
(s, 1H), 7.38 (d, J = 1.6 Hz, 1H), 7.54 (d, J = 1.6 Hz, 1H), 7.84(s, 1H), 7.96 (s, 1H), 9.71 (s,
13
1H), 11.67 (s, 1H), 12.55 (s, 1H), 12.83 (bp, 1H). C-NMR (DMSO-d6, 100 MHz): δ 28.3,
37.2, 79.6, 107.9, 117.7, 120.7, 122.3, 142.6, 145.1, 158.6, 158.8, 159.6, 160.1, 162.7. EI-MS
18

565
566
(20 eV): 492 (M+, 0.01), 349 (C15H17N4O4S•, 10), 249 (C10H9N4O2S•,100), 241
(C9H11N3O3S•, 5). IR KBr ν (cm-1) = 3148, 2971, 2932, 1663, 1491,1311.
567
568
569
4.6. Biological methods
4.6.1. Viability assays for T. b. brucei and murine macrophages
570
571
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573
574
575
576
577
578
579
580
581
582
583
584
585
586
587
The bloodstream form of T. b. brucei (monomorphic strain 427) expressing an ectopic copy
of the redox biosensor hGrx1-roGFP2 [29] (cell line 449_Grx-roGFP2) [19] was grown in
HMI-9 medium complemented with 10 % v/v Fetal Bovine Serum (FBS, GIBCO®), 10
U/mL penicillin, 10 µg/mL streptomycin, 0.2 µg/mL bleomycin and 5 µg/mL hygromycin.
Expression of the biosensor was induced by adding oxytetracycline (end concentration 1
µg/mL) to the culture medium. Cells were incubated aerobically in a humidified incubator
containing 5% CO2 at 37 ºC. Working solutions of the compounds were prepared at different
concentrations in 100% (v/v) DMSO and the concentration of vehicle in the assay never
exceeded 1% (v/v). The screening was performed at a fixed compound concentration of 5 µM
whereas for EC50 determinations the concentrations tested ranged from 0.0001 to 120 µM.
Controls included cells treated with DMSO 1% (v/v), 10 µM nifurtimox or 78 nM suramin.
Parasite viability was evaluated as described by Maiwald et al. [52]. Briefly, 200 µL of a cell
suspension containing 5×105 parasites/mL in exponential growth phase was seeded per well
in a 96-well culture plate, then 2 µL from each compound was added per well and the culture
plates were incubated at 37 ºC, 5% CO2 for 24 h. Next, 50 µL from each well was transferred
to a 96 U bottom well plate containing 100 µL of PBS 1X glucose [1% (w/v)], 2 µg/mL
propidum iodide (PI) and analyzed in an Accuri™C6 (BD) flow cytometer (laser/filter pairs:
λex/em = 488 nm / 540±85 nm). The data were analyzed with the Accuri C6 software (BD).
588
589
590
591
592
Murine macrophages (cell line J774, ATCC® TIB-67TM) were cultivated in DMEM medium
supplemented with FBS [10% (v/v)], 10 U/ mL penicillin and 10 µg/mL streptomycin, at 37
ºC, 5% CO2 in a humidified incubator. Cell viability was determined from triplicates of 6-
point concentrations of compounds, using the WST-1 reagent and the protocol described by
Demoro et al. [53].
593
594
595
For all assays, at least three experimental replicates were analyzed and cell viability was
calculated as follows: viability(%) = 100 x (number of cells for compound Y at concentration
X/ number of cells in the DMSO-treated control).
19

596
597
EC50 values were obtained from dose/response curves using a nonlinear fitting. The error is
expressed as S.D. (corresponding to σn-1).
598
4.6.2. Membrane permeability assay
599
600
601
602
603
Two million parasites/mL were incubated with 16 (12 nM) and 17 (7 nM), DMSO [1% (v/v),
negative control] and Triton X-100 [0.001% (v/v), positive control]. Culture samples were
taken at different time points, diluted with PBS 1X glucose [1% (w/v)], added of PI (final
concentration of 2 µg/mL) and analyzed by flow cytometry as described above. Results are
expressed as mean ± S.D. (n = 3).
604
4.6.3. Intracellular redox state assay
605
606
607
608
609
610
611
612
Two million parasites/mL were incubated with 16 (59 and 12 nM), 17 (36 and 7 nM) or
DMSO [1% (v/v)] for 4 h. For calibrating the biosensor, non-treated parasites were incubated
for 20 min with DTT (1 mM), menadione (250 µΜ) or diamide (250 µM). In order to exclude
dead cells from the analysis, PI was added at 2 µg/mL immediately prior to sample analysis
by flow cytometry (Accuri™C6, BD). The following laser/filter pairs: λex/em = 488 nm /
530±33 nm for hGrx-roGFP2 and λex/em = 488 nm / 613 ± 30 nm for PI were used. The data
were processed and analyzed with the Accuri C6 software. Results are expressed as mean ±
S.D. (n = 3).
613
4.6.4. Cell cycle analysis
614
615
616
617
618
619
620
621
622
623
624
625
Parasites in exponential growth phase were plated at a density of 5×105 cells/mL and
incubated for 6 h with hidroxyurea at 10 µg/mL. Next, cells were centrifuged (2000 g, 10 min
at room temperature), washed twice with PBS 1X (10 mL) and then seeded back on a 6-well
plate at a density of 5×105 parasites/mL. 16 (7 nM), 17 (14 nM), DAC (41 nM) or DMSO
[1% (v/v)] were immediately added and cultures incubated for 18 h and 24 h at 37°C / 5%
CO2. Cells were centrifuged and washed thrice with PBS 1X (1 mL). Then the pellet was
resuspended gently in EtOH [70% (v/v) in PBS 1X] and incubated overnight at 4º C. Finally,
parasites were centrifuged, washed twice with PBS 1X (1 mL), resuspended in PBS 1X
containing RNAse A (30 µg/mL, Fermentas) and incubated for 30 min at 37 ºC. PI was added
at 2 µg/mL to stain nucleic acids. The samples were analyzed by flow cytometry as described
above. Cell cycle analysis was performed using the ModFit LT software. Results are
expressed as mean ± S.D. (estimated as σn-1, n=2).
626
4.6.5. Epifluorescence microscopy
20

627
628
629
630
631
632
633
634
635
636
Parasites cultured as described before and treated with DMSO [1% (v/v)], DAC (41 nM), 16
(12 nM) or 17 (7 nM) for 24 h were centrifuged (2000 g, 10 min) and washed twice with PBS
1X (1 mL). The pellet was resuspended in paraformaldehyde [4% (v/v) in PBS 1X] to a cell
density of 4×104 parasites/µL and further incubated for 18 min at room temperature. After
washing thrice with PBS 1X (1 mL), parasites were resuspended in PBS 1X at a density of
4×104 cells/µL and smeared on a glass slide. The slides were incubated protected from light
for about 18 h at 4 ºC and then mounted using a FluoroshieldTM (Sigma-Aldrich) solution
containing DAPI. Samples were analyzed by epifluorescence microscopy using 60X and
100X objectives with an OLYMPUS IX81 microscope. At least 50 cells were analyzed for
each sample.
637
4.6.6. DNA-binding competition assays
638
639
640
641
642
643
644
645
646
647
648
649
Synthesis of kDNA and insertion into pUC18 plasmid (construct pUC18 kDNA) was ordered
to GENSCRIPT. pUC18 and pUC18 kDNA (ampicillin resistance) were produced and
isolated from Escherichia coli XL-1 using the PureLinkTM Maxiprep Extraction Kit
(Invitrogen). Purity and concentration were analyzed in a NanoDrop 1000 Spectrophotometer
(Thermo Scientific) at 260 nm and by agarose gel electrophoresis [1% (w/v)] in TAE 0.5 X
buffer at 135 mV, using λHindIII (Thermo Scientific) and a 1-Kb DNA-ladder (Invitrogen).
Plasmids were dissolved in PBS 1X buffer (pH = 7.4) at 5 nM and incubated for 0.5 and 2 h
with 20 µM DAC, 16, 17, or vehicle alone [DMSO 1% (v/v)]. Then DAPI (500 nM final
concentration) was added and the emission spectra was recorded in a Cary Spectrofluorimeter
(λex/em = 358 nm / 368-600 nm, using a 5 nm slit filter and the PMT at 700 mV). The
fluorescence of the test samples was normalized against the control sample containing DNA,
DMSO and DAPI, set at 100 %. Results are expressed as mean ± S.D. (σn-1) for n = 3.
650
4.6.7. Therapeutic efficacy study
651
652
653
654
655
656
657
658
Animal assays were carried employing procedures approved by the Animal Use and Ethic
Committee (CEUA) of the Institut Pasteur de Montevideo (Protocol 004-12), which are in
accordance with the Federation of European Laboratory Animal Science Association
(FELASA) guidelines and the National law for Laboratory Animal Experimentation (Law nr.
18.611). Six to eight weeks-old female BalB/cJ mice (n = 24) weighing about 20 g were
infected intraperitoneally with 104 parasites (bloodstream form of T. b. brucei strain AnTaT
1.1). At day 4th post-infection, mice were randomly divided in 4 groups (n = 6/group) and
administered intraperitoneally with 300 µL (prepared in 1 X PBS) of 17 at 1000X (8.36
21

659
660
661
662
663
mg/Kg × day × 7 days) or 250X (2.09 mg/Kg × day × 7 days), DMSO 3.3 mg/Kg × day ×7
days or a single dose of DAC at 40 mg/Kg. Parasitemia (determined by cell counting in a
light microscope of blood samples extracted from the submandibular vein) and animal health
status were controlled regularly as described earlier [54]. Animals displaying an impaired
health status or parasitemia higher than 109 parasites/mL were sacrificed for ethical reasons.
664
4.6.8. Inactivation of 17 by serum samples
665
666
667
668
669
A solution of 17 at 100 µM prepared in fresh or heat-inactivated (54 ºC for 45 min) serum
containing a maximal DMSO concentration of 1% (v/v) was incubated at 37 ºC for 24 h. The
biological activity of both samples of 17 against T. brucei (cell line 449-hGrx-roGFP2) was
tested at a final compound concentration of 10 and 1000 nM, and according to the method
described in section 4.6.1.
670
4.6.9. Statistical Analysis
671
672
673
All data here reported were plotted and analysed using the GraphPad Prism software (version
6.01). The statistical tests applied and the p values obtained are indicated in the respective
figures.
674
675
676
677
678
679
680
681
Acknowledgements
J.F. acknowledges the support of ANII – Uruguay (POS_NAC_2014_1_102739). The
technical support of staff from the Cell Biology Unit, Institut Pasteur de Montevideo, is
gratefully acknowledged. The support of CSIC Grupos (UdelaR) and PEDECIBA (Uruguay)
is gratefully acknowledged by L.S.. All funding sources were not involved in study design,
collection, analysis and interpretation of data; in the writing of the paper; and in the decision
to submit the article for publication.
682
683
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Figure legends
853
854
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856
Scheme 1 Building block synthesis i) TFAA, Et2O, 0 ºC, 6 h, 98 % ii) Ac2O, HNO3, 10 ºC, 3
h, 80 % iii) NaH, DMF:H2O, 60 ºC, 3 h, 86 % iv) (Boc)2O, TEA, 4-DMAP, CH2Cl2, reflux, 6
h, Y7 : 100 %, 72 h Y8= 74 % v) KOH, H2O:THF; Y9,10 = 100 % vi) Ac2O, Py, reflux, 6 h,
Y = 100 %.
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Scheme 2 i) HBTU, DIPEA, 4-DMAP,5 or 6 CH2Cl2, rt, 24 h Y13 = 87 % Y14 = 62 % ii) a)
H2/Pd (C), MeOH, rt, 3 h b) HBTU, DIPEA, 4-DMAP, 9, 10 or 12, CH2Cl2, rt, 24 h Y15 = 30
%, Y16 = 40 %, Y17 = 35 % iii) LiOH, MeOH:H2O, rt, 24 h, Y 18,19,20 = 100 %.
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Figure 1. Distamycin and analogues.
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Figure 2. Plasma membrane integrity and intracellular redox state of African
trypanosomes treated with N-methyl pyrrole bis-thiazoles. The bloodstream form of T. b.
brucei (2×106 parasites/mL) was incubated 4 h with 16 and 17 at their EC50 (12 nM and 7
nM, respectively) or 5X EC50. Thereafter, A) membrane integrity was assayed by the
incorporation of propidium iodide and B) the intracellular redox state was assessed with a
redox biosensor. Controls included parasites treated with vehicle (DMSO 1 %, v/v), Triton X-
100 (0.001 %, v/v), a reducing (DTT 1 mM) and an oxidizing agent (Menadione 250 µM).
For details in the protocol see section 4.6.3. The results are expressed as mean ± S.D (n = 3).
The asterisks denote the probability index (**** p < 0.0001; ONE WAY ANOVA analysis)
compared to DMSO.
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Figure 3. Cell cycle analysis of African trypanosomes treated with N-methyl pyrrole bis-
thiazoles. The bloodstream form of T. b. brucei (5×105 parasites/mL) was incubated for 24 h
with DMSO [1% (v/v)], 17 (7 or 14 nM) or with DAC (41 nM), and the cell populations with
different DNA content analyzed by flow cytometry prior staining with PI. The results are
represented as the average corresponding to three experimental replicates with an associated
error in the determination ≤ 15%. The asterisks denote statistical difference (****, p <
0.0001) vs. DMSO according to a χ2 contingency test.
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Figure 4. DNA-content analysis by epifluorescence microscopy. T. b. brucei parasites
(5×105 parasites/mL) were treated with DMSO (1 %, v/v), DAC (41 nM), 16 (12 nM) and 17
(7 nM) for 24 h, then stained with DAPI and analyzed by epifluorescence microscopy. A)
Parasite populations classified according to their DNA-phenotype. Representative images
(superposition of bright field and fluorescence) for DAPI-stained parasites treated with B)
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DMSO, C) 17 and D) DAC. At least 50 parasites were analyzed for each condition. N=
nucleous, K= kinetoplast.
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Figure 5. DAPI-competition assays for different DNA samples. Five nM DNA (pUC18 or
pUC18kDNA dissolved in PBS 1X) were incubated with DMSO (1% v/v), 20 µM DAC, 16
or 17 (all prepared in PBS 1X with 1 % v/v DMSO) for A) 30 min and B) 120 min, and then
DAPI was added at a final concentration of 0.5 µM. The emission spectra was recorded in a
spectrofluorimeter (λex/em = 358 nm/368-600 nm). The fluorescence is expressed as
percentage normalized against the maximum value (λem = 461 nm) obtained for the
corresponding DNA + DMSO samples set at 100 %. Multiple comparison two-way ANOVA
test were performed, where ** and ## denote statistical significance (p <0.01) when plasmids
(pUC18 vs. pUC18kDNA) and incubation time (30 vs. 120 min), respectively, is compared
for each treatment.
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Figure 6. Parasitemia of T. b. brucei-infected mice treated with 17 and the control drug
DAC. BalbC/J mice (n=6/group) were infected with 104 bloodstream T. b. brucei (strain
Antat 1.1) and at day 4 post-infection were treated intraperitoneally with DMSO (3.3 mg/Kg
× day/7 days), a single dose of DAC (40 mg/Kg) or (at 8.36 mg/Kg or 2.09 mg/Kg both doses
applied daily during 7 days). The lower and higher dose of 17 are referred here as the 250X
and 1000X, respectively, EC50 values determined in vitro. Parasitemia was assessed at
different time-points by microscopy using a Neubauer chamber. The probability values are
shown for groups with differences statistically significant with respect to the vehicle control
group (DMSO), according to a non-parametric Kruskal-Wallis test.
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