Synthesis and antikinetoplastid evaluation of bis(benzyl)spermidine derivatives

Article abstract of DOI:10.1016/j.ejmech.2018.02.087

This study describes the synthesis and the biological evaluation of twenty-four original bis(benzyl)spermidines. Structural modifications of the polyamine scaffold were performed in order to avoid easily metabolized bonds. Some bis(benzyl)polyamine derivatives have demonstrated promising activity in vitro against Trypanosoma brucei gambiense and Leishmania donovani. From the enzymatic experiments on trypanothione reductase, we observed that this enzyme was not targeted by our compounds. In vivo evaluation on Swiss mice model infected by T. b. gambiense or L. donovani was done with the most interesting compound of the series.

Full text of DOI:10.1016/j.ejmech.2018.02.087

Accepted Manuscript
Synthesis and antikinetoplastid evaluation of bis(benzyl)spermidine derivatives
Elodie Jagu, Sébastien Pomel, Alba Diez-Martinez, Estelle Rascol, Stéphanie Pethe,
Philippe M. Loiseau, Raphaël Labruère
PII:
S0223-5234(18)30234-4
10.1016/j.ejmech.2018.02.087
EJMECH 10264
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 29 November 2017
Revised Date: 27 February 2018
Accepted Date: 28 February 2018
Please cite this article as: E. Jagu, Sé. Pomel, A. Diez-Martinez, E. Rascol, Sté. Pethe, P.M. Loiseau,
Raphaë. Labruère, Synthesis and antikinetoplastid evaluation of bis(benzyl)spermidine derivatives,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.087.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and antikinetoplastid evaluation of
bis(benzyl)spermidine derivatives
Elodie Jagua, Sébastien Pomelb, Alba Diez-Martineza, Estelle Rascola, Stéphanie Pethea, Philippe M.
Loiseaub*, Raphaël Labruèrea*
a- Institut de Chimie Moléculaire et des Matériaux d’Orsay (ICMMO), CNRS, Univ Paris Sud, Université
Paris-Saclay, 15 rue Georges Clemenceau, 91405 Orsay Cedex, France
b- Chimiothérapie antiparasitaire, UMR 8076 BioCis, CNRS, Univ Paris Sud, Université Paris-Saclay, 5
rue Jean-Baptiste Clément, 92290 Châtenay-Malabry, France
Keywords: Antikinetoplastids, Trypanosoma brucei, Leishmania donovani, Polyamines
* Corresponding authors
E-mail addresses: philippe.loiseau@u-psud.fr (P. M. Loiseau), raphael.labruere@u-psud.fr (R. Labruère).
ABSTRACT
This study describes the synthesis and the biological evaluation of twenty-four original
bis(benzyl)spermidines. Structural modifications of the polyamine scaffold were performed in
order to avoid easily metabolized bonds. Some bis(benzyl)polyamine derivatives have
demonstrated promising activity in vitro against Trypanosoma brucei gambiense and Leishmania
donovani. From the enzymatic experiments on trypanothione reductase, we observed that this
enzyme was not targeted by our compounds. In vivo evaluation on Swiss mice model infected by
T. b. gambiense or L. donovani was done with the most interesting compound of the series.
INTRODUCTION
Infections by Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense and
Leishmania sp. cause more than 100,000 deaths per year and the World Health Organization
(WHO) has classified these infections as challenging neglected disease.1 These kinetoplastids
are responsible for Human African Trypanosomiasis (HAT) and leishmaniasis, respectively. Only
few drugs are currently used for the treatment of these infectious diseases and the development
of resistant strains to these existing drugs is quite concerning. Moreover, the drugs

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administrated to patients are essentially old and present serious side effects.2 In regard of the
lack of efficacy of the current treatments, the discovery of new and safe antikinetoplastid
molecules is clearly needed.3,4 In order to limit the adverse effects, the new drugs should target
specifically the parasite by preferably impairing a metabolic pathway only found in kinetoplastids.
In kinetoplastids, polyamine and trypanothione metabolisms are essential for parasite survival
and infectivity.5,6 The trypanothione is a metabolite made of two glutathione molecules linked to
one spermidine, which plays a key role against oxidative stress in these protozoan parasites.
The trypanothione synthetase (TryS) is involved in the trypanothione anabolism by catalyzing
the coupling of glutathione and spermidine. Furthermore, trypanothione reductase (TryR) allows
trypanothione to be preserved in its reduced form in the presence of NADPH. Since this
compound is not biosynthesized in mammalian cells, the trypanothione pathway is a promising
target for drug discovery in kinetoplastids.7
Numerous natural polyamine analogs or conjugates have been studied for their antikinetoplastid
activity.8 In most cases, the target was not studied nor mentioned, but, when a target was
assayed, TryR was often disrupted by the polyamine derivatives. However, other target recently
revealed such as methionyl-tRNA synthetase could be also impaired by the same polyamine
derivatives.9 From the literature, we noted that antikinetoplastid activity was significant when a
polyamine chain was linked to a benzyl moiety.8 Among them, the bis(benzyl)polyamines were
outstanding inhibitors of TryR often associated with a good in vitro activity against both L.
donovani and T. cruzi.10 These compounds displayed a high selectivity index and were markedly
effective when the two benzyl groups were attached to the same nitrogen of the polyamine. In
our previous efforts regarding the development of new antikinetoplastid agents,11,12 spermidine-
aryl conjugates exhibited the most interesting antiparasitic activity, in particular, derivatives
bearing aromatic substituent on the N8-nitrogen atom.12 With these observations, we decided to
design spermidine disubstituted on the N8-nitrogen by different aryl groups as potential
antikinetoplastid compounds. Some bisbenzyl derivatives were inspired from existing literature.
Indeed, thiophene,13 benzyloxybenzyl, diphenyl14 and naphthalene15 have also been identified as
efficient substitutents for trypanocidal activity. Therefore, we elaborated different spermidine
derivatives bearing these scaffolds (Figure 1).

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Figure 1: General structure of linear bis(benzyl)spermidines.
Additionally, the different aryl-polyamine previously tested in vivo were described as rapidly
metabolized or excreted.16,17,18,19 We hypothesized that adding structural modification to these
derivatives could increase the bioavailability: (i) we introduced a fluorine atom on the para
position of the phenyl ring which is well known to avoid rapid metabolization;20 (ii) we also
decided to prepare some polyamine derivatives bearing a rigidified polyamine chain since
constraint structures generate better bioavailability. 21 It has been shown that polyamine
derivatives bearing substituent in α-position of the amine were not hydroxylated by the spermine
oxidase in mammals cells.22,23 Consequently, we introduced a cyclohexyl ring on the polyamine
skeleton (Figure 2) in order to combine rigidity of the structure and prevent fast degradation of
the compound. Furthermore, we will be able to compare the linear and the cyclic polyamine
structure toward the biological effect.
Figure 2: General structure of the rigidified spermidine derivatives.
Thus, we investigated each of the compounds for potential activity against both T. brucei and L.
donovani in vitro, and the most promising compound was tested in vivo in mice models infected
either by trypanosomes or Leishmania. Preliminary experiments in regard of the in vitro
metabolism of selected compounds were carried out. We evaluated the cytotoxicity of the
compounds on human macrophages in order to establish the selectivity index. We also tested all

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the compounds for activity against T. brucei TryR since this enzyme was a probable target of
this series.
RESULTS AND DISCUSSION
Synthesis. Spermidine derivatives 5a-g were synthesized in four steps, starting from
commercially available benzyl chloride or benzyl bromide 1a-g (Figure 3). Firstly, preparation of
N-diaryl-aminoalcohol 2a-g was carried out using 3-aminopropanol 2 and benzyl halides 1a-g in
presence of potassium carbonate in different solvent according to the benzyl halides 1a-g
solubility (acetone, DCM or DMF, see experimental section). The hydroxyl group of 2a-g was
mesylated to provide activated compounds 3a-g. The diaminobutane monoprotected with a Boc
group 4 was prepared according to protocol described by Roblot et al. 24 Nucleophilic
replacement of mesylate with the primary amine of 4 in the presence of Hünig's base provided
4a-g in 12-41% yield on two steps. Disubstitution was occasionnally observed during this
reaction and afforded byproducts 6a and 6b. These compounds were isolated and included in
the study for biological evaluation. The Boc protecting group was then removed by treatment
with 4M HCl in dioxane. Thus, the final compounds 5a-g, 7a and 7b were obtained as
hydrochloride salts in quantitative yield.

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Figure 3: Synthesis of diaryl derivatives. Reagents and conditions: (a) K2CO3, acetone or DCM or
DMF, rt, 12-16 h; (b) CH3SO2Cl, DIEA, DMAP, DCM, 0 °C to rt, 16h; (c) DIEA, ACN , reflux, 16h; (d) 4M
HCl/Dioxane, rt, 16 h.
In an attempt to confer a degree of structural rigidity, analogs of 5a and 5b featuring restricted
rotation in the central polyamine chain were designed. This was accomplished by incorporating a
trans-cyclohexane ring into the polyamine skeleton (Figure 4). Thus, trans-1,4-
diaminocyclohexane 8 was monosubstituted by a Boc protecting group following a slightly
modified method previously described.25 Boc-diaminohexane 9 was then coupled to two different
mesylated intermediates 3a and 3b to give diarylated cyclohexane-1,4-diamine 10a and 10b.
During this substitution reaction with the fluorinated derivative 3b, a second product was
obtained. This latter compound is derived from disubstitution of the mesylate 3b conducting to
the tertiary amine 12b. This byproduct was isolated, then deprotected in acidic condition as
mentioned above to afford compound 13b which was included in the biological assay. Likewise,
10a and 10b were also deprotected to give the hydrochloride salts 11a and 11b.

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Figure 4: Synthesis of ((diaryl)aminopropyl)cyclohexanediamine. Reagents and conditions: (a)
Boc2O, DCM, 0 °C to rt, 16 h; (b) DIEA, ACN, reflux, 16 h; (c) 4M HCl/Dioxane, rt, 16 h.
In vitro antikinetoplastid evaluation. Boc-protected and -deprotected molecules have been
evaluated in vitro against both T. b. gambiense trypomastigotes and Leishmania donovani
axenic and intramacrophage amastigotes. The results of the in vitro screening are shown in
Tables 1, 2 and 3.

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Regarding the difference of activity between the Boc protected and the free amine compounds, it
is difficult to infer a general trend that either increase or not the activity. In most cases, the
protecting group did not influence the biological effect, except for the couples Boc-protected
polyamine/free polyamine: 4a/5a, 6b/7b and 4f/5f on T. b. gambiense (Tables 1 and 2). Against
the same parasite, ten compounds of this series have displayed an IC50 below 1 µM and five
below 0.5 µM (5f, 5g, 6a, 7b, 11b). Furthermore, these latter molecules were assayed on non-
infected macrophages and all were moderately cytotoxic with only 7b displaying an IC50 in the
low micromolar range (Table 2). Hence, all these derivatives had a selectivity index (SI) over 10.
Substituents on the aromatic ring were never detrimental on the trypanocidal activity except with
the meta-phenyl group (IC50 (4e/5e) > 8 µM) (Table 1). We further noted that attaching a second
aryl nucleus (naphthalene 5g and para-biphenyl 5f) was rather enhancing the trypanocidal
activity providing that this additional ring is attached linearly in a rigid configuration to the first
one. Indeed, 5g (naphtyl substituent) was 20 times more active than the corresponding phenyl
derivatives 5a and, para-biphenyl 5f was at least 20 times more trypanocidal than both meta-
biphenyl 5e (non-linear/rigid) and para-benzyloxyphenyl 5d (linear/non-rigid). Interestingly,
O’Sullivan et al. reported a positional isomer of N1,N1-bis(2-naphtylmethyl)-spermidine 5g where
each naphtyl group were borne by one terminal nitrogen of a spermidine molecule (i.e. N1,N8-
bis(2-naphtylmethyl) isomer) that was shown as active as 5g against four strains of T. brucei
(IC50 = 0.2-0.6 µM).17 When compound 5a is compared with the rigidified cyclohexane-containing
analog 11a, an improvement of the antikinetoplastid activity could be noted as well as an
enhancement of the cytotoxic effect. However, the corresponding fluorobenzyl derivative 11b
was 5 times more active against T. b. gambiense than 11a with an IC50 of 0.4 µM and a
selectivity index of 12 (Table 3).
Concerning the activity against Leishmania donovani, we observed that the vast majority of the
compounds displayed similar or better activity on intramacrophage than on axenic amastigotes.
Among the synthesized compounds, the tetra-aryl derivatives were the most potent, 6a was
indeed remarkable since it displayed IC50 value of 2.5 µM against L. donovani axenic
amastigotes and, interestingly, an IC50 of 1.5 µM against the intramagrophage ones (Table 2).
7b, a Boc-deprotected and fluorinated analog of 6a, was equally active against axenic
amastigotes and slightly less active on intramacrophage amastigotes, but this compound had a
strong cytotoxicity on macrophages (IC50 = 1.5 µM). The thiophenes 4c, 5c and the
benzyloxybenzyls 4d, 5d did not show any activity against L. donovani. Remarkably, we
observed that the meta-biphenyl derivative 5e was not leishmanicidal whereas the para-biphenyl
5f had a moderate activity against both axenic and intramacrophage amastigotes (IC50 of 3.9

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and 7.1 µM respectively) (Table 1). In regards of reported compounds displaying a structure
close to our derivatives, a series of N1,N1-bisaryl-diamines were described by Labadie et al.14 In
the latter study, two bisbenzyl-diamines (2- or 3-carbon chain) were as leishmanicidal as the
N1,N1-bisbenzyl-spermidine 5a (IC50 ~ 25 µM). However, N1,N1-bisbenzyloxybenzyl-diamines (2-
or 3-carbon chain) were active in the low micromolar range14 whereas N1,N1-
bisbenzyloxybenzyl-spermidine
5d
was
inactive
(IC50
>
3.9
µM).

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Table 1: Antikinetoplastid activity and cytotoxicity of the linear bis(benzyl)spermidines.
[a] SI: Selectivity Index expressed as the ratio CC50/IC50 ; SITb: relative to IC50 on T. b. gambiense; SIaa: relative to IC50 on L. donovani axenic
amastigotes; SIia: relative to L. donovani intramacrophage amastigotes
[b] 50% growth inhibitory concentration
L. d. LV9
Cytotoxicity on
L. d. LV9 axenic
amastigotes IC50
(µM) ± SD
T. b. g. IC50
(µM) ± SD
intramacrophage
amastigotes
macrophages
CC50 ± SD
(µM)[b]
[a]
[a]
[a]
SITb
SIaa
SIia
Ar
Compounds
IC50 ±SD (µM)[b]
0.6 ± 0.1
2.0 ± 0.2
1.5 ± 0.1
20.8
4.3
14.6 ± 3.6
14.7 ± 0.5
25.3 ± 0.5
-
-
-
3.4 ± 1.2
14.5 ± 0.9
21.1 ± 1.0
3.7
12.5
-
-
8.7 ± 0.2
16.4 ± 0.2
10.9
4.7 ± 0.2
7.1
26.4 ± 1.6
1.3
42.0 ± 6.2
-
33.3 ± 2.8
4
R = Boc
33.9 ± 0.6
14.4 ± 2.2
> 2.9
> 6.9
79.5 ± 5.5
38.5 ± 4.6
> 1.2
> 2.6
> 100
-
> 100
> 100
41.7 ± 1.
> 2.4
0.8 ± 0.0
8.8 ± 2.2
5.4
2.0
3.8 ± 0.1
> 100
1.1
-
6.1 ± 2.9
-
-
4.3 ± 0.1
5
27.4 ± 2.7
17.7 ± 0.6
R = H

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3.5 ± 0.5
6.1 ± 0.9
9.4
8.6
70.3 ± 2.3
>100
-
-
35.8 ± 4.2
59.1 ± 3.3
-
-
33.1 ± 0.2
52.8 ± 1.1
35.5 ± 0.5
> 2.8
72.2 ± 8.5
> 1.4
> 100
-
> 100
8.7 ± 0.6
0.2 ± 0.0
3.5
35.7 ± 1.3
3.9 ± 0.1
-
27.8 ± 5.4
7.1 ± 1.4
1.1
-
30.3 ± 3.0
4.2 ± 0.0
21.0
1.1
0.4 ± 0.0
12.2
5.0 ± 0.4
-
8.7 ± 0.3
-
4.9 ± 1.1

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Table 2: Antikinetoplastid activity and cytotoxicity of the linear disubstituted bis(benzyl)spermidines.
[a] SI: Selectivity Index expressed as the ratio CC50/IC50 ; SITb: relative to IC50 on T. b. gambiense; SIaa: relative to IC50 on L. donovani axenic
amastigotes; SIia: relative to L. donovani intramacrophage amastigotes
[b] 50% growth inhibitory concentration
L. d. LV9
Cytotoxicity on
L. d. LV9 axenic
amastigotes IC50
(µM) ± SD
T. b. g. IC50
(µM) ± SD
intramacrophage
amastigotes
macrophages
CC50 ± SD
(µM)[b]
[a]
[a]
[a]
SITb
SIaa
SIia
Ar
Compounds
IC50 ±SD (µM)[b]
0.3 ± 0.1
1.9 ± 0.2
0.9 ± 0.2
0.1 ± 0.0
41.7
6.3
2.5 ± 0.7
6.6 ± 0.1
57.3 ± 4.1
2.0 ± 0.8
5.0
1.8
-
1.5 ± 0.2
16.6 ± 4.2
22.4 ± 5.8
6.5 ± 1.6
8.3
12.5
6
R = Boc
-
-
-
12.0 ± 3.2
1.2 ± 0.2
1.5 ± 0.3
1.3
7
R = H
15.0
-

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Table 3: Antikinetoplastid activity and cytotoxicity of the rigidified bis(benzyl)spermidines.
[a] SI: Selectivity Index expressed as the ratio CC50/IC50 ; SITb: relative to IC50 on T. b. gambiense; SIaa: relative to IC50 on L. donovani axenic
amastigotes; SIia: relative to L. donovani intramacrophage amastigotes
[b] 50% growth inhibitory concentration
L. d. LV9
Cytotoxicity on
L. d. LV9 axenic
amastigotes IC50
(µM) ± SD
T. b. g. IC50
(µM) ± SD
intramacrophage
amastigotes
macrophages
CC50 ± SD
(µM)[b]
[a]
[a]
[a]
SITb
SIaa
SIia
Ar
Compounds
IC50 ±SD (µM)[b]
0.7 ± 0.1
0.5 ± 0.0
7.3
8.4
14.2 ± 2.1
6.8 ± 0.2
-
-
5.4 ± 1.8
6.8 ± 0.4
-
-
5.1 ± 1.4
4.2 ± 0.0
10
R = Boc
2.1 ± 0.0
0.4 ± 0.0
3.6
11
>100
-
-
6.6 ± 1.2
1.1
-
7.6 ± 2.2
4.4 ± 0.0
11
R = H
44.4 ± 2.0
12.4 ± 3.5
12
R = Boc
4.2 ± 0.2
> 23.8
10.2 ± 0.3
15.7 ± 1.3
> 9.8
17.2 ± 6.6
4.2 ± 1.6
> 5.8
1.1
> 100
13
R = H
10.4 ± 1.2
-
-
4.5 ± 1.0

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Enzymatic assay. As mentioned earlier, TryR is a potential drug target for polyamine
derivatives since large number of different TryR inhibitors reported so far displayed such
scaffold attached to a hydrophobic aryl nucleus. All the compounds have been evaluated on
TryR from T. brucei. A first screening at a fixed concentration of inhibitor at 40 µM in assay
buffer containing 5% DMSO has been carried out. The activity was measured in the
presence of 40 and 100 µM TS2, respectively, and the percentage of inhibition was
calculated. The results of assay using 40 µM TS2 were depicted in the Figure 5.
Figure 5: Inhibition of T. b. TryR. The activity was measured following the NADPH consumption
at 340 nm as described in the Experimental Section. The assays contained a fixed
concentration of 100 or 40 µM of TS2 and 40 µM of inhibitor. Figure shows the average
percentage inhibition at the two different substrate concentrations. The controls contained the
same amount of DMSO used to dissolve the compounds.
Three compounds displayed an activity higher than 50% inhibition at 40 µM (5a, 7b and 4g)
and compound 5g has shown 47% inhibition at the same concentration. However, compound
7b seems to present substrate dependent inhibition. Indeed, 7b showed more than 60% of
inhibition at a TS2 concentration of 40 µM, and around 30% of inhibition when substrate
concentration is reaching 100 µM. Compound 4g seems to have the same behavior when
concentration substrate increases (60% inhibition at 40 µM against 45% at 100 µM). In
contrast, percentage of inhibition for compound 5a was independent of the substrate
concentration which means that this derivative acts as pure competitive ligand.
In view of this preliminary screening, we determined the inhibitory constant and the type of
inhibition of compound 5a. We observed that 5a behaves as mixed-type inhibitors (data not

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shown), with calculated Ki- and Ki’-value of 7.5 µM and 20 µM, respectively. This activity on
T. b. TryR is relatively low and could explain the poor IC50 of 8.8 µM against T. b. gambiense
in vitro. In light of the weak inhibition obtained on T. b. TryR with this series of compounds,
their trypanocidal effect is therefore due to their action on a different target.
In vivo evaluation. Among the most active compounds, three deprotected polyamines 5a,
5f and 11b were assayed for their in vitro metabolic stability both in plasma (hydrolytic
enzymes) and liver S9 fraction (phase I and II drug metabolizing enzymes). It was observed
that the three molecules were stable over 12 h in human plasma. Compound 11b which has
a fluorine atom on the benzyl groups and for which the polyamine chain has been rigidified
was among the most trypanocidal derivative in vitro, bearing rather hydrophobic moieties and
arduously breakable bond by classical metabolic transformations. The ability of this
compound to remain stable in the S9 fraction was therefore evaluated and no metabolic
transformations were noted in presence or absence of NADPH. The microsome-predicted
hepatic extraction ratios obtained based on the relative rates of test compound degradation
was relatively high (= 0.9). In view of these results, we decided to evaluate 11b in vivo on the
Trypanosoma brucei/Swiss mice model. Compound 11b was administered by intraperitoneal
injection at doses of 100 mg/kg and 10 mg/kg. The results are gathered in Table 4.
Table 4 : In vivo evaluation of compound 11b at the single dose of 10 mg/kg administered by
intraperitoneal route on mice infected with Trypanosoma brucei. (+) 1-50 parasites / µL blood;
(++) 50-500 parasites / µL blood; (+++) 500-5000 parasites / µL blood; (++++) 5000-50000
parasites / µL blood. Mean survival time of excipient treated mice = 3.5 ± 0.5 days.
Mean survival
time minus
Dose
Parasitemia at
Toxicity: number
of dead mice
Compound
mean control
survival time
(Days ± SD)
Day Day
Day
[day of death
µmol/kg mg/kg
2
3
4
post-treatment]
217
100
10
/
/
/
-2.3 ± 0.5
0.0 ± 1.1
2[Day 1]; 4[Day 2]
11b
1[Day 2]; 3[Day 4];
2[Day 5]
21.7
+
++
++++
Pentamidine di-
isethionate
100
100
60
50
-
-
-
-
-
-
> 30
> 30
No toxicity
No toxicity
Melarsamine
dihydrochloride

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8
Excipient
/
/
+++ ++++ mice
dead
0
No toxicity
Although no prolongation of survival has been observed, the polyamine derivative 11b
induces a delay in the development of parasitemia rate, proving a trypanocidal activity at 10
mg/kg even though parasites are still present at this dose. However, this compound was
toxic at 100 mg/kg. Afterwards, 11b has been also tested in vivo on Balb/c mice model
infected with Leishmania donovani. Considering that no toxicity was observed on the T. b.
brucei mice model at 10 mg/kg in a single dose, we used a dose at 2 mg/kg/day by
intraperitoneal route on five consecutive days, corresponding to the classical protocol for the
in vivo leishmaniasis model (protocol and data not shown). Therefore, the total dose
evaluated for the in vivo model of Leishmania donovani was 10 mg/kg and no specific action
on parasites was observed at this dose.
CONCLUSION
Twenty-four new polyamines derivatives have been synthesized and evaluated in vitro
against T. b. gambiense and L. donovani. Many of them displayed a good trypanocidal
activity on T. b. gambiense. In spite of a high level of toxicity against non-infected
macrophages, some derivatives were promising for the development of new antikinetoplastid
compounds, in particular, compounds 6a and 11b which displayed an IC50 against T. b.
gambiense below 0.5 µM and significant selectivity indices. Some compounds showed an
antikinetoplastid activity without inhibitory effect on TryR. Therefore, further studies are
required to determine the mechanism of action, and the target(s) of these compounds.
Regarding the activity against L. donovani, we highlighted new structures which were
efficient against the intramacrophage form of the parasite. The major disadvantage
mentioned in the literature for the polyamine derivatives is the weak in vivo activity. It was
suggested that lack of trypanocidal activity on infected animal is probably due to either rapid
excretion of polyamine derivatives or fast metabolism in mammalian host. In our study, we
tried to optimize the polyamine structure but unfortunately compound 11b was not sufficiently
effective in vivo on infected mice although it causes a slowdown in T. brucei development.
The next challenge will be now the possibility to develop new antikinetoplastid polyamines
displaying no toxicity toward the host.

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EXPERIMENTAL SECTION
Chemistry. All chemical reagents were of analytical grade, obtained from Acros, Alfa Aesar,
or Aldrich, and used without further purification. Solvents were obtained from SDS or VWR-
Prolabo. Dichloromethane was dried on molecular sieves and used immediately.
Chromatography was performed using silica gel (35-70 ꢀm, Merck). Concentration of
solutions was performed under reduced pressure at temperature below 40°C using rotary
evaporator. Analytical TLC was performed using Silica Gel 60 F254 pre-coated aluminum
plates (Merck). Spots were visualized by treatment with ninhydrine revelator followed by
heating and/or by absorbance of UV light at 254 nm. NMR spectra were collected on Bruker
DRX 250 (1H at 250 MHz and 13C at 62.5 MHz), DRX 300 (1H at 300 MHz and 13C at 75
MHz) or DRX 360 (1H at 300 MHz and 13C at 90 MHz) spectrometers using MestReNova
software. Chemical shifts are reported in ppm (δ) and coupling constants in Hz (J). 1H NMR
spectra were performed in CDCl3, MeOD or D2O. High-resolution mass spectrometry
(HRMS) analyses were performed by electrospray with positive (ESI+). The purity of all
compounds used for biological activity test was checked by reverse phase analytical HPLC
and confirmed to be ≥ 95%.
General procedure for the synthesis of the aminoalcohols 2a-g:
A mixture of the corresponding benzyl chloride/bromide (2.1 eq.), 3-aminopropanol (1.0 eq.)
and K2CO3 (5.0 eq.) in acetone, dichloromethane or dimethylformamide was stirred at room
temperature. After completion of the reaction (12-16 h) monitored by TLC, the reaction
mixture was filtered and the filtrate was concentrated under reduced pressure. The residue
was purified by silica gel chromatography to afford the corresponding aminoalcohol.
2a: 3-(dibenzylamino)propan-1-ol
Conditions: Benzylbromide in acetone. Yellow oil, (70%). Rf (Cyclohexane/EtOAc 8:2) = 0.26.
1H NMR (300 MHz, MeOD): δ = 7.35-7.21 (m, 10H), 3.56-3.53 (m, 6H), 2.55 (t, J = 6.5 Hz,
13
2H), 1.76 (m, 2H) ppm. C NMR (75 MHz, D2O): δ = 138.1, 128.9, 128.2, 63.4, 58.3, 52.7,
27.9 ppm. HRMS-ESI(+): calcd for C17H21NO: 255.1623, found: 256.1629 [M+H]+.
2b: 3-(bis(4-fluorobenzyl)amino)propan-1-ol
Conditions: 4-Fluorobenzyl chloride in acetone. Yellow oil, (32%). Rf (Cyclohexane/EtOAc
8:2) = 0.32. 1H NMR (300 MHz, CDCl3): δ = 7.28 (m, 4H), 7.04 (t, J = 8.7 Hz, 4H), 4.46 (brs,
1H), 3.65 (t, J = 6.5 Hz, 2H), 3.54 (s, 4H), 2.64 (t, J = 6.5 Hz, 2H), 1.81-1.74 (m, 2H). 13C
NMR (75 MHz, CDCl3): δ = 162.1 (d, J = 244 Hz), 133.8, 130.7 (d, J = 10 Hz), 115.3 (d, J =

ACCEPTED MANUSCRIPT
21 Hz), 63.8 57.7, 52.9, 29.7. HRMS-ESI(+): calcd for C17H19F2NO: 291.1435, found:
292.1516 [M+H]+.
2c: 3-(bis(thiophen-3-ylmethyl)amino)propan-1-ol
Conditions: 3-(bromomethyl)thiophene in DCM. White solid, (74%). Rf (Cyclohexane/EtOAc
1:1) = 0.43. 1H NMR (300 MHz, CDCl3): δ = 7.29 (dd, J = 4.9, 3.0 Hz, 2H), 7.14 (dd, J = 3.0,
0.8 Hz, 2H), 7.06 (dd, J = 4.9, 0.8 Hz, 2H), 5.06 (br s, 1H), 3.70 (t, J = 5.2 Hz, 2H), 3.61 (s,
4H), 2.65 (t, J = 5.8 Hz, 2H), 1.76 (qt, J = 5.5 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 138.9,
128.3, 126.0, 123.2, 64.2, 53.5, 53.0, 28.0 ppm. HRMS-ESI(+): calcd for C13H17NOS2:
268.0752, found: 268.0815 [M+H]+.
2d: 3-(bis(4-(benzyloxy)benzyl)amino)propan-1-ol
Conditions: 4-benzyloxy(benzyl) chloride in DMF. White solid, (47%). Rf (Cyclohexane/EtOAc
1
1:1) = 0.51. H NMR (300 MHz, CDCl3): δ = 7.46-7.38 (m, 10H), 7.27 (d, J = 8.6 Hz, 4H),
6.97 (d, J = 8.6 Hz, 4H), 5.08 (s, 4H), 3.67 (m, 2H), 3.54 (s, 4H), 2.67 (m, 2H), 1.81 (m, 2H).
13C NMR (75 MHz, CDCl3): δ = 158.1, 137.0, 130.4, 128.6, 127.9, 127.5, 114.8, 70.0, 64.1,
57.7, 53.0, 27.8 ppm. HRMS-ESI(+): calcd for C31H33NO3: 467.2460, found: 468.2513
[M+H]+.
2e: 3-(bis([1,1'-biphenyl]-3-ylmethyl)amino)propan-1-ol
Conditions: 3-phenylbenzyl bromide in DMF. White solid, (78%). Rf (Cyclohexane/EtOAc 6:4)
= 0.54. 1H NMR (300 MHz, CDCl3): δ = 7.64-7.60 (m, 8H), 7.46-7.38 (m, 10H), 4.70 (brs, 1H),
3.72 (s, 4H), 2.76 (m, 2H), 1.85 (m, 2H), 1.61 (m, 2H). 13C NMR (75 MHz, CDCl3): δ = 144.7,
140.6, 138.9, 130.1, 128.8, 127.9, 128.1, 128.0, 127.5, 125.3, 60.2, 59.4, 53.8, 28.8 ppm.
HRMS-ESI(+): calcd for C29H29NO: 407.2249, found: 408.2313 [M+H]+.
2f: 3-(bis([1,1'-biphenyl]-4-ylmethyl)amino)propan-1-ol
Conditions: 4-phenylbenzyl bromide in DMF. White solid, (58%). Rf (Cyclohexane/EtOAc
65:35) = 0.55. 1H NMR (300 MHz, CDCl3): δ = 7.63 - 7.54 (m, 8H), 7.50 - 7.29 (m, 10H), 4.70
(brs, 1H), 3.73 (t, J = 5.3 Hz, 2H), 3.67 (s, 4H), 2.72 (t, J = 5.7 Hz, 2H), 1.83 (qt, J = 5.7 Hz,
13
2H). C NMR (75 MHz, CDCl3): δ = 141.0, 140.3, 137.49, 129.7, 128.9, 127.3, 127.2, 64.1,
58.5, 53.5, 28.2. HRMS-ESI(+): calcd for C29H29NO: 407.2249, found: 408.2309 [M+H]+.
2g: 3-(bis(naphthalen-2-ylmethyl)amino)propan-1-ol

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Conditions:
2-(bromomethyl)naphthalene
in
DCM.
White
solid,
(86%).
Rf
1
(Cyclohexane/EtOAc 65:35) = 0.46. H NMR (300 MHz, CDCl3): δ = 7.91 – 7.75 (m, 8H),
7.56 – 7.46 (m, 6H), 4.68 (br s, 1H), 3.79 (s, 4H), 3.67 (t, J = 5.3 Hz, 2H), 2.74 (t, J = 5.8 Hz,
2H), 1.83 (qt, J = 5.5 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 136.0, 133.4, 132.9, 128.4,
128.2, 127.8, 127.2, 126.2, 125.9, 63.9, 59.0, 53.3, 28.3. HRMS-ESI(+): calcd for C25H25NO:
355.1936, found: 356.2000 [M+H]+.
General procedure for the synthesis of the methanosulfonates 3a-g:
Methanesulfonyl chloride (2.0 eq.) in DCM (0.22 M) was cooled at 0 °C using an ice bath. A
mixture of the corresponding aminoalcohol (1.0 eq.), di-isopropylethylamine (5.0 eq.) and
DMAP (10 mol%) in DCM (0.44 M) was added dropwise under stirring. The reaction mixture
was stirred at low temperature for 5 min and then, at room temperature for 16 h. Then, the
dark red-brown reaction mixture was poured into a mixture of ice-water and DCM and the
organic layer was separated. The aqueous phase was extracted with DCM three times. The
combined organic layers were washed with water and brine, dried with MgSO4 and
evaporated to give the mesylated intermediate as pale yellow gummy solid. The product was
used without further purification.
3a: 3-(dibenzylamino)propyl methanesulfonate
Rf (Cyclohexane/EtOAc 8:2) = 0.28. 1H NMR (300 MHz, CDCl3): δ = 7.35-7.32 (m, 10H), 4.24
(t, 2H, J = 6.5 Hz), 3.55 (s, 4H), 2.82 (s, 3H), 2.55 (t, 2H, J = 6.5 Hz), 1.92 (qt, 2H, J = 6.5
Hz). 13C NMR (75 MHz, CDCl3): δ = 138.6, 128.8, 127.4, 126.7, 67.6, 61.0, 52.8, 37.6, 26.7.
3b: 3-(bis(4-fluorobenzyl)amino)propyl methanesulfonate
Rf (Cyclohexane/EtOAc 8:2) = 0.33. 1H NMR (300 MHz, CDCl3): δ = 7.28 (m, 4H), 7.01 (t, J =
8.7 Hz, 4H), 4.23 (t, J = 6.5 Hz, 2H), 3.51 (s, 4H), 2.88 (s, 3H), 2.53 (t, J = 6.5 Hz, 2H), 1.90
(qt, J = 6.5 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 161.9 (d, J = 244 Hz), 130.3, 115.2 (d, J
= 21 Hz), 67.8, 57.6, 49.2, 37.3, 26,7. HRMS-ESI(+): calcd for C18H21F2NO3S: 369.1210,
found: 370.1312 [M+H]+.
3c: 3-(bis(thiophen-3-ylmethyl)amino)propyl methanesulfonate
Rf (Cyclohexane/EtOAc 1:1) = 0.62. 1H NMR (300 MHz, CDCl3): δ = 7.35 - 7.25 (m, 2H), 7.17
- 7.11 (m, 2H), 7.09 - 7.04 (m, 2H), 4.27 (t, J = 6.4 Hz, 2H), 3.60 (s, 4H), 2.92 (s, 3H), 2.56 (t,
J = 6.6 Hz, 2H), 1.91 (qt, J = 6.6Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 140.2, 128.4, 125.8,
122.6, 68.2, 53.3, 49.4, 37.4, 27.3. HRMS-ESI(+): calcd for C14H19NO3S3: 345.0527, found:
346.0582 [M+H]+.

ACCEPTED MANUSCRIPT
3d: 3-(bis(4-(benzyloxy)benzyl)amino)propyl methanesulfonate
1
Rf (Cyclohexane/EtOAc 6:4) = 0.73. H NMR (300 MHz, CDCl3): δ = 7.47-7.38 (m, 10H),
7.36-7.23 (m, 4H), 6.96-6.93 (m, 4H), 5.07 (s, 4H), 4.24 (t, J = 6.4 Hz, 2H), 3.49 (s, 4H), 2.82
13
(s, 3H), 2.53-2.48 (m, 2H), 1.91-1.83 (m, 2H). C NMR (75 MHz, CDCl3): δ = 158.3, 137.4,
131.2, 129.6, 125.7, 122.6, 69.9, 61.5, 54.0, 49.0, 38.4, 26.0. HRMS-ESI(+): calcd for
C32H35NO5S: 545.2236, found: 546.2208 [M+H]+.
3e: 3-(bis([1,1'-biphenyl]-3-ylmethyl)amino)propyl methanesulfonate
1
Rf (Cyclohexane/EtOAc 6:4) = 0.77. H NMR (300 MHz, CDCl3): δ = 7.78-7.44 (m, 18H),
4.30-4.24 (m, 2H), 3.69 (s, 4H), 2.76 (s, 3H), 2.67-2.61 (m, 2H), 2.02-1.93 (m, 2H). 13C NMR
(75 MHz, CDCl3): δ = 154.8, 141.4, 137.1, 132.3, 129.8, 126.7, 125.0, 124.8, 124.5, 123.9,
64.2, 62.1, 52.0, 37.6, 27.9. HRMS-ESI(+): calcd for C30H31NO3S: 485.2025, found: 486.2065
[M+H]+.
3f: 3-(bis([1,1'-biphenyl]-4-ylmethyl)amino)propyl methanesulfonate
1
Rf (Cyclohexane/EtOAc 65:35) = 0.42. H NMR (300 MHz, CDCl3): δ = 7.65 - 7.54 (m, 8H),
7.51-7.41 (m, 8H), 7.39-7.30 (m, 2H), 4.30 (t, J = 6.4 Hz), 3.65 (s, 4H), 2.82 (s, 3H), 2.63 (t, J
= 6.5 Hz, 2H), 1.97 (qt, J = 6.5 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 141.0, 140.1, 138.5,
129.4, 128.9, 127.3, 127.2, 127.1, 68.2, 58.4, 49.5, 37.3, 27.2. HRMS-ESI(+): calcd for
C30H31NO3S: 485.2025, found: 486.2077 [M+H]+.
3g: 3-(bis(naphthalen-2-ylmethyl)amino)propyl methanesulfonate
1
Rf (Cyclohexane/EtOAc 65:35) = 0.50. H NMR (300 MHz, CDCl3): δ = 7.89 - 7.72 (m, 8H),
7.54 (m, 2H), 7.50 - 7.44 (m, 4H), 4.24 (t, J = 6.4 Hz, 2H), 3.76 (s, 4H), 2.65 (t, J = 6.6 Hz,
2H), 2.61 (s, 3H), 1.95 (qt, J = 6.6 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 137.0, 133.5,
132.9, 128.2, 127.8, 127.7, 127.3, 126.2, 125.8, 68.1, 59.1, 49.5, 37.1, 27.3. HRMS-ESI(+):
calcd for C26H27NO3S: 433.1712, found: 434.1776 [M+H]+.
General procedure for the synthesis of the NHBoc amines 4a-g, 6a, 6b:
To a solution of the corresponding methanesulfonate (3a-g) (1.0 eq.) and di-
isopropylethylamine (2.0 eq.) in ACN (0.2 M) at room temperature, the diaminobutane 4 (1.5
eq.) was added dissolved in the minimum amount of ACN. The reaction mixture was stirred
at 60 °C overnight. After evaporation of the solven t at reduced pressure, the residue was

ACCEPTED MANUSCRIPT
dissolved in water and DCM and the organic layer was separated. The aqueous phase was
extracted with DCM three times. The combined organic layers were washed with water and
brine, dried with MgSO4 and evaporated. The crude product was purified by silica gel
chromatography to afford the corresponding Boc-protected compounds. For compounds 4a
and 4b the first fraction of purification was isolated, concentrated and characterized to afford
compounds 6a and 6b.
4a: tert-butyl (4-((3-(dibenzylamino)propyl)amino)butyl)carbamate
Yellow oil, (37% in two steps). Rf (EtOAc/MeOH 9:1) = 0.60. 1H NMR (300 MHz, CDCl3): δ =
7.34-7.24 (m, 10H), 5.18 (s, 1H), 3.54 (s, 4H), 3.09 (s, 2H), 2.63 (t, 2H, J = 6.5 Hz), 2.49 -
2.44 (m, 4H), 1.78 - 7.72 (m, 2H), 1-57 - 1.53 (m, 4H), 1.45 (s, 9H). 13C NMR (75 MHz,
CDCl3): δ = 164.2, 138.2, 128.9, 128.2, 126.9, 76.9, 58.5, 51.4, 49.1, 48.0, 36.2, 28.5, 27.4,
27.1, 25.5. HRMS-ESI(+) calcd for C26H39NO2: 425.3042, found: 426.3126 [M+H]+.
6a: tert-butyl (4-(bis(3-(dibenzylamino)propyl)amino)butyl)carbamate
Yellow oil, (16% in two steps). Rf (EtOAc/MeOH 9:1) = 0.68. 1H NMR (300 MHz, CDCl3): δ =
7.43 - 7.27 (m, 20H), 4.98 (s, 1H), 3.60 (s, 8H), 3.11-3.09 (m, 2H), 2.49-2.37 (m, 10H), 1.70-
1.64 (m, 4H), 1.51 (s, 9H), 1.42-1.34 (m, 2H). HRMS-ESI(+): calcd for C43H58N4O2: 662.4560,
found: 663.4655 [M+H]+.
4b: tert-butyl (4-((3-(bis(4-fluorobenzyl)amino)propyl)amino)butyl)carbamate
Yellow oil, (12% in two steps). Rf (EtOAc/MeOH 9:1) = 0.80. 1H NMR (300 MHz, CDCl3): δ =
7.26 (dd, J = 8.2, 5.8 Hz, 4H), 7.00 (t, J = 8.6 Hz, 4H), 4.98 (s, 1H), 3.48 (s, 4H), 3.10 (d, J =
4.8 Hz, 2H), 2.79 - 2.59 (m, 4H), 2.46 (t, J = 6.3 Hz, 2H), 1.88 (t, J = 6.7 Hz, 2H), 1.63 (d, J =
7.0 Hz, 2H), 1.52 (d, J = 7.0 Hz, 2H), 1.43 (s, 9H). 13C NMR (75 MHz, CDCl3): δ = 162.1 (d, J
= 244 Hz), 155.8, 135.3, 130.9 (d, J = 6 Hz), 115.2 (d, J = 17 Hz), 78.4, 59.0, 50.7, 49.8,
47.9, 32.5, 28.7, 28.3, 27.2, 24.9. HRMS-ESI(+) calcd for C26H38F2N3O2: 461.2854, found:
462.2926 [M+H]+.
6b: tert-butyl (4-(bis(3-(bis(4-fluorobenzyl)amino)propyl)amino)butyl)carbamate
1
Yellow oil, (13% in two steps). Rf (Cyclohexane/EtOAc 5:5) = 0.55. H NMR (300 MHz,
CDCl3): δ = 7.28 (dd, J = 8.4, 5.7 Hz, 8H), 6.99 (t, J = 8.7 Hz, 8H), 4.85 (brs, 1H), 3.48 (s,
8H), 3.08 (m, 2H), 2.35 (m, 10H), 1.57 (m, 4H), 1.45 (s, 9H), 1.37 (m, 4H). 13C NMR (75
MHz, CDCl3): δ = 161.9 (d, J = 204 Hz), 155.8, 135.3, 130.1 (d, J = 6 Hz), 115.0 (d, J = 17

ACCEPTED MANUSCRIPT
Hz), 79.5, 57.5, 53.7, 52.0, 51.7, 40.2, 28.4, 28.1, 24.60, 24.40. HRMS-ESI(+) calcd for
C43H54F4N4O2: 734.4183, found: 735.4354 [M+H]+.
4c: tert-butyl (4-((3-(bis(thiophen-3-ylmethyl)amino)propyl)amino)butyl)carbamate
Yellow oil, (29% in two steps). Rf (EtOAc/MeOH 9:1) = 0.20. 1H NMR (360 MHz, CDCl3): δ =
7.28 (dd, J = 4.3, 3.0 Hz, 2H), 7.11 (dd, J = 2.8, 0.8 Hz, 2H), 7.05 (dd, J = 4.5, 1.0 Hz, 2H),
4.98 (brs, 1H), 3.57 (s, 4H), 3.20 - 3.03 (m, 2H), 2.61 (t, J = 6.8 Hz, 2H), 2.54 (t, J = 6.7 Hz,
2H), 2.46 (t, J = 6.7 Hz, 2H), 1.70 (qt, J = 6.6 Hz, 2H), 1.53-1.42 (m, 13H). 13C NMR (90
MHz, CDCl3): δ = 156.2, 140.6, 128.4, 125.5, 122.3, 79.7, 53.1, 51.4, 49.7, 48.2, 40.6, 28.6,
28.1, 27.7, 27.2. HRMS-ESI(+) calcd for C22H35N3O2S2: 438.2171, found: 438.2242 [M+H]+.
4d: tert-butyl (4-((3-(bis(4-(benzyloxy)benzyl)amino)propyl)amino)butyl)carbamate
Yellow oil, (14% in two steps). Rf (EtOAc/MeOH 9:1) = 0.13. 1H NMR (300 MHz, CDCl3): δ =
7.48 - 7.32 (m, 10H), 7.26 (d, J = 8.5 Hz, 4H), 6.92 (d, J = 8.6 Hz, 4H), 5.04 (s, 4H), 3.48 (s,
4H), 3.07 (s, 2H), 2.40-2.36 (m, 6H), 1.66-1.60 (m, 4H), 1.44 (s, 9H), 1.28-1.24 (m, 2H). 13C
NMR (75 MHz, CDCl3): δ = 157.7, 156.0, 137.2, 132.1, 129.9, 128.5, 127.9, 127.5, 114.5,
70.0, 57.5, 53.2, 52.8, 50.1, 37.3, 28.4, 26.5, 25.2 ppm. HRMS-ESI(+): calcd for C40H51N3O4:
637.3880, found: 638.3921 [M+H]+.
4e: tert-butyl(4-((3-(bis([1,1'-biphenyl]-3-ylmethyl)amino)propyl)amino)butyl)carbamate
Yellow oil, (40% in two steps). Rf (EtOAc/MeOH 8:2) = 0.24. 1H NMR (300 MHz, CDCl3): δ =
7.69-7.33 (m, 18H), 4.75 (s, 1H), 3.63 (s, 4H),3.14-3.12 (m, 2H), 2.67-2.64 (m, 2 H), 2.55-
2.42 (m, 4H), 1.76-1.70 (m, 4H), 1.37-1.23 (m, 2H), 151-1.47 (m, 2H), 1.44 (s, 9H). 13C NMR
(75 MHz, CDCl3): δ = 155.8, 141.2, 141.0, 140.4, 128.7, 128.6, 127.7, 127.5, 127.2, 127.1,
125.7, 58.4, 52.1, 51.8, 47.2, 28.4, 26.8, 25.0 ppm. HRMS-ESI(+): calcd for C38H47N3O2:
577.3668, found: 578.3731 [M+H]+.
4f: tert-butyl (4-((3-(bis([1,1'-biphenyl]-4-ylmethyl)amino)propyl)amino)butyl)carbamate
Yellow oil, (34% in two steps). Rf (EtOAc/MeOH 9:1) = 0.23. 1H NMR (300 MHz, MeOD-d4): δ
= 7.66 - 7.53 (m, 8H), 7.48 - 7.36 (m, 8H), 7.35 - 7.26 (m, 2H), 3.58 (s, 4H), 3.04-2.88 (m,
13
2H), 2.60-2.39 (m, 6H), 1.78 - 1.66 (m, 2H), 1.50 - 1.32 (m, 13H) ppm. C NMR (75 MHz,
MeOD-d4): δ = 160.7, 142.2, 141.3, 140.0, 130.7, 129.9, 128.3, 127.9, 59.2, 52.5, 50.3, 48.8,
41.1, 28.8, 27.8, 27.3 ppm. HRMS-ESI(+): calcd for C38H47N3O2: 577.3668, found: 578.3733
[M+H]+.
4g: tert-butyl (4-((3-(bis(naphthalen-2-ylmethyl)amino)propyl)amino)butyl)carbamate

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Yellow oil, (25% in two steps). Rf (EtOAc/MeOH 9:1) = 0.18. 1H NMR (360 MHz, CDCl3): δ =
7.86 - 7.74 (m, 8H), 7.58 - 7.51 (m, 2H), 7.51 - 7.40 (m, 4H), 4.84 (brs, 1H), 3.73 (s, 4H),
3.07 - 2.95 (m, 2H), 2.63 - 2.50 (m, 4H), 2.43 (t, J = 6.9 Hz, 2H), 1.75 (qt, J = 6.5 Hz, 2H),
1.44 (s, 9H), 1.40 - 1.28 (m, 4H). 13C NMR (90 MHz, CDCl3): δ = 156.1, 137.5, 133.5, 132.9,
128.0, 127.8, 127.6, 127.4, 126.1, 125.7, 58.9, 51.4, 49.6, 48.0, 40.6, 28.6, 27.9, 27.5, 27.1.
HRMS-ESI(+): calcd for C34H43N3O2: 525.3355, found: 526.3422 [M+H]+.
10a: tert-butyl (4-((3-(dibenzylamino)propyl)amino)cyclohexyl)carbamate
Yellowish solid, (41%). Rf (EtOAc/MeOH 9:1) = 0.19. 1H NMR (250 MHz, CDCl3): δ = 7.46 -
7.16 (m, 10H), 4.35 (m, 1H), 3.52 (s, 4H), 3.39 (m, 1H), 2.59 (t, J = 6.8 Hz, 2H), 2.44 (t, J =
6.6 Hz, 2H), 2.28 (brs, 1H), 1.99-1.95 (m, 2H), 1.85-1.81 (m, 2H), 1.71-1.65 (m, 2H), 1.44 (s,
13
9H),1.21-0.98 (m, 2H). C NMR (62.5 MHz, CDCl3): δ = 155.1, 139.7, 128.8, 128.2, 126.8,
59.6, 58.4, 55.6, 51.3, 45.0, 32.2, 31.9, 28.4, 27.1. HRMS-ESI(+) calcd for C28H41N3O2:
451.3199, found: 452.3297 [M+H]+.
10b: tert-butyl (4-((3-(bis(4-fluorobenzyl)amino)propyl)amino)cyclohexyl)carbamate
1
Yellow oil. (29%) Rf (EtOAc/MeOH 8:2) = 0.09. H NMR (250 MHz, CDCl3): δ = 7.11 - 7.08
(m, 4H), 6.87 - 6.81 (m, 4H), 4.21 - 4.20 (m, 1H), 3.31 (s, 2H), 3.25 (s, 1H), 2.28 (t, J = 6.9
13
Hz, 2H), 2.28 (t, J = 6.2 Hz, 2H), 1.74 - 1.62 (m, 6H), 1.28 (s, 9H), 1.14 - 1.10 (m, 4H). C
NMR (62.5 MHz, CDCl3): δ = 161.9 (d, J = 244 Hz), 155.1, 134.9, 130.3 (d, J = 7 Hz), 115.1
(d, J = 20 Hz), 79.3, 57.5, 56.1, 51.1, 49.0, 44.6, 31.7, 30.4, 28.4, 25.9 HRMS-ESI(+): calcd
for C28H39F2N3O2: 487.3010, found: 488.3080 [M+H]+.
12b: tert-butyl (4-(bis(3-(bis(4-fluorobenzyl)amino)propyl)amino)cyclohexyl)carbamate
1
Yellow oil, (25% in two steps). Rf (Cyclohexane/EtOAc 5:5) = 0.22. H NMR (250 MHz,
CDCl3): δ = 7.29 (m, 8H), 7.00 (m, 8H), 4.38 (m, 1H), 3.48 (s, 8H), 3.33 (m, 1H), 2.37 (m,
6H), 2.01 (m, 4H), 1.67 (m, 6H), 1.47 (s, 9H), 1.28 (m, 4H). 13C NMR (62.5 MHz, CDCl3): δ =
161.9 (d, J = 244 Hz), 155.2, 135.3, 130.1 (d, J = 7 Hz), 115.0 (d, J = 21 Hz), 79.4, 57.6,
51.6, 48.7, 42.3, 32.9, 31.5, 28.4, 26.5. HRMS-ESI(+): calcd for C45H56F4N4O2: 760.4339,
found: 761.4440 [M+H]+.
General procedure for synthesis of compounds 5a-g, 7a-b, 11a-b and 13b:
Boc-protected polyamines were deprotected using an excess of 4M HCl in dioxane. The
mixture was stirred overnight at room temperature and the solvent was removed under
reduced pressure to afford the final product as pure hydrochloride salt.

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5a: N1-(3-(dibenzylamino)propyl)butane-1,4-diamine
1
Yellowish solid, (quantitative yield). H NMR (300 MHz, MeOD): δ = 7.67 - 7.58 (m, 10H),
3.55 (s, 4H), 2.80 - 2.76 (m, 6H), 1.76 - 1.59 (m, 6H), 1.42 - 1.37 (m, 2H) ppm. 13C NMR (75
MHz, MeOD): δ = 139.2, 128.7, 128.5, 126.9, 59.1, 52.4, 49.7, 48.4, 40.8, 31.14, 27.3, 27.0
ppm. HRMS-ESI(+): calcd for C21H31N3: 325.2518, found: 326.2611 [M+H]+.
7a: N1,N1-bis(3-(dibenzylamino)propyl)butane-1,4-diamine
1
Yellowish solid, (quantitative yield). H NMR (300 MHz, MeOD): δ = 7.76 - 7.71 (m, 20H),
3.59 (s, 8H), 2.91-2.88 (m, 8H), 1.65 - 1.52 (m, 10H), 1.36 - 1.29 (m, 2H) ppm. 13C NMR (75
MHz, MeOD): δ = 140.2, 138.2, 129.0, 126.3, 60.5, 59.5, 58.5, 55.3, 53.09, 52.9, 40.2, 31.3,
25.0, 24.2 ppm. HRMS-ESI(+): calcd for C38H50N4: 562.4035, found: 563.4121 [M+H]+.
5b: N1-(3-(bis(4-fluorobenzyl)amino)propyl)butane-1,4-diamine
Yellowish solid, (quantitative yield). 1H NMR (300 MHz, MeOD): δ = 7.70 - 7.66 (m, 4H), 7.24
(t, J = 8.4 Hz, 4H), 4.46 (s, 4H), 3.36-3.30 (m, 2H), 3.07 - 3.01 (m, 6H), 2.35 - 2.33 (m, 2H),
1.83 - 1.80 (m, 4H) ppm. 13C NMR (75 MHz, MeOD): δ = 163.6 (d, J = 244 Hz), 133.6, 125.2,
115.9 (d, J = 21 Hz), 56.2, 49.3, 44.5, 38.6, 24.1, 22.8, 20.6 ppm. HRMS-ESI(+): calcd for
C21H29F2N3: 361.2330, found: 362.2391 [M+H]+.
7b: N1,N1-bis(3-(bis(4-fluorobenzyl)amino)propyl)butane-1,4-diamine
Yellowish solid, (quantitative yield). 1H NMR (300 MHz, MeOD): δ = 7.50 - 7.47 (m, 8H), 7.26
- 7.23 (m, 8H), 4.43 (s, 8H), 3.19 - 3.09 (m, 12H), 2.18 - 2.13 (m, 4H), 1.75 - 1.70 (m, 4H).
13C NMR (75 MHz, MeOD): δ = 164.0 (d, J = 246 Hz), 133.8, 125.3, 116.9 (d, J = 21 Hz),
57.5, 52.9, 50.2, 49.6, 39.2, 24.3, 21.0, 19.4. HRMS-ESI(+): calcd for C38H46F4N4: 634.3659,
found: 635.3742 [M+H]+.
5c: N1-(3-(bis(thiophen-3-ylmethyl)amino)propyl)butane-1,4-diamine
Yellowish solid, (quantitative yield). 1H NMR (360 MHz, MeOD): δ = 7.87 (d, J = 1.9 Hz, 2H),
7.61 (dd, , J = 4.9, 2.8 Hz, 2H), 7.35 (d, J = 4.9 Hz, 2H), 4.45 (s, 4H), 3.24 - 3.13 (m, 2H),
3.13 - 2.93 (m, 6H), 2.39 - 2.24 (m, 2H), 1.91 - 1.71 (m, 4H). 13C NMR (90 MHz, MeOD): δ =
130.8, 130.5, 130.2, 128.9, 52.4, 50.3, 48.2, 45.8, 40.0, 25.5, 24.2, 22.0. HRMS-ESI(+):
calcd for C17H27N3S2: 337.1646; found: 338.1713 [M+H]+.
5d: N1-(3-(bis(4-(benzyloxy)benzyl)amino)propyl)butane-1,4-diamine

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1
Yellowish solid, (quantitative yield). H NMR (300 MHz, MeOD): δ = 7.45 - 7.33 (m, 14H),
7.11 - 7.08 (m, 4H), 5.14 (s, 4H), 4.35 (s, 4H), 3.04 - 2.72 (m, 6H), 2.41 - 2.38 (m, 4H), 1.83 -
1.78 (m, 4H). 13C NMR (75 MHz, MeOD): δ = 162.3, 139.5, 133.7, 130.7, 129.7, 125.7,
117.8, 117.6, 72.2, 59.3, 57.0, 48.6, 44.9, 29.9, 28.4, 24.3. HRMS-ESI(+): calcd for
C35H43N3O2: 537.3355, found: 538.3417 [M+H]+.
5e: N1-(3-(bis([1,1'-biphenyl]-3-ylmethyl)amino)propyl)butane-1,4-diamine
1
Yellowish solid, (quantitative yield). H NMR (300 MHz, MeOD): δ = 7.68 - 7.34 (m, 18H),
4.66-4.55 (m, 4H), 3.36 - 3.32 (m, 4H), 2.98 - 2.87 (m, 2H), 2.56 - 2.53 (m, 4H), 1.86 - 1.74
(m, 4H). 13C NMR (75 MHz, MeOD): δ = 142.1, 139.7, 129.8, 129.5, 128.6, 128.2, 127.5,
126.8, 57.5, 52.7, 50.6, 49.6, 38.6, 24.1, 20.7, 19.0. HRMS-ESI(+): calcd for C33H39N3:
477.3144, found: 478.3179 [M+H]+.
5f: N1-(3-(bis([1,1'-biphenyl]-4-ylmethyl)amino)propyl)butane-1,4-diamine
1
Yellowish solid, (quantitative yield). H NMR (360 MHz, MeOD): δ = 7.81-7.51 (m, 12H),
7.48-7.23 (m, 6H), 4.48 (s, 4H), 3.29-3.21 (m, 2H), 3.13-2.87 (m, 6H), 2.56-2.30 (m, 2H),
1.96-1.65 (m, 4H). 13C NMR (90 MHz, MeOD): δ = 144.0, 140.1, 133.1, 130.0, 129.3, 129.0,
128.8, 128.0, 58.2, 50.8, 48.3, 45.9, 40.0, 25.5, 24.2, 22.0. HRMS-ESI(+): calcd for C33H39N3:
477.3144, found: 478.93 [M+H]+.
5g: N1-(3-(bis(naphthalen-2-ylmethyl)amino)propyl)butane-1,4-diamine
Yellowish solid, (quantitative yield). 1H NMR (300 MHz, MeOD): δ = 8.15 - 8.09 (m, 2H), 8.01
- 7.85 (m, 6H), 7.68 (d, J = 7.8 Hz, 2H), 7.61 - 7.50 (m, 4H), 4.66 (s, 4H), 3.43 - 3.21 (m, 2H),
3.16 - 2.90 (m, 6H), 2.51 - 2.33 (m, 2H), 1.94 - 1.66 (m, 4H). 13C NMR (75 MHz, MeOD): δ =
135.2, 134.6, 132.8, 130.4, 129.4, 128.8, 128.6, 128.0, 127.8, 58.9, 51.1, 48.3, 45.9, 40.0,
25.5, 24.2, 22.1. HRMS-ESI(+): calcd for C29H35N3: 425.2831, found: 426.2904 [M+H]+.
11a: N1-(3-(dibenzylamino)propyl)cyclohexane-1,4-diamine
Yellowish solid, (quantitative yield). 1H NMR (300 MHz, MeOD): δ = 7.55 - 7.50 (m, 4H), 7.48
- 7.44 (m, 6H), 4.39 (s, 4H), 3.28 - 3.25 (m, 2H), 3.16 - 3.12 (m, 2H), 3.01 - 2.99 (m, 2H),
2.31 - 2.26 (m, 4H), 2.19 - 2.16 (m, 2H), 1.53 - 1.49 (m, 4H). 13C NMR (75 MHz, MeOD): δ =
139.3, 129.1, 128.9, 127.9, 126.5, 59.0, 58.1, 53.3, 50.5, 49.7, 33.2, 30.6, 27.3. HRMS-
ESI(+): calcd for C23H33N3: 351.2674, found: 352.2641 [M+H]+.
11b: N1-(3-(bis(4-fluorobenzyl)amino)propyl)cyclohexane-1,4-diamine

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Yellowish solid, (quantitative yield). 1H NMR (300 MHz, MeOD): δ = 7.75 - 7.72 (m, 4H), 7.29
- 7.25 (m, 4H), 5.01 (s, 4H), 4.55 - 4.49 (m, 3H), 3.28 - 3.25 (m, 1H), 3.15 - 3.13 (m, 2H),
13
2.31 - 2.24 (m, 6H), 1.62 - 1.57 (m, 4H). C NMR (75 MHz, MeOD): δ = 167.7 (d, J = 244
Hz), 137.7, 129.2, 119.9 (d, J = 20 Hz), 60.3, 53.3, 45.8, 32.1, 30.5, 24.7, 22.3. HRMS-
ESI(+): calcd for C23H31F2N3: 387.2486, found: 388.2547 [M+H]+.
13b: N1,N1-bis(3-(bis(4-fluorobenzyl)amino)propyl)cyclohexane-1,4-diamine
Yellowish solid, (quantitative yield). 1H NMR (300 MHz, MeOD): δ = 7.69 (dd, J = 8.7, 5.2 Hz,
8H), 7.25 (t, J = 8.7 Hz, 8H), 4.51 (s, 1H), 4.47 (s, 8H), 3.27 (m, 6H), 2.53 (m, 4H), 2.22 (m,
4H), 1.74 (m, 4H), 1.31 (m, 2H). 13C NMR (75 MHz, MeOD): δ = 167.7 (d, J = 244 Hz), 135.2,
130.4, 119.9 (d, J = 20 Hz), 58.9, 51.1, 48.3, 45.9, 40.0, 25.5, 24.2, 22.1. HRMS-ESI(+):
calcd for C40H48F4N4: 660.3815, found: 661.3877 [M+H]+.
Culture of Leishmania donovani. MHOM/ET/67/HU3, also called LV9, promastigote forms
were grown in M-199 medium supplemented with 40 mM HEPES, 100 mM adenosine, 0.5
mg.mL-1 haemin, 10% heat-inactivated foetal bovine serum (FBS) and 50 mg.mL-1
gentamycin at 26 °C in a dark environment. Differen tiation of promastigotes into axenic
amastigotes was achieved by dilution of 1.106 promastigotes in 5 mL of axenic amastigote
media M-199 (15 mM KCl; 8 mM glucose; 5 mM glutamine, 2.5% BBLTM trypticaseTM
peptone, 4 mM haemin, and 20% fetal bovine serum). The pH was adjusted to pH 5.5.
Axenic amastigotes were grown at 37 °C under an atm osphere of 5% CO2. All the
experiments were performed with parasites in their logarithmic phase of growth.
In vitro evaluation of Leishmania donovani axenic amastigotes. The evaluations of
activity on axenic amastigotes were adapted from the protocols previously described.26 Two
fold serial dilutions (100 to 0.02 µM) of the compounds were performed in 100 µL of complete
medium (see above) in 96-well microplates. Axenic amastigotes were then added to each
well at a density of 106/mL in a 200 µL final volume. After 72 h of incubation at 37 °C with 5
% CO2, 20 µL of resazurin (450 µM) was added to each well and further incubated in the
dark for 24 h at 37 °C. In living cells, resazurin is reduced in resorufin and this conversion is
monitored by measuring OD570nm (resorufin) and OD600nm (resazurin) (Lab systems Multiskan
MS). The activity of the compounds was expressed as IC50 in µM (concentration of drug
inhibiting 50% of the parasite growth, comparatively to the controls treated with the excipient
only). Miltefosine was used as the reference drug.
In vitro evaluation on Leishmania donovani intramacrophage amastigotes. The
evaluations of activity on intramacrophage amastigotes were adapted from the protocols

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previously described.26 RAW 264.7 macrophages (from ATCC) were plated in 96-well
microplates at a density of 2.104 cells per well and incubated for 24 h at 37 °C wit h 5% CO2.
Axenic amastigotes were differenciated as described above, centrifuged at 2,000 g for 10
min, resuspended in DMEM complete medium, and added to each well to reach a 16:1
parasite to macrophage ratio. After 24 h of infection at 37 °C with 5% CO 2, extracellular
parasites were removed, and DMEM complete medium (100 µL) containing two fold serial
dilutions of the compounds from a maximal concentration of 100 µM was added to each well.
After 48 h of treatment, the medium was removed and replaced by DirectPCR Lysis Reagent
(100 µL; Euromedex) before 3 freeze-thaw cycles at room temperature, addition of 50 µg/ml
proteinase K, and a final incubation at 55°C overni ght to allow cell lysis. 10 µL of each cell
extract was then added to 40 µL of DirectPCR Lysis reagent containing Sybr Green I (0.05
%; Invitrogen). DNA fluorescence was monitored using Mastercycler® realplex (Eppendorf).
This method detects both host cell DNA and parasite DNA. Total DNA of treated infected
macrophages were compared with total DNA of untreated infected macrophages.
Mastercycler® realplex detects significant differences allowing a measurement of compound
activities. The activity of the compounds was expressed as IC50. Miltefosine was used as the
reference drug.
Culture of Trypanosoma brucei gambiense and in vitro trypanocidal evaluation. Strain
FéoITMAP/1893 was grown in a medium constituted of prepacked Iscove’s modified
Dulbecco's medium (Gibco, BRL) supplemented with 36 mM NaHCO3, 1 mM hypoxanthine,
0.05 mM bathocuproine, 0.16 mM thymidine, 0.2 mM 2-mercaptoethanol, 1.5 mM L-cysteine,
10 % heat-inactivated foetal bovine serum, 100 IU penicillin and 100 ꢀg.mL-1 streptomycin.
Two fold serial dilutions of the compounds were performed in 100 µL of the same medium in
96-well microplates. Trypanosomes were then added to each well at 4.104/mL in 200 µL final
volume. After 72 h of incubation at 37 °C with 5% C O2 in the dark, 20 µL of 450 µM resazurin
in aqueous solution was added to each well and further incubated for 6 h at 37 °C with 5%
CO2. Cell viability was then monitored as described above. In this evaluation, the activity was
expressed in IC50 (concentration inhibiting the parasite growth by 50%) and the reference
drug used was pentamidine diisethionate.
Cytotoxicity assays. Cytotoxicity was evaluated on RAW 264.7 macrophages as previously
described.26 Briefly, cells were plated in 96-well microplates at a density of 2.104 cells per
well. After an incubation of 24 h at 37 °C with 5% CO2, the medium was removed in each
well, and 100 µL of DMEM complete medium containing two fold serial dilutions of the
compounds was added to each well. After 48 h of incubation at 37 °C with 5% CO 2, 10 µL of
resazurin (450 µM) was added to each well, and further incubated in the dark for 4 h at 37 °C
with 5% CO2. Cell viability was then monitored as described above. The cytotoxicity of the

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compounds was expressed as CC50 (Cytotoxic Concentration 50%: concentration inhibiting
the macrophages growth by 50%).
Metabolic assays. Human plasma was diluted 10-fold in PBS buffer. 150 µL of 1 mM
solution of compounds 5a, 5f and 11b were incubated for 0, 2 and 12 h at 37 °C. After e ach
time point, 150 µL of ice-cold acetonitrile were added. Precipitated proteins were removed by
centrifugation for 20 min at 3000 rpm before analysis. The supernatants were filtered through
0.2 µm syringe filters. HPLC analyses were performed on an Agilent 1260 series using an
analytical “Zorbax Eclipse Plus C18 column” (3.5 µm, 100 x 4.6 mm) maintained at 25°C.
Human pooled S9 fractions were diluted 20-fold in PBS buffer (1 mg/ml of proteins). 100 µM
and 1 mM solutions of 11b were incubated for 0 and 12 h at 37 °C for a final volume of 150
µL. Metabolization by hepatic enzymes, such as cytochromes or cytosolic enzymes was
investigated in presence or absence of 1 mM NADPH.
Enzymatic assays. Recombinant T. brucei TryR was prepared and assayed as described
previously.27 Stock solutions of the inhibitors were prepared in DMSO. The activity was
measured at 25 °C in a total volume of 1 mL of 40 m M Hepes, 1 mM EDTA, pH 7.5 (Assay
Buffer) in the presence of 100 ꢀM NADPH and 5-10 mU TryR containing 5% DMSO. The
reaction was started by adding TS2 and NADPH consumption was followed
spectrophotometrically at 340 nm. To determine the percentage of inhibition, the assay
contained 100 ꢀM and 40 ꢀM TS2, respectively, in the absence and presence of a fixed
concentration (40 µM) of inhibitor. The type of inhibition was determined by a Lineweaver-
Burk plot. The experiment was performed in triplicates and the results are expressed as
mean value ± standard deviation.
In vivo evaluation.
In vivo, Swiss mice were infected intraperitoneally with 10,000 trypanosomes (T. brucei
brucei CMP strain) and then randomly allocated to groups and treated by intraperitoneal
route with compound 11b at the single dose of 100 mg/kg or 10 mg/kg. Six mice were used
per batch. Control mice were treated with the reference compounds (pentamidine and
melarsamine) or the excipient. The follow-up of parasitemia was monitored at days 2, 3, 4,
and 30. The efficiency was evaluated with the survival time in comparison with control mice
(mean survival time, 3.5 ± 0.5 days).
ACKNOWLEDGMENTS

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This work was supported by Initiative d’Excellence Paris-Saclay (IdEx) and Laboratoire
d’Excellence en Recherche sur le Médicament et l’Innovation Thérapeutique (LabEx
LERMIT). The authors are grateful to the COST Action CM1307 for the collaboration
established between Heidelberg and Paris-Sud laboratories. The authors would like to thank
P. Grellier who kindly provided the strain FéoITMAP/1893 of Trypanosoma brucei
gambiense. R. Luise Krauth-Siegel is also acknowledged for providing the T. b. TryR
enzyme.
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