Bioorganic & Medicinal Chemistry 25 (2017) 84–90

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Synthesis and in vitro antikinetoplastid activity

of polyamine–hydroxybenzotriazole conjugates
Elodie Jagu a, Sébastien Pomel b, Alba Diez-Martinez a, Florence Ramiandrasoa a, R. Luise Krauth-Siegel c,
Stéphanie Pethe a, Casimir Blonski a, Raphaël Labruère a,⇑, Philippe M. Loiseau b,⇑
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
c
Center of Biochemistry of Heidelberg University, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany

a r t i c l e i n f o a b s t r a c t

Article history: Thirteen new polyamine derivatives coupled to hydroxybenzotriazole have been synthesized and
Received 4 July 2016 evaluated for their in vitro antikinetoplastid activity. Trypanosoma Trypanothione reductase (TryR) was
Revised 20 September 2016 envisioned as a potential target. Among all tested molecules, only one compound, a N3-spermidine–
Accepted 10 October 2016
benzotriazole derivative, displayed relevant inhibitory activity on this enzyme but was not active on
Available online 12 October 2016
parasites. The corresponding Boc-protected spermidine–benzotriazole was however trypanocidal against
Trypanosoma brucei gambiense with an IC50 value of 1 lM and was completely devoid of cytotoxicity. On
Keywords:
the intramacrophage amastigotes of Leishmania donovani, a N2-spermidine conjugate of this series,
Antikinetoplastids
Trypanosoma brucei
exhibited an interesting IC50 value of 3 lM associated with both low cytotoxicity against axenic
Leishmania donovani Leishmania donovani. These new compounds are promising leads for the development of antikinetoplastid
Polyamines agents and their targets have to be deciphered.
Benzotriazole Ó 2016 Elsevier Ltd. All rights reserved.
Trypanothione reductase

1. Introduction regulated by or imply these cationic organic compounds. It was

observed that Kinetoplastid activity is interrupted by targeting
Human African trypanosomiasis and leishmaniasis, caused by metabolism and transport of polyamines by efficient drugs, and
kinetoplastid parasites (Trypanosoma brucei gambiense, Try- thus polyamine metabolism and transporter have been validated
panosoma brucei rhodesiense and Leishmania sp., respectively) are as efficient drug targets in kinetoplastids.7 Many natural or syn-
considered as challenging neglected diseases by the World Health thetic polyamine derivatives have been studied for their antipara-
Organization (WHO).1 The diseases are responsible for 120,000 sitic ability and most of them were found as disruptors of the
deaths per year and for the disability of many more. The wide trypanothione metabolism.8–10 This antioxidant system is specific
spread of parasite strains that are resistant to current drugs and for trypanosomes and involves the Trypanothione reductase (TryR)
insecticides is one of the major concerns for parasitologists. Fur- as a main enzyme,11 which is responsible for the reduction of
thermore, a very small number of drugs are currently available dithiol form of trypanothione into the disulfide form. As trypanoth-
and most of these cause severe host toxicity. Since the trypanocidal ione—the natural substrate of TryR—bears spermidine backbone in
and antileishmanial drugs in human medicine exhibit such draw- its structure, many spermidine derivatives have been studied and
backs, the discovery of safe compounds is urgently necessary.2,3 identified as potential inhibitors of TryR, such as the natural prod-
However, the new drugs should be more specific in their action, ucts kukoamine A and lunarine and also synthetic derivatives.4–6,8–
10,12,13
targeting well identified biological mechanisms in the parasites Antikinetoplastid activity was mainly observed with conju-
in order to prevent or minimize adverse effects. gates bearing a hydrophobic moiety, particularly an aryl group,
In the last thirty years, trypanocidal and antileishmanial polya- directly linked to the polyamine.4–6 Nonetheless, trypanocidal
mine conjugates were developed as polyamine metabolism inhibi- and antileishmanial activities were independent of the length
tors.4–6 Polyamines are simple linear polycations, detected in all and the nature (diamine, triamine, . . .) of the polyamine
living organisms. An impressive number of cellular processes are conjugates.
In line with our program toward the development of polya-
mine-based antikinetoplastid agents14 as well as published
⇑ Corresponding authors.
data,4–6 we decided to couple polyamines to a heteroaryl moiety
E-mail addresses: [email protected] (R. Labruère), philippe.loiseau@
u-psud.fr (P.M. Loiseau). found in several antiparasitic agents. The benzotriazole scaffold

http://dx.doi.org/10.1016/j.bmc.2016.10.013
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
 E. Jagu et al. / Bioorg. Med. Chem. 25 (2017) 84–90 85

1,3-propanediamine 1a, putrescine 1b, spermidine 1c, and

spermine 1d—were coupled to the benzotriazole moiety through
an amide bond (Fig. 1 and Scheme 1). Only one hydroxybenzotria-
zole group was inserted in each case. Spermine was linked to the
benzotriazole exclusively via its primary amine group. Regarding
the dissymmetric polyamine, spermidine 1c, N1-, N2- and
N3-spermidine conjugates were prepared through the linkage of
either the primary or secondary amines. The latter compounds
were designed to study the biological importance of the
benzotriazole position on the spermidine.
Polyamine derivatives were synthetized from previously
reported tert-butyloxycarbamate (Boc) protected polyamines.
Many carbamates have been designed as prodrugs24 as they have
chemical stability and capability to permeate cell membranes.25
Also, some carbamates have been found to be more active than
Figure 1. Targeted benzotriazole–polyamine conjugates.
the corresponding free amines (i.e. the antileishmanial activity).26
Literature data on the metabolic hydrolysis of therapeutic carba-
(Fig. 1) was chosen since it is widely used by medicinal chemists mates showed that those derived from aliphatic amines had longer
for the development of drug candidates,15,16 and was found as life-span27 than the corresponding free amines, maybe due to an
antiprotozoal agent against amoeba17 and Kinetoplastids.18 This increase of the bioavailability into the organism. In addition, we
heterocycle was originally incorporated as a bioisosteric replace- targeted Trypanosoma b. gambiense trypomastigotes and Leishma-
ment of imidazole nucleus or the corresponding benzene-fused nia donovani amastigote, two parasites that apparently do not
compound, benzimidazole, a common pharmacophore clinically express polyamine transporters.28–31 For all these reasons, we eval-
used in anthelminthic drugs.19 The benzotriazole scaffold is able uated the Boc protecting intermediates, as well as the final free
to bind various targets via non-covalent interactions such as amine derivatives. The Boc derivatives, as more lipophilic com-
hydrogen bonds, van der Waals interactions, and p stacking. Com- pounds, could cross more easily the parasite plasma membrane.
pounds bearing this scaffold display diverse pharmacological activ-
ities often associated with good water solubility and bioavailability 2. Results and discussion
as well as limited toxicity. Among pharmacologically active benzo-
triazoles, the particular 1-hydroxybenzotriazole motif (Fig. 1) was 2.1. Synthesis
found having antibacterial,20,21 antiviral22 and antiproliferative
activities as histone deacetylase (HDAC) inhibitors.23 In these com- The key synthons 3a–d were obtained through the conjugation
pounds, either ether or ester bonds link various molecular scaffolds by peptidic coupling of hydroxybenzotriazole acetic acid 6 and
to 1-hydroxybenzotriazole through its hydroxyl group. Given their polyamines 2a–d protected at appropriate positions (Scheme 1).
structure, these aryl scaffolds coupled to polyamines could display The carboxylic acid 6 was prepared in 62% yield by the alkyla-
antikinetoplastid activity through inhibition of TryR. tion of 1-hydroxybenzotriazole 5 with chloroacetic acid in alkaline
In this study, hydroxybenzotriazole group was considered as a medium using the method published by Boido et al.32 In order to
privileged scaffold since it can be readily incorporated starting react the benzotriazole scaffold at desired positions of the polyami-
from the commercially available peptide coupling reagent nes, selective N-Boc protection of other functional amino groups
1-hydroxybenzotriazole. Four polyamines of biological interest— was made. The various N-Boc protected polyamines 2a–d were

Scheme 1. Synthesis of benzotriazoles conjugates. Reagents and conditions: (a) ClCH2COOH, NaOH, EtOH, 40 °C, 16 h (62%); (b) BOP, NEt3, DCM/DMF, rt, 16 h (22–80%); (c)
HCl/dioxane (4 M), rt, 16 h (50%-quant).
86 E. Jagu et al. / Bioorg. Med. Chem. 25 (2017) 84–90

synthesized according to previously reported procedures.33–35 most of the tested polyamines. The conjugates bearing a diamine
Amide bond formation between N-Boc polyamines 2a–d and car- moiety either Boc-protected or not (3a–b and 4a–b) lacked any sig-
boxylic acid 6 yielded the protected intermediates 3a–d in 10– nificant biological activity.
92% yield. Removal of the Boc group was carried out in a 4 M Four compounds exhibited IC50 values below 5 lM against
hydrochloric solution in dioxane, to afford hydrochloride salts of T. b. gambiense. The most active compounds with IC50 less than
the polyamine–hydroxybenzotriazole conjugates 4a–d. 2 lM proved to be Boc-protected spermidine derivatives conju-
gated with the benzotriazole at one of their primary amines (3c
2.2. Bioactivity of compounds and 3c0 ). Interestingly, compound 3c0 was specifically trypanocidal
as it did not exert any in vitro antileishmanial activity. This speci-
Thirteen compounds (Fig. 2) were tested in vitro against both ficity was also found although slightly less pronounced for com-
T. b. gambiense (strain FéoITMAP/1893) trypomastigotes according pound 3c. Their free counterparts, compounds 4c and 4c0 led to a
to the protocol by Pomel et al.36 and Leishmania donovani dramatic decrease of the in vitro trypanocidal activity. In particular,
(MHOM/ET/67/HU3, also called LV9) axenic and intramacrophage compound 4c was sixty times less active than compound 3c
amastigotes according to the protocols by Balaraman et al.37 (Boc-protected). This is also valid, although to a lesser extent, in
Pentamidine and miltefosine were included in the assay as the case of the spermidine derivatives branched at the central nitro-
reference drugs for T. b. gambiense and L. donovani, respectively. gen (3c00 and 4c00 ). The lipophilic Boc-protected conjugates (in
The cytotoxic effect of the compounds on RAW 264.7 mammalian agreement with predictive C log P values shown in Table 1) exerted
cells was evaluated in order to determine the selectivity index (SI) a better activity against T. b. gambiense than the corresponding
toward parasitic cells according to Balaraman et al.37 The results conjugates with free amines. In contrast to 3c00 which is slightly
are depicted in Table 1. cytotoxic (12.5 ± 1.4 lM), the spermidine derivatives linked to the
Firstly, spermidine has been described in the literature as pos- benzotriazole via their primary amines (3c and 3c0 ) have very inter-
sibly activating trypanosome multiplication in one T. b. gambiense esting in vitro trypanocidal activity with an SI higher than 50.
strain (Wellcome strain) whereas this effect was not significantly The same trend was observed with the spermine conjugates (3d
observed in T. b. brucei ILtat 1.4 strain.39 In our study, we observed and 4d) but the differences between Boc- and non-protected
that the IC50 of spermidine was about 3 lM on our T. b. gambiense derivatives were only weak.
Féo strain. These apparent contradictory results could be ascribed Regarding L. donovani axenic amastigotes, the two most active
to the trypanosome polymorphism leading to a large variability compounds, 3c00 (Boc-protected) and 4d (without Boc), exhibited
of drug susceptibility in this parasite genus, as it is observed for IC50 of about 10 lM. The presence of Boc did not enhance the
the clinically used drug, eflornithine. in vitro antileishmanial activity since 3d is three times less active
Secondly, we verified that the common nucleus, (benzotria- than the unprotected spermine 4d. It should be noted that all
zolyl)oxyacetic acid 6, was not active against the tested parasites. active compounds against L. donovani were also among the most
This compound was also not cytotoxic and this was the case for active ones against T. b. gambiense.

Figure 2. Polyamine-benzotriazole derivative structures of 6, 3a–d and 4a–d.

 E. Jagu et al. / Bioorg. Med. Chem. 25 (2017) 84–90 87

Table 1
Antiparasitic activities and cytotoxicity of benzotriazole conjugates

Compounds C log Pa Trypanosoma brucei SITbc Leishmania donovani LV9 SIaac Leishmania donovani LV9 SIiac Cytotoxicity on
gambiense IC50 ± SDb axenic amastigotes IC50 ± SD intramacrophage amastigotes macrophages
(lM) (lM) IC50 ± SDb (lM) CC50 ± SDb (lM)
Spermidine 1.2 3.3 ± 1.6 29 8.3 ± 3.6 12 >100 >100
6 0.8 >100 — >100 — >100 — >100
3a 1.5 28.8 ± 8.3 3 >100 — 56.7 ± 1.9 2 >100
4a 0.2 >100 — >100 — 29.5 ± 2.1 3 >100
3b 1.6 >100 — >100 — >100 — >100
4b 0.3 9.5 ± 6.9 11 >100 — >100 — >100
3c 1.8 1.2 ± 0.2 85 60.5 ± 10.5 1.6 67.8 ± 15.2 1 >100
4c 0.3 72.0 ± 12.2 1 >100 1.5 >100 — >100
3c0 1.8 2.0 ± 0.5 50 >100 — 41.4 ± 4.9 2 >100
4c0 0.3 38.7 ± 4.4 3 >100 9 >100 — >100
3c00 2.8 3.2 ± 0.3 4 11.5 ± 1.8 1 3.1 ± 0.6 4 12.5 ± 1.4
4c00 0.1 25.3 ± 5.3 4 >100 — 23.4 ± 2.0 4 >100
3d 3.8 3.5 ± 0.5 10 27.0 ± 0.9 1.4 34.7 ± 5.7 1 37.4 ± 1.0
4d 0.3 13.8 ± 4.1 7 10.9 ± 1.6 9 38.6 ± 12.2 3 >100
Miltefosine ND — 1.9 ± 0.6 1.5 ± 0.3 >100
Pentamidine 0.0021 ± 0.0015 ND ND >100

ND: not determined.

a
C log P: Calculated log P with Virtual Computational Chemistry Laboratory, http://www.vcclab.org, 2005.38
b
50% growth inhibitory concentration.
c
SI: Selectivity Index calculated from Cytotoxicity Concentration 50% (CC50)/Inhibitory Concentration 50% (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.

Only compound 3c00 was active on intramacrophage amastig- C o m p o u n d [4 0 µ M ]

otes of L. donovani with an IC50 value of 3 lM and a SI of 4. Inter- 100
estingly, the compound displayed a higher activity on
intramacrophage amastigotes of L. donovani than on the axenic 80
amastigotes. This enhanced activity could be due to physiological
% In h ib it io n

modifications of the parasite once inside the macrophage.

60
As opposed to trypanocidal activity, no clear-cut relationships
could be established between the antileishmanial activity and
the lipophilicity of the compounds. Among the different targets 40
in kinetoplastid affected by polyamine derivatives, TryR is the most
frequently identified.4–6 Functional TryR is essential to combat 20
oxidative stress in these parasites. This NADPH-dependent flavoen-
zyme reduces trypanothione disulfide (TS2) and displays structural
0
and functional similarities to glutathione reductase (GSR), the clos-
)

O
est related enzyme in mammalian cells. However, TryR and human
2

2
S

S
c
c

GSR have mutually exclusive substrate specificities.40 Based on its M

4
4

D
o

preference for positively charged ligands,41,42 we decided to study

M

M
µ

µ
0

our compounds as putative inhibitors of recombinant T. brucei

0

(4
(1

TryR.
All compounds were subjected to TryR assays using a fixed con- Figure 3. Inhibition of TbTryR by 4c. The activity was measured following the
centration of 40 lM in assay buffer containing 5% DMSO. The activ- NADPH consumption at 340 nm as described in the Section 4. The assays contained
a fixed concentration of 100 or 40 lM of TS2 and 40 lM of inhibitor. The controls
ity was measured in the presence of 40 and 100 lM TS2,
contained the same amount of DMSO used to dissolve the compounds. 4c displayed
respectively, and the percentage of inhibition was calculated. Only the same degree of inhibition at both substrate concentrations.
one compound showed a noticeable activity at 40 lM (Fig. 3). The
spermidine derivative 4c inhibited the TryR to 80% under these
conditions. The finding that the degree of inhibition was indepen-
dent of the substrate concentration indicated that this compound
did not act as pure competitive ligand.
In light of the interesting preliminary activity of this spermidine
derivative, we determined the inhibitor constant and type of inhi-
bition. The Lineweaver–Burk plot showed that 4c behaves as a
mixed-type inhibitor, with calculated Ki- and Ki0 -values of 6.7 lM
and 16.8 lM, respectively (Fig. 4).
This type of inhibition was already observed on TryR.12
Moreover, inhibitors with such a good Ki value also display strong
trypanocidal activity in vitro and surprisingly, 4c was not active
against T. b. gambiense.4 Interestingly, the Boc-protected
counterpart of this polyamine, 3c, exhibited high activity against Figure 4. Lineweaver–Burk plot of compound 4c.The assays contained 0, 10, and
T. b. gambiense but was not an inhibitor of TryR. As described 40 lM inhibitor, respectively, and the TS2 concentration was varied. The data are
above, the trypanocidal activity correlated with the lipophilicity the mean of three determinations ± standard deviations.
88 E. Jagu et al. / Bioorg. Med. Chem. 25 (2017) 84–90

of the polyamine. Thus, we may hypothesize that the more lipophi- to give 6 as a white powder (4.45 g, 62%); mp 169.5 °C (170.5–
lic Boc-protected polyamine (3c) diffuses via hydrophobic interac- 171.5 °C lit.16); 1H NMR (300 MHz, MeOD): d = 8.01–7.81 (m, 2H),
tion through the membrane of the parasite and once inside the cell, 7.61 (t, J = 7.5 Hz, 1H), 7.47 (t, J = 7.5 Hz, 1H), 5.28 ppm (s, 2H);
the Boc moieties would be removed to provide the free polyamine HRMS-ESI(+): calcd for C8H7N3O3: 194.0417, found: 194.0414 [M
4c. +H]+.
Even though less active towards the parasite, the spermidine 4c0
and the spermine 4d followed a comparable trend, in line with a 4.3. General procedure for synthesis of compounds 3a–d
prodrug-like effect supposed for 3c to 4c. Indeed, the polyamines
4c0 and 4d were not trypanocidal whereas their Boc-protected To a solution of carboxylic acid 6 in DCM/DMF (1:1) was added
equivalent (3c0 and 3d) displayed significant activity against T. b. successively the protected polyamine (0.5 mmol, 1 equiv), BOP
gambiense. Since 4c0 and 4d are not inhibitors of TryR, we assume reagent (0.5 mmol, 1 equiv) followed by the addition of triethy-
that these deprotected polyamines have other target(s) within the lamine (2 mmol, 4 equiv). The mixture was stirred overnight at
parasite. Similarly, the increased activity of 3c00 on intra- room temperature, the solvent was then evaporated under reduced
macrophage amastigotes compared to axenic L. donovani could pressure and the residue was taken up with brine and ethyl acet-
be due to structural modifications of this polyamine such as Boc ate. The organic phase was separated and washed successively
removal by macrophage metabolism. with 5% aq citric acid, 5% aq NaHCO3 and water, dried over Na2SO4,
and evaporated. Purification was carried out by column chro-
3. Conclusion matography (Cyclohexane/Ethyl Acetate).

We have designed and synthesized an original series of polya- 4.3.1. Compound 3a

mine–hydroxybenzotriazole conjugates. Only 4c showed real TryR Column chromatography (Cyclohexane/Ethyl Acetate 5:5);
inhibition activity meaning that this enzyme is not the main target Light-colored oil (20%). 1H NMR (300 MHz, CDCl3): d = 8.00 (d,
of our derivatives. In vitro evaluation against two parasites, T. b. J = 8.0 Hz, 1H), 7.73 (d, J = 8.0 Hz, 1H), 7.58 (t, J = 7.5 Hz, 1H), 7.43
gambiense and L. donovani allowed identification of two polyamine (t, J = 7.5 Hz, 1H), 5.00 (s, 2H), 3.47 (q, J = 6.3 Hz, 2H), 3.23 (q,
derivatives as potential antikinetoplastid agents. Compound 3c J = 6.3 Hz, 2H), 1.78–1.68 (m, 2H), 1.44 (s, 9H) ppm; 13C NMR
appeared as the most active derivative against T. b. gambiense with (90 MHz, CDCl3): d = 165.9, 156.7, 128.6, 125.2, 120.2, 110.2, 79.8,
an IC50 value of 1 lM and without any cytotoxicity up to 100 lM. 77.9, 36.1, 29.9, 24.2 ppm; HRMS-ESI(+): calcd for C16H23N5O4:
Another compound of this series, 3c00 , exhibited an IC50 value of 372.1642, found: 372.1631 [M+Na]+.
3 lM towards intramacrophage amastigotes of L. donovani which
are particularly difficult to target. We will further investigate these 4.3.2. Compound 3b
two compounds through microsomal stability and then through Column chromatography (Cyclohexane/Ethyl Acetate 5:5);
in vivo antikinetoplastid activity on experimental African try- Light-colored oil (54%). 1H NMR (300 MHz, CDCl3): d = 8.00 (d,
panosomiasis (for 3c) and leishmaniasis mice models (for 3c00 ). J = 7.5 Hz, 1H), 7.60 (t, J = 7.5, 1.5 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H),
7.44 (t, J = 7.5, 1.5 Hz, 1H), 4.95 (s, 2H), 3.45 (q, J = 7.5, 1.5 Hz,
2H), 3.16 (q, J = 7.5, 1.5 Hz, 2H), 1.59 (m, 4H), 1.43 (s, 9H) ppm;
4. Experimental 13
C NMR (90 MHz, CDCl3): d = 164.9, 157.1, 128.8, 124.3, 121.2,
110.8, 79.7, 79.1, 39.1, 28.4, 26.5 ppm; HRMS-ESI(+): calcd for
4.1. General information C17H25N5O4: 386.1799, found: 386.1789 [M+Na]+.

All chemical reagents were of analytical grade, obtained from 4.3.3. Compound 3c
Acros, Alfa Aesar, or Aldrich, and used without further purification Column chromatography (Cyclohexane/Ethyl Acetate 5:5);
except for hydroxybenzotriazole which was recrystallized from Light-colored oil (10%). 1H NMR (300 MHz, CDCl3): d = 7.98 (d,
diethyl ether. Solvents were obtained from SDS or VWR-Prolabo. J = 8.0 Hz, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.38
Dichloromethane was dried on molecular sieves and used immedi- (t, J = 7.5 Hz, 1H), 5.32 (s, 2H), 3.15–2.88 (m, 6H), 1.85–1.82 (m,
ately. Chromatography was performed using silica gel (35–70 lm, 2H), 1.48–1.40 (m, 6H), 1.43 (s, 18H) ppm; 13C NMR (90 MHz,
Merck). Concentration of solutions was performed under dimin- CDCl3): d = 165.2, 155.6, 153.5, 128.2, 124.7, 119.8, 110.3, 79.3,
ished pressure at temperature below 40 °C using rotary evaporator. 78.7, 39.6, 36.9, 28.4, 26.2 ppm; HRMS-ESI(+): calcd for
Analytical TLC was performed using Silica Gel 60 F254 pre-coated C25H40N6O6: 543.2902, found: 543.2880 [M+Na]+.
aluminum plates (Merck). Spots were visualized by treatment with
ninhydrin revelator followed by heating and/or by absorbance of 4.3.4. Compound 3c0
UV light at 254 nm. NMR spectra were collected on a Bruker DRX Column chromatography (Cyclohexane/Ethyl Acetate 5:5);
300 (1H at 300 MHz and 13C at 75 MHz), or 360 NMR Avance 360 Light-colored oil (21%). 1H NMR (300 MHz, MeOD): d = 8.00 (d,
(1H at 360 MHz and 13C at 90 MHz), spectrometer using MestRe- J = 8.0 Hz, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 7.5 Hz, 1H), 7.48
Nova software. Chemical shift are reported in ppm (d) and coupling (t, J = 7.5 Hz, 1H), 5.08 (s, 2H), 3.19–3.16 (m, 6H), 3.05–2.99 (m,
constants in Hz (J). 1H NMR spectra were performed in CDCl3, CD3- 2H), 1.69–1.66 (m, 2H), 1.44–1.42 (m, 22H) ppm; 13C NMR
OD or D2O. High-resolution mass spectrometry (HRMS) analyses (90 MHz, MeOD): d = 168.4, 157.4, 156.4, 128.5, 125.3, 118.9,
were performed by electrospray with positive ionization (ESI+). 109.2, 79.7, 78.7 36.8, 27.3, 25.1 ppm; HRMS-ESI(+): calcd for
C25H40N6O6: 543.2902, found: 543.2874 [M+Na]+.
4.2. Synthesis of (benzotriazolyl)oxyacetic acid (6)
4.3.5. Compound 3c00
To a solution of hydroxybenzotriazole (5 g, 37 mmol) in ethanol Column chromatography (Cyclohexane/Ethyl Acetate 3:7);
(100 mL), was added 3.48 g of chloroacetic acid (1 equiv) and 3 g of Light-colored oil (17%). 1H NMR (300 MHz, CDCl3): d = 7.94 (d,
sodium hydroxide (2 equiv). The solution was stirred overnight at J = 8.0 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.34
40 °C. After solvent evaporation, the residue was dissolved in water (t, J = 7.5 Hz, 1H), 4.04 (s, 2H), 3.32–3.28 (m, 4H), 3.15–3.07 (m,
and washed with ether. Then the aqueous layer was acidified with 4H), 1.68–1.66 (m, 6H), 1.39 (s, 18H) ppm; 13C NMR (90 MHz,
1 N HCl and the precipitate was collected and washed with water CDCl3): d = 169.4, 156.4, 155.9, 128.2, 124.8, 119.7, 110.0, 79.6,
 E. Jagu et al. / Bioorg. Med. Chem. 25 (2017) 84–90 89

78.8, 32.4, 28.1, 27.6, 24.5 ppm; HRMS-ESI(+): calcd for 4.4.6. Compound 4d
C25H40N6O6: 543.2902, found: 543.2925 [M+Na]+. Light-colored oil (quantitative yield). 1H NMR (360 MHz, D2O):
d = 7.85–7.26 (m, 4H), 5.11 (s, 2H), 3.34–3.30 (m, 2H), 3.15–3.05
4.3.6. Compound 3d (m, 4H), 2.08–2.04 (m, 4H), 2.01–1.90 (m, 2H), 1.88–1.75 (m, 8H)
Column chromatography (Cyclohexane/Ethyl Acetate 4:6); ppm; 13C NMR (90 MHz, D2O): d = 169.6, 130.2, 127.5, 119.9,
Light-colored oil (92%). 1H NMR (300 MHz, CDCl3): d = 8.02 (d, 110.3, 78.3, 46.9, 46.8, 44.9, 44.5, 36.5, 36.0, 25.3, 23.7, 22.7 ppm;
J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.5 Hz, 1H), 7.40 HRMS-ESI(+): calcd for C18H31N7O2: 378.2612, found: 378.2599
(t, J = 7.5 Hz, 1H), 5.00 (s, 2H), 3.25–3.23 (m, 2H), 3.13–3.10 (m, [M+H]+.
8H), 1.89–1.87 (m, 2H), 1.68–1.66 (m, 8H), 1.42 (s, 27H) ppm;
13
C NMR (90 MHz, CDCl3): d = 165.6, 156.6, 156.3, 155.6, 143.6, 4.5. Culture of Leishmania donovani MHOM/ET/67/HU3, also
128.5, 125.1, 109.2, 79.9, 77.9, 47.0, 44.3, 43.8, 43.4, 37.8, 37.4, called LV9
35.7, 29.8, 28.6, 27.8, 26.1, 26.0 ppm; HRMS-ESI(+): calcd for
C33H55N7O8: 678.4112, found: 678.4078 [M+H]+. Promastigote forms were grown in M-199 medium supple-
mented with 40 mM HEPES, 100 mM adenosine, 0.5 mgmL1 hae-
4.4. General procedure for synthesis of compounds 4a–d min, 10% heat-inactivated fetal bovine serum (FBS) and
50 mgmL1 gentamycin at 26 °C in a dark environment. Differen-
Boc-protected polyamines were deprotected using an excess of tiation of promastigotes into axenic amastigotes was achieved by
4 M HCl in dioxane. The mixture was stirred overnight at room dilution of 1106 promastigotes in 5 ml of axenic amastigote media
temperature and the solvent was removed under reduced pressure M-199 (15 mM KCl; 8 mM glucose; 5 mM glutamine, 2.5% BBLTM
to afford the final product as pure hydrochloride salt. trypticaseTM peptone, 4 mM haemin, and 20% Fetal Bovine Serum).
The pH was adjusted to pH 5.5. Axenic amastigotes were grown
4.4.1. Compound 4a at 37 °C under an atmosphere of 5% CO2. All the experiments were
Light-colored solid (quantitative yield). 1H NMR (300 MHz, performed with parasites in their logarithmic phase of growth.
D2O): d = 7.89 (d, J = 7.9 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.57 (t,
J = 7.5 Hz, 1H), 7.45 (t, J = 7.5 Hz, 1H), 5.03 (s, 2H), 3.26–3.24 (m, 4.6. In vitro evaluation of Leishmania donovani axenic
2H), 2.88–2.85 (m, 2H), 1.79–1.76 (m, 2H) ppm; 13C NMR amastigotes
(90 MHz, D2O): d = 170.3, 131.3, 130.8, 128.3, 127.6, 120.6, 114.3,
110.9, 79.1, 46.8, 37.7, 28.0 ppm; HRMS-ESI(+): calcd for Amastigote forms were suspended to yield 107 cellsml1. The
C11H15N5O2: 250.1291, found: 250.1294 [M+H]+. maximum final compound concentrations used were 100 lM. Trip-
licates were used for each concentration. Cultures were incubated
4.4.2. Compound 4b at 37 °C for 72 h in the dark and under a 5% CO2 atmosphere, and
Light-colored solid (72%). 1H NMR (300 MHz, D2O): d = 8.00 (d, then the viability of the amastigotes was assessed. Parasite growth
J = 7.5 Hz, 1H), 7.60 (t, J = 7.5, 1.5 Hz, 1H), 7.55 (d, J = 7.5 Hz, 1H), was determined by using SYBR Green I, a dye with marked fluores-
7.44 (t, J = 7.5, 1.5 Hz, 1H), 4.94 (s, 2H), 3.11–3.07 (m, 2H), 2.79– cence enhancement upon contact with parasite DNA. Parasites
2.66 (m, 2H), 1.48–1.39 (m, 4H) ppm; 13C NMR (90 MHz, D2O); were lysed following Direct PCR-Cell Genotyping without DNA iso-
d = 165.5, 126.8, 123.6, 117.5, 109.8, 75.8, 46.6, 37.4, 24.4 ppm; lation protocol (Euromedex, France). 10 lL of lysed parasite solu-
HRMS-ESI(+): calcd for C12H17N5O2: 264.1455, found: 264.1445 tion of each well was added to 40 lL of PCR-Cell reagent
[M+H]+. containing the SYBR green I in a qPCR plate of 96 wells, and the
contents were mixed. Fluorescence was measured with Mastercy-
4.4.3. Compound 4c cler ep realplex (Eppendorf, France). Fluorescence obtained was
Light-colored solid (50%). 1H NMR (300 MHz, D2O): d = 7.82 (d, compared to those from the range obtained with different parasite
J = 7.9 Hz, 1H), 7.77 (d, J = 7.9 Hz, 1H), 7.69 (t, J = 7.5 Hz, 1H), 7.58 densities. The antileishmanial activity was expressed as IC50 in lM
(t, J = 7.5 Hz, 1H), 4.31 (s, 1H), 4.12 (s, 1H), 3.09–3.02 (m, 8H), (concentration of drug inhibiting 50% of the parasite growth, com-
2.10–2.01 (m, 2H), 1.76–1.73 (m, 4H) ppm; 13C NMR (90 MHz, paratively to the controls treated with the excipient only). Milte-
D2O): d = 172.0, 130.9, 127.4, 113.5, 78.5 39.4, 24.5 ppm; HRMS- fosine was used as reference compound.
ESI(+): calcd for C15H24N6O2: 321.2034, found: 321.2031 [M+H]+.
4.7. In vitro evaluation on intramacrophage amastigotes
4.4.4. Compound 4c0
Light-colored solid (quantitative yield). 1H NMR (300 MHz, The mouse monocyte/macrophage cell line RAW 264.7 was
D2O): d = 7.90 (d, J = 7.9 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.66 (t, maintained in DMEM supplemented with 10% heat-inactivated
J = 7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 5.02 (s, 1H), 4.04 (s, 1H), fetal bovine serum. RAW 264.7 cells were seeded into a 96-well
3.19–3.06 (m, 2H), 3.04–2.94 (m, 6H), 2.08–2.02 (m, 2H), 1.75– microtiter plate at a density of 5103 cells/well in 100 ll of
1.50 (m, 4H) ppm; 13C NMR (90 MHz, D2O): d = 168.8, 129.9, DMEM. After incubation in a 5% CO2 incubator at 37 °C for
127.3, 119.7, 109.9, 78.5, 44.8, 38.8, 24.3 ppm; HRMS-ESI(+): calcd 24 h, the culture medium was replaced with 100 lL of fresh
for C15H24N6O2: 321.2034, found: 321.2026 [M+H]+. DMEM containing a suspension of amastigote forms of 106
cellsmL1. After incubation in a 5% CO2 incubator at 37 °C for
4.4.5. Compound 4c00 24 h, the culture medium was replaced with 100 lL of fresh
Light-colored solid (quantitative yield). 1H NMR (300 MHz, DMEM containing the test compounds for a new incubation of
D2O): d = 7.92 (d, J = 7.9 Hz, 1H), 7.85 (d, J = 7.9 Hz, 1H), 7.57 (t, 48 h. The viability of the amastigotes into macrophages was then
J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 4.22 (s, 2H), 3.43–3.49 (m, assessed using the SYBR Green I (Invitrogen, France) incorpora-
4H), 2.98–2.93 (m, 4H), 1.95–1.92 (m, 2H), 1.71–1.60 (m, 4H) tion method. Fluorescence obtained was compared to those from
ppm; 13C NMR (90 MHz, D2O): d = 158.3, 130.9, 127.5, 120,2, the range obtained with parasite infected cell and non-infected
110.2, 78.3, 45.6, 37.2, 24.3 ppm; HRMS-ESI(+): calcd for cell densities. Results were expressed as IC50. Miltefosine was
C15H24N6O2: 321.2034, found: 321.2022 [M+H]+. used as reference compound.
90 E. Jagu et al. / Bioorg. Med. Chem. 25 (2017) 84–90

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Supplementary data (1H NMR spectra for all compounds) asso-

ciated with this article can be found, in the online version, at
http://dx.doi.org/10.1016/j.bmc.2016.10.013.