Synthesis, biological, and photophysical studies of molecular rotor-based fluorescent inhibitors of the trypanosome alternative oxidase

Article abstract of DOI:10.1016/j.ejmech.2021.113470

We have recently reported on the development and trypanocidal activity of a class of inhibitors of Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to lipophilic cations via C14 linkers to enable optimal interaction with the enzyme's active site. This strategy resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has not been formally established. We therefore synthesized a series of fluorescent analogues to visualize accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and their photophysical and biological properties were studied. These probes display a red emission, with a high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver) mitochondria was estimated to 66 ± 20% of the total. The compounds inhibited recombinant TAO enzyme in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity (SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected, presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins, whereas the probe was located, virtually exclusively, in the trypanosome's mitochondrion.

Full text of DOI:10.1016/j.ejmech.2021.113470

European Journal of Medicinal Chemistry 220 (2021) 113470
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, biological, and photophysical studies of molecular rotor-
based fluorescent inhibitors of the trypanosome alternative oxidase
,
, c,
Eduardo J. Cueto-Díaz a 1, Godwin U. Ebiloma b d, Ibrahim A. Alfayez c,
Marzuq A. Ungogo c , Leandro Lemgruber , M. Carmen Gonzalez-García ,
,
e
c
f
ꢀ
g
g
a
b
ꢀ
ꢀ
Maria D. Giron , Rafael Salto , Francisco Jose Fueyo-Gonzalez , Tomoo Shiba ,
f
f
f
c
ꢀ
ꢀ
Juan A. Gonzalez-Vera , Maria Jose Ruedas Rama , Angel Orte , Harry P. de Koning ,
Christophe Dardonville a
,
*
a
ꢀ
Instituto de Química Medica, IQMeCSIC, Juan de la Cierva 3, Ee28006, Madrid, Spain
b Graduate School of Science and Technology, Department of Applied Biology, Kyoto Institute of Technology, Kyoto, 606-8585, Japan
c Institute of Infection, Immunity and Inflammation, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom
d School of Health and Life Sciences, Teesside University, Middlesbrough, United Kingdom
e Department of Veterinary Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ahmadu Bello University, Zaria, Nigeria
f Departamento de Fisicoquimica, Facultad de Farmacia, Universidad de Granada, C. U. Cartuja, Ee18071, Granada, Spain
g Departamento de Bioquimica y Biologia Molecular II. Facultad de Farmacia, Universidad de Granada, C. U. Cartuja, Ee18071, Granada, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
We have recently reported on the development and trypanocidal activity of a class of inhibitors of
Trypanosome Alternative Oxidase (TAO) that are targeted to the mitochondrial matrix by coupling to
lipophilic cations via C14 linkers to enable optimal interaction with the enzyme’s active site. This strategy
resulted in a much-enhanced anti-parasite effect, which we ascribed to the greater accumulation of the
compound at the location of the target protein, i.e. the mitochondrion, but to date this localization has
not been formally established. We therefore synthesized a series of fluorescent analogues to visualize
accumulation and distribution within the cell. The fluorophore chosen, julolidine, has the remarkable
extra feature of being able to function as a viscosity sensor and might thus additionally act as a probe of
the cellular glycerol that is expected to be produced when TAO is inhibited. Two series of fluorescent
inhibitor conjugates incorporating a cationic julolidine-based viscosity sensor were synthesized and
their photophysical and biological properties were studied. These probes display a red emission, with a
high signal-to-noise ratio (SNR), using both single- and two-photon excitation. Upon incubation with
T. brucei and mammalian cells, the fluorescent inhibitors 1a and 2a were taken up selectively in the
mitochondria as shown by live-cell imaging. Efficient partition of 1a in functional isolated (rat liver)
mitochondria was estimated to 66 20% of the total. The compounds inhibited recombinant TAO enzyme
in the submicromolar (1a, 2c, 2d) to low nanomolar range (2a) and were effective against WT and
multidrug-resistant trypanosome strains (B48, AQP1-3 KO) in the submicromolar range. Good selectivity
(SI > 29) over mammalian HEK cells was observed. However, no viscosity-related shift could be detected,
presumably because the glycerol was produced cytosolically, and released through aquaglyceroporins,
whereas the probe was located, virtually exclusively, in the trypanosome’s mitochondrion.
Received 1 February 2021
Received in revised form
26 March 2021
Accepted 10 April 2021
Available online 16 April 2021
Keywords:
Trypanosome alternative oxidase (TAO)
inhibitor
Trypanosoma brucei
Molecular rotor
Fluorescent probe
2,4-dihydroxybenzoic acid derivative
Julolidine
© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-
NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Sleeping sickness, a parasitic disease caused by African try-
panosomes (i.e. T. brucei gambiense and T. b. rhodesiense), is a
neglected tropical disease that put millions of people at risk in sub-
Saharan Africa [1]. The parasites are transmitted by the bite of
infected tsetse flies and the illness takes place in two phases: an
* Corresponding author.
E-mail address: dardonville@iqm.csic.es (C. Dardonville).
Current affiliation: Centro de Astrobiología, CSICeINTA, Eꢀ28850 Torrejon de
1
ꢀ
Ardoz, Spain.
https://doi.org/10.1016/j.ejmech.2021.113470
0223-5234/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
early hemolymphatic stage with few specific symptoms that is
followed by a central nervous system infection leading to the death
of the patient. Treatment options for sleeping sickness have relied
for decades on relatively toxic drugs that require long administra-
tion protocols and hospitalization of the patients [2]. In the last
years, a new orally active drug, fexinidazole, has been registered for
both stages of gambiense sleeping sickness. Even though the
approval of fexinidazole constitutes important progress in the
battle against the disease, the possible occurrence of cross-
resistance between the two nitroimidazole drugs fexinidazole
and nifurtimox (both used for late-stage gambiense HAT) should not
be overlooked. Moreover, rhodesiense sleeping sickness is still the
most neglected disease with only suramin (early stage) and the
extremely toxic arsenical melarsoprol in propylene glycol for the
late-stage disease [2]. Moreover, brucei-group trypanosomes
including T. b. brucei, T. vivax, T. congolense, T. evansi and
T. equiperdum are involved in diseases of valuable livestock, not just
in Africa but also in South America and Asia [3]. Hence, the search
for new compounds with different modes of action against T. brucei
and related species remains an important goal in tropical diseases
research.
Bloodstream form (BSF) trypomastigotes, the human-infective
form of T. brucei, depend on aerobic glycolysis for their energy
supply because they have no functional oxidative phosphorylation
pathway [4]. In its place, they use a plant-like alternative oxidase
(trypanosome alternative oxidase, TAO) to re-oxidize the NADPH
that is produced during glycolysis [5]. This enzyme, which is an
interfacial inner mitochondrial membrane protein [6,7], is unique
and essential to BSF trypanosomes; inhibitors of TAO have shown
in vitro and in vivo activity in mouse models of trypanosomiasis,
showing the potential of this target for chemotherapy [8].
In earlier studies, we showed that it was possible to enhance the
trypanocidal activity of TAO inhibitors using lipophilic cations (LC;
triphenylphosphonium or quinolinium salts), which are used as
mitochondrion-targeting moieties (Chart 1). The inhibitor and LC
group must be separated by a long flexible chain, typically con-
sisting of 14 methylene units, to allow optimal interaction with the
TAO catalytic site [8e11]. However, to date we have not been able to
verify the extent to which these LC-coupled inhibitors, featuring
the unusual long linkers, do distribute to the trypanosome mito-
chondrion, nor the dynamics of the cellular uptake of the drugs by
the parasites.
its photophysical properties (red emitting fluorescence) [12].
Moreover, as a molecular rotor, this group can function as a vis-
cosity sensor, which might allow the detection of the cellular
glycerol produced as a metabolic consequence of TAO inhibition,
thereby reporting on both the localization and the effectiveness of
the probe as an on-target inhibitor.
Molecular rotors are fluorescent probes known to form twisted
intramolecular charge-transfer (TICT) complexes in the excited
state, the fluorescence quantum yield of which is dependent on the
nearby milieu [13]. The new probes 1a and 2aed generated a red,
high signal-to-noise ratio (SNR) emission and single-/two-photon
excitation fluorescence that allowed precise mitochondrial locali-
zation in trypanosomes and human cells. The rotational properties
make these TAO inhibitors sensitive to viscosity changes (i.e. inside
the mitochondrion or in the presence of glycerol): a weak emission
was observed in non-viscous media whereas a higher fluorescence
was observed in high viscosity medium due to the restriction of the
free rotation of the molecular rotor [14]. As the inhibition of TAO
leads to a shift in trypanosomal glucose metabolism and the pro-
duction of large amounts of glycerol as a glycolytic end product
[8,15], we investigated the possibility that these molecular probes
would allow not only the study of the uptake and distribution of LC-
coupled TAO inhibitors in trypanosomes, but also the real-time
monitoring of TAO inhibition itself, through the production of
cellular glycerol.
2. Results and discussion
2.1. Synthesis of compounds 1a, 2aed, and 12
Compound 1a was prepared in five synthetic steps from
commercially available orcinol 3 (Scheme 1). Formylation of 3 un-
der Vilsmeier-Haack conditions led to 4 in good yields (73%); sub-
sequent Pinnick oxidation yielded 5a. In the next step, selective
chlorination of 5a was problematic as a mixture of mono- (5c,
~30%) and di-chlorinated (5b, ~70%) compounds was obtained.
Isolation of pure 5c was not achieved as both 5b and 5c displayed
similar Rfs. Different protocols were tried (e.g. working at ꢀ20 ꢁC,
modifying reaction time or equiv. of reagents, etc.) to obtain the
pure mono-chlorinated compound 5c following this strategy
[16,17]. However, we were unable to reproduce the reported results
(i.e. 92% yield of the monochlorinated compound 5b after column
chromatography) [17] after several attempts. However, when
freshly purchased acetaldehyde was used in the reaction, the di-
chlorinated derivative 5b was isolated in 90% yield without
further purification. Esterification of benzoic acid 5a using an
excess of 1,14-dibromotetradecane [18] (2.5 eq) afforded ester 6a
after silica chromatography. The same protocol was used to syn-
thesize 6c starting from the 70/30 mixture of 5b/5c obtained in the
previous step. In this way, the mono-chlorinated analogue 6c was
Here, we report the synthesis, photophysical characterization,
and biological activity of fluorescent TAO inhibitor probes (1a,
2aed) that incorporate a julolidine-based molecular rotor with a
cationic pyridinium salt as mitochondrial targeting moiety (Fig. 1).
(E)-4-(2-(8-hydroxy-julolidine-9-yl)vinyl-1-methylpyridine-1-ium
bromide (HJVPI) was chosen for its compact and lipophilic nature,
which would not meaningfully interfere with cellular distribution
engendered by the LC group, and its low cytotoxicity, as well as for
Fig. 1. Structures of fluorescent TAO inhibitor conjugates 1a, 2a, 2c, and 2d bearing a molecular rotor as viscosity sensor (in red) and a TAO inhibitor pharmacophore (in blue).
2

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
Scheme 1. Synthesis of julolidine derivative 1a and chloro intermediates 7b,c.
Reagents and conditions: (i) POCl3, DMF, r.t, 18 h; (ii) NaClO2, Na2HPO4, DMSO/H2O, 0 ꢁC then r.t, 18 h; (iii) H3NSO3, CH2O, NaClO2, H2O/THF, 0 ꢁC, 20 min; (iv) 1,14-
dibromotetradecane, NaHCO3, CH3CN, 80 ꢁC, 72 h (5a and 5c); (v) 14-bromotetradecan-1-ol [19] Ph3P, DIAD, THF, 0/rt, 3 h (5b); (vi) 4-methylpyridine, CH3CN, 80 ꢁC, 3 days;
(vii) 9-Formyl-8-hydroxyjulolidine, pyridine, 70 ꢁC, 24 h.
isolated in low yield (12%) by silica chromatography. Compound 6b
was synthesized with 47% yield from 5b to 14-bromotetradecan-1-
ol [19] using the Mitsunobu protocol. Quaternary pyridinium salts
intermediates 7a-c were produced in quantitative yields after 3
days of reaction with 4-methylpyridine. Finally, Knoevenagel
condensation between 7a and 9-formyl-8-hydroxyjulolidine yiel-
ded the target compound 1a incorporating the julolidine-based
viscosity sensor. This last reaction, which is low yielding (typi-
cally 20e30%), was a limiting step of this synthetic route. We found
that the best results were obtained with 0.6e0.7 eq of piperidine,
EtOH as solvent, working at 65e70 ꢁC. The reaction time should not
exceed 2 days to avoid degradation of the product and promotion of
other by-products. Hence, part of the starting material was recov-
ered during the purification step.
structure-activity relationships (SAR) of these series.
In order to obtain a carboxylic group in the pharmacophore, the
strategy was modified so that the C14 methylene chain is grafted to
OH in position 4 via Mitsunobu reaction [9]. The synthetic pathway
leading to compounds 2a-d is depicted in Scheme 2. Compound 8
was synthesized from ethyl 2,4-dihydroxy-6-methylbenzoate and
bromotetradecan-1-ol using the Mitsunobu protocol as reported
[9]. Reaction of 8 with 4-methylpyridine in refluxing CH3CN gave
the quaternary pyridinium salt 9 (71%). Saponification of 9 with
3.5 M KOH at 50 ꢁC yielded the carboxylic acid 10 in 72% yield.
Knoevenagel condensation between 9-formyl-8-hydroxyjulolidine
and 9 yielded the expected ethyl ester target compound 2a (21%).
In contrast, Knoevenagel condensation between 9-formyl-8-
hydroxyjulolidine and 10 gave the decarboxylated by-product 2c
instead of 2b. The methyl ester 2d, obtained from 2a by trans-
esterification with MeOH in aqueous KOH, was isolated by pre-
parative HPLC whereas 2b could not be isolated in pure form.
The julolidine molecular rotor lacking the TAO pharmacophoric
moiety (compound 12) was synthesized in two steps from 4-
methylpyridine (Scheme 3). The low yield (7%) of the second step
of this synthesis was attributed to the formation of the 4-methyl-1-
(14-(piperidin-1-yl)tetradecyl)pyridin-1-ium by-product.
Finally, due to the difficulties encountered with the selective
chlorination of 2,4-dihydroxy-6-methylbenzoic acid (5a), the syn-
thesis of the mono-chlorinated analogue of 1a (R1 ¼ Cl, R2 ¼ OH)
was dropped. However, the biological activity of the chlorinated
intermediates was determined (Table 1) to complement the
Table 1
Photophysical properties of 1a.
2.2. Photophysical characterization of TAO inhibitor conjugates
Tau (ns)b
a
Solvent
FF
Water
EtOH
0.0013 0.0005
0.0057 0.0006
0.0094 0.0004
0.027 0.002
0.049 0.007
0.080 0.010
0.129 0.019
0.137 0.010
0.065 0.007
0.077 0.002
0.097 0.002
0.175 0.002
0.26 0.03
0.36 0.06
0.47 0.09
0.59 0.15
Compounds 1a and 2aed incorporate the same molecular rotor
in their structure, separated from the TAO pharmacophore by a C14
methylene linker. Hence, they should display similar photophysical
properties. Therefore, only the photophysical properties of 1a were
characterized in detail. For comparison, the photophysical proper-
ties of 2a are given in Table S1.
Emission properties are sensitive to viscosity changes. Com-
pound 1a holds a typical DepeA chromophore structure, which
usually results in marked solvatochromic behavior due to polar
solvents stabilizing the excited-state through dipoleedipole in-
teractions and hydrogen bonding [20]. Even more important is the
EtOH:Glycerol 8:2
EtOH:Glycerol 1:1
EtOH:Glycerol 3:7
EtOH:Glycerol 2:8
EtOH:Glycerol 1:9
Glycerol
a
Calculated using two different concentrations and two different excitation
wavelengths. The employed reference was Rhodamine 101 (in EtOH).
b
Fluorescence lifetimes are amplitude-weighted average values from tri-
exponential decay functions, globally fitted to five experimental decay traces ob-
tained with lex ¼ 530 nm and at the emission wavelengths 570, 590, and 610 nm.
3

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
Scheme 2. Synthesis of julolidine derivatives 2a-d. Reagents and conditions: (i) DIAD, PPh3, HOe(CH2)14eBr, THF, r.t., 18 h; (ii) 4-methylpyridine, CH3CN, 80 ꢁC, 3 days; (iii) 1) 3.5 M
KOH, H2O/MeOH (2/1), 50 ꢁC, 48 h, 2) 3.5 M HCl; (iv) 9-formyl-8-hydroxyjulolidine, pyridine, 75 ꢁC, 24 h; (v) KOH, H2O/MeOH, r.t., 24 h.
Scheme 3. Synthesis of compound 12. Reagents and conditions: (i) Bre(CH2)14eBr, CH3CN, 48 h; (ii) 9-Formyl-8-hydroxyjulolidine, piperidine, 75 ꢁC, 24 h.
fact that the HJVPI moiety behaves as a common rotor fluorophore,
thus exhibiting viscosity dependence of its photophysical proper-
ties [12]. The absorption spectrum in the visible range, attributed to
the HJVPI rotor, showed a maximum ranged from circa 540 nm in
organic alcohols to 620 nm in water, and usually exhibiting dual-
band absorption (Figure S1 in the Supplementary Materials). The
fact that the spectrum is clearly blue-shifted in organic alcohols
indicated a specific interaction with hydroxyl groups. Interestingly,
a change in the absorption spectrum was observed when different
amounts of glycerol (ranging from 1 to 20%) were added to a so-
lution of 1a in H2O (Figure S2). This behavior was ascribed to the
conformational switch from a twisted structure (lmax ¼ 540 nm) to
a planar form of 1a (lmax ¼ 620 nm), probably caused by steric
hindrance of the hydrogen bonds between organic alcohols and the
HJVPI hydroxyl group. This steric hindrance makes the twisted
structure more stable in alcohols, whereas in water, a small mole-
cule, the planar structure is more stable in the ground state.
Importantly, the rotor behavior of the HJVPI chromophore
Fig. 2. Emission spectra of 1a (normalized by the absorbance at the excitation wave-
length, lex ¼ 530 nm) in EtOH:glycerol mixtures.
resulted in a large dependence of the emission properties with the
environment viscosity. Fast rotation of the fluorophore in the
excited state resulted in low fluorescence emission efficiency of 1a
as evidenced in water and EtOH (see Table 1).
three different exponential components. Interestingly, in the red-
edge of the emission band, the short decay time exhibited nega-
tive pre-exponential factors in high viscosity mixtures, indicating
the presence of excited-state dynamics in which the more fluo-
rescent form is formed. This was also supported by a slight spectral
shift in the emission maximum from 627 nm in EtOH to 632 nm in
glycerol. The marked change in the fluorescence of 1a was attrib-
uted to the geometry relaxation to a state with substantial intra-
molecular charge transfer (see more details in Computational
Results section) [21].
In contrast, as expected for a molecular rotor-based fluorophore,
the fluorescence properties of 1a were sensitive to the viscosity of
the environment as evidenced in the emission spectra of 1a in
different mixtures EtOH:glycerol, gathered in Fig. 2. The emission
quantum yield, FF, of 1a was enhanced 24-fold in more viscous
systems containing a higher proportion of glycerol (from 0.006 in
8:2 EtOH to 0.14 in pure glycerol). The enhancement with solvent
viscosity in the average fluorescence lifetime ranged from 77 ps in
EtOH to 590 ps in pure glycerol (Table 1). In any case, these average
lifetime values represent fast deactivation dynamics, composed by
Given that the fluorophore part of molecules 2a-d is the same
4

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
than that in 1a, similar results are expected for the other com-
pounds. Indeed, 2a exhibited very similar photophysical properties
to 1a in water, ethanol and ethanol:glycerol mixtures (Table S1,
Figure S3).
The fluorescence studies of 1a and 2a recorded in
glyceroleethanol mixtures agree with the interconverting ‘twisted’
and ‘planar’ conformers [22] of compound 1a, respectively. In
organic alcohols, such fluorophore undergoes [23] fast intra-
molecular electron transfer from the donor to the acceptor part of
the molecule. This electron transfer is accompanied by intra-
molecular DeA twisting around the single bond (Fig. 3) and pro-
duces a relaxed perpendicular structure.
2.3. Ab initio calculations
Fig. 4. Optimized (a) ground excited state (So) and (b) first excited state (S1) structures.
The julolidine in the ground state minimum is almost planar with f2 ¼ 17ꢁ. The long
The optimized geometries of 1a obtained at B3LYP/6-31 þ G**
level of theory for the ground (a) and excited (b) states are shown in
Fig. 4 and Table S2. DFT calculations give a slightly twisted
axis of the molecular rotor is almost straight and is bent with an angle
a
¼ 130ꢁ. In the
first excited state, the molecular rotor is twisted f2 ¼ 90ꢁ between the donor and the
(
f2 ¼ 17ꢁ) optimized geometry for the ground state whereas a
acceptor with an angle
a
¼ 122ꢁ.
larger dihedral angle (f2 ¼ 90ꢁ) between the julolidine and the
pyridinium salt is found in the excited state. In addition, the angle
a
for B48 and AQP1-3 KO, respectively (P < 0.001). Pentamidine is
transported by both P2/TbAT1 [27] and HAPT1, the latter of which
has been identified as TbAQP2 [28].
for C7e23CeC24 is slightly different from the ground state to the
excited state (130ꢁ and 122ꢁ respectively). Of note is the shorter
C4eC7 bond length in the excited state, as a result of the favored
charge transfer (CT) from the nitrogen of the julolidine moiety to
the pyridinium group (Table S2).
As regards to enzyme inhibition, the carboxylate probes 2a-
d
were more potent inhibitors of recombinant TAO
(IC50 ¼ 1.6e145 nM) than the benzoate derivative 1a (360 nM). The
higher inhibitory potency of ethyl and methyl ester substituents
(2a, 2d) vs H (2c) agreed with the SAR results observed previously
for analogous series lacking the fluorescent julolidine-based vis-
cosity sensor [9]. This could indicate that these compounds bind to
TAO in a similar fashion.
The charge delocalization of the cationic moiety seemed to play
a role in TAO inhibition as shown by the micromolar IC50 values of
the pyridinium intermediates (7a, 10) compared to the sub-
micromolar inhibition displayed by the julolidine-based com-
pounds (1a, 2a-d) or the triphenylphosphonium and quinolinium
analogues reported before (Chart 1) [9,10]. In line with previous
reports [8,29,30], the addition of one (7c) or two chlorine atoms
(7b) in the TAO-binding pharmacophore enhanced TAO inhibition
(7c z 7b > 7a). However, and unexpectedly, the addition of chlo-
rine atoms correlated negatively with the activity against T. brucei
(7b < 7c < 7a). Since 7a is the most cytotoxic compound of the
Further mechanical computations were conducted to investi-
gate the role of frontier molecular orbitals (FMO) crucial to un-
derstand and acquire information about the molecule after
excitation. As shown in Figure S4, the HOMO electronic distribution
of 1a is mainly located on the julolidine core whereas the electron
density of the LUMO/LUMOþ1 is localized on the electron with-
drawing pyridinium group, which indicates effective charge
transfer from donor to acceptor group. The major contribution to
the transition bands involves the excitation from HOMO-1 to LUMO
(coefficient value ~ 0.65) and the minor contribution HOMO-LUMO
(coefficient value ~ 0.65).
2.4. Biological activity
The trypanocidal activity of the fluorescent probes 1a and 2a-
d was determined in vitro against wild type (s427) and drug
resistant strains of T. brucei (i.e. B48, AQP1-3 KO) with a resazurin-
series (CC50 ¼ 0.76
mM), the observed nanomolar trypanocidal ef-
fect could be due, to some extent, to unspecific toxicity upon
accumulation to high levels into trypanosomes.
based assay [9,10]. The EC50 values ranged from 0.5 to 1.0
mM
against WT parasites with selectivity indices over mammalian cells
ranging from 29 to >100 (Table 2). Importantly, the compounds
were equipotent against the multi-drug resistant strain B48 that
lacks the P2/TbAT1 and High Affinity Pentamidine Transporter
(HAPT1) drug transporters (P > 0.05 by Student’s unpaired t-test)
[24]. Interestingly, however, several of the analogues were signifi-
cantly (P < 0.05, P < 0.01) more active against a strain lacking all
three T. b. brucei aquaporins [25], indicating potential sensitization
under conditions where glycerol, a metabolic waste product in T. b.
brucei [26], accumulates to higher levels in the cell. The reference
drug pentamidine displayed resistance factors (RF) of 102 and 6.9
As expected, the julolidine derivative 12 lacking the 2,4-
dihydroxy-6-methylbenzoic acid pharmacophore did not inhibit
TAO although it showed submicromolar activity against T. brucei
(Table 2). Compound 12 holds the lipophilic pyridino-julolidine
conjugated cation like 1a, 2c, and 2d; thus, the observed activity
possibly reflects a different mechanism of action via other mito-
chondrial targets of T. brucei. For the whole series, it is expected that
accumulation of these compounds in the T. brucei mitochondrion
will affect the mitochondrial membrane potential Jm because of
Fig. 3. Twisted and planar conformations of compound 1a.
5

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
Table 2
Biological activity of fluorescent TAO inhibitor conjugates 1a and 2aed, and synthetic intermediates 6c, 7a-c, 10 and 12: in vitro activity against T. brucei wild-type (WT) and
drug-resistant lines, cytotoxicity against mammalian cells, and inhibition of TAO.
T. b. brucei EC50
(
m
M)
Cytotoxicity CC50
HEKe
(
m
M)
rTAO IC50 (m
M)g
Cmpd
S427 (WT)a
B48b
RFc
AQP1-3 KOd
RFc
SIf
1a
2a
2c
2d
12
6c
7a
7b
1.01 0.04
0.80 0.05
0.50 0.07
0.54 0.02
0.43 0.01
6.84 0.60
0.055 0.008
2.55 0.17
0.24 0.05
19.7 0.4
1.19 0.05
0.85 0.05
0.63 0.05
0.59 0.01
0.49 0.04
10.1 0.9
0.06 0.01
2.64 0.18
0.22 0.04
23.3 0.8
1.17
1.07
1.25
1.10
1.16
1.48
1.04
1.03
1.53
1.18
102
1.048 0.050
0.663 0.054
0.43 0.05
0.44 0.04
0.34 0.02*
13.1 0.3**
NTh
1.03
0.83
0.85
0.81
0.79
1.91
>100
>100
29.1
44.1
43.6
62.6
8.2
13.8
1.7
182
>10
0.36 0.07
23.3 0.1
22.2 2.2
23.6 1.5
26.7 0.6
55.9 0.7
0.76 0.14
4.4 0.5i
43.7 2.7
>200
0.0016 0.0002
0.145 0.015
0.042 0.02
>5
0.75 0.19
>5
0.91 0.08
0.72 0.20
1.80 0.13
NT
7c
10
0.19 0.02
16.6 0.9*
0.034 0.03
0.80
0.84
6.9
Pentamidine
Phenylarsine oxide
0.0049 0.0004
0.50 0.05
0.46 0.04
*, P < 0.05 and **, P < 0.01 from WT control EC50 by Student’s unpaired t-test; n ¼ 3.
a
Trypomastigotes of T. b. brucei s427 (n ꢂ 3).
b
T. brucei cell line from which the TbAT1 gene was knocked out, followed by in vitro adaptation to pentamidine [24].
Resistance factor relative to WT.
c
d
e
f
T. brucei cell line from which all aquaporins were knocked out [25] (n ꢂ 3).
Cytotoxicity on human embryonic kidney cells (n ¼ 3).
Selectivity index (SI) ¼ CC50/EC50 (T. brucei WT).
g
h
i
Purified recombinant trypanosome alternative oxidase (
D
MTS-TAO) [10] from T. b. brucei (n ¼ 3).
Not tested.
Partial inhibition.
the (1) accumulation of cations in the mitochondrial matrix and (2)
disruption of mitochondrial functions involved in maintaining the
ion gradients. Similar effects have been shown for various dia-
midines [31], choline-derived dications [32], phenanthridines [33]
and bisphosphonium compounds [34,35]. The latter were shown to
act on the mitochondrial FoF1 ATPase of T. brucei [36]. Consequently,
the lack of correlation between TAO inhibition and efficacy against
T. brucei of this series is probably the consequence of various factors
including multi-target activity.
null) and which is extra sensitized to TAO inhibitors [25]. However,
Fig. 5B reveals no difference between the fluorescence of aqp1-3
null cells incubated with 12 in the presence of TAO38 or TAO47 or
the same volume of PBS. As even the addition of 10 mM glycerol to
the culture medium failed to elicit a significant fluorescence
response (P > 0.05 for both strains), we conclude that the metab-
olite is very effectively excluded from entering the mitochondrion.
This is consistent with the latest insights into T. brucei mitochon-
drial metabolism, as there is no known metabolic role for glycerol
in the mitochondrion, and no known mitochondrial carrier for it.
Moreover, although cardiolipin biosynthesis, which is essential in
T. brucei [40,41], is finalized inside the mitochondrion, the glycerol-
utilising step is conducted in the cytosol [42].
2.4.1. Real-time monitoring of TAO inhibitor accumulation
In our previous reports on the trypanocidal effects of TAO in-
hibitors it was never possible to monitor the rate at which the drug
is accumulated in the trypanosome. However, the coupling to the
julolidine fluorophore allowed us to monitor the concentrative
uptake into the cells in real-time, using a fluorimeter. Fig. 5A shows
that the uptake of compound 2a was very rapid, eliciting a
detectable signal immediately after uptake. It was also dose-
2.5. Fluorescence imaging in live cells
2.5.1. One/two-photon excitation fluorescence microscopy in
human preosteoblast cells
dependent, as at 0.2
m
M the fluorescence increased by 3.86 A U./
An important step in the biological activity of TAO inhibitors is
that their activity concentrates in the mitochondria. Hence, the
chimeric design of compounds 1a and 2a-d allows direct imaging of
the localization of the drug at a cellular and/or microorganism level.
First, we tested the feasibility of 1a, 2a and 2c for 1- and 2-
photon absorption fluorescence imaging in live cells, and studied
colocalization with mitochondria, using different commercial
mitochondria trackers (MitoTracker Green, MTG, or MitoTracker
Deep Red, MTDR).
For single-photon excitation fluorescence imaging, we used
dual-color fluorescence microscopy, allowing pulsed-interleaved
excitation with 561-nm and 640-nm lasers [43]. This configura-
tion permits 2-color colocalization studies avoiding spectral
crossover due to direct excitation of one dye into the other detec-
tion channel. For these experiments, we excited 1a, 2a and 2c with
the 561-nm laser whereas we used MTDR, excited with the 640-nm
laser, for mitochondria imaging. Fig. 6 shows that the three com-
pounds exhibited a good mitochondrial localization, with average
Pearson coefficients of 0.7 0.1, 0.5 0.1, and 0.40 0.05 for 1a, 2a
and 2c, respectively. Figures S5, S6, and S7 in the Supplementary
Material shows further examples of colocalization of the three dyes.
min and at 2 M the rate was 33.04 A U./min by linear regression
m
(average of triplicate). As 2a is a potent inhibitor of TAO, this strong
fluorescence might in part result from the production of glycerol
that TAO inhibition is known to engender. To separate the signal of
the accumulation from the extra fluorescence associated with any
changes in viscosity, we utilized an identical probe without the TAO
pharmacophore head group, 12, which we allowed to accumulate
for 1 h followed by the application of a known TAO inhibitor, TAO38
or TAO47 (Table S3) [10], followed by a further recording of 1 h
(Fig. 5B). Neither inhibitor affected the fluorescence relative to the
control (same volume of PBS pH7.4).
However, the glycerol that is produced after inhibition of TAO
derives from the de-phosphorylation of Glycerol-3-phosphate by
glycerol kinase [37], which is located in the glycosomes [38]. The
glycerol so produced is believed to escape to the cytosol, from
which it is released as a waste product to the extracellular envi-
ronment, via aquaglyceroporins (AQPs) [39], and no passive diffu-
sion of glycerol can be detected across the T. brucei cell membrane
[28]. As such, the experiment was repeated with a T. b. brucei strain
from which all three AQP-encoding genes were deleted (aqp1-3
6

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
Fig. 5. Real-time monitoring of fluorescence in T. b. brucei bloodstream forms. A. Fluorescence of T. b. brucei s427WT incubated with 2a, added to the indicated concentrations at
14 min into the recording. B. Fluorescence of 12, added at 20 min to a final concentration of 0.2 M, was recorded in parallel for strain 427WT and aqp1-3 null, on the same plate. At
M TAO38 or TAO47 in PBS was added to the appropriate wells. Every trace is the average of 3 replicate wells on the same
m
80 min, 20
plate.
mL of PBS pH 7.4, 100 mM glycerol/PBS or of 2 m
We also tested compound 1a in colocalization studies with
mitochondria using two-photon excitation microscopy (see
Experimental section for details). Multi-photon excitation is a
nonlinear phenomenon that enhances resolution of fluorescence
images by reducing the excitation cross-section, reduces back-
ground, and allows for deeper penetration of the excitation light in
tissues, since near infrared radiation is employed. For these ex-
periments, we used 850-nm excitation radiation, suitable for
simultaneously exciting the mitochondrial tracker MTG and 1a.
Fig. 7 confirms the excellent mitochondrial localization of the dye
(Pearson coefficient of 0.74 0.07, as well as its suitability for two-
photon absorption imaging.
specifically in the mitochondria of mammalian and trypanosome
cells, are fluorescent probes that are sensitive to the viscosity of the
milieu. They show a high SNR in one and two photon absorption
spectroscopy. The lack of a detectable increase in fluorescence with
this probe upon inhibition of TAO shows that the T. brucei mito-
chondrial membrane is impervious to glycerol, consistent with
current models of its metabolism.
4. Experimental part
2.5.2. Quantification of accumulation in mitochondria
4.1. Chemistry
To confirm mitochondrial accumulation, we also quantified the
partition of 1a in functional isolated rat liver mitochondria using
fluorescence spectroscopy [44]. By comparing the absorption and
fluorescence emission of 1a in the supernatant after partition in
extract containing functional mitochondria and subsequent sepa-
ration by centrifugation and washing (see Experimental section for
details), we estimated that 66 20% of the total was delivered into
rat liver mitochondria. This confirms an efficient partition of the
dye in such organelles. We could not perform similar experiments
with the other dyes because of their very low quantum yield in
water, so that the associated error in measuring the fluorescence
emission in the supernatants was very large.
Anhydrous solvents were purchased to ACROS Organics in
AcroSeal® bottles and used as received. Thin Layer chromatography
(TLC) was performed on silica gel 60 F254 aluminum TLC plates
(MERCK). Chromatography was performed on silica gel 60
(0.040e0.063 mm, 230e400 mesh ASTM, MERCK). LC-MS spectra
were recorded on a WATERS apparatus integrated with a HPLC
separation module (2695), PDA detector (2996) and Micromass ZQ
spectrometer using electrospray ionization (ESþ). Analytical HPLC
was performed with
a
SunFire C18e3.5
mm
column
(4.6 mm ꢃ 50 mm). Mobile phase A: CH3CN þ 0.08% formic acid and
B: H2O þ 0.05% formic acid. UV detection was carried over
190e440 nm. Accurate mass were measured with an Q-TOF 6520
spectrometer (Agilent Technologies) using electrospray ionization.
1H NMR and 13C NMR spectra were registered on a Bruker Avance-
300, Varian Inova-400, Varian-Mercury-400, and Varian-500.
Chemical shifts of the 1H NMR spectra were referenced to the re-
2.5.3. Fluorescence microscopy in T. brucei
To attest the localization of the drug in T. b. brucei, live parasites
were imaged using a high-resolution microscope. Cells were
immobilized with PBS-primed CyGEL, using MTG to stain the par-
asite’s single mitochondria and Hoechst 33,342 to stain DNA (the
nucleus and the mitochondria). Compound 1d was observed to
stain the same structure as the MTG, confirming the mitochondrial
localization of 1d in live trypanosomes (Fig. 8).
sidual peak of the deuterated solvent:
d 7.26 ppm (CDCl3), 3.31 ppm
(Methanol-d4), and 2.50 ppm (DMSO‑d6). Chemical shifts of the 13
C
NMR spectra were referenced at 77.16 ppm (CDCl3), 49.0 ppm
(CD3OD), and 39.5 ppm (DMSO‑d6). Peak multiplicities are defined
as follows: br (broad peak), s (singlet), d (doublet), t (triplet), q
(quadruplet), p (quintuplet), m (multiplet), and combinations of the
same. Coupling constants J are given in hertz (Hz). Melting points
were measured with a Reichert-Jung Thermovar apparatus and are
uncorrected. All of the biologically tested compounds were ꢂ95%
pure by HPLC.
3. Conclusions
Three fluorescent TAO inhibitors with submicromolar activity
against T. brucei were synthesized and their photophysical prop-
erties were determined. The inhibitors, which accumulate
7

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
Fig. 6. Colocalization experiments between compounds 1a, 2a and 2c (green channel) and MTDR as mitochondrial tracker (red channel) in the preosteoblast cell line MC3T3-E1
using single-photon excitation fluorescence microscopy. The overlay images show colocalized pixels in white. Insets represent zoomed sections of the overall images.
4.2. Synthesis protocols
(1.48 g, 16.4 mmol) in water (4.5 mL). The reaction mixture was
stirred at room temperature for 48 h. The reaction mixture was
basified with aqueous K2CO3 and extracted with EtOAc. The
aqueous phase was acidified to pH ¼ 1 with 37% HCl, then it was
extracted with EtOAc (3 ꢃ ). The organic phase was dried (MgSO4)
and the pure product was isolated by crystallization from a mixture
of 30% AcOH in water. Off-white solid (386 mg, 37%). HPLC
2,4-dihydroxy-6-methylbenzaldehyde (4). Compounds 4 and
5a were synthesized following a reported procedure [45]. A solu-
tion of orcinol (6.9 g, 55.6 mmol) in anhydrous DMF (23 mL) was
added slowly to a stirred solution of phosphorous oxychloride
(10.4 mL, 110 mmol) in anhydrous DMF (26 mL) cooled down to
10 ꢁC. Then, the reaction mixture was kept under stirring 18 h at
room temperature. The flask was cooled with an ice-bath and the
reaction was cautiously quenched with cold water (150 mL) and
basified to pH z 10 with 10% aqueous NaOH solution. The reaction
mixture was heated to reflux for 10 min, cooled to room temper-
ature, and acidified to pH 1 with concentrated HCl. The yellowish
precipitate was collected by filtration, washed with water and dried
under vacuum to give 4 as pale yellow powder (6.2 g, 73%). 1H NMR
(UV) > 95%. 1H NMR (300 MHz, DMSO‑d6)
d 2.39 (s, 3H), 6.11 (s, 1H),
6.17 (s, 1H), 10.13 (brs, 1H), 12.06 (br, 1H). LRMS (ESIꢀ) m/z 167
(M ꢀ H)-.
3,5-dichloro-2,4-dihydroxy-6-methylbenzoic acid (5b). Under
inert atmosphere p-orsellinic acid 5a (300 mg, 1.8 mmol) and sul-
famic acid (871 mg, 8.9 mmol) were suspended in 12 mL of a
mixture H2O/THF (2:1). The mixture was cooled down to 0 ꢁC and
freshly purchased acetaldehyde (100
mL, 1.78 mmol) was added.
(300 MHz, DMSO‑d6)
d
(ppm): 2.45 (s, 3H), 6.12 (d, J ¼ 3.0 Hz, 1H),
Thereafter, NaClO2 (807 mg, 8.9 mmol) dissolved in H2O (1 mL) was
introduced and the reaction was warm up to room temperature and
stirred for 20 min. Then, the mixture was quenched with saturated
NH4Cl aqueous solution (50 mL) and extracted with ethyl acetate
(100 mL). The organic layer was dried over MgSO4 and
6.20 (d, J ¼ 3.0 Hz, 1H), 10.05 (s, 1H), 10.68 (br, 1H), 12.05 (brs, 1H).
2,4-dihydroxy-6-methylbenzoic acid (5a). To a solution of 4
(1.0 g, 6.6 mmol) and NaH2PO4 (1.97 g, 16.4 mmol) in DMSO (21 mL)
and water (4.5 mL) at 0 ꢁC was slowly added a solution of NaClO2
8

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
Fig. 7. Two representative images of colocalization experiments between 1a (red channel) and MTG as mitochondrial tracker (green channel) in the preosteoblast cell line MC3T3-
E1 using 850-nm two-photon excitation fluorescence microscopy.
Fig. 8. Mitochondrial localization of 1a in T. brucei. The first column is Hoechst 33342 (stains DNA), the second Mitotracker Green, third compound 1a and the last one the overlay
(Hoechst in cyan, Mitotracker in yellow and 1a (40 mM, 1 h) in magenta). All scale bars indicate 5 mm.
9

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
down to room temperature, a white precipitate appeared. The
precipitate was collected by filtration and washed with cold Et2O to
give 7a (63 mg, 97%) as colorless solid. HPLC (UV) > 95%. mp
118e120 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 1.23e1.37 (m, 20H),
1.65e1.69 (m, 2H), 1.83e1.91 (m, 2H), 2.30 (s, 3H, Ors-CH3), 2.60 (s,
3H, Pyr-CH3), 4.22 (t, J ¼ 6.5 Hz, 2H), 4.51 (t, J ¼ 7.4 Hz, 2H), 6.15 (d,
J ¼ 2.3 Hz, 1H), 6.17 (d, J ¼ 2.3 Hz, 1H), 7.98 (d, J ¼ 6.3 Hz, 2H), 8.92
(d, J ¼ 6.8 Hz, 2H), 10.00 (s, 1H), 10.86 (s, 1H). 13C NMR (100 MHz,
DMSO‑d6)
d
170.1, 161.6, 161.2, 158.7, 143.7 (Nþ-CH), 140.9, 128.3
(Nþ-CH-CH-), 110.3, 107.1, 100.5, 64.7(-CH2-OCO-), 59.9 (-CH2-Pyr),
30.5, 28.94, 28.88, 28.85, 28.7, 28.5, 28.3, 28.0, 25.5, 25.3, 22.3 (Ors-
CH3), 21.3 (Pyr-CH3). ESI-HMRS: m/z 456.3114 [M]þ (C28H42NO4
requires: 456.3114).
1-(14-((3,5-dichloro-2,4-dihydroxy-6-methylbenzoyl)oxy)
tetradecyl)-4-methylpyridin-1-ium bromide (7b). Under inert
Chart 1. Example of previous 2,4-dihydroxybenzoate-based TAO inhibitors [8,10].
Ph3Pþ
: : quinolin-1-ium; rTAO: recombinant TAO
triphenylphosphonium; Quinþ
enzyme.
atmosphere, 6b (25 mg, 49
mmol) and 4-methylpyridine (5.5
mL,
56
m
mol) were dissolved in anhydrous CH3CN (1.5 mL). The reaction
was heated up at 80 ꢁC and kept under stirring for 2 days. The
solvent was evaporated and the residue was washed several times
with hot Et2O to give 7b (10.8 mg, 36%) as pale brownish solid. HPLC
(UV) > 95%. mp 126e130 ꢁC. 1H NMR (300 MHz, Methanol-d4)
concentrated under reduced pressure leading to a pale brown solid
(380 mg, 90%) that was used for the next step without further
purification. HPLC (UV) > 93%. 1H NMR (400 MHz, Methanol-d4)
d
2.63 (s, 3H). 13C NMR (100 MHz, Methanol-d4)
d 174.1, 159.3, 155.0,
d
1.29e1.76 (m, 20H), 1.76e1.81 (m, 2H), 1.96e2.01 (m, 2H), 2.53 (s,
138.9, 115.6, 108.2, 108.1, 19.6. ESI-HMRS: m/z 235.9646[M]þ
(C8H6Cl2O4 requires: 235.9643).
3H), 2.67 (s, 3H), 4.38 (t, J ¼ 6.5 Hz, 2H), 4.55 (t, J ¼ 7.5 Hz, 2H), 7.93
(d, J ¼ 6.1 Hz, 2H), 8.80 (d, J ¼ 6.2 Hz, 2H). 13C NMR (100 MHz,
14-bromotetradecyl 2,4-dihydroxy-6-methylbenzoate (6a).
Compound 6a was synthesized following a reported procedure [11].
Methanol-d4)
d 171.6, 161.3, 157.2, 154.6, 144.8, 137.4, 129.9, 115.6,
110.4, 108.5, 67.3, 62.2, 32.4, 30.65, 30.60, 30.56, 30.51, 30.47, 30.45,
30.12, 30.09, 29.5, 27.2, 27.1, 22.0, 19.5. ESI-HMRS: m/z 524.2323
[M]þ (C28H40Cl2NO4 requires 524.2334).
1-(14-((3-chloro-4,6-dihydroxy-2-methylbenzoyl)oxy)tetra-
decyl)-4-methylpyridin-1-ium bromide (7c). Under inert atmo-
14-bromotetradecyl
3,5-dichloro-2,4-dihydroxy-6-
methylbenzoate (6b). Under inert atmosphere, 5b (44 mg,
0.2 mmol), 14-bromotetradecan-1-ol [19] (50 mg, 0.17 mmol) and
PPh3 (45 mg, 0.19 mmol) were dissolved in THF (3 mL). Then, the
reaction was cooled down to 0 ꢁC and DIAD (37
mL, 0.19 mmol) was
sphere, 6c (40 mg, 84
mmol) and 4-methylpyridine (10 mL,
added. Thereafter, the mixture was warm up to room temperature
and was kept under stirring for 2.5 h. The solvent was removed
under high vacuum, and the crude solid was purified by flash
chromatography column on silica gel (stepwise gradient from 0 to
20% EtOAc in Hexanes). Compound 6b was obtained as off-white
amorphous solid (41 mg, 47%). 1H NMR (300 MHz, CDCl3/Meth-
0.10 mmol) were dissolved in anhydrous CH3CN (1.8 mL). The re-
action mixture was heated up at 80 ꢁC, and kept under stirring for 3
days. After cooling down to room temperature, a white precipitate
appeared. The precipitate was collected by filtration and washed
with cold Et2O to give 7c (35 mg, 73%) as colorless solid. HPLC
(UV) > 95%. mp 137e139 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
anol-d4)
d 1.04e1.33 (m, 20H), 1.56e1.76 (m, 4H), 2.47 (s, 3H), 3.26
d
1.23e1.37 (m, 20H), 1.61e1.64 (m, 2H), 1.83e1.91 (m, 2H), 2.20 (s,
(t, J ¼ 6.8 Hz, 2H), 4.23 (t, J ¼ 6.6 Hz, 1H). 13C NMR (75 MHz, CDCl3)
3H, Ors-CH3), 2.60 (s, 3H, Pyr-CH3), 4.18 (t, J ¼ 6.5 Hz, 2H), 4.50 (t,
J ¼ 7.4 Hz, 2H), 6.43 (s, 1H), 7.97 (d, J ¼ 6.3 Hz, 2H), 8.91 (d,
J ¼ 6.7 Hz, 2H), 9.99 (s, 1H), 10.35 (s, 1H). 13C NMR (100 MHz,
d
171.2, 158.4, 152.4, 138.0, 114.0, 107. 6, 106.8, 66.8, 34.2, 33.0, 29.7,
29.65, 29.6, 29.3, 28.9, 28.6, 28.3, 26.2, 19.9. ESI-HMRS: m/z:
510.0932 [M]þ (C22H33Cl2O4 requires 510.0939).
DMSO‑d6) d 167.8, 158.8, 154.9, 154.7, 143.7, 134.7, 128.4, 113.9, 111.3,
14-bromotetradecyl
3-chloro-4,6-dihydroxy-2-
101.3, 64.7, 60.0, 48.7, 30.9, 29.01, 28.95, 28.92, 28.8, 28.6, 28.4, 28.1,
25.45, 25.41, 21.4, 17.5. ESI-HMRS: m/z: 490.2730 [M]þ
(C28H41ClNO4 requires 490.2724).
(E)-1-(14-((2,4-dihydroxy-6-methylbenzoyl)oxy)tetradecyl)-4-
(2-(8-hydroxy-2,3,6,7-tetrahydroe1H,5H-pyrido[3,2,1-ij]quinolin-
9-yl)vinyl)pyridin-1-ium bromide (1a). Under inert atmosphere, 7a
(340 mg, 0.63 mmol) and 9-formyl-8-hydroxyjulolidine (165 mg,
0.76 mmol) were dissolved in absolute EtOH (5.0 mL). Thereafter,
methylbenzoate (6c). Under inert atmosphere, 5c (177 mg,
0.88 mmol; used as approximately 70/30 mixture of 5b/5c) was
added to a suspension of NaHCO3 (81 mg, 0.97 mmol) in anhydrous
CH3CN (3 mL).Then, 1,14-dibromotetradecane [18] (806 mg,
2.2 mmol) was added. The reaction was heated up at 80 ꢁC and kept
under stirring for one day. After cooling down to room temperature,
the solvent was removed under high vacuum. The crude solid was
purified by flash chromatography column on silica gel (stepwise
gradient from 0 to 20% EtOAc in Hexanes) leading to 6c (50 mg,12%)
as a pale off-white solid. mp 43e46 ꢁC. 1H NMR (400 MHz, Meth-
piperidine (43.0
mL, 0.44 mmol) was added and the reaction
mixture was stirred at 70 ꢁC for 24 h. After cooling down to room
temperature the solvent was removed under high-vacuum. The
crude solid was purified by flash chromatography column on silica
gel (stepwise gradient from 0 to 50% MeOH in EtOAc) leading to 1a
(95 mg, 31%) as dark red solid (NOTE: the product gives a visible
reddish spot in TLC that vanishes with time upon exposure to
sunlight). HPLC (UV) > 92%. mp 198e202 ꢁC. 1H NMR (300 MHz,
anol-d4) d 1.28e1.43 (m, 20H), 1.74e1.84 (m, 4H), 2.53 (s, 3H, -CH3),
3.41 (t, J ¼ 6.8 Hz, 2H), 4.32 (t, J ¼ 6.5 Hz, 1H), 6.34 (s, 1H). 13C NMR
(100 MHz, Methanol-d4)
d 171.9, 161.6, 158.7, 139.7, 115.1, 109.5,
102.4, 66.7, 34.4, 34.0, 30.65, 30.61, 30.58, 30.53, 30.49, 30.46, 30.2,
29.8, 29.6, 29.1, 27.2, 19.6. ESI-HMRS: m/z: 476.1316 [M]þ
(C22H34BrClO4 requires 476.1329).
1-(14-((2,4-dihydroxy-6-methylbenzoyl)oxy)tetradecyl)-4-
methylpyridin-1-ium bromide (7a). Under inert atmosphere, 14-
bromotetradecyl 2,4-dihydroxy-6-methylbenzoate (6a) [11]
Methanol-d4) d 1.23e1.54 (m, 24H), 1.62e1.84 (m, 4H), 2.46 (s, 3H),
2.64 (t, J ¼ 6.5 Hz, 2H), 2.70 (t, J ¼ 6.3 Hz, 2H), 3.21e3.29 (m, 4H),
4.20e4.43 (m, 4H), 6.10e6.31 (m, 2H), 6.98 (d, J ¼ 15.7 Hz, 1H), 7.19
(s, 1H), 7.78 (d, J ¼ 6.6 Hz, 2H), 8.17 (d, J ¼ 15.7 Hz, 1H), 8.39 (d,
J ¼ 6.6 Hz, 2H). 13C NMR (126 MHz, acetic acid-d4)
d
173.5, 166.8,
(58 mg, 0.13 mmol) and 4-methylpyridine (15 mL, 0.16 mmol) were
dissolved in anhydrous CH3CN (1.8 mL). The reaction mixture was
163.4, 156.8, 155.3, 148.7, 145.2, 144.1, 139.9, 127.3, 123.3, 117.2, 113.1,
112.8, 108.9, 106.4, 102.6, 66.9, 61.5, 51.6, 50.8, 32.6, 31.2, 31.1, 31.0,
heated up at 80 ꢁC, and kept under stirring for 3 days. After cooling
10

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
30.93, 30.89, 30.6, 30.5, 30.0, 28.8, 27.6 (2C), 25.3, 23.3, 22.4. ESI-
J ¼ 6.4 Hz, 2H), 4.35e4.42 (m, 4H), 6.29 (d, J ¼ 6.6 Hz, 2H), 6.99 (d,
J ¼ 15.8 Hz, 1H), 7.20 (s, 1H), 7.80 (d, J ¼ 6.7 Hz, 2H), 8.17 (d,
J ¼ 15.8 Hz, 1H), 8.43 (d, J ¼ 6.5 Hz, 2H). 13C NMR (101 MHz,
HMRS: m/z 655.4117 [M]þ (C41H55N2O5 requires: 655.4111).
Ethyl
4-((14-bromotetradecyl)oxy)-2-hydroxy-6-
methylbenzoate (8). Under inert atmosphere, ethyl 2,4-
dihydroxy-6-methylbenzoate (300 mg, 1.53 mmol), bromote-
tradecan-1-ol (373 mg, 1.27 mmol), and PPh3 (401 mg, 1.53 mmol)
were dissolved in anhydrous THF (26 mL) and the flask was cooled
with an ice-bath. DIAD (0.3 mL, 1.53 mmol) was added to the so-
lution, the ice-batch was removed and the reaction mixture was
kept under stirring for 2.5 h at room temperature. The organic
solvent was removed under vacuum and the crude oil was washed
with HCl (0.1 N, 35 mL) and extracted with AcOEt (2 ꢃ 75 mL). The
combined organic layers were successively washed with water
(2 ꢃ 100 mL) and brine (2 ꢃ 100 mL). The solvent was dried over
MgSO4, filtered and evaporated. The crude solid was purified by
flash column chromatography (SiO2; gradient from 0 to 5% EtOAc in
hexanes) to give 8 as a colorless solid (395 mg, 55%). Spectroscopic
data agreed with the reported ones [9].
Methanol-d4) d 181.6, 172.9, 166.0, 164.8, 156.8, 155.6, 144.1, 143.8,
143.6, 140.2, 127.0, 122.6, 120.8, 116.57, 115.5, 112. 8, 112.0, 108.3,
106.7, 100.3, 69.1, 62.3, 51.2, 50.5, 32.1, 30.6, 30.5, 30.40, 30.35,
30.31, 30.1, 30.0, 28.5, 28.3, 27.0, 24.4, 23.76, 23.1, 22.9, 22.27, 22.24,
19.2, 14.6. ESI-HMRS: m/z 683.4419 [M]þ (C43H59N2O5 requires
683.4424).
(E)-4-(2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-
ij]quinolin-9-yl)vinyl)-1-(14-(3-hydroxy-5-methylphenoxy)tet-
radecyl)pyridin-1-ium bromine (2c). Compound 2c was the sole
product isolated from the reaction of 10 and 9-formyl-8-
hydroxyjulolidine when following the same protocol described
for the synthesis of 2a. Purple solid (30 mg, 19%). HPLC (UV) > 95%.
mp 158e162 ꢁC. 1H NMR (400 MHz, Methanol-d4)
d 1.49e1.23 (m,
22H), 1.76e1.64 (m, 2H), 1.97e1.90 (m, 4H), 2.21 (s, 3H), 2.70e2.63
(m, 4H), 3.27e3.23 (m, 4H), 3.86 (t, J ¼ 6.4 Hz, 2H), 4.34 (t, J ¼ 7.3 Hz,
2H), 4.58 (bs, 1H), 6.13 (s, 1H), 6.20 (m, 2H), 6.97 (d, J ¼ 15.8 Hz, 1H),
7.19 (s, 1H), 7.78 (d, J ¼ 6.6 Hz, 2H), 8.18 (d, J ¼ 15.8 Hz, 1H), 8.40 (d,
1-(14-(4-(ethoxycarbonyl)-3-hydroxy-5-methylphenoxy)tet-
radecyl)-4-methylpyridin-1-ium bromide (9). Under inert atmo-
sphere, 8 (100 mg, 0.21 mmol) and 4-methylpyridine (25
mL,
J ¼ 6.7 Hz, 2H). 13C NMR (126 MHz, Methanol-d4)
d 181.5, 161.7,
0.25 mmol) were dissolved in anhydrous CH3CN (2 mL). The reac-
tion was stirred at 80 ꢁC for 3 days. After cooling down to room
temperature, the solvent was removed under vacuum. The crude
solid was triturated in Et2O and the supernatant was discarded.
Compound 9 was obtained as white solid (73 mg, 71%). HPLC
(UV) > 95%. mp 50e54 ꢁC. 1H NMR (400 MHz, Methanol-d4)
159.3, 156.9, 155.8, 148.6, 143.6, 141.1, 140.3, 132.4, 127.0, 122.5,
116.6, 115.4, 112.8, 109.5, 108.4, 107.7, 100.0, 68.8, 60.7, 30.8, 30.7,
30.6, 30.5, 30.4, 30.34, 30.28, 30.1, 30.0, 28.5, 27.14, 27.05, 23.9, 23.1,
22.3, 22.2, 21.7. ESI-HMRS: m/z 611.4215 [M]þ (C40H55N2O3 requires
611.4213).
(E)-4-(2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]
quinolin-9-yl)vinyl)-1-(14-(3-hydroxy-4-(methoxycarbonyl)-5-
methylphenoxy)tetradecyl)pyridin-1-ium bromide (2d). Secondary
product from the saponification of 2a in MeOH/H2O using KOH. To a
solution of 2a (70 mg, 0.092 mmol) dissolved in methanol (1 mL)
was added a solution of potassium hydroxide (102 mg, 1.8 mmol) in
water (2 mL). The blueish reaction mixture was heated at 50 ꢁC for
24 h to give a mixture of 2b and 2d as observed by HPLCeMS. The
mixture was acidified with 1 M aqueous HCl solution until pH z 4.
The precipitate was washed with water and the crude product was
purified by semi-preparative HPLCeMS. Compound 2b was not
isolated pure whereas 2d was obtained as pure purple solid. HPLC
(UV) > 95%. mr 80e100 ꢁC (dec.). 1H NMR (400 MHz, Methanol-d4)
d
1.30e1.46 (m, 23H), 1.73e1.79 (m, 2H), 1.99e2.01 (m, 2H), 2.49 (s,
3H, Ors-CH3), 2.68 (s, 3H, Pyr-CH3), 3.97 (t, J ¼ 6.4 Hz, 2H), 4.39 (q,
J ¼ 7.1 Hz, 2H, eCOOeCH2-), 4.55 (t, J ¼ 6.4 Hz, 2H), 6.28e6.31 (m,
2H), 7.93 (d, J ¼ 6.3 Hz, 2H), 8.80 (d, J ¼ 6.7 Hz, 2H). 13C NMR
(100 MHz, Methanol-d4)
d 172.9, 166.1, 164.8, 161.3, 144.8, 144.1,
129.9, 112.0, 106.7, 100.3, 69.1, 62.3, 32.4, 30.67 (2C), 30.62, 30.58,
30.5, 30.4, 30.2, 30.1, 27.2, 27.0, 24.4, 22.0, 14.6. ESI-HMRS: m/z:
484.3432 [M]þ (C30H46NO4 requires 484.3427).
1-(14-(4-carboxy-3-hydroxy-5-methylphenoxy)tetradecyl)-
4-methylpyridin-1-ium bromide (10). A solution of ethyl ester 9
(250 mg, 0.44 mmol) and KOH (198 mg, 3.5 mmol) in a 2:1 mixture
of H2O/MeOH (3 mL) was stirred at 50 ꢁC for 2 days. The reaction
was neutralized with aq. HCl and the solvents were evaporated to
dryness. Silica chromatography of the crude solid with stepwise
gradient from 0 to 50% MeOH (þfew drops of AcOH) in EtOAc
d
1.29e1.47 (m, 24H), 1.71e1.76 (m, 2H), 1.90e1.97 (m, 6H), 2.47 (s,
3H), 2.64 (t, J ¼ 6.5 Hz, 2H), 2.70 (t, J ¼ 6.3 Hz, 2H), 3.23e3.30 (m,
4H-partially masked), 3.90 (s, 3H), 3.94 (t, J ¼ 6.4 Hz, 2H), 4.33 (t,
J ¼ 7.3 Hz, 2H), 6.28 (d, J ¼ 2.6 Hz, 1H), 6.30 (d, J ¼ 2.6 Hz, 1H), 6.98
(d, J ¼ 15.7 Hz, 1H), 7.19 (s, 1H), 7.78 (d, J ¼ 6.7 Hz, 2H), 8.17 (d,
J ¼ 15.8 Hz, 1H), 8.39 (d, J ¼ 6.7 Hz, 2H). ESI-HMRS: m/z 669.4264
[M]þ (C42H57N2O5 requires 669.4268).
yielded 10 as brownish solid (170 mg, 72%). HPLC (UV) > 95%. 1
H
NMR (400 MHz, DMSO‑d6)
d
1.09e1.42 (m, 20H), 1.64 (p, J ¼ 6.7 Hz,
2H), 1.82e1.93 (m, 2H), 2.46 (s, 3H), 2.60 (s, 3H), 3.84 (t, J ¼ 6.4 Hz,
2H), 4.51 (t, J ¼ 7.4 Hz, 2H), 5.91 (dd, J ¼ 25.2, 2.6 Hz, 2H), 7.98 (d,
J ¼ 6.3 Hz, 2H), 8.95 (d, J ¼ 6.3 Hz, 2H). 13C NMR (101 MHz,
1-(14-bromotetradecyl)-4-methylpyridin-1-ium (11). Under
inert atmosphere, 4-methylpyridine (0.34 mL, 3.5 mmol) and 1,14-
dibromotetradecane (3.0 g, 8.4 mmol) were dissolved in 10 mL of
anhydrous CH3CN. The reaction was stirred at 80 ꢁC for 72 h.
Thereafter, the mixture was allowed to cool down at 0 ꢁC. The white
precipitate obtained, was triturated with Et2O (50 mL) and filtered
over a sintered funnel. The crude solid was purified by flash chro-
matography column on silica gel (stepwise gradient from 0 to 50%
MeOH in EtOAc) to give compound 12 as a white solid (1.0 g, 64%).
DMSO‑d6) d 172.2, 167.0, 161.4, 159.6, 158.7, 143.7, 142.0,128.3,106.5,
98.8, 66.6, 59.9, 40.2, 30.6, 28.9, 28.8, 28.7, 28.6, 28.3, 25.42, 25.36,
23.44, 23.39, 21.4. ESI-LRMS: m/z 456 [Mþ, 100%].
(E)-1-(14-(4-(ethoxycarbonyl)-3-hydroxy-5-methylphenoxy)
tetradecyl)-4-(2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido
[3,2,1-ij]quinolin-9-yl)vinyl)pyridin-1-ium bromide (2a). Under
inert atmosphere,
hydroxyjulolidine (592 mg, 2.73 mmol) were dissolved in abso-
lute EtOH (5.0 mL) and piperidine (175 L, 1.78 mmol) was added to
9
(1.4 g, 2.4 mmol) and 9-formyl-8-
m
mp 89e91 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 1.20e1.37 (m, 20H),
the mixture. The reaction was heated up at 80 ꢁC and kept under
stirring for 24 h. After cooling down to room temperature the sol-
vent was removed under high-vacuum. The crude solid was puri-
fied by flash chromatography column on silica gel (stepwise
gradient from 0 to 50% MeOH in EtOAc) to give 2a (405 mg, 21%) as
a reddish solid. HPLC (UV) > 95%. mr 178e198 ꢁC. 1H NMR
1.65e1.69 (m, 2H),1.83e1.91 (m, 2H), 2.30 (s, 3H), 2.61 (d, J ¼ 4.0 Hz,
3H, Pyr-CH3), 3.49e3.54 (m, 2H), 4.52e4.57 (m, 2H), 7.98e8.01 (m,
2H), 8.96e8.99 (m, 2H), 10.00 (s, 1H), 10.86 (s, 1H). 13C NMR
(100 MHz, DMSO‑d6)
d
158.7 (-C-CH3), 143.7 (Nþ-CH), 128.3 (Nþ-
CH-CH-), 59.8 (Pyr-CH2-), 35.2 (BreCH2-), 32.2 (BreCH2eCH2-),
30.6 (PyreCH2eCH2-), 29.0, 28.9, 28.9, 28.8, 28.4, 28.1, 27.5, 25.4,
21.4 (Pyr-CH3). ESI-HMRS: m/z 368.1955 [M]þ(C20H35BrN requires
368.1953).
(400 MHz, Methanol-d4)
d 1.29e1.46 (m, 20H), 1.71e1.79 (m, 2H),
1.90e1.99 (m, 6H), 2.48 (s, 3H), 2.65 (t, J ¼ 6.4 Hz, 2H), 2.70 (t,
J ¼ 6.5 Hz, 2H), 3.23e3.30 (m, 4H-partially masked), 3.94 (t,
(E)-1-(14-bromotetradecyl)-4-(2-(8-hydroxy-2,3,6,7-
11

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
tetrahydro-1H,5H-pyrido[3,2,1-ij]quinolin-9-yl)vinyl)pyridin-1-
ium (12). Under inert atmosphere, compound 11 (530 mg,
1.18 mmol) and 9-formyl-8-hydroxyjulolidine (283 mg, 1.3 mmol)
wild-type (s427 WT), and the drug resistant strains B48 and AQP1-
3 KO; B48 is derived from s427 WT by subsequently deleting the
TbAT1 drug transporter gene [50] and in vitro adaptation to high
concentrations of pentamidine [24], whereas AQP1-3 KO is a strain
from which all aquaporin genes were deleted [25], including the
pentamidine- and arsenicals-transporting TbAQP2 [28]. Cells were
grown at 37 ꢁC in a 5% CO2 atmosphere in Hirumi’s Modifed Iscove’s
medium 9 (HMI-9; Life Technologies) supplemented with 10% (vol/
vol) heat-inactivated Fetal Calf Serum (FCS; Biosera), 3.0 g/L sodium
were dissolved in 6.0 mL of EtOH. Then, piperidine (58
mL,
0.59 mmol) was added to the solution. The reaction was stirred at
80 ꢁC for 1.5 h. After cooling down at room temperature, the solvent
was removed under high vacuum. The crude solid was purified by
flash chromatography column on silica gel (stepwise gradient from
0 to 10% MeOH in EtOAc) to yield 12 as a red-like solid (30 mg, 7%).
mp > 120 ꢁC (dec.). 1H NMR (400 MHz, CDCl3)
d
1.20e1.37 (m, 20H),
hydrogen carbonate (Sigma) per litre of medium, and 14
mercaptoethanol (Sigma), pH 7.4 (complete HMI-9).
mL/L b-
1.56e1.69 (m, 2H), 1.78e1.89 (m, 2H), 1.91e1.98 (m, 4H), 2.67 (t,
J ¼ 6.3 Hz, 2H), 2.81 (t, J ¼ 6.3 Hz, 2H), 3.20e3.27 (m, 4H), 3.40 (t,
J ¼ 6.9 Hz, 2H, Pyr-CH2-), 4.25 (t, J ¼ 7.4 Hz, 2H, BreCH2-), 6.56 (d,
J ¼ 15.4 Hz,1H), 7.04 (s,1H), 7.84 (d, J ¼ 6.5 Hz, 2H), 8.11 (bs,1H, OH),
8.21 (d, J ¼ 6.6 Hz, 2H), 8.40 (d, J ¼ 15.4 Hz, 1H). 13C NMR (100 MHz,
A double dilution of the test compounds was prepared in 100
mL
of the same medium over 2 rows (23 wells), with the 24th well
receiving 100 mL of medium as drug-free control. The cell density
was adjusted to 2 ꢃ 105 cells/mL in complete HMI-9 after which
CDCl3)
d
155.3, 155.1, 147.9, 141.5, 140.6, 125.0, 122.2, 115.4, 113.5,
100 mL of this suspension was added to each well of the 96-well
111.0, 107.8, 59.9, 50.4, 49.9, 34.3, 33.0, 31.4, 29.7, 29.7, 29.6, 29.6,
29.5, 29.2, 29.0, 28.9, 28.3, 27.6, 26.3, 22.1, 21.7, 21.2. ESI-HMRS: m/z
567.2956 [M]þ (C33H48BrN2O requires 567.2950).
plate containing the serially diluted test compounds.
The plate containing trypanosomes and test compound was
incubated at 37 ꢁC in a 5% CO2 atmosphere for a period of 48 h,
followed by the addition of 20
mL of a 500 mM filter-sterilised
resazurin sodium salt (Sigma) solution in PBS (pH 7.4), and a
further 24-h incubation. The standard trypanocide pentamidine
(Sigma) was used as positive control. Fluorescence was measured in
a FLUOstar Optima plate reader (BMG Labtech, Durham, NC, USA) at
wavelengths of 544 nm for excitation, 590 for emission. EC50 values
were determined by non-linear regression using an equation for a
sigmoidal dose-response curve with variable slope (GraphPad
Prism 7.0, GraphPad Software Inc., San Diego, CA, USA). The EC50
values were obtained from at least 3 independent experiments.
4.3. Photophysical characterization
Absorption spectra were recorded employing a Lambda 650
double-beam UV spectrophotometer (PerkinElmer, USA), steady-
state fluorescence emission were recorded by an FP-8500 spec-
trofluorimeter (Jasco, Japan), and fluorescence lifetimes were ob-
tained by collecting five fluorescence decay traces, between 570
and 650 nm (Dl ¼ 20 nm), using a FluoTime 200 time-resolved
fluorometer (PicoQuant, Germany) employing a 530-nm pulsed
diode laser (LDH-530, PicoQuant, Germany) with a repetition rate
of 20 MHz. Average fluorescence lifetimes, tav, were obtained by
global fitting the decay traces to tri-exponential decay functions,
and using the equation
4.5.2. Cell culture and cytotoxicity of the test compounds on human
embryonic kidney cells
The inhibitors were tested for their toxicity on Human Embry-
onic Kidney (HEK) cells (strain 293T), essentially as described
previously [51]. HEK cells were grown as a monolayer in a standard
filtered-sterile culture that comprised 500 mL Dulbecco’s Modified
Eagle’s Medium (DMEM; Sigma), glutamine (0.58 g/L), and 10 mL/L
of 100 ꢃ penicillin and streptomycin (PenStrep; Thermo Fisher
Scientific, Waltham, MA, USA), supplemented with 50 mL heat-
inactivated foetal bovine serum (Thermo Fisher Scientific). The
cells were cultured and incubated at 37 ꢁC under a humidified 5%
CO2 atmosphere, and were passaged when reaching 80e85%
confluence in a vented flask.
P
p,i ti
i
tav
¼
P
pi i
Where ti are the individual fluorescence decay times and pi are
the corresponding preexponential factors, to estimate the average
at each emission wavelength. We then obtained the average of the
tav values for only the wavelengths in which all the preexponential
factors were positive.
Quantum yield values were estimated measured using the
relative method and rhodamine 101 in methanol (ɸ ¼ 1) as the
reference standard [46].
For the toxicity assay, cells were adjusted to the desired density
of 3 ꢃ 105 cells/mL, of which 100
mL was added to each well of a 96-
well plate. To allow cell adhesion, the plate was incubated for 24 h.
Serial drug dilutions of the test compounds (130
separate 96-well sterile plate. Using a multichannel pipette, 100
mL) were made in a
4.4. Ab initio calculations
mL
of these dilutions (test compounds) were transferred to the corre-
sponding wells of the 96-well sterile plate containing the cells, with
All molecular orbital calculations were performed using the
Gaussian 09 quantum chemical package. Ground state geometry of
the molecule 1a was optimized using the DFT hybrid functional,
B3LYP [47,48]. Pople’s basis set with both diffuse and polarization
functions, 6-31 þ G** was used for all the atoms. The compound of
interest 1a was subjected of geometry optimization in the i) ground
state using B3LYP/6-31 þ G** level of theory and ii) excited state
using time dependent density functional theory (TD-DFT) with
similar hybrid functional. Both optimizations used the same
IEFPCM solvation in EtOH.
the last well receiving 100
mL of medium as drug-free control.
Phenylarsine oxide (PAO) was used as positive control. The plate
containing the cells and test compounds was then incubated for an
additional 30 h at 37 ꢁC/5% CO2. After this, 10
mL of resazurin so-
lution (125 mg/L in PBS) was added to every well of the plate before
a final 24-h incubation (37 ꢁC/5% CO2). The plate was read in a
FLUOstar OPTIMA fluorimeter at wavelengths 530 nm for excitation
and 590 nm for emission. Analysis of the data to determine the EC50
values was done using GraphPad Prism 7.0.
4.5. Biology
4.5.3. Ubiquinol oxidase/TAO inhibitory assay
The Ubiquinol oxidase activity of recombinant TAO lacking the
4.5.1. Drug sensitivity assay in bloodstream forms T. b. brucei
The resazurin (Alamar blue) assay [49] was used to determine
the susceptibilities of bloodstream trypomastigotes of T. b. brucei:
mitochondrial targeting signal (
cording to a previously published protocol for TAO inhibition assay
[10]. Briefly, MTS TAO activity was determined using a V-630 Jasco
DMTS) was carried out exactly ac-
D
12

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
UVeVis spectrophotometer (Jasco Corporation, Tokyo, Japan) by
measuring the absorbance change of the substrate-ubiquinol
(ε278 ¼ 15,000 Mꢀ1 cmꢀ1) at 278 nm over a period of 2 min in a
1 cm cuvette. The enzyme was pre-incubated for 2 min in a 50 mM
TriseHCl (pH 7.4) buffer containing the detergent octaethylene
glycol monododecylether (0.05% (w/v)) in a total reaction volume
of 1 mL at 25 ꢁC. Reactions were initiated by the addition of ubiq-
uinol to the cuvette.
The inhibition reaction was carried out by pre-incubating a fixed
amount of rTAO with varying amounts of the inhibitor for 2 min in
the same buffer before adding the substrate. DMSO and Ascofur-
anone/SHAM were used as negative and positive controls, respec-
tively. Control experiments were also carried out throughout the
experiment to verify that there was no auto-oxidation of ubiquinol
in the medium. Residual activities were plotted against the corre-
sponding inhibitor concentration to generate the IC50 using
GraphPad Prism.
and 540 nm excitation. For comparison, the dyes were dissolved in
the mitochondrial suspension at the same concentration and
incubated for 20 min at room temperature. The suspension was
then centrifuged at 10,000ꢃg for 10 min and the supernatant was
separated and its emission spectra were measured at the same
instrumental conditions. The relative amount of partitioned dye in
mitochondria was estimated by comparing the total area under the
spectral curve in the supernatant and in the control solution. We
report the average value of two different repeated experiments
(using different mitochondrial extracts) and averaged at the
different excitation wavelengths employed. Reliable results were
only obtained with 1a. For the other dyes, the quantum yield in
aqueous solution was so low that made this method impractical.
4.5.7. Fluorescence microscopy in human cells
MC3T3-E1 preosteoblast (ECACC 99072810) cell line was pro-
vided by the Cell Culture Facility, University of Granada. MC3T3-E1
cells were grown in alpha minimum essential medium (
containing 10% (v/v) fetal bovine serum, 2 mM glutamine,100 U/mL
penicillin, and 0.1 g/mL streptomycin in a humidified 5% CO2
aMEM)
4.5.4. Real-time measurement of fluorescence in T. b. brucei
Cultures of T. b. brucei bloodstream forms in mid-log growth
phase of either 427WT or aqp1-3 null strains were centrifuged for
10 min at 800ꢃg, washed twice in assay buffer by centrifugation at
2400 rpm and resuspended at a density of 108 cells/mL and the cell
m
incubator. For microscopy experiments, MC3T3-E1 cells were
€
seeded onto
m
-Slide 8 Well Glass Bottom (Ibidi GmbH, Grafelfing,
Germany) at a density of 25,000 cells/well.
suspension was distributed into wells of a 96-well plate (200
m
L/
For single-photon excitation mitochondrial localization studies
we employed an Abberior Expert Line Confocal Microscope
(Abberior, Germany) recording data into two different channels: a
first channel with a 561-nm pulsed diode laser as excitation source
and a detection window of 580e630 nm and a second channel with
an excitation source of 640-nm pulsed diode laser and a detection
window of 650e720 nm. Data were collected employing alter-
nating excitation for each channel to avoid crosstalk interferences.
well). The plate was transferred to a microplate reader (FLUOstar
Optima, BMG Labtech) equipped with atmospheric controls set at
37 ꢁC and 5% CO2, and fluorescence read using 544/620 excitation
and emission filters. After a pre-set time to record background
fluorescence, the reading was paused and 20
12 was added into respective wells to a final concentration of
0.2 M or 2 M as indicated, followed by continuation of the
reading. For some experiments the measurement was paused again
at 80 min and 20 L of either PBS pH7.4, TAO47, TAO38 (to final
concentration of 0.2 M) or 100 mM glycerol (10 mM final con-
mL of compound 2a or
m
m
Cells were preincubated with 1a, 2a and 2c 0.6 mM, respectively
m
30 min at 37 ꢁC and, after that, Mitotracker Deep Red (Thermo-
Fisher Scientific, USA) were added to obtain a final concentration of
16 nM and incubated 30 min at 37 ꢁC before measure. Pearson’s
coefficients were estimated using dedicated macros on FiJi (an
ImageJ distribution) [52], having reported the average value from
12 different images for 1a, 7 images for 2a and 5 images for 2c.
Errors in the Pearson’s coefficient values are expressed as standard
deviation.
m
centration) was added to respective wells and reading continued.
4.5.5. Mitochondrial staining in T. brucei
1 mL T. b. brucei cells prepared at a density of 2 ꢃ 106 cells/mL
were incubated with 4 mM, 8 mM and 40 mM of compound 1a in
complete HMI-9 for 1 h (37 ꢁC, 5% CO2). MitoTracker™ Green FM
(Invitrogen, M7514) was added to the cells to a final concentration
of 100 nM, followed by further incubation for 10 min. The cells were
then centrifuged at 1000ꢃg for 10 min, and the pellet was washed
Two-photon absorption fluorescence imaging was performed
on a Leica TCS-SP5 II (Leica, Germany), equipped with a MaiTai
Ti:Sapphire fs-pulsed tunable laser (Spectra Physics, USA). We
employed excitation at 850 nm, which permitted simultaneous
excitation of 1a and Mitotracker Green (MTG), and two channels of
detection, 500e550 nm for MTG and 600e650 for collecting
emission of TAO inhibitors. For these experiment cells were seeded
onto coverslips in 12 well-plates with a density of 9 ꢃ 105 cell/well
and incubated at 37 ꢁC during 24 h. Cells were preincubated with
16 nM MTG for 30 min before the addition of 1a, and further 30 min
incubation at 37 ꢁC. Then, cells were fixed with 2% of para-
formaldehyde in PBS during 15 min at room temperature.
once in 1 mL of 1 ꢃ sterile PBS pH 7.4, and resuspended in 50
mL
PBS. The sample was then prepared and incubated in 5 mg/mL of
Hoechst 33342 Staining Dye Solution (Abcam, ab228551) in the
dark at room temperature for 10 min. This was followed by another
wash in 1 mL of 1 ꢃ cold sterile PBS and the cells were re-
suspended in 20 mL of ice-chilled PBS-primed CyGEL™ (Abcam,
ab109204) in order to gently immobilise the trypanosomes. The
mixture was transferred into a clean slide on ice, covered with
coverslip and sealed with nail varnish. Images were obtained
immediately under DeltaVision microscope (GE Healthcare) using
softWoRx software and processed using ImageJ software.
Declaration of competing interest
4.5.6. Accumulation of 1a in rat liver mitochondria
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
We homogenized rat liver in 20 mM HEPES (4-(2-
hydroxyethyl)-1-piperazineethanesulfonic acid) 250 mM sucrose
pH 7.4 buffer (1:10; m:w) using a mechanical potter. After two
cycles of centrifuging at 800ꢃg and washing to precipitate nuclei
and undamaged cells, the supernatant was centrifuged at 9000ꢃg
to obtain a fraction enriched in mitochondria that were resus-
pended in HEPES-sucrose buffer.
Acknowledgements
This work was supported by the Spanish Ministerio de Econo-
mia y Competitividad (grant SAF2015-66690-R), the Spanish Min-
The dyes were dissolved at 3 ꢃ 10ꢀ7 M in HEPES-sucrose buffer,
pH ¼ 7.4, and the emission spectra were measured with both 470
isterio de Ciencia, Innovacion y Universidades (MCIU/AEI/FEDER,
ꢀ
UE; grants RTI2018-093940-B-I00 to CD, and CTQ2017-85658-R to
13

E.J. Cueto-Díaz, G.U. Ebiloma, I.A. Alfayez et al.
European Journal of Medicinal Chemistry 220 (2021) 113470
(2014) 2771e2779.
AO) and the Japan Society for the promotion of Science (JSPS grant-
17F17420 to GUE). MAU is funded through a studentship from the
Petroleum Technology Development Fund (PTDF), Abuja, Nigeria.
IAA was funded through a Ph.D. studentship from the Ministry of
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