Structure-activity relationships and mechanistic studies of novel mitochondria-targeted, leishmanicidal derivatives of the 4-aminostyrylquinoline scaffold

Article abstract of DOI:10.1016/j.ejmech.2019.03.007

A new class of quinoline derivatives, bearing amino chains at C-4 and a styryl group at C-2, were tested on Leishmania donovani promastigotes and axenic and intracellular Leishmania pifanoi amastigotes. The introduction of the C-4 substituent improves the activity, which is due to interference with the mitochondrial activity of the parasite and its concomitant bioenergetic collapse by ATP exhaustion. Some compounds show a promising antileishmanial profile, with low micromolar or submicromolar activity on promastigote and amastigote forms and a good selectivity index.

Full text of DOI:10.1016/j.ejmech.2019.03.007

Accepted Manuscript
Structure-activity relationships and mechanistic studies of novel mitochondria-
targeted, leishmanicidal derivatives of the 4-aminostyrylquinoline scaffold
Matteo Staderini, Marta Piquero, María Ángeles Abengózar, Montserrat Nachér-
Vázquez, Giulia Romanelli, Pilar López-Alvarado, Luis Rivas, Maria Laura Bolognesi,
J. Carlos Menéndez
PII:
S0223-5234(19)30218-1
DOI:
https://doi.org/10.1016/j.ejmech.2019.03.007
EJMECH 11177
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 December 2018
Revised Date: 8 February 2019
Accepted Date: 4 March 2019
Please cite this article as: M. Staderini, M. Piquero, Marí.Á. Abengózar, M. Nachér-Vázquez,
G. Romanelli, P. López-Alvarado, L. Rivas, M.L. Bolognesi, J.C. Menéndez, Structure-activity
relationships and mechanistic studies of novel mitochondria-targeted, leishmanicidal derivatives of
the 4-aminostyrylquinoline scaffold, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.03.007.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-activity relationships and mechanistic studies of novel
mitochondria-targeted,
leishmanicidal
derivatives
of
the
4-
aminostyrylquinoline scaffold
Matteo Staderini,1,‡ Marta Piquero,1‡ María Ángeles Abengózar,2,‡ Montserrat
2
1
1
2,
Nachér-Vázquez, Giulia Romanelli, Pilar López-Alvarado, Luis Rivas, * Maria
3,
1,
Laura Bolognesi, * J. Carlos Menéndez *
1Unidad de Química Orgánica y Farmacéutica, Departamento de Química en
Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense, 28040
Madrid, Spain.
2Physico-Chemical Biology, Centro de Investigaciones Biológicas, CSIC, Ramiro de
Maeztu 9, 28040 Madrid, Spain.
3Department of Pharmacy and Biotechnology, Alma Mater Studiorum – University
of Bologna, Via Belmeloro 6, 40126 Bologna, Italy.
ABSTRACT
A new class of quinoline derivatives, bearing amino chains at C-4 and a styryl
group at C-2, were tested on Leishmania donovani promastigotes and axenic and
intracellular Leishmania pifanoi amastigotes. The introduction of the C-4
substituent improves the activity, which is due to interference with the
mitochondrial activity of the parasite and its concomitant bioenergetic collapse by
ATP exhaustion. Some compounds show a promising antileishmanial profile, with
low micromolar or submicromolar activity on promastigote and amastigote forms
and a good selectivity index.

ACCEPTED MANUSCRIPT
KEY WORDS
Leishmanicidal compounds
4
-Aminoquinolines
-Styrylquinolines
2
Mitochondrial metabolism

ACCEPTED MANUSCRIPT
INTRODUCTION
Among protozoal diseases, leishmaniasis is only superseded by malaria in terms of
its impact on human health. Leishmaniasis, a neglected tropical disease, affects
more than 2 million patients worldwide, most of them living in low-income
1
countries in East Africa, India and South America. Visceral leishmaniasis, caused
by Leishmania donovani, L. infantum and L. chagasi, is fatal if untreated.2,3
No effective human leishmaniasis vaccine is available so far. Current
pharmacological therapy is based on a few drugs, often with important side effects
and/or high costs, the main ones being pentavalent antimonials (sodium
stibogluconate, meglumine antimoniate), amphotericin B, particularly in a
liposomal formulation, pentamidine, miltefosine and paromomycin (aminosidine).
With the exception of oral miltefosine and topical paromomycin for the cutaneous
ulcers, these drugs require parenteral administration with hospitalization of the
patient. Furthermore, drug resistance threatens the effectiveness of
chemotherapy.4,5 Thus, there is an urgent need to discover new antileishmanial
drugs.
In spite of the steady advances in the knowledge of Leishmania biology over the
last two decades, their translation into the practical implementation of new drugs
in the clinic has been poor.6,7 This obeys to several reasons: (a) The return of
investment in developing new leads from scratch for this disease is meager, as
most of the patients live in poor-income areas. (b) Pharmacological treatment of
leishmaniasis is intrinsically challenging, since an ideal therapy should provide a
high effectiveness for all endemic regions, achieve cure in less than 10 days, be
orally active, and cost less than $10 per course of treatment.8

ACCEPTED MANUSCRIPT
Quinolines offer a high potential to develop effective and affordable oral
9
treatments for leishmaniasis. Among them, some 2-substituted derivatives have
shown a high in vitro activity, low toxicity and a higher metabolic stability than
their non-substituted counterparts.10 The presence of a conjugated double bond on
the side chain seems beneficial for activity, and in this context a class of
styrylquinolines has shown activity at micromolar concentrations against L. major
amastigotes infecting human monocyte-derived macrophages.11-13 The quinoline
derivative 1 (Figure 1), with an improved antileishmanial activity over the
reference drug miltefosine and a good selectivity index (SI), entered the Drugs for
Neglected Diseases initiative (DNDi) pipeline for in vivo evaluation.14 Recently, the
complex quinaldine derivative 2, designed by pharmacophore-based virtual
screening methods on the Leishmania infantum type 2 NADH dehydrogenase
(NDH2), was experimentally confirmed to inhibit the enzyme and also to exhibit
good activity against L. infantum axenic amastigotes and promastigotes.15
Figure 1. Structures of two leishmanicidal quinoline derivatives and the established anti-
leishmanial drug miltefosine

ACCEPTED MANUSCRIPT
RESULTS AND DISCUSSION
Compound design
There is much evidence showing that 8-aminoquinoline-based polyamines act on
mitochondria. The most relevant example is sitamaquine (3), an orally active
quinoline acting on the succinate dehydrogenase (complex II) at the Leishmania
respiratory chain,16,17 which has reached phase IIb clinical trials for the oral
treatment of visceral leishmaniasis in India and Africa. Tafenoquine (4) is another
quinoline bearing an 8-polyamino chain that targets the respiratory chain at the
18
Leishmania mitochondrion, leading to an apoptosis-like death (Figure 2). Other
examples of antitrypanosomatid agents acting at mitochondria include
naphthoquinone-cadaverine19
compounds.
and
dibenzosuberyl–polyamine20
hybrid
Figure 2. Structures of antileishmanial 8-aminoquinolines having mitochondrial targets.
Using the mitochondrial activity of 8-aminoquinoline-based polyamines as the
starting point, we reasoned that an increase in the positive charge of the
styrylquinoline nucleus would endow the resulting molecule with a more effective
accumulation in the mitochondrion. This effect would be driven by the
mitochondrial electrochemical potential (∆Ψ ), which is the highest among
m

ACCEPTED MANUSCRIPT
intracellular organelles. In L. donovani promastigotes ∆Ψ has a value of = -150
m
mV. Furthermore, mitochondrial dysfunction is particularly noxious for
trypanosomatids such as Leishmania, which harbour a single mitochondrion, as
their energy metabolism is based mostly on oxidative phosphorylation. Thus,
glycolysis only accounts for 30% of ATP production in L. donovani
promastigotes,21 and a higher dependence of L. mexicana intracellular amastigotes
on mitochondrial metabolism with respect to promastigotes has been reported.22
Furthermore, glycolysis retooling to offset a dysfunctional mitochondrial function
is quite limited, as demonstrated for tafenoquine-resistant L. major strains.23 This
is in contrast to higher eukaryotes, which may contain hundreds of mitochondria
in a single cell.24 Additionally, a drug targeting the mitochondrial synthesis of ATP
ought to jeopardize the bioenergetic requirements of Leishmania, as 70% of them
are fulfilled through oxidative phosphorylation. This is in line with a growing body
of evidence that underscores the effectiveness of drugs acting on the Leishmania
mitochondrion.21
To achieve our goal, we designed a new family of compounds obtained by
introducing a polyamine chain at the C-4 position of the 2-styrylquinoline
structure. The 4-aminoquinoline structural fragment would lead to an increased
basicity at the heterocyclic nitrogen and, importantly, to a positive charge
delocalized by resonance across the N
1
=C
2
-C
3
=C
4
-NH fragment. This feature is
2
expected to enhance the behavior of the molecule as a lipophilic cation, key for its
potential-driven transport across the inner mitochondrial membrane and
accumulation into the mitochondrial matrix.25 Surprisingly, 4-aminoquinolines, a
mainstay in other fields of antiprotozoal therapy (e.g. in the case of malaria), have
received scant attention in leishmaniasis.15,26 On the other hand, the presence of

ACCEPTED MANUSCRIPT
two amino groups in the polyamine chain could lead to an increased compound
retention by the acidic environment of the phagolysosomes, where the amastigote
forms multiply.27 The design of our compounds sought to find out which of these
two effects takes precedence. Thus, the choice of substituents at the end of the
polyamine chain (represented by Z) aimed to modulate the basicity of the nitrogen
atoms at C-4, and it was also guided by their presence in previously reported
antileishmanial compounds, as in the case of the amodiaquine analogue 21.28,29
and indolylquinoline 22.30 A number of compounds representing structural
fragments of our proposed structures, namely 17-20 and 2-styrylquinoline 23,
devoid of the C-4 substituent, served as a controls.
The main features of the criteria used for the design of our compounds are
summarized in Figure 3, and the structures of the styrylquinoline derivatives
studied (compounds 11a-15b) are shown in Figure 4.
Figure 3. A summary of our criteria for compound design

ACCEPTED MANUSCRIPT
Z
HN
N
n
H
N
Ph
n
Cmpd.
1a
n
2
Z
Cmpd.
Z
O-tBu
O-tBu
N
1
14g
2
4
O
O
O
N
Ph
11b
4
N
O
H
14h
1
2a
2b
2
4
Ph
Ph
1
4i
4j
2
4
N
H
1
H
O
O
CH3
N
1
4a
4b
2
2
N
H
1
O
O
CH3
1
N
CH3
Cl
Cl
Cl
CH3
N
14k
2
4
1
4c
4d
2
2
H
N
N
N
N
O
O
O
O
Cl
1
N
14i
H
O
O
O
1
4e
4f
2
2
OEt
OEt
O
N
15a
2
4
1
15b
O
O
CH3
O
O
NH2
N
Me
NH
NH2
Me
N
Me
N
Ph
Me
N
Ph
N
Ph
17
20
1
8
19
OH
NEt2
HN
HN
N
Ph
23
N
Ph
N
Ph
21
22
Figure 4. Structures of the compounds studied in this work.
Preliminary ADME-tox computational studies of the designed compounds were
carried out using SwissADME.31 For all our compounds, a high gastrointestinal
absorption was predicted, which is an important factor for oral bioavailability.
Moreover, only compounds 14 and 22 were found to violate Lipinski’s rule of five,

ACCEPTED MANUSCRIPT
and only compound 21 showed a PAINS alert. See the full details of this study in
Table S2.
Synthesis
Our synthetic strategy involved the initial generation of a functionalized
styrylquinoline core, followed by its decoration with the selected basic chains at C-
4. We started with the preparation of the known 4-chloro-2-styrylquinoline 6 by
two alternative methods, which differ from the previous one found in the
literature32 and are summarized in Scheme 1. For route A, the commercially
available 4-chloroquinaldine 5 was submitted to a microwave-promoted aldol
condensation with benzaldehyde in the presence of zinc chloride. Alternatively
(
route B), a similar aldol condensation of the known 2-methylquinolin-4(1H)-one,
this time in the presence of acetic anhydride, afforded compound 8, which was
treated with POCl to furnish 6.
3
Scheme 1. Synthesis of the starting material, compound 6

ACCEPTED MANUSCRIPT
After preparation of the suitable monoprotected polyamine chains (10a and 10b),
compound 6 was treated with an excess of these compounds in the presence of
phenol to promote a nucleophilic aromatic substitution that yielded compounds
11a and 11b. These intermediates were deprotected using a solution of
trifluoroacetic acid in methanol, giving 12a and 12b. Compound 12a was also
obtained by treating 6 with a large excess of ethylenediamine under focused
microwave irradiation, conditions that were designed to avoid a competing double
substitution. In the case of compound 12b, this procedure was not feasible due to
the impossibility of evaporating the diamine in excess. Compounds 12a and 12b
were then transformed into the corresponding halides 13 by treatment with
bromoacetyl bromide at -5 °C. In the final step, the reaction of 13 with a variety of
amines under microwave irradiation in the presence of Cs
2
CO yielded the target
3
compounds 14 a-h. Similarly, the treatment of 13 with ethanol under the same
reaction conditions gave compounds 15 a-b (Scheme 2).
This method failed for the preparation of the aniline derivatives 14i-l, owing to the
low nucleophilicity of aromatic amines. In search for an alternative procedure, we
prepared N-arylglycine derivatives, having the desired aryl substitution patterns
(compounds 16a-b), by reacting the suitable anilines with ethyl bromoacetate in
the presence of sodium hydride, followed by ester saponification. Compounds 16a-
b were then coupled to the previously mentioned primary amines 12a-b using
EDC/HOAt as activating agents (Scheme 3).

ACCEPTED MANUSCRIPT
Scheme 2. Synthetic route for the preparation of compounds 14a-h and 15a-b
1
. Br-CH -CO Et,
2 2
NaH, DMF, 12 h, rt
. NaOH, EtOH-
H O, reflux, 1 h
2
O
2
H
N
NH2
R
OH
R
R
R
O
R
H
1
6a, 51% R = H
6b, 16% R = Cl
EDC·HCl,
N
1
HN
N
n
N
HOAt, Et N
3
H
THF, 12 h, reflux
R
Ph
HN
N
n
NH2
1
1
1
4i, 34%
4j, 26%
4k, 50%
Ph
14l, 67%
12a (n = 2)
12b (n = 4)
Scheme 3. Synthetic route for the preparation of compounds 14i-l.
In order to evaluate the influence of the polyaminoalkyl chain, the reference
compound 20 was designed. For its preparation, the commercially available 4-
amino-2-methylquinoline 17 was condensed with benzaldehyde in the presence of

ACCEPTED MANUSCRIPT
zinc chloride and acetic anhydride, giving a mixture of compounds 18 and 19, at
different levels of N-acetylation. Although both compounds were purified for their
biological evaluation, for the preparation of the target compound 20, the mixture
was submitted to amide hydrolysis using KOH dissolved in methanol-water under
microwave irradiation (Scheme 4).
Scheme 4. Synthetic route for the preparation of compound 20.
The amodiaquine analogue 21 and the 4-(3-indolyl) derivative 22 were prepared
using our reported conditions for C-N and C-C bond formation at the quinoline C-4
position, that involve the use of InCl as a Lewis acid catalyst, under focused
3
microwave irradiation.33 By applying this method, treatment of 4-
chlorostyrylquinoline 6 with 4-amino-2-(diethylaminomethyl)phenol or indole
afforded compounds 21 and 22 in 54% and 58% yield, respectively (Scheme 5).
Finally, the reference non-substituted 2-styrylquinoline 23 was synthesized
following a known method based on the use of a type II vinylogous Povarov
reaction.34

ACCEPTED MANUSCRIPT
Scheme 5. Synthetic route for the preparation of compounds 21 and 22
Antileishmanial activity studies
In order to test the mitochondrial tropism of our compounds, we decided to use
Leishmania promastigotes as a model system. At this stage of the parasite life cycle,
mitochondrial synthesis accounts for nearly 70% of the total production of
ATP,21,24,35 and therefore the study of promastigote forms facilitates the
identification of drugs affecting mitochondrial ATP production. Thus, the activity of
compounds 11-23 was evaluated against Leishmania donovani promastigotes
(strain MHOM/SD/00/1S-2D) in a phenotypic screening. All compounds, except
the control 23, inhibited the proliferation of this parasite form, showing IC50 values
in the 0.2 – 35.1 μM range (Table 1). Notably, most of our compounds were more
potent than 2, a marketed antileishmanial drug.36 The low activity (IC50 > 50 µM)
of 2-styrylquinoline 23, devoid of the amino tail at its C-4 position, showed that the
latter structural fragment is critical for activity, and the comparison of compounds
18 and 19 with 20 confirmed the starting hypothesis that the basicity of the
quinoline core is critical for activity. Moreover, the lower activity of compound 17
compared to 20 proved the importance of the styryl fragment. This was further
underscored by comparison of compound 12a with a related quinoline derivative
lacking the styryl substituent, namely 7-chloro-4-(2-aminoethylamino)quinoline,
which showed no activity against L. amazonensis and L. brasilensis promastigotes.37

ACCEPTED MANUSCRIPT
Motivated by these promising results, we studied the activity of our compounds
against Leishmania pifanoi axenic amastigotes (strain MHOM-ET-67/L82), with the
results summarized in Table 1. The choice of this model was driven by the
extensive documentation for this axenic line and its similarity with those
amastigotes obtained from lesions.38 A good inhibition of amastigote proliferation
was found for most compounds and some of them, namely 14b and 14c, showed
submicromolar IC50 values, being only slightly less potent than our miltefosine
standard. It is worth noting that the amastigote stages had similar or even higher
sensitivity towards our styrylquinolines in comparison with promastigotes. This is
a relevant finding, as there are remarkable differences between these two forms
with respect to biochemical and mitochondrial bioenergetics, and many anti-
39
leishmanial compounds active on promastigotes fail to inhibit amastigotes. The
mammalian cell toxicity of compounds 11-23 was then assessed on macrophages,
the host cell for Leishmania, using the tumoral murine macrophage cell line J774, a
model extensively used to test drug activity on intracellular amastigotes while
avoiding the activation of the macrophages by phorbol esters required for the
differentiation of promonocytic and monocytic cell lines.40 The IC50 values thus
obtained were used to calculate a selectivity index (SI). As shown in Table 1, many
of our compounds showed some macrophage toxicities at leishmanicidal
concentrations, and had selectivity indexes below 10. However, 14a, 14c, 14k and
15a, displaying potent antileishmanial activities, showed respectable SI values,
allowing to regard them as relatively selective, nontoxic chemotypes. It is
interesting to note that, in those cases allowing comparison (14c vs. 14h, 14i vs.
14j, 14k vs. 14l, 15a vs. 15b), the two-carbon spacer chain led to better results
than the four-carbon spacer. While several compounds had good activity against

ACCEPTED MANUSCRIPT
amastigotes, 14k had the best overall profile, with micromolar activity against
amastigotes and a SI above 41.6. The fact that this is the compound with the least
basic side chain, but carrying the 4-amino substituent, suggests that protonation of
the quinoline moiety is important for activity and has implications for the design of
future antileishmanial quinoline derivatives.
Table 1. Leishmanicidal activities, cytotoxicities and selectivity indexes of compounds 11-23 and
miltefosine (2)a
L. donovani
promastigotes, IC50 (µM)
0.45 ±0. 32
0.56 ±0.12
2.4 ±0.3
L. pifanoi amastigotes,
IC50 (µM)
4.3 ± 1.2
4.9 ±0.8
>50
J774, IC50
(µM)
Cmpd.
SIb
1
1a
1b
2a
2b
4a
4b
4c
4d
4e
4f
4g
4h
6.6 ± 1.8
3.7 ± 0.5
4.6 ± 0.2
6.6 ± 0.6
15.0 ±2.7
0.7 ± 0.1
12.5 ±3.5
4.7 ± 1.0
> 50
4.7
0.8
1
1
<0.1
<0.3
13.6
0.8
1
0.44 ± 0.1
1.6 ± 0.0
> 25
1
1.1 ± 0.1
0.9 ± 0.1
0.9 ± 0.1
1.2 ± 0.1
> 25
1
1.6 ± 0.0
1
2.1 ± 0.2
13.9
3.4
1
2.1± 0.2
1
1.3 ± 0.1
2.0
1
3.5 ± 0.3
3.5 ± 0.3
1.5 ± 0.2
5.3 ± 0.8
4.3 ± 2.4
6.9 ± 1.5
1.2 ± 0.8
12.3 ± 3.2
1.6 ± 0.4
4.5 ± 1.2
> 50
34 ± 1.2
4.5 ± 0.2
7.1 ±1.0
7.8 ± 1.2
3.3 ± 0.8
>50
9.7
1
2.1 ± 0.1
3.0
1
0.2 ± 0.0
1.4
1
1
4i
3.3 ± 0.8
1.8
4j
1.6 ±0.4
0.48
> 41.6
0.6
1
4k
4l
5a
5b
8.4 ± 2.4
1
2.1 ± 0.2
7.3 ± 1.2
22.2 ± 4.6
7.2 ± 0.9
> 50
1
3.4 ± 0.2
13.8
1.6
1
1.06 ± 0.11
10.9 ± 2.2
35.1 ± 4.6
ND.
1
7
8
9
0
-
1
13.4 ±3.8
9.5 ±2.2
8.9 ±2.0
>25
>1.86
>2.6
0.5
1
2
>25
0.5 ±0.1
4.4 ± 0.2

ACCEPTED MANUSCRIPT
21
22
23
9.6 ± 0.4
9.0 ± 0.6
1.0 ± 0.5
> 50
37 ± 2.2
4.1 ±1.0
> 50
4.1
4.1
1.5 ± 0.6
>50
ND
2
4.4 ± 0.7
6.2 ± 1.6
> 50
> 8.1
a
All results are representative of two experiments performed independently. b SI =
IC50(J774)/IC50 (L. pifanoi amastigotes).
We next planned to investigate the leishmanicidal activity of one selected
compound against macrophage cells infected with L. pifanoi amastigotes. Several of
our compounds (14a-d, 14g, 14k, 15a, 22) had shown similar inhibitory activities
against amastigote forms in the low micromolar level and were therefore
considered suitable for these studies. Among them, we chose 15a, due to its better
synthetic accessibility. Murine peritoneal macrophages are terminally
differentiated non-transformed cells, and were used instead of J774 in order to
avoid replication of the host cells, as well as additional toxicity due to the tumoral
character of the J774 line. Parasites were fluorescently labelled with
carboxyfluorescein succinimidyl ester (CFSE) prior to the infection to facilitate
their fluorescent visualization inside the macrophages. The infection index
(number of parasites/macrophage) decreased from 5.3 ± 2.5 in untreated
macrophages to 1.6 ± 0.8 after incubation with 15a at 10 µM concentration. A
visual assessment of this activity is shown in Figure 4.

ACCEPTED MANUSCRIPT
Figure 4. Leishmanicidal activity of 15a on murine peritoneal macrophages infected with L. pifanoi
amastigotes. Infected macrophages were treated with 15a (10 µM) for 12 h. Infection was observed
under a confocal microscope. Amastigotes were prelabelled intracellularly with CFSE (λexc = 488
nm/ λem = 520 nm, green fluorescence). Intracellular nucleic acids were stained with DAPI (λexc
50 nm/ λem = 460 nm, blue fluorescence).
=
3
Mechanistic studies
The proposed mitochondrial involvement in the leishmanicidal mechanism of our
compounds was corroborated by two complementary approaches. First, as the
mitochondrion is the main contributor to ATP synthesis in Leishmania, the
variation in the levels of intracellular ATP with our compounds was monitored
using L. donovani promastigotes from the 3-Luc strain.41 These parasites express a
cytoplasmic form of firefly luciferase that is episomally encoded. The free
cytoplasmic ATP was the limiting substrate for in vivo luminescence, as luciferin
supply was in excess, administered as the caged membrane-permeable substrate
DMNPE-luciferin. This strategy has been previously employed to characterize the
involvement of mitochondrion in leishmanicidal mechanisms.19,42 At 50 µM
concentration, all studied compounds caused a decrease in luminescence to a
variable extent (Table S1). The dependence of this effect with concentration is

ACCEPTED MANUSCRIPT
represented in Figure S1 for the case of 15a, which showed a threshold of 3.12 µM.
The inhibition endpoint was reached in less than 5 min after compound addition.
Inhibition of luciferase in vitro at the highest concentration (50 µM) was lower
than 2% in every case (data not shown). This dramatic drop in ATP levels can be
feasibly due either to permeabilization of the cell membrane or to mitochondrial
dysfunction. To check the first mechanism, we studied whether a selection of our
compounds were able to induce plasma membrane permeabilization. To this end,
the entry of the vital dye SYTOX Green, included in the incubation medium, was
monitored after addition of the styrylquinoline by examining the increase in its
fluorescence due to its binding to intracellular nucleic acids. Fully permeabilized
cells were obtained by addition of 0.1% Triton X-100. None of the compounds was
found to alter the plasma membrane permeability (Figure S2).
Once plasma membrane permeabilization was discarded, loss of mitochondrial
functionality was addressed. Variations in the mitochondrial electrochemical
potential (∆Ψ
m
), essential to drive ATP synthesis, were monitored after addition of
, was
selected compounds. The accumulation of rhodamine 123, driven by ∆Ψ
m
tested by flow cytometry (Figure S3). Compounds 14d, 14h, and 15a showed the
highest inhibition and were selected for a concentration dependency study. At 25
µM, these styrylquinolines markedly decreased rhodamine 123 levels, matching
the decrease induced by 20 mM KCN, a control for full mitochondrial
depolarization. The combined results show that exposure to our compounds
induces mitochondrial dysfunction.
To further corroborate our initial hypothesis, the mitochondrial localization of
compound 15a inside Leishmania was assessed on axenic amastigotes by confocal
microscopy. At 20 µM, the accumulation of 15a appeared as a punctuated

ACCEPTED MANUSCRIPT
fluorescence pattern (Figure 5, panel A). Fluorescence was considerably inhibited
upon addition of 1 mM KCN, a concentration able to fully depolarize mitochondrion
while preserving parasite viability,43 underscoring the role of ∆Ψ
in the
m
intracellular compound accumulation. In a double-labeling experiment, 15a was
incubated together with the fluorescent mitochondrial probe MitoTracker Red
(MTR). A high degree of overlapping for both probes was observed (Figure 5, panel
B).
Figure 5. Confocal microscopy of L. pifanoi axenic amastigotes incubated with compound 15a. A:
Uptake and intracellular distribution of 15a in L. pifanoi amastigotes. Parasites were incubated with
15a at 20 µM for 1 h and observed by confocal microscopy. Amastigotes with a depolarized
mitochondrion were obtained by incubation with 1 mM KCN 15 min prior to addition of 15a. B: Co-
localization of 15a with MTR. Amastigotes were successively labeled with 0.05 µM MTR, 20 µM 15a,
and 5 µg/ml DAPI. Fluorescence settings: λexc = 488 nm/λem = 520 nm for 15a; λexc = 350 nm/ λem
60 nm for DAPI; and λexc = 570 nm/ λem = 590 nm for MTR. Fluorescence overlapping for 15a and
MTR was expressed as Pearson coefficient (r); r = 0.53 ± 0.03. Magnification bar = 15 µm.
=
4

ACCEPTED MANUSCRIPT
As the Leishmania mitochondrion is involved in programmed cell death,44 cells at
the late stages of apoptosis, characterized by a fragmented chromatin, can be
identified by a lower degree of staining with propidium iodide (subG1 population).
As shown in Figure S4, the subG1 population for L. donovani promastigotes
incubated with 15a (4.3%) was similar to untreated parasites (4.6%), whereas
upon treatment with 2, an inducer of apoptosis–like process in Leishmania, it
reached 22.5%.45 The concentration of 15a in this experiment (20 µM) exceeded
that required for a full elimination of the promastigotes. Therefore, compound 15a
is not an apoptosis inducer.
The morphological damage inflicted by 15a on L. donovani promastigotes was
assessed by transmission electron microscopy (Figure 6). Parasites treated with
15a showed a swollen mitochondrion and strong vacuolization of the cytoplasm,
together with the appearance of distended acidocalcisomes, identified by the
presence of electron-dense material, mostly Zn2+ and Fe in these organelles.
2
2
+
Acidocalcisomes, in fact, work as an acidic storage for divalent (Ca ) and heavy
metal (Zn2+ and Fe ) cations, due to their high polyphosphate content.46 In view of
this result, the role of acidocalcisomes as a feasible target of 15a was further
investigated, by using acridine orange (AO) dye as an acidocalcisome pH
indicator.47 Not unexpectedly, the intraorganellar acidic pH was quenched by 15a,
but not by the less basic (and less active) 23, according to the quantitative
differences in fluorescence of AO inside the parasites (Figure S5, panel A) and
visualization by confocal microscopy (Figure S5, panel B). Nevertheless, the
importance of acidocalcisomes in terms of the leishmanicidal mechanism of 15a is
questionable, for two reasons. First, the polyphosphate content was not reduced
after treatment with 15a (5 µM), as evaluated by DAPI fluorescence. Second,
2+

ACCEPTED MANUSCRIPT
acidocalcisomes were discarded as a target for tafenoquine (4), even though it
caused a more severe dysfunction than 15a, including a decrease in polyphosphate
content, because parasites deficient in acidocalcisome biogenesis were equally
susceptible to 4.48
These studies notwithstanding, additional targets for styrylquinolines cannot be
ruled out, as frequently found for other antileishmanial drugs, such as
miltefosine49,50 or paromomycin.51,52 Even for drugs with traditionally well-
established lethal mechanisms, additional effects are often uncovered, mostly
through analysis of drug-resistant strains. A case in point is amphotericin B; its
lethal mechanism is mostly based on the plasma membrane permeabilization of
the parasite by formation of an aqueous pore. Nevertheless, subtle additional
effects contributing to its lethal action have been recently described, including
triggering of signal transduction pathways and induction of oxidative stress, not
only for pathogens, but also for their host cells.53
Figure 6. Transmission electron microscopy of L. donovani promastigotes treated with 15a (5 µM).
Legend: (K) kinetoplast, (M) mitochondrion, (N) nucleus, (FP) flagellar pocket, (*) acidocalcisome.

ACCEPTED MANUSCRIPT
CONCLUSIONS
Our results confirm the mitochondrial druggability by aminoquinolines, adding the
-amino-2-styrylquinoline family to the previously known 8-aminoquinolines, that
4
also kill Leishmania by inhibition of mitochondrial functionality.17,18 Some of our
compounds, especially 14a, 14c, 14k and 15a, exhibit a promising antileishmanial
profile, with a similar activity against promastigote and amastigote forms to the
drug miltefosine, used as a reference, and a good selectivity index.
EXPERIMENTAL SECTION
Chemistry
General experimental information
All reagents (Aldrich, Fischer, Alpha Aesar, SDS) and solvents (Scharlau, Fischer,
SDS) were of commercial quality and were used as received. Reactions under
microwave irradiation were carried out in a CEM Discover SP microwave reactor.
Reactions were monitored by thin layer chromatography on aluminium plates
coated with silica gel and fluorescent indicator (Macherey-Nagel Xtra SIL
G/UV254). Separations by flash chromatography were performed on silica gel
(Scharlau 40–60 µm, 230–400 mesh ASTM) or neutral alumina (Merck S22).
Melting points were determined using a Stuart Scientific apparatus, SMP3 Model,
and are uncorrected. Infrared spectra were recorded with an Agilent Cary630 FTIR
spectrophotometer with a diamond accessory for solid and liquid samples. NMR
spectroscopic data were recorded using a Bruker Avance 250 spectrometer
operating at 250 MHz for H NMR and 63 MHz for 13C NMR maintained by the NMR
facility of Universidad Complutense (CAI de Resonancia Magnética Nuclear);
1

ACCEPTED MANUSCRIPT
chemical shifts are given in ppm and coupling constants in Hertz. High-resolution
mass spectra (HRMS) were recorded on a mass spectrometer fitted with an
electrospray detector (ESI) by the mass spectral facility of Universidad
Complutense (CAI de Espectrometría de Masas) and elemental analyses were
determined by the microanalysis facility of Universidad Complutense (CAI de
Microanálisis Elemental), using a Leco 932 combustion microanalyzer. The
synthesis of compounds 6 and 10 is described in the Supporting Information.
General procedure for the synthesis of protected polyamino styrylquinolines
(
11)
(E)-4-Chloro-2-styrylquinoline 6 (1 eq), the corresponding monoprotected
diamine 10 (1.5 eq) and phenol (6 eq) were placed in a round bottom flask and
stirred at 110 °C for 6 h. The reaction mixture was basified with an aqueous
solution of NaOH (5M) and extracted with ethyl acetate (3 × 10 ml). The combined
organic phases were dried with anhydrous Na
2
SO and evaporated. The resulting
4
crude was purified by flash chromatography through a silica gel column eluting
with hexane: ethyl acetate (2:1, v/v) as the mobile phase to give the desired
products.
tert-Butyl (E)-(2-((2-styrylquinolin-4-yl)amino)ethyl) carbamate (11a).
Prepared from (E)-4-chloro-2-styrylquinoline 6 (0.654 g, 2.46 mmol), tert-butyl
(2-aminoethyl) carbamate 10a (0.591 g, 3.69 mmol) and phenol (2.1 g, 22.3
mmol); yield: 0.678 g (71%); brown solid. Mp: 71-73 °C. IR (neat): 3346, 2925,
-1 1
1
7
1
682, 1585, 1534, 1161 cm . H NMR (250 MHz, MeOD) δ 8.00 (d, J = 8.3 Hz, 1H),
.82 (d, J = 8.3 Hz, 1H), 7.77 – 7.59 (m, 4H), 7.44 – 7.32 (m, 4H), 7.25 (d, J = 16.5 Hz,
H), 7.03 (s, 1H), 3.53 (t, J = 5.8 Hz, 2H), 3.42 (t, J = 5.8 Hz, 2H), 1.44 (s, 9H) ppm.

ACCEPTED MANUSCRIPT
13C NMR (63 MHz, MeOD) δ 159.6, 157.6, 153.6, 148.3, 138.3, 136.7, 131.6, 130.3 (3
C), 129.0, 128.7 (2 C), 128.1, 126.1, 122.6, 119.9, 96.6, 80.7, 45.1, 40.4, 29.2 (3c)
ppm. Elemental analysis (%) calcd for C24
H
27
N
3
O : C 74.01, H 6.99, N 10.79; found:
2
C 73.85, H 6.84, N 10.46.
tert-Butyl (E)-(4-((2-styrylquinolin-4-yl)amino)butyl) carbamate (11b).
Prepared from (E)-4-chloro-2-styrylquinoline 6 (0.6 g, 2.25 mmol),tert-butyl (4-
aminobutyl) carbamate 10b (0.637 g, 3.38 mmol) and phenol (1.91 g, 20.3 mmol);
yield: 0.627 g (74%); orange solid. Mp: 93- 95 °C. IR (neat): 3338, 2923, 1681,
-1 1
1
586, 1535, 1162 cm . H NMR (250 MHz, MeOD) δ 8.07 (d, J = 8.4 Hz, 1H), 7.84 (d,
J = 8.4 Hz, 1H), 7.69 – 7.57 (m, 4H), 7.45 – 7.24 (m, 5H), 6.84 (s, 1H), 3.48 (t, J = 6.7
Hz, 2H), 3.14 (t, J = 6.7 Hz, 2H), 1.87 – 1.74 (m, 2H), 1.72 – 1.59 (m, 2H), 1.41 (s, 9H)
ppm. 13C NMR (63 MHz, MeOD) δ 159.1, 158.2, 153.1, 149.6, 138.5, 135.5, 131.1,
1
4
7
30.3, 130.3 (2 C), 130.0, 129.0, 128.6 (2C), 125.7, 122.5, 120.2, 96.9, 80.3, 44.1,
1.5, 29.2 (3C), 27.2 ppm. Elemental analysis (%) calcd for C26
H
31
N
3
O : C 74.79, H
2
.48, N 10.06; found: C 74.54, H 7.51, N 9.70.
General procedure for the synthesis of deprotected polyamino
styrylquinolines (12)
The suitable styrylquinoline 11 was dissolved in a mixture of metanol:
trifluoroacetic acid (2:1, v/v) and stirred at room temperature for 1h. Once the
reaction was finished, the reaction mixture was basified with aqueous NaOH (5 M)
and extracted with a mixture of CH Cl : MeOH (10:1, v/v), which was dried with
2
2
anhydrous Na
2
SO and evaporated in vacuo to give the pure product.
4
1
(
E)-N -(2-Styrylquinolin-4-yl)ethane-1,2-diamine (12a). Prepared from
compound 11a (0.578 g, 1.49 mmol); yield: 0.374 g (87%); yellow solid. Mp: 85-
-1 1
8
6 °C. IR (neat): 3250, 2921, 1583, 1531, 753 cm . H NMR (250 MHz, MeOD) δ

ACCEPTED MANUSCRIPT
8.06 (d, J = 8.3 Hz, 1H), 7.84 (d, J = 8.3 Hz, 1H), 7.68 – 7.55 (m, 4H), 7.43 – 7.22 (m,
5
H), 6.87 (s, 1H), 3.51 (t, J = 6.3 Hz, 2H), 3.01 (t, J = 6.3 Hz, 2H) ppm. 13C NMR (63
MHz, MeOD) δ 158.2, 153.1, 149.5, 138.5, 135.6, 131.2, 130.3 (3 C), 130.1, 129.1,
28.6 (2 C), 125.9, 122.5, 120.3, 96.9, 46.7, 41.5 ppm. Elemental analysis (%) calcd
1
for C19
H
19
N : C 78.86, H 6.62, N 14.52; found: C 78.56, H 6.72, N 14.32.
3
1
(
E)-N -(2-Styrylquinolin-4-yl)butane-1,4-diamine (12b).
Method A. Prepared from compound 11b (0.150 g, 0.359 mmol) according to the
general procedure described above; yield: 0.100 g (88%); yellow solid. Mp: 68-69
-1 1
°
C. IR (neat): 3261, 2918, 2850, 1580, 1531, 752 cm . H NMR (250 MHz, MeOD) δ
8.05 (d, J = 8.3 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.68 – 7.53 (m, 4H), 7.44 – 7.22 (m,
5H), 6.78 (s, 1H), 3.41 (t, J = 6.7 Hz, 2H), 2.70 (t, J = 6.7 Hz, 2H), 1.78 (dd, J = 13.8,
6
.9 Hz, 2H), 1.64 (dd, J = 13.8, 6.9 Hz, 2H) ppm. 13C NMR (63 MHz, MeOD) δ 158.2,
153.1, 149.6, 138.5, 135.5, 131.1, 130.4, 130.3 (2 C), 130.0, 129.1, 128.6 (2C),
125.8, 122.5, 120.2, 96.9, 44.2, 42.6, 31.5, 27.4 ppm. Elemental analysis (%) calcd
for C21
H
23
N : C 79.46, H 7.30, N 13.24; found: C, 79.12; H, 6.93; N, 13.13.
3
Method B. Ethylenediamine (1.130 g, 18.8 mmol) was added to 4-chloro-2-
styrylquinoline 6 (0.250 g, 0.94 mmol) in a pressure-tight microwave tube
containing a stirring bar. The mixture was heated under microwave irradiation for
1
h at 120 °C, with an irradiation power of 200 W, using a CEM Discover SP
microwave reactor. Thereafter, the cooled reaction mixture was treated with 10%
KOH aqueous solution, extracted with CH Cl , dried with anhydrous Na SO and
2
2
2
4
evaporated in vacuo. The excess of ethylenediamine was removed by distillation in
the presence of EtOH. The resulting residue was subsequently crystallized in
Methanol and hexane to give compound 12b as a yellow solid 0.109 g (40%).

ACCEPTED MANUSCRIPT
(
E)-2-Bromo-N-(4-((2-styrylquinolin-4-yl)amino)butyl)acetamide (13a). A
solution of 2-bromoacetyl bromide (1 eq, 0.53 mmol) in CH Cl (20 ml) was slowly
2
2
added (30 min) using a syringe pump to a solution of compound 12a (0.170 g, 0.53
mmol) in dichlorometane (20 ml). Once the addition was finished, the reaction
mixture was stirred at -5 °C for an additional 30 min period. Then, the resulting
crude was basified with an aqueous solution of NaOH (1M) and extracted with a
mixture of CH Cl : MeOH (10:1, v/v), dried with anhydrous Na
SO
2 4
and
2
2
evaporated in vacuo, to give the desired product, which was used in the next step
without further purification.
The same procedure was used to obtain (E)-2-bromo-N-(2-((2-styrylquinolin-4-
yl)amino)ethyl)acetamide (13b) starting from 12b.
General procedure for the synthesis of 4-aminostyrylquinolines 14 and 15.
Aliphatic amine derivatives 14.
Compound 13 (80 mg, 0.195 mmol), the suitable amine (1 eq) and Cs
2
CO (0.127 g,
3
0.39 mmol, 2 eq) were suspended in EtOH (1 ml) in a pressure-tight microwave
tube containing a stirring bar. The mixture was heated under microwave
irradiation for 30 minutes at 120 °C, with an irradiation power of 200 W, using a
focused microwave reactor. Thereafter, the cooled reaction mixture was dissolved
in CH
Cl
2 2
and washed with water, which was re-extracted with CH
2
Cl . The
2
combined organic layers were dried with anhydrous Na
2
SO and evaporated in
4
vacuo. The resulting crude products were subsequently crystallized from MeOH-
hexane mixtures or purified via a neutral alumina column.
(
(
E)-2-(Dimethylamino)-N-[2-(2-styrylquinolin-4-ylamino)ethyl]acetamide
14a). Prepared from 13a (80 mg, 0.195 mmol) and dimethylamine (0.02 ml,

ACCEPTED MANUSCRIPT
0.195 mmol); the compound was crystallized from a MeOH-hexane mixture to give
14a (0.052 g, 72%) as an orange powder. Mp 57- 60 °C. IR (neat): 3346, 2923,
-
1 1
2
8
7
2
1
1
C
850, 2780, 1660, 1588, 1365, 1044 cm . H NMR (250 MHz, CDCl ) δ 7.99 (d, J =
3
.4 Hz, 1H), 7.85 (d, J = 8.4 Hz, 1H), 7.76 (t, J = 6.7 Hz, 1H), 7.71 – 7.60 (m, 4H),
.47-7.32 (m, 5H), 6.62 (s, 1H), 6.55 (br s, 1H), 3.86-3.75 (m, 2H), 3.60 – 3.47 (m,
H), 3.03 (s, 2H), 2.30 (s, 6H) ppm. 13C NMR (63 MHz, CDCl
) δ 174.0, 156.7, 150.7,
3
37.3, 133.7, 133.5, 130.5, 129.8 (2C), 129.1 (2C), 128.7, 127.6 (2C), 125.0, 120.6,
18.7, 96.3, 63.3, 46.5 (2C), 46.2, 38.9 ppm. Elemental analysis (%) calcd for
23
H
N
26 4
O: C 73.77, H 7.00, N 14.96; found: C 73.61, H 7.16, N 14.40.
E)-2-(Diethylamino)-N-[2-(2-styrylquinolin-4-ylamino)ethyl]acetamide
14b). Prepared from 13a (80 mg, 0.195 mmol) and diethylamine (14 mg, 0.195
mmol); the compound was crystallized from a MeOH-hexane mixture to give 14b
(
(
(0.056 g, 71 %) as an orange powder. Mp 54- 57 °C. IR (neat): 3341, 2959, 2905,
-1 1
2
850, 1658, 1588, 1430, 1367, 756 cm . H NMR (250 MHz, CDCl ) δ 7.99 (d, J = 8.4
3
Hz, 1H), 7.86 (d, J = 7.6 Hz, 1H), 7.71-7.59 (m, 4H), 7.49 – 7.30 (m, 5H), 6.62 (s, 1H),
3
0
1
9
C
.85 – 3.75 (m, 2H), 3.60 – 3.46 (m, 2H), 3.12 (s, 2H), 2.56 (q, J = 7.1 Hz, 4H), 1.04 –
.93 (t, J = 7.1 Hz, 6H) ppm. 13C NMR (63 MHz, CDCl
3
) δ 175.6, 156.6, 150.8, 137.2,
33.6, 130.3, 130.3, 129.8, 129.6, 129.1 (2C), 128.7, 127.6 (2C), 125.1, 120.6, 118.7,
6.2, 57.8, 49.2 (2C), 46.2, 38.9, 12.7 (2C) ppm. Elemental analysis (%) calcd for
25
H
N
30 4
O: C 74.59, H 7.51, N 13.92; found: C 74.52, H 7.10, N 13.50.
E)-2-(pyrrolidin-1-yl)-N-(2-(2-styrylquinolin-4-ylamino)ethyl)acetamide
14c). Prepared from 13a (80 mg, 0.195 mmol) and pyrrolidine (14 mg, 0.195
mmol); the compound was crystallized from a MeOH-hexane mixture to give 14c
(
(
(0.054 g, 69 %) as a red solid. Mp 68- 71 °C. IR (neat): 3346, 2918, 2848, 2809,
-1 1
1
655, 1588, 1539, 1365 cm . H NMR (250 MHz, CDCl ) δ 7.98 (d, J = 7.7 Hz, 1H),
3

ACCEPTED MANUSCRIPT
7.85 (d, J = 8.1 Hz, 1H), 7.70 – 7.60 (m, 4H), 7.48-7.32 (m, 5H), 6.62 (s, 1H), 6.59 (br
s, 1H), 3.85-3-78 (m, 2H), 3.60 – 3.46 (m, 2H), 3.26 (s, 2H), 2.67-2.53 (m, 4H), 1.85-
1
1
5
C
.75 (m, 4H) ppm. 13C NMR (63 MHz, CDCl
) δ 174.4, 156.7, 150.7, 148.9, 137.3,
3
33.5, 130.5, 129.8 (2C), 129.1 (2C), 128.7, 127.6 (2C), 125.0, 120.6, 118.7, 96.3,
9.4, 55.1 (2C), 46.2, 38.9, 24.4 (2C) ppm. Elemental analysis (%) calcd for
25
H
N
28 4
O: C 74.97, H 7.05, N 13.99; found: C 74.04, H 6.98, N 13.83.
E)-2-(Piperidin-1-yl)-N-(2-(2-styrylquinolin-4-ylamino)ethyl)acetamide
14d). Prepared from 13a (80 mg, 0.195 mmol) and piperidine (17 mg, 0.195
mmol); the compound was crystallized from a MeOH-hexane mixture to give 14d
(
(
(0.057 g, 70 %) as a yellow solid. Mp 71- 74 °C. IR (neat): 3348, 2936, 2851, 1659,
-1 1
1
563, 1538, 1366, 1129 cm . H NMR (250 MHz, CDCl
3
) δ 8.10 (d, J = 8.2 Hz, 1H),
7.97 (d, J = 8.2 Hz, 1H), 7.8-7.72 (m, 4H), 7.6-7.4 (m, 5H), 6.74 (s, 1H), 3.99 – 3.84
(m, 2H), 3.72-3.58 (m, 2H), 3.14 (s, 2H), 2.62-2.48 (m, 4H), 1.70 – 1.67 (m, 4H),
1
1
9
.57 – 1.55 (m, 2H) ppm. 13C NMR (63 MHz, CDCl
) δ 174.2, 156.6, 150.8, 137.2,
3
33.6, 130.3 (2C), 129.8, 129.7, 129.1 (2C), 128.7, 127.6 (2C), 125.1, 120.6, 118.7,
6.2, 62.6, 55.4 (2C), 46.2, 38.9, 26.6 (2C), 24.0 ppm. Elemental analysis (%) calcd
for C26
H
30 4
N O: C 75.33, H 7.29, N 13.52; found: C 75.66, H 7.05, N 13.70.
(
E)-2-Morpholino-N-[2-(2-styrylquinolin-4-ylamino)ethyl]acetamide (14e).
Prepared from 13a (80 mg, 0.195 mmol) and morpholine (17 mg, 0.195 mmol);
the compound was crystallized from a MeOH-hexane mixture to give 14e (0.048 g,
59%) as an orange powder. Mp 67- 70 °C. IR (neat): 3297, 2918, 2850, 1656, 1599,
-1 1
1
560, 1538, 1464 cm . H NMR (250 MHz, CDCl ) δ 8.03 (d, J = 8.0 Hz, 1H), 7.88 (d,
3
J = 8.5 Hz, 1H), 7.79 (br s, 1H), 7.72 -7.57 (m, 4H), 7.48 – 7.31 (m, 5H), 6.59 (s, 1H),
.88 3.77 (m, 2H), 3.77 – 3.65 (m, 4H), 3.62 – 3.45 (m, 2H), 3.10 (s, 2H), 2.60 – 2.49
3
13
(
m, 4H) ppm. C NMR (63 MHz, CDCl ) δ 173.2, 153.1, 151.5, 147.6, 136.8, 131.4,
3