In vitro phenotypic screening of 7-chloro-4-amino(oxy)quinoline derivatives as putative anti-Trypanosoma cruzi agents

Article abstract of DOI:10.1016/j.bmcl.2013.12.071

In this study, a series of 22 pre-synthesized 7-chloro-4-amino(oxy) quinoline derivatives was assayed in vitro as potential antichagasic agents. A primary screening against Trypanosoma cruzi epimastigotes and a non-specific cytotoxicity assay on murine fibroblasts were simultaneously performed, resulting quinolines 3, 7 and 12 with great selectivity (SI) on the extracellular parasite (SI₇, SI₃, SI₁₂ and SI_BZ >9.44). Therefore, the activity of these derivatives was evaluated on intracellular amastigotes, achieving derivative 7 the best SI (SI = 12.73). These results, supported by the in silico prediction of a good oral bioavailability and a suitable risk profile, propose the 4-amino-7- chloroquinoline scaffold as a potential template for designing trypanocidal prototypes.

Full text of DOI:10.1016/j.bmcl.2013.12.071

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1209–1213
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
In vitro phenotypic screening of 7-chloro-4-amino(oxy)quinoline
derivatives as putative anti-Trypanosoma cruzi agents
c
Cristina Fonseca-Berzal a,b, , Fernando A. Rojas Ruiz , José A. Escario a,b, Vladimir V. Kouznetsov c,
⇑
Alicia Gómez-Barrio a,b
a CEI Campus Moncloa, UCM-UPM & CSIC, Madrid, Spain
b Departamento de Parasitología, Facultad de Farmacia, Universidad Complutense de Madrid, Pza. Ramón y Cajal s/n, 28040 Madrid, Spain
c Laboratorio de Química Orgánica y Biomolecular, Escuela de Química, Universidad Industrial de Santander, A.A. 678 Bucaramanga, Colombia
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study, a series of 22 pre-synthesized 7-chloro-4-amino(oxy)quinoline derivatives was assayed
in vitro as potential antichagasic agents. A primary screening against Trypanosoma cruzi epimastigotes
and a non-specific cytotoxicity assay on murine fibroblasts were simultaneously performed, resulting
quinolines 3, 7 and 12 with great selectivity (SI) on the extracellular parasite (SI7, SI3, SI12 and SIBZ
>9.44). Therefore, the activity of these derivatives was evaluated on intracellular amastigotes, achieving
derivative 7 the best SI (SI = 12.73). These results, supported by the in silico prediction of a good oral bio-
availability and a suitable risk profile, propose the 4-amino-7-chloroquinoline scaffold as a potential tem-
plate for designing trypanocidal prototypes.
Received 18 November 2013
Revised 17 December 2013
Accepted 18 December 2013
Available online 9 January 2014
Keywords:
Trypanosoma cruzi
Chloroquinoline derivatives
Cytotoxicity
Ó 2014 Elsevier Ltd. All rights reserved.
Lipinski’s rule
OSIRIS software
Although more than a century has elapsed since the discovery
of Chagas disease (American trypanosomiasis), the etiological
treatment of a neglected tropical disease that is currently endemic
in poor rural areas of 21 countries along Central and South America
is still unsatisfactory.1 The parasitic illness caused by the hemofla-
gellate Trypanosoma cruzi and endemically transmitted by triato-
mine vectors, has in the last years emerged in non-endemic
countries outside Latin America since the increase of international
migrations and the existence of non-vectorial mechanisms of
transmission (e.g., congenital route),2,3 infecting this way about
10 million people worldwide.1 Focusing on the chemotherapy, nif-
urtimox (a 5-nitrofuran) and benznidazole (a 2-nitroimidazole),
the only two drugs up to now commercialized for the specific
treatment of Chagas disease, were introduced more than forty
years ago and their accessibility to patients has been discontinued
through this time.4 Although they are effective in the early stages
of the trypanosomiasis, both display a limited activity during the
chronic infection.5 Moreover, the toxicity associated to these
drugs6 and the existence of T. cruzi strains naturally resistant to
them,7 also hinder the effectiveness of these treatments. The epi-
demiological data, together with the lack of either a suitable che-
motherapy or a vaccine, supports the priority in the development
of new prototypes of anti-T. cruzi agents.
In the present work, a first series of 16 different 7-chloroquino-
line molecules substituted at the C-4 position by either benzyl-
amino fragment or N-(aminoalkyl)-1,3-thiazolidin-4-one moiety,
previously synthesized and tested against Plasmodium falciparum,8
has been evaluated as potential antichagasic compounds according
to the trypanocidal activity exhibited by some thiazolidine deriva-
tives9,10 and diverse aminoquinolines.11,12 Concretely, the biologi-
cal properties associated to the substituted 1,3-thiazolidin-4-ones
have led to the introduction of this structure whether in several
antimicrobial agents13 as well as in trypanocidal prototypes with
promising in vitro activity.14,15 Nevertheless, data of neither 7-
chloroquinoline-thiazolidinone derivatives (called chloroquine hy-
brids) nor other 4-arylaminomethylquinolines tested on T. cruzi
models have been found in literature. Likewise, a second series of
4-aryloxy-7-chloroquinoline molecules16 also assessed as antima-
larial drug prototypes (data not published), has been in vitro eval-
uated as potential trypanocidal agents.
Both series of compounds were synthesized by employing
straightforward and efficient procedures.8,16 Benzylamino or
aryloxy fragments were connected to the 4,7-chloroquinoline ring
(DCQ) by nucleophilic substitution SNAr using commercial benzyl-
amines or substituted phenols to give compounds 1–4 and com-
pounds 17–22, respectively. Chloroquinoline–thiazolidinone
hybrids 5–16 were assembled via a synthetic two-step protocol
that includes the preparation of diamines based on DCQ ring
⇑
Corresponding author. Tel.: +34 913941818; fax: +34 3941815.
E-mail address: crfonseca@pdi.ucm.es (C. Fonseca-Berzal).
through nucleophilic substitution of DCQ and a,x-diaminoalkanes
0960-894X/$ - see front matter Ó 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.071

1210
C. Fonseca-Berzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1209–1213
NH2(CH2)nCH2NH2 (n = 1–3) and the one-pot three component
reaction of these diamines, diverse (hetero)aromatic aldehydes
Regarding the trypanocidal activity of the 7-chloroquinoline
derivatives, quinolines 3, 7 and 12 proved to be the most active
compounds over T. cruzi epimastigotes (Table 1). These results, to-
gether with the LogP values predicted, confirm the importance of
an appropriate lipophilicity that allows these derivatives to access
into the parasitic cell19,22 and consequently, interact with the
hydrophobic binding site of an enzyme or receptor.23 Conversely,
the presence of 4-hydroxy-3-methoxyphenyl (compounds 13–15)
or 3-hydroxy-4-methoxyphenyl radicals (compound 16) on the
thiazolidinone-based hybrids, seems to decrease these LogP values
below the estimated average (4.095) and to significantly modify
the activity of these derivatives on the extracellular parasite when
compared with the unsubstituted phenyl ones (compounds 11 and
12). However, the para introduction of hydroxyl groups on the phe-
nyl ring (compounds 13–15) not only turns this series into the less
active compounds from the first group,24 but also into the less
harmful for fibroblasts. Otherwise, such an effect was not observed
when the phenolic hydroxyl group is introduced in meta position
(compound 16) (Table 1). Details of the growth inhibition assay
on T. cruzi epimastigotes are described in the Supplementary
material.
Since the severe side effects frequently suffered by treated Cha-
gas patients25,26 often lead to a lack of treatment adherence, indi-
rectly encouraging the development of T. cruzi resistance towards
nifurtimox and benznidazole,27 other molecular descriptors point-
ing to the presence of structural fragments generally responsible
for mutagenic, tumorigenic, irritant or reproductive effects28 were
also evaluated. The toxicity risk profile assessment was performed
employing the OSIRIS free software (http://www.organic-chemis-
try.org/prog/peo)29 and prediction results were valued and color-
coded, showing those properties with high risks of undesired
effects in red (e.g., mutagenicity or poor intestinal absorption),
whereas a green color indicates drug-conform behavior. Virtually
exploring the potential toxicity associated, all the synthesized 7-
chloroquinoline derivatives represent low risks, with the exception
of compounds 17–20, in which the introduction of the formyl
group as fragment in the structure induces a negative effect over
the safety of these molecules. Even though, the fragments and
topology of both reference compounds present potential risk as
well (Table S2, Supplementary material).
Besides, the in vitro unspecific cytotoxicity of all these quino-
line derivatives was simultaneously evaluated on NCTC-929 fibro-
blasts (Experimental procedures are detailed in the Supplementary
material). It was specially noticed that compounds previously de-
fined as cytotoxic on J774 murine macrophages (3, 7 and 8), did
not show such an obvious effect on NCTC-929 fibroblasts and
HepG2.8 Moreover, these compounds also achieved remarkable
selectivity indexes on epimastigotes (SI >5) (Table 1). This different
cytotoxicity could occur as a result of a higher drug intake by mac-
rophages (phagocytic cell line) compared with that of fibroblasts
and hepatocytes (both non-phagocytic cell lines).30 Moreover, sev-
eral studies reviewed in31 lead to the idea that the process of
autophagy observed in mammalian cells, and usually involved in
survival pathways, can also act as a non-apoptotic mechanism of
cell death. In fact, the toxicity of chloroquine on both mouse mac-
rophages and L-strain fibroblasts by inducing autophagy has been
studied in detail, being the toxic effect of the antimalarial drug
more uniform for the first mammalian cell line,32,33 what also ex-
plains the differences registered in the toxicity caused by our chlo-
roquine derivatives on NCTC-929 fibroblasts (clone of strain L).
Finally, we also used the OSIRIS program (http://www.organic-
chemistry.org/prog/peo) for the prediction of drug-score and drug-
likeness parameters. According to this analysis, compounds 1–16
revealed promising values when compared with both reference
drugs. Nevertheless, compounds 17–22 proved to be the less
drug-like structures among the quinoline derivatives synthesized,
and
a-mercaptoacetic acid with ratios 1:2.5:2.5 respectively, in
dry acetonitrile (reflux, 12 h) to get solid products, which can be
filtered and recrystallized in ethanol from the reaction mixture
(Scheme 1).
All 7-chloro-4-amino(oxy)quinoline derivatives 1–22 were
purified by column chromatography and obtained as stable pow-
dered substances, which were fully characterized and their chem-
ical purity corroborated by the analysis of spectroscopic methods
(i.e., IR, 1H NMR, 13C NMR and GC–MS), and agree with previous
published data.8,16
The 22 synthesized quinolines have been distributed in two
groups according to their chemical structure, mainly based on
the chemical nature of the C-4 substituent attached to the
quinoline nucleus. Group 1 includes the simple 4-N-benzyl-
amino-7-chloroquinolines 1–4 and the 7-chloroquinoline-1,
3-thiazolidin-4-one conjugates 5–16 (Fig. 1). Group 2 includes 4-
aryloxyquinoline derivatives (compounds 17–22) (Fig. 2).
Molecular design was achieved based on structure–activity
relationships (SAR) studies and virtual screening analysis reported
in literature. The pre-screening for hit identification from libraries
of synthetic compounds was based on the oral bioavailability esti-
mated using the Lipinski’s rules concepts,17 through the analysis of
the rule of five by employing the free online software Molinspira-
tion (http://www.molinspiration.com/services/). The principal
molecular properties, including the number of hydrogen donors
(nNHOH), number of hydrogen acceptors (nNO), number of rotable
bonds (nRB), molecular weight (MW) and lipophilicity (LogP) were
calculated. The topological surface area (TPSA),18 another recog-
nized parameter for the membrane permeation and prerequisite
for the bioavailability, was also considered (Table S1, Supplemen-
tary material).
According to this analysis, the vast majority of the quinoline
derivatives did not present any violation for the oral activity
and therefore, are expected to display high bioavailability. Only
two 4-aryloxyquinolines (compounds 21 and 22) violated Lipin-
ski’s rule exhibiting LogP values higher than 5.0. In fact, the LogP
values estimated for compounds 1–20 (LogP 3.3–4.6) predict an
auspicious entry into the parasitic cell by penetrating across bio-
logical membranes.19,20 However, the antichagasic reference
drugs (nifurtimox and benznidazole) are considerably more
hydrophilic molecules exhibiting LogP below 1.0 value. Likewise,
the TPSA parameters obtained show acceptable values (24.919–
74.960 Å2) (Table S1, Supplementary material), signifying TPSA
values lower than 142 Å2 a good membrane permeability and
lower than 60 Å2 a good penetration through the blood–brain
barrier.21
Scheme 1. Reagents and conditions: (a) DCQ (4,7-dichloroquinoline) (2.5 mmol),
N-benzylamine or phenols (5.10 mmol), and K2CO3 (5.01 mmol), DMF, 140 °C, 10 h;
(b) DCQ (20.2 mmol), NH2(CH2)nCH2NH2 (101 mmol), 80 °C for 1 h, 140–150 °C for
6–7 h; (c) diamines based on DCQ, ArCHO, HSCH2COOH, PhMe, reflux for 1 h.

C. Fonseca-Berzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1209–1213
1211
Figure 1. Chemical structures of 4-N-benzylamino-7-chloroquinolines (compounds 1–4) and 7-chloroquinoline-1,3-thiazolidin-4-one conjugates (compounds 5–16) (Group
1).
Table 1
Antiepimastigote activity (%AE) and unspecific cytotoxicity on NCTC-929 fibroblasts
(%C) of 7-chloroquinoline derivatives (1–16) and 4-aryloxyquinoline derivatives (17–
22) expressed as the percentage of growth inhibition at the highest concentration
tested,a IC50
(l
M) and LC50
(
lM), respectively
b
c
c
Compound
%AE
IC50
(
l
M)
%C
LC50
2.28
14.81
42.78
7.90
(
l
M)
SI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
95.08
96.73
99.37
100
6.55
5.93
1.09
6.76
7.38
7.15
1.90
<8
71.43
89.20
69.39
89.76
82.06
85.59
74.50
74.87
68.19
ND
3.71
2.50
39.33
1.17
2.24
3.29
63.52
>5.19
ND
2.85
1.59
>14.06
<6.74
ND
>3.67
5.63
ND
1.36
1.95
2.14
2.94
ND
96.39
97.77
99.83
99.77
96.34
84.35
89.14
91.13
87.34
54.29
ND
93.07
57.83
94.40
97.28
96.92
99.22
89.95
100
16.49
23.55
120.65
41.53
178.49
19.49
11.18
14.06
215.54
ND
NDd
6.84
7.02
<1
92.41
89.85
60.55
ND
>32
Figure 2. Chemical structures of 4-aryloxy-7-chloroquinoline molecules (Group 2).
235.89
69.66
4.34
9.00
>256
88.80
63.05
86.23
87.44
88.09
ND
24.46
218.20
153.57
25.65
26.74
12.00
ND
likewise the antichagasic drug benznidazole (Fig. S1, Supplemen-
tary material).
17
18
19
20
21
22
NDd
113.22
13.18
12.50
2.94
6.17
3.61
Some antimalarial agents, such as quinacrine, display notable
in vitro activity against the causative agent of Chagas disease as
trypanothione reductase (TR) inhibitors, by interacting with the
enzyme active site through a ring nitrogen atom, a chlorine atom,
a methoxy group and a planar aromatic system.34 According to our
results, those 4-aryloxyquinolines derived from (iso)vainillin cores
(compounds 19 and 20) displayed an important improvement in
ND
Nifurtimox
Benznidazole
26.83
17.38
>256
>256
>70.91
>9.44
82.01
27.12
Selectivity indexes (SI) for epimastigotes were also estimated.
a
256
l
M for both experiments.
b
the activity profile on epimastigotes (IC50 ca. 12–13
with their respective derivatives with methoxy groups absence
(compounds 17 and 18) (IC50 >110 M) (Table 1). This fact, to-
lM) compared
Estimated by plotting drug concentrations versus percentages of antiepimas-
tigote activity and unspecific cytotoxicity, respectively.
c
Each concentration was tested by triplicate. SD less than 10% in all cases.
Not determined, because of solubility problems.
l
d
gether with the SAR approaches aforementioned, suggests TR as
a presumed target molecule for these derivatives. Moreover, the
most active compounds of this second group correspond to the
4-aryloxyquinolines 21 and 22, in which (iso)eugenol moieties
with a three carbon alkenyl side chain are incorporated (Table 1),
as well as other quinolines previously assayed on T. cruzi.35 On

1212
C. Fonseca-Berzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1209–1213
Table 2
Antiamastigote activity of promising derivatives expressed as the percentage of growth inhibition (%AA) at the selected concentrationsa,b and IC50
(
l
M)
M)
c
Compound
32
lM
16
—
100
—
100
100
lM
8
l
M
4
l
M
2
l
M
1
l
M
0.5
l
M
IC50
(
l
SI
3
7
12
—
—
—
100
100
—
38.67
—
96.63
100
41.93
29.95
33.15
74.71
100
29.89
—
26.65
64.37
100
—
—
20.24
55.64
100
—
—
—
41.64
96.54
5.34
9.48
6.61
0.80
8.01
12.73
2.13
>320
>512
Benznidazole
Nifurtimox
<0.50
Selectivity indexes (SI) for amastigotes were also estimated.
a
According to the criteria established in Ref. 30.
Each concentration was tested by triplicate. SD less than 10% in all cases.
Extrapolated by plotting drug concentrations versus percentages of antiamastigote activity.
b
c
the other hand, the low SI shown by derivatives 17–21, allow spec-
ulating about the absence of T. cruzi specific mechanisms of action,
assuming that the presence of methoxy groups on the phenyl ring
(compounds 19–22) causes an increase in the cytotoxicity of these
derivatives (Table 1). However, some tricyclic heterocycle-based
drugs with great affinity to T. cruzi TR (i.e., some phenothiazines)
also exert other effects such as an anti-calmodulin action or a mito-
chondrial disruption in epimastigotes and trypomastigotes,36,37
not only generating these damages upon the parasite, but also
interfering in vitro on the viability of mammalian cultured
cells.38,39
ferrocenic 4-aminoquinoline ureas resulted by the introduction
of a benzyl moiety as a secondary amine substituent. Indeed,
we have recently proposed the outstanding influence that ben-
zylamino fragments have over the effectiveness of trypanocidal
prototypes against the intracellular stage of T. cruzi,53 as this
moiety is also present in the reference drug benznidazole. Addi-
tionally, the higher LogP values for these three derivatives are
(Table S1, Supplementary material), the more active on the intra-
cellular parasite they become (IC50 value on amastigotes com-
pound 3 < compound 12 < compound 7), bearing out the
positive impact that relative lipophilicity has over biological
activity. Actually, these three quinolines showed good correlation
between their theoretical LogP values and the trypanocidal activ-
ity displayed on the intracellular parasite (r2 = 0.9885). However,
epimastigotes were more sensitive to these three derivatives
than intracellular amastigotes, as demonstrated by their IC50 val-
ues (Tables 1 and 2), suggesting that the host cell may function
as a barrier to these molecules.30 In spite of the fact that these
three molecules displayed outstanding IC50 values on the intra-
cellular parasite, resulting derivative 7 the most selective one
(SI = 12.73), no compound more active than benznidazole
The kind of phenotypic screening followed in this study enables
the identification of chemical protoypes with activity on multiple
parasite proteins or biochemical pathways, by the exposition of
whole organisms to the compounds.40 The existence of an intracel-
lular epimastigote-like form as an intermediate stage within the
mammalian host, morphologically and biochemically similar to
the bona fide one,41–43 supports the preliminary screening of try-
panocidal compounds on the non-infectious stage of the parasite.
However, the sensitivity of the extracellular forms (both epim-
astigotes and trypomastigotes) to drugs is usually higher than that
of the intracellular parasite and therefore, a growth inhibition as-
say over intracellular amastigotes must be carried out in order to
avoid any false positive found during the epimastigote susceptibil-
ity assay.44,45 The great capability of T. cruzi to invade and replicate
it as amastigote inside a wide variety of mammalian cells, allows to
employ several cell lines to reproduce in vitro the intracellular cy-
cle of the parasite.46,47 Our research group has great experience in
developing this kind of pharmacological assays by using either
phagocytic45,48 or non-phagocytic mammalian cells.22,49 However,
previous studies have revealed diverse effects displayed by chloro-
quine that interfere with the intracellular cycle of T. cruzi, affecting
both parasite and target cell. In fact, Hrabák et al.50 suggested that
chloroquine irreversibly blocks the NO production in murine peri-
toneal macrophages and therefore, increases the rate of infected
cells by T. cruzi. Moreover, Stecconi-Silva et al.51 proposed a differ-
ent behavior throughout the parasite kinetics in chloroquine-trea-
ted Vero cells infected with trypomastigotes. Considering these
findings, together with the scaffolds selected to test and the rela-
tive cytotoxicity on J774 macrophages we previously reported,8
NCTC-929 fibroblasts were here selected as the preferred host cell
for the amastigote susceptibility assay (see the Suplementary
material for the experimental procedures).
(IC50 = 0.80
lM and SI >320) and nifurtimox (IC50 <0.50 lM and
SI >512) was found.
The preliminary in vitro results compiled in the present work
propose the antiplasmodial 4-amino-7-chloroquinoline scaffold,
with derivative 7 (SI for epimastigotes = 63.52, SI for amastig-
otes = 12.73) as prototype of anti-T. cruzi agents, supported by
the in silico prediction of a good oral bioavailability and a suit-
able risk profile. However, the low selectivity displayed by some
chloroquinoline derivatives, prompts us to optimize the lead
compounds of this series in order to ameliorate their unspecific
cytotoxicity over T. cruzi host cells and therefore, to enhance
their potential antichagasic effectiveness. Finally, further kinetic
assays directed to explore the putative cellular target for this
series are required in order to assert the hypothesis here
presented.
Acknowledgments
Partial support of this work by the Spanish Ministries of Sci-
ence and Innovation (MICINN, ref. SAF2009-10399) and Foreign
Affairs and Cooperation (AP/036539/11), by Universidad Complu-
tense de Madrid (Proyecto de Cooperación al Desarrollo, ref. 9/12
and UCM-BSCH Research Group ‘Terapia Antiparasitaria’, ref.
911120) (C.F.-B, J.A.E and A.G.-B), and by Patrimonio Autónomo
Fondo Nacional de Financiamiento para la Ciencia, la Tecnología
y la Innovación, Francisco José de Caldas (contract RC-0572-
2012) (V.V.K.), is gratefully acknowledged. C.F.-B acknowledges
Moncloa Campus of International Excellence (UCM-UPM & CSIC)
for a PICATA predoctoral fellowship. The authors thank to LAFEPE,
Pernambuco, Brazil, for kindly providing benznidazole raw
product.
The activity on the intracellular parasite was only determined
for compounds that after the primary screening resulted at least
as selective as the cut-off estimated for benznidazole (SI >10).
According to this, only three derivatives (3, 7 and 12) from the
initial 22 molecules were tested on intracellular amastigotes, by
applying the methodology we previously reported.22 As Table 2
reflects, derivative 3 achieved the best trypanocidal profile on
amastigotes (IC50 = 5.34
lM). Similar results were reported by
Blackie et al.52 since the most active compound of a series of

C. Fonseca-Berzal et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1209–1213
1213
23. Paula, F. R.; Jorge, S. D.; de Almeida, L. V.; Pasqualoto, K. F.; Tavares, L. C. Bioorg.
Med. Chem. 2009, 17, 2673.
Supplementary data
24. da Silva, R. B.; Loback, V. B.; Salomão, K.; de Castro, S. L.; Wardell, J. L.; Wardell,
S. M.; Costa, T. E.; Penido, C.; Henriques, Md.; Carvalho, S. A.; da Silva, E. F.;
Fraga, C. A. Molecules 2013, 18, 3445.
25. Castro, J. A.; Díaz de Toranzo, E. G. Biomed. Environ. Sci. 1988, 1, 19.
26. Viotti, R.; Vigliano, C.; Lococo, B.; Álvarez, M. G.; Petti, M.; Bertocchi, G.;
Armenti, A. Exp. Rev. Anti Infect. Ther. 2009, 7, 157.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.071.
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