Transmembrane anion transport and cytotoxicity of synthetic tambjamine analogs

Article abstract of DOI:10.1039/c3ob42341g

Ten synthetic analogs of the marine alkaloids tambjamines, bearing aromatic enamine moieties, have been synthesized. These compounds proved to be highly efficient transmembrane anion transporters in model liposomes. Changes in the electronic nature of the

Full text of DOI:10.1039/c3ob42341g

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
PAPER
Transmembrane anion transport and cytotoxicity
of synthetic tambjamine analogs†
Cite this: Org. Biomol. Chem., 2014,
12, 1771
Elsa Hernando,a Vanessa Soto-Cerrato,b Susana Cortés-Arroyo,b
Ricardo Pérez-Tomásb and Roberto Quesada*a
Ten synthetic analogs of the marine alkaloids tambjamines, bearing aromatic enamine moieties, have
been synthesized. These compounds proved to be highly efficient transmembrane anion transporters in
model liposomes. Changes in the electronic nature of the substituents of the aromatic enamine or the
alkoxy group of the central pyrrole group did not affect this anionophore activity. The in vitro activity of
these compounds has also been studied. They trigger apoptosis in several cancer cell lines with IC50
values in the low micromolar range as well as modify the intracellular pH, inducing the basification of
acidic organelles.
Received 22nd November 2013,
Accepted 15th January 2014
DOI: 10.1039/c3ob42341g
www.rsc.org/obc
We recently reported the anion transport activities of some
Introduction
tambjamine derivatives.8 They proved to be very efficient anion
The development of small molecules capable of facilitating the exchangers in model liposomes, promoting both chloride and
transmembrane transport of ions is an active field of bicarbonate transport. All naturally occurring tambjamines
research.1 These compounds can mimic the activity of natural present alkyl substituted imine groups. Nevertheless, during
ionophores, which are widely used as antibiotics and tools for our previous studies, we identified the synthetic tambjamine
biomembrane research.2 Their potential as future anticancer analog 2, bearing an aromatic substituent, outperforming the
drugs and chemotherapeutics under conditions related to the naturally occurring derivatives studied.8 Prompted by this
dysfunction of natural transport mechanisms is starting to be result, we decided to explore the anion transport and antiproli-
recognized.3 The majority of these compounds facilitate the ferative activities of synthetic tambjamine analogs bearing aro-
transmembrane transport of cations, and anion selective iono- matic substituents in the enamine moiety as well as the effect
phores are relatively scarce, the prodiginines being the most of varying the substitution of the alkoxy substituent of the
representative examples.4 These compounds display intriguing bipyrrole.
pharmacological activities, and there is growing evidence
linking the ionophoric activity of these molecules and their
cytotoxicity.5 These molecules can permeabilize cellular mem-
branes, upsetting the normal ionic balance and modifying the
Results and discussion
intracellular pH, triggering apoptosis.
Compounds 1–10 were synthesised by acid catalyzed conden-
sation of the 4-alkoxy-2,2′-bipyrrole aldehyde and the corres-
ponding amine (Fig. 1).9 The products were obtained in good
yields as orange-yellow solids in the form of hydrochloric salts.
In their protonated form, these compounds are stable for
several weeks in organic solutions. We decided to explore aro-
matic enamine groups bearing both electron donating and
electron withdrawing substituents as well as a heteroaromatic
(pyridine) group. We also decided to modify the substitution
of the pyrrole ring replacing the methoxy group characteristic
of the naturally occurring derivatives by a benzyloxy group.
Compounds 1 and 2 were previously reported by us.8
We decided to study the marine alkaloids tambjamines.
These naturally occurring compounds are structurally related
to the prodiginines, being characterized by an imine substi-
tuted 4-methoxy-1H,1′H-2,2′-bipyrrole moiety.6 Likewise, these
compounds have been shown to display antimicrobial and
antitumor activities.7 The mechanism of action of these com-
pounds is unclear but may involve their ionophoric activity.5
aDepartamento de Química, Facultad de Ciencias, Universidad de Burgos,
09001 Burgos, Spain. E-mail: rquesada@ubu.es
bDepartment of Pathology and Experimental Therapeutics, Cancer Cell Biology
1
The H NMR spectra of these compounds provide evidence
Research Group, Universidad de Barcelona, Barcelona, Spain. E-mail: rperez@ub.edu
†Electronic supplementary information (ESI) available: Spectral characterization
data, details of anion binding and anion transport experiments. See DOI:
10.1039/c3ob42341g
of a strong interaction of the chloride anion with the tamb-
jamine derivative through the hydrogen cleft formed by the
bypyrrole-enamine moiety. Chloride binding in solution was
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1771–1778 | 1771

View Article Online
Paper
Organic & Biomolecular Chemistry
Fig. 2 Stack plot of partial 1H NMR of compound 4 in DMSO-d6–
0.5H2O solution with the addition of increasing amounts of tetrabutyl-
ammonium chloride.
Fig. 1 Synthetic tambjamine analogs 1–10.
1
This result is in agreement with the involvement of the C–H′
3
studied by H NMR titration experiments of perchlorate salts
proton in the binding of chloride. Gale and colleagues have
described a similar anion binding behaviour for a pyrrole
group.11
of compounds 1–10 with tetrabutylammonium chloride
(TBACl) in DMSO-d6–0.5% water. Perchlorate salts of 1–10 were
prepared by washing crude reaction mixtures with diluted
aqueous perchloric acid. Addition of TBACl resulted in the
effective replacement of the perchlorate anion by chloride, as
Anion transport assays
1
evidenced by the H NMR spectra indistinguishable from that
The transmembrane anion transport activity of compounds
1–10 was explored in 1-palmitoyl-2-oleoyl-sn-glycero-3-phos-
phocholine (POPC) liposomes. Using a chloride selective elec-
trode, the chloride efflux from chloride loaded vesicles was
monitored over time, according to reported methods.12 The
EC50, defined as the concentration of carrier needed to induce
a 50% chloride release in the time scale of our experiments
(300 seconds), was calculated using hill plot analyses (Table 2).
All the compounds proved to be very efficient anion transpor-
ters with EC50 values in the submicromolar range and down
to nanomolar levels for the most efficient derivatives in the
chloride/nitrate exchange assay.
of the corresponding hydrochloric salt. Fitting of the data
obtained in the 1H NMR titration experiments using
WinEQNMR2 software provided a quantitative assessment of
the chloride binding affinity for 1–10 under these conditions
(Table 1).10 The values of the association constants were found
to be similar for all the compounds. Thus the electronic char-
acter of the aromatic substituent as well as the nature of the
alkoxy substitution of the central pyrrole ring had little influ-
ence on the calculated Ka value. Although the electronic nature
of the aromatic substituent should influence the binding capa-
bility of the molecule, it should be noted that the binding
process involved both the dissociation of the perchlorate anion
and the coordination of chloride. Thus, since perchlorate is
also bound by these compounds, this effect was not reflected
in the values of the calculated association constants.
Table 2 Transport activities expressed as EC50 (nM) of compounds
1–10
Interestingly, the chemical shift of the NH′ proton (see
1
Hill
Hill
ESI† for two-dimensional NMR data) remained almost un-
altered during the titration experiments with tetrabutyl-
ammonium chloride whereas that of the C–H group in the 3′
position experienced an important downfield shift (Fig. 2).
EC50 (nM)
parameter
EC50 (nM)
parameter
Compound NO3−/Cl− a n NO3−/Cl− HCO3−/Cl− b
n HCO3−/Cl−
1
2
3
4
5
6
7
8
9
10
40
50
140
70
2
8
6
5
9
1.17 0.09
1.14 0.3
1.18 0.07
1.39 0.2
0.92 0.3
1.32 0.3
1.20 0.09
1.04 0.09 11 920 1000 1.11 0.1
1.45 0.1
1.24 0.1
460 70
240 40
890 80
460 60
260 30
370 50
880 300
0.89 0.1
1.19 0.2
1.04 0.09
0.90 0.1
1.05 0.08
1.12 0.2
0.67 0.1
20
Table 1 Association constants (Ka, M−1
) of compounds 1–10 with
chloride (added as tetrabutylammonium salt) determined from 1H NMR
80 10
60
720 70
3
titration experiments in DMSO-d6–0.5H2O at 293 K
70
40
3
2
430 100
170 20
0.99 0.1
1.10 0.1
Compound
Ka (M−1
)
Compound
Ka (M−1
)
a Vesicles loaded with 488 mM NaCl dispersed in 488 mM NaNO3
(5 mM phosphate buffer, pH 7.2). b Vesicles loaded with 451 mM NaCl
dispersed in 150 mM Na2SO4 (20 mM phosphate buffer, pH 7.2) upon
addition of a NaHCO3 pulse to make the extravesicular bicarbonate
concentration 40 mM.
1
2
3
4
5
1592 25
1990 122
1789 222
1681 107
1510 157
6
7
8
9
1428 30
1498 48
1491 59
1836 114
1960 219
10
1772 | Org. Biomol. Chem., 2014, 12, 1771–1778
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
The transport activity was found to be dependent on the –OBn derivatives were observed. This was an unexpected
nature of the extravesicular anion. Thus, the EC50 values were result. We anticipated that this change would affect the overall
found to be roughly an order of magnitude lower when nitrate lipophilicity of the molecule, a parameter that has been shown
was used as an external anion instead of bicarbonate. This by us and others to profoundly influence the activity of trans-
result reflected the higher lipophilicity of nitrate compared to membrane anion transporters.13 It could be that in this case
bicarbonate. The lack of significant chloride efflux when the lipophilicity range of these molecules is adequate to
sulfate was the only extravesicular anion further supported achieve an important activity as carriers and the modifications
anion exchange as the transport mechanism accounting for introduced in the central pyrrole ring did not result in lipophi-
the activity of these compounds (Fig. 3). A Hill parameter licity values outside this adequate range.
value around 1 was consistent with a non-cooperative effect
and a carrier mechanism for the transmembrane transport
activity of these compounds (Table 2).
Biological studies
We decided to study the in vitro antitumor activity of com-
pounds 1–10. A single point screening (10 µM) on a panel of
Similarly to the results observed in the quantification of the
chloride binding affinity, the nature of the aromatic substitu-
different cancer cell lines (human melanoma (A375), human
ent had little effect on the ionophoric efficiency of these com-
lung carcinoma (A549), human colorectal adenocarcinoma
pounds. Compounds bearing both electron withdrawing and
(SW620),
and
human
mammary
adenocarcinoma
electron donor substituents promoted anion transport with
excellent activity. The trifluoromethyl substituted compounds
3 and 8 were found to be the less active derivatives, with 8 dis-
playing the highest EC50 values. When performing transport
experiments using this compound, precipitation of the added
carrier was evident. It could be possible that the comparatively
lower efficiency of 8 was due to the failure to partition into the
liposome membranes. Compound 5 was found to be the most
efficient chloride/nitrate exchanger with a calculated EC50 of
20 nM. This derivative outperforms our previously reported
carrier 2 (see Fig. S106†).
(MDA-MB-231)) using the MTT assay revealed the important
cytotoxicity of these derivatives (Fig. 4).
The concentration that causes 50% growth inhibition (IC50
values) was determined for 1–10 on melanoma (A375) and
mammary adenocarcinoma (MDA-MB-231) human cancer cell
lines as well as human mammary epithelial (MCF-10A) cell
lines (Table 3). Compounds 2 and 7 were found to be the most
cytotoxic compounds in these assays, with IC50 values ranging
from 1.10 to 2.49 µM. On the other hand compound 5
displayed significantly higher IC50 values, around 18 µM.
The replacement of the methoxy group of the central
pyrrole ring, found in all the naturally occurring derivatives, by
a benzyloxy group did not result in an important modification
of the transport efficiency of these derivatives. For this set
of compounds, comparable results for the parent –OMe and
Fig. 4 Single point screening of synthetic tambjamine analogs 1–10
(10 µM) on a panel of cancer cell lines (A375, A549, SW620, and
MDA-MB-231). Cell viability measured using the MTT assay after 48 h
treatment.
Table 3 IC50 of compounds 1–10 on human melanoma (A375), human
mammary adenocarcinoma (MDA-MB-231) and human mammary
epithelial (MCF-10A) cell lines
Fig. 3 Comparative of chloride efflux induced by compound 4 (0.5 µM,
0.1% mol) from: (a) POPC liposomes loaded with NaCl (488 mM NaCl
and 5 mM phosphate buffer, pH 7.2) immersed in NaNO3 (494 mM
NaNO3 and 5 mM phosphate buffer, pH 7.2); (b) vesicles containing NaCl
(451 mM NaCl and 20 mM phosphate buffer, pH 7.2) immersed in
Na2SO4 (150 mM, Na2SO4; 40 mM HCO3− and 20 mM phosphate buffer,
pH 7.2); (c) vesicles loaded with NaCl (451 mM NaCl and 20 mM phos-
phate buffer, pH 7.2) immersed in Na2SO4 (150 mM, Na2SO4; 20 mM
phosphate buffer, pH 7.2); (d) control experiment (10 µL DMSO): vesicles
loaded with NaCl (451 mM NaCl and 20 mM phosphate buffer, pH 7.2)
immersed in Na2SO4 (150 mM, Na2SO4; 20 mM phosphate buffer, pH
7.2). All traces are an average of at least three independent experiments.
IC50 (µM)
A375
IC50 (µM)
MDA-MB-231
IC50 (µM)
MCF-10A
Compound
1
2
3
4
5
6
7
8
9
10
8.19 2.90
1.10 0.01
5.20 0.08
8.30 2.44
17.48 1.94
1.56 0.69
1.18 0.04
1.29 0.11
6.75 2.85
2.74 0.53
6.58 1.01
2.28 0.56
8.13 1.56
13.67 4.31
18.11 1.16
4.71 2.87
2.49 0.65
3.85 1.39
19.11 0.67
3.43 1.68
10.90 4.34
2.36 0.01
4.12 1.13
26.27 8.76
19.86 1.79
4.90 1.53
2.80 0.72
3.90 1.28
36.50 4.72
4.70 1.79
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1771–1778 | 1773

View Article Online
Paper
Organic & Biomolecular Chemistry
Replacement of the O–Me group by an O–Bn substituent
resulted in an enhancement of the cytotoxicity of these com-
pounds with average IC50 values of 8.90 µM for the –OMe substi-
tuted 1–5 and 4.71 µM for the –OBn substituted 6–10 respectively
for the cancer lines examined. This effect was especially marked
for the pyridine substituted pair 5 and 10. Little discrimination
between normal and cancerous cell lines was observed. However,
non-cancerous human mammary epithelial cells MCF-10A were
less sensitive to the studied compounds than cancerous cells.
Average IC50 values on this cell line for compounds 1–5 were
12.70 µM and 10.56 µM for compounds 6–10 respectively.
In order to analyze the cell death induced by these com-
pounds we used Hoechst 33342 staining (Fig. 5). This DNA
binding dye allows the differentiation of apoptosis from other
cell death mechanisms. In contrast to untreated cells (control),
cells treated with compounds 1–10 showed stronger blue fluo-
rescence, indicating chromatin condensation, fragmentation
and apoptotic bodies formation, along with “bean” shaped
nuclei. All these features are hallmarks of apoptosis.
Finally, vital staining with acridine orange (AO) was used to
evaluate the effects of these compounds on the intracellular
Fig. 6 Acridine orange staining on melanoma (A375) human cancer cell
line. (a) Untreated (control) cells; (b)–(k) cells treated with compounds
1–10. Treated cells showed significant disappearance of orange fluo-
rescence due to basification of acidic organelles.
pH levels. Protonation and accumulation of this dye in acidic
compartments such as lysosomes result in a characteristic
orange fluorescence emission, whereas it emits green fluo-
rescence at higher pH.14 Human melanoma (A375) cells were
stained with AO and granular orange fluorescence was
observed in the cytoplasm, corresponding to acidic organelles
(Fig. 6). Treatment of these cells with compounds 1–10
resulted in the disappearance of the orange fluorescence,
suggesting the basification of acidic organelles. The observed
qualitative results correlate well with the antiproliferative
activity of these derivatives. The effect was more evident for
the most cytotoxic compounds whereas the action of the less
toxic derivatives such as 5 led only to minimal changes and
appearance similar to untreated cells.
Conclusions
Synthetic analogs of tambjamine alkaloids bearing aromatic
enamine substituents represent privileged structures for
the development of highly efficient transmembrane anion
Fig. 5 Hoechst 33342 staining on melanoma (A375) human cancer cell
line. (a) Untreated (control) cells; (b)–(k) cells treated with compounds
1–10. Treated cells showed typical features of apoptotic processes.
1774 | Org. Biomol. Chem., 2014, 12, 1771–1778
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
transporters. These compounds efficiently promoted nitrate 111.57 (CH), 92.40 (CH), 59.03 (CH3); HRMS (EI) m/z calcd for
and bicarbonate/chloride exchange in model liposomes at very [C16H15N3O] 265.1215; found: 265.1208.
low concentrations. At this stage it is unclear which para-
(Z)-N-(4-(tert-Butyl)phenyl)-1-(4-methoxy-1H,1′H -[2,2′-bipyrrol]-
meters make these compounds so effective as anionophores, 5-yl)methanimine (2). Yield 95%. 1H NMR (300 MHz, CDCl3): δ
and studies aimed to shed light on this matter are currently = 13.94 (s, NH, 1H), 11.22 (d, NH, J = 14.4 Hz, 1H), 10.70 (s,
underway in our laboratories. In vitro studies showed that they NH, 1H), 7.77 (d, NH, J = 14.6 Hz, 1H), 7.40 (d, J = 8.6 Hz, 2H),
were able to alter the intracellular pH levels, triggering apopto- 7.32 (d, J = 8.4 Hz, 2H), 7.25 (s, 1H), 7.13 (s, 1H), 6.81 (s, 1H),
sis in different cancer cell lines with IC50 values in the low 6.32 (d, J = 1.5 Hz, 1H), 6.00 (s, 1H), 3.98 (s, 3H), 1.31 (s, 9H);
micromolar range. The toxicity of these compounds is an 13C NMR (75 MHz, CDCl3): δ = 165.01 (C), 149.16 (C), 143.95
important issue to be addressed in order to continue their (C), 135.97 (C), 130.15 (CH), 126.71 (CH), 124.99 (CH), 122.53
development as potential anticancer drugs. The tolerance of (C), 116.88 (CH), 114.67 (CH), 113.17 (C), 111.27 (CH), 92.06
this motif regarding changes in both the electronic nature of (CH), 58.79 (CH3), 34.58 (C), 31.31 (CH3); HRMS (EI) m/z calcd
the aromatic enamine substituent as well as the alkoxy group for [C20H23N3O] 321.1841; found: 321.1844.
of the central pyrrole ring suggests that it could be possible
(Z)-1-(4-Methoxy-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-(4-(trifluoro-
to introduce modifications aimed to increase the selectivity methyl)phenyl)methanimine (3). Yield 80%. 1H NMR
toward cancer cells without losing their anionophoric (300 MHz, DMSO-d6): δ = 13.29 (s, NH, 1H), 12.58 (d, NH, J =
properties.
13.9 Hz, 1H), 12.13 (s, NH, 1H), 8.31 (d, J = 13.7 Hz, 1H), 7.77
(s, 4H), 7.29 (s, 2H), 6.61 (s, 1H), 6.36 (s, 1H), 4.01 (s, 3H). 13C
NMR (75 MHz, DMSO): δ = 166.37 (C), 145.47 (C), 142.22 (C),
131.32 (CH), 127.00 (c, JC–F = 3.7 Hz CH), 126.12 (CH), 124.99
(c, JC–F = 32.2 Hz, C) 124.22 (c, JC–F = 271.1 Hz, CF3) 121.95 (C),
117.53 (CH), 114.00 (CH), 113.90 (C), 111.57 (CH), 92.98 (CH),
59.13 (CH3). HRMS (EI) m/z calcd for [C17H14F3N3O] 333.1089;
found: 333.1088.
(Z)-1-(4-Methoxy-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-(4-methoxy-
phenyl)methanimine (4). Yield 92%. 1H NMR (300 MHz,
CDCl3): δ = 13.65 (s, 1H), 11.17 (d, J = 13.7 Hz, 1H), 10.58 (s,
1H), 7.64 (d, J = 13.8 Hz, 1H), 7.36–7.22 (m, 2H), 7.07 (s, 1H),
6.93–6.81 (m, 2H), 6.76 (d, J = 1.3 Hz, 1H), 6.26 (d, J = 1.7 Hz,
1H), 5.94 (s, 1H), 3.91 (s, 3H), 3.77 (s, 3H). 13C NMR (75 MHz,
CDCl3) δ = 164.71 (C), 157.83 (C), 143.42 (C), 131.86 (C), 130.43
(CH), 124.88 (CH), 122.65 (C), 118.59 (CH), 115.05 (CH), 114.35
(CH), 112.89 (C), 111.24 (CH), 91.91 (CH), 58.76 (CH3), 55.65
(CH3). HRMS (EI) m/z calcd for [C17H17N3O2] 295.1321; found:
295.1322.
Experimental section
General procedures and methods
Commercial reagents were used as received without any
further purification. NMR spectra were recorded on Varian
Mercury-300 MHz and Varian Unity Inova-400 MHz spectro-
meters. Chemical shifts are reported in ppm using the residual
solvent peak as a reference and coupling constants are
reported in Hz. High resolution mass spectra (HRMS) were
recorded on a MicromassAutospec S-2 spectrometer using
EI at 70 eV. 4-Methoxy-2,2′-bipyrrole-5-carboxaldehyde and
4-benzyloxy-2,2′-bipyrrole-5-carboxaldehyde were prepared as
described.15 1H NMR titration experiments were performed in
DMSO-d6–H2O 99.5 : 0.5 mixtures at 303 K. Data fitting was
carried out using WinEQNMR2 software (see the ESI† for
details).9
(Z)-1-(4-Methoxy-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-(pyridin-2-yl)-
methanimine (5). Yield 65%. H NMR (300 MHz, CDCl3): δ =
Synthesis of tambjamine analogs
1
Compounds 1–10 were synthesised using modifications of a 13.85 (s, NH, 1H), 11.41 (d, J = 13.1 Hz, NH, 1H), 10.73 (s, NH,
previously reported method.9 To a mixture of the 2,2′-bipyr- 1H), 8.63 (d, J = 13.4 Hz, 1H), 8.33 (d, J = 4.0 Hz, 1H), 7.69 (s,
role-5-carboxaldehyde (190 mg, 1 mmol) and the corres- 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.16 (s, 1H), 7.08 (dd, J = 7.3,
ponding amine (1.3–3 mmol, 1.3–3 equivalents) in 10 ml of 5.0 Hz, 1H), 6.88 (s, 1H), 6.34 (s, 1H), 6.02 (d, J = 1.8 Hz, 1H),
chloroform, 40 µL of acetic acid were added. The mixture was 4.00 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 166.68 (C), 150.02
stirred at 60 °C until TLC showed disappearance of the starting (C), 148.49 (CH), 145.97 (C), 138.92 (CH), 128.83 (CH), 126.23
material. The reaction mixture was then diluted with 40 ml of (CH), 122.40 (C), 120.69 (CH), 116.04 (CH), 114.73 (C), 113.85
dichloromethane and washed with HCl 1 M (3 × 25 ml). The (CH), 111.79 (CH), 92.47 (CH), 58.98 (CH3). HRMS (EI) m/z
organic fraction was dried over Na2SO4 and the solvent evapor- calcd for [C15H14N4O] 266.1168; found: 266.1168.
ated to yield 1–10 as yellow-orange solids in good to excellent
yields.
(Z)-1-(4-(Benzyloxy)-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-phenyl-
methanimine (6). Yield 94%. H NMR (300 MHz, CDCl3): δ =
1
(Z)-1-(4-Methoxy-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-phenylmethan- 14.03 (s, NH, 1H), 11.28 (d, NH, J = 14.5 Hz, 1H), 10.73 (s, NH,
imine (1). Yield 94%. 1H NMR (300 MHz, DMSO-d6): δ 13.61 1H), 7.83 (d, J = 14.6 Hz, 1H), 7.45 (d, J = 1.4 Hz, 4H), 7.41–7.38
(s, NH, 1H), 11.07 (d, NH, 1H, J = 14.3 Hz), 10.63 (s, NH, 1H), (m, 3H), 7.26 (dd, J = 1.5, 0.6 Hz, 2H), 7.20 (s, 1H), 7.15 (s, 1H),
7.61 (d, 1H, J = 14.1 Hz), 7.28 (t, 4H, J = 5.2 Hz), 7.13–7.05 (m, 6.83 (s, 1H), 6.33 (dt, J = 3.5, 2.1 Hz, 1H), 6.06 (s, 1H), 5.20 (s,
1H), 7.01 (d, 1H, J = 1.1 Hz), 6.76 (s, 1H), 6.21 (m, 1H), 5.95 (s, 2H). 13C NMR (75 MHz, CDCl3): δ = 164.17 (C), 144.49 (C),
1H), 3.86 (s, 3H);13C NMR (75 MHz, CDCl3): δ 165.47 (C), 138.52 (C), 134.79 (C), 130.21 (CH), 129.94 (CH), 129.07 (CH),
144.58 (C), 138.60 (C), 130.02 (CH), 129.96 (CH), 125.88 (CH), 128.97 (CH), 128.26 (CH), 125.93 (CH), 125.46 (CH), 122.55 (C),
125.34 (CH), 122.65 (C), 117.17 (CH), 115.17 (CH), 113.60 (C), 117.27 (CH), 115.11 (CH), 113.81 (C), 111.45 (CH), 93.13 (CH),
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1771–1778 | 1775

View Article Online
Paper
Organic & Biomolecular Chemistry
73.88 (CH2). HRMS (EI) m/z calcd for [C22H19N3O] 341.1528; pH 7.2 or 451 mM NaCl and 20 mM phosphate buffer, pH 7.2),
found: 341.1515. followed by careful vortexing. The lipid suspension was then
(Z)-N-((4-(Benzyloxy)-1H,1′H-[2,2′-bipyrrol]-5-yl)methylene)-4- subjected to nine freeze–thaw cycles and twenty-nine extru-
1
(tert-butyl)aniline (7). Yield 92%. H NMR (300 MHz, CDCl3): sions through a 200 nm polycarbonate nucleopore membrane
δ = 13.83 (s, NH, 1H), 11.23 (d, NH, J = 14.5 Hz, 1H), 10.69 using a LiposoFast Basic extruder (Avestin, Inc.). The resulting
(s, NH, 1H), 7.73 (d, J = 14.4 Hz, 1H), 7.61–7.13 (m, 9H), 7.05 unilamellar vesicles were dialyzed against NaNO3 solution
(s, 1H), 6.78 (s, 1H), 6.27 (s, 1H), 6.07 (s, 1H), 5.18 (s, 2H), 1.29 (488 mM NaNO3 and 5 mM phosphate buffer, pH 7.2) or a
(s, 9H). 13C NMR (75 MHz, CDCl3): δ = 163.86 (C), 149.36 (C), Na2SO4 solution (150 mM Na2SO4 and 20 mM phosphate
143.97 (C), 136.01 (C), 134.84 (C), 130.53 (CH), 129.03 (CH), buffer, pH 7.2) to remove unencapsulated chloride.
128.95 (CH), 128.22 (CH), 126.79 (CH), 125.22 (CH), 122.59 (C),
ISE transport assays
117.10 (CH), 114.75 (CH), 113.49 (C), 111.32 (CH), 93.00 (CH),
73.77 (CH2), 34.66 (C), 31.36 (CH3). HRMS (EI) m/z calcd for Unilamellar vesicles (200 nm mean diameter) composed of
[C26H27N3O] 397.2154; found: 397.2165.
POPC containing an encapsulated solution of 488 mM NaCl
(Z)-1-(4-(Benzyloxy)-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-(4-(trifluoro- and 5 mM phosphate buffer, pH 7.2 or 451 mM NaCl and
methyl)phenyl)methanimine (8). Yield 44%. 1H NMR 20 mM phosphate buffer, pH 7.2, were suspended in a solution
(300 MHz, CDCl3): δ = 14.03 (s, NH, 1H), 11.32 (d, NH, J = of 494 mM NaNO3 and 5 mM phosphate buffer, pH 7.2 or
13.3 Hz, 1H), 10.73 (s, NH, 1H), 7.78 (d, J = 14.1 Hz, 1H), 7.60 150 mM Na2SO4 and 20 mM phosphate buffer, pH 7.2 respect-
(d, J = 8.4 Hz, 2H), 7.17 (s, 1H), 6.88 (s, 1H), 6.35 (s, 1H), 6.07 ively, for a final lipid concentration of 0.5 mM and a total
(s, 1H), 5.20 (s, 2H). 13C NMR (75 MHz, CDCl3): δ = 164.93 (C), volume of 5 ml. A DMSO solution of the carrier molecule, typi-
146.14 (C), 141.55 (C), 134.49 (CH), 129.33 (CH), 129.12 (CH), cally 5 μL to avoid the influence of the solvent molecules in
128.90 (C), 128.35 (CH), 127.38 (c, JC–F = 33.68 Hz, C), 127.25 the assay, was added and the chloride release from vesicles
(c, JC–F = 3.7 Hz, CH), 126.63 (CH), 123.96 (c, JC–F = 271.8 Hz, was monitored using a symphony combination chloride elec-
CF3), 122.36 (C), 117.04 (CH), 116.41 (CH), 115.05 (C), 111.98 trode. At the end of the experiment the vesicles were lysed with
(CH), 93.50 (CH), 74.19 (CH2). HRMS (EI) m/z calcd for detergent (Triton-X 10% dispersion in water, 20 μL) to release
[C23H18F3N3O] 409.1402; found: 409.1402.
all chloride ions; the resulting value was considered to rep-
(Z)-1-(4-(Benzyloxy)-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-(4-methoxy- resent 100% release and was used as such. For the bicarbonate
phenyl)methanimine (9). Yield 97%. 1H NMR (300 MHz, anion exchange assays, to the vesicles suspended in a Na2SO4
DMSO): δ = 13.12 (s, 1H), 12.58 (d, J = 14.5 Hz, 1H), 11.96 (s, solution (150 mM in Na2SO4 buffered to pH 7.2 with 20 mM
1H), 8.23 (d, J = 14.4 Hz, 1H), 7.69–7.50 (m, 4H), 7.40 (ddd, J = sodium phosphate salts), NaHCO3 was added for a final bi-
8.5, 7.7, 3.6 Hz, 3H), 7.24–7.12 (m, 2H), 7.00 (d, J = 9.0 Hz, 2H), carbonate concentration of 40 mM and the chloride efflux was
6.61 (d, J = 1.9 Hz, 1H), 6.30 (d, J = 2.2 Hz, 1H), 5.30 (s, 2H), monitored for another 5 minutes before they were lysed with
3.75 (s, 3H). 13C NMR (75 MHz, DMSO): δ = 163.40 (C), 157.45 detergent to release all chloride ions; the resulting value was
(C), 142.47 (C), 135.53 (C), 132.88 (CH), 131.85 (C), 128.55 considered to represent 100% release and used as such. For
(CH), 128.37 (CH), 127.95 (CH), 124.56 (CH), 122.16 (C), 119.12 the experiments using NaNO3 as an external solution, vesicles
(CH), 114.93 (CH), 112.49 (C), 112.24 (CH), 110.89 (CH), 93.25 containing an encapsulated solution of 488 mM NaCl and
(CH), 72.82 (CH2), 55.47 (CH3). HRMS (EI) m/z calcd for 5 mM phosphate buffer were suspended in a 488 mM NaNO3
[C23H21N3O2] 371.1634; found: 371.1635.
and 5 mM phosphate buffer, pH 7.2, solution.
(Z)-1-(4-(Benzyloxy)-1H,1′H-[2,2′-bipyrrol]-5-yl)-N-(pyridin-2-yl)-
methanimine (10). Yield 46%. H NMR (300 MHz, DMSO): δ =
1
Cell lines and culture conditions
13.21 (s, 1H), 12.82 (s, 1H), 12.20 (s, 1H), 8.50 (s, 1H), 8.38 (dd, Human melanoma (A375), human lung carcinoma (A549),
J = 4.9, 1.5 Hz, 1H), 7.87 (td, J = 7.7, 1.6 Hz, 1H), 7.55 (dd, J = human colorectal adenocarcinoma (SW620), human mammary
7.9, 1.6 Hz, 2H), 7.43 (dd, J = 11.1, 3.8 Hz, 3H), 7.34–7.15 (m, adenocarcinoma (MDA-MB-231) and human mammary epi-
4H), 6.70 (s, 1H), 6.37 (s, 1H), 5.32 (s, 2H). 13C NMR (75 MHz, thelial (MCF-10A) cell lines were purchased from the American
DMSO): δ = 165.39 (C), 149.82 (C), 148.80 (CH), 145.75 (C), Type Culture Collection (ATCC, Manassas, VA). Cells were cul-
139.48 (CH), 135.13 (C), 128.71 (CH), 128.49 (CH), 127.75 (CH), tured in DMEM medium (Biological Industries) supplemented
126.28 (CH), 121.86 (C), 120.98 (CH), 114.11 (CH), 113.98 (C), with 10% heat-inactivated foetal bovine serum (FBS; Life
112.92 (CH), 111.64 (CH), 93.89 (CH), 73.38 (CH2). HRMS (EI) Technologies, Carlsbad, CA), 100 U ml−1 penicillin, 100 µg
m/z calcd for [C21H18N4O] 342.1481; found: 342.1490.
ml−1 streptomycin, and 2 mM L-glutamine, all from Biological
Industries. MDA-MB-231 cell line was cultured in DMEM–
F12 media (1 : 1, Biological Industries) supplemented with
Preparation of phospholipid vesicles
A
chloroform solution of 1-palmitoyl-2-oleoyl-sn-glycero- 10% heat-inactivated FBS, 100 U ml−1 penicillin, 100 µg ml−1
3-phosphocholine (POPC) (20 mg ml−1) (Sigma-Aldrich) was streptomycin, and 2 mM L-glutamine. MCF10A cell line was
evaporated in vacuo using a rotary evaporator and the lipid cultured in DMEM–F12 media (1 : 1) supplemented with 5%
film obtained was dried under high vacuum for at least horse serum (Life Technologies), 20 ng ml−1 EGF, 0.5 µg ml−1
2 hours. The lipid film was rehydrated by addition of a sodium hydrocortisone, 100 ng ml−1 cholera toxin, and 10 µg ml−1
chloride solution (488 mM NaCl and 5 mM phosphate buffer, insulin, all from Sigma-Aldrich Chemical Co. (St. Louis, MO)
1776 | Org. Biomol. Chem., 2014, 12, 1771–1778
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
and 100 U ml−1 penicillin, 100 µg ml−1 streptomycin, and
2 mM L-glutamine. Cells were grown at 37 °C under a 5% CO2
atmosphere.
2013, 29, 9031–9040; (c) S. Matile, A. Vargas Jentzsch,
J. Montenegro and A. Fin, Chem. Soc. Rev., 2011, 40, 2453–
2474; (d) J. T. Davis, O. Okunola and R. Quesada, Chem.
Soc. Rev., 2010, 39, 3843–3862; (e) C. J. E. Haynes and
P. A. Gale, Chem. Commun., 2011, 47, 8203–8209;
(f) T. M. Fyles, Chem. Soc. Rev., 2007, 36, 335–347;
(g) A. P. Davis, D. N. Sheppard and B. D. Smith, Chem. Soc.
Rev., 2007, 36, 348–357; (h) S. Hussain, P. R. Brotherhood,
L. W. Judd and A. P. Davis, J. Am. Chem. Soc., 2011, 133,
1614–1617; (i) S. Bahmanjah, N. Zhang and J. T. Davis,
Chem. Commun., 2012, 48, 4432–4434; ( j) W. A. Harrell, Jr.,
M. L. Bergmeyer, P. Y. Zavalij and J. T. Davis, Chem.
Commun., 2010, 46, 3950–3952.
Cell viability assay
Cells (1 × 105 cells per ml) were seeded in 96-well plates and
allowed to grow for 24 h. Afterwards, they were treated with
10 µM of each compound for single point experiments and
dose–response curves were performed ranging from 0.39
to 50 µM for 48 h to calculate the inhibitory concentration
of 50% of cell population (IC50) values of the compounds.
Cell viability was determined by the MTT assay. After
treatment, 10 µM of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (Sigma-Aldrich) was added to each well
for an additional 4 h. DMSO was added in control cells. Media
was aspirated and the blue MTT formazan precipitate was dis-
solved in 100 µl of DMSO. The absorbance at 570 nm was
measured on a multiwell plate reader. Cell viability was
expressed as a percentage of control cells, and data are shown
as the mean value S.D. of three independent experiments
performed in triplicate for single point evaluation or in dupli-
cate for dose–response curves. IC50 values were calculated with
GraphPad Prism 5 software.
2 (a) M. M. Faul and B. E. Huff, Chem. Rev., 2000, 100, 2407–
2473; (b) I. Alfonso and R. Quesada, Chem. Sci., 2013, 4,
3009–3019.
3 (a) X. Li, B. Shen, X.-Q. Yao and D. Yang, J. Am. Chem. Soc.,
2009, 131, 13676–13680; (b) B. Shen, X. Li, X. Yao and
D. Yang, PLoS ONE, 2012, 7, e34694; (c) C.-R. Elie,
M. Charbonneau and A. R. Schmitzer, MedChemComm,
2012, 3, 1231–1234; (d) S. Rastogi, E. Marchal, I. Uddin,
B. Groves, J. Colpitts, S. A. McFarland, J. T. Davis and
A. Thompson, Org. Biomol. Chem., 2013, 11, 3834–3845;
(e) S. J. Moore, M. Wenzel, M. E. Light, R. Morley,
S. J. Bradberry, P. Gomez-Iglesias, V. Soto- Cerrato, R. Perez-
Tomas and P. A. Gale, Chem. Sci., 2012, 3, 2501–2508.
4 (a) A. Fürstner, Angew. Chem., Int. Ed., 2003, 42, 3582–3603;
(b) N. R. Williamson, P. C. Fineran, F. J. Leeper and
G. P. C. Salmond, Nat. Rev. Microbiol., 2006, 4, 887–899;
(c) N. R. Williamson, P. C. Fineran, T. Gristwood,
S. R. Chawrai, F. J. Leeper and G. P. C. Salmond, Future
Microbiol., 2007, 2, 605–618; (d) M. S. Melvin,
M. W. Calcutt, R. E. Noftle and R. A. Mandeville, Chem. Res.
Toxicol., 2002, 15, 742–748; (e) R. Pérez-Tomás,
B. Montaner, E. Llagostera and V. Soto-Cerrato, Biochem.
Pharmacol., 2003, 66, 1447–1452.
5 (a) P. A. Gale, R. Peréz-Tomás and R. Quesada, Acc. Chem.
Res., 2013, 46, 2801–2813; (b) B. Díaz de Greñu, P. Iglesias
Hernández, M. Espona, D. Quiñonero, M. E. Light,
T. Torroba, R. Pérez Tomás and R. Quesada, Chem.–Eur. J.,
2011, 17, 14074–14083.
6 (a) B. Carté and D. J. Faulkner, J. Org. Chem., 1983, 48,
2314–2318; (b) A. J. Blackman and C. P. Li, Aust. J. Chem.,
1994, 47, 1625–1629; (c) R. A. Davis, A. R. Carroll and
R. J. Quinn, Aust. J. Chem., 2001, 54, 355–359.
Acridine orange staining
A375 cells (105 cells per ml) were seeded on glass slices and
48 h later they were treated with IC50 concentrations of the
studied compounds for 1 : 30 h. DMSO was added in control
cells. Afterwards, cells were washed twice with PBS and incu-
bated in 5 µg ml−1 acridine orange solution for 30 min at
room temperature. Finally, they were washed three times with
PBS–10% FBS and examined by fluorescence in a NIKON
eclipse E800 microscope (SCT filter 330/380 nm).
Hoechst staining
A375 cells (105 cells per ml) were seeded in 12-well plates,
allowed to grow for 24 h and then treated with 10 μM of each
compound for 48 h. DMSO was added in control cells. They
were washed with PBS, resuspended in 2 µg ml−1 Hoechst
33342 (Sigma-Aldrich Chemical Co.) and incubated for 30 min
at 37 °C in the dark. Finally, cells were washed with PBS and
examined by fluorescence in a NIKON eclipse E800 microscope
(SCT filter 330/380 nm).
Acknowledgements
7 (a) M. Carbone, C. Irace, F. Costagliola, F. Castelluccio,
G. Villani, G. Calado, V. Padula, G. Cimino, J. L. Cervera,
R. Santamaria and M. Gavagnin, Bioorg. Med. Chem. Lett.,
2010, 20, 2668–2670; (b) L. N. Aldrich, S. L. Stoops,
B. C. Crews, L. J. Marnett and C. W. Lindsley, Bioorg. Med.
Chem. Lett., 2010, 20, 5207–5211; (c) D. M. Pinkerton,
M. G. Banwell, M. J. Garson, N. Kumar, M. O. de Moraes,
B. C. Cavalcanti, F. W. A. Barros and C. Pessoa, Chem. Bio-
diversity, 2010, 7, 1311–1324; (d) F. Ballestriero, T. Thomas,
C. Burke, S. Egan and S. Kjelleberg, Appl. Environ. Micro-
biol., 2010, 76, 5710–5717.
The authors gratefully acknowledge Andrea Sancho for contri-
butions to the synthesis. This research was supported by
Consejería de Educación de la Junta de Castilla y León (Project
BU340U13) and Fundació la Maratón de TV3.
Notes and references
1 (a) N. Busschaert and P. A. Gale, Angew. Chem., Int. Ed.,
2013, 52, 1374–1382; (b) N. Sakai and S. Matile, Langmuir,
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 1771–1778 | 1777

View Article Online
Paper
Organic & Biomolecular Chemistry
8 P. Iglesias Hernández, D. Moreno, A. Araujo Javier, 13 (a) V. Saggiomo, S. Otto, I. Marques, V. Félix, T. Torroba and
T. Torroba, R. Pérez-Tomás and R. Quesada, Chem.
Commun., 2012, 48, 1556–1558.
9 D. M. Pinkerton, M. G. Banwell and A. C. Willis, Org. Lett.,
2007, 9, 5127–5130.
10 M. J. Hynes, J. Chem. Soc., Dalton Trans., 1993, 311–312.
11 I. E. D. Vega, P. A. Gale, M. E. Light and S. J. Loeb, Chem.
Commun., 2005, 4913–4915.
12 A. V. Koulov, T. N. Lambert, R. Shukla, M. Jain, J. M. Boon,
B. D. Smith, H. Li, D. N. Sheppard, J.-B. Joos, J. P. Clare
R. Quesada, Chem. Commun., 2012, 48, 5274–5276;
(b) N. Busschaert, S. J. Bradberry, M. Wenzel,
C. J. E. Haynes, J. R. Hiscock, I. L. Kirby, L. E. Karagiannidis,
S. J. Moore, N. J. Wells, J. Herniman, G. J. Langley,
P. N. Horton, M. E. Light, I. Marques, P. J. Costa, V. Félix,
J. G. Frey and P. A. Gale, Chem. Sci., 2013, 4, 3036–3045.
14 A. C. Allison and M. R. Young, Lysosomes in Biology and
Pathology, North-Holland Publishing Co., Amsterdam,
1969, vol. 2.
and A. P. Davis, Angew. Chem., 2003, 115, 5081–5083, 15 K. Dairi, S. Tripathy, G. Attardo and J.-F. Lavallee, Tetra-
(Angew. Chem., Int. Ed., 2003, 42, 4931–4933). hedron Lett., 2006, 47, 2605–2606.
1778 | Org. Biomol. Chem., 2014, 12, 1771–1778
This journal is © The Royal Society of Chemistry 2014