Perenosins: A new class of anion transporter with anti-cancer activity

Article abstract of DOI:10.1039/c6ob00002a

A new class of anion transporter named 'perenosins' consisting of a pyrrole linked through an imine to either an indole, benzimidazole or indazole is reported. The indole containing members of the perenosin family function as effective transmembrane Cl^-/NO₃^- antiporters and HCl cotransporters in a manner similar to the prodigiosenes. The compounds reduce the viability of MDA-MB-231 and MCF-7.

Full text of DOI:10.1039/c6ob00002a

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Perenosins: a new class of anion transporter with
anti-cancer activity†
Cite this: DOI: 10.1039/c6ob00002a
Wim Van Rossom, Daniel J. Asby, Ali Tavassoli and Philip A. Gale*
Received 1st January 2016,
Accepted 26th January 2016
A new class of anion transporter named ‘perenosins’ consisting of a pyrrole linked through an imine to
either an indole, benzimidazole or indazole is reported. The indole containing members of the perenosin
family function as effective transmembrane Cl−/NO3 antiporters and HCl cotransporters in a manner
−
DOI: 10.1039/c6ob00002a
www.rsc.org/obc
similar to the prodigiosenes. The compounds reduce the viability of MDA-MB-231 and MCF-7.
compounds including the tambjamines12,13 and obatoclax,14
having similar structures, have been shown to exhibit similar
Introduction
Prodigiosin is naturally occurring tripyrrolic compound,1 pro- biological properties (Fig. 1). Obatoclax mesylate GX15-070, an
duced by a group of microorganisms including Serratia marcescens indole-based prodigiosin analogue, is currently in clinical
(Fig. 1).2 Although the compound was first isolated in trials, being evaluated in solid tumors and hematological
pure form in 1929,3 it was not until 1977 that Fullan and co- neoplasms.
workers demonstrated that prodigiosin has anti-tumour
Pyrrole and indole groups are found in many synthetic
activity. Since that time the anti-cancer properties of many anion receptor systems. Examples that have been employed in
different natural and synthetic prodigiosenes have been lipid bilayer anion transport include amidopyrrole functiona-
explored.4,5 Prodigiosin has been shown to passively transport lized with a basic methylimidazole group that was shown to co-
HCl across lipid bilayer membranes and it is proposed that the transport HCl,15 calixpyrrole-based transporters16 including
anti-cancer properties of this class of compounds may be strapped systems that trigger apoptosis in cells due to influx of
linked to this process.6,7 Many prodigiosenes have also shown NaCl,17 and indole functionalized thioureas.18 This latter class
potent antimicrobial,8 antimalarial9 and immunosuppressive of compound have also been used as carboxylate transporters.19
activity.10 Unfortunately, the high toxicity of prodigiosin and
In this paper we report the synthesis of a new class of anion
its analogues prevents their use in the clinic.11 Closely related transporter with structures inspired by prodigiosin. Known as
‘perenosins’20 these compounds contain a pyrrole hydrogen
bond donor linked through an imine to an indole, benzimida-
zole or indazole. Compounds with a range of lipophilicities
have been prepared and their anion complexation and trans-
port properties studied.
Results and discussion
Synthesis and characterization
Perenosins 1a–e, 2 and 3 (Fig. 2) were prepared using a con-
Fig. 1 Prodigiosin, tambjamine C and obatoclax.
densation reaction (EtOH, MgSO4, room temperature, 24 h) of
3,5-dimethylpyrrole-2-carboxaldehyde with a reduced 7-nitro-
indole, 7-nitrobenzimidazole or 7-nitroindazole (H2, Pd/C
10%, EtOH, room temperature, 5 h). The non-commercial
7-nitroindoles (except for R = MeO) were readily obtained from
the respective 2-nitroaniline starting material through an iodi-
nation – Sonagashira reaction – base assisted cyclization
pathway. For 5-methoxy-7-nitroindole an alternative Fisher
indole synthesis – decarboxylation route was followed. Further
details are provided in the ESI.†
Department of Chemistry, University of Southampton, Southampton, SO17 1BJ, UK.
E-mail: philip.gale@soton.ac.uk
†Electronic supplementary information (ESI) available: Syntheses details, 1H
NMR and 13C NMR spectra, X-ray crystallographic data, vesicle transport assay
details, Hill plots, hydrolysis data, in vitro assays details, and other supporting
figures. Data underlying this publication are available see: DOI: 10.5258/SOTON/
386610. CCDC 1441636. For ESI and crystallographic data in CIF or other elec-
tronic format see DOI: 10.1039/C6OB00002A
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Fig. 2 The structures of the perenosins reported in this paper.
Compounds 1a–e, 2 and 3 all obey ‘Lipinski’s rule of 5’
(except the non-protonated form of compound 1d which has a
clog P slightly over 5).23 The clog P of 1a–e, 2, 3 were calculated
with VCCLabs (Table 1).22 Although initially hypothesised by
J. T. Davis et al., no direct correlation between the basicity of
prodigiosenes and their anti-cancer properties was found.24
pKa is, however, an indication at which pH the compounds are
protonated and therefore at what point an increased affinity
for anions is to be expected. The apparent pKa values for 1a–e
and 2 were determined via a spectrophotometric method, pre-
viously described by Manderville (Fig. 3 and Table 1).25 In solu-
tion, protonated perenosins are dark yellow to orange with
absorbance maxima above 380 nm. As free-base the com-
pounds are mostly lightly yellow and absorb at a lower wave-
length. Gradual variation of pH allows monitoring of the
change in ionization state and determination of the pKa values
from plots of log(Abs) versus pH (see ESI†).
Fig. 3 UV-Absorbance spectra for 1c (added as DMSO solution) as a
function of pH in phosphate buffer at 20 °C (0.1 M NaCl). Due to solubi-
lity issues minor spectral deviations were observed at some pH values.
X-ray crystallographic analysis
A single crystal of 1a with HCl suitable for X-ray analysis was
obtained via slow evaporation of a CDCl3 solution of 1a in the
presence of a small excess of HCl. The structure (Fig. 4) reveals
that the protonated perenosin 1a forms three hydrogen bonds
to chloride to form a 1 : 1 complex (N–Cl distances 3.169(4)–
Fig. 4 X-ray crystal structure of [1a + HCl]; (a) indicating the distance
(given in Å) between donor and acceptor; (b) side view (Cl− omitted for
clarity).
Table 1 Experimentally determined EC50 270 s, Hill coefficient (n), and
pKa and calculated log P (clog P) (free-base and protonated form) for
perenosins 1a–e, 2 and 3
3.223(3) Å, N–H⋯Cl angles 164.3–175.5°). The compound
clog P
adopts
a planar conformation allowing for conjugation
b
EC50 270 s
(mol%)
clog P
protonated
throughout the molecule and a more rigid structure (Fig. 4b).
Transporter pKa
n
(error)c
(error)c
1H NMR titration studies
1a
1b
1c
1d
1e
2
3
6.84
5.38
6.81
6.65
7.11
7.18
n.d.d
0.0773
1.1922
0.0301
0.0299
0.1859
6.4084
5.4305
1.11 2.98 ( 0.60) 2.05 ( 1.20)
1.52 3.84 ( 0.61) 3.37 ( 0.59)
1.00 3.36 ( 0.66) 2.41 ( 1.17)
1.00 5.16 ( 0.98) 4.02 ( 1.46)
1.33 2.85 ( 0.74) 2.00 ( 1.16)
1.93 2.32 ( 0.44) 1.58 ( 1.38)
2.21 2.44 ( 0.50) 1.48 ( 1.26)
1.00 4.12 ( 0.78) 3.28 ( 1.15)
To assess the affinity for the biologically important chloride
and bicarbonate anions, 1H NMR titration studies were per-
formed to obtain association constants (1 × 10−5 M DMSO-d6/
0.5% H2O, 298 K). The apparent association constants were
calculated using WinEqNMR 2 26 by fitting the titration data to
a 1 : 1 binding model, as was found from Job plot analysis27
supported by the single-crystal X-ray analysis (Table 2). Upon
Prodigiosin
7.16a 0.0002
a Literature value.21 b Cl−/NO3 assay using POPC/chol (7 : 3) at pH 7.2.
c Values calculated with VCCLabs.22 d not determined.
−
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Table 2 Association constants Ka (M−1) for 1 : 1 complexation between
1a–e, 2, 3 (1 × 10−5 M, 298 K) and anionic guests in DMSO-d6/0.5% H2Oa
suspended in an isotonic sodium nitrate solution (489 mM
with 5 mM phosphate buffer at pH 7.2). Perenosins 1a–e, 2, 3
were added as a DMSO solutions and the resulting chloride
efflux was monitored by a chloride selective electrode. At the
end of the experiment detergent (octaethylene glycol monodo-
decyl ether) was added to lyse the liposomes and calibrate the
electrode to 100% chloride release.
Receptor
Cl– (M–1) (1 equiv. HPF6)b
Cl– (M–1
)
HCO3– (M–1
)
1a
1b
1c
1d
1e
2
1340
4030
1670
1650
2320
318
<5c
6.98
<5c
<5c
<5c
8.98
Deprot.
9.50
11.5
14.4
Deprot.
Deprot.
This assay and others described below are evidence that the
perenosins are mediating chloride/nitrate antiport in this case.
Through the addition of transporter 1a in various concen-
3
433
<5c
a Calculated using WinEqNMR2.26 Maximum error estimated to be
trations,
a
Hill plot29 was derived giving EC50 270
of
s
15%. b The exchange between PF6 and Cl− is observed. c Only minor
−
0.0773 mol% (carrier to lipid). The more electron-deficient
analogue 1b (R = CF3), having a higher affinity for chloride but
a lower pKa, proved to be a less efficient chloride transporter
with EC50 270 s of 1.1922 mol% (Fig. 5). The introduction of a
methyl or pentyl substituent in the 5-position of the indole
moiety provided a decrease of the EC50 270 s value (EC50 270 s
0.0301 and 0.0299 mol%, respectively). Presumably the
increase in clog P with respect to 1a resulted in improved
chloride efflux. A higher pKa via the use of methoxy-derivative
1e which should be protonated more easily and is therefore
expected to transport more efficiently, resulted in a less
effective transporter than 1a–d (EC50 270 s 0.1859 mol%) most
probably attributed to the receptor’s lower clog P value
(Table 1). The benzimidazole and indazole derivatives were
shown to be poor lipid bilayer chloride transporters. Perenosin
spectral changes were observed under these conditions.
stepwise addition of chloride to the protonated host [1a +
HPF6] the pyrrole N–H (11.91 to 13.28 ppm), iminium N–H
(11.82 to 13.00 ppm) and indole N–H (11.37 to 11.57 ppm)
proton resonances shifted downfield. In addition, a substan-
tial downfield shift for the indole proton in the 6-position
(7.33 to 7.59 ppm) and a modest downfield shift for the imine
C–H proton (8.64 to 8.71 ppm) was observed. For the remain-
ing protons no significant changes in chemical shift were
observed. Addition of TEAHCO3 or TBAH2PO4 to the proto-
nated [1a + HPF6] resulted in deprotonation of the receptor,
whereas upon addition of TBANO3 no notable changes in
chemical shift were observed. Similar results have previously
been observed with prodigiosin.28 Receptors 1b–e responded
in a similar manner to the addition of chloride and were
shown to possess a similar affinity for chloride. For com-
pounds 2 and 3, having benzimidazole and indazole function-
alities instead of the indole group, respectively, the association
constant with chloride was 10 times lower presumably due to
the different resonance forms present (see ESI†).
1d with the lowest EC50 270 was found to be two orders of
s
magnitude slower than prodigiosin (EC50 270
0.0002, respectively).
0.0299 and
s
To investigate the effect of the pH on the transport activity
of perenosins 1a–e and 2, chloride/nitrate antiport was fol-
lowed at different pH values (pH 4.0, 6.2, 7.2 and 8.2; Fig. 6).
Upon decreasing the pH from 7.2 to 6.2, an increase in trans-
port is observed corresponding to the increased amount of
Addition of TBACl to 1a–e, 2, 3 (1 × 10−5 M DMSO-d6/0.5%
H2O, 298 K) only perturbed the proton resonances minimally
not allowing for an association constant to be calculated in
this competitive solvent mixture (Table 2). Addition of
TEAHCO3 to the free-base form of the receptors revealed the
presence of a very weak interaction, presumably due to the
more basic nature of the anion and the potential binding of
the anion’s proton to the imine functionality.
Transmembrane chloride transport studies
The anti-cancer properties of the prodigiosenes have been
linked to their ability to transport passively chloride or H+/Cl−
across vesicle and cell membranes.6,7 Consequently, the ability
of the perenosins to facilitate chloride and proton transport
across lipid bilayers was assessed using a combination of ion
selective electrode (ISE) and fluorescence assays. To quantify
Fig. 5 Chloride efflux promoted by a DMSO solution of compound 1a–e,
the chloride efflux rate Hill plots were determined for 200 nm 2, 3 (1 mol% carrier to lipid) from unilamlellar POPC : cholesterol vesicles
loaded with 489 mM NaCl buffered to pH 7.2 with 5 mM sodium
phosphate salts. The vesicles were dispersed in 489 mM NaNO3
buffered to pH 7.2 with 5 mM sodium phosphate salts. At the end of the
experiments, detergent was added to lyse the vesicles and calibrate the
POPC : cholesterol liposomes (Table 1; see ESI†). Typically, uni-
lamellar vesicles were prepared from 1-palmitoyl-2-oleoyl-sn-
glycero-3-phosphocholine (POPC) and cholesterol (7 : 3 ratio),
containing an intravesicular sodium chloride solution
ISE to 100% chloride efflux. Each point represents the average of three
(489 mM with 5 mM phosphate buffer at pH 7.2), were trials. DMSO was used as a control.
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Fig. 6 Anion exchange assay promoted by a DMSO solution of com-
pound 1d (1 mol% carrier to lipid) from unilamlellar POPC : cholesterol
vesicles loaded with 489 mM NaCl buffered to a given pH with 5 mM
sodium phosphate salts (pH 7.2 and 8.2), piperazine (pH 6.2) or citric
acid (pH 4.0). The vesicles were dispersed in 489 mM NaNO3 buffered
to a given pH with 5 mM sodium phosphate salts (pH 7.2 and 8.2), piper-
azine (pH 6.2) or citric acid (pH 4.0). At the end of the experiments,
detergent was added to lyse the vesicles and calibrate the ISE to 100%
chloride efflux. Each point represents the average of three trials. DMSO
was used as a control.
Fig. 7 Change of extravesicular chloride concentration over time for
1a–e, 2 (1 mol% carrier to lipid) of unilamlellar POPC : cholesterol vesi-
cles loaded with 489 mM NaCl buffered to pH 7.2 with 20 mM sodium
phosphate salts. The vesicles were dispersed in 167 mM Na2SO4
buffered to pH 7.2 with 20 mM sodium phosphate salts. At t = 120 s, a
solution of NaHCO3 spike was introduced such that the external
NaHCO3 concentration was 40 mM. At the end of the experiments,
detergent was added to lyse the vesicles and calibrate the ISE to 100%
chloride efflux. Each point represents the average of three trials.
1 mM 8-hydroxy-1,3,6-pyrenetrisulfonate (HPTS), a pH sensi-
tive fluorescent dye were prepared.31 The vesicles were sus-
pended in a solution of sodium sulfate (167 mM) and the
HPTS fluorescence measured upon addition of a DMSO solu-
tion of compounds 1a–e, 2 (Fig. 8). An increase in pH was
observed, corresponding to the deacidification of the vesicles
via a H+/Cl− co-transport mechanism (with Cl−/OH− antiport
being ruled out due to the decrease in transport observed at
higher pH and with basic anions such as bicarbonate).
Prodigiosenes have been shown to transport HCl across the
lipid bilayer via a mobile carrier mechanism.1 The Hill coeffi-
cients found for all indole-based perenosins and prodigiosin
have a value of approximately 1 evidence in support of
the hypothesis that the transport of a chloride ion can be
receptor molecules being protonated. At pH 8.2 there is a sig-
nificant drop in transport activity as presumably a significant
proportion of the transporters are not protonated. This is
evidence that only the protonated form of this perenosin is
capable of transporting anions.
To further explore the transport mechanism operating in
this system, a variety of ISE and fluorescence vesicle assays
were performed altering the bilayer and the intra- or extravesi-
cular solution composition. To probe whether metal ion–anion
symport occurs POPC vesicles were loaded with different group
1 metal (Na+, K+, Cs+) chloride salts (see ESI†). The metal was
found to have no effect on the rate of chloride efflux from the
vesicles upon addition of compound 1a, evidence in support
of a transport mechanism not involving metal cations.
The sulfate ion is highly hydrophilic and is more challen-
ging to transport across the lipid bilayer than nitrate.30 Upon
addition of perenosin to vesicles loaded with sodium chloride
suspended in a sodium sulfate solution, no chloride efflux was
observed (see ESI†). Upon addition of bicarbonate to the extra-
vesicular solution, a chloride/bicarbonate antiport mechanism
may be initiated. After the bicarbonate pulse at t = 120 s,
a modest increase in extravesicular chloride concentration was
noted (Fig. 7). The compounds proved to be quite poor bicar-
bonate transporters presumably due to deprotonation of the
protonated perenosin. This is evidence in support of the proto-
nated form of the receptor being the species that is capable of
transporting anions across the bilayer.
In the sulfate assays no anion transport was observed,
however due to the very small intravesicular volume, HCl co-
transport along a pH gradient using ion-selective electrode
assays is very hard to quantify. The possible presence of HCl
co-transport was studied by fluorescence using a pH gradient
assay. Vesicles containing sodium chloride (489 mM) and
Fig. 8 Fluorescence assay (λex = 403 and 460 nm, λem = 510 nm) for
1a–e, 2 (1 mol% carrier to lipid) using unilamellar POPC : cholesterol
vesicles loaded with 489 mM NaCl buffered to pH 7.2 with 20 mM
sodium phosphate salts and 1 mM HPTS. The vesicles were dispersed in
167 mM Na2SO4 buffered to pH 7.2 with 20 mM sodium phosphate salts.
Each point represents the average of three trials.
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performed by a single carrier molecule.32 The non-indole pere-
nosins 2 and 3 have a Hill coefficient of 1.93 and 2.21, respect-
ively, evidence in support of cooperative mechanism involving
two carrier molecules transporting one chloride ion.
Table 3 IC50 (μM) values for 1a–e, 2 on MDA-MB-231, MCF-7 and
MCF-10A cells
Compound
MDA-MB-231 (μM)
MCF-7 (μM)
MCF-10A (μM)
1a
1b
1c
1d
1e
2
9.07 1.30
3.67 0.05
5.10 1.08
4.39 0.80
9.92 0.80
28.78 2.33
6.02 1.27
4.13 0.22
4.38 0.30
3.84 0.12
5.61 1.48
20.22 7.35
15.43 5.75
12.93 3.23
11.92 3.37
22.37 7.74
18.28 6.69
51.20 15.31
Evidence for a carrier mechanism was derived from U-tube
experiments.33 Transporters 1a, 1c–e, 2 as a solution in chloro-
form (1 mM) were kept between two aqueous phases as a
membrane model mimicking a vesicle assay (see ESI†). The
source aqueous phase was loaded with sodium chloride
(489 mM buffered to pH 7.2 with 5 mM sodium phosphate
salts) and the receiving aqueous phase was loaded with
sodium nitrate (489 mM buffered to pH 7.2 with 5 mM sodium
phosphate salts). The large separation between the two
aqueous phases rules out the possibility of transport via
channel formation. Chloride transport was monitored using
an ISE and showed that all the tested perenosins yielded an
increase in chloride concentration in the receiving phase over
time (5 days). These results support the hypothesis of a mobile
carrier mechanism being the most likely mode of transport in
this case.
the viability of breast carcinoma MDA-MB-231 (invasive) and
MCF-7 (non-invasive) model cell lines, as well as MCF-10A
normal mammary model cells (Table 3). The cell lines were
treated with increasing doses of perenosins for 24 h and the
effect on the degree of cell viability was determined by MTT
assays giving a dose–response curve (see ESI Fig. S85–S90†)
that was used to determine the IC50 values for each compound
in each cell line.
All indole-based perenosins 1a–e were cytotoxic to the two
malignant cell lines at low µM, with 1b, 1c and 1d being most
potent. All the molecules tested here were less potent in the
normal MCF-10A cells. Interestingly, 1d showed the largest
selectivity (∼5.5 fold) for the cancerous cell lines tested here.
The benzimidazole derivative 2 was found to be substantially
less active (IC50 24.32 μM) than the other molecules. The most
active compounds in cells (1b and 1d) were also the most lipo-
philic in the series (clog P 3.84 and 5.06, respectively). The
reduced cytotoxicity observed in the normal MCF-10A cells
suggests a potential mechanism for selective targeting of
cancer cells with more potent derivatives of these molecules.
Transport studies with the prodigiosenes show that the pKa
24
of the transporter correlates well with the EC50 270s
,
however
no clear correlation could be found between the pKa of pereno-
sins and their EC50 270s. However, compounds with a pKa value
higher than 6.65 and a clog P between than 2.85 and 5.16,
(supported by the log P range stipulated by Quesada et al.,13b
)
appear to exhibit the best chloride transport properties.
Taking all the transport studies together the results show
that the indole perenosins behave similarly to prodigiosin
namely functioning as both a HCl cotransporter and a Cl−/
NO3− antiporter6 and forming a 1 : 1 complex with the anion.
Hydrolysis studies
The rate of hydrolysis is an important factor within the set of
pharmokinetic properties, the collective of bioavailability and
processes of absorption, distribution, metabolism and elimin-
ation. As a model for all perenosins, the hydrolysis rate of 1a
in phosphate buffer (0.1 M NaCl) was determined following
the perturbation of the UV/Vis spectroscopic data over time
(see ESI†). At pH 7.2 the half-life time of 1a was calculated, fol-
lowing first-order kinetics, to be 10.8 h.34 Lowering the pH to
4.0 shortened the half-life time of 1a to 6.6 h, consistent with
the presence of the imine linker. Entrapment of 1a inside a
vesicular lipid bilayer at pH 7.2 extended the life span of pere-
nosin 1a six fold (half-life 60.2 h). Vesicles that fuse with the
cancer cell membrane, potentially decorated with cancer cell
selective receptors and fluorophores, therefore offer a potential
route to administer future anti-cancer agents based upon the
perenosin scaffold. An additional benefit would be the
Conclusions
The perenosins represent a new class of highly effective anion
transporters based upon the structure of prodigiosin. It has
been demonstrated that indole-based perenosins are highly
efficient chloride transporters and function as a mobile-carrier
by an antiport and H+/Cl− symport mechanism of anion trans-
port. The most lipophilic derivatives affect the viability of two
breast cancer cell lines with ∼5.5-fold selectivity over normal
breast cells. These compounds therefore represent excellent
lead structures for further exploration of the potentially selec-
tive anti-cancer activities of this new class of molecules.
Acknowledgements
reduced toxicity for highly active compounds when adminis- W. V. R. and P. A. G. thank the European Union for a Marie
tered whilst embedded inside liposomes.35
Curie Career Integration Grant. P. A. G. thanks the Royal
Society and the Wolfson Foundation for a Royal Society
Wolfson Research Merit Award. D. J. A. and A. T. thank the
Cell-based analysis
We performed preliminary studies to assess the effect of pere- Engineering and Physical Sciences Research Council (for EP/
nosins on the viability of cancerous and non-cancerous model H04986X/1). The authors thank Dr Ethan Howe for assistance
cell lines. Compounds 1a–e, 2 were assessed for their effect on with the preparation of this manuscript.
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