Antitumor Activity of Ionic Liquids Based on Ampicillin

Article abstract of DOI:10.1002/cmdc.201500142

Significant antiproliferative effects against various tumor cell lines were observed with novel ampicillin salts as ionic liquids. The combination of anionic ampicillin with appropriate ammonium, imidazolium, phosphonium, and pyridinium cations yielded ac

Full text of DOI:10.1002/cmdc.201500142

DOI: 10.1002/cmdc.201500142
Communications
Antitumor Activity of Ionic Liquids Based on Ampicillin
Ricardo Ferraz,[a, b] Jo¼o Costa-Rodrigues,*[c] Maria H. Fernandes,[c] Miguel M. Santos,[a]
Isabel M. Marrucho,[d] Luís Paulo N. Rebelo,[d] Cristina PrudÞncio,[b, e] Jo¼o Paulo Noronha,[a]
[a]
[a]
ˇ
Zeljko Petrovski,* and Luís C. Branco*
Significant antiproliferative effects against various tumor cell
lines were observed with novel ampicillin salts as ionic liquids.
The combination of anionic ampicillin with appropriate ammo-
nium, imidazolium, phosphonium, and pyridinium cations
yielded active pharmaceutical ingredient ionic liquids (API-ILs)
that show potent antiproliferative activities against five differ-
ent human cancer cell lines: T47D (breast), PC3 (prostate),
HepG2 (liver), MG63 (osteosarcoma), and RKO (colon). Some
API-ILs showed IC50 values between 5 and 42 nm, activities that
stand in dramatic contrast to the negligible cytotoxic activity
level shown by the ampicillin sodium salt. Moreover, very low
cytotoxicity against two primary cell lines—skin (SF) and gingi-
val fibroblasts (GF)—indicates that the majority of these API-ILs
are nontoxic to normal human cell lines. The most promising
combination of antitumor activity and low toxicity toward
healthy cells was observed for the 1-hydroxyethyl-3-methylimi-
dazolium–ampicillin pair ([C2OHMIM][Amp]), making this the
most suitable lead API-IL for future studies.
or potassium form, have been developed to improve aqueous
solubility. Ionic liquids (ILs) are a peculiar class of organic salts
with melting points below 1008C which have been the focus
of increasing interest in the scientific community and industry;
the large number of potential cation–anion combinations
allows a variety of tunable interactions and applications.[1] One
of the most promising applications of ILs is in the so-called
third generation of ILs, that is, their arrangement with APIs to
generate API-ILs.[2–6] Successful buffer-controlled procedures for
the preparation of API-ILs containing the anion of ampicillin (a
well-known antibiotic) combined with members of four organ-
ic cation classes (ammonium, imidazolium, phosphonium, and
pyridinium) have been reported.[7–9] These API-IL combinations
can be an innovative solution to the polymorphic behavior of
certain drugs, and can also improve water solubility, permeabil-
ity, and therefore bioavailability. The appropriate arrangement
of a biocompatible anion or cation with a specific drug can be
relevant in modifying the corresponding biopharmaceutical
drug classification system (BCS)[2,4,10–12] as well as their drug for-
mulation process.
Researchers in the pharmaceutical industry are currently facing
a series of challenges in the discovery of innovative and effec-
tive drugs and their subsequent therapeutic applications. Most
active pharmaceutical ingredients (APIs) are available in solid
form, a state that frequently suffers from polymorphic conver-
sion, low solubility, and a variety of other factors that affect
the final bioavailability. Various pharmaceutical salts, in sodium
The important question regarding the toxicity of the API
counter-ion in ILs has been the major cause behind the de-
layed entry of ILs into the wider biosciences field.[5,13] Toxicities
toward microorganisms and eukaryotic cell cultures cover the
entire range of biocidal potencies, from rather inactive molecu-
lar solvents that are biocompatible, up to high concentrations
in aqueous solutions for pharmaceutical applications,[2,4,12] and
even as potential in vitro anticancer agents.[8,10] Antimicrobial
activities against Escherichia coli (susceptible and resistant),
Klebsiella pneumoniae, Staphylococcus aureus, and Enterococcus
faecium have been reported for some ampicillin organic salts
in comparison with sodium ampicillin ([Na][Amp]).[9,14] Conse-
quently, the development of novel anticancer drugs that are
less toxic than currently available chemotherapeutic treat-
ments can also be pursued with ionic liquids.[3,4,12] Herein we
describe the antiproliferative effects of several previously syn-
thesized ampicillin ILs (Figure 1) against various tumor cell
lines.
ˇ
[a] Dr. R. Ferraz, Dr. M. M. Santos, Prof. J. P. Noronha, Dr. Z. Petrovski,
Dr. L. C. Branco
Departamento de Química, REQUIMTE-CQFB, Faculdade de CiÞncias e Tec-
nologia da Universidade Nova de Lisboa, 2829-516 Caparica (Portugal)
E-mail: l.branco@fct.unl.pt
z.petrovski@fct.unl.pt
[b] Dr. R. Ferraz, Prof. C. PrudÞncio
CiÞncias Químicas e das BiomolØculas, Escola Superior de Tecnologia da
Safflde do Porto do Instituto PolitØcnico do Porto, Rua Valente Perfeito 322,
4400-330 Vila Nova de Gaia (Portugal)
[c] Prof. J. Costa-Rodrigues, Prof. M. H. Fernandes
Laboratório de Farmacologia e Biocompatibilidade Celular, Faculdade de
Medicina Dentµria, Universidade do Porto, Rua Dr. Manuel Pereira da Silva,
4200-393 Porto (Portugal)
Table 1 summarizes the antitumor activities (IC50 and LD50) of
the ampicillin-anion-based ILs against two primary human cell
types: skin (SF) and gingival fibroblasts (GF). [Na][Amp] was
used as control, and as is evident from the data listed in
Table 1, it is one of the least toxic. The IC50 value was not de-
tected in the concentration range used against SF, and the IC50
value toward GF was 109.1 mm. In comparing the other synthe-
sized compounds with [Na][Amp], the most toxic toward the
healthy human cell lines are [P6,6,6,14][Amp] and [C16Pyr][Amp]
(lowest IC50 values). Ammonium-based ILs [TEA][Amp] and
E-mail: jrodrigues@fmd.up.pt
[d] Dr. I. M. Marrucho, Prof. L. P. N. Rebelo
Instituto de Tecnologia Química e Biológica, ITQB, Universidade Nova de
Lisboa, Av. da Repfflblica Estażo Agronómica Nacional, 2780-157 Oeiras
(Portugal)
[e] Prof. C. PrudÞncio
Centro de Farmacologia e Biopatologia Química (U38-FCT), Faculdade de
Medicina da Universidade do Porto (FMUP) (Portugal)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201500142.
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Communications
ter-ions) can significantly assist the transfer of highly polar
molecules (different APIs) through prokaryotic and eukaryotic
membranes, thereby improving drug activity. However, we
have also shown that the lipophilicity of the counter-ion alone
does not necessarily solve all API problems, but can contribute
to the improvement of certain physicochemical properties
such as aqueous solubility and octanol–water partition coeffi-
cients (Kow).[5] In this context, the outstanding antitumor activi-
ty of ampicillin salts can be explained by the stronger and
more stable cation–anion pairings brought about by the ex-
pected hydrogen bonding and p–p interactions, as well as
electrostatic interactions that can be established between the
ampicillin anion and certain organic cations. For example,
[C2OHMIM] can establish hydrogen bonding and p–p interac-
tions, as illustrated in Figure 1. As indicated in Figure 1 and
Table 2, the best results were observed for the cases of stron-
ger and more stable cation–anion interactions, where the ap-
propriate counter-ion acts as a more lipophilic drug carrier.
Ampicillin sodium salt ([Na][Amp]), which is a standard anti-
biotic in current use, only shows cytotoxic activity toward PC3
and RKO cell lines, with respective IC50 values greater than
0.597 and 2.406 mm. Moreover, its antiproliferative activity is
very weak toward the cell lines studied, with the exception of
HepG2. However, it is quite well tolerated by primary fibro-
blasts, namely GF. Markedly different antitumor results have
been observed by pairing distinct organic cations with ampicil-
lin. For instance, whereas [TEA][Amp] is highly cytotoxic
toward MG63 and T47D cells, [Cholin][Amp] was found to be
particularly selective for the RKO cell line. In addition, this API-
IL only shows significant antiproliferative behavior toward
MG63 cells. In contrast to other bulky cations, the high cyto-
toxic effect of [Cholin][Amp] toward MG63 cancer cells and
high selectivity away from noncancerous cells can be partially
attributed to its high water solubility and partition coeffi-
cient,[5] which facilitate the transport of the xenobiotic (in this
case, ampicillin) to the cytosol of cancer cells. Moreover, this
capacity is particularly enhanced in these cells, as the [Cholin]
cation is a nutrient.[3,16] On the other hand, [EMIM][Amp] is par-
ticularly selective against RKO, with an IC50 value >0.269 mm.
Considering the previously reported physicochemical proper-
ties of these compounds,[5] the enhanced cytotoxic activity
toward particular cancer cell lines as well as low toxicity
against primary fibroblasts can be related to the greater lipo-
philic character of these cations than the sodium analogue.
[C2OHMIM][Amp] was found to be a very potent antitumor
agent against all cancer cell lines studied, with IC50 and LD50
values in the ranges of 0.146–0.738 mm and 0.297–4.197 mm, re-
Figure 1. Structures of ampicillin salts and their possible cation–anion inter-
actions. Details of their synthesis are available in the Supporting Informa-
tion.
Table 1. Antitumor activities of the prepared ILs against two primary
human cell types.
Compd
Cell line
SF[b]
GF[c]
ND[d]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
ND[d]
ND[d]
>0.249
>0.249
>0.032
>0.815
[TEA][Amp]
>58.040
>0.173
[P6,6,6,14][Amp]
[C16Pyr][Amp]
[Cholin][Amp]
[EMIM][Amp]
[C2OHMIM][Amp]
[Na][Amp][a]
>0.176
>0.012
45.510Æ0.131
48.480Æ0.571
49.790Æ0.368
>6.366
ND[d]
ND[d]
>0.853
>9.357
>0.462
>6.366
>5.084
>22.600
ND[d]
>8.104
30.470Æ0.428
>109.100
>109.100
[a] [Na][Amp] was used as a control. [b] Skin fibroblasts. [c] Gingival fibro-
blasts. [d] Not detected in the concentration range used. [Amp]=ampicil-
lin anion, [TEA]=tetraethylammonium, [P6,6,6,14]=trihexyltetradecylphos-
phonium, [C16Pyr]=cetylpyridinium, [Cholin]=choline, [EMIM]=1-ethyl-3-
methylimidazolium,
[C2OHMIM]=1-hydroxyethyl-3-methylimidazolium.
Data were obtained from three independent experiments, and each assay
was performed in three replicates; for all experiments the associated
errors (ÆSD) did not exceed 5%.
[Cholin][Amp] were found to be the least toxic, with imidazoli-
um-based ILs [EMIM][Amp] and [C2OHMIM][Amp] having IC50
values in between. These results are in agreement with pub-
lished work showing that higher hydrophobicity and increased
alkyl chain length lead to increased toxicity.[13,15]
spectively. A similar observation was made for the [P6,6,6,14
]
Table 2 summarizes the antitumor activities of the ampicillin-
based ILs against several human cancer cell lines: T47D
(breast), PC3 (prostate), HepG2 (liver), MG63 (osteosarcoma),
and RKO (colon). Figure 2 shows some representative experi-
mental data used to calculate these values listed. Among all
compounds tested, the 3-(2-hydroxyethyl)-1-methylimidazoli-
um–ampicillin salt ([C2OHMIM][Amp]) possesses the greatest
selectivity and antiproliferative activity against these five
cancer cell lines. Some lipophilic phase-transfer agents (coun-
[Amp] IL; however, it is 90- to 173-fold more toxic toward pri-
mary fibroblasts. This finding is not surprising, as the very high
toxicity of compounds with this cation toward other cell lines
was described previously, with similar IC50 and LD50 values.[17,18]
[C16Pyr][Amp], which showed IC50 values in the low nanomolar
range toward MG63 and T47D cells, was also shown to be
fairly toxic toward fibroblasts, namely SF. This observation is in
line with that previously reported for the [C16Pyr] cation,[19,20]
despite its frequent application in pharmaceutical formulations.
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Figure 2. Experimental IC50 data for the most representative compounds toward A) RKO, B) PC3, C) HepG2, and D) T47D cell lines. Data are the mean ÆSD de-
termined from three separate experiments; each assay was performed in three replicates.
Table 2. Antitumor activities of the prepared ILs against several human cancer cell lines.
Compd
Cell line[b]
T47D
MG63
>0.030
HepG2
ND[d]
0.095Æ0.008
>0.322
>0.322
ND[d]
ND[d]
PC3
RKO
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
IC50 [mm]
LD50 [mm]
>0.042
>35.650
>58.920
>0.354
>56.700
68.370Æ0.511
>0.180
[TEA][Amp]
1.368Æ0.020
0.312Æ0.019
>43.990
>0.264
>4.970
>0.005
ND[d]
>110.900
>110.900
ND[d]
[P6,6,6,14
[Amp]
]
>0.312
9.335Æ0.179
>11.860
>0.226
[C16Pyr]
[Amp]
[Cholin]
[Amp]
[EMIM]
[Amp]
[C2OHMIM]
[Amp]
>0.011
>183.200
>0.017
>0.031
>1.122
>1.122
>0.738
>1.240
ND[d]
132.700Æ3.593
>132.700
>0.982
59.170Æ0.095
>1.619
>5.765
>24.270
>24.270
>0.319
>0.4040
ND[d]
ND[d]
17.160Æ0.514
>0.707
>0.269
>29.990
32.370Æ0.121
>31.020
>0.297
>209.700
>0.359
>0.146
>0.498
ND[d]
>0.297
>0.362
[Na]
[Amp][a]
>0.597
>2.406
>506.600
ND[d]
>47.790
560.900Æ0.515
>920.800
[a] [Na][Amp] was used as a control. [b] Cancer cell lines: T47D (breast), PC3 (prostate), HepG2 (liver), MG63 (osteosarcoma), and RKO (colon). [d] Not de-
tected in the concentration range used. [Amp]=ampicillin anion, [TEA]=tetraethylammonium, [P6,6,6,14]=trihexyltetradecylphosphonium, [C16Pyr]=cetyl-
pyridinium, [Cholin]=choline, [EMIM]=1-ethyl-3-methylimidazolium, [C2OHMIM]=1-hydroxyethyl-3-methylimidazolium. Data were obtained from three in-
dependent experiments, and each assay was performed in three replicates; for all experiments the associated errors (ÆSD) did not exceed 8%.
The particularly high toxicity toward cancerous and/or normal
cells shown by these three API-ILs is probably related to their
high amphiphilic character relative to the analogous sodium
salt;[5] this promotes cell membrane permeation and conse-
quently their efficiency.
midazolium-based ionic liquids only show cytotoxic activity if
the alkyl side chain contains more than 11 methylene units.
In conclusion, the combination of ampicillin with organic
cations in place of sodium has yielded novel ionic liquids that
show low IC50 and LD50 values against five different cancer cell
lines: T47D (breast), PC3 (prostate), HepG2 (liver), MG63 (osteo-
sarcoma), and RKO (colon). Furthermore, very low toxicity
against two primary cell lines—skin (SF) and gingival fibro-
blasts (GF)—indicates that the majority of these compounds
are nontoxic to certain healthy human cells. Outstanding cyto-
toxic and antiproliferative activities against several tumor cell
Notably, choline salts with acetate, propanoate, butanoate,
hexanoate, and pivalate anions showed IC50 values >10 mm
toward MCF-7 lung cancer cells which increase with the length
of the carbon chain.[21] Usually, other ionic liquids containing
quaternary ammonium cations are not cytotoxic toward cancer
cell lines.[8] As previously reported by Malhotra et al.,[9] methyli-
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LD50 is the median lethal dose and is the amount of material that
causes the death of 50% of a population.[16]
lines, with IC50 and LD50 values in the low micromolar range,
associated with low activity against primary fibroblasts was ob-
served for [C2OHMIM][Amp]. On the other hand, [TEA][Amp],
[Cholin][Amp], and [EMIM][Amp] showed IC50 values in the
nanomolar or low micromolar range against some of the
cancer cell lines tested, as well as very low toxicity toward
normal cells. These data make these API-ILs the most promis-
ing lead compounds for future in vivo studies. Importantly, se-
lection of the appropriate counter-ions can explain these anti-
tumor activities due to the stronger and more stable cation–
anion interactions relative to the sodium analogue. Therefore,
suitable organic cations can establish efficient ion pairs, acting
as lipophilic drug carriers as well as improving water solubility,
permeability, and bioavailability of the final pharmaceutical
salts.
Keywords: active pharmaceutical ingredients · ampicillin ·
antitumor agents · ionic liquids · toxicity
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Received: April 1, 2015
Revised: May 5, 2015
Published online on July 16, 2015
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