Cytotoxic Activity of Salicylic Acid-Containing Drug Models with Ionic and Covalent Binding

Article abstract of DOI:10.1021/acsmedchemlett.5b00258

Three different types of drug delivery platforms based on imidazolium ionic liquids (ILs) were synthesized in high preparative yields, namely, the models involving (i) ionic binding of drug and IL; (ii) covalent binding of drug and IL; and (iii) dual binding using both ionic and covalent approaches. Seven ionic liquids containing salicylic acid (SA-ILs) in the cation or/and in the anion were prepared, and their cytotoxicity toward the human cell lines CaCo-2 (colorectal adenocarcinoma) and 3215 LS (normal fibroblasts) was evaluated. Cytotoxicity of SA-ILs was significantly higher than that of conventional imidazolium-based ILs and was comparable to the pure salicylic acid. It is important to note that the obtained SA-ILs dissolved in water more readily than salicylic acid, suggesting benefits of possible usage of traditional nonsoluble active pharmaceutical ingredients in an ionic liquid form.

Full text of DOI:10.1021/acsmedchemlett.5b00258

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Letter
Cytotoxic activity of salicylic acid-containing
drug models with ionic and covalent binding
Ksenia S. Egorova, Marina M. Seitkalieva, Alexandra V.
Posvyatenko, Victor N. Khrustalev, and Valentine P Ananikov
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00258 • Publication Date (Web): 28 Sep 2015
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Cytotoxic activity of salicylic acid-containing drug models
with ionic and covalent binding
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Ksenia S. Egorova1*, Marina M. Seitkalieva1, Alexandra V. Posvyatenko2,3, Victor N. Khrustalev 4,5 and Valentine
P. Ananikov1,6*
1N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky prospect 47, Moscow, 119991 Russia
2Institute of Gene Biology, Russian Academy of Sciences, Vavilova str. 34/5, Moscow, 119334 Russia
3D. Rogachev Federal Scientific Clinical Centre of Pediatric Hematology, Oncology and Immunology, Ministry of Health of Russian
Federation, Samory Mashela str., Moscow, 117198 Russia
4Peoples’ Friendship University of Russia, 6 Miklukho-Maklay Street, Moscow 117198, Russian Federation
5A.N. Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, 28 Vavilov Street, Moscow 119991, Rus-
sian Federation
6Saint Petersburg State University, Universitetsky Prospekt 26, Stary Petergof, 198504, Russia
KEYWORDS: drug development, ionic liquids, active pharmaceutical ingredients, salicylic acid, API-IL, cytotoxicity
ABSTRACT: Three different types of drug delivery platforms based on imidazolium ionic liquids (ILs) were synthesized in high preparative
yields, namely the models involving: i) ionic binding of drug and IL; ii) covalent binding of drug and IL; and iii) dual binding using both ionic
and covalent approaches. Seven ionic liquids containing salicylic acid (SA-ILs) in the cation or/and in the anion were prepared, and their
cytotoxicity towards the human cell lines CaCo-2 (colorectal adenocarcinoma) and 3215 LS (normal fibroblasts) was evaluated. Cytotoxicity
of SA-ILs was significantly higher than that of conventional imidazolium-based ILs and was comparable to the pure salicylic acid. It is im-
portant to note, that the obtained SA-ILs dissolved in water more readily than salicylic acid, suggesting benefits of possible usage of traditional
non-soluble active pharmaceutical ingredients in an ionic liquid form.
Most of active pharmaceutical ingredients (API) currently used
in modern medicine are solid substances suffering from several
significant disadvantages. Being crystalline compounds, API may
exist as various polymorphs or pseudopolymorphs with different
solubility and bioavalability, and the determination of a particular
form of an API in the drug composition is a difficult and important
task.1,2 One of the most common strategies for overcoming this
issue is the conversion of neutral API into salts with sodium, chlo-
ride, phosphate, citrate and other counter-ions. Ionic-core micelles
were also proposed to be used for targeted drug delivery.3-5 Recent-
ly, it has been suggested that ionic liquids, or liquid salts, also can be
used as ionizing agents in pharmaceutics.2,6
ILs might be introduced into the drug formula to relieve admin-
istration of insoluble preparations into the organism in the form of
active pharmaceutical ingredient ionic liquids, or API-ILs.15-18 Being
unique tunable chemicals, ILs are ideal candidates for incorporat-
ing various functional groups,19 including biologically active sub-
stances.20 One of the latest areas of IL application in pharmaceutics
involves using them as functionalization agents for non-ionizable
active substances.6,16,20 Figure 1 shows most remarkable structural
features, which make ILs valuable potential candidates for new-age
drug delivery platforms.
Presently, there are two main strategies of combining API and
ILs: (a) using API as an anion or a cation (ionic transport, Fig. 1A)
and (b) covalently linking API to IL (covalent transport, Fig. 1B).
Moreover, the cationic-anionic structure of IL allows combining
API with different properties within one molecule (both cation an
anion may bear different or same API, Fig. 1C).21 Poorly soluble
API may be transformed into readily soluble API-ILs.2,22 Synthesis
and activity of several drugs, such as ampicillin,23,24 aspirin25 and
salicylic acid,26 in the IL form have been investigated. Particularly,
ampicillin ILs demonstrated dramatically higher antibiotic activity
than conventional sodium ampicillin.23
Ionic liquids (ILs) comprise an outstanding class of chemicals,
which during the last several decades have found application in
such diverse scientific and industrial fields as organic synthesis,7
nanoparticle research,8 catalysis,9 biomass conversion,10 electro-
chemistry,11 enzymatic reactions,12 extraction,13 and medicine.14,15
Initially ILs, being non-volatile and non-flammable substances,
were considered environmentally benign, but results of intense
studies proved otherwise.14 As biological activity of ILs has become
apparent, their potential application in medicine has attracted
much attention. First, ILs were explored as alternative reaction
media for synthesis of various drugs.16 Then it became evident that
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analogous compounds. The imidazolium ring is connected with the
–
)
salicylic fragment via the alkane bridge, which adopts the t-g(+)-t-g(
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(t – trans, g – gauche) conformation. It is well-known that, in ionic
liquids, a 2-position imidazolyl [NC(H)N] proton possesses the
highly acidic nature and is able to compete with active hydrogen
atoms for the formation of hydrogen bonding interactions with
halogenide anions.30 In the case of [PrMIM-OSal][Cl], however,
the hydrogen atom of the hydroxyl group in the sidearm functional-
ized with salicylic acid forms a strong O3─H3…Cl1 hydrogen
bond with a chloride anion [O…Cl 2.969(2), H…Cl 2.14(4) Å,
O─H…Cl 172(3) ], whereas the acidic imidazolyl proton links
two O1 and O3 oxygen atoms of a neighboring cation by the bifur-
cate hydrogen bond (see the Supporting information). Thus, cati-
ons and anions of [PrMIM-OSal][Cl] are bound by the above
hydrogen bonds into zigzag chains towards [010] (Figure S19, see
the Supporting information). The chains are packed in stacks along
the a axis.
Salicylic acid is a known cytotoxin with anti-cancer effects.31,32
Therefore, to investigate whether SA retains its activity upon in-
corporation into an IL, we used the MTT assay in two human cell
lines, CaCo-2 (colorectal adenocarcinoma) and 3215 LS (normal
fibroblasts). The resultant data on cytotoxicity of the SA-ILs, as
well as conventional imidazolium-based ILs and pure salicylic acid,
are shown in Table 1 (expressed as 24-h IC50, half maximal inhibi-
tory concentration after 24-hour exposure). Details on experi-
mental procedures and statistical analysis are provided in the Sup-
porting information.
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Figure 1.Three types of drug development platforms based on ILs:
API-IL containing ionic API as its anion (A), covalently linked API
within its cation (B), or both binding options (C).
Our first observation was that the studied ILs, both conventional
and newly synthesized ones, demonstrated similar toxicity in the
cancer and normal cell lines. Several ILs, such as [BMIM][BF4],
[Ala-OMe][BF4], [EMIM][Sal], and [BMIM][Sal], exhibited
significantly higher toxicity towards normal fibroblasts, as com-
pared with cells of colorectal adenocarcinoma (Table 1, entries 4-
7). Salicylic acid also did not distinguish between the two cell lines
and was even less toxic towards the cancerous one; thus, we could
not determine IC50 in CaCo-2, because the saturated solution of SA
did not kill 100% cells (Table 1, entry 13).
In this work, we used salicylic acid (SA) as a model drug. SA is a
relatively simple molecule, and its biological activity has been stud-
ied thoroughly.27 We synthesized seven imidazolium-based ionic
liquids (SA-ILs), which corresponded to the three API-IL classes
stated in Figure 1: ILs bearing SA in their anion ([EMIM][Sal],
[BMIM][Sal], [HMIM][Sal]); ILs with SA covalently linked to
the cation ([EMIM-OSal][Cl], [PrMIM-OSal][Cl], [EMIM-
OSal][BF4]), and an IL bearing SA both in the anion and cation
([EMIM-OSal][Sal]). Formulae of SA-ILs and conventional ILs
used in the study are shown in Figure 2.
Synthesis of [EMIM][Sal]26,28 and [BMIM][Sal]28,29 has been
described earlier, whereas preparation of [HMIM][Sal], [EMIM-
OSal][Cl], [PrMIM-OSal][Cl], [EMIM-OSal][BF4], and [EMIM-
OSal][Sal] have, to our knowledge, not been published before. As a
representative example, the scheme of synthesis of [EMIM-
OSal][Sal] is shown in Figure 3. The preparation procedure in-
volves three stages: (1) synthesis of chloroalkyl ethers of salicylic
acid; (2) incorporation of imidazole core; and (3) anion exchange
reaction with sodium salicylate. Structures and purity of the synthe-
sized ILs were confirmed using 1H, 13C NMR spectroscopy, and
mass spectrometry; the melting temperature was measured for
solid substances. The corresponding experimental details and char-
acterization data for all synthesized compounds are provided in the
Supporting information.
Introduction of salicylic acid into imidazolium-based ILs dramat-
ically increased their cytotoxicity. The replacement of the chloride
anion with salicylate led to a significant decrease of 24-h IC50 (Ta-
ble 1, entries 1-3 and 6-8). [BMIM][Sal] was even more cytotoxic
than [BMIM][BF4], for which rather high toxicity had been
demonstrated in this and other33 studies (Table 1, entries 4 and 7).
Introduction of salicylic acid into the cation resulted in a similar
increase of cytotoxicity (Table 1, entries 9-11).
The molecular structure of [PrMIM-OSal][Cl] was established
by X-ray analysis (Figure 4). The geometry of the imidazolium
cation in this salt is comparable to that observed in other reported
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Figure 2. Ionic liquids with various combinations of anions and cations utilized in the present study. (See Figure 1 for types A-C.)
Figure 3. Synthetic procedure for the preparation of [EMIM-OSal][Sal].
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Table 1. Cytotoxicity of studied ILs towards cell lines 3215 LS and
CaCo-2
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IL
3215 LS (24-h
IC50, mM)*
CaCo-2 (24-h
IC50, mM)*
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1
2
[EMIM][Cl]
46.15 (45.70-
46.59)
32.1033
[BMIM][Cl]
30.08 (19.47-
40.70)
18.8533
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Figure 4. Molecular structure of [PrMIM-OSal][Cl] determined by X-
ray analysis. (N1─C11 1.329(3) Å, N1─C13 1.377(3) Å, N2─C11
1.330(3) Å, N2─C12 1.372(3) Å, C12─C13 1.357(4) Å,
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[HMIM][Cl]
[BMIM][BF4]
[Ala-OMe][BF4]
[EMIM][Sal]
[BMIM][Sal]
[HMIM][Sal]
7.54 (2.55-12.54)
4.88 (4.27-5.50)
2.63 (2.32-2.94)
1.39 (0.64-2.14)
0.64 (0.19-1.09)
2.00 (0.69-3.31)
6.2133
11.1933
N1─C11─N2
O2─C8─C9─C10
108.2(2) ,
64.0(3) ,
C1─O2─C8─C9
C8─C9─C10─N1
-179.5(2) ,
166.7(2) ,
6.24 (4.32-8.17)
5.97 (4.51-7.91)
4.34 (2.48-7.61)
1.96 (0.79-3.14)
4.77 (3.27-6.27)
3.82 (3.34-4.31)
2.55 (2.08-3.13)
C9─C10─N1─C11 -55.8(4) .)
Of note, cytotoxicity of the ILs with SA in the cation was compa-
rable to that of the amino acid-based IL [Ala-OMe][BF4] (Table 1,
entries 5 and 9-11). In our previous work, we demonstrated that
the presence of an amino acid led to unexpectedly high cytotoxicity
of ILs with the tetrafluoroborate anion, possibly due to specific
interactions between the amino acid and transporter proteins on
the surface of the cell.33 In the case of salicylic acid, the anion had
insignificant influence on the cytotoxicity. In contrast to the con-
ventional [BMIM][Cl] and [BMIM][BF4], [EMIM-OSal][BF4]
and [EMIM-OSal][Cl] possessed similar toxicity (Table 1, entries
2 and 4, 9 and 10), suggesting the inherent toxicity of the anion to
be overwhelmed by that of the cation.
[EMIM-O][Sal] BF4 4.12 (2.51-5.73)
10 [EMIM-OSal][Cl]
11 [PrMIM-OSal][Cl]
2.85 (1.30-4.40)
3.19 (0.84-5.54)
0.71 (0.58-0.84)
**
[EMIM-OSal][Sal]
12
-
13 Salicylic acid
1.14 (0.67-1.94)
5-7***
* 95% confidence interval is shown in parentheses.
** No data available.
*** Approximate value (see the text).
We also studied the cytotoxicity of [EMIM-OSal][Sal], which
contained salicylic acid both in its cation (covalent linkage) and
anion (ionic interaction) (Table 1, entry 12). Its IC50 was 0.71 mM
and did not differ significantly from the SA-ILs bearing SA in the
anion only (Table 1, entries 6-8). This observation suggests that in
the case of imidazolium-based ILs, one part of salicylic acid per an
IL molecule is enough for reaching the maximum possible cytotox-
icity, and that increasing the SA content does not lead to higher
biological activity. In general, IC50 of SA-ILs belonging to the three
structural API-ILs types did not differ significantly, though SA-IL
bearing covalently linked salicylic acid within their cation tended to
demonstrate lower toxicity (IC50 2.85-4.12 mM), than SA-ILs with
salicylate anions (IC50 0.64-2.00 mM). This observation may be
possibly explained by known allosteric activity of SA.34 The cova-
lent linkage between SA and IL may hinder binding of the former
to its cell targets; on the other hand, the ionic linkage allows for
more flexible interactions.
Salicylic acid is known to cause apoptosis in vitro and in
vivo,31,32,36 but the exact mechanisms of its action just begin to
emerge. SA was shown to impact mitochondrial activity37 and to
inhibit protective catalase in malignant cells directly, therefore
activating the signaling mediated by reactive oxygen species
(ROS), which triggers apoptosis in the cell.32 SA decreased glucose
consumption by breast cancer cells MCF-738 and increased caspase
activity in CaCo-2 cells when applied at such low concentration as
10 M, the latter effect being dependent on the oxygen availabil-
ity.31
The fact that SA retains its ability to cause cell death upon cova-
lent or ionic linking to an IL suggests that its mechanism of action
remains unperturbed and does not include metabolic modifica-
tions.
In summary, we investigated cytotoxicity of salicylic acid-
containing imidazolium-based ILs (SA-ILs) belonging to three
currently available structural classes of API-ILs: API-ILs with ionic
API as their anion (type A); API-ILs with covalently linked API
within their cation (type B); and API-ILs bearing API both in the
anion and cation (type C). Cytotoxicity of the studied SA-ILs was
significantly higher than that of conventional imidazolium-based
ILs and was comparable to that of the pure salicylic acid. In contrast
to salicylic acid, SA-ILs possessed relatively high water solubility.
According to our results, API-ILs of types A and C demonstrated
similar biological activity, which was higher than that of type B.
Interestingly, in most cases the cytotoxicity of SA-ILs did not dif-
fer significantly from that of the pure salicylic acid (Table 1, entries
6-13). We were unable to determine the exact 24-h IC50 for SA in
CaCo-2 cells (at maximum concentration, SA killed about 95%
cells) and estimated it as 5-7 mM; in 3215 LS, 24-h IC50 for SA was
1.14 mM. Therefore, the introduction of SA into an ionic liquid did
not impede its cytotoxic action, but significantly increased its solu-
bility. For [EMIM-OSal][Cl], solubility in water was 267 g/L (r.t.),
as compared to 2.20 g/L (r.t.)35 for pure salicylic acid. All SA-ILs
were readily dissolved in water in the concentration range used for
the testing (see Table S1 in the Supporting information).
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promising candidates for future studies. The obtained results con-
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ASSOCIATED CONTENT
Supporting Information: materials, experimental procedures, details on
X-ray, MS, and NMR studies.
9
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eco(cyto)activity as complicated, but unavoidable parameter for task-
specific optimization. ChemSusChem 2014, 7, 336–360.
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AUTHOR INFORMATION
Corresponding Author
(15) Williams, H. D.; Sahbaz, Y.; Ford, L.; Nguyen, T. H.; Scammells, P. J.;
Porter, C. J. Ionic liquids provide unique opportunities for oral drug
delivery: structure optimization and in vivo evidence of utility. Chem.
Commun. (Cambridge, U. K.) 2014, 50, 1688–1690.
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pharmaceutical applications. Annu. Rev. Chem. Biomol. Eng. 2014, 5, 527–
546.
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ingredients in topical drug delivery systems. Int. J. Pharm. (Amsterdam,
Neth.) 2013, 441, 620–627.
E-mail: egorova-ks@ioc.ac.ru, val@ioc.ac.ru.
Author Contributions
The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.
Funding Sources
This work was supported by the Russian Foundation for Basic Re-
search (project 14-03-31478) and by the Grant of the President of the
Russian Federation (project MK-5053.2015.3). Mechanistic studies
were supported by the Russian Science Foundation (RSF grant 14-50-
00126).
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Muller-Buschbaum, K.; Sakalis, A.; Luhmann, T.; Berghausen, J.;
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ACKNOWLEDGMENT
The authors thank E. Kopantsev (M.M. Shemyakin and Yu.A. Ovchin-
nikov Institute of Bioorganic Chemistry RAS) for the 3215 LS cell
culture.
ABBREVIATIONS
IL, ionic liquid; API-IL, active pharmaceutical ingredient ionic liquid;
SA-IL, salicylic acid-containing ionic liquid; IC50, half maximal inhibi-
tory concentration.
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Lay Summary: Three different drug development platforms involving ionic binding of the drug components, covalent binding of
the drug components and dual ionic/covalent binding were explored in model imidazolium ionic liquids. Tunable nature of ionic
liquids and readily accessible synthetic procedures provide excellent opportunities for diverse applications.
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