Enhancement of water solubility of poorly water-soluble drugs by new biocompatible N-acetyl amino acid N-alkyl cholinium-based ionic liquids

Article abstract of DOI:10.1016/j.ejpb.2019.03.004

The major challenge of the pharmaceutical industry is to find potential solvents for poorly water-soluble drug molecules. Ionic liquids (ILs) have attracted this industry as (co-) solvents due to their unique physicochemical and biological properties. Herein, a straightforward approach for the enhancement of the water solubility of paracetamol and sodium diclofenac is presented, using new biocompatible N-acetyl amino acid N-alkyl cholinium-based ionic liquids as co-solvents (0.2–1 mol%). These new ionic liquids were able to increase the water solubility of these drugs up to four times that in pure water or in an inorganic salt solution. In the presence of these ILs, the drugs lipophilicity (log P was not significantly changed for paracetamol, but for sodium diclofenac it was possible to decrease significantly its lipophilicity. Concerning cytotoxicity in human dermal fibroblasts it was observed that ILs did not show a significant toxicity, and were able to improve cell viability compared with the respective precursors.

Full text of DOI:10.1016/j.ejpb.2019.03.004

Accepted Manuscript
Enhancement of water solubility of poorly water-soluble drugs by new biocom-
patible N-acetyl amino acid N-alkyl cholinium-based ionic liquids
Ana R. Jesus, Mário R.C. Soromenho, Luís R. Raposo, José M.S.S. Esperança,
Pedro V. Baptista, Alexandra R. Fernandes, Patrícia M. Reis
PII:
S0939-6411(18)31488-7
https://doi.org/10.1016/j.ejpb.2019.03.004
EJPB 13002
DOI:
Reference:
European Journal of Pharmaceutics and Biophar-
maceutics
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 November 2018
4 February 2019
1 March 2019
Please cite this article as: A.R. Jesus, M.R.C. Soromenho, L.R. Raposo, J. M.S.S. Esperança, P.V. Baptista, A.R.
Fernandes, P.M. Reis, Enhancement of water solubility of poorly water-soluble drugs by new biocompatible N-
acetyl amino acid N-alkyl cholinium-based ionic liquids, European Journal of Pharmaceutics and
Biopharmaceutics (2019), doi: https://doi.org/10.1016/j.ejpb.2019.03.004
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Enhancement of water solubility of poorly water-soluble drugs
by new biocompatible N-acetyl amino acid N-alkyl cholinium-
based ionic liquids
Ana R. Jesus,[a] Mário R. C. Soromenho,[a] Luís R. Raposo,[b] José M. S. S.
Esperança,[a] Pedro V. Baptista,[b] Alexandra R. Fernandes,[b] and Patrícia M. Reis*[a]
a LAQV, Requimte, Departamento de Química, Faculdade de Ciências e Tecnologia,
Universidade Nova de Lisboa, Caparica, 2829-516, Portugal, email: preis@fct.unl.pt
b
UCIBIO, Life Sciences Department, Faculdade Ciências e Tecnologia, Universidade
Nova de Lisboa, Caparica, 2829-516, Portugal
Abstract: The major challenge of the pharmaceutical industry is to find potential solvents for poorly water-soluble drug
molecules. Ionic liquids (ILs) have attracted this industry as (co-) solvents due to their unique physicochemical and
biological properties. Herein, a straightforward approach for the enhancement of water solubility of paracetamol and
sodium diclofenac is presented, using new biocompatible N-acetyl amino acid N-alkyl cholinium-based ionic liquids as
co-solvents (0.2 - 1 mol%). These new ionic liquids were able to increase water solubility of these drugs up to four times
higher than in pure water or in an inorganic salt solution. In the presence of these ILs the drugs lipophilicity (log Kow)
was not significantly changed for paracetamol, but for sodium diclofenac it was possible to decrease significantly its
lipophilicity. Concerning cytotoxicity in human dermal fibroblasts it was observed that ILs did not show a significant
toxicity, and were able to improve cell viability compared with the respective precursors.
Keywords: Amino acids • Cytotoxicity • Ionic Liquids • Drug Delivery • Bioavailability
Introduction
In pharmaceutical industry, the introduction of new drugs in the market is a very challenging goal, mainly due to
the fact that most drugs are poorly water-soluble and therefore present low gastro-intestinal absorption1. The
methods reported in literature2,3 to enhance the solubility of such drugs include a) pH modification and salt
formation,4 b) co-solvency and surfactant solubilization,2,5 c) amorphous forms, solid dispersions and cocrystals
formation,6 d) polymeric micelles formation,7 e) inclusion complexation,8 f) size reduction and nanonization,9,10 g)
solid lipid nanoparticles formation,11 h) encapsulation in liposomes and proliposomes,12 and i) microemulsions
and self-emulsifying drug delivery systems.13,14
The solubility enhancement process of hydrophobic drugs plays a key role in the formulation development to
achieve the bioavailability and therapeutic action of the drug at the target site. Poorly water-soluble drugs lead to
poor drug absorption and poor bioavailability via oral drug administration, due to their high hydrophobicity. The
common polar organic solvents excipients that are used to enhance the poor solubility of drugs are pyridine,
dimethylformamide (DMF) and dimethyl sulfoxide (DMSO).14,15 However, they are volatile, flammable, and toxic
solvents, hence not ideal as pharmaceutical ingredients.
Ionic liquids (ILs), i.e. ionic compounds with melting point below 100 ºC, have shown interesting properties that
can be controlled by the tunability of both cation and/or anion, with a multidisciplinary application in chemical,
biological, engineering, environmental and material sciences.16
The application of these new materials in pharmaceutical industry and medicine are emerging, as well as their
biological application as active pharmaceutical ingredients.17,18 The ability of ionic liquids to dissolve poorly
water-soluble drugs has gained more attention from researchers as a new tool for drug delivery.1,19,20 ILs can be
tailored to promote the dissolution of a specific drug or a class of drugs (e.g. poorly soluble oral drugs),
potentially increasing their concentration for delivery and enhancing drug permeation to boost therapeutic
efficacy.
Ionic liquids have showed potential to be used as co-solvents to solve similar problems.19,21-23 They possess dual
capability of dissolving both polar and nonpolar species and the most useful feature is that they do not evaporate
even at high temperatures. The use of ILs can markedly improve the pharmacokinetic and pharmacodynamics
properties of drugs.24 Many drug molecules show poor permeability, thus an excipient, such as lipid-based
formulations, may be added externally to enhance permeation transiently by facilitating drug permeability through

the intestinal epithelium or by stimulating chylomicron secretin, which results in enhanced drug uptake through
lymphatic system, bypassing the initial metabolism in the liver.25
The major challenge is the development of non-toxic and more biodegradable ILs with high dissolution
capability.1 Cholinium-based ILs are some of the most biodegradable, less toxic, easily accessible and
cost-effective ILs, when compared with other ILs such as pyridinium and imidazolium-based ILs.26,27
Cholinium amino acid based ionic liquids have sparked recent interest throughout the scientific
community due to their allegedly low cost and natural origin. They are prepared from non-toxic
renewable feedstocks, endowed with solubilizing power, sometimes selective and always really eco-
friendly.28
Amino acids have been reported as biocompatible anions for ionic liquids with multiple applications.29-33
The thermal stability of acetyl amino acids is greater than the corresponding amino acids.34 For this
reason our work was focused in the synthesis of new and non-toxic N-acetyl amino acid N-alkyl
cholinium-based ionic liquids, and in the improvement of water solubility of oral drugs, as paracetamol
and sodium diclofenac using these ILs as co-solvents.
In this study, we have prepared 20 new biocompatible ionic liquids based on the N-alkyl cholinium cation (Fig.
1A) and N-acetyl amino acids anions (Fig. 1B), aiming the improvement on the water solubility of two drugs,
paracetamol (Fig. 2A) and sodium diclofenac (Fig. 2B).
Fig. 1 (A) N-Alkyl cholinium-based cations, (B) Racemic N-Acetyl amino acid-based anions
Fig. 2 Chemical structure (A) Paracetamol, (B) Sodium Diclofenac
Results and Discussion
Solubility assays
Several studies to improve solubility of poorly water-soluble drugs in ionic liquids and/or deep eutectic
solvents have already been reported.21,35 However, these studies focus only on the determination of the
maximum solubility of drugs in pure ionic liquids/deep eutectic solvents. Herein, we propose the
improvement of water solubility of two commercial drugs using a small percentage (0.1-1 mol%) of N-
acetyl amino acid N-alkyl cholinium-based ionic liquids as co-solvents.
In this study, we have determined the solubility of paracetamol and sodium diclofenac in pure water at
25 ºC. Our results were 15.0 mg/mL and 20.4 mg/mL, which are very similar to those reported in
literature, 14.9 mg/mL 36 and 19.4 mg/mL, 37 respectively.
To mimic the body temperature, we have measured the solubility of these drugs in water at 37 ºC. At this
temperature the maximum solubility of paracetamol and sodium diclofenac were 18.0 mg/mL and 34.5
mg/mL, respectively. Then, the same approach for the determination of maximum solubility of both drugs
in the presence of 0.2-1 mol% of N-acetyl amino acid N-alkyl cholinium-based ionic liquids was used and
the results are presented accordingly.
Regarding the results obtained for paracetamol (Fig. 3), all the ionic liquids promoted a higher solubility
of the drug (29- 65 mg/mL) when compared to pure water or using an aqueous solution of an inorganic

salt (e.g. KBr). For instance, with only 1 mol% of ionic liquid, such as [C5Ch][AcAla], [C4Ch][AcPhe] or
[C5Ch][AcPhe], it was possible to achieve total paracetamol solubilization of about 55-65 mg/mL, which
represents a 3-fold improvement on the drug solubility in comparison to pure water.
Analysing the effect of the alkyl chain in the cation moiety in the solubility of paracetamol (Fig. 3) it was
possible to observe that ILs containing an acetyl phenylalanine-based anion contributed in general to the
highest values of solubility, independently of the cation moiety. Likewise, ILs containing a C5-cholinium
cation, for any type of anion also contributed to a significant improvement in solubility of paracetamol.
To understand the influence of IL concentration in the solubility of paracetamol, the ionic liquids, which
have shown better results, namely [C5Ch][AcAla], [C5Ch][AcPhe], and [C4Ch][AcPhe], were also used
at a smaller IL concentration (0.5 mol%). Figure 4 shows that, even with half amount of IL, it is possible
to achieve more than double the water solubility of paracetamol in pure water (18.0 mg/mL) or in the
presence of the same % of an inorganic salt (18.5 mg/mL).
Sodium diclofenac is more soluble in pure water, at 37 ºC, than paracetamol (34.5 mg/mL vs 18.0
mg/mL) and it was decided to start at a lower IL concentration (0.2 mol%).
The results obtained for this drug are presented in Fig. 5. The ILs affording the best solubility values (92-
100 mg/mL) at only 0.2 mol%, were [C4Ch]Br, [C4Ch][AcGly], [C5Ch]Br, [C3Ch][AcPhe], [C5Ch][AcGly],
[C5Ch][AcMet], and [C5Ch][AcPhe], representing a 3-fold improvement on sodium diclofenac solubility in
comparison to pure water.
70
60
50
40
30
20
10
0
[Br]
[AcGly]
[AcAla]
[AcPhe]
[AcCys]
[AcMet]
[C2Ch]
[C3Ch]
[C4Ch]
[C5Ch]
Fig. 3 Solubility of paracetamol (horizontal line corresponds to the water solubility)
70
60
50
40
0.5%
30
1.0%
20
10
0
KBr
[C5Ch][AcAla] [C5Ch][AcPhe] [C4Ch][AcPhe]
Fig. 4. Solubility of paracetamol at 0.5 mol% vs 1 mol% IL (horizontal line corresponds to the water solubility)
Analysing the effect of the alkyl chain in the cation moiety in the solubility of sodium diclofenac, (Fig. 5) it
was possible to conclude that, C5Ch and C4Ch-based ILs were the best co-solvents for this drug.
Moreover, in general, ILs with an [AcMet] anion promoted higher solubilization of sodium diclofenac with
any type of N-alkyl cholinium cation.
The relevance of IL amount in the solubility of the sodium diclofenac was also evaluated for the best
candidates, and therefore the IL amount was reduced to half, 0.1%. The selected ionic liquids were
[C4Ch]Br, [C5Ch]Br and [C4Ch][AcGly]. Again, Fig. 6 shows that reducing the amount of IL to half
resulted in the decrease of solubility values, yet still up 2.5 times higher (68 – 75 mg/mL) that with pure
water (34.5 mg/mL) or in the presence of the same amount of an inorganic salt (26.9 mg/mL).
Both paracetamol and sodium diclofenac are taken at 6-8 h intervals, hence we have studied their
solubility in selected ionic liquids aqueous solutions after 6h. After the 6h period it was observed that the
solubility was practically the same than after 24 h. (see graphics in ESI)
Octanol-water partition coefficient and log P
In the context of pharmacokinetics the partition coefficient (log P) can be useful to estimate the
distribution profile of a drug within the body, which has a great influence in its ADME profile. It also helps

scientist to determine the most likely way of administration. In the case of an orally absorbed drug it has
to pass first through lipid bilayers in the intestinal epithelium. Therefore, it is necessary a certain
lipophilicity to partition into that bilayer but also require some hydrophilicity so that will not partition out
again.38
According to literature, paracetamol has a log P value ranging from 0.30 to 0.56,39 which means that it is
primarily found in the blood serum; on the other hand, sodium diclofenac has a log P higher than 4,
meaning that is distributed to hydrophobic areas such as lipidic bilayers of cells.
In this study, we have showed that N-acetyl amino acids N-alkyl cholinium-based ionic liquids were able
to improve water solubility of paracetamol and sodium diclofenac. However, a question remained,
whether is it possible to improve pharmacokinetic parameters of these drugs to meliorate their ADME
profile. To answer this question, we have determined the log P value of these drugs using ionic liquids
as co-solvents.
120
100
[Br]
80
60
40
20
0
[AcGly]
[AcAla]
[AcPhe]
[AcCys]
[AcMet]
[C2Ch]
[C3Ch]
[C4Ch]
[C5Ch]
Fig. 5 Solubility of sodium diclofenac (horizontal line corresponds to water solubility)
120
100
80
0.1%
0.2%
60
40
20
0
KBr
[C4Ch][Br]
[C5Ch][Br]
[C4Ch][AcGly]
Fig. 6 Solubility of sodium diclofenac at 0.2% vs 0.1% IL (horizontal line corresponds to water solubility)
We have selected ionic liquids that promoted the maximum solubility for each drug and the
corresponding halogenated precursor, [C5Ch][AcAla], [C5Ch][AcPhe] and [C5Ch]Br.
Initially, it was necessary to prepare a calibration curve for each drug in octanol-saturated water, by UV-
Vis spectrophotometry. The absorbance of a range of known concentrations of each drug was measure,
maintaining absorbance values below 1.
Then, an amount within the solubility range of each drug was mixed with equal amounts of mutually
saturated water and octanol. After mixing and separation of layers, the absorbance of water-rich phase
was measured, and concentration of drug was calculated based on the calibration curve.
Log P values for each drug in aqueous solution, and in the presence of the corresponding percentage of
ionic liquid was determined using equations 1 and 2, and results are presented in Table 1. However, log
P of sodium diclofenac was not determined due to the fact that the separation of octanol-water phases
was not possible.42

Table 1. Log P values for paracetamol and sodium
diclofenac
System/Media
Log P
0.423
-
Paracetamol/water
Sodium diclofenac/water

0.015
Paracetamol/ water + 1 mol%
[C5Ch]Br
0.034
0.093


0.006
0.017
Paracetamol/water + 1 mol%
[C5Ch][AcAla]
Paracetamol/water + 1 mol%
[C5Ch][AcPhe]
- 0.051

0.120
Sodium diclofenac/water + 0.2
mol% [C5Ch]Br
1.81
1.93
1.93



0.16
Sodium diclofenac/water + 0.2
mol% [C5Ch][AcAla]
0.20
0.01
Sodium diclofenac/water + 0.2
mol% [C5Ch][AcPhe]
The results obtained for paracetamol indicate that its log P decreases slightly in the presence of a 1
mol% ionic liquid solution. This indicates that in ILs aqueous solutions paracetamol becomes even more
hydrophilic, and therefore with a higher affinity for the aqueous phase. As mentioned previously there
must be a hydrophilicity/lipophilicity balance to achieve an ideal oral drug. This methodology, however
did not afford the desired results for paracetamol as it only meliorated its solubility (although paracetamol
is already considered a soluble drug) but did not improved its distribution coefficient.
However, the most outstanding results were obtained with sodium diclofenac with log P values below 2.
It is important to highlight that in ionic liquid solutions we did not encounter the same problems described
for the determination of log P of sodium diclofenac in water.
In literature there is not much information regarding the log P of sodium diclofenac but the log P of the
active compound diclofenac (acidic form) is higher than 4.43 In this study, ionic liquids not only
significantly improve the solubility of this drug but also meliorate its log P value down to 1.91-1.93.
According to literature the optimum log P values are between 0 and 3.44
Cytotoxicity evaluation
We have chosen only few ionic liquids to study their cytotoxicity vs the cytotoxicity of their halogen
precursors. Therefore, [C3Ch][AcGly], [C4Ch][AcPhe], [C5Ch][AcAla], and [C5Ch][AcPhe], which
afforded the best results in terms of solubility, were selected as well as their precursors [C3Ch]Br,
[C4Ch]Br, and [C5Ch]Br.
The results presented in Fig. 7 B), D), F) and G) and in table 2 show that the ILs displayed IC50 values
ranging from 19.3 to 122.7 mM. Also, these ILs showed less toxicity compared to the respective
precursors (IC50 ranging from 17.5 mM to 75.1 mM) as observed in Fig. 7 A), C) and E) and Table 2.

Fig. 7 Cytotoxicity of the precursors and respective derived ionic liquids (A) [C3Ch]Br, (B) [C3Ch][AcGly], (C) [C4Ch]Br, (D)
[C4Ch][AcPhe], (E) [C5Ch]Br, (F) [C5Ch][AcAla], and (G) [C5Ch][AcPhe] in normal human dermal fibroblasts after 48 h
exposure to different concentrations of the respective ILs. The results are expressed as the average ± SD from three
independent biological assays. The symbol * indicates that the viabilities are statistically significant with p < 0.01 when
compared to the control.
As the N-alkyl chain of the cation moiety increases there is a decrease in the IC50 value, indicating a
higher lipophilicity, and therefore a higher ability to permeate the cell membrane. Moreover, for the same
cation (C5Ch) a phenylalanine anion also induces a decrease in the IC50 value for being a more
hydrophobic anion than alanine itself.
Interestingly, these IC50 values for the tested ILs are similar or higher than those previously described in
the literature for paracetamol45 reinforcing their low cytotoxicity in normal human dermal fibroblasts.
Table 2. Relative IC50 of the precursors and respective
derived ionic liquids [C3Ch]Br, [C3Ch][AcGly],
[C4Ch]Br, [C4Ch][AcPhe], [C5Ch]Br, [C5Ch][AcAla],
[C5Ch][AcPhe] after 48 h exposure in normal human
dermal fibroblasts.
Compound
[C3Ch]Br
IC50 (mM)
75.1
[C3Ch][AcGly]
[C4Ch]Br
122.7
33.3
[C4Ch][AcPhe]
[C5Ch]Br
46.4
17.5
[C5Ch][AcAla]
[C5Ch][AcPhe]
28.4
19.3

Conclusions
This work proved that a small percentage of N-acetyl amino acid N-alkyl cholinium-based ionic liquids
can improve solubility of paracetamol and sodium diclofenac by 2- to 4-fold as compared to their
solubility in water or in the presence of the same percentage of an inorganic salt. More specifically,
sodium diclofenac which is an oral drug that belongs to BCS class II (poor aqueous solubility), is
converted in a class I drug by the addition of small quantities of these new ILs, that lower the lipophilicity
of the drug, mimicking a high permeability and high solubility compound.
In addition, it was possible to observe that the cytotoxicity of the ILs in human dermal fibroblasts
decreased in comparison with the corresponding precursors.
Thus, these new biocompatible ionic liquids are promising vehicles for aqueous administration of poorly
water-soluble drugs.
Experimental Section
Detailed analysis data of all the synthesized N-acetyl amino acids and ionic liquids are given in the ESI.
Materials and methods
Amino acid phenyl-L-alanine (99%), N-acetyl-L-glycine (99%), N-acetyl-DL-methionine (99%), N-acetyl-L-cysteine (≥ 99.5%),
N,N-Dimethyl ethanolamine (≥ 99.5 %), and acetic anhydride (≥ 98%) were purchased from Aldrich and used as received.
Glacial acetic acid (100%) was purchased from Merck. Amino acid DL-alanine (99%), bromoethane (98%), 1-bromopropane
(99%), 1-bromobutane (≥ 98%) and 1-bromopentane (99%) were purchased from Alfa Aesar and used as received.
General procedure for the synthesis of N-acetyl amino acids. Amino acid (10 g) was mixed with glacial acetic acid (50
mL). Acetic anhydride (1.1 equiv.) was added to the reaction vessel and the mixture was kept at 70 ºC for 10-30 min,
until total dissolution of the initial product. Acetic acid was then removed under vacuum and the N-acetyl amino acid
was recrystallized from ethyl acetate. The final compound was obtained in 95-99% yield and dried in high vacuum
overnight and high purity was confirmed by NMR experiments.
General Procedure for the synthesis of [CnCh]Br. The synthesis was carried out as reported elsewhere.46 In general,
N,N-Dimethyl ethanolamine (0.2 mmol) was mixed with the alkylating agent, [CnH2n+2]Br (2.0 equiv.), and n-hexane (50
mL) in a 100 mL pressure reaction vessel. The mixture was kept at 80-90 ºC for 24 h for shorter alkyl chains (n=2,3)
and 48 h for longer alkyl chains (n=4,5), and then allowing it to cool down to room temperature. The precipitated
bromide salts were thoroughly washed with EtOAc and/or acetone to remove unreacted amine and/or alkylating agent.
Ionic liquids were then dried in the vacuum for 1-2 days, and their purity confirmed by NMR. The bromide salts were
obtained in 95-99% yield.
General procedure for the metathesis reaction. The synthesis was carried out as reported elsewhere.47 An
aqueous solution of N-alkyl choline bromide (10 mmol) was passed slowly through an anion exchange column
AmberliteTM IRN-78. Then, the corresponding hydroxide solution was slowly added to an aqueous solution of N-acetyl
amino acid (1.0 equiv.). The reaction mixture was stirred at room temperature for 2-3 h prior to water removal by
evaporation. Ionic liquids were obtained in quantitative yield and dried in high vacuum for 3-4 days at 50 - 60 ºC prior
to the coulometric assays to guarantee minimum water content. Coulometric Karl-Fischer titrations yielded final water
contents below 1000-1500 ppm depending on the IL. Moreover, AgNO3 test was used to confirm the absence of
halogen presence in the final IL.
Solubility assays. A calibration curve was prepared for each drug, in water, at their maximum absorbance
wavelength, 243 nm for paracetamol and 277 nm for sodium diclofenac, at 25 ºC. Absorbance values were kept below
1.
General procedure for the determination of solubility limit of Paracetamol. Aqueous solutions (5 mL) of ionic
liquids (1 mol%) were prepared and paracetamol (325 mg) was added to each sample (at this concentration we are
above the solubility limit for all the samples). The solution was stirred for 24 h at 37ºC until equilibrium was reached.
Keeping the solutions in a 37 ºC bath, the non-dissolved drugs were filtered off through a 0.25
m syringe filter, and 10

L of each filtered solution was diluted to 50 mL of MilliQ® water. Triplicates were prepared for each sample and
maximum absorbance was measured at 243 nm. Solubility of paracetamol in the presence of each ionic liquid was
calculated based on the calibration curve.
General procedure for the determination of solubility limit of Sodium Diclofenac. Aqueous solutions (5 mL) of
ionic liquids (0.2 mol%) were prepared and sodium diclofenac (500 mg) was added to each sample (at this
concentration we are above the solubility limit for all the samples). The solution was stirred for 24 h at 37ºC until
equilibrium was reached. Keeping the solutions in a 37 ºC, the non-dissolved drugs were filtered off through a 0.25

m syringe filter, and 10 L of each filtered solution was diluted to 50 mL of MilliQ® water. Triplicates were prepared

for each sample and maximum absorbance was measured at 277 nm. The solubility of sodium diclofenac in the
presence of each ionic liquid was calculated based on the calibration curve.
General procedure for the determination of partition coefficient and log P. The Kow and log P values of
paracetamol and sodium diclofenac in the presence of ionic liquids were determined using the shake-flask method.
Water and octanol were both saturated prior to the experiments. Saturation was achieved by mixing equals amounts of
water and octanol and stirring for 3 days at room temperature. Then, the mixture was transferred to a separation
funnel, and layers were allowed to completely separate for 2 days. The octanol-saturated aqueous layer was used to
prepare the ionic liquid solutions at 0.2 or 1 mol%, prior to the assays. The experimental setup consisted of 15 mL
glass vials equipped with a magnetic stirrer, in which random but known amounts of drug were dissolved in 1 g of IL
solution and mixed with 1 g of water-saturated octanol. The solutions stirred vigorously for 18-24 h and then sat for
some hours until full separation of organic and aqueous layers. Three independent experiments were performed.
Octanol and water layers were separated and centrifuged for 30 minutes at 2000 rpm. The drug concentration in the
water-rich phase was analysed in a UV-Vis VWR® spectrophotometer, model UV-6300PC, using a previously
prepared calibration curve. Samples were diluted until the absorbance was smaller than 1.
The concentration of drug in the octanol-rich phase was directly calculated by subtracting the amount in the water-rich
phase to the initial amount dissolved.
Kow and log P of the three samples were determined using equation 1 and 2, and the mean value was taken.
Kow = [drug]oct/[drug]aq
log P = log Kow
(1)
(2)
Cytotoxicity evaluation
Cell culture. Normal primary dermal fibroblasts, ATCC® PCS-201-010, (ATCC, Manassas, VA, USA), were cultivated
in Dulbecco’s modified Eagle’s medium (DMEM) (Thermofisher Scientific, Waltham, Ma, EUA) supplemented with 10%
(v/v) Fetal Bovine Serum, FBS, (Thermofisher Scientific) 100 U/mL penicillin and 100 mg/mL streptomycin
(Thermofisher Scientific) at 37 ºC in an atmosphere with 5% (v/v) CO2 and 99% (v/v) relative humidity.
Viability assays. The cellular viability was evaluated as previously described.48 Fibroblast cells were incubated in 96-
well plates (SPL Life Sciences, Co., Ltd. Naechon-myeon, South Korea) at a concentration of 7500 cells per well, for
48 h with different concentrations of ILs [C5Ch][AcAla], [C3Ch][AcGly], [C4Ch][AcPhe], [C5Ch][AcPhe], or the
precursors [C5Ch]Br, [C4Ch]Br and [C3Ch]Br, ranging from 5 to 500 mM. Cell viability was evaluated using the
CellTiter 96® AQueous One Solution Cell Proliferation Assay System (Promega, Fitchburg, Wi, USA) according to the
manufacturer’s instructions. Viable cells produce formazan by reduction of the 3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS). The formazan product was quantified by
measuring the absorbance at 490 nm in an Infinite M200 microplate reader (Tecan, Mannedorf, Switzerland).
GraphPad Prism v6.01 (GraphPad Software Inc, La Jolla, California) was used to perform statistical analysis. Data is
expressed as average ± standard deviation from at least 3 independent experiments.
Acknowledgements
The authors thank Fundação para a Ciência e Tecnologia, FCT/ MCTES (Portugal) for financial support
through Investigator contracts (IF/00355/2012 – J.M.S.S. Esperança and IF/00621/2015 – P.M. Reis),
and through project IF/00621/2015.
This work was also partially supported by the Associated Laboratory Research Unit for Green Chemistry,
Technologies and Clean Processes, LAQV/Requimte (financed by national funds from FCT/MCTES,
UID/QUI/50006/2019 and co-financed by the ERDF under the PT2020, POCI-01-0145-FEDER-007265)
and by the Unidade de Ciências Biomoleculares Aplicadas - UCIBIO (financed by national funds from
FCT/MCTES, UID/Multi/04378/2019 and co-financed by the ERDF under the PT2020 Partnership
Agreement, POCI-01-0145-FEDER-007728).
The NMR spectrometers are part of The National NMR Facility, supported by FCT/MCTES (RECI/BBB-
BQB/0230/2012).
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Graphical abstract
O
O
OH
nN
R
NHAc
[AcAA]
[CnCh]
Drug in water + IL
Paracetamol: up to 3.2-fold higher
Sodium Diclofenac: up to 3.2-fold higher
Drug in water
Paracetamol: 18 mg/mL
Sodium Diclofenac: 34.5 mg/mL
* enhanced solubility
* log P improvement
* small % of IL
* poor water solubility
* non-toxic ILs