Synthesis and antibacterial activity of ionic liquids and organic salts based on penicillin g and amoxicillin hydrolysate derivatives against resistant bacteria

Article abstract of DOI:10.3390/pharmaceutics12030221

The preparation and characterization of ionic liquids and organic salts (OSILs) that contain anionic penicillin G [secoPen] and amoxicillin [seco-Amx] hydrolysate derivatives and their in vitro antibacterial activity against sensitive and resistant Escherichia coli and Staphylococcus aureus strains is reported. Eleven hydrolyzed β-lactam-OSILs were obtained after precipitation in moderate-to-high yields via the neutralization of the basic ammonia buffer of antibiotics with different cation hydroxide salts. The obtained minimum inhibitory concentration (MIC) data of the prepared compounds showed a relative decrease of the inhibitory concentrations (RDIC) in the order of 100 in the case of [C2OHMIM][seco-Pen] against sensitive S. aureus ATCC25923 and, most strikingly, higher than 1000 with [C16Pyr][seco-Amx] against methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300. These outstanding in vitro results showcase that a straightforward transformation of standard antibiotics into hydrolyzed organic salts can dramatically change the pharmaceutical activity of a drug, including giving rise to potent formulations of antibiotics against deadly bacteria strains.

Full text of DOI:10.3390/pharmaceutics12030221

Article
Synthesis and Antibacterial Activity of Ionic Liquids
and Organic Salts Based on Penicillin G and
Amoxicillin hydrolysate Derivatives
against Resistant Bacteria
1,2, ,†
3,†
1,2
1
3
Ricardo Ferraz * , Dário Silva , Ana Rita Dias , Vitorino Dias , Miguel M. Santos ,
3
1,4
3
Luís Pinheiro , Cristina Prudêncio , João Paulo Noronha ,
3,
3,
Željko Petrovski * and Luís C. Branco *
1
Ciências Químicas e das Biomoléculas (CQB) e Centro de Investigação em Saúde e Ambiente (CISA),
Escola Superior de Saúde do Instituto Politécnico do Porto, 4400‐330 Porto, Portugal;
anaritadias3@gmail.com (A.R.D.); solchemar@gmail.com (V.D.); cps@estsp.ipp.pt (C.P.)
LAQV‐REQUIMTE, Departamento de Química e Bioquímica, Faculdade de Ciências, Universidade do
2
Porto, Rua do Campo Alegre 687, 4169‐007 Porto, Portugal
LAQV‐REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia da Universidade Nova
3
de Lisboa, 2829‐516 Caparica, Portugal; dmv.silva@campus.fct.unl.pt (D.S.);
miguelmsantos@fct.unl.pt (M.M.S.); l.pinheiro@campus.fct.unl.pt (L.P.); jpnoronha@fct.unl.pt (J.P.N.)
i3S, Instituto de Inovação e Investigação em Saúde, Universidade do Porto, 4099‐002 Porto, Portugal
4
* Correspondence: ricardoferraz@eu.ipp.pt (R.F.); z.petrovski@fct.unl.pt (Z.P.); l.branco@fct.unl.pt (L.C.B.)
† These two authors contribute equally to this work.
Received: 31 December 2019; Accepted: 19 February 2020; Published: 2 March 2020
Abstract: The preparation and characterization of ionic liquids and organic salts (OSILs) that contain
anionic penicillin G [secoPen] and amoxicillin [seco‐Amx] hydrolysate derivatives and their in vitro
antibacterial activity against sensitive and resistant Escherichia coli and Staphylococcus aureus strains
is reported. Eleven hydrolyzed β‐lactam‐OSILs were obtained after precipitation in moderate‐to‐
high yields via the neutralization of the basic ammonia buffer of antibiotics with different cation
hydroxide salts. The obtained minimum inhibitory concentration (MIC) data of the prepared
compounds showed a relative decrease of the inhibitory concentrations (RDIC) in the order of 100
in the case of [C OHMIM][seco‐Pen] against sensitive S. aureus ATCC25923 and, most strikingly,
2
higher than 1000 with [C16Pyr][seco‐Amx] against methicillin‐resistant Staphylococcus aureus
(MRSA) ATCC 43300. These outstanding in vitro results showcase that a straightforward
transformation of standard antibiotics into hydrolyzed organic salts can dramatically change the
pharmaceutical activity of a drug, including giving rise to potent formulations of antibiotics against
deadly bacteria strains.
Keywords: active pharmaceutical ingredients‐ionic liquids and organic salts (API‐OSILs); penicillin
G and amoxicillin hydrolysate derivatives; sensitive bacteria; resistant bacteria
1. Introduction
Bacterial resistance to antibiotics has been increasing in Europe over the last few years [1–3].
New classes of antibiotics have not been introduced recently [4–7], and, thus, more resistances to old
drugs are developing daily [8–10]. Recent efforts and huge investments being made in this field by
big pharma companies such as GlaxoSmithKline, Merck, Pfizer and Wyeth [3–5,11,12] have had
disappointing returns from their R&D departments, including clinical trials. This is a significant
Pharmaceutics 2020, 12, 221; doi:10.3390/pharmaceutics12030221
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factor to allocate anti‐infective R&D resources into other fields of investigation and thus remain
highly competitive [3–5,13]. Considering the disappointing results on genomics and the exodus of
big pharma, the problem of bacteria resistance has continued to evolve, reaching alarming
dimensions [3,8,10].
For the last 12 years, organic salts and ionic liquids (OSILs) from active pharmaceutical
ingredients (APIs), or simply API‐OSILs [14–22], have been studied at the academic level
[14,18,19,23–25]. Ionic liquids (ILs) are salts with melting points below 100 °C (some of them are liquid
at room temperature) that result from the pairing of organic cations with organic and inorganic
anions [14,20,23]. When the melting point is above 100 °C, these compounds are simply designated
by organic salts [24]. Nowadays, there is a significant increase in the scope of both the physical and
chemical properties of OSILs [19,26–29], and, thus, their application in several topics of science and
technology is currently being studied [15–18,20,23,26].
In the case of API‐OSILs, it is known that the interaction between an ionic API with selected
counter‐ions may significantly improve the pharmaceutical activity of the former [17,30–32]. In
addition, this combination may also boost the stability and solubility of the API in physiological
media, as well as enhance the bioavailability and modify the pharmacokinetics and delivery mode of
the drug [21,27–29,33–35]. Consequently, the biopharmaceutics drug classification system (BCS) for
API‐ILs can be significantly modified in comparison with the parent drugs [24], meaning that this
new salt of the old API can be treated as a new chemical entity and thus be independently patented
[17,26,30,36]. Furthermore, the polymorphism of a given API can be severely mitigated or even
eliminated if it becomes liquid, hence tackling one of the most important problems in the
pharmaceutical industry that can dramatically alter a drug’s solubility and dosages [26,30,37–40]. In
fact, solid forms of drugs can suffer from several limitations such as low solubility, polymorphic
conversion, and low bioavailability [20,26,36,41].
The inherent properties of ILs could be of extreme importance to overcome such difficulties of
solid form drugs [20,36,41]. Recent works have shown that API‐OSILs possess many attractive
properties when compared to conventional drugs [14,16,20,23]. Our group recently studied the
relevant pharmacological properties of ampicillin‐ and primaquine‐based API‐OSILs such as water
solubility, the octanol–water partition coefficient, the hexadecylphosphocholine (HDPC) micelle–
water partition coefficient, and critical micelle concentration [14–17,30]. In the case of ampicillin‐
based API‐OSILs, the data are clearly consistent with a greater potential of API‐ILs in comparison
with the parent API, specifically regarding their solubility in water, as well as more specific properties
such as membrane affinity and permeation. In fact, the accurate selection of the organic cation allows
for the fine‐tuning of some important physical and thermal properties like water solubility,
membrane permeation, melting point, and thermal stability [17]. In another study, we found that
primaquine API‐OSILs had a particular affinity to intercalate negatively‐charged lipid bilayers
(membrane models of Plasmodium infected erythrocyte) and also, to a lesser extent, zwitterionic lipid
bilayers (membrane models of healthy cells), in comparison with the parent drug [14,42].
A large quantity of recent communications and reviews have referred to the toxicity and activity
of ILs against microorganisms and cell cultures, especially antimicrobial activity, and as novel forms
of bioactive materials and as drug delivery systems [14,20,43–46]. Recently, OSILs have been studied
to fight multi‐drug resistance [16,47] and as agents for microbial biofilms [32,45,48−51], and they have
shown a potent, broad spectrum activity against a variety of clinically significant microbial
pathogens, including methicillin‐resistant Staphylococcus aureus (MRSA) [32,49,52]. The 2011 outbreak
of multi drug‐resistant Escherichia coli O104 in Germany as well as as Gram‐negative
Enterobacteriaceae due to presence of the New Delhi metallo‐β‐lactamase [53,54] are examples of an
increasingly documented major public health problem.
Therefore, there is an increasing demand to develop new drugs to address multi‐resistant
infections and to develop more efficient tools so that new resistances are not developed. In this
context, the results up to this point given by API‐OSILs have followed these demands [20].
Thus, following our work on ampicillin [16–18] and fluoroquinolone‐based API‐OSILs [30], we
herein describe the synthesis of ILs based on amoxicillin and penicillin G through a synthetic strategy

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that was optimized by us in the past for the preparation of ampicillin‐OSILs [18]. The synthetic
method consists on the deprotonation of an API with different hydroxides of organic cations in an
ammonium buffer (buffer‐controlled reaction procedure) [18]. In all cases, the antibiotic API is
combined as an anion with organic cations that contain imidazolium, ammonium, phosphonium and
pyridinium structures.
2. Results and Discussion
2.1. Chemistry
Ionic liquids and organic salts based on the ammonia hydrolysate anion of penicillin G and
amoxicillin (α‐amide of benzyl penicilloic acid and of amoxicillin penicilloic acid, respectively),
abbreviated here as [seco‐Pen] and [seco‐Amx], respectively, were prepared by an ammonia buffer
reaction procedure that was recently developed by us for the synthesis of ampicillin API‐OSILs [18].
Our original idea was to test a new method for preparation of API‐OSILs based on parent penicillin
and amoxicillin in the anionic form. However, due to ammonolysis (β‐lactam ring opening with the
formation of an amide group) of amoxicillin and penicillin G under the employed reaction conditions,
ionic liquids and organic salts of [seco‐Pen] and [seco‐Amx] anions were prepared instead (see Figure
1). The prefix seco (Latin verb secare) is used in antibiotics nomenclature [55], and it means to cut.
Two penicilloic acids of penicillin G and amoxicillin are already well known in the literature [56–58],
and some of their stable amides—products of the amino‐ and ammonolysis of β‐lactam ring of parent
antibiotics—have also already been described [59–61].
While amoxicillin (Amx) was used as provided (in trihydrate form), penicillin G, which was
supplied as a potassium salt ([K][Pen]), was previously converted to the corresponding ammonium
salt [NH4][Pen] by following the procedure of Brown et al. [62]. The experimental procedure
consisted on the reaction of hydroxide of the selected cations with amoxicillin and penicillin
ammonium salt. Halide (chloride, bromide) salts of selected organic cations were converted into the
corresponding hydroxides on Amberlite resin (OH form), and the highly basic solution that was
obtained was then added to the solution of the API in an aqueous ammonia buffer in order to provide
the compounds [18]. [Na][seco‐Amx] was prepared through a reaction with sodium hydroxide
(instead of organic hydroxide), while a [K][seco‐Pen] derivative was directly obtained by
ammonolysis from [K][Pen].
Hydrolyzed amoxicillin (seco‐Amx) and penicillin G (seco‐Pen) derivatives in the anionic form
were combined with the following organic cations (see Figure 1): 1‐ethyl‐3‐methylimidazolium
[EMIM], 1‐hydroxy‐ethyl‐3‐methylimidazolium [C2OHMIM], (2‐hydroxyethyl)trimethylammonium
[Choline],
tetraethylammonium
[TEA],
cetylpyridinium
[C16Pyr]
and
trihexyltetradecylphosphonium [P6,6,6,14]. These cations were selected due to their low toxicity, except
for [P6,6,6,14], which was chosen because it usually produces room temperature ionic liquids (RTILs).
Cetylpyridinium chloride is also already used in some types of mouthwashes and toothpastes [53],
although it is irritant in higher concentrations [54]. For control purposes in the biological activity
studies, we also prepared the corresponding sodium and potassium salts of the hydrolyzed
antibiotics by following the same synthetic procedure.

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Figure 1. Schematic synthetic methodology for the preparation of metallic active pharmaceutical
ingredients (API) salts and [seco‐Amx] and [seco‐Pen] ionic liquids and organic salts (OSILs).
The isolation of these compounds was performed similarly to the previously reported
ampicillin‐based API‐OSILs [18]. Briefly, the excess reactant was filtered‐off after crystallization from
acetonitrile/methanol (9:1), and this was followed by solvent evaporation. Table 1 shows the reaction
yields, physical states and melting points of the prepared compounds. From the eleven synthesized
API‐OSILs, two were organic salts and nine were ionic liquids, including four RTILs.
Table 1. Yield, physical state, and melting point of the synthesized API‐OSILs.
Compound
Yield
Physical State
Melting Point/°C
[EMIM][seco‐Amx]
[C2OHMIM][seco‐Amx] 60% Yellow solid
[P6,6,6,14][seco‐Amx]
[C16Pyr][seco‐Amx]
77% Yellow solid
84–86
109–111
‐
92% Yellow viscous liquid
94% Yellow solid
96–98

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[Choline][seco‐Amx]
[Na][seco‐Amx]
93% Yellow solid
96% Yellow solid
143–144
137–139
[EMIM][seco‐Pen]
[C2OHMIM][seco‐Pen]
[Choline][seco‐Pen]
[P6,6,6,14][seco‐Pen]
[C16Pyr][seco‐Pen]
81% Colorless viscous liquid
83% Yellow solid
95% Yellow solid
97% Yellow viscous liquid
89% Yellow solid
48–50
69–71
‐
76–78
‐
[TEA][seco‐Pen]
[K][seco‐Pen]
90% Yellow viscous liquid
97% White solid
193–195
The strict 1:1 cation–anion stoichiometry of the prepared API‐OSILs, as well as the structural
1
integrity of both components, was confirmed by H NMR spectroscopy. Further characterization was
13
performed by C NMR and FTIR spectroscopies, as well as specific rotation, elemental analysis, and
mass spectrometry. The ammonolysis of the β‐lactam rings of the original APIs was confirmed by
mass spectrometry. In the acquired electrospray ionization mass spectra (ESI‐MS) spectra of all
−
analyzed API‐OSILs in the negative mode, the base peak corresponded to [M + 17] (m/z), which was
consistent with the β‐lactam ring opening with the consequent formation of an amide group and a
secondary amine at the thiazolidine group.
2.2. Biological Activity
In the present study, all prepared compounds were tested against several sensitive and resistant
Gram‐positive and Gram‐negative bacteria strains: Staphylococcus aureus ATCC 25923, Escherichia coli
ATCC 25922, methicillin resistant Staphylococcus aureus (MRSA ATCC 43300) and E. coli CTX M2 and
CTX M9.
The minimum inhibitory concentration (MIC) values were determined from three assays in
triplicate by the broth micro dilution method in a 96‐well microtiter plate by using tryptic soy broth
(TSB) and adapted methodology from the Clinical Laboratory Standard Institute (CLSI) [62]. The
strains were grown individually on tryptic soy agar for 24 h at 37 °C prior to each antibacterial test.
Preceding MIC determination, each inoculum density was adjusted in TSB to 0.5 McFarland
standards with a photometric device [62]. This resulted in a suspension that contained approximately
8
8
−1
®
1 × 10 to 2 × 10 colony forming units (CFUmL ) for E. coli ATCC25922 [60]. A similar approach
was used for the other strains. Then, 0.5 μL of the suspension was added to each well to have 5000–
−1
25,000 CFUmL . Bacteria were exposed to API‐OSIL concentrations of 5, 2.5, 0.5, 0.05, 0.005, 0.0005,
and 0.00005 mM. All compounds were dissolved in water except for OSILs that contained [P6,6,6,14]
and [C16Pyr] cations, which were diluted in 1% dimethyl sulfoxide (DMSO). Their activity was
determined in aqueous media, and the results of their activity were compared with bacteria that had
been grown in TSB broth in the presence of 1% DMSO. The MIC for each compound was recorded as
lowest molar concentration, showing no turbidity after 24 h of incubation at 37 °C [63,64]. The
presence of turbidity was an indication of microbial growth, and the corresponding concentration of
antibacterial agent was considered ineffective. The pharmacological activity of the prepared
compound was then compared to the parent commercial API in terms of the relative decrease of
inhibitory concentration (RDIC) as described earlier [16]. Herein, the RDIC value was calculated by
dividing the MIC of the commercial API (penicillin G potassium salt or amoxicillin trihydrate) by the
MIC of the corresponding synthesized compound.
2.3. Studies on Sensitive Bacteria
Table 2 shows data from the bioactivity study of the prepared compounds on S. aureus and E.
coli sensitive strains.

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Table 2. Minimum inhibitory concentrations (mM) and relative decrease of inhibitory concentrations
(RDIC) of the new compounds that were produced on sensitive strains.
S. aureus
ATCC25923
E. coli
ATCC25922
Compound
RDIC
RDIC
[EMIM][seco‐Amx]
[C2OHMIM][seco‐Amx]
[P6,6,6,14][seco‐Amx]
[C16Pyr][seco‐Amx]
[Choline][seco‐Amx]
Na[seco‐Amx]
5.0
0.050
>5.0
>5.0
>5.0
>5.0
0.050
>5.0
0.005
>5.0
>5.0
>5.0
0.01
1
<0.01
<0.01
<0.01
<0.01
1
<0.1
100
<0.1
<0.1
<0.1
2.5
5.0
0.5
0.050
>5.0
>5.0
0.005
>5.0
>5.0
5.0
0.002
0.001
0.01
0.1
<0.001
<0.001
1
<0.1
<0.1
0.1
Amx
[EMIM][seco‐Pen]
[C2OHMIM][seco‐Pen]
[Choline][seco‐Pen]
[P6,6,6,14][seco‐Pen]
[C16Pyr][seco‐Pen]
>5.0
>5.0
<0.1
0.1
[TEA][seco‐Pen]
K[seco‐Pen]
K[Pen]
>5.0
>5.0
0.500
0.05
>5.0
2.5
<0.1
<0.1
1
0.050
>5.0
0.500
>5
10
<0.1
1
[EMIM][Br]
[C2OHMIM][Cl]
[P6,6,6,14][Cl]
[C16Pyr][Cl]
[Choline][Cl]
[TEA][Br]
‐‐‐
‐‐‐
‐‐‐
5.0
‐‐‐
‐‐‐
2.5
‐‐‐
0.5
‐‐‐‐
‐‐‐
0.5
‐‐‐‐
‐‐‐
2.5
>5.0
>5.0
2.5
‐‐‐
‐‐‐
The data gathered in Table 2 show that, on a first approach, the hydrolyzed salts of both Amx
and Pen lost all antimicrobial activity against the tested sensitive strains. In fact, this was true for the
vast majority of prepared OSILs. It has been a consistently and firmly established belief that the
antimicrobial activity of β‐lactam antibiotics relies on the sacrificial action of these functional groups
[65] on transpeptidases that are responsible for the last cross‐linking step of peptidoglycan synthesis
in the bacterial cell wall. For that purpose, the transpeptidase active site (Ser residues in the case of
D, and D‐ and Cys residues in the case of L,D‐transpeptidases) nucleophilically attacks the
nucleophilicity the carbonyl of the β‐lactam ring, resulting in its opening of the ring and the
irreversible formation of a covalent and stable acyl‐enzyme complex [66,67]. However, this premise
is widely accepted but poorly demonstrated. Recently, it was shown that even the acylation reactions
of Cys residue of L,D‐transpeptidases can be reversible, thus leading to limited antibacterial activity
[68].
The same result was obtained for the majority of the prepared salts, irrespective of the cation
polarity, resembling an ion trapping effect [69], i.e., ionic compounds such as [seco‐Amx] and [seco‐
Pen] and their conjugated acids are subject to a variety of processes, such as dissociation, electrical
interactions with organic matter, and changes in their partitioning in hydrophobic/hydrophilic
media. These processes depend on pH, ionic strength, their polarity, and pKa, and they ultimately
lead to their accumulation in certain zones of the bacterial cell, i.e., the ion‐trap effect. On the other
hand, highly polar cations are more prone to closely interact with them in anionic form, anchoring

Pharmaceutics 2020, 12, 221
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them in the polar solution. This effect has also been seen by us with ampicillin‐based API‐OSILs
against sensitive Gram‐positive and Gram‐negative species [16] and seem to adversely affect the
activity of those compounds. In the case of highly hydrophobic cations, the micelles of API‐OSILs
may be formed, thus reducing their antimicrobial activity [70].
The only observed exceptions were [C2OHMIM][seco‐Amx], [C2OHMIM][seco‐Pen], and
[TEA][seco‐Pen]. While the first one showed no advantage over Amx (RDIC = 1) against S. aureus, the
second and third seco‐Pen OSILs recorded RDICs of 100 (S. aureus) and 10 (E. coli), respectively.
‐
+
While inactive in the Cl salt form, the [C2OHMIM] cation was the only selected cationic entity
that allowed for the enhancement of the antimicrobial activity of both seco‐Amx and seco‐Pen against
this sensitive S. aureus strain. These results most probably come from specific intermolecular
interactions between the cation and the anion, thereby enabling the deactivating interactions of
crucial PBPs. PBP:API‐OSILs interaction studies will be conducted in the future and published
accordingly.
In the case of the sensitive Gram‐negative E. coli strain, its outer membrane can hinder drug
delivery, as proposed above. In fact, the activity of [seco‐Amx] and [seco‐Pen] towards this strain did
not seem to be enhanced by the combination with [C2OHMIM] or with any of the other cations, with
the exception of [TEA] (RDIC = 10). In truth, [TEA][seco‐Pen] was found to be ten times more effective
than the parent K[Pen]. These results are probably related with augmented hindrance of the porin
entrance and/or uncompetitive phase transfer delivery through the outer membrane in comparison
with the free antibiotic.
Similar results were also recorded in a past study regarding ampicillin‐based API‐ILs with
sensitive Gram‐positive and Gram‐negative bacteria [16]. Recent results in the literature regarding
API‐OSILs as antibiotics against sensitive bacteria have referred to discrete interactions with the
bacteria cell wall or membrane [71–80]. In particular, pyridyl cationic‐modified benzylidene
cyclopentanone photosensitizers (PSs) that were developed by Wu et al. [78] showed that Gram‐
positive bacteria are more sensitive than Gram‐negative bacteria to photodynamic therapy because
their walls are more porous and all types of PSs can readily diffuse through them. In contrast, Gram‐
negative bacteria possess an additional negatively charged outer layer that serves as a permeability
barrier, so neutral and anionic PSs often fail to effectively inactivate Gram‐negative bacteria, while
cationic PSs can still strongly bind to their outer membrane and damage their integrity. In addition,
antimicrobial studies that were supported by FTIR spectroscopy experiments revealed that nalidixic
acid salts with particular ammonium cations exhibit enhanced antimicrobial activity against six
different Gram‐negative Salmonella species and two nalidixic acid‐resistant S. typhimurium strains by
displaying different modes of action towards proteins, carbohydrates, and lipids within the cell
membrane [79]. One final example concerning FTIR bioassays revealed that hydrophobic N‐
alkyltropinium bromide surfactants preferably interact with the fatty acids and amide groups within
the cell envelope of Gram‐negative E. coli and with the peptidoglycan multilayer of the Gram‐positive
Listeria innocua cells [80]. The interaction of the ILs based on penicillin and amoxicillin, as well as
[seco‐Pen] and [seco‐Amx] anions with the cell membrane of Gram‐positive and Gram‐negative
bacteria strains, will be studied soon and published accordingly.
2.4. Studies on Resistant Bacteria
The prepared OSILs from hydrolyzed amoxicillin and penicillin antibiotics were also studied
against resistant Gram‐negative E. coli strains CTX M9 and CTX M2, as well as the methicillin‐
resistant S. aureus ATCC 43,300 (see Table 3).

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Table 3. Minimum inhibitory concentrations (mM) and and relative decrease of inhibitory
concentration (RDIC) of the new compounds that were produced on resistant strains.
MRSA
ATCC
43300
E. coli
CTX M9
E. coli
CTX M2
Compound
RDIC
RDIC
RDIC
[EMIM][seco‐Amx]
[C2OHMIM][seco‐
Amx]
[P6,6,6,14][seco‐Amx]
[C16Pyr][seco‐Amx]
[Choline][seco‐Amx]
Na[seco‐Amx]
>5
>5
‐
‐
>5
‐
‐
>5
5
‐
> 5
>1
0.05
0.05
0.5
>5
>100
>100
>10
‐
1.0
0.05
0.05
>5
>5
>100
>100
‐
1
> 5
0.005
0.5
>5
‐
>1000
10
‐
1
Amx
>5
1
>5
>5
[EMIM][seco‐Pen]
[C2OHMIM][seo‐Pen]
[Choline][seco‐Pen]
[P6,6,6,14][seco‐Pen]
[C16Pyr]seco‐[Pen]
>5
>5
1.0
0.5
0.5
‐
‐
>5
>5
>5
0.5
0.5
‐
‐
‐
>5
>5
1.0
>5
‐
‐
>5
>5
>10
>10
>10
>10
‐
0.05
>100
[TEA][seco‐Pen]
K[seco‐Pen]
K[Pen]
>5
>5
>5
‐
> 5
>5
>5
‐
>5
>5
>5
‐
‐
‐
‐
1
1
1
As expected, parent antibiotics (Amx and [K][Pen]), as well as the sodium and potassium salts
of their ammonia hydrolysates, were found to be inactive against all tested resistant bacteria strains.
For E. coli, five OSILs that contained the [seco‐Amx] anion and three that contained the [seco‐Pen]
anion showed increased activity (RDIC values between >5 and >100) against the parent
antimicrobials. The highest activity (RDIC > 100) was recorded for [C16Pyr][seco‐Amx] against both
resistant E. coli strains, while [P6,6,6,14][seco‐Amx] and [Choline][seco‐Amx] were selective towards only
one of the strains—CTX M9 and CTX M2, respectively.
These results were somewhat similar to those regarding ampicillin‐based API‐OSILs against
resistant Gram‐negative E. coli species that were previously described by us [16], where
[C16Pyr][Amp] and [P6,6,6,14][Amp] showed the highest antimicrobial activities of all compounds
tested. Therein we suggested that the drug delivery of the APIs is enhanced in some resistant E. coli
strains by the lipophilic counter‐ions through the permeation of the outer layer. This postulation is
supported by results from other authors. More specifically, Vincent et al. [81] and Langgartner, J. et
al. [82] similarly demonstrated that transport across biological membranes bearing a highly polar
anionic framework can be facilitated if the APIs are paired with lipophilic ammonium ions that act
as phase transfer agents. Additionally, Rogers et al. [83] recently demonstrated on synthetic
membrane models that API‐OSILs that contain lipophilic cations, preferably with established
hydrogen bonds, exhibit an increased membrane transport as compared to API‐OSILs with weaker
electrostatic interactions or even traditional halide or metal salts. Finally, it is important to note that
both hydrophobic and hydrophilic ionic liquids have been recently studied as penetration enhancers
[16,20,84,85]. Various nanocarriers can serve as antimicrobial enhancers because they incorporate into
lipid membranes or cell walls, leading to membrane or wall disruptions in both Gram‐positive and
Gram‐negative bacteria strains [78,86–92], therefore increasing the drug’s efficiency.
The activity of prepared OSILs against the Gram‐positive MRSA ATCC 43,300 strain seems even
more peculiar. IC50 values as low as 5 and 50 μM, respectively, were obtained with [C16Pyr][seco‐Amx]
and [C16Pyr][seco‐Pen], which corresponded to RDICs higher than 1000 and 100, respectively. The
only other measurable value was obtained with [Choline][seco‐Pen] (RDIC > 5). In the former cases,

Pharmaceutics 2020, 12, 221
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the contribution from the [C16Pyr] cation was unquestionable. Literature data [93] have shown that
the MIC for [C16Pyr][Cl] is five times higher in the methicillin‐resistant than in methicillin‐sensitive
S. aureus strains, suggesting that the contribution of this cation to the antibacterial activity of these
particular API‐OSILs [93] is particularly significant as opposed to the parent antibiotics or their
hydrolyzed analogues (see Table 3). In other words, the amplified activity of [C16Pyr][seco‐Amx] and
[C16Pyr][seco‐Pen] can only be achieved due to a synergic effect of both ionic species. Such a strong
+
influence of [C16Pyr] seems quite in contrast with the unspecific activity of so‐called enhancers (such
+
as [C2OHMIM] for sensitive S. aureus above) and prompt us to consider it as a β‐lactam antibiotic
potentiator [63,72]. Similar results from the literature have shown that berberine in the presence of
ampicillin and oxacillin markedly lowers their MICs against MRSA despite berberine alone
exhibiting no bactericidal activity. Later, it was shown that, in fact, berberine affected MRSA biofilm
development and the dissemination of biofilm‐associated infections [94,95]. This effect is most likely
related with the formation of salt bridges at an allosteric site of the PBP2a in MRSA ATCC 43300 [93].
Curiously, similar interactions were also found for another alkylpyridinium compound, namely the
antibiotic ceftaroline [96]. Thus, an analogous ionic allosteric effect at PBP2a may be occurring with
the described [C16Pyr]‐based OSILs. Further studies, namely molecular dynamic and docking
simulations, must be performed in the future to confirm this assumption. Regardless of the
mechanism, the pronounced increase in RDIC values of the prepared API‐OSILs, particularly against
resistant species, seems very interesting for a potential drug combination strategy. In spite of some
controversy, the combination of antimicrobials with non‐active compounds may provide a quite
promising strategy to address the widespread emergence of antibiotic‐resistant bacteria strains [97–
99].
3. Conclusions
The present work highlights that organic salts and ionic liquids that contain ammonia
hydrolysates of amoxicillin and penicillin G (seco‐Amx and seco‐Pen), in particular [C2OHMIM][seco‐
Amx], [C2OHMIM][seco‐Pen], [TEA][seco‐Pen], [C16Pyr][seco‐Amx], abd [Choline][seco‐Amx],
[C16Pyr][seco‐Pen] display a very strong antibacterial effect on sensitive and resistant E. coli and
MRSA strains, respectively. The gathered data suggest that the adequate ionic pairing of such
hydrolyzed antimicrobials with an ion‐pair effect is vital to enhance or promote antibiotic activity,
with possible alterations in their mechanism of action according to the selected counter‐ion. In
particular, the hydrophobic combination [C16Pyr][seco‐Amx] demonstrated the highest efficiency
towards resistant bacteria strains, with particular emphasis to MRSA ATCC 43300. The combination
of [C16Pyr] with [seco‐Pen] was also very effective against the latter. These results are clearly
promising and point towards a beneficial effect on the drug delivery of the modified APIs when
combined with hydrophobic organic cations. In this way, the antimicrobial resistance to these
standard β‐lactam antibiotics can be drastically reduced in vitro.
Our results also show that future developments of novel APIs‐OSILs must not only focus on the
toxicity and hydrophobicity of the counter ion—they must also look at the outcome. More
specifically, [P6,6,6,14][seco‐Amp] follows the trend of [C16Pyr][Amp] on E. coli resistant strains, which
suggests that there may be other factors at stake to be considered.
The virtually limitless number of ionic pairs that can be assembled as API‐OSILs makes this area
of research particularly interesting and potentially thriving. In addition, the straightforward
synthetic procedure adds virtually no barriers to its future industrial up‐scaling and will thus
eventually lead to an effective combination therapy model to tackle the ever‐emerging bacterial
resistance towards antibiotics.
4. Experimental Section
4.1. Synthesis
Commercially available reagents were purchased from Aldrich, BDH—the Frilabo and
Solchemar laboratory reagents were used as received. The solvents were from Valente & Ribeiro and

Pharmaceutics 2020, 12, 221
10 of 23
distilled before use. Whenever necessary, the solvents were dried by standard procedures, distilled
under nitrogen and stored over molecular sieves.
‐1
The basic anion‐exchange resin Amberlite IRA‐400‐OH (ion‐exchange capacity 1.4 eq.mL ) and
1
13
Amberlyst A‐26 resins were purchased from Supelco. H and C‐NMR spectra in (CD3)2SO, CD3OD
or D2O (from Euriso‐Top) were recorded on a Bruker AMX400 spectrometer at room temperature
unless specified otherwise. To perform NMR, 5 mm borosilicate tubes were used, and the sample
1
13
concentration was, approximately, 7 mg/mL for H‐NMR and 37 mg/mL for C‐NMR. Chemical
shifts are reported downfield in parts per million (ppm).
ESI‐MS were acquired with an API‐ION TRAP(PO03MS), ITQB, Oeiras, Portugal, operating in
both positive‐ and negative ion modes and equipped with a Z‐spray source. Source and desolvation
temperatures were 80 and 100 °C, respectively. The ionic liquid solutions in methanol at
–3
–1
concentrations ∼10–4 mol dm were introduced at a 10 μl min flow rate. The capillary and the cone
voltage were 2600 and 25 V, respectively. Nitrogen was used as a nebulization gas and argon was
used as a collision gas. ESI‐MS‐MS were acquired by selecting the precursor ion with the quadrupole
and then performing collisions with argon at energies from 3 to 20 eV in the hexapole.
IR spectra were measured on a Perkin Elmer 683 by using KBr sample disks. Optical rotations
were recorded on a Perkin Elmer 241MC. The melting temperature (mT) was determined with a
melting point apparatus (Stuart Scientific). The elemental analysis experiments were performed in a
CHNS Series Thermo Finnigan‐CE Instruments Flash EA 1112 under standard conditions (T
combustion reactor 900 °C, T GC column furnace 65 °C, multiseparation SS GC column, He2 flow 130
mL/min, O2 flow 250 mL/min). The penicillin G potassium was transformed in penicillin G
ammonium by the adaptation of the method of Salivar, C. J. et al. [63].Figures 2 to 16 illustrate the
chemical structures of all prepared OSILs based APIs.
4.1.1. Synthesis of seco‐Pen‐Based OSILs
The preparation of ammonium (2S,5R,6R)‐3,3‐dimethyl‐7‐oxo‐6‐(2‐phenylacetamido)‐4‐thia‐1‐
azabicyclo [3.2.0]heptane‐2‐carboxylate [NH4][Pen] was done as indicated by Salivar et al method..
Figure 2. [NH4][Pen]
The penicillin G potassium was transformed in penicillin G ammonium following the method
of Salivar, C. J. et al. [63] before the reaction with hydroxide reactants.
Preparation of [NH4][seco‐Pen]
Figure 3. [NH4][seco‐Pen]

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Potassium penicillin (1 g; 2.6 mmol) was dissolved in 15 mL of a 1.0 M aqueous ammonium
solution. To the solution was added Amberlyst A‐26 resin (5 eq.) that was previously stirred in a 2.0M
aqueous ammonium solution for 1 hour. The reaction mixture was stirred at room temperature for
an additional 1 h. The resin was filtered off, and the was solvent evaporated to provide the desired
1
product as a grey solid (0.828 g; 83%); H NMR (400 MHz, D2O) δ(ppm): 7.33–7.43 (m, 5H); 4.99 (d, J
13
= 8 Hz, 1H); 4.81(s, 1H); 4.41 (d, J = 7.6Hz, 1H); 3,69 (s, 2H); 3.50 (s, 1H); 1.56 (s, 3H); 1.25 (s, 3H);
C
NMR (100 MHz, D2O) δ(ppm): 177.55; 177.40; 176.65; 137.54; 132.14; 131.79; 130.22; 77.42; 67.24; 61.68;
60.93; 44.87; 29.56; 29.30.IR:υ = 3171; 2964; 1644; 1571; 1494; 1454; 1381; 1188; 1130; 1073; 1032; 784;
692; 502. Elemental analysis calculated for C16H24N4O4S0.8H2O: C 50.19; H 6.74; N 14.63. Found: C
50.11; H 6.67; N 14.97.
Preparation of [K][seco‐Pen]
Figure 4. [K][seco‐Pen]
Potassium penicillin (0.137g; 0.36 mmol) was dissolved in a 1.0 M aqueous ammonium solution.
The mixture was stirred at room temperature for 4 h. The solvent was evaporated to provide the
1
desired product as a yellow solid (0.139 g; 97%); m.p. 193–195 °C; H NMR (400 MHz, D2O) δ(ppm):
7.34–7.44 (m, 5H); 4.98 (d, J = 7.6 Hz, 1H); 4.83 (t, J = 1.6 Hz, 1H); 4.39 (d, J = 8 Hz, 1H); 3.70 (s, 2H);
13
3.47 (s, 1H); 1.57(s, 3H); 1.25 (s, 3H); C NMR (100 MHz, D2O) δ(ppm): 177.82; 177.44; 176.79; 137.56;
131.77; 132.13; 130.19; 77.58; 67.42; 62.05; 61.20; 44.85; 29.49; 29.37; IR: υ = 3288; 2969; 2925; 1648; 1582;
1496; 1454; 1382; 1364; 1257; 1128; 877; 791; 694; 502. Elemental analysis calculated for
C16H20KN4O5S3H2O: C 44.22; H 5.80; N 9.67. Found: C 44.45; H 5.54; N 9.70.
Preparation of [Na][seco‐Amx]
Figure 5. [Na][seco‐Amx]
Amoxicillin (0.127 g; 0.3 mmol) was dissolved in a 1.0M aqueous ammonium solution. After 20
min, NaOH (0.012 g; 0.3 mmol) was added, and the mixture was stirred at room temperature for 4 h.
The solvent was evaporated to provide the desired product as a yellow solid (0.127 g; 96%); m.p. 137–
1
139 °C; H NMR (400 MHz, D2O) δ(ppm): 7.30 (d, J = 8 Hz, 2H); 6.89 (d, J = 8.4 Hz, 2H); 4.35 (d, J = 7.2
13
Hz, 1H); 3.29 (s, 1H); 1.38 (s, 3H); 1.17 (s, 3H); C NMR (100 MHz, D2O) δ(ppm): 177.77; 177.05; 176.60;
158.88; 131.63; 118.81; 77.61; 67.33; 61.91; 60.77; 60.23; 29.05; 28.85; IR: υ = 3277; 2969; 2919; 1648; 1575;
1510; 1433; 1383; 1322; 1245; 1173; 1127; 981; 865; 817; 780. Elemental analysis calculated for
C16H21N4NaO5S3H2O: C 41.92; H 5.94; N 12.22. Found: C 41.87; H 6.00; N 11.99.
Preparation of [TEA][seco‐Pen]

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Figure 6. [TEA][seco‐Pen]
Tetraethylammonium bromide (0.420 g; 2.00 mmol) was dissolved in methanol and passed
−1
−1
through Amberlite IRA‐400‐OH an ion‐exchange column [18,100] (5 eq., flux rate 0.133 mLmL min
−1
= 8 BVh ). Then, the tetraethylammonium hydroxide solution that was formed was slowly added to
the ammonium penicillin G (0.751 g; 2.14 mmol) that was dissolved in the 1.0 M aqueous ammonium
−1
solution (50 mgmL ). The reaction mixture was stirred at room temperature for 1 h. After solvent
evaporation, the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100] and
left refrigerated overnight (4 °C) [18,100] to induce the precipitation of the excess of reagents. When
the reagent crystals were filtered out, the solution was evaporated, and the rest was dried in a vacuum
25
for 24 h to provide the desired product as a yellow viscous liquid (0.856 g; 90%). [α]D = 104.0  6.1 (c
−1
1
= 2 mgmL in methanol), H‐NMR (400.13 MHz, CD3OD) δ = 7.31–7.22 (m, 5H), 5.46 (s, 1H), 4.17 (s,
1H) 3.70 (s, 1H), 3.63‐3.58 (m, 2H), 3.51 (bs, 1H), 3.27–3.26 (m, 8H), 1.63 (s, 3H), 1.55 (s, 3H), 1.30–1.24
13
(m, 12H); C‐NMR (100.62 MHz, CD3OD) δ = 174.72, 174.37, 174.15, 140.88, 136.73, 130.31, 130.25,
129.63, 127.96, 75.73, 66.68, 60.19, 59.27, 53.29, 46.65, 43.83, 29.41, 28.67, 28.38, 27.66, 7.64 ppm; IR
(KBr): υ = 3420, 2981, 2924, 2862, 1840, 1736, 1721, 1648, 1560, 1543, 1490, 1459, 1432, 1396, 1367, 1173,
−1
+
+
1130, 1053, 1027, 1001, 785, 734, 696, 619, 539 cm ; (ESI ) m/z calculated for C8H20N : 130.1 found 130.0;
‐
‐
(ESI ) m/z calculated for C16H20N3O4S 350.1 found 349.8.
Preparation of [P6,6,6,14][seco‐Pen]
Figure 7. [P6,6,6,14][seco‐Pen].
Trihexyl(tetradecyl)phosphonium chloride (1.000 g; 1.92 mmol) was dissolved in methanol and
passed through an Amberlite IRA‐400‐OH [18,100] ion‐exchange column (5 eq., flux rate 0.133
−1
−1
−1
mLmL min = 8 BVh ). Then, the trihexyl(tetradecyl)phosphonium hydroxide solution that was
formed was slowly added to ammonium penicillin G (0.853 g; 2.43 mmol) and dissolved in a 1.0 M
−1
aqueous ammonium solution (50 mgmL ). The mixture was stirred at room temperature for 1 h.
After solvent evaporation, the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9)
[18,100] and left refrigerated overnight (4 °C) [14] to induce the precipitation of the excess reagent.
When the reagent crystals were filtered out, the solution was evaporated, and the rest was dried in a
25
vacuum for 24 h to provide the desired product as a yellow viscous liquid (1.560 g; 97%). [α]D = 67.7
−1
1
 3.0 (c = 2 mgmL in methanol); H‐NMR (400.13 MHz, CD3OD) δ = 7.34–7.22 (m, 5H), 4.97 (1H, d, J
= 6.7 Hz), 4.34 (dd, 1H, J = 6.7 Hz), 3.60 (d, 2H, J = 8.2 Hz), 3.50 (s, 1H), 2.20 (m, 8H), 1.56–1.25 (m,
13
54H), 0.96–0.88 (m, 12H) ppm ; C‐NMR (100.62 MHz, CD3OD) δ = 175.30, 174.83, 173.92, 136.76,
130.37, 129.59, 127.87, 76.81, 66.78, 60.14, 54.86, 43.68, 33.11, 32.19, 31.92, 31.84, 30.80, 30.51, 30.45,
29.91, 27.88, 23.77, 23.49, 22.36, 19.53, 19.05, 14.51, 14.38 ppm; IR (KBr): ν = 3308, 3028, 2951, 2923, 2853,
1737, 1669, 1607, 1536, 1496, 1456, 1418, 1379, 1262, 1201, 1113, 1031, 986, 860, 810, 761, 722, 694, 617,
−1
+
+
−
454, 439, 424 cm ; (ESI ) m/z calculated for C32H68P : 483.4 found 483.8; (ESI ) m/z calculated for
‐
C16H20N3O4S 350.1, found 349.9.

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Preparation of [C16Pyr][seco‐Pen]
Figure 8. [C16Pyr][seco‐Pen]
Procedure I
Cetylpyridinium chloride (0.822 g; 2.30 mmol) was dissolved in methanol and passed through
−1
−1
an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133 mLmL min ). Then,
the cetylpyridinium hydroxide solution that was formed was slowly added to ammonium penicillin
−1
G (0.973 g; 2.77 mmol) that was dissolved in a 1.0 M aqueous ammonium solution (50 mgmL ). The
mixture was stirred at room temperature for 1 h. After solvent evaporation, the residue was dissolved
in a 20 mL solution (methanol/acetonitrile 1:9) [18,100] and left refrigerated overnight (4 °C) [18,100]
to induce the precipitation of the excess reagent. When the reagent crystals were filtered out, the
solution was evaporated, and the rest was dried in a vacuum for 24 h to provide the desired product
25
−1
1
as a yellow solid (1.332 g; 89%). m.p. 76–78 °C; [α]D = 47.3  3.6 (c = 2 mgmL in methanol); H‐NMR
(400.13 MHz, CD3OD) δ = 9.01 (d, 2H,J = 5.7 Hz), 8.59 (t, 1H, J = 7.8 Hz), 8.12 (t, 2H, J = 6.8 Hz), 7.33–
7.21 (m, 5H), 4.95 (d, 1H, J = 7.1 Hz), 4.63 (t, 2H, J = 7.5Hz,), 4.35 (d, 1H, J = 7.0 Hz), 3.60 (2H, d, J = 7.5
13
Hz), 3.50 (s, 1H), 1.56 (m, 3H), 1.42‐1.09 (m, 31H), 0.90 (t, 3H, J = 6.6 Hz) ppm; C‐NMR (100.62 MHz,
CD3OD) δ = 175.16, 174.78, 173.98, 146.87, 146.00, 136.73, 130.40, 129.80, 129.60, 127.93, 76.34, 66.64,
63,15, 60.14, 43.67, 33.12, 32.55, 30.81, 30.67, 30.17, 27.86, 27.24, 23.78, 14.49 ppm; IR (KBr): δ = 3041,
3059, 2914, 2848, 1739, 1658, 167, 1601, 1542, 1528, 1508, 1487, 1472, 1397, 1368, 1322, 1270, 1209, 1177,
−1
+
+
1128, 1078, 1032, 987, 960, 926, 818, 777, 716, 686, 619, 574, 475 cm ; (ESI ) m/z calculated for C21H38N :
‐
‐
304.3 found 304.2; (ESI ) m/z calculated for C16H20N3O4S 350.1, found 349.9.
Procedure II
Cetylpyridinium chloride (0.145g; 0.43 mmol) was dissolved in methanol and was added
Amberlyst A‐26 (3 eq.) The mixture was stirred for 1 h at room temperature. Then, the
cetylpyridinium hydroxide solution that was formed was slowly added to [NH4][seco‐Pen] (0.150 g;
0.43 mmol) that was dissolved in a 2.0 M aqueous ammonium solution, and the mixture was stirred
at room temperature for 1 h. The solvent was evaporated to p rovide the desired product as a white
solid (0.263 g; 94%).
Preparation of [Choline][seco‐Pen]
Figure 9. [Choline][seco‐Pen]
(2‐hydroxyethyl)trimethylammonium chloride (0.277 g; 1.99 mmol) was dissolved in methanol
and passed through an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133
−1
−1
−1
mLmL min = 8 BVh ). Then, the hydroxide solution that was formed was slowly added to
ammonium penicillin G (0.848 g; 2.41 mmol) that was dissolved in a 1.0 M aqueous ammonium

Pharmaceutics 2020, 12, 221
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−1
solution (50 mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation,
the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100]and left refrigerated
overnight (4 °C) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals
were filtered out, the solution was evaporated, and the rest was dried in a vacuum for 24 h to provide
25
−1
the desired product as a yellow solid (0.856 g; 95%). m.p. 69–71 °C; [α]D = 47.3  3.6 (c = 2 mgmL in
1
methanol); H‐NMR (400.13 MHz, CD3OD) δ = 7.33–7.29 (m, 5H), 4.95 (d, 1H, J1 = 7.0 Hz), 4.35 (d, 1H,
J1 = 7.0 Hz), 4.02–3.98 (m, 2H), 3.66–3.56 (m, 2H), 3.50‐3.47 (m, 3H), 3.20 (s, 9H) 1.56 (s, 3H), 1.25 (s,
13
3H) ppm; C‐NMR (100.62 MHz, CD3OD) δ = 174.72, 174.37, 174.15, 140.88, 136.73, 130.31, 130.25,
129.63, 127.96, 75.73, 69.06, 66.67, 60.20, 60.04, 59.53, 57.10, 46.65, 27.78, 27.47 ppm; IR (KBr): υ = 3468,
3074, 2966, 1652, 1496, 1479, 1461, 1396, 1356, 1279, 1204, 1131, 1083, 1054, 1010, 955, 887, 867, 796, 734,
−1
+
+
−
702, 670, 620, 540, 476 cm ; (ESI ) m/z calculated for C5H14NO : 104.1, found 104.1; (ESI ) m/z calculated
‐
for C16H20N3O4S 350.1, found 349.9.
Preparation of [EMIM][seco‐Pen]
Figure 10. [EMIM][seco‐Pen]
1‐ethyl‐3‐methylimidazolium bromide (0.578 g; 3.03 mmol) was dissolved in methanol and
passed through an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133
−1
−1
−1
mLmL min = 8 BVh ). Then, the hydroxide solution that was formed was slowly added to
ammonium penicillin G (1.11 g; 3.16 mmol) that was dissolved in an aqueous 1.0 M ammonium
−1
solution (50 mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation,
the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100]and left refrigerated
overnight (4 °C) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals
were filtered out, the solution was evaporated, and the rest was dried in a vacuum for 24 h to provide
25
−1
the desired product as a colorless viscous liquid (1.136 g; 81%). [α]D = 89.0  7.0 (c = 2 mgmL in
1
methanol); H‐NMR (400.13 MHz, CD3OD) δ = 8.99 (bs, 1H), 7.63 (s, 1H) 7.53 (s, 1H), 7.34–7.20 (m,
5H), 4.95 (d, 1H, J = 7.0 Hz), 4.35 (d, 1H, J = 7.0 Hz), 4.24 (q, 2H, J = 7.3 Hz), 3.92 (s, 3H), 3.60 (d, 2H, J
13
= 7.2 Hz), 3.50 (s, 1H), 1,60–1.50 (m, 6H), 1.25 (s, 3H) ppm; C‐NMR (100.62 MHz, CD3OD) δ = 175.06,
174.83, 173.99, 136.80, 130.65, 130.39, 129.60, 127.90, 124.94, 123.25, 76.51, 60.13, 60.18, 60.11, 59.54,
46.02, 43.66, 36.51, 27.81, 27.65, 15.61 pm; IR (KBr): υ = 3468, 3368, 2970, 1660, 1540, 1501, 1456, 1395,
−1
+
1456, 1395, 1354, 1300, 1258, 1169, 1131, 1301, 965, 918, 862, 828, 729, 700, 651, 620, 545 cm ; (ESI ) m/z
+
−
‐
calculated for C6H11N2 : 111.1, found 111.0; (ESI ) m/z calculated for C16H20N3O4S 350.1, found 349.9.
Preparation of [C2OHMIM][seco‐Pen]
Figure 11. [C2OHMIM][seco‐Pen]

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3‐(2‐hydroxyethyl)‐1‐methylimidazolium chloride (0.328 g; 2.03 mmol) was dissolved in
methanol and passed through an Amberlite IRA‐400(OH) ion‐exchange column [14,41] (5 eq., flux
−1
−1
−1
rate 0.133 mLmL min = 8 BVh ). Then, the hydroxide solution that was formed was slowly added
to ammonium penicillin G (0.754 g; 2.14 mmol) that was dissolved in a 1.0 M aqueous ammonium
−1
solution (50 mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation,
the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100]and left refrigerated
overnight (4 °C) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals
were filtered out, the solution was evaporated, and the rest was dried in a vacuum for 24 h to provide
25
−1
the desired product as a yellow solid (0.799 g; 83%). m.p. 48–50 °C; [α]D = 41.3  6.0 (c = 2 mgmL in
1
methanol); H‐NMR (400.13 MHz, CD3OD) δ = 8.98, (s, 1H), 7.61 (s, 1H), 7.54 (s, 1H), 7.33–7.21 (m,
5H), 4.94 (d, 1H, J = 7.1 Hz), 4.36 (d, 1H, J = 7.1 Hz), 4.29 (t, 2H, J = 4.9 Hz), 3.92, (s, 3H), 3.86 (t, 2H, J =
13
4.9 Hz), 3.59 (d, 2H, J = 7.1 Hz), 3.50 (s, 1H), 1.55 (s, 3H), 1.24 (s, 3H) ppm; C‐NMR (100.62 MHz,
CD3OD) δ = 175.04, 174.82, 174.00, 136.78, 130.63, 130.51, 130.40, 129.61, 129.85, 129.61, 127.91, 124.68,
124.00, 76.50, 66.76, 61.10, 60.12, 59.53, 53.26, 43.65, 36.45, 27.84 ppm; IR (KBr): υ = 3418, 2965, 2931,
2108, 1644, 1585, 1499, 1455, 1398, 1356, 1260, 1167, 1127, 1076, 1034, 879, 798, 734, 704, 668, 619, 464,
−1
+
+
−
445, 432, 424 cm ; (ESI ) m/z calculated for C6H11N2O : 127.2, found 127.0; (ESI ) m/z calculated for
‐
C16H20N3O4S 350.1, found 349.9.
4.1.2. Synthesis of seco‐Amx‐Based OSILs
Preparation of [EMIM][seco‐Amx]
Figure 12. [EMIM][seco‐Amx]
1‐ethyl‐3‐methylimidazolium chloride (0.385 g; 2.01 mmol) was dissolved in methanol and
passed through an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133
−1
−1
−1
mLmL min = 8 BVh ). Then, the hydroxide solution that was formed was slowly added to
amoxicillin trihydrate (0.930 g; 2.22 mmol) that was dissolved in an aqueous 1.0 M ammonium
−1
solution (50 mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation,
the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100]and left refrigerated
overnight (4 °C) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals
were filtered out, the solution was evaporated, and the rest was dried in a vacuum for 24 h to provide
25
−1
the desired product as a yellow solid (0.768 g; 77%). m.p. 84–86 °C; [α]D = 48.3  5.0 (c = 2 mgmL in
1
methanol); H‐NMR (400.13 MHz, CD3OD) δ = 7.64 (s, 1H), 7.56 (s, 1H), 7.27 (d, 2H, J = 8.2 Hz), 6.74
(d, 2H, J = 8.4 Hz), 5.00 (d, 1H, J1 = 5.9 Hz), 4.74 (s, 1H), 4.30 (d, 1H, J1 = 5.9 Hz), 4.25 (1, 2H, J = 7.3 Hz),
3.77 (bs, 1H), 3.92 (s, 3H), 3.73, (bs, 1H), 3.43 (bs, 1H), 3.35 (s, 1H, s), 1.55–1.48 (m, 6H); 1.22 (s, 3H)
13
ppm; C‐NMR (100.62 MHz, CD3OD) δ = 175.57 , 175.15, 174.84, 141.24, 129.83, 129.12, 128.50, 124.96,
123.31, 77.12, 66.67, 60.18, 60.11, 59.54, 46.03, 36.46, 27.78, 27.47, 15.63 ppm; IR (KBr): υ = 3461, 2921,
−1
2852, 1706, 1688,1656, 1636, 1560, 1541, 1508, 1461, 1403, 1348, 1260, 1170, 1130, 673, 620, 474, 422 cm ;
+
+
−
‐
(ESI ) m/z calculated for C6H11N2 : 111.1, found 111.0; (ESI ) m/z calculated for C16H21N4O5S 381.1,
found 380.8.
Preparation of [P6,6,6,14][seco‐Amx]

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Figure 13. [P6,6,6,14][seco‐Amx]
Trihexyl(tetradecyl)phosphonium chloride (1.042 g; 2.01 mmol) was dissolved in methanol and
passed through an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133
−1
−1
−1
mLmL min = 8 BVh ). Then, the trihexyl(tetradecyl)phosphonium hydroxide solution that was
formed was slowly added to amoxicillin (0.988 g; 2.36 mmol) that was dissolved in a 1.0 M aqueous
−1
ammonium solution (50 mgmL ). The mixture was stirred at room temperature for 1 h. After solvent
evaporation, the residue was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100] and
left refrigerated overnight (4 °C) [18,100] to induce the precipitation of the excess reagent. Then, the
reagent crystals were filtered out, the solution was evaporated, and the rest was dried in a vacuum
25
for 24 h to provide the desired product as a yellow viscous liquid (1.586 g; 92%). [α]D = 22.0  5.8 (c
−1
= 2 mgmL in methanol); (400.13 MHz, CD3OD) δ = 7.28 (d, 2H, J = 8.4 Hz), 6.76 (d, 2H, J = 8.4 Hz),
5.00 (d, 1H, J = 5.9 Hz), 4.53 (s, 1H), 4.31 (d, 1H, J = 5.9 Hz), 3.42 (s, 1H), 2.23–2.16 (m, 8H), 1.60–1.22
13
(m, 54H), 0.95–0.88 (m, 12H) ppm; C‐NMR (100.62 MHz, CD3OD) δ = 175.35, 174.22, 142.01, 129.62,
128.92, 116.59, 77.12, 66.57, 60.17, 54.94, 43.76, 33.18, 32.27, 31.93, 31.56, 30.89, 30.58, 30.01, 27.96, 23.85,
23.57, 22.45, 19.62, 19.15, 14.58, 14.46 ppm; IR (KBr): ν = 3419, 3921, 2107.38, 1638, 1560, 1506, 1459,
−1
+
+
1398, 1270, 1130, 1000, 668, 619, 570, 476, 456, 433, 412 cm ; (ESI ) m/z calculated for C32H68P : 483.8
−
‐
found 483.6; (ESI ) m/z calculated for C16H21N4O5S 381.1, found 381.0.
Preparation of [C16Pyr][seco‐Amx]
Figure 14. [C16Pyr][seco‐Amx]
Cetylpyridinium chloride (0.456 g; 1.28 mmol) was dissolved in methanol and passed through
−1
−1
an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133 mLmL min = 8
−1
BVh ). Then, the cetylpyridinium hydroxide solution that was formed was slowly added to
amoxicillin (0.587 g; 1.40 mmol) that was dissolved in a 1.0 M aqueous ammonium solution (50
−1
mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation, the residue
was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100]and left refrigerated overnight
(4 ºC) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals were
filtered out, the solution was evaporated, and the rest was dried in a vacuum for 24 h to provide the
25
−1
desired product as a yellow solid (0.402 g; 52%). m.p. 96–98 ºC; [α]D = 77.0  5.8 (c = 2 mgmL in
1
methanol); H‐NMR (400.13 MHz, CD3OD) δ = 8.98 (d, 2H, J = 5.8 Hz), 8.58 (t, 1H, J = 7.74 Hz), 8.10 (t,
2H, J = 6.7 Hz), 7.26 (d, 2H, J = 8.5 Hz), 6.73 (d, 2H, J = 8.4 Hz), 5.01 (d, 1H, J = 6.0 Hz), 4.62 (t, 2H, J =
7.5 Hz), 4.46 (s, 1H), 4.30 (d, 1H, J = 6.0 Hz), 3.44 (s, 1H), 2.02, (t, 2H, J = 6.9 Hz), 1.48 (s, 3H), 1.38–1.26
13
(m, 28H), 1.22 (s, 3H) 0.90 (3H, t, J = 6.7 Hz) ppm; C‐NMR (100.62 MHz, CD3OD) δ = 176.32, 175.60,
174.94, 158.32, 150.28, 146.87, 145.91, 132.86, 129.54, 116.55, 77.13, 66.61, 63,17, 60.14, 33.11, 32.53, 30.80,

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30.67, 30.52, 30.16, 27.24, 23.77, 14.49 ppm; IR (KBr): υ = 3440, 2914, 2849, 1685, 1651, 1636, 1560, 1488,
−1
+
1472, 1400, 1384, 1260, 1175, 1128, 847, 778, 720, 687, 621, 498, 476 cm ; (ESI ) m/z calculated for
+
−
‐
C21H38N : 304.3 found 304.4; (ESI ) m/z calculated for C16H21N4O5S 381.1, found 380.9.
Preparation [choline][seco‐Amx]
Figure 15. [choline][seco‐Amx]
(2‐hydroxyethyl)trimethylammonium chloride (0.179 g; 1.28 mmol) was dissolved in methanol
and passed through an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux rate 0.133
−1
−1
−1
mLmL min = 8 BVh ). Then, the hydroxide solution that was formed was slowly added to
amoxicillin (0.587 g; 1.40 mmol) that was dissolved in a 1.0 M aqueous ammonium solution (50
−1
mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation, the residue
was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100]and left refrigerated overnight
(4 °C) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals were
filtered out, the solution was evaporated, and the rest was dried in a vacuum for 24 h to provide the
25
−1
desired product as a yellow solid (0.570 g; 93%). m.p. 143–144 °C; [α]D = 104.0  3.4 (c = 2 mgmL in
1
methanol); H‐NMR (400.13 MHz, CD3OD) δ = 7.36 (d, J = 8.1 Hz, 2H), 6.93 (d, J = 8.1 Hz, 2H), 5.06 (d,
J = 6.8 Hz, 1H), 4.64 (s, 1H), 4.40 (d, J = 6.8 Hz, 1H), 4.13–4.04 (m, 2H), 3.53 (t, J = 4.6 Hz, 2H), 3.37 (s,
13
1H), 3.22 (s, 9H), 1.46 (s, 3H), 1.24 (s, 3H) ppm; C‐NMR (100.62 MHz, CD3OD) δ = 176.14, 175.48,
174.46, 156.70, 131.47, 129.19, 116.54, 75.51, 67.98, 65.21, 59.78, 58.63, 58.46, 56.16, 54.48, 54.44, 54.40,
26.98, 26.79 ppm; IR (KBr): υ = 3300, 2964, 2927, 1673, 1594, 1513, 1435, 1389, 1251, 1118, 1130, 1087,
−1
956, 837, 780 cm .
Preparation of [C2OHMIM][seco‐Amx]
Figure 16. [C2OHMIM][seco‐Amx]
3‐(2‐hydroxyethyl)‐1‐methylimidazolium chloride (0.456 g; 1.28 mmol) was dissolved in
methanol and passed through an Amberlite IRA‐400‐OH ion‐exchange column [18,100] (5 eq., flux
−1
−1
−1
rate 0.133 mLmL min = 8 BVh ). Then, the hydroxide solution that was formed was slowly added
to amoxicillin (0.525 g; 1.44 mmol) that was dissolved in a 1.0 M aqueous ammonium solution (50
−1
mgmL ). The mixture was stirred at room temperature for 1 h. After solvent evaporation, the residue
was dissolved in a 20 mL solution (methanol/acetonitrile 1:9) [18,100] and left refrigerated overnight
(4 °C) [18,100] to induce the precipitation of the excess reagent. When the reagent crystals were
filtered from the solution, the solution was evaporated, and the rest was dried in a vacuum for 24 h
25
to provide the desired product as a yellow solid (0.359 g; 60%). m.p. 109‐111 °C; [α]D = 47.3  3.6 (c

Pharmaceutics 2020, 12, 221
18 of 23
−1
1
= 2 mgmL in methanol); H‐NMR (400.13 MHz, CD3OD) δ = 7.61 (s, 1H), 7.55 (s, 1H), 7.27 (d, 2H, J =
8.4 Hz), 6.74 (d, 2H, J = 8.4 Hz), 5.00 (d,1H, J = 6.0 Hz), 4.47 (s, 1H), 4.30–4.27 (m, 3H), 3.92 (s, 3H), 3.86
13
(t, 2H, J = 4.86 Hz), 3.43 (s, 1H), 1.48 (s, 3H), 1.22 (s, 3H) ppm; C‐NMR (100.62 MHz, CD3OD) δ =
176.33, 175.62, 174.95, 158.34, 132.89, 129.54. 124.75,124.04, 116.56, 77.17 66.64, 61.11, 60.15, 59.83, 59.50,
53.81, 36.45, 27.78, 27.47 ppm; IR (KBr): υ = 3420, 2970, 2921, 1722, 1690, 1655, 1599, 1577, 1545, 1509,
−1
+
1436, 1386, 1322, 1251, 1170, 1132, 1108, 1067, 877, 840, 820, 778, 652, 621, 535, 474 cm ; (ESI ) m/z
+
−
‐
calculated for C6H11N2O : 127.2, found 127.0; (ESI ) m/z calculated for C16H21N4O5S 381.1, found 380.9.
Author Contributions: The manuscript was written through contributions of all authors. All authors have read
and agree to the published version of the manuscript. Conceptualization, R. Ferraz; Z. Petrovski and L.C. Branco;
methodology, .R. Ferraz; Z. Petrovski and L.C. Branco; validation, R. Ferraz, M. M. Santos and Z. Petrovski;
investigation, R. Ferraz, D. Silva, A. R. Dias, V. Dias, L. Pinheiro.; writing—original draft preparation, R. Ferraz,
Z. Petrovski and M. M. Santos; writing—review and editing, L. C. Branco; visualization, C. Prudêncio and J. P.
Noronha. The first two authors contribute equally for the manuscript (share in equal parts as first author). All
authors have read and agreed to the published version of the manuscript.
Funding:
Acknowledgments: This research was funded by by Fundação para a Ciência e Tecnologia through
projects (PTDC/QUI‐QOR/32406/2017, PEst‐C/LA0006/2013, PTDC/CTM/103664/2008) and PTDC/BTM‐
SAL/29786/2017, and one contract under Investigador FCT (L.C.B.). ZP thanks to Fundacão para a Ciência e a
Tecnologia, MCTES, for the Norma transitória DL 57/2016 Program Contract. This work was also supported by
the Associate Laboratory for Green Chemistry, which is financed by national funds from FCT/MEC
(UID/QUI/50006/2013), UIDB/50006/2020
and co‐financed by the ERDF under the PT2020 Partnership
Agreement (POCI‐01‐ 0145‐FEDER‐007265). The authors also thank Solchemar company for support.
Conflicts of Interest: The authors declare no conflicts of interest.
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