A novel class of cationic and non-peptidic small molecules as hits for the development of antimicrobial agents

Article abstract of DOI:10.3390/molecules23071513

Cationic and non-peptide small molecules containing a total of six positive charges arranged on one side and a long aliphatic tail on the other have been synthesized and tested against Gram-positive and Gram-negative bacteria. The positive charges have be

Full text of DOI:10.3390/molecules23071513

molecules
Article
A Novel Class of Cationic and Non-Peptidic Small
Molecules as Hits for the Development of
Antimicrobial Agents
Aranza Jiménez 1,†,‡, Pablo García 2,†, Sofia de la Puente 1, Andrés Madrona 1,§
María José Camarasa 1 ID , María-Jesús Pérez-Pérez 1 ID , José-Carlos Quintela 3,
,
Francisco García-del Portillo 2,
*
and Ana San-Félix 1,
*
ID
ID
1
Instituto de Química Médica, Consejo Superior de Investigaciones Científicas (IQM, CSIC),
Juan de la Cierva 3, 28006 Madrid, Spain; Aranzaj@hotmail.com (A.J.); sofdelapuen@hotmail.com (S.d.l.P.);
andres.madrona@novartis.com (A.M.); mj.camarasa@iqm.csic.es (M.J.C.); mjperez@iqm.csic.es (M.-J.P.-P.)
Laboratorio de Patógenos Bacterianos Intracelulares, Centro Nacional de Biotecnología,
Consejo Superior de Investigaciones Científicas (CNB, CSIC), Darwin 3, 28049 Madrid, Spain;
pgarcia@cnb.csic.es
2
3
Natac Biotech S.L., Parque Científico de Madrid, Campus de Cantoblanco, 28049 Madrid, Spain;
jcquintela@natac.es
*
Correspondence: fgportillo@cnb.csic.es (F.G.-d.P.); anarosa@iqm.csic.es (A.S.-F.);
Tel.: +34-91-2587617 (A.S.-F.); Fax: +34-91-5644853 (A.S.-F.)
†
‡
These authors contributed equally to this work.
Current address: Agencia Española de Medicamentos y productos Sanitarios, Campezo 1, Edificio 8,
28022 Madrid, Spain.
§
Current address: Novartis Farmacéutica S.A. Gran Vía de les Corts Catalanes, 764, 08013 Barcelona, Spain.
Academic Editor: Jóhannes Reynisson FRSC
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 6 June 2018; Accepted: 20 June 2018; Published: 22 June 2018
Abstract: Cationic and non-peptide small molecules containing a total of six positive charges
arranged on one side and a long aliphatic tail on the other have been synthesized and tested against
Gram-positive and Gram-negative bacteria. The positive charges have been contributed by two
aminophenol residues. These molecules have showed remarkable antimicrobial activity against
Gram-positive bacteria including multidrug-resistant strains. Our structure–activity relationship
studies demonstrated the importance of the length and flexibility of the hydrophobic tail for
the antimicrobial activity. Importantly, these compounds are non-toxic to eukaryotic cells at the
concentration affecting growth in bacteria, reflecting an acceptable margin of safety. The small size
and easy synthetic accessibility of our molecules can be of interest for the further development of
novel antimicrobials against Gram-positive bacterial pathogens, including multidrug-resistant strains.
Keywords: antimicrobial agents; antibiotic resistance; antimicrobial peptides
1. Introduction
At present, microbial resistance to conventional antibiotics is becoming a growing problem that
affects, in particular, to hospitals and other health care centers. This crisis has been attributed to
the failure to correctly administrate antibiotics or to strictly use them when necessary, to feed farm
animals with antibiotics as well as to the lack of investment of the pharmaceutical companies in the
development of new antibiotics [1–3].
According to a recent report of the World Health Organization, if no initiatives are taken the cost
of resistance to antibiotics could exceed $100 billion and lead to the premature death of 300 million
Molecules 2018, 23, 1513; doi:10.3390/molecules23071513
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1513
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people in 2050 [4]. Therefore, it is of critical importance to develop new antimicrobial agents in order
to substitute or complement currently available antibiotics.
It is well known that many multicellular organisms (animal and plants) produce a variety of
peptides, namely antimicrobial peptides (AMPs) and host defense peptides (HDPs), which are effective
defensive weapons against a wide range of pathogens including, bacteria, fungi, enveloped viruses,
and protozoa [5,6].
Naturally occurring AMPs are amphipathic molecules having both hydrophobic residues and
hydrophilic positive charges (cationic amino acids), which allow them to bind simultaneously at
several sites on the biological membranes [5–8]. In the particular case of bacteria, the main driving
force leading to an efficient binding of these amphipathic molecules is the interaction between the
positive charges of AMPs and the negatively charged phospholipid head-groups present on the surface
of bacterial membranes [
with the lipid component of the bacterial membrane, leading to its perturbation, and in certain cases,
internalization of the peptide damaging critical intracellular targets [ ]. The ability of AMPs
9–12]. This is followed by interaction of the hydrophobic residues of AMPs
5,6,9
to distinguish between bacterial and mammalian cells is mainly due to the differences in the lipid
components of their respective cell membranes. In bacteria, the surface exposed to the outer world is
heavily populated by lipids with negatively charged phospholipid head-groups, while the membranes
of plant and animals is composed principally of lipids with no net charge [5,9–12].
With respect to conventional antibiotics, AMPs showed several advantages. First, the emergence
of resistance against AMPs is less probable than in the case of conventional antibiotics [
In addition, AMPs are able to modulate the immune response though a variety of mechanisms to fight
infections [ 14 16]. Moreover, AMPs also show a broad antimicrobial spectrum acting against
a variety of pathogens other than bacteria such as viruses, fungi, and protozoa [ 14 16]. Despite all of
these advantages, AMPs also show several disadvantages that limit their clinical use. Among them,
poor bioavailability, potential lability to proteases and high cost of manufacturing [ ]. In fact, although
5,12,13].
5,6,9, –
5,
–
6
some AMPs are currently in clinical trials, there are still few of them available for clinical use—those
being polymyxin B, colistin, gramicidin S, daptomycin, and nisin [17,18].
Inspired by the antimicrobial activities showed by the natural AMPs, different synthetic
mimics have been described over the past few years. Most of them are peptidomimetics but also
polymers or oligomers (molecular weight > 1000 Da) [19,20]. With the aim of overcoming the
problems associated with the large size of these molecules, more manageable downsized compounds
(molecular weight < 1000 Da) have been also synthesized [21–25].
Having all of this in mind, we decided to synthesize small molecules (molecular weight < 1000 Da
)
of general formula (Figure 1) as AMPs mimics. These synthetic molecules bear two aminophenol ‘heads’
I
on one side and a long aliphatic tail of different length on the other. In the design of these small molecules,
some essential structural characteristics of the natural AMPs, such as the simultaneous presence of cationic
charges and hydrophobic groups, have been taken into consideration. In our case, the hydrophobicity
was provided by the two aromatic rings of the ‘heads’ and the length of the tail, while hydrophilicity
by the six positive charges (+6) of the quaternary ammonium groups of the ‘heads’. The length of the
alkyl chain, -(CH2)nCH3, was varied systematically through variation of the number of methylene
groups (n = 6 to n = 16). Finally, in order to determine the role of the conformational flexibility in the
antibacterial activity, analogues in which the aliphatic ‘tail’ has been replaced with an unsaturated
chain with one or four double bonds were prepared.
For these compounds, inhibition of representative drug-sensitive and multidrug-resistant bacterial
pathogens has been determined and is herein described.

Molecules 2018, 23, 1513
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Figure 1. General structure of the synthesized compounds.
2. Results and Discussion
2.1. Chemical Results
The synthesis of the proposed final compounds was achieved in four steps.
The aromatic ‘head’
With this purpose, commercially available methyl 3,4,5-trihydroxybenzoate
2-(Boc-amino) ethyl bromide in the presence of NaI and Cs2CO3 (Scheme 1) to afford intermediate
(69%). Saponification of the methyl ester present on 2, using LiOH/H2O, afforded compound
(84% yield), with a free carboxylic acid on the focal point (Scheme 1). It should be mentioned that the
synthesis of has been previously described [26] using potassium carbonate (K2CO3) as a base. In our
3
, containing three Boc-protected amino groups was prepared first (Scheme 1).
1
was treated with
2
3
2
hands, this method led to a poor yield of this compound (10%). However, when cesium carbonate and
sodium iodide were used, the yield of 2 improved until 69%.
O
O
O
O
HN
O
HN
O
O
O
O
OH
HN
HN
O
HO
OH
ii
i
MeOOC
HOOC
O
O
O
O
COOMe
NH
O
NH
O
O
O
1
3
(84%)
2 (69%)
Scheme 1. Synthesis of the tris-Boc-aminoethoxy benzoate
conditions: (i) Br(CH2)2NHBoc, Cs2CO3, NaI, acetone, 65 C (ii) LiOH·H2O, then HCl 1M.
3 (aromatic «head»). Reagents and
◦
Next, reaction of the commercially available 2-amino-1,3-propanediol (serinol)
4
(Scheme 2) with
the corresponding acyl chloride, in the presence of trimethylamine at
−
20 ◦C, afforded the N-acyl
intermediates 5–9 in moderate to good yields (57–89%).
Subsequent condensation of
presence of DCC, as coupling reagent and DMAP as base, afforded the Boc protected derivatives 10
5
–
9
with the 2-(Boc-amino) ethyl-protected galloyl acid
3
in the
14
–
in low to moderate yields (10–43%) (Scheme 3).
Catalytic hydrogenation of 13 at atmospheric pressure, in the presence of 10% Pd/C in ethyl
acetate, afforded the saturated derivative 15 in quantitative yield (Scheme 4).

Molecules 2018, 23, 1513
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Finally, treatment of the Boc-protected derivatives 10
–
15 with TFA or HCl gave the deprotected
final compounds 16–21 (45–90%) as their corresponding salts (Scheme 5).
O
i
OH
HO
OH
NH
OH
R
+
Cl
n
NH2
O
4
5, R = (CH2)6CH3, (75%)
6,
R = (CH2)10CH3 (80%)
7, R = (CH2)14CH3 (57%)
8,
R = (CH2)7HC=CH(CH2)7CH3 (64%)
9, R = (CH2)3HC=CH(CH2CH=CH)3(CH2)4CH3 (89%)
Scheme 2. Synthesis of the N-acyl serinol derivatives
Et3N, −20 C to room temperature.
5–
9. Reagents and conditions: (i) MeOH:THF,
◦
O
O
O
O
N
O
HN
H
N
O
H
R
O
O
O
OH
O
HN
O
NH
OH
R
O
i
O
O
+
O
NH
HOOC
O
H
O
O
N
O
O
NH
O
O
O
O
NH
H
N
O
O
5-9
O
O
O
O
O
NH
O
O
3
10, R = (CH2)6CH3 (28%)
11, R = (CH2)10CH3 (43%)
12,
13,
14,
R = (CH2)14CH3 (10%)
R = (CH2)7HC=CH(CH2)7CH3 (41%)
R = (CH2)3HC=CH(CH2CH=CH)3(CH2)4CH3 (36%)
Scheme 3. Synthesis of intermediates 10–14. Reagents and conditions: (i) DCC, DMAP, dry dichloromethane.
O
O
O
H
N
O
H
i
O
N
O
O
N
H
O
O
O
O
O
N
H
O
O
O
O
O
O
HN
O
O
O
HN
O
O
O
O
O
O
O
HN
HN
HN
N
H
O
O
O
O
O
O
O
O
O
O
O
O
HN
HN
NH
O
NH
O
O
O
O
O
15 (
quant)
13
Scheme 4. Synthesis of the fully saturated derivative 15. Reagents and conditions: (i) H2, Pd/C (10%),
EtOAc, rt.
Scheme 5. Synthesis of the deprotected final derivatives 16–21. Reagents and conditions: (i) TFA or
HCl, dichloromethane, rt.

Molecules 2018, 23, 1513
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2.2. Biological Results
2.2.1. Antibacterial Activity
The antimicrobial activities of the new aminophenol derivatives (16
–
21) were tested against
representative drug-sensitive and multidrug-resistant bacterial pathogens. As drug-sensitive bacteria
Salmonella enterica serovar Typhimurium SV5015 (S. Typhimurium SV5015) (Gram-negative),
Listeria monocytogenes EGD-e (L. monocytogenes EGD-e) (Gram-positive), and Staphylococcus aureus
Newman (S. aureus Newman) (Gram-positive) have been chosen. As drug-resistant bacteria
Staphylococcus aureus SC-1 (S. aureus SC-1) and Staphylococcus aureus USA-300 (S. aureus USA-300)
have been used.
Initial assays performed in liquid culture showed that none of these compounds 16
inhibitory against the Gram-negative bacteria S. Typhimurium SV5015 up to 50
g mL−1 (Figure 2A).
However, with the exception of 16, the rest of compounds 17 21 showed inhibitory effects against the
Gram-positive bacteria L. monocytogenes EGD-e (only 20, as representative of the active compounds, is
showed in Figure 2B). Interestingly, 17 21 also showed inhibitory effects against the multidrug-resistant
–21 proved
µ
–
–
bacteria S. aureus USA-300 (only 20, as representative of the active compounds, is showed in Figure 2C).
Figure 2. Antimicrobial assays in liquid culture using different aminophenol compounds and bacterial
species. Bacteria were cultured in media containing the aminophenol for 18 h overnight culture.
(
A
) Lack of effect of the test compounds (50
µ
g mL−1) in the Gram-negative bacterial pathogen
S. Typhimurium strain SV5015; ( ) Differential effect of aminophenol compounds 16 and 20 in the
B
Gram-positive bacterial pathogen L. monocytogenes strain EGD-e. Note the lack of effect of compound
16 and the inhibitory effect of compound 20. Similar results as for compound 20 were obtained for
compounds 17, 18, 19, and 21 (not shown); (C) Differential effect of aminophenol compounds 16 and 20
in the multidrug resistant clinical isolate S. aureus USA-300 strain. Note the lack of effect of compound
16 and the inhibitory effect of compound 20. Similar results as for compound 20 were obtained for
compounds 17
, 18, 19, and 21 (not shown). The compounds 17, 18, 19, 20, and 21 also displayed
inhibitory capacity against S. aureus strains Newman and SC-1 (not shown, see text for details).

Molecules 2018, 23, 1513
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Next, the minimal inhibitory concentrations (MIC) of 16
–
21 have been determined. In this
experiment, kanamycin—a conventional aminoglycoside antibiotic that inhibits the synthesis of
bacterial proteins—was used as control [27]. Table 1 summarizes the results of this evaluation.
Table 1. Antibacterial activity and cytotoxicity of the test compounds.
−1
MIC a (µg mL
)
CC b
(µg mL
Drug Sensitive Strains
Multidrug Resistant Strains
−1
)
S. Typhimurium
L. monocytogenes
S. aureus
S. aureus
S. aureus
Compound
SV5015
EGD-e
Newman
SC-1
USA-300
16
17
18
19
20
21
>50 (*)
>50
>50
>50
>50
>50
12.5
3.13
3.13
3.13
12.5
2.34
>50
50
12.5
12.5
12.5
50
>50
50
12.5
12.5
12.5
50
>50
12.5
3.13
50
12.5
12.5
>150
≥100
>100 (**)
≥100
≥100
≥100
>50
n/d
≥100
Kanamycin
9.4
>150
n/d
(*) no effect in bacterial growth, even at the highest concentration used (50
µ
g mL−1). All values are in g mL−1
µ
and are a summary of multiple dose-response curves (>2) in multiple (>1) experiments. n/d: Not determined.
a
MIC: minimum concentration of the compound that inhibits bacterial proliferation after overnight incubation.
CC: minimum concentration of the compound that inhibits human HeLa epithelial cells or rat fibroblasts NRK-49F
b
proliferation after overnight incubation. (**) In the case of compound 17, toxicity in NRK-49F fibroblasts was not
observed at the highest concentration tested (100 µg mL−1).
As it was shown in Table 1, the N-octanoyl derivative 16, with an acyl chain containing only
6 methylenes, did not show significant activity up to 50
g mL−1 against any of the drug-sensitive
µ
bacteria (Salmonella enterica serovar Typhimurium strain SV5015, Listeria monocytogenes strain
EGD-e, and Staphylococcus aureus strain Newman). The N-dodecanoyl analogue, compound 17,
with 10 methylenes, displayed improved antibacterial activity against L. monocytogenes EGD-e and
S. aureus Newman (MIC: 12.5 and 50 µ
g mL−1, respectively). Further increase in the length of the
aliphatic chain (14 methylenes) yielded compound 18 (N-hexadecanoyl analogue) that displayed much
improved antibacterial activity against L. monocytogenes EGD-e and S. aureus Newman (MIC: 3.13 and
12.5 µ
g mL−1, respectively). However, the highest long chain analogue 19 (N-octadecanoyl analogue,
16 methylenes) did not display any significant change in activity with respect to 18, indicating that the
additional two methylene groups present in the aliphatic chain of 19 are superfluous.
The effect of increasing the rigidity of the molecule in the antibacterial activity was analyzed
with compounds 20 and 21, with one and four double bonds, respectively, in the aliphatic chain.
The unsaturated analogue 20, with only one double bond in the aliphatic chain, was found to be as
active against L. monocytogenes EGD-e and S. aureus Newman as the saturated counterparts 18 and 19
,
whereas compound 21, containing four double bonds was less active, suggesting that an excessive
conformational rigidity in the aliphatic chain is detrimental for the antibacterial activity.
The new aminophenol derivatives 16
clinically isolated multidrug-resistant (MDR) bacteria such as S. aureus strains SC-1 and USA-300 [28].
Compounds 17 and 21 only showed moderate activity (MIC: 50
g mL−1) against the
drug-resistant strain S. aureus SC-1, while they showed significant activity (MIC: 12.5
g mL−1
against the drug-resistant strain S. aureus USA-300. Just the opposite was observed for compound 19,
which displayed moderate activity (MIC: 50
g mL−1) against S. aureus USA-300 strain and significant
(MIC: 12.5
g mL−1) against S. aureus SC-1. Interestingly, the activity showed by compounds 18 and
–21 were also tested for their in vitro inhibitory effects on
µ
µ
)
µ
µ
20 against both resistant strains was remarkable. It contrasted with the lack of activity showed by the
conventional antibiotic kanamycin, which was used as control. Similar to the results found for the
drug-sensitive strains, compound 16, with the shortest aliphatic chain (octanoyl chain), did not show
significant activity up to 50 µg mL−1 against any of the multidrug-resistant bacteria.

Molecules 2018, 23, 1513
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The saturated compound 18, with a long aliphatic chain (C16) and 20 with a long chain (C18)
containing only one unsaturation, were therefore the most potent compounds of this series against all
the tested bacteria strains (drug-sensitive and multidrug-resistant).
It should be noted that the activity showed by 18 and 20 against Gram-positive drug-sensitive
bacteria is very similar to that of the conventional antibiotic used as control, kanamycin. Moreover,
18 and 20 resulted active against drug-resistant bacterial strains for which kanamycin was inactive.
In summary, the structure–activity relationship (SAR) studies reveal the significance of our design
for achieving Gram-positive antimicrobial activity, especially against drug-resistant bacterial strains.
Our molecules, that display the most important characteristic of most AMPs, like cationic facial
amphiphilicity and positive charges, have several advantages over the natural counterparts. Among
them, non-peptidic character, low-cost synthesis, and possibility to fine-tuning their potency and safety.
Based on that, they can be useful hits for the further design of novel antimicrobial agents against
Gram-positive bacteria.
2.2.2. Antimicrobial Kinetics
Next, the effect of the newly synthesized compounds over the bacterial growth in function of time
was determined. Bacterial growth (cells per volume unit), estimated from the turbidity of the culture,
was measured using a spectrophotometer at a wavelength of 600 nm and it was represented as optical
density (OD600).
First, we monitored the growth over time of the drug-sensitive bacteria L. monocytogenes EGD-e
in the presence of compounds 17
their respective MICs (4 MIC) (see Table 1). The aminoglycoside kanamycin at 30
used as relevant control since it rapidly arrests growth in actively proliferating bacteria [27]. As it
was shown in Figure 3A the curves for compounds 17 19, and 20 are very similar to those of the
kanamycin. Thus, this experiment clearly shows that the treatment with aminophenols 17 19, and 20
,
19, and 20, used at a concentration that was four-fold higher than
µ
g mL−1 was
,
,
resulted in rapid arrest of bacteria growth (Figure 3A) and, consequently, these compounds behave as
potent antimicrobials.
Figure 3. The aminophenol compounds arrest efficiently growth of L. monocytogenes and S. aureus.
(
A
) Growth curve of a culture of L. monocytogenes strain EGD-e exposed to the indicated
compounds. Time 0 in the graphic corresponds to the time the compound was added to the culture
OD600 ~0.15–0.20). All antimicrobial was used at four-fold of their respective MIC value for each
of the indicated bacterial strains (see Table 1); ( ) Growth curves of L. monocytogenes strain EGD-e
(
B
and S. aureus cultures exposed to the amino phenol 18. Amino phenol 18 was used at four-fold of its
respective MIC value for each of the indicated bacterial strains (see Table 1).

Molecules 2018, 23, 1513
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Next, the growth over time of the drug-sensitive bacteria L. monocytogenes EGD-e and S. aureus
Newman, together with the multidrug-resistant bacteria S. aureus strains SC1 and USA300 was
analyzed in the presence of the most potent aminophenol derivative 18 (4 MIC) (Figure 3B). Our results
showed that 18 is able of arresting the growth of the four studied bacterial strains. Apparently, S. aureus
Newman (drug-sensitive) and S. aureus SC-1 (multidrug-resistant) strains were more sensitive to the
action of 18 than the other bacterial strains (Figure 3B).
2.2.3. Cytotoxicity Evaluation
Finally, the cytotoxic effects of the aminophenol derivatives 17–21 were determined in two
different eukaryotic cell types, HeLa epithelial cells (Figure 4) and NRK-49F fibroblasts (Figure 5).
Figure 4. Toxicity assay of the series of compounds 17 to 21 in human HeLa epithelial cells. The cell
−1
culture was exposed to the different compounds for 18 h at different concentrations (only 100
µg mL
−1
or 20
µ
g mL was indicated). Cells were fixed and imaged in an inverted microscopy as described in
Section 4.
Different concentrations of the aminophenol compounds 17–21 were used in these experiments.
After an overnight exposure to the compounds, toxicity—denoted by detachment of cells and drastic
morphological changes—was evident only at 100
g mL−1 while no toxicity was found at lower
concentrations. Importantly, the highest concentration (100
g mL−1) was 8-fold or 32-fold higher
21 in the antimicrobial assays (see Table 1) reflecting an acceptable
µ
µ
than the MIC value observed for 17
–
margin of safety.

Molecules 2018, 23, 1513
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Figure 5. Toxicity assay of the series of compounds 17–21 in rat fibroblasts NRK-49F. The cell culture
was exposed to the different compounds for 18 h at different concentrations (only two concentrations
−1 −1
100 µg mL or 20 µg mL are indicated). Cells were fixed and imaged in an inverted microscopy as
described in Materials and Methods. In the case of compound 17, toxicity in NRK-49F fibroblasts was
not observed at the highest concentration tested (100 µg mL−1).
3. Conclusions
In this study, we have developed an efficient strategy for the synthesis of novel aminophenol
derivatives 17 21. All of these compounds have the same hydrophilic portion consisting in two
–
cationic ‘heads’ with three positive charges each (six positive charges in total) and a lipophilic ‘tail’
of different length and conformational flexibility. These compounds were synthesized in four simple
synthetic steps.
The synthesized compounds showed antimicrobial activity against different Gram-positive
bacteria (L. monocytogenes and S. aureus) while probed to be inactive against Gram negative
bacteria (S. Typhimurium). Interestingly 17–21 also showed inhibitory effects against Gram-positive
multidrug-resistant strains such as S. aureus SC-1 and S. aureus USA-300.
In this respect, it should be noted that defined AMPs have been reported to be only active
against Gram-positive bacteria while others are not selective and showed broad activity against both
Gram-negative and Gram-positive bacteria [29–31]. Despite their mode of action not being fully
understood and the basis for their selectivity being unknown, many studies reveal the importance of
the membrane components of bacteria—lipid content in particular—in the activity and selectivity of
these molecules [29–31].

Molecules 2018, 23, 1513
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These precedents support the idea of a mechanism of action for the compounds here described
directly related to a defined structure in the bacterial cell envelope. Further studies are required to
decipher their mode of action and selectivity towards microbial cells.
Presence of long chains (C16 or C18) is an important structural requirement for antimicrobial
activity. Introduction of one double bond in a long alkyl chain (C18) renders molecules that also
showed good antibacterial activity while the incorporation of four double bonds led to much less active
molecules. This finding emphasized the importance of the length and flexibility of the hydrophobic
alkyl portion (‘tail’) for the antibacterial activity.
Compounds 18 (C16) and 19 (C18) with saturated long chains, together with 20, that incorporates
one double bond in its long chain (C18), were found to be the most potent antibacterial agents among
this series. These compounds are non-toxic to eukaryotic cells at the concentration affecting growth in
bacteria, reflecting an acceptable margin of safety.
Taken together, these data proved that the newly synthesized aminophenol compounds can be
promising hits for the further development of antimicrobial agents against Gram-positive bacteria
(drug-sensitive and multidrug-resistant). However, further research is needed to elucidate the
pharmacophore group(s) and their mechanism of action.
4. Materials and Methods
4.1. Synthesis
Commercial reagents and solvents were used as received from the suppliers without further
purification unless otherwise stated. The solvents used in some reactions were dried prior use.
DMF dry was commercially available (Sigma-Aldrich Quimica SL, Madrid, Spain).
Analytical thin-layer chromatography (TLC) was performed on aluminum plates pre-coated with
silica gel 60 (F254, 0.25 mm). Products were◦visualized using an ultraviolet lamp (254 and 365 nm
)
or by heating on a hot plate (approx. 200 C), directly or after treatment with a 5% solution of
phosphomolybdic acid or vanillin in ethanol.
The compounds were purified by: (a) High Performance Flash Chromatography (HPFC) with
a system “Isolera One” (Biotage, Uppsala, Sweden) in reverse phase using water/acetonitrile
(100:0 to 0:100) as eluent; (b) flash column chromatography on silica gel (60 Merck 230–400 mesh);
and (c) preparative centrifugal circular thin layer chromatography (CCTLC) on a chromatotron®
(Kiesegel 60 PF254 gipshaltig, Merck, Dramstand, Germany) layer thickness 1 mm, flow rate
2–4 mL/min.
For HPLC analysis, an Agilent Technologies 1120 Compact LC with a reverse phase column ACE
5 C18-300 (4.6
×
150 mm, 3.5 µm) equipped with a PDA (photodiode array) detector Waters 2996 was
used. Acetonitrile was used as mobile phase A, and water with 0.05% of TFA was used as mobile
phase B at a flow rate of 1 mL min−1. All retention times are quoted in minutes and the gradients are
specified for each compound in the experimental data.
For high resolution mass spectrometry (HRMS) was used an Agilent 6520 Accurate Mass QTOF
(quadrupole time-of-flight) coupled with LC/MS using an electrospray interface (ESI) working in the
positive-ion (ESI+) and negative-ion (ESI−) mode.
NMR spectra (1H and 13C NMR) were recorded on a Varian UNIT INOVA-300 (300 MHz)
(now Agilent, Santa Clara, CA, USA), Bruker AVANCE 300 (300 and 75 MHz) (Bruker, Billerica,
MA, USA), Varian INOVA-400 (400 and 100 MHz) (now Agilent, Santa Clara, CA, USA), Varian
MERCURY-400 (now Agilent, Santa Clara, CA, USA) (400 and 100 MHz), and Varian-500 (now Agilent,
Santa Clara, CA, USA) (500 and 125 MHz) spectrometers, using DMSO-d6 and CDCl3 as solvents.
Chemical shift (δ) values are reported in parts per million (ppm) relative to tetramethylsilane (TMS)
in 1H and CDCl3 (δ = 77.0) in 13C NMR. Coupling constant (J values) are reported in hertz (Hz) and
multiplicities of signals are indicated by the following symbol: s (singlet), d (doublet), t (triplet),

Molecules 2018, 23, 1513
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q (quadruplet), m (multiplet), and bs (broad singlet). Some two-dimensional spectra (COSY, HSQC,
and HMBC) were performed to identify the structure.
Final compounds were lyophilized using a Telstar 6–80 systhem.
4.1.1. Methyl 3,4,5-tris[2-(Boc-amino)-1-ethoxy]benzoate (2)
A mixture of methyl 3,4,5-trihydroxybenzoate
1 (250 mg, 1.35 mmol), Cs2CO3 (1.75 g, 5.4 mmol),
NaI (203 mg, 1.35 mmol) and 2-(Boc-amino)ethyl bromide (1.2 g, 5.4 mmol) in acetone (10 mL) was
◦
refluxed overnight at 65 C and then evaporated to dryness. The residue was dissolved in ethyl acetate
(20 mL) and washed with aqueous solutions of citric acid (10%) (3
The organic phase was dried over anhydrous Na2SO4, filtered and evaporated to dryness. The residue
was purified on a Biotage HPFC (high performance flash chromotography) purification system in
× 20 mL) and brine (2 × 20 mL).
a reverse phase using water/acetonitrile (100:0 to 0:100) to afford 576 mg (69%) of
2 as a yellow foam.
1H NMR (400 MHz, DMSO-d6)
δ
7.20 (s, 2H, Ar), 6.96 (t, J = 5.7 Hz, 2H, NHCO), 6.63 (t, J = 5.7 Hz
,
1H, NHCO), 4.01 (t, J = 5.7 Hz, 4H, CH2O), 3.96 (t, J = 5.9 Hz, 2H, CH2O), 3.83 (s, 3H, CH3O),
3.35 (m, 4H, CH2NH), 3.22 (m, 2H, CH2NH), 1.35 (s, 18H, Boc), 1.36 (s, 9H, Boc). HPLC (tR) [gradient:
A:B, 10–100% of A in 10 min]: 9.54 min.
4.1.2. 3,4,5-tris[2-(Boc-amino-1-ethoxy]benzoate (3)
◦
To a solution at 0 C containing
2 (333 mg, 0.54 mmol) in THF (15 mL), a second solution
of LiOH H2O (45 mg, 1.08 mmol) in water (7 mL) was added. The mixture was stirred at room
·
temperature overnight. Then, an aqueous solution of 1N hydrochloric acid was added to reach
pH = 3–4, and the mixture was evaporated to dryness. The residue was dissolved in ethyl acetate
(20 mL) and washed with H2O (3
filtered and evaporated to dryness to afford 272 mg (84%) of
1H NMR (300 MHz, DMSO-d6)
7.21 (s, 2H, Ar), 6.77 (br s, 2H, NHCO), 6.45 (br s, 1H, NHCO),
×
20 mL). The organic phase was dried over anhydrous Na2SO4,
3
as an amorphous white solid.
δ
4.01 (m, 6H, CH2O), 3.35 (m, 4H, CH2NH), 3.23 (m, 2H, CH2NH), 1.37 (s, 18H, Boc), 1.36 (s, 9H, Boc).
HPLC (tR) [gradient: A:B, 10–100% of A in 10 min]: 5.32 min.
4.1.3. General Procedure for the Synthesis of the N-acyl Serinol Derivatives 5–9
A stirred solution of 2-amino-1,3-propanediol (serinol)
4 (1 eq) and triethylamine (TEA) in
MeOH (10 mL) was cooled at
−
20 ◦C (carbon dioxide snow) and added dropwise a solution
of the corresponding acyl chloride (1.1 eq) in THF (5 mL). The reaction mixture was allowed to
warm to room temperature and stirred overnight. Then it was poured into brine and extracted
with dichloromethane (3
×
10 mL). The combined organic phases were washed with brine, dried
over MgSO4 and concentrated under reduced pressure. The residue was purified by flash column
chromatography on silica gel using as eluent EtOAc/MeOH (20:1 to 7:1) to provide the corresponding
N-acyl serinol derivatives.
4.1.4. N-octanoyl Serinol (5)
According to the general procedure serinol
4
(150 mg, 1.64 mmol) was treated with octyl chloride
1
(1.81 mmol, 0.3 mL) and TEA (0.4 mL) to give 267 mg (75%) of
(400 MHz, DMSO-d6)
5
as an amorphous white solid. H NMR
δ
7.40 (d, J = 8.1 Hz, 1H, NHCO), 4.55 (t, J = 5.5 Hz, 2H, OH), 3.69 (m, 1H, CHNH),
3.37 (t, J = 5.6 Hz, 4H, CH2OH), 2.06 (t, J = 7.4 Hz, 2H, CH2CO), 1.46 (m, 2H, CH2), 1.22 (m, 8H, CH2),
0.86 (t, J = 6.8 Hz, 3H, CH3).
4.1.5. N-dodecanoyl Odecanoyl Serinol (6)
According to the general procedure serinol
chloride (625 mg, 2.8 mmol) and TEA (0.6 mL) to give 478 mg, (80%) of
Spectroscopic data of this compound are consistent with those found in the literature [32].
4
(200 mg, 2.19 mmol) was treated with dodecanoyl
6
as an amorphous white solid.

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4.1.6. N-hexadecanoyl Exadecanoyl Serinol (7)
According to the general procedure serinol
4
(150 mg, 1.64 mmol) was treated with hexadecanoyl
chloride (1.81 mmol, 0.5 mL) and TEA (0.4 mL) to give 309 mg (57%) of
7 as an amorphous
1
white solid. H NMR (400 MHz, DMSO-d6)
δ 7.39 (d, J = 8.1 Hz, 1H, NHCO), 4.55 (t, J = 5.5 Hz,
2H, OH), 3.66 (m, 1H, CHNH), 3.35 (t, J = 5.6 Hz, 4H, CH2OH), 2.03 (t, J = 7.4 Hz, 2H, CH2CO),
1.43 (m, 2H, CH2), 1.21 (m, 24H, CH2), 0.83 (t, J = 6.6 Hz, 3H, CH3).
4.1.7. N-oleoyl Serinol (8)
According to the general procedure serinol
chloride (oleoyl chloride) (541.6 mg, 0.6 mL) and TEA (0.4 mL) to give 375 mg (64%) of
4
(150 mg, 1.64 mmol) was treated with 9-octadecenoyl
as
8
an amorphous white solid. Spectroscopic data of this compound are consistent with those found in the
literature [33].
4.1.8. N-arachidonoyl Serinol (9)
A solution of Z,Z,Z,Z-5,8,11,14-eicosatetraenoic acid (arachidonic acid) (510 mg, 1.5 mmol) and
thionyl chloride (87 mmol, 6.3 mL) in dry dichloromethane (10 mL) was stirred at room temperature
under argon atmosphere until the reaction is complete. The solvent was evaporated to dryness and the
residue was used immediately for the next step.
According to the general procedure, serinol
mentioned arachidonic chloride (193 mg, 0.6 mmol) and TEA (0.1 mL) to give 182 mg (89%) of
as an amorphous white solid. 1H NMR (400 MHz, DMSO-d6)
7.42 (d, J = 8.1 Hz, 1H, NHCO),
4 (49 mg, 0.54 mmol) was treated with the above
9
δ
5.34 (m, 8H, CH=), 4.54 (t, J = 5.6 Hz, 2H, OH), 3.68 (m, 1H, CHNH), 3.38 (t, J = 5.4 Hz, 4H, CH2OH),
2.79 (m, 6H, CH2CH=), 2.08 (t, J = 7.5 Hz, 2H, CH2CO), 2.02 (m, 4H, CH2CH=), 1.53 (m, 2H, CH2),
1.40–1.16 (m, 6H, CH2), 0.85 (t, J = 6.7 Hz, 3H, CH3).
4.1.9. General Coupling Procedure for the Synthesis of the Boc Protected Intermediates 10–14
A solution of the Boc protected gallic acid derivative
in dry CH2Cl2 (7.5 mL) was stirred at room temperature for 15 min and then added dropwise to
a second solution, also stirred for 15 min, containing the corresponding serinol derivate (1 eq) and
3 (2.2 eq), DMAP (1.6 eq), and DCC (3.2 eq)
5–9
DMAP (1.6 eq) in dry CH2Cl2 (7.5 mL). To dissolve all components, an extra amount of DMF (2 mL)
were added. The solution was stirred at 30 ◦C for 24 h and then evaporated to dryness. The residue
was dissolved in ethyl acetate (20 mL). Urea by-product was filtered and the organic residue was
evaporated. This process was repeated several times until no precipitation was observed. The residue
was purified on a Biotage HPFC purification system in a reverse phase using water:acetonitrile (100:0 to
0:100) to give the corresponding intermediate. Note that all the solid reactives are pre-dried overnight
by storage inside a vacuum desiccator with a drying agent like phosphorous pentoxide.
4.1.10. Bis-O-[3,4,5-tris(2-N-Boc-amino-1-ethoxy)benzoyl]-N-octanoyl Serinol (10)
Following the general procedure,
to afford 47.3 mg (28%) of 10 as an amorphous white solid. 1H NMR (400 MHz, DMSO-d6)
8.08 (d, J = 8.4 Hz, 1H, NHCO), 7.24 (s, 4H, Ar), 6.98 (t, J = 5.7 Hz, 4H, NHCO), 6.64 (m, 2H, NHCO),
5 (25 mg, 0.1 mmol) was treated with 3 (203.1 mg, 0.34 mmol)
δ
4.58 (m, 1H, CHNH), 4.40 (dd, J = 11.2, 5.1 Hz, 2H, CH2O), 4.30 (dd, J = 11.1, 6.8 Hz, 2H, CH2O),
3.99 (m, 12H, CH2O), 3.36 (m, 8H, CH2N), 3.23 (m, 4H, CH2N), 2.09 (t, J = 7.3 Hz, 2H, CH2CO),
1.45 (t, J = 7.2 Hz, 2H, CH2), 1.37 (s, 54H, Boc), 1.20-1.01 (m, 8H, CH2), 0.77 (t, J = 7.1 Hz, 3H, CH3).
HPLC (tR) [isocratic of acetonitrile A:B, 0–100%]: 2.3 min.
4.1.11. Bis-O-[3,4,5-tris(2-N-Boc-amino-1-ethoxy)benzoyl]-N-dodecanoyl Serinol (11)
Following the general procedure,
to afford 112.5 mg (43%) of 11 as an amorphous white solid. H NMR (300 MHz, DMSO-d6)
6
(50 mg, 0.2 mmol) was treated with
3
(240 mg, 0.4 mmol)
1

Molecules 2018, 23, 1513
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δ
8.11 (d, J = 8.3 Hz, 1H, NHCO), 7.23 (s, 4H, Ar), 7.01 (t, J = 5.5 Hz, 4H, NHCO), 6.68 (br s, 2H,
NHCO), 4.57 (m, 1H, CHNH), 4.37 (m, 2H, CH2O), 4.30 (m, 2H, CH2O), 3.98 (m, 12H, CH2O),
3.36 (m, 8H, CH2N), 3.21 (m, 4H, CH2N), 2.09 (t, J = 7.2 Hz, 2H, CH2CO), 1.36 (m, 56H, CH2 and Boc),
1.26–1.03 (m, 16H, CH2), 0.84 (t, J = 6.5 Hz, 3H, CH3). HPLC (tR) [isocratic of acetonitrile A:B, 0–100%]:
2.7 min.
4.1.12. Bis-O-[3,4,5-tris(2-N-Boc-amino-1-ethoxy)benzoyl]-N-hexadecanoyl Serinol (12)
Following the general procedure,
to afford 11.8 mg (10%) of 12 as an amorphous white solid. 1H NMR (300 MHz, DMSO-d6)
8.11 (d, J = 8.3 Hz, 1H, NHCO), 7.23 (s, 4H, Ar), 7.00 (t, J = 5.5 Hz, 4H, NHCO), 6.66 (t, J = 5.6
7 (25 mg, 0.07 mmol) was treated with 3 (127 mg, 0.21 mmol)
δ
Hz, 2H, NHCO), 4.56 (m, 1H, CHNH), 4.36 (m, 2H, CH2O), 4.27 (m, 2H, CH2O), 3.94 (m, 12H, CH2O),
3.33 (m, 8H, CH2N), 3.20 (m, 4H, CH2N), 2.06 (t, J = 7.2 Hz, 2H, CH2CO), 1.36 (m, 56H, CH2 and Boc),
1.25–1.05 (m, 24H, CH2), 0.84 (t, J = 6.3 Hz, 3H, CH3). HPLC (tR) [isocratic of acetonitrile A:B, 0–100%]:
3.5 min.
4.1.13. Bis-O-[3,4,5-tris(2-N-Boc-amino-1-ethoxy)benzoyl]-N-oleoyl Serinol (13)
Following the general procedure,
to afford 87 mg (41%) of 13 as an amorphous white solid. 1H NMR (300 MHz, DMSO-d6)
8.10 (d, J = 8.5 Hz, 1H, NHCO), 7.24 (s, 4H, Ar), 7.00 (t, J = 5.7 Hz, 4H, NHCO), 6.65 (d, J = 5.8 Hz
8 (50 mg, 0.14 mmol) was treated with 3 (236.1 mg, 0.39 mmol)
δ
,
2H, NHCO), 5.28 (dd, J = 4.0, 1.5 Hz, 2H, CH=), 4.58 (m, 1H, CHNH), 4.41 (dd, J = 10.9, 4.9 Hz, 2H,
CH2O), 4.30 (dd, J = 11.1, 6.8 Hz, 2H, CH2O), 3.99 (m, 12H, CH2O), 3.33 (m, 8H, CH2N), 3.22 (m, 4H,
CH2N), 2.10 (t, J = 7.4 Hz, 2H, CH2CO), 1.93 (m, 4H, CH2CH=), 1.37 (m, 56H, CH2 and Boc), 1.29–1.12
(m, 20H, CH2), 0.84 (t, J = 6.7 Hz, 3H, CH3). HPLC (tR) [isocratic of acetonitrile A:B, 0–100%]: 3.5 min.
4.1.14. Bis-O-[3,4,5-tris(2-N-Boc-amino-1-ethoxy)benzoyl]-N-arachidonoyl Serinol (14)
Following the general procedure,
0.51 mmol) to afford 113 mg (36%) of 14 as an amorphous white solid. 1H NMR (400 MHz,
DMSO-d6) 8.12 (d, J = 8.3 Hz, 1H, NHCO), 7.24 (s, 4H, Ar), 6.98 (t, J = 5.8 Hz, 4H, NHCO),
9 (78 mg, 0.20 mmol) was treated with 3 (308 mg,
δ
6.64 (d, J = 6.6 Hz, 2H, NHCO), 5.30 (m, 8H, CH=), 4.56 (m, 1H, CHNH), 4.40 (t, J = 6.4 Hz, 2H,
CH2O), 4.30 (dd, J = 11.1, 6.6 Hz, 2H, CH2O), 3.98 (m, 12H, CH2O), 3.33 (m, 8H, CH2N), 3.23 (m, 4H,
CH2N), 2.73 (m, 4H, CH2CH=), 2.70 (m, 2H, CH2CH=), 2.11 (m, 2H, CH2CO), 2.00 (m, 4H, CH2CH=),
1.50 (m, 2H, CH2), 1.37 (s, 54H, Boc), 1.25 (m, 6H, CH2), 0.83 (t, J = 6.7 Hz, 3H, CH3). HPLC (tR)
[isocratic of acetonitrile A:B, 0–100%]: 3.8 min.
4.1.15. Bis-O-[3,4,5-tris(2-N-Boc-amino-1-ethoxy)benzoyl]-N-octadecanoyl Serinol (15)
A solution of 13◦ (114 mg, 7.05 mmol) in ethyl acetate containing 30% wt. of 10% Pd/C was
hydrogenated at 30 C for 4–6 h under atmospheric pressure using a reaction balloon filled with
hydrogen gas and a glass flask as the reaction vessel. The Pd/C was filtered through Whatman®
filter paper 42 and the solvent was removed under reduced pressure to give 110.3 mg (quant) of the
1
title compound as an amorphous white solid. H NMR (400 MHz, DMSO-d6)
δ 8.08 (d, J = 8.3 Hz,
1H, NHCO), 7.24 (s, 4H, Ar), 6.98 (m, 4H, NHCO), 6.63 (m, 2H, NHCO), 4.57 (m, 1H, CHNH),
4.40 (dd, J = 11.2, 5.1 Hz, 2H, CH2O), 4.30 (dd, J = 11.1, 6.8 Hz, 2H, CH2O), 3.99 (m, 12H, CH2O),
3.32 (m, 8H, CH2N), 3.21 (m, 4H, CH2N), 2.08 (t, J = 7.3 Hz, 1H, CH2CO), 1.48 (m, 2H, CH2),
1.38 (s, 54H, Boc), 1.27–1.01 (m, 28H, CH2), 0.85 (t, J = 6.8 Hz, 3H, CH3). HPLC (tR) [isocratic of
acetonitrile A:B, 0–100%]: 4.5 min.
4.1.16. Synthesis of the Deprotected Serinol Derivatives 16–21
To a solution containing the corresponding Boc protected derivative (10
–15) in CH2Cl2 (10 mL),
TFA was added. After stirring at room temperature overnight the solution was evaporated to dryness

Molecules 2018, 23, 1513
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and co-evaporated successively with CH2Cl2 and MeOH. The residue was lyophilized to afford the
final products.
4.1.17. Bis-O-[3,4,5-tris(2-ammonium-1-ethoxy)benzoyl]-N-octanoyl Serinol Trifluoroacetate (16)
Following the general procedure, a solution of 10 (41.4 mg, 0.08 mmol) was treated with TFA
(0.2 mL) to afford 38 mg (86%) of 16 as an amorphous white solid .1H NMR (400 MHz, DMSO-d6)
δ
8.48 (bs, 18H, NH3+), 7.36 (s, 4H, Ar), 4.56 (m, 1H, CHNH), 4.46–4.23 (m, 16H, CH2O), 3.56–3.15
(m, 12H, CH2N), 2.16 (t, J = 7.3 Hz, 2H, CH2CO), 1.47 (m, 2H, CH2), 1.17 (m, 8H, CH2), 0.80 (t, J = 6.4 Hz
,
3H, CH3). 13C NMR (100 MHz, DMSO-d6)
δ
170.0 (CONH), 156.8 (Ar), 145.0 (Ar), 130.2 (Ar), 113.6 (Ar),
74.6 (CH2O), 71.0 (CH2O), 69.2 (CH2O), 51.9 (CHNH), 43.5 (CH2N), 40.7 (CH2N), 36.2, 33.6, 33.6, 30.5,
27.1 (CH2), 19.0 (CH3). HPLC (tR) [gradient: A:B, 10–100% of A in 10 min]: 2.13 min. HRMS (ESI+)
m/z: Calc. for C37H61N7O11 780.4556. Found 779.4425.
4.1.18. Bis-O-[3,4,5-tris(2-ammonium-1-ethoxy)benzoyl]-N-dodecanoyl Serinol Chloride (17)
Following the general procedure, a solution of 11 (112.5 mg, 0.08 mmol) was treated with TFA
(0.4 mL) and co-evaporated several times with HCl 1M in methanol to afford 59.4 mg (50%) of 17 as
δ
an amorphous dark yellow solid. 1H NMR (400 MHz, DMSO-d6) 8.45 (bs, 18H, NH3+), 7.36 (s, 4H, Ar),
4.55 (m, 1H, CHNH), 4.40 (m, 2H, CH2O), 4.36 (m, 2H, CH2O), 4.33–4.23 (m, 12H, CH2O), 3.33 (m, 8H,
CH2N), 3.18 (m, 4H, CH2N), 2.16 (t, J = 7.4 Hz, 2H, CH2CO), 1.46 (m, 2H, CH2), 1.19 (m, 16H, CH2),
0.84 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
δ 173.1 (CONH), 165.3 (COO), 151.7 (Ar),
140.6 (Ar), 125.3 (Ar), 108.6 (Ar), 69.1 (CH2O), 65.8 (CH2O), 63.7 (CH2O), 46.7 (CHNH), 40.68 (CH2N),
35.4 (CH2CO), 31.3 (CH2), 29.1, 28.9, 28.8, 28.6, 25.3, 22.1 (CH2), 14.0 (CH3). HPLC (tR) [gradient: A:B,
10–100% of A in 10 min]: 5.26 min. HRMS (ESI+) m/z: Calc. for C41H69N7O11 836.5152. Found 835.5097.
4.1.19. Bis-O-[3,4,5-tris(2-ammonium-1-ethoxy)benzoyl]-N-hexadecanoyl Serinol Chloride (18)
Following the general procedure, a solution of 12 (55 mg, 0.03 mmol) was treated with TFA
(0.2 mL) and co-evaporated several times with HCl 1M in methanol to afford 27.3 mg (45%) of
18 as an amorphous dark yellow solid. 1H NMR (400 MHz, DMSO-d6)
δ
8.41 (bs, 18H, NH3+),
7.32 (s, 4H, Ar), 4.56 (m, 1H, CHNH), 4.42 (m, 2H, CH2O), 4.35–4.20 (m, 14H, CH2O), 3.34 (m, 8H,
CH2N), 3.16 (m, 4H, CH2N), 2.15 (t, J = 7.4 Hz, 2H, CH2CO), 1.44 (m, 2H, CH2), 1.19 (m, 24H, CH2),
0.85 (t, J = 6.5 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
δ 172.6 (CONH), 164.8 (COO), 151.7 (Ar),
140.5 (Ar), 125.3 (Ar), 108.6 (Ar), 69.2 (CH2O), 65.9 (CH2O), 63.8 (CH2O), 46.7 (CHNH), 40.7 (CH2N),
35.4 (CH2CO), 31.2 (CH2), 29.1, 29.0, 28.9, 28.8, 28.7, 28.6, 25.4, 22.1 (CH2), 14.0 (CH3). HPLC (tR)
[gradient: A:B, 10–100% of A in 10 min]: 6.52 min. HRMS (ESI+) m/z: Calc. for C45H77N7O11 892.5803.
Found 891.5714.
4.1.20. Bis-O-[3,4,5-tris(2-ammonium-1-ethoxy)benzoyl]-N-octadecanoyl Serinol Trifluoroacetate (19
)
Following the general procedure, a solution of 13 (50.4 mg, 0.03 mmol) was treated with TFA
1
(0.2 mL) to afford 37 mg (69.6%) of 19 as an amorphous white solid. H NMR (400 MHz, DMSO-d6)
δ
8.17 (bs, 18H, NH3+), 7.33 (s, 4H, Ar), 4.58 (m, 1H, CHNH), 4.42 (dd, J = 11.2, 5.3 Hz, 2H, CH2O),
4.31 (dd, J = 11.2, 6.8 Hz, 2H, CH2O), 4.25 (t, J = 5.1 Hz, 8H, CH2O), 4.15 (t, J = 5.1 Hz, 4H, CH2O),
3.34 (m, 8H, CH2N), 3.19 (t, J = 4.7 Hz, 4H, CH2N), 2.10 (t, J = 7.3 Hz, 2H, CH2CO), 1.45 (m, 2H, CH2),
1.19 (m, 28H, CH2), 0.84 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
δ 172.7 (CONH),
164.8 (COO), 151.7 (Ar), 140.4 (Ar), 125.4 (Ar), 108.4 (Ar), 69.4 (CH2O), 65.8 (CH2O), 63.7 (CH2O),
46.8 (CHNH), 40.7 (CH2N), 35.5 (CH2CO), 31.3, 29.0, 28.9, 28.8, 28.5, 25.3, 22.1 (CH2), 14.0 (CH3).
HPLC (
920.6101. Found 919.6006.
t
R) [gradient: A:B, 10–100% of A in 10 min]: 7.06 min. HRMS (ESI+) m/z: Calc. for C47H81N7O11

Molecules 2018, 23, 1513
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4.1.21. Bis-O-[3,4,5-tris(2-ammonium-1-ethoxy)benzoyl]-N-oleoyl Serinol Trifluoroacetate (20)
Following the general procedure, a solution of 14 (65.1 mg, 0.08 mmol) was treated with TFA
(0.4 mL) to afford 65.8 mg (95%) of 20 as an amorphous white solid.1H NMR (400 MHz, DMSO-d6)
δ
8.16 (bs, 18H, NH3+), 7.31 (s, 4H, Ar), 5.27 (m, 2H, CH=), 4.56 (m, 1H, CHNH), 4.39 (m, 2H, CH2O),
4.29 (m, 2H, CH2O), 4.23 (t, J = 5.0 Hz, 8H, CH2O), 4.13 (t, J = 5.0 Hz, 4H, CH2O), 3.32 (m, 12H,
CH2N), 2.08 (t, J = 7.4 Hz, 2H, CH2CO), 1.91 (m, 4H, CH2CH=), 1.43 (m, 2H, CH2), 1.17 (m, 20H, CH2),
0.82 (t, J = 6.4 Hz, 3H, CH3). 13C NMR (100 MHz, DMSO-d6)
δ 172.6 (CONH), 164.8 (COO), 151.7 (Ar),
140.6 (Ar), 129.6 (CH=), 130.0 (CH=), 125.2 (Ar), 108.6 (Ar), 69.2 (CH2O), 65.9 (CH2O), 63.7 (CH2O),
46.8 (CHNH), 40.7 (CH2N), 35.2 (CH2CO), 31.3, 29.5, 29.3, 29.1, 29.0, 28.9, 27.0, 26.6, 25.3, 22.1 (CH2),
13.9 (CH3). HPLC (tR) [gradient: A:B, 10–100% of A in 10 min]: 6.74 min. HRMS (ESI+) m/z: Calc. for
C47H79N7O11 918.5827. Found 917.5874.
4.1.22. Bis-O-[3,4,5-tris(2-ammonium-1-ethoxy)benzoyl]-N-octadecanoyl Serinol Trifluoroacetate (21
)
Following the general procedure, a solution of 15 (50 mg, 0.03 mmol) in CH2Cl2 (10 mL),
was treated with TFA (0.25 mL) to afford 26.3 mg (50%) of 21 as an amorphous solid of cream color.
1H NMR (400 MHz, DMSO-d6)
δ 8.20 (bs, 18H, NH), 7.34 (s, 4H, Ar), 5.31 (m, 8H, CH=), 4.58 (m, 1H,
CHNH), 4.43 (m, 2H, CH2O), 4.33 (m, 2H, CH2O), 4.25 (m, 8H, CH2O), 4.17 (m, 4H, CH2O), 3.32 (m, 8H,
CH2N), 3.19 (m, 4H, CH2N), 2.75 (m, 4H, CH2CH=), 2.65 (m, 4H, CH2CH=), 2.13 (m, 2H, CH2CO) 1.96
(m, 4H, CH2CH=), 1.49 (m, 2H, CH2), 1.25 (m, 6H, CH2), 0.85 (t, J = 8.0, 3H, CH3). 13C NMR (100 MHz,
DMSO-d6)
δ 172.9 (CONH), 166.2 (COO), 152.1 (Ar), 140.8 (Ar), 130.4 (CH=), 129.7 (CH=), 128.6 (CH=),
128.5 (CH=), 128.3 (CH=), 128.2 (CH=), 128.0 (CH=), 127.9 (CH=), 125.8 (Ar), 108.8 (Ar), 69.8 (CH2O),
66.3 (CH2O), 64.3 (CH2O), 47.2 (CHNH), 40.7 (CH2N), 35.4 (CH2CO), 31.3, 29.1, 28.2, 27.0, 26.6, 25.7,
25.6, 25.5, 22.4 (CH2), 14.3 (CH3). HPLC (tR) [gradient: A:B, 10–100% of A in 10 min]: 6.65 min. HRMS
(ESI+) m/z: Calc. for C49H77N7O11 939.5735. Found 938.6043.
4.2. Biological Methods
4.2.1. Bacterial Strains
The bacterial strains used in this study included: Salmonella enterica serovar Typhimurium
SV5015 [34] Listeria monocytogenes EGD-e [35], Staphylococcus aureus strain Newman [36]. Staphylococcus
aureus SC-1 (gift of Dr. Daniel López, Centro Nacional de Biotecnología, CNB-CSIC, Madrid, Spain)
and USA-300 [28] have been reported as multidrug-resistant (MDR) isolates. These bacteria were
grown at 37 ◦C and shaking conditions (150 rpm) in the following nutrient-rich media: Luria-Bertani
(LB) medium for S. Typhimurium; brain heart infusion (BHI) medium for L. monocytogenes;
and, trypticase-soy broth (TSB) for S. aureus. Under these growth conditions, the overnight cultures
reached an optical density (OD600) of 2.0–3.0 (S. Typhimurium and L. monocytogenes) and 10.0–12.0
(S. aureus).
4.2.2. Determination of Minimal Inhibitory Concentrations (MIC) of Aminophenol Compounds
Overnight cultures were diluted 1:18,000 (S. Typhimurium and L. monocytogenes) or 1: 60,000
(S. aureus) in fresh media. A volume of 50
medium containing serial dilutions (1:4) of the aminophenol compound. The aminoglycoside antibiotic
kanamycin was tested in parallel as control. Starting concentrations used were 50
g mL−1 for the
aminophenol compounds and 150
g mL−1 for kanamycin. After an overnight culture in shaking
µL of this culture was further used to inoculate 2 mL of
µ
µ
conditions, minimal inhibitory concentration (MIC) was determined as the minimum inhibitory
concentration of the compound inhibiting bacterial proliferation (lack of turbidity in the culture).

Molecules 2018, 23, 1513
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4.2.3. Monitoring of Bacterial Growth in the Presence of Aminophenol Compounds
Overnight cultures were diluted 1:100 (L. monocytogenes) or 1:500 (S. aureus) in fresh medium.
After aprox. 1.5–2 h of incubation when cultures reached an OD600 ~0.2, bacteria were exposed to
four-fold the MIC value of the specific antimicrobial to be tested. OD600 where then measured every
30 min.
4.2.4. Toxicity Assays in Eukaryotic Cell Cultures
Cultures of normal rat fibroblasts NRK-49F (ATCC CRL-1570) and HeLa human epithelial cells
(ATCC CCL-2) were used to assess putative toxicity effects of the aminophenol-derivate compounds.
The cells were seeded in 24-well plates and propagated in Dulbecco’s modified Eagle’s medium
(DMEM) or Eagle’s minimum essential medium containing 10% (v/v) fetal bovine serum (FBS) at 37 ◦C
in a 5% CO2 atmosphere. Following adherence of the cells to the substrate, the medium was replaced
with fresh medium containing different concentrations of the compounds. To 0.5 mL of tissue culture
medium per well either 5
added. This was equivalent to a final concentration of 100 to 20
containing 5 L of the solvent DMSO was analyzed in parallel. The toxicity potential of the compounds
µL to 1.25
µ
L of a 10 mg mL−1 stock solution of the compound to test, were
g mL−1, respectively. A control
µ
µ
was evaluated based on two main parameters: morphological changes in the fibroblast or epithelial
cells and fraction of the monolayer culture that lost adherence to the substrate. Changes in these
parameters were monitored by microscopy. Cells were fixed with 3% (v/v) paraformaldehyde (PFA)
and processed as described [37]. Images were acquired on an inverted Leica DMI 6000B microscope
with an automated CTR/7000 HS controller (Leica Microsystems, Wetzlar, Germany) and an Orca-R2
CCD camera (Hamamatsu Photonics, Hamamatsu, Japan).
4.2.5. Phase-Contrast Microscopy
To determine probable effect of the aminophenol-derivate compounds in bacterial morphology,
bacteria were grown to mid-exponential phase (OD600 ~0.2–0.3). At this time, bacteria were exposed
for 1 h to a concentration of the compound equivalent to 4 MIC and the OD600 measured every 30 min.
To monitor probable morphological changes not appreciable by OD600, the bacteria were harvested
by centrifugation (4300
microscopy as described [38].
×
g, 5 min, RT), washed in PBS, fixed with 3% (v/v) PFA, and processed for
Supplementary Materials: Supplementary materials are available online. Copies of representative 1H and
13C NMR spectra are included.
Author Contributions: Authors declare no potential conflict of interest. All co-authors participated sufficiently in
the work to take responsibility for the content and all co-authors approved the final version. Conceptualization:
M.J.C., M.-J.P.-P., J.-C.Q., F.G.-d.P., and A.S.-F.; Data Curation: F.G.-d.P., and A.S.-F.; Investigation: F.G.-d.P. and
A.S.-F.; Methodology: A.J., P.G., S.d.l.P., and A.M.; Supervision: F.G.-d.P. and A.S.-F.; Writing—Original Draft
Preparation: F.G.-d.P. and A.S.-F.; Writing—Review and Editing: F.G.-d.P. and A.S.-F.
Funding: This research was funded by the Spanish “Plan Nacional de Cooperación Público-Privada, Subprograma
INNPACTO” project number IPT-2012-0213 060000, cofinanced by the FEDER program (to J.-C.Q., F.G.-d.P, A.S.-F.
and M.-J.P.-P.), “Ministerio de Economia, Industria y Competitividad, Gobierno de España” and “European
Regional Development Funds” projects numbers SAF2015-64629-C2-1-R (MINECO/FEDER) (to A.S.-F., M.-J.C.
and M.-J.P.-P.) and BIO2016-77639-P (MINECO/FEDER) (to F.G.-d.P.). The Spanish MEC/MINECO is also
acknowledged for Garantía Juvenil contracts to A.J. and S.d.l.P.
Acknowledgments: We thank Daniel López (CNB-CSIC) for providing us with the S. aureus strains sensitive and
resistant to drugs and María Angeles Bonache for help with the editing of the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).