Synthesis and antimicrobial studies of new antibacterial azo-compounds active against staphylococcus aureus and listeria monocytogenes

Article abstract of DOI:10.3390/molecules22081372

Some novel (phenyl-diazenyl)phenols (4a–m) were designed and synthesized to be evaluated for their antibacterial activity. Starting from an active previously-synthesized azobenzene chosen as lead compound, we introduced some modifications and optimization of the structure, in order to improve solubility and drug conveyance. Structures of all newly-synthesized compounds were confirmed by ¹H nuclear magnetic resonance (NMR), mass spectrometry, and UV-Vis spectroscopy. Antibacterial activity of the new compounds was tested with the dilution method against the bacteria strains Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa PAO1. All the compounds were selectively active against Gram-positive bacteria. In particular, compounds 4d, 4h, and 4i showed the highest activity against S. aureus and Listeria monocytogenes, reaching remarkable MIC₁₀₀ values of 4 μg/mL and 8 μg/mL. The relationship between antimicrobial activity and compound structure has suggested that the presence of hydroxyl groups seems to be essential for antimicrobial activity of phenolic compounds.

Full text of DOI:10.3390/molecules22081372

molecules
Article
Synthesis and Antimicrobial Studies of New
Antibacterial Azo-Compounds Active against
Staphylococcus aureus and Listeria monocytogenes
Stefano Piotto 1, Simona Concilio 2,*, Lucia Sessa 1, Rosita Diana 3, Gabriel Torrens 4,
Carlos Juan 4, Ugo Caruso 3 and Pio Iannelli 1
1
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano (SA), Italy;
piotto@unisa.it (S.P.); lucsessa@unisa.it (L.S.); piannelli@unisa.it (P.I.)
Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II 132, 84084
2
Fisciano (SA), Italy
3
Department of Chemical Science, University of Napoli “Federico II”, Cupa Nuova Cintia, Napoli 21-80126 ,
Italy; rosita.diana@libero.it (R.D.); ugo.caruso@unina.it (U.C.)
Servicio de Microbiología, Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de
4
Palma (IdISPa), Ctra. Valldemossa 79, 07010 Palma de Mallorca, Spain; gabriel.torrens@ssib.es (G.T.);
carlos.juan@ssib.es (C.J.)
*
Correspondence: sconcilio@unisa.it; Tel.: +39-089-964-115
Received: 24 July 2017; Accepted: 18 August 2017; Published: 19 August 2017
Abstract: Some novel (phenyl-diazenyl)phenols (4a–m) were designed and synthesized to be
evaluated for their antibacterial activity. Starting from an active previously-synthesized azobenzene
chosen as lead compound, we introduced some modifications and optimization of the structure, in
order to improve solubility and drug conveyance. Structures of all newly-synthesized compounds
were confirmed by 1H nuclear magnetic resonance (NMR), mass spectrometry, and UV-Vis
spectroscopy. Antibacterial activity of the new compounds was tested with the dilution method
against the bacteria strains Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and
Pseudomonas aeruginosa PAO1. All the compounds were selectively active against Gram-positive
bacteria. In particular, compounds 4d
Listeria monocytogenes, reaching remarkable MIC100 values of 4
,
4h, and 4i showed the highest activity against S. aureus and
g/mL and 8 g/mL. The relationship
µ
µ
between antimicrobial activity and compound structure has suggested that the presence of hydroxyl
groups seems to be essential for antimicrobial activity of phenolic compounds.
Keywords: azo-compound; Gram-positive antibacterial; listeria monocytogenes; synthesis
1. Introduction
Substituted azobenzene generally have a color ranging from yellow to red, due to the extensive
conjugated system and presence of various electron-withdrawing or electron-donating substituents on
the aromatic rings. A key feature of azobenzene and other molecules characterized by a high conjugated
pattern is its ability to absorb light with a consequent modification of its structure through the transition
from cis isomeric form to the trans form. This feature is widely-used in light-responsive materials [
liquid crystals [ ], electronic devices [ 11], or in NLO chromophores [12 13]. In recent papers, we
reported a series of azobenzene-based molecules with antimicrobial properties [14 17]. For example,
1–5],
6,
7
8
–
,
–
studies carried out in our labs showed that A4 (Figure 1) is one of the most active of the series of
compounds synthesized so far, showing antimicrobial activity against Staphylococcus aureus (MIC100
20
against Candida albicans (MIC0 17
the growth of 100% of fungi), and Listeria monocytogenes (MIC100 25 µg/mL) [16].
µg/mL, the Minimum Inhibitory Concentration required to inhibit the growth of 100% of bacteria),
µg/mL, the Minimum Inhibitory Concentration required to inhibit
Molecules 2017, 22, 1372; doi:10.3390/molecules22081372
www.mdpi.com/journal/molecules

Molecules 2017, 22, 1372
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Figure 1. Chemical structure of lead compound A4 [16].
Listeria monocytogenes is one of the most dangerous food pathogens. It shows the ability to
survive adverse conditions such as vacuum, low temperature, wide range of pH (4–9), ultraviolet
rays and is resistant to conventional pasteurization [18
by L. monocytogenes serogroup 4 [22 23]. The ingestion of products contaminated with this organism
may be a potential health threat to high-risk populations, such as the immune-suppressed, pregnant
women, and the elderly [24 25]. Azo-compounds that possess antilisterial properties may therefore
–21]. Human listeriosis is most often caused
,
,
be expected to become valuable additives as consumer concern with preventing L. monocytogenes
infections increases.
In an attempt to improve the bioavailability of these molecules, their effectiveness and/or
specificity of action, various structural modifications have been proposed of the lead compound
A4, in order to obtain compounds with better physicochemical properties, while maintaining their
antimicrobial activity [26]. In order to evaluate the effect of different substituents on the azobenzene
structure, we introduced some new structural changes.
By moving the hydroxyl group from one azobenzene ring to the other and/or adding substituents
on both the rings, we obtained compounds with improved antimicrobial activity, with remarkable
MIC100 value of 10 µg/mL against S. aureus, and MIC0 of 3 µg/mL against C. albicans [26]. Nevertheless,
for some new compounds reported here (like 4l), a tendency to crystallize from aqueous solution was
also observed during the period of incubation, and in these cases, it was not possible to reach high
concentration of active molecule in the dilution tests. For these molecules, the presence of aromatic
rings with hydroxyl groups induces a slow crystallization of the molecule, probably due to the easy
packing of the aromatic structures.
Starting from these observations, we decided to improve the activity of these molecules and to
achieve a better understanding of the structure-activity relationship of this class of compounds. The
changes include the introduction of alkyl derivatives from four to eight carbon atoms such as butyl,
isobutyl, neopentyl, isopentyl, and hexyl moieties, in order to reduce lattice energies in the crystals.
The main goal was to prevent the formation of highly ordered crystals that can be an obstacle in
reaching the most convenient solubility for pharmaceutical applications.
2. Results and Discussion
2.1. Chemistry
The chemical structures of substituted azobenzene derivatives (4a–m) are reported in Figure 2.
The synthetic scheme of substituted-azobenzene (4a–m) derivatives is reported in Figure 3.

Molecules 2017, 22, 1372
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R1
R2
R3
R4
R5
4a
4b
4c
4d
4e
4f
4g
4h
4i
O(CH2)3CH3
OCH2CH(CH3)2
OCH2C(CH3)3
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
OH
OH
OH
OH
OH
OCH3
OH
OH
OH
OH
OH
OH
OH
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
OCH2CH2CH(CH3)2
OCH2CH(CH2CH3)CH2(CH2)2CH3
H
OCH2CH(CH3)2
OCH2C(CH3)3
OCH2CH2CH(CH3)2
OCH2CH(CH2CH3)CH2(CH2)2CH3
OH
H
OH
OH
OH
OH
OH
OH
OH
4j
4k
4l
CH3
OCH3
H
H
H
H
4m
Figure 2. Substituted-(phenyl-diazenyl) phenols derivatives (4a–m).
Figure 3. Synthetic route for compounds 4a–m. i: Alkylhalide, K2CO3, DMF reflux, 3 h; ii: SnCl2,
ethanol reflux, 2 h; iii: 0–5 ◦C, NaNO2 (aq), HCl 37%; iv: 10–15 ◦C, 1,3-dimethylphenol (for 4a
resorcinol (for 4g–m), NaOH (aq). For Rn moieties, see Figure 2.
–f) or
2.2. Thermal and Optical Properties
In Table 1, thermal and optical properties of the synthesized compounds are reported. Analogues
4b–m showed only a melting peak in the first heating run and they were not able to crystallize from
the melt. Only compound 4a, after the melting peak at 88.7 ◦C, shows a crystallization phenomenon
◦
during the cooling run, at 53.3 C. When heated in the second run, it showed the same melting peak as
◦
the first. Compound 4c showed three melting peaks in the first heating run at 85.0, 116.7, and 126.0 C,
ascribed to a polymorphism, as confirmed by optical observation under polarized light, but it was not
possible to observe any crystallization from the melt.

Molecules 2017, 22, 1372
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Table 1. Thermal and optical properties of compounds 4a–m.
Thermal Characterization
Optical Characterization
Molecule
◦
◦
−1
−1
)
Tm ( C)
∆Hm (J/g)
Tc ( C)
∆Hc (J/g)
λ
(nm)
ε
max
(L mol cm
max
4a
4b
88.7
126.5
85.00
116.7
126.0
74.2
59.2
-
156.3
179.3
125.3
82.6
235
76.5
74.3
52.6
19.5
10.8
88.4
56.1
-
54.2
80.2
73.6
47.2
75.4
91.5
24.8
53.35
-
66.17
-
360
360
26,000
25,000
-
-
26,100
4c
360
4d
4e
4f *
4g
4h
4i
4j
4k
4l
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
361
361
38,000
33,100
384
384
384
384
382
447
344
3300
29,500
35,700
3000
8300
7800
238
160
4m
4600
Tm = melting temperature, from DSC analysis, 10 ◦C/min, nitrogen flow; Tc = crystallization temperature, from
DSC cooling run; Instrument error Hm Hc = melting/crystallization enthalpy, evaluated by integration
0.5 ◦C.
of the peak. Experimental error 5%. λmax = wavelength at the principal absorption maximum, εmax = molar
extinction coefficient at absorption maximum. * Compound 4f is an oil at room temperature.
±
∆
/∆
±
The spectral region 650–240 nm was investigated by UV-Vis spectrophotometry at a concentration
of about 3.0
10−5 mol L−1 of azo-compound in acetonitrile solution (Table 1). The UV-visible spectra
for 4a are mainly dependent only on the azobenzene unit, which is the same for all compounds.
×
–
m
The UV-Vis absorption spectra of all the analogues in solution, showed the typical absorption bands
of the electronic transitions of the azobenzene chromophore, with absorption maxima ranging from
360 nm to 447 nm.
2.3. Antibacterial Activity
The MIC100 of all synthesized compounds was determined by the microbroth dilution method for
Staphylococcus aureus ATCC 29213, a Listeria monocytogenes clinical strain, Pseudomonas aeruginosa PAO1,
and Escherichia coli MG1655 reference strains (Table 2).
Table 2. Antimicrobial activity of 4a–m analogues, expressed as MIC100 (µg/mL).
MIC100 (µg/mL) after 24 h
Staphylococcus aureus
Listeria monocytogenes
Escherichia coli
Pseudomonas aeruginosa
4a
4b
4c
4d
4e
4f
4g
4h
4i
32
128
>128
4
>128
128
>128
8
48
>128
96
8
8
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>32 *
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>32 *
>128
12
>128
>128
4
4
16
>128
>32 *
16
4j
4k
4l
>128
>32 *
16
4m
MIC100: Minimum Inhibitory Concentration required inhibiting the growth of 100% of organisms after 24 h. The
values are the geometric mean of at least three determinations. * Dissolution problems: For these compounds, the
initial solution was prepared to achieve 128 g/mL, but in the end, a maximum MIC of 32 g/mL was reached,
before observing a crystallization of the compound.
µ
µ

Molecules 2017, 22, 1372
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For six of the analyzed compounds, a strong activity against S. aureus and L. monocytogenes was
measured, with MIC100 values up to 4 g/mL and 8 g/mL, respectively (for 4d 4h and 4i). To our
µ
µ
,
knowledge, these MIC100 values are the lowest ever reached for antimicrobial azobenzene compounds.
On the contrary, the activity against gram-bacteria E. coli and P. aeruginosa was not detectable for any
of the synthesized compound, confirming the trend already observed in our previous reports [16,26].
In particular, the high antibacterial activity against Listeria is particularly noteworthy, because of the
ability of this bacterium to survive in several adverse conditions. Since we do not know the mechanism
of action, it is not easy to rationalize the activity of all the analogues 4a–m. Nevertheless, some
general considerations can be done, regarding their behavior. The presence of long and disordered
aliphatic chains produced the expected effect on the tendency to crystallization for many of the
obtained compounds, making them more soluble and available in the incubation medium, compared
to analogues 4k–m. For these, it was not possible to achieve high concentration in the dilution tests,
due to their easy precipitation from the solution. Another consideration is that by completely removing
all hydroxyl groups from the azobenzene moiety, we obtained a complete loss of antibacterial activity
(compound 4f); this indicates that the possibility of making hydrogen bonds is probably fundamental
for the interaction of these antibiotics with their target.
3. Materials and Methods
3.1. General
All reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy) and used without
further purification. Optical observations were performed by using a Jenapol (Zeiss S.p.A., Milano,
Italy) microscope fitted with a Linkam THMS 600 hot stage (Linkam, Waterfield, Epsom, Tadworth,
UK). Phase transition temperatures and enthalpies were measured using a DSC scanning calorimeter
◦
Perkin Elmer Pyris 1 (PerkinElmer, Waltham, MA, USA) at a scanning rate of 10 C/min, under
◦
nitrogen flow. UV absorption spectra of the samples were recorded at 25 C in acetonitrile solution, on
a Perkin Elmer Lambda 19 spectrophotometer. The spectral region 650–240 nm was investigated using
−5 −1
10 mol L was
a cell path length of 1.0 cm. Azobenzene chromophore concentration of about 3.0
×
1
used. H-NMR and 13C-NMR spectra were recorded with a Bruker DRX/400 Spectrometer (Bruker,
Billerica, MA, USA).
High-resolution mass spectra were acquired on a LTQ-Orbitrap instrument (Thermo-Fisher,
Waltham, MA, USA) operating in negative (compounds 4a–e, 4g–m) or positive (compound 4f) ion
mode. Each compound was singularly dissolved in methanol at a concentration of 0.1 mg/mL and
injected into the MS ion source. Spectra were acquired in the 150–400 m/z range.
3.2. General Method of the Synthesis of (Phenyl-diazenyl)phenols Derivatives 4a–m
3.2.1. Synthesis of 1-Alkyloxy-4-nitrobenzene (2a–e, 2g–j)
A suspension of 0.0216 mol of p-nitrophenol in 25 mL of DMF was prepared. Then, 6.0 g of K2CO3
and 0.0264 mol of alkyliodide or bromide were added, and the solution refluxed for 3 h. The solution
was then filtered in water and the crude precipitate was recovered and dried under vacuum. Yield
ranged between 60 and 90%.
1
1-Butoxy-4-nitrobenzene (2a). H-NMR CDCl3: (
δ, ppm) = 8.15 (d, 2H), 7.25 (d, 2H), 4.06 (t, 2H), 1.76 (m,
2H), 1.47 (m, 2H), 0.99 (t, 3H). Yield 85%.
1
1-(Isobutoxy)-4-nitrobenzene (2b
,
2g). H-NMR (DMSO-d6): (
δ, ppm) = 8.19 (d, 2H), 7.15 (d, 2H), 3.90 (d,
2H), 2.01 (m, 1H), 0.91 (d, 6H). Yield 80%.
1
1-(Neopentyloxy)-4-nitrobenzene (2c
,
2h). H-NMR (DMSO-d6): (
δ, ppm) = 8.19 (d, 2H), 7.15 (d, 2H), 3.90
(s, 2H), 1.05 (s, 9H). Yield 60%.

Molecules 2017, 22, 1372
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1
1-(Isopentyloxy)-4-nitrobenzene (2d
,
2i). H-NMR (DMSO-d6): (
δ, ppm) = 8.16 (d, 2H), 7.10 (d, 2H), 4.10
(t, 2H), 1.78 (m, 3H), 0.91 (d, 6H). Yield 60%.
1
1-((2-Ethylhexyl)oxy)-4-nitrobenzene (2e
,
2j). H-NMR (DMSO-d6): (
δ
, ppm) = 8.18 (d, 2H), 7.15 (d, 2H),
4.01 (d, 1H), 3.95 (d, 1H), 1.71 (m, 1H), 1.38 (m, 4H), 1.32 (m, 4H), 0.88 (m, 6H). Yield 65%.
3.2.2. Synthesis of 4-Alkyloxyaniline (3a–e, 3g–j)
The appropriate 1-alkyloxy-4-nitrobenzene (0.0270 mol) was dissolved in 70 mL of ethanol. Tin
chloride (0.119 mol) was dissolved in 30 mL of ethanol and was added dropwise. The reaction was
set to take place for two hours at 80 ◦C. The crude product was extracted twice in a mixture of ethyl
acetate/water (1:1), in which 60 g of potassium carbonate was dissolved. The organic solvent was
evaporated at reduced pressure and the product was obtained as a yellow oil. Yield ranged between
65 and 85%.
1
4-Butoxyaniline (3a). H-NMR (DMSO-d6): (
δ
, ppm) = 6.64 (d, 2H), 6.52 (d, 2H), 4.56 (s, 2H), 3.58 (t, 2H),
1.75(m, 2H), 1.39 (m, 2H), 0.99 (t, 3H). Yield 65%.
1
4-Isobutoxyaniline (3b
,
3g). H-NMR (DMSO-d6): (
δ, ppm) = 6.64 (d, 2H), 6.50 (d, 2H), 4.56 (s, 2H), 3.46
(d, 2H), 2.22 (m, 1 H), 0.96 (s, 6H). Yield 65%.
1
4-Neopentyloxyaniline (3c
,
3h). H-NMR (DMSO-d6): (
δ, ppm) = 6.74 (d, 2H), 6.66 (d, 2H), 4.03 (s, 2H),
3.39 (s, 2H), 1.55 (s, 9H). Yield 85%.
1
4-Isopentyloxyaniline (3d
,
3i). H-NMR (DMSO-d6): (
δ
, ppm) = 8.16 (d, 2H), 7.10 (d, 2H), 4.10 (t, 2H),
3.42 (s, 2H), 1.74 (m, 1H), 1.63 (m, 2H), 0.91 (d, 6H). Yield 83%.
1
4-((2-Ethylhexyl)oxy)aniline (3e
,
3j). H-NMR (DMSO-d6): (
δ, ppm) = 6.74 (d, 2H), 6.66 (d, 2H), 4.03 (d,
2H), 3.40 (d, 2H), 1.98 (m, 1H), 1.55 (m, 2H), 1.25 (m, 6H), 0.90 (m, 6H). Yield 85%.
3.2.3. Synthesis of Azobenzene Derivatives 4a–m
Azo compounds 4a–m were synthesized according to the classic scheme of diazo coupling
reactions, as reported in ref. [16], and illustrated in Figure 3. The general procedure was the following:
0.0052 mol of the selected alkyloxyaniline (3a–m) were suspended in a solution containing 4.0 mL
of water and 1.1 mL of HCl 37% (w/w). The solution was cooled at 0–5 ◦C in a water/ice bath. A
solution of 0.42 g of sodium nitrite (0.006 mol) dissolved in 1.2 mL of water was added drop-wise,
obtaining a suspension of the diazonium salt (solution A). Separately, a solution containing 1.72 g
of NaOH (0.0420 mol) in 16 mL of water with 0.0052 mol of the proper phenol (depending on 4a–m)
◦
was prepared (solution B). Solution A was added drop-wise to solution B, under stirring at 12 C,
adjusting the pH at 9–10, with addition of NaOH, if necessary. The system was left reacting for 30 min.
Then, 1.2 mL of acetic acid were added, in order to reach a pH value of 5–6, under stirring. A reddish
precipitate of the azo compound formed. The crude precipitate was filtered and dried under vacuum.
Yields ranged between 45 and 90%.
4-((4-Butoxyphenyl)diazenyl)-2,6-dimethylphenol 4a. 4-Butoxyaniline (3a) and 2-6-dimethylphenol were
used as starting reagents. The crude product was crystallized from water/ethanol (10:1) to give pure
1
4a as gold yellow crystals. Final yield 85%. H-NMR (DMSO-d6): (
δ
, ppm) = 9.01 (broad, 1H), 7.76 (d,
2H), 7.49 (s, 2H), 7.07 (d, 2H), 4.06 (t, 2H), 2.25 (s, 6H), 1.76 (m, 2H), 1.47 (m, 2H), 0.99 (t, 3H). 13C-NMR
(DMSO-d6): (δ, ppm) = 163.3; 161.2; 148.5; 146.3; 127.7; 122.7; 116.2; 70.6; 31.3; 20.7; 17.4, 14.7. HR-MS
(m/z): 298.17 [M − H]−.
4-((4-Isobutoxyphenyl)diazenyl)-2,6-dimethylphenol 4b. 4-Isobutoxyaniline (3b) and 2-6-dimethylphenol
were used as starting reagents. The crude product was crystallized from water/ethanol (10:1) to give
1
pure 4b as yellow crystals. Final yield 90%. H-NMR (DMSO-d6): (
δ
, ppm) = 7.76 (d, 2H), 7.49 (s, 2H),
7.07 (d, 2H), 4.06 (d, 2H), 3.85 (s, 1H), 2.25 (s, 6H), 2.04 (m, 1H), 1.03 (d, 6H). 13C-NMR (DMSO-d6): (
δ,

Molecules 2017, 22, 1372
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ppm) = 163.4; 161.0; 148.6; 146.1; 127.6; 124.6; 124.3; 116.5; 75.0; 27.5; 20.3; 17.2. HR-MS (m/z): 298.17
[M − H]−.
2,6-Dimethyl-4-((4-(neopentyloxy)phenyl)diazenyl)phenol 4c
.
4-Neopentyloxyaniline (3c) and 2-6-
dimethylphenol were used as starting reagents. The crude product was crystallized from water/ethanol
1
(10:1) to give pure 4c as dark yellow crystals. Final yield 40%. H-NMR (acetone-d6): (
δ
, ppm) = 7.80 (d,
2H), 7.53 (s, 2H), 7.05 (d, 2H), 4.06 (s, 2H), 2.29 (s, 6H), 1.03 (s, 9H). 13C-NMR (DMSO-d6): (
163.6; 161.0; 148.9; 146.0; 127.5; 124.5; 124.0; 117.0; 79.5; 32.6; 26.6; 17.1. HR-MS (m/z): 312.18 [M
δ, ppm)−=
−
H] .
4-((4-(Isopentyloxy)phenyl)diazenyl)-2,6-dimethylphenol 4d
.
4-Isopentyloxyaniline (3d) and 2-6-
dimethylphenol were used as starting reagents. The crude product was crystallized from water/ethanol
1
(10:1) to give pure 4d as dark yellow crystals. Final yield 85%. H-NMR (acetone-d6): (
δ, ppm) = 7.83
(d, 2H), 7.56 (s, 2H), 7.05 (d, 2H), 4.14 (t, 2H), 2.32 (s, 6H), 1.87 (m, 1H), 1.72 (m, 2H), 0.98 (d, 6H).
13C-NMR (DMSO-d6): (
δ, ppm) = 163.1; 161.0; 148.3; 146.1; 127.6; 124.6; 115.9; 70.8; 38.0; 28.1; 23.3; 17.2.
HR-MS (m/z): 311.18 [M − H]−.
4-((4-((2-Ethylhexyl)oxy)phenyl)diazenyl)-2,6-dimethylphenol 4e. 4-((2-Ethylhexyl)oxy)aniline (3e) and
2-6-dimethylphenol were used as starting reagents. The crude product was crystallized from
water/ethanol (10:1) to give pure 4e as orange crystals. Final yield 60%. 1H-NMR (acetone-d6):
(δ, ppm) = 7.83 (d, 2H), 7.55 (s, 2H), 7.08 (d, 2H), 4.10 (t, 2H), 2.32 (s, 6H), 1.75 (m, 1H), 1.52 (m, 2H),
1.45 (m, 6H), 0.93(m, 6H). 13C-NMR (DMSO-d6): (
δ, ppm) = 163.4; 161.0; 148.6; 146.1; 127.6; 124.5; 124.2;
116.4; 73.4; 39.7; 31.8; 30.5; 24.9; 24.0; 17.2; 14.6; 12.2. HR-MS (m/z): 354.23 [M − H]−.
1-(4-Methoxy-3,5-dimethylphenyl)-2-phenyldiazene 4f. The procedure for obtaining 4f was a simple
methylation of compound 3b, described in ref [26]. Methyl iodide (0.55 mL) was added to a solution
containing 1.00 g of 3b and 1.22 g of potassium carbonate in 15.0 mL of DMF. The reaction was
conducted under reflux for 5 h. The final mixture was then poured in 100 mL of cold distilled water.
The organic layer was then extracted in chloroform, dried with sodium sulfate, and the solvent was
1
removed under reduced pressure. Final yield 57%. H-NMR (DMSO-d6): (
δ
, ppm) = 7.95 (s, 2H), 7.85
, ppm) = 162.6; 153.1; 148.3;
130.7; 130.0; 129.7; 125.8; 123.0; 61.2; 17.1. HR-MS (m/z): 241.13 [M + H]+.
(s, 2H), 7.62 (m, 3H), 3.74 (s, 3H), 2.33 (s, 6H). 13C-NMR (DMSO-d6): (
δ
4-((4-Isobutoxyphenyl)diazenyl)benzene-1,3-diol 4g. 4-Isobutoxyaniline (3g) and resorcinol were used as
starting reagents. The crude product was purified by column chromatography (ethyl acetate/hexane
1
3:7) to give pure 4g as red crystals. Final yield 40%. H-NMR (Acetone-d6): (
δ
, ppm) = 7.85 (d, 2H),
7.71 (d, 1H), 7.10 (d, 2H), 6.59 (d, 2H), 6.40 (s, 1H), 3.88 (d, 2H), 1.05 (d, 6H). 13C-NMR (DMSO-d6): (
δ,
ppm) = 164.5; 163.4; 161.2; 148.6; 134.6; 125.1; 124.2; 116.4; 110.6; 103.7; 75.0; 27.5; 20.3. HR-MS (m/z):
286.13 [M − H]−.
4-((4-(Neopentyloxy)phenyl)diazenyl)benzene-1,3-diol 4h. 4-Neopentyloxyaniline (3h) and resorcinol
were used as starting reagents. The crude product was purified by column chromatography (ethyl
1
acetate/hexane 3:7) to give pure 4h as brilliant red crystals. Final yield 40%. H-NMR (Acetone-d6):
(δ, ppm) = 7.83 (d, 2H), 7.71 (d, 1H), 7.11 (d, 2H), 6.59 (d, 1H), 6.40 (s, 1H), 3.76 (s, 2H), 1.06 (s, 9H).
13C-NMR (DMSO-d6): (
δ, ppm) = 164.5; 163.6; 161.2; 148.9; 134.7; 125.1; 124.0; 117.1; 110.6; 103.5; 79.3;
32.4; 26.4. HR-MS (m/z): 300.15 [M − H]−.
4-((4-(Isopentyloxy)phenyl)diazenyl)benzene-1,3-diol 4i. 4-Isopentyloxyaniline (3i) and resorcinol were
used as starting reagents. The crude product was purified by column chromatography (ethyl
acetate/hexane 3:7) to give pure 4i as dark red crystals. Final yield 45%. 1H-NMR (Acetone-d6):
(δ, ppm) = 7.84 (d, 2H), 7.71 (d, 1H), 7.10 (d, 2H), 6.60 (d, 1H), 6.40 (s, 1H), 4.15 (t, 2H), 1.81(m, 1H), 1.71
(m, 2H), 0.98 (d, 6H). 13C-NMR (DMSO-d6): (
δ, ppm) = 164.2; 162.7; 160.8; 147.9; 134.3; 124.7; 124.1;
115.5; 110.3; 103.4; 70.4; 37.6; 27.8; 22.9. HR-MS (m/z): 300.15 [M − H]−.
4-((4-((2-Ethylhexyl)oxy)phenyl)diazenyl)benzene-1,3-diol 4j
.
4-((2-Ethylhexyl)oxy)aniline (3j) and
resorcinol were used as starting reagents. The crude product was purified by column chromatography

Molecules 2017, 22, 1372
8 of 10
1
(ethyl acetate/hexane 3:7) to give pure 4j as red crystals. Final yield 35%. H-NMR (Acetone-d6): (
ppm) = 7.80 (d, 2H), 7.67 (d, 1H), 7.08 (d, 2H), 6.56 (d, 1H), 6.36 (s, 1H), 3.98 (d, 2H), 2,01(m, 1H), 1.74
(m, 2H), 1.48(m, 2H), 1.32 (m, 4H), 0,90 (m, 6H). 13C-NMR (DMSO-d6): (
, ppm) = 163.8; 162.8; 160.5;
δ,
δ
147.9; 133.9; 124.3; 123.5; 115.9; 110.1; 103.2; 72.6; 39.1; 31.2; 29.9; 24.3; 23.6; 14.1; 11.6. HR-MS (m/z):
342.19 [M − H]−.
4-((4-Hydroxyphenyl)diazenyl)benzene-1,3-diol 4k. 4-Aminophenol and resorcinol were used as starting
reagents. The crude product was crystallized from water/methanol (10:1) to give pure 4k as red
1
crystals. Final yield 73%. H-NMR (DMSO-d6): (
δ
, ppm) = 10.37, 10.18, 7.74 (d, 2H), 7.62 (d, 1H), 6.91
(d, 2H), 6.50–6.46 (dd, 1H), 6.32 (s, 1H). 13C-NMR (DMSO-d6): (
δ, ppm) = 164.1; 160.9; 160.6; 146.8;
134.1; 124.7; 124.4; 116.9; 110.1; 103.1. HR-MS (m/z): 230.07 [M − H]−.
4-(p-Tolyldiazenyl)benzene-1,3-diol 4l. p-Toluidine and resorcinol were used as starting reagents. The
crude product was crystallized from boiling n-octane and then from water/methanol (10:1) to give
1
pure 4l as red crystals. Final yield 81%. H-NMR (DMSO-d6): (
δ
, ppm) = 7.63 (d, 2H); 7.50 (d, 2H); 7.44
, ppm) = 163.7; 160.4; 149.2;
(d, 1H); 7.34 (d, 2H); 7.27 (d, 2H); 2.33 (d, 3H). 13C-NMR (DMSO-d6): (
δ
140.7; 134.1; 129.7; 124.4; 122.7; 110.1; 103.1; 21.3. HR-MS (m/z): 228.09 [M − H]−.
4-((4-Methoxyphenyl)diazenyl)benzene-1,3-diol 4m. 4-Methoxyaniline and resorcinol were used as starting
reagents. The crude product was crystallized from boiling n-octane and then from water/methanol
1
(10:1) to give pure 4m as red crystals. Final yield 45%. H-NMR (DMSO-d6): (
δ
, ppm) = 10.39 (s, 1H);
7.83 (d, 2H); 7.62 (d, 2H); 7.08 (d, 2H); 7.47 (d, 1H); 6.34 (s, 1H); 3.83 (s, 3H). 13C-NMR (DMSO-d6): (
δ,
ppm) = 163.7; 162.1; 160.4; 147.2; 133.9; 124.4; 114.8; 110.2; 103.2; 56.2. HR-MS (m/z): 244.08 [M
−
H]−.
3.3. Antimicrobial Tests
Bacterial strains and antimicrobial compounds susceptibility determinations.
Four bacterial species were used as models for susceptibility testing; two Gram-negative:
Pseudomonas aeruginosa PAO1 and Escherichia coli MG1655 reference strains; and two Gram-positive:
Staphylococcus aureus ATCC 29213 strain and a Listeria monocytogenes clinical strain isolated in Hospital
Son Espases, Palma de Mallorca, Spain. These strains were cultured on blood agar plates, at 37 ◦C
the day before susceptibility determination, which was assessed through the Minimum Inhibitory
Concentration (MIC). The MICs of the different antimicrobial peptides were determined by the Müller
Hinton Broth (MHB) microdilution method, according to the Clinical and Laboratory Standards
Institute (CLSI) guidelines [27]. Briefly, a stock solution of each compound was prepared, following
their particular solubility patterns (DMSO, MilliQ water, etc.). This stock solution was then diluted
at least 1:50 in MHB to the appropriate concentration of antimicrobial. The MHB containing the
appropriate concentrations of antimicrobial compounds was poured into 96-wells plates (100
well), making 1:2 dilutions with fresh MHB on each column of the plate (concentrations ranging
from 128 g/mL to 0.0625 g/mL). To inoculate each well, 0.5 McFarland bacterial suspensions were
prepared in saline solution. Afterwards, 1:100 dilutions in MHB were obtained from those to yield an
approx. concentration of 1 L of these suspensions were used to inoculate
106 CFU/mL. Finally, 100
µL per
µ
µ
×
µ
the corresponding wells, mixing them with the previously-added MHB containing the antimicrobial
compound. Positive (MHB with bacteria but without antimicrobial compound) and negative (MHB
alone) controls were routinely prepared in several wells of the plates. Once inoculated, the plates were
incubated overnight at 37 ◦C, and the lowest antimicrobial concentration well in which the bacteria
did not grow was considered the MIC. The experiments were always performed in duplicate, in at
least two independent occasions, and median MIC values were considered.
4. Conclusions
Some novel derivatives of (phenyl-diazenyl)phenol (4a–m) were designed, synthesized, and
biologically evaluated as antibacterial agents. Many of the synthesized compounds exhibited
a significant Gram-positive specific antibacterial activity, in particular against S. aureus and L.

Molecules 2017, 22, 1372
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monocytogenes, and they were inactive against Gram-negative bacteria. Three of the synthesized
analogues show the best antibacterial activity ever measured for azobenzene compounds.
4-((4-(isopentyloxy)phenyl)diazenyl)-2,6-dimethylphenol (4d), 4-((4-(neopentyloxy)phenyl)diazenyl)
benzene-1,3-diol (4h), and 4-((4-(isopentyloxy)phenyl)diazenyl)benzene-1,3-diol (4i) were able to
inhibit the growth of 100% of S. aureus and L. monocytogenes at concentrations three times lower than
the lead compound.
The most active antibacterial compounds contain hydroxyl groups, together with long and
branched aliphatic chains in their structure. A complete loss of activity was registered for derivative
4f, which lacks in hydroxyl groups. These observations suggest that the possibility of making
hydrogen bonds is probably fundamental for the interaction of these antibiotic molecules with their
target receptor.
Thanks to the introduction of particularly bulky and flexible side chains, the molecules presented
here could be easily incorporated in DPC micelles, to further increase the drug release. Finally, we
could observe that the complete removal of all hydroxyl groups from the azobenzene moiety led to the
complete loss of antibacterial activity (compound 4f).
Acknowledgments: This research work was financially supported by the Italian Minister of Instruction,
University and Research (MIUR)–300395FRB16. The authors are grateful to Fabrizio dal Piaz for the mass
spectral measurements.
Author Contributions: Simona Concilio, Stefano Piotto and Pio Iannelli designed the idea, and the protocol of
the study. Simona Concilio and Stefano Piotto helped in synthesizing the compounds and wrote the manuscript.
Lucia Sessa, Rosita Diana and Ugo Caruso synthesized and characterized the compounds, wrote the experimental
parts and interpreted the experimental data. Gabriel Torrens and Carlos Juan performed the biological studies
and wrote the experimental data. All the authors revised the whole manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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2017 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/).