Structure modification of an active azo-compound as a route to new antimicrobial compounds

Article abstract of DOI:10.3390/molecules22060875

Some novel (phenyl-diazenyl)phenols 3a-g were designed and synthesized to be evaluated for their antimicrobial activity. A previously synthesized molecule, active against bacteria and fungi, was used as lead for modifications and optimization of the structure, by introduction/removal or displacement of hydroxyl groups on the azobenzene rings. The aim of this work was to evaluate the consequent changes of the antimicrobial activity and to validate the hypothesis that, for these compounds, a plausible mechanism could involve an interaction with protein receptors, rather than an interaction with membrane. All newly synthesized compounds were analyzed by ¹H-NMR, DSC thermal analysis and UV-Vis spectroscopy. The in vitro minimal inhibitory concentrations (MIC) of each compound was determined against Gram-positive and Gram-negative bacteria and Candida albicans. Compounds 3b and 3g showed the highest activity against S. aureus and C. albicans, with remarkable MIC values of 10 μg/mL and 3 μg/mL, respectively. Structure-activity relationship studies were capable to rationalize the effect of different substitutions on the phenyl ring of the azobenzene on antimicrobial activity.

Full text of DOI:10.3390/molecules22060875

molecules
Article
Structure Modification of an Active Azo-Compound
as a Route to New Antimicrobial Compounds
Simona Concilio 1,*, Lucia Sessa 2, Anna Maria Petrone 2,3, Amalia Porta 2, Rosita Diana 4,
Pio Iannelli 2 and Stefano Piotto 2
1
Department of Industrial Engineering, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy
Department of Pharmacy, University of Salerno, Via Giovanni Paolo II 132, 84084 Fisciano, Italy;
2
lucsessa@unisa.it (L.S.); Anpetrone@unisa.it (A.M.P.); aporta@unisa.it (A.P.); piannelli@unisa.it (P.I.);
piotto@unisa.it (S.P.)
PhD Program in Drug Discovery and Development, University of Salerno, Via Giovanni Paolo II 132,
3
84084 Fisciano, Italy
4
Department of Chemical Science, University of Napoli “Federico II”, Cupa Nuova Cintia 21, 80126 Napoli,
Italy; rosita.diana@libero.it
*
Correspondence: sconcilio@unisa.it; Tel.: +39-089-964-115
Academic Editors: Krasimir Vasilev, Alex Cavallaro and Peter Zilm
Received: 4 May 2017; Accepted: 22 May 2017; Published: 25 May 2017
Abstract: Some novel (phenyl-diazenyl)phenols 3a–g were designed and synthesized to be evaluated
for their antimicrobial activity. A previously synthesized molecule, active against bacteria and fungi,
was used as lead for modifications and optimization of the structure, by introduction/removal or
displacement of hydroxyl groups on the azobenzene rings. The aim of this work was to evaluate
the consequent changes of the antimicrobial activity and to validate the hypothesis that, for these
compounds, a plausible mechanism could involve an interaction with protein receptors, rather than
1
an interaction with membrane. All newly synthesized compounds were analyzed by H-NMR,
DSC thermal analysis and UV-Vis spectroscopy. The in vitro minimal inhibitory concentrations
(MIC) of each compound was determined against Gram-positive and Gram-negative bacteria and
Candida albicans. Compounds 3b and 3g showed the highest activity against S. aureus and C. albicans,
with remarkable MIC values of 10 µg/mL and 3 µg/mL, respectively. Structure-activity relationship
studies were capable to rationalize the effect of different substitutions on the phenyl ring of the
azobenzene on antimicrobial activity.
Keywords: azo-compound; antimicrobial; synthesis; QSAR
1. Introduction
Antimicrobial therapy is a powerful tool for the treatment of several diseases, and is a keystone of
modern medicinal practice. Infections by pathogenic microorganisms are of great concern in many
fields, particularly in medical devices, hospital surfaces, surgery equipment, health care products,
food packaging and storage. Infections are generally combated with antimicrobial agents [
some bacteria, upon mutating their genes, have become resistant to common antibiotics and this
makes difficult their elimination [ ]. Consequently, the increased resistance of microorganisms to the
1], however,
2–6
currently used antimicrobials has led to the evaluation of other agents that might have antimicrobial
activity [7,8].
Recently, our research group described the synthesis and properties of some azobenzene-based
compounds and their antimicrobial properties both as single molecules [
matrix [10 12]. In one of these works, we also selected the best candidates by preliminary in-silico
test of ADMET properties and then we synthesized the azo compounds with lowest in-silico toxicity
9] and dispersed in a polymer
–
Molecules 2017, 22, 875; doi:10.3390/molecules22060875
www.mdpi.com/journal/molecules

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values according to the classic diazo coupling reaction scheme [
9
,13]. The majority of synthesized
compounds exhibited high antibacterial activity against S. aureus and antifungal activity against
C. albicans, but they were inactive against Gram-negative bacteria such as Pseudomonas aeruginosa and
Salmonella typhimurium. Because of the structural similarity with stilbenes, these compounds can share
a similar mechanism of action. They could inhibit ATP synthase binding at the interface between
α
and γ subunits [9].
Azobenzene-based derivatives possess interesting features that warrant further investigation.
Among others, the presence of photo-inducible isomerism can be exploited in remodeling lipid
membranes [14] or to modulate the membrane interaction of peptides [15]. Currently, we are evaluating
the possibility to exploit azobenzene-based derivatives to synthesize tunable metal complexes [16
,17]
having antimicrobial activity.
In this work, we introduced some structure modifications to enhance the biological activity of the
previously obtained azo-compounds. In particular, we aimed to evaluate the change in activity related
to different types of substituents linked on the azobenzene structure.
We chose compound A4 from [9], one the best antibacterial azo-compound we synthesized,
as lead compound to synthesize several analogues (see Figure 1). We introduced simple molecular
modifications on both azobenzene rings, in order to evaluate the role of the phenolic hydroxyl group in
the mechanism of action. The substituents are joined to the fixed moiety azobenzene: the modifications
involved moving of the phenolic hydroxyl group from the para- to the meta-position, or removal of the
phenolic hydroxyl group, and replacing the phenolic hydroxyl group by methoxy and methyl groups,
to study and compare their antimicrobial activity.
Quantitative Structure-Activity Relationships (QSAR) are mathematical frameworks that relate
the molecular structures of compounds with their natural activities in a quantitative way [7]. The main
success of the QSAR method is the possibility to estimate the properties of new chemical compounds
without the need to synthesize and test them. One of the most efficient and popular technique is the
Genetic Algorithm Search (GAS). GAS are inspired by natural evolution principles where variables
play the role of genes (in this case a set of descriptors) in an individual of the species. A group of
random individuals (population) evolves according to a fitness function (in this case the antimicrobial
activity) that determines the persistence of the individuals. The algorithm exploits selection, mutation,
and crossover operations on the genes. The QSAR correlates biological activity with structural and
chemical properties. The results of the QSAR analysis permitted to develop a model of activity
in which electronics transition are crucial. This approach will permit a more efficient design of
azobenzene-based antibiotics.
2. Results and Discussion
2.1. Chemistry
The substituted-(phenyldiazenyl)phenols 3a–g derivatives are reported in Figure 1.
Figure 1. Substituted-(phenyldiazenyl)phenol derivatives 3a–g.

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All derivatives 3a–g were synthesized according to the methods outlined in Schemes 1 and 2.
Scheme 1. Synthetic route for compounds 3a–d.
Scheme 2. Synthetic route for compounds 3e–g.
Synthesis of compounds 3a
scheme. An aromatic amine (depending on the A4 analogue) was suspended in an acid solution
and cooled in an ice bath. Sodium nitrite was added, obtaining a suspension of the diazonium salt
–g was accomplished according to the classic diazo coupling reaction
1
2.
Separately, an aqueous solution of NaOH containing the appropriate phenol was prepared and the two
solutions mixed under stirring, maintaining the pH values around 10 for 30 min. The final solution
1
was acidified until a dark red precipitate of the azo compound was formed. H-NMR experiments
were performed to confirm all structures.
2.2. Thermal and Optical Properties
In Table 1, the thermal and optical properties of the synthesized compounds are reported. Analogues
3a, 3b, 3d and 3e showed only a melting peak in the first heating run and they were not able to
crystallize from the melt. In particular, 3a melts at 114.4 ◦C, 3b at 87.7 ◦C, 3d at 89.3 ◦C and 162.0 ◦C.
Compound ◦3c showed a sharp melting peak in the first heating run at 128.3 ◦C and a crystallization
peak at 94.6 C in the cooling run. When heated in the second run, it showed the same melting peak as
in the first run. Compound 3f showed a sharp melting peak in the first heating run at 147.3 ◦C and a
◦
crystallization peak at 110.6 C in the cooling run; when heated in the second run, it showed the same
melting peak as in the first run. 3g showed a sharp melting peak in the first heating run at 158.7 ◦C

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and a crystallization peak at 90.7 ◦C in the cooling run. When heated in the second run, it showed the
same melting peak as in the first run.
Table 1. Thermal and optical properties of compounds 3a–g.
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
3a
3b
3c
3d
3e
3f
114.4
87.7
128.3
89.3
162.0
147.3
158.7
5.7
78.0
98.1
86.1
149.8
123.3
139.4
-
-
358
9300
-
94.6
-
-
92.1
-
344
360
354
344
355
358
18,300
14,000
11,400
14,470
14,200
14,350
-
-
110.6
90.7
104.5
83.7
3g
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.
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 3a are qualitatively only depend on the azobenzene unit, which is the same for all compounds.
–g
The UV absorption spectra of A4 analogues in trans configuration, showed the typical absorption
bands of the electronic transitions of the azobenzene chromophore [18].
2.3. Antimicrobial Activity
The MICs of the synthesized analogues and of lead compound A4 were determined by the
microbroth dilution for S. aureus A170, P. aeruginosa ATCC-27853 strains and C. albicans SC5314 (Table 2).
Table 2. Antimicrobial and antifungal activity of A4 analogues. Geometric mean MICs (µg/mL) after 24 h.
S. aureus
P. aeruginosa
C. albicans
Molecule
MIC50
MIC2
A4 (lead)
15 (MIC100 20 *)
>35
>35
>35
>35
>35
>35
>35
>35
15 (MIC0 17 *)
3a
3b
3c
3d
3e
3f
30
25
10
>35
>35
20
7 (MIC100 10)
>35
>35
25
25
25
MIC0 3
3g
7 (MIC100 20)
MIC50: Minimum Inhibitory Concentration required to inhibit the growth of 50% of bacteria. MIC100: Minimum
Inhibitory Concentration required to inhibit the growth of 100% of bacteria. MIC2: Minimum Inhibitory
Concentration required to inhibit the growth of 50% of fungus. MIC0: Minimum Inhibitory Concentration required
to inhibit the growth of 100% of fungus. The values are the geometric mean of at least three determinations. * Data
from ref. [9].
Analogues 3b and 3g are the most active antimicrobial analogue, exhibiting activity at a
concentration lower than the lead compound concentration. In particular, 3b is the only analogue able
to inhibit the growth of 100% of S. aureus at a concentration of 10
µg/mL; indeed for other compounds
only the MIC50 values are reported (Table 2). Compound 3g is the most active antifungal analogue,
showing an MIC0, which means 100% of inhibition of C. albicans duplication already at 3 µg/mL
(see also Figure 2).
Compared to the reference molecule, other analogues such as 3a, 3e and 3f show lower activities.
Nevertheless, they still possess antimicrobial and antifungal activity, even if at concentrations higher
than A4. Compounds 3c and 3d lose the antibacterial and antifungal activity and none of the analogues

Molecules 2017, 22, 875
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exhibit antimicrobial activity against P. aeruginosa, as already observed for previously synthesized
similar compounds [9].
Interestingly, all the compounds show antibacterial activity only against Gram-positive bacteria.
This can be correlated with the different composition and structure of the Gram-negative bacteria.
Figure 2 shows the total inhibition of germination and hyphae formation of azo compound 3g on C. albicans,
at 3
µg/mL. In the absence of azo compound, an extensive hyphae formation was observed, whereas
when 3g was present, hyphae formation was severely hampered in a concentration-dependent manner.
Figure 2. Inhibition of hyphae formation in Candida albicans at different concentrations (
µg/mL) of A4
and 3g (20× optical zoom).
2.4. QSAR Models
QSAR models were performed to predict the activity of azobenzene molecules against S. aureus.
We have generated several QSAR models using different training and test sets and we have retained
only statistically significant models. The efficacy of the molecules are estimated as the inverse of MIC50.
Therefore, higher efficacy corresponds to higher values of 1/MIC50. After careful evaluation of all
models, we have chosen the following:
1
= 0.0789·VR1Dzp − 0.0580·SpMin8Bhi + 0.116
(1)
MIC50
where VR1_Dzp is the Randic-like eigenvector-based index from Barysz matrix/weighted by
polarizabilities and standardized by range, and SpMin8_Bhi is the smallest absolute eigenvalue
of Burden modified matrix—n 8/weighted by relative first ionization potential standardized by range.
These descriptors belong to the Barysz distance matrix-based descriptors, and to the Burden
modified matrix. The best model correlates polarizabilities and ionization potential with the biocidal
activity and, therefore, suggests an interaction with a protein receptor rather than with the bacterial
cell membrane. The comparison between the experimental biocidal activity against S. aureus and the
predicted MIC50 calculated with Equation (1) is shown in Table 3.

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Table 3. Comparison of experimental and calculated antimicrobial activity against S. aureus.
MIC50
Experimental
Actual
Values for
C:1/MIC50
Equation (1): Equation (1):
MIC50
Predicted
Molecule
ALogP
Predicted
Values
Residual
Values
(µg/mL)
A4
3a
3b
3c
3d
3e
3f
15
30
7
80 *
80 *
25
25
7
17
15
0.067
0.033
0.143
0.013
0.013
0.040
0.040
0.143
0.059
0.067
0.071
0.056
0.013
0.013
2.8
2.8
5.0
3.0
7.2
1.8
0.7
3.2
0.8
2.6
0.2
4.3
0.3
1.1
0.0513
0.0520
0.1460
0.0214
0.0357
0.0452
0.0909
0.0950
0.0756
0.0822
0.0699
0.0625
0.0142
0.0142
0.0154
−0.0187
−0.0030
−0.0089
−0.0232
−0.0052
−0.0509
0.0479
19
19
7
50
30
22
11
10
13
12
14
16
70
70
3g
A1
A2
A3
A5
B10
B11
−0.0168
0.0155
14
18
80 *
80 *
0.0015
−0.0069
−0.0017
−0.0017
* Molecules that did not show activity for concentration larger than 35
to perform the GFA.
µg/mL, have been arbitrarily assigned to 80
The validation tests used to check the predictive ability of the model are listed in Table 4.
Table 4. Validation of the genetic function results.
Parameters
Value
Friedman LOF
R-squared
Adjusted R-squared
0.00302
0.802
0.766
0.654
Yes
Cross validated R-squared
Significant Regression
Significance-of-regression F-value
Critical SOR F-value (95%)
Replicate points
22.321
5.2341
0
Computed experimental error
Number or points for Lack-of-fit test
Min expt. error for non-significant LOF (95%)
0.000
11
0.0201
The validation tests indicate good predictive capability of the model, but there are a few molecules
showing a large error. In particular, molecule 3d displays the highest error. This could be associated
to the low solubility of 3d that, in fact, has the largest value of AlogP. AlogP is a thermodynamic
descriptor and represents hydrophobicity [19].
3. Materials and Methods
3.1. General Information
All the reagents and solvents were purchased from Sigma-Aldrich (Milan, Italy) and used without
further purification. Optical observations were performed by using a Jenapol microscope (Zeiss S.p.A.,
Milano, Italy) fitted with a THMS 600 hot stage (Linkam, Waterfield, Epsom, Tadworth, UK). Phase
transition temperatures and enthalpies were measured using a Pyris 1 DSC scanning calorimeter
◦
(PerkinElmer, Waltham, MA, USA) at a scanning rate of 10 C/min, under nitrogen flow. UV-Vis
absorption spectra of the samples were recorded at 25 ◦C in acetonitrile solution, on a Perkin Elmer
Lambda 19 spectrophotometer. The spectral region 650
length of 1.0 cm. Azobenzene chromophore concentration of about 3.0
−
240 nm was investigated by using cell path
10−5 mol L−1 was used.
×

Molecules 2017, 22, 875
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1H-NMR spectra were recorded with a DRX/400 spectrometer (Bruker, Billerica, MA, USA). Chemical
shifts are reported relative to the residual solvent peak (dimethylsulfoxide-d6: H = 2.50 ppm). HPLC
runs were carried out on a C18 column (Jupiter, 5
Torrance, CA, USA) at a flow rate of 1.0 mL/min. The gradient [solution A: TFA (0.1%); solution B:
TFA (0.07%), H2O (5%), CH3CN (95%)] was 20 70% B over 20 min. HPLC analysis was performed
µm, 300 Å, LC column 150
×
4.60 mm, Phenomenex,
−
on an 1260 Infinity Series system (Agilent, Santa Clara, CA, USA) with UV detection at 280, 320, 350
and 380 nm.
3.2. General Method of the Synthesis of (Phenyl-Diazenyl)Phenols Derivatives 3a–d
A suspension of the proper aromatic amine (0.0183 mol) in a solution containing water (38 mL)
◦
and HCl 37% (w/w, 4 mL) was cooled at 0
−
5 C in a water-ice bath. A solution of sodium nitrite (1.39 g,
0.0202 mol) dissolved in water (8.0 mL) was added dropwise, giving a suspension of the diazonium
salt (solution A). Separately, a solution containing NaOH (0.7 g, 0.0175 mol) in water (50 mL) with
2,6-dimethylphenol (2.23 g, 0.0183 mol) was prepared (solution B). Solution A was added dropwise to
◦
solution B, under stirring at 12 C. The system was left reacting for 30 min, maintaining the pH = 10
−
11.
Then the final solution was slowly added to 103 mL of an acid solution (100 mL of water and 3 mL
of acetic acid), and then stirred for 30 min at 15 ◦C. A dark red precipitate of the azo compound was
formed. The crude precipitate was filtered and dried under vacuum. Yields ranged between 30%
and 40%.
0
3 -Hydroxy-(4-hydroxy-3,5-dimethyl)-azobenzene (3a): 3-Aminophenol and 2,6-dimethylphenol were used
as starting reagents. The crude product was extracted and crystallized from boiling n-octane (100 mL)
and dried. Final crystallization from boiling water/ethanol (3:1) gave pure 3a as orange crystals.
1
Polarized optical microscopy showed a needle-like crystalline form. Yield: 30%. H-NMR (DMSO-d6):
δ
(ppm) = 7.55 (s, 2H), 7.34 (m, 2H), 7.19 (d, 1H), 6.90 (d, 1H), 2.27 (s, 6H). HPLC chromatogram of 3a
shows a single peak, ensuring a purity >95% (Supplementary Materials Figure S1).
4-Hydroxy-3,5-dimethylazobenzene (3b): Aniline and 2,6-dimethylphenol were used as starting reagents.
The crude product was extracted and crystallized from boiling water/ethanol (3:1) and dried, to give
pure 3b as amber crystals. Polarized optical microscopy showed a needle crystalline habitus. Yield:
35%.1H-NMR (DMSO-d6):
δ (ppm) = 7.82 (d, 2H), 7.55 (m, 5H), 2.27 (s, 6H). HPLC chromatogram of 3b
shows a single peak, ensuring a purity >95% (Supplementary Materials Figure S2).
40-Methoxy-(4-hydroxy-3,5-dimethyl)azobenzene (3c): p-Anisidine and 2,6-dimethylphenol were used as
starting reagents. The extraction and crystallization of the crude product from boiling water/ethanol
(3:1) gave pure 3c as yellow crystals. Polarized optical microscopy showed a needle-like crystalline
1
form. Yield: 40%. H-NMR (DMSO-d6):
δ (ppm) = 7.81 (d, 2H), 7.51 (s, 2H), 7.11 (d, 2H), 3.85 (s, 3H),
2.26 (s, 6H). HPLC chromatogram of 3c shows a single peak, ensuring a purity >95% (Supplementary
Materials Figure S3).
40-Methyl-(4-hydroxy-3,5-dimethyl)azobenzene (3d): p-Toluidine and 2,6-dimethylphenol were used as
starting reagents. The extraction and crystallization of the crude product from boiling water/ethanol
(3:1) gave pure 3d as orange crystals. Polarized optical microscopy showed a plate-like crystalline
1
form. Yield: 38%. H-NMR (DMSO-d6):
δ (ppm) = 7.72 (d, 2H), 7.54 (s, 2H), 7.36 (d, 2H), 2.39 (s, 3H),
2.27 (s, 6H). HPLC chromatogram of 3d shows a single peak, ensuring a purity >95% (Supplementary
Materials Figure S4).
3.3. General Method of the Synthesis of (Phenyl-Diazenyl)Phenols Derivatives 3e–g
A suspension of the proper aromatic amine (0.0183 mol) in a solution containing water (38 mL)
◦
and HCl 37% (w/w, 4 mL) was cooled at 0
−
5 C in a water-ice bath. A solution of sodium nitrite (1.39 g,
0.0202 mol) dissolved in water (8 mL) was added dropwise, giving a suspension of the diazonium salt
(solution A). Separately, a solution containing NaOH (0.7 g, 0.0175 mol) in water (100 mL, pH = 14)

Molecules 2017, 22, 875
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with phenol (1.72 g, 0.0183 mol) was prepared (solution B). Solution A was added dropwise to solution
B, under stirring at 12 ◦C. The system was left reacting for 30 min, maintaining the pH = 11. The final
solution was slowly added to 300 mL of an aqueous solution of acetic acid (pH = 5) and then stirred
for 30 min at 15 ◦C. In this step, to facilitate the precipitation of the azo compound I sodium acetate
powder (0.5 g) was added and the mixture stirred for 20 min in water-ice bath. A dark precipitate
of the azo compound was obtained, filtered and dried under vacuum. Yields ranged between 50%
and 60%.
40-Hydroxyazobenzene (3e): Aniline and phenol were used as starting reagents. The crude product was
extracted and crystallized from boiling n-octane (100 mL) and dried. Final crystallization from boiling
water gave pure 3e as orange/yellow crystals. Polarized optical microscopy allowed the observation
1
of needle-like crystals of the azo compound. Yield: 56%. H-NMR (DMSO-d6):
δ (ppm) = 7.81 (t, 4H),
7.54 (m, 3H), 6.96 (d, 2H). HPLC chromatogram of 3e shows a single peak, ensuring a purity >95%
(Supplementary Materials Figure S5).
0
4 -Hydroxy-4-methoxyazobenzene (3f): p-Anisidine and phenol were used as starting reagents. The crude
product was extracted and crystallized from boiling n-octane (100 mL) and dried. Final crystallization
from boiling water/ethanol (3:1) gave pure 3f as dark orange crystals. Polarized optical microscopy
1
showed a plate-like crystalline form. Yield: 58%. H-NMR (DMSO-d6):
δ (ppm) = 7.80 (dd, 4H), 7.51
(s, 2H), 7.11 (d, 2H), 6.93 (d, 2H), 3.86 (s, 3H). HPLC chromatogram of 3f shows a single peak, ensuring
a purity >95% (Supplementary Materials Figure S6).
40-Hydroxy-4-methylazobenzene (3g): p-Toluidine and phenol were used as starting reagents. The crude
product was extracted and crystallized from boiling n-octane (100 mL) and dried. Final crystallization
from boiling water/ethanol (3:1) gave pure 3g as brilliant yellow crystals. Polarized optical microscopy
1
showed a needle-like crystalline form. Yield: 60%. H-NMR (DMSO-d6):
δ (ppm) = 7.75 (dd, 4H), 7.54
(s, 2H), 7.35 (d, 2H), 6.93 (d, 2H), 2.39 (s, 3H). HPLC chromatogram of 3g shows a single peak, ensuring
a purity >95% (Supplementary Materials Figure S7).
3.4. Antimicrobial Tests
3.4.1. Bacterial Strains and Minimum Inhibitory Concentrations
The in vitro minimal inhibitory concentrations (MIC) of each compound was determined against
Candida albicans SC5314 by the micro-broth dilution method in 96-well plates according to the guidelines
suggested by the Clinical and Laboratory Standards Institute (CLSI) [20] using three separate plates
each containing the same batch of azo compounds.
Microtiter plates containing 100
µ
L of two-fold serial dilutions of azo compounds in RPMI 1640
medium were inoculated with 100 L of cells containing 2.5
µ
×
103 yeast/mL and incubated at 35 ◦C
for 24 h. The resulting MICs were visually read as the lowest concentration of compound causing a
reduction or an absence of growth (optically clear) in comparison to the drug-free growth control.
For S. aureus A170 a clinically isolated gentamicin resistant strain (kindly provided by Prof.
R. Capparelli from the University of Naples, Naples, Italy) and P. aeruginosa ATCC-27853, MIC values
of each compound were determined by the serial broth microdilution method as reported by Patton [21].
Therefore, flat-bottom polystyrene microtiter plates containing 100
(six replicates per dilution) of azo compounds were inoculated with 100
µ
L of two-fold serial dilutions
µL of ~5
×
105 CFU/mL of
each bacterium grown in Mueller–Hinton broth 2. The controls were the wells contained broth only
(negative control) and bacteria a◦nd broth (positive control).
Plates were incubated at 37 C with shaking at 160 rpm for 24 h. Data were analyzed according to
Patton et al. [21]. The optical density (OD) was determined just before the incubation (T0) and again
after 24 h incubation (T24) at 600 nm. The OD for each replicate at T0 was subtracted from the OD for
each replicate at T24. The adjusted OD of each control well was then assigned a value of 100% growth.

Molecules 2017, 22, 875
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The MIC is reported as the lowest concentration of azo compounds, which results in 100% or 50%
inhibition of growth.
3.4.2. Candida Albicans Morphological Analysis
Hyphal growth of Candida-treated cells was induced using RPMI 1640 medium. Stationary yeast
cells were inoculated into a fresh pre-warmed medium at a density of 6
×
106 cells/mL in a flat-bottom
96 well microtiter plates. Different concentrations of azo compounds (ranging from 1 to 50 µg/mL),
were added to each well. After incubation at 37 ◦C for 24 h, each microtiter plate was examined using
an inverted microscope to monitor phenotypic modification and hyphae formation.
3.5. QSAR Analysis
For the series of 14 azobenzenes molecules we have calculated 3780 chemical descriptors by
means of the PaDel tool [22] and the Genetic algorithms available in the program Materials Studio [23].
The 3780 parameters calculated have been standardized by range between
was made using the inverse of MIC50 against S. aureus.
−
1 and 1. The regression
The number of descriptors in the regression equation was set to 2, and Population and Generation
were set to 5000 and 50,000, respectively. Mutation probability was 0.1, and the smoothing parameter
was 0.5. The choice of keeping the number of descriptors low was intended to avoid overfitting and
to determine the most important molecular descriptors only, whereas all the other parameters have
been chosen as recommended by the software developers. In all simulations, the algorithm reached
convergence within 270 and 380 generations.
The choice of the models was based on Friedman’s Lack of Fit (LOF) calculated as follows:
SSE
LOF =
(2)
h
ꢀ
ꢁi
2
c + dp
M 1 − λ
M
where SSE is the sum of squares of errors, c is the number of terms in the model, other than the constant
term, d is a scaled smoothing parameter, p is the total number of descriptors contained in all model
terms (again ignoring the constant term), M is the number of samples in the training set, and
λ is a
safety factor, with a value of 0.99, to ensure that the denominator of the expression can never become
zero, and so the LOF is always well-defined.
An adjusted R2 estimation, where the variance is reduced in proportion to the size of the estimated
model. The adjusted R2 is calculated as follows:
SSE/(n − p)
1 −
(3)
SST/(n − 1)
where p is the number of parameters in the regression equation. SST is the total sum of squares, and n is
the number of data points from which the model is built. Compared to R2, this measure penalizes large
equations. The cross validated R-squared (R2(CV)) value is also a key measure of the predictive power
of a model. The closer the value is to 1.0, the better the predictive power. For a good model, R2(CV)
should be fairly close to R2. If R2(CV) is much less than R2, the model equation is probably overfitting
the data. A model with an R2(CV) value of 0.0 or less has no predictive power at all, according to the
cross-validation criterion. The R2(CV) is calculated as follows:
PRESS
SST
1 −
(4)

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where PRESS is the predictive sum of squares of a model. After a required group of data is deleted, the
remaining data in x is used to produce a new model for y. These predicted values of y are compared
with the exact values that have been excluded:
n
ꢂ
ꢃ
2
PRESS (n) =
yi − y0
(5)
∑
i
i=0
A model is produced for each row or group of rows excluded. An indication of whether or not
the regression is statistically significant: Yes if F > Fcr, No if not. F is the significance-of-regression F
value and Fcr is the critical SOR F value (95%).
The F test is a standard statistical test for the equality of the variances of two populations having
normal distributions. Here, it is used to test whether the variance in the data which is explained by the
regression is much larger than the variance remaining due to errors. If this is the case, then the model
is said to be significant, rather than one which simply fits the noise [24]. The significance-of-regression
F value is defined as:
SSR/(p − 1)
(6)
SSE/(n − p)
This parameter is used to determine whether or not the regression is statistically significant.
The critical significance-of-regression (SOR) F value is the critical point of the F distribution of degrees
n
−
p and p
−
1 evaluated for probability 0.05 (at 95% confidence level). In conjunction with the
significance-of-regression F value, it is used to determine whether or not the regression is significant.
4. Conclusions
Some novel derivatives of (40-hydroxy-(4-hydroxy-3,5-dimethyl)azobenzene) were designed,
synthesized, and biologically evaluated as antimicrobial agents. The majority of the synthesized
compounds exhibited significant antibacterial activity against S. aureus and antifungal activity against
C. albicans, but they were inactive against Gram-negative bacteria such as P. aeruginosa. The best
results were obtained for 4-hydroxy-3,5-dimethylazobenzene (3b), which showed higher antibacterial
activity against S. aureus than lead compound. The best antifungal activity was obtained for
40-hydroxy-4-methylazobenzene (3g) that was able to inhibit the growth of 100% of C. albicans at
a concentration five times lower than the lead compound.
Structure-activity relationship studies could rationalize the effect of different substitution patterns
on the phenyl ring of the azobenzene on antimicrobial activity. Changing the electronic nature and the
position of the substituent group attached to the aromatic ring led to changes in the observed MIC.
These observations suggest that the killing ability depends mainly by the interaction with a not yet
identified receptor. The best QSAR model explains the role of polarity and ionizability in determining
the killing capacity of the synthetized compounds and suggests that these molecules interact with
protein receptors and that the interaction with membranes is of minor importance. The solubility and
the partition coefficient may rationalize the apparent poor activity of the compound 3d. Taken together,
these results can be extremely promising for the design of novel azobenzene-based antibiotics.
Supplementary Materials: The Supplementary Materials are available online.
Acknowledgments: This research work was financially supported by the Italian Minister of Instruction, University
and Research (MIUR)–300395FRB15.
Author Contributions: S. Concilio, S. Piotto and P. Iannelli designed the idea, and the protocol of the study.
S. Concilio helped in synthesizing the compounds and wrote the manuscript. L. Sessa and R. Diana synthesized
the compounds, wrote the experimental parts and interpreted the experimental data. S. Piotto performed the
computational analysis and wrote the manuscript. A. Porta and A.M. Petrone 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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Sample Availability: Samples of the compounds are not available from the authors.
©
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/).