Effect of photodynamic antibacterial chemotherapy combined with antibiotics on Gram-positive and gram-negative bacteria

Article abstract of DOI:10.3390/molecules23123152

The well-known and rapidly growing phenomenon of bacterial resistance to antibiotics is caused by uncontrolled, excessive and inappropriate use of antibiotics. One of alternatives to antibiotics is Photodynamic Antibacterial Chemotherapy (PACT). In the present study, the effect of PACT using a photosensitizer Rose Bengal alone and in combination with antibiotics including methicillin and derivatives of sulfanilamide synthesized by us was tested against antibiotic-sensitive and antibiotic-resistant clinical isolates of Gram-positive S. aureus and Gram-negative P. aeruginosa. Antibiotic-sensitive and resistant strains of P. aeruginosa were eradicated by Rose Bengal under illumination and by sulfanilamide but were not inhibited by new sulfanilamide derivatives. No increase in sensitivity of P. aeruginosa cells to sulfanilamide was observed upon a combination of Rose Bengal and sulfanilamide under illumination. All tested S. aureus strains (MSSA and MRSA) were effectively inhibited by PACT. When treated with sub-MIC concentrations of Rose Bengal under illumination, the minimum inhibitory concentrations (MIC) of methicillin decreased significantly for MSSA and MRSA strains. In some cases, antibiotic sensitivity of resistant strains can be restored by combining antibiotics with PACT.

Full text of DOI:10.3390/molecules23123152

molecules
Article
Effect of Photodynamic Antibacterial Chemotherapy
Combined with Antibiotics on Gram-Positive and
Gram-Negative Bacteria
Yana Ilizirov 1, Andrei Formanovsky 2, Irina Mikhura 2, Yossi Paitan 3, Faina Nakonechny 1
and Marina Nisnevitch 1,
*
1
Department of Chemical Engineering, Biotechnology and Materials, Ariel University, Ariel 4070000, Israel;
yanailizirov1@gmail.com (Y.I.); fainan@ariel.ac.il (F.N.)
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, 117977 Moscow,
Russia; formanovsky@yandex.ru (A.F.); ivmikhura@yandex.ru (I.M.)
Clinical Microbiology Laboratory, Meir Medical Center, Kfar Saba 4428164, Israel; yossi.paitan@clalit.org.il
Correspondence: marinan@ariel.ac.il; Tel.: +972-39-14-3040
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 30 October 2018; Accepted: 27 November 2018; Published: 30 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: The well-known and rapidly growing phenomenon of bacterial resistance to antibiotics
is caused by uncontrolled, excessive and inappropriate use of antibiotics. One of alternatives to
antibiotics is Photodynamic Antibacterial Chemotherapy (PACT). In the present study, the effect
of PACT using a photosensitizer Rose Bengal alone and in combination with antibiotics including
methicillin and derivatives of sulfanilamide synthesized by us was tested against antibiotic-sensitive
and antibiotic-resistant clinical isolates of Gram-positive S. aureus and Gram-negative P. aeruginosa.
Antibiotic-sensitive and resistant strains of P. aeruginosa were eradicated by Rose Bengal under
illumination and by sulfanilamide but were not inhibited by new sulfanilamide derivatives.
No increase in sensitivity of P. aeruginosa cells to sulfanilamide was observed upon a combination of
Rose Bengal and sulfanilamide under illumination. All tested S. aureus strains (MSSA and MRSA)
were effectively inhibited by PACT. When treated with sub-MIC concentrations of Rose Bengal under
illumination, the minimum inhibitory concentrations (MIC) of methicillin decreased significantly for
MSSA and MRSA strains. In some cases, antibiotic sensitivity of resistant strains can be restored by
combining antibiotics with PACT.
Keywords: antibiotic resistance; Rose Bengal; PACT; sulfanilamide derivatives
1. Introduction
Controlling microbial infections is one of the most serious problems faced by mankind.
For decades, antibiotics were considered as the most powerful medications against infections. However,
the emergence of bacterial resistance to antibiotics changed this situation dramatically. The appearance
of antibiotic-resistant bacterial strains is a global problem, since once they appeared in one region,
resistant cells spread rapidly all over the globe [
when they are not killed during the course of a standard antibiotic treatment [
1
]. Bacterial strains are considered to be resistant
]. The main reasons
2
for bacterial resistance lie in misuse, inappropriate use and overuse of antibiotics in humans and
animals, including agricultural and veterinary applications that cause antibiotic contamination
of the environment [
alternatives to antibiotic therapy. For example, Herry et al. [
3
]. The phenomenon of antibiotic resistance necessitated a search for new
] applied artificial liposomes composed
4
of cholesterol and sphingomyelin to combat Gram-positive pathogens such as Staphylococcus aureus
and Streptococcus pneumoniae which produce pore-forming cytotoxins. These liposomes bind toxins,
Molecules 2018, 23, 3152; doi:10.3390/molecules23123152
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3152
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thus preventing their binding to host cells. The treatment with liposomes was tested in vivo on mice
and proven to be effective. The authors propose applying the liposomes either alone or conjugated with
antibiotics. Horn et al. [5] suggested using statins and showed that statins in general and simvastatin in
particular, protect patients from host cell invasion by a common agent of sepsis, Staphylococcus aureus.
Simvastatin restricted localization of phosphoinositide 3-kinase (isoform p85) and inhibited actin
dynamics needed for bacterial endocytosis. Another approach included repurposing drugs, such as
non-steroidal anti-inflammatory agents, for treatment of microbial infections [6]. This approach seems
to be time- and manpower-conserving and exhibits some advantages over the development of new
drugs for this purpose. In some methods, bacteria were not eradicated but lost their ability to harm the
host cells. For example, anti-adhesion therapy proposed by Cozens et al. [7] prevented adherence of
bacteria to host cells and tissues and enabled the avoidance of infection, since in many cases adherence
is a prerequisite for the infection.
Photodynamic eradication of bacteria, called photodynamic antimicrobial chemotherapy (PACT),
holds a special place among the alternatives to antibiotics [8–10]. PACT is based on the excitation
of a photosensitizer (PS) upon illumination, causing transition of the PS from a low-energy ground
state to a high-energy excited state. After excitation, PS molecules participate in either Type I or
Type II reactions. The PS either reacts with organic substrates with production of free radicals or
radical ions (Type I mechanism) or passes its energy to dissolved molecular oxygen producing reactive
oxygen species, ROS (Type II mechanism). In both cases, active products of the reactions interact
with bacterial membrane phospholipids and proteins. This causes membrane leakage and leads
to cell death [11,12]. PS belong to various chemical groups, such as xanthene and phenothiazine
dyes, porphyrin derivatives, phthalocyanines, psoralens, perylenequinonoids and other macrocyclic
molecules containing a large number of conjugated double bonds [13]. PACT was found to be effective
in the eradication of Gram-positive and Gram-negative bacteria [8,9,13–23]. Use of PS has several
advantages over antibiotics: 1. PS can eradicate not only Gram-positive and Gram-negative bacteria
but also protozoa, viruses and fungi, whereas antibiotics often have a very narrow spectrum of
antibacterial activity; 2. Contrary to antibiotics, the use of PS does not induce resistance in bacteria;
3. The probability of causing mutagenic effects upon PS application is very low; 4. PS were found to be
active not only against planktonic antibiotic-sensitive bacteria but also against resistant strains and
biofilm-forming bacterial species [24].
Another approach to bacterial inhibition is based on a powerful idea of combining antimicrobials
with different cytotoxic mechanisms. Konaté et al. [25] tested the antimicrobial action of alkaloid
compounds from Cienfuegosia digitata Cav. (Malvaceae) in combination with methicillin and ampicillin.
This combined treatment exhibited a synergistic inhibitory effect against microorganisms resistant to
these antibiotics (MRSA/ARSA). Several works reported use of antibiotics combined with PS. In some
cases, this combination exhibited a synergistic effect and showed a principle possibility of recovering
the bacterial cells’ sensitivity to antibiotics [26–30].
In the present work, the sensitivity of resistant hospital isolates of Staphylococcus aureus and
Pseudomonas aeruginosa to the photosensitizer Rose Bengal alone and in combination with other
antimicrobial agents was studied.
2. Results
2.1. Characterization of Sulfanilamide Derivatives
Yields, melting points and NMR data for the synthesized sulfanilamide derivatives (IIa
–n) and
their precursors (Ia–n) are given in Appendix A.
2.2. Characterization of Hospital Isolates of P. aeruginosa and S. aureus
Two different bacterial species, one Gram-negative and one Gram-positive were used in the
study. P. aeruginosa and S. aureus were chosen since both species represent major human pathogens

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which are often associated with multi-drug resistant strains, difficult to treat. Bacteria S. aureus belong
to the most common human Gram-positive commensal pathogens and are generally divided into
two groups according to their resistance to the antibiotic methicillin: methicillin-resistant S. aureus
(MRSA) and methicillin-sensitive S. aureus (MSSA). Both groups are related in our study. P. aeruginosa
is a common Gram-negative opportunistic human pathogen of considerable medical importance.
In general, P. aeruginosa isolates are divided according to their antibiotic resistant patterns to sensitive or
multi-drug resistant isolates, often according to their resistance to ceftriaxone and other 3rd generation
cephalosporins. We therefore decided to study both ceftriaxone sensitive and multi-drug ceftriaxone
resistant P. aeruginosa isolates.
Bacterial strains used in this study were obtained from different clinical or screening samples.
Isolates of P. aeruginosa and S. aureus were obtained from hospital in-patients hospitalized due to
different infections or from MRSA screening samples in the case of MRSA. The isolated strains
were tested for sensitivity to different antibiotics as described in the Materials and Methods section.
Five isolates of P. aeruginosa strains (strains 68147, 68161, 68166, 68172 and 64606) were sensitive to
ceftriaxone and to most other antibiotics and five other isolates (strains 63917, 63982, 62817, 610656 and
611908) were ceftriaxone resistant and exhibited multidrug resistance to 13 to 15 antibiotics. One MSSA
isolate (strain 611971) was sensitive to the tested antibiotics and 5 other MRSA isolates (strains 69654,
69740, 69430, 69621 and 69397) were resistant to 2 or 4 antibiotics. Two ATCC strains (ATCC 25668
P. aeruginosa and ATCC 11541 MSSA) were used as control strains.
2.3. PACT against MSSA and MRSA Strains
S. aureus cells were examined for their sensitivity to Rose Bengal under exposure to white light
and minimum inhibitory concentration (MIC) values of Rose Bengal were determined for sensitive
and antibiotic-resistant strains (Table 1). The tested strains exhibited sensitivity to the PACT treatment.
However, the MIC of Rose Bengal under illumination was the lowest for the control MSSA (0.625 mg/L),
whereas the MIC values for the hospital isolates ranged from 1.25 to 2.5 mg/L. In the dark controls,
MIC of Rose Bengal was 20 mg/L for both MSSA and MRSA strains.
Table 1. MIC of Rose Bengal, of methicillin alone and in the presence of sub-MIC concentrations of
Rose Bengal under illumination for MSSA and MRSA.
MIC of Methicillin, mg/L 1, in the
Presence of Sub-MIC
Concentration of Rose Bengal
Strain of
S. aureus
MIC of Rose
MIC of Methicillin,
mg/L 1
Sub-MIC of Rose
Bengal, mg/L 1
Bengal, mg/L 1
MSSA 11541
MSSA 611971
MRSA 69430
MRSA 69397
MRSA 69654
MRSA 69740
MRSA 69621
0.625 ± 0.009
1.25 ± 0.02
2.50 ± 0.03
1.25 ± 0.02
1.25 ± 0.02
1.25 ± 0.02
1.25 ± 0.02
1.19 ± 0.02
2.38 ± 0.03
>19.0 ± 0.3
19.0 ± 0.3
>19.0 ± 0.3
19.0 ± 0.3
>19.0 ± 0.3
0.30 ± 0.01
0.15 ± 0.01
0.313 ± 0.004
0.625 ± 0.009
1.25 ± 0.02
0.30 ± 0.004
<0.0091 ± 0.0002
<0.0091 ± 0.0002
0.15 ± 0.01
0.625 ± 0.009
0.625 ± 0.009
0.625 ± 0.009
0.625 ± 0.009
<0.0091 ± 0.0002
1
Data are presented as a value ± a standard error.
2.4. PACT Combined with Methicillin against MSSA and MRSA
The MIC of methicillin was determined for MSSA and MRSA strains in the absence and in the
presence of Rose Bengal, in order to examine whether a combination of photodynamic and antibiotic
treatments with different antibacterial mechanisms can increase the sensitivity of bacterial strains to
antibiotics. The combined experiments were carried out in the presence of sub-MIC concentrations
of Rose Bengal, chosen as half of the MIC value for each strain, in order to exclude any masking
effect of Rose Bengal. The results presented in Table 1 show that MIC values of methicillin were 8
to more than 16-fold higher for the MRSA strains than for the MSSA strains. Upon application of
sub-MIC concentrations of Rose Bengal to both MSSA and MRSA cells, the MIC of methicillin dropped

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very drastically, whereas in control experiments Rose Bengal alone at the same concentrations caused
no inhibition.
Since in the case of MRSA strains the MIC of methicillin decreased very significantly, even below
the initial MIC value for MSSA, it may be concluded that it is possible to restore the MRSA cells’
sensitivity to methicillin. MRSA 69397 cells, which showed a very high response to the combined
treatment, were examined by the SEM technique. For this purpose, the cells were treated only by
methicillin at the sub-MIC concentration, only by PACT at the sub-MIC concentration of Rose Bengal
and by the combined antibiotic-PACT treatment at sub-MIC concentrations of both Rose Bengal and
methicillin, when the latter was determined in the presence of sub-MIC concentration of Rose Bengal
(Table 1). In the two first cases, the cells remained intact and were unaffected by each of the factors
separately (Figure 1a,b). However, when they were treated at sub-MIC concentrations of Rose Bengal
and by methicillin at very low concentration (9.1
hardly found (Figure 1c).
µg/L), the bacteria were inhibited and live cells were
Figure 1. SEM micrographs of MRSA 69397 cells in the presence of 9.5 mg/L of methicillin (
a),
0.625 mg/L of Rose Bengal (b) and 9.1 µg/L of methicillin combined with 0.625 mg/L of Rose
Bengal (c).
In order to determine whether the sensitivity of MRSA cells to PACT and to the combined
antibiotic-PACT treatment depended on the number of antibiotics to which these cells were resistant,
MIC values of Rose Bengal alone and of methicillin in the presence of sub-MIC concentrations of
Rose Bengal were presented against the number of antibiotics (Figure 2a,b, respectively). Figure 2
shows that in both cases, the sensitivity was found to be independent of the number of antibiotics to

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which the bacteria are resistant (R2 = 0.3437, Figure 2a and R2 = 0.1405, Figure 2b). Moreover, the type
of antibiotics to which MRSA strains are resistant does not affect the restorability of cell sensitivity
to methicillin.
a
3
2
1
0
0
0
2
2
4
4
4
Number of antibiotics
b
0.4
0.3
0.2
0.1
0
0
0
2
2
4
4
4
Number of antibiotics
Figure 2. MIC values of Rose Bengal (
a) and of methicillin in the presence of sub-MIC concentrations
of Rose Bengal (
standard errors.
b) for MSSA and MRSA strains resistant up to 4 antibiotics. Error bars represent
2.5. Antibacterial Activity of Sulfanilamide Derivatives Alone and Combined with Rose Bengal
The antibacterial activity of sulfanilamide and its derivatives was tested against hospital isolates
of P. aeruginosa. First, the antibiotic-sensitive and resistant strains were tested for sensitivity to
sulfanilamide alone. Figure 3 shows that MIC values vary for both sensitive and resistant strains.
As can be seen from Figure 3, the MIC values of sulfanilamide were 3.5 g/L or less for only two
sensitive and one resistant strains, whereas the rest of the cells had a MIC of 7 g/L or more, which is
above the solubility of sulfanilamide in the aqueous phase.
It was interesting to evaluate whether the sensitivity of the cells to sulfanilamide depended on
their resistance to other antibiotics and whether the MIC values of sulfanilamide were related to the

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number of antibiotics to which the cells were resistant (Figure 3). In addition, the correlation coefficient
was shown to be very low (R2 = 0.055). It can therefore be concluded that there is no correlation
between the cells’ sensitivity to sulfanilamide and to other antibiotics.
8
6
4
2
0
0
0
0
0
0
0
13
14
14
15
15
Number of antibiotics
Figure 3. MIC values of sulfanilamide for P. aeruginosa strains resistant up to 15 antibiotics. Error bars
represent standard errors.
Because of the poor response of most of the P. aeruginosa cells to sulfanilamide, the sensitivity of
cells to Rose Bengal alone and in combination with sulfanilamide was studied for only two strains—the
sensitive 25668 strain and the resistant 63917 strain, which showed low MIC values of sulfanilamide.
Table 2 shows that both strains were inhibited by PACT and that the MIC of Rose Bengal was lower
for the sensitive strain 25668. When treated with sulfanilamide combined with PACT in the presence
of sub-MIC concentrations of Rose Bengal, the MIC values of sulfanilamide remained unchanged.
Contrary to the combined antibiotic-PACT treatment, a combination of sulfanilamide and PACT did
not exhibit any decrease in the MIC of sulfanilamide.
Table 2. MIC values of antibacterial agents for the P. aeruginosa antibiotic-sensitive strain 25668 and the
antibiotic-resistant strain 63917.
MIC, g/L 1
Applied Antimicrobials
Strain of P. aeruginosa
63917
25668
Sulfanilamide
1.75 ± 0.02
3.50 ± 0.08
Rose Bengal
Sulfanilamide in the presence of Rose Bengal
at the sub-MIC concentration
1
0.78 ± 0.01
0.39 ± 0.01
1.75 ± 0.02
3.50 ± 0.08
Data are presented as a value ± a standard error.
The effect of sulfanilamide and its new derivatives IIa
63917 strain, which was chosen due to its highest sensitivity for sulfanilamide. The experiments
for determination of MIC values of sulfanilamide and compounds IIa were performed with their
saturated solutions in the initial test tube. For sulfanilamide it was 7 g/L, which corresponded to its
solubility [31]. The experiments showed that none of the IIa compounds inhibited the P. aeruginosa
–n was studied only for the resistant
–n
–
n
cells and in all cases the MIC values were higher than the solubility of these compounds in aqueous
solutions (Table 3).

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Table 3. MIC values of sulfanilamide and its derivatives for the P. aeruginosa antibiotic-resistant
strain 63917.
Sulfanilamide Derivative
MIC
1.75 ± 0.02 g/L 1
Sulfanilamide
IIa
IIb
IIc
IId
IIe
IIf
IIg
IIh
IIi
>2.68 g/L
>53.7 mg/L
>0.963 g/L
>21.3 mg/L
>8.40 mg/L
>0.102 g/L
>0.157 g/L
>2.74 g/L
>35.7 mg/L
>0.372 g/L
>9.07 mg/L
>26.7 mg/L
>0.447 g/L
>2.10 g/L
IIj
IIk
IIl
IIm
IIn
1
Presented as a value ± a standard error.
3. Discussion
Two approaches were studied in the present work in an attempt to promote methods for the
treatment of antibiotic-resistant strains: PACT and a combination of PACT with methicillin in the case
of MRSA strains and with sulfanilamide in the case of multi-resistant P. aeruginosa. As demonstrated
here, multidrug resistant strains of S. aureus can be successfully inhibited by PACT, where the MIC
values of Rose Bengal for resistant strains were of the same order of magnitude as for sensitive strains.
Sensitive and resistant strains of P. aeruginosa however were inhibited at much higher concentrations of
Rose Bengal when compared to S. aureus. The efficiency of PACT against resistant bacteria was a good
reason for a trial to restore their sensitivity to antibiotics by combining them with a PS.
MRSA cells differ from MSSA cells on the genetic level. MRSA cells bear an additional gene,
mecA, in their chromosome, which encodes for a penicillin-binding protein PBP-2a that has low
affinity for methicillin and remains inactivated upon exposure of the cell to the antibiotic, contrary to
four other PBPs (PBP1-PBP4) normally present in S. aureus cells [32–34]. The normal PBPs catalyze
crosslinking reactions of transpeptidation which occur on the external surface of the cytoplasmic
membrane. In MSSA cells, methicillin binds to the transpeptidation region of normal PBPs and,
as a consequence, causes inhibition of the transpeptidation reaction. The lack of crosslinking of the
peptidoglycan leads to mechanical weakening of the cell envelope, release of the cytoplasmic content
and cell death. In MRSA cells, after inhibition of normal PBP proteins by methicillin, PBP-2a maintains
its activity and takes over the functions of PBP, ensuring crosslinking of the glycan chains in the
peptidoglycan. Although PBP-2a does not completely compensate for the normal functioning of the
PBP1-PBP4 and the cells growing in the presence of methicillin have reduced crosslinking of the
peptidoglycan, this limited rate of crosslinking is sufficient for providing not only survival of the cells
but also their propagation [32,33]. Since PACT is known to affect first of all components of the bacterial
membrane, a combination of methicillin and PACT seemed to us to be a powerful idea, where the
role of methicillin was to inhibit normal cell PBP1-PBP4 and the role of the PS was to interfere with
the functioning of PBP-2a by destroying the weakened cell peptidoglycan. Indeed, the results of the
present study show that treatment of the MRSA cells by methicillin combined with Rose Bengal at
sub-MIC concentrations led to a recovery of the cells’ sensitivity to methicillin, where in most cases the
MIC values of methicillin dropped drastically, even below the MIC level of MSSA cells. The observed
phenomenon probably exhibited a synergistic character, thus enabling a reduction in applied doses of
both drugs and preventing the appearance of resistant cells.
Other successful attempts to combine antibiotics and PACT have been reported recently by others
and by us [26–30,35]. Barra et al. [36] showed the combined action of PS and antibiotics against biofilms

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of clinical isolates of gentamicin-resistant Gram-positive bacteria of the Staphylococcus genus: S. aureus,
S. epidermidis and S. hemolyticus. Gentamicin was applied in combination with 5-aminolevulinic
acid (5-ALA) which does not exhibit photodynamic activity but acts as a metabolic precursor for the
endogenous PS protoporphyrin IX (PpIX). Pre-treatment of Gram-positive bacteria biofilms with 5-ALA
under illumination was found to make them more sensitive to gentamicin. The authors explain the
observed phenomenon by the profound damage to membranes, protein breakdowns and crosslinking,
sugar modifications, peroxidation of lipids and harm to the sugars and bases of ribonucleic acids
caused by the photodynamic treatment. Due to these effects, penetration of gentamicin through the
bacterial membranes was significantly improved. This enabled the antibiotic to bind the 30S subunit of
the bacterial ribosome, thus depressing protein synthesis and causing cell death [35].
Efficient eradication of S. aureus biofilms was achieved after treating cells first with meso-tetra
(N-methyl-4-pyridyl) porphine tetra tosylate under illumination and then with vancomycin [27].
Amplification of antibiotic activity by PACT was shown for S. aureus and P. aeruginosa cells
treated with methylene blue or meso-tetra(N-methyl-4-pyridyl)porphine tetra tosylate at sub-lethal
concentrations [36]. Similar results were achieved when planktonic and biofilm cultures of
Burkholderia cepacian were exposed to PS combined with tobramycin, ceftazidime, chloramphenicol or
ciprofloxacin [26].
Several attempts were made to conjugate antibiotics with PS and these conjugate molecules
were shown to have amplified antibacterial activity compared to each component separately.
Xing et al. [37] synthesized a porphyrin-vancomycin conjugate, which was found promising for
inactivation of not only vancomycin-sensitive but also vancomycin-resistant enterococcal strains.
Enhanced photodynamic inactivation of bacteria was attributed to effective adhesion of vancomycin to
bacterial envelopes, so that vancomycin was used as an affinity ligand. Cahan et al. [38] demonstrated
that Rose Bengal linked covalently to penicillanic acid or kanamycin was effective against pathogenic
S. aureus and E. coli bacteria. The achieved effect enabled the authors to propose that the use of
antibiotic-PS conjugates may not only help solve the problem of bacterial drug resistance but may
also reduce side effects of antibiotics in patients. Combined antibiotic-PACT therapy has good
prospects for wide applications in the treatment of local and systemic external infections. For internal
infections, photodynamic excitation of PS should be replaced by alternative methods, for instance
chemiluminescent or sonodynamic activation [17,23].
The antimicrobial activity of compounds from the sulfanilamide group is based on competitive
inhibition of the enzyme dihydropteroate synthase, which catalyzes conversion of a para-aminobenzoic
acid into folic acid necessary for cell replication [39]. Although the inhibitory mechanism of the
sulfanilamide compounds is totally different from that of PACT, a combination of sulfanilamide and
Rose Bengal did not yield any increase in the sensitivity of antibiotic-sensitive and antibiotic-resistant
P. aeruginosa cells to sulfanilamide. Contrary to the effect of the combined application of methicillin
and Rose Bengal when these two factors were targeted against the cytoplasmic membrane of S. aureus,
the combined application of sulfanilamide and Rose Bengal against P. aeruginosa was not effective.
The absence of any additional effect of a combination between sulfanilamide and PACT may be
explained by very low rates of bacterial envelope damage by Rose Bengal at sub-MIC concentrations,
which was not sufficient for enhancing the action of sulfanilamide. It is probably preferable to choose
antimicrobials which affect the same cell sites in order to achieve amplification of their activity.
4. Materials and Methods
4.1. Materials and Bacterial Strains
Rose Bengal, sulfanilamide, reagents for synthesis and solvents were purchased from
Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). Methicillin was purchased from TOKU-E
(Bellingham, WA, USA), growth media Brain Heart broth (BH) and Brain Heart agar (BHA) were
purchased from Merck (Darmstadt, Germany) and Antibiotic medium 3 for minimal inhibitory

Molecules 2018, 23, 3152
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concentration (MIC) studies was purchased from Becton Dickinson & Company (Le Pont de Claix,
France). Antibiotic discs were purchased from Abtek Biologicals Ltd. (Liverpool, UK). Antibiotic
sensitive and resistant strains of S. aureus and P. aeruginosa were hospital isolates from the Meir Medical
Center (Kfar Saba, Israel). S. aureus strain ATCC 11541 and P. aeruginosa ATCC 25668 strains were
purchased from ATCC (Manassas, VA, USA).
4.2. Synthesis of Sulfanilamide Derivatives
New sulfanilamide derivatives IIa–n were synthetized according to the scheme presented in
Figure 4.
Figure 4. Scheme of the synthesis of sulfanilamide derivatives (IIa–n).
Details of the synthesis of compounds from series I and II are specified below.
4.2.1. N-Methyl-4-acetylamino Benzenesulfonyl Amide (Ia)
5.4 g (80 mmol, 2.0 equiv.) CH3NH2 HCl were added to a solution of 8.48 g (80 mmol, 2.0 equiv.)
·
Na2CO3 in 85 mL of H2O at 0 ◦C. Then, 9.34 g (40 mmol, 1 equiv.) 4-acetylamino benzenesulfonyl
chloride were added in small portions (0.5 g each) for 1 h under stirring. The reaction mixture was
stirred for 1.5 h at room temperature and 0.5 h at 80 ◦C. After the end of the reaction, the reaction
mixture was adjusted to pH 6 with 1 M HCl solution. The precipitate was separated, washed with
H2O (500 mL) and dried in vacuum under KOH.
4.2.2. General Procedure for Synthesis of Compounds (Ib–n)
3.24 mL (23 mmol, 0.77 equiv.) of Et3N was added to 30 mmol (1 equiv.) of substituted amine in
30 mL of dry DMF. 32 mmol (1.1 equiv.) of 4-acetylamino benzenesulfonyl chloride were then added in
small portions (5 portions for 1 h) under stirring and cooling in a water bath (10–15 ◦C). The reaction
mixture was stirred for 12 h at room temperature. After the end of the reaction, the reaction mixture
was poured into 100 mL of 1 M HCl and 50 mL of a saturated NaCl solution were added under cooling
(10 ◦C). The precipitate was separated, washed with 200 mL H2O and dried in vacuum under KOH.
4.2.3. Sulfanilic Acid N-Methyl Amide (IIa)
12 mL of concentrated HCl were added to a suspension of 5.50 g (24.12 mmol) Ia in 40 mL of
MeOH and were refluxed for 1 h in a flask equipped with a reflux condenser. After the end of the

Molecules 2018, 23, 3152
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reaction, the reaction mixture was poured into 250 mL of 10% NaHCO3 in H2O. The precipitate was
separated, washed with 200 mL H2O and dried in vacuum under KOH.
4.2.4. General Procedure for Synthesis of Compounds (IIb–n)
10 mL of concentrated HCl were added to a suspension of 25 mmol N-substituted 4-acetylamino
benzenesulfonyl amide in 40 mL of MeOH and the reaction mixture was refluxed for 1 h in a flask
equipped with a reflux condenser. After the end of the reaction, the reaction mixture was poured into
150 mL of 10% NaHCO3 in H2O. The precipitate was separated, washed with 200 mL H2O and dried
in vacuum under KOH.
4.3. Characterization of the Synthesized Sulfanilamides
All compounds were characterized by melting point measurement, by NMR spectra and by HPLC
chromatography. Melting points were determined with a Boetius micro melting point apparatus
and not corrected. 1H NMR spectra were measured with a Brucker HX-270 spectrometer (Bruker
Instruments, Billerica, MA, USA) (300 MHz) in acetone-d6, DMSO-d6 and CD3OD at 298 K. Chemical
shifts (δ) are reported in parts per million (ppm) and are referenced to residual signals for deuteriated
acetone (
δ
= 2.07 ppm) and DMSO ( = 2.50 ppm) and CD3OD ( = 3.34 ppm) as internal standards.
δ
δ
Multiplicities are reported as follows: s = singlet, d = doublet, q = quartet, m =multiplet, br = broad,
Ar = aromatic, Ph = phenyl, Im = imidazole and coupling constants (J) are given in Hertz. Purity was
tested by HPLC analysis using Jasco LC model (Tokyo, Japan) on a RP-18 column YMC-Triart C18,
75 × 3.0 mm, bead size 1.9
20 mM ammonium acetate: acetonitrile: methanol. Chromatograms of compounds IIa
in Appendix B (Figure A1). Solubility of the compounds IIa in water was determined in their
µm in the isocratic regime using an eluent composed of 10:40:50 v/v of
–
n
are presented
–
n
saturated at ambient temperature aqueous solutions by analyzing saturated aqueous solutions with
the help of HPLC using solutions of IIa–n with known concentrations in DMSO as standards.
4.4. Characterization of Bacterial Strains
Isolation and identification of strains was preformed according to SOPs following standard CLSI
procedures. Identification of isolated colonies was performed by the Vitek II system (BioMérieux,
Durham, NC, USA) or by matrix-assisted laser desorption ionization–time of flight mass spectrometry
(MALDI-TOF MS, BioMérieux). Antibiotic resistance assay was performed in the clinical laboratory
by Vitek II system (BioMérieux) using the AST-N270 card and by standard disk diffusion test
for P. aeruginosa isolates and by standard disk diffusion test for S. aureus isolates. Antibiotic
resistance/sensitivity tests were performed and interpreted following CLSI guidelines.
4.5. Bacterial Cell Growth
Cultures of S. aureus and P. aeruginosa were grown on brain-heart agar (BHA; Acumedia, Lansing,
MI, USA) for 24 h, then transferred into brain-heart broth (BH; Acumedia) and grown overnight at
37 ◦C and shaking at a 170 rpm. Cell cultures were first diluted by the Antibiotic Medium 3 to obtain
an OD600 = 0.1, which corresponded to an initial cell concentration of 108 CFU/mL. This concentration
was confirmed by a viable cell count [40] in which 100 µL volumes of bacterial samples at various
decimal dilutions were evenly spread over BHA plates with a Drigalsky spreader. The plates were
incubated at 37 ◦C overnight and colony forming units (CFU) were counted using a colony counter
Scan 500 (Interscience, Saint Nom la Bretèche, France).
4.6. PACT Experiments
The minimum inhibitory concentrations (MIC) of Rose Bengal for bacteria were determined by
a PACT procedure in transparent glass test tubes as described earlier by us [16]. In brief, Rose Bengal
in 2 mL of Antibiotic Medium 3 (hereinafter–medium 3) was distributed by a double dilution method

Molecules 2018, 23, 3152
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when the Rose Bengal concentration in the first test tube was 40 mg/L. Bacterial suspensions with
a concentration of 108 CFU/mL (Section 4.5) were diluted 1:100 with medium 3 and 100
L of the
µ
latter suspensions were then added to 2 mL of medium 3, thus yielding a cell concentration of
5 × 104 CFU/mL. The test tubes, containing bacteria and Rose Bengal, were illuminated for 30 min
at ambient temperature by a 18 W white luminescent lamp with an emissions range of 400–700 nm
and a light intensity of 1.25 mW/cm2 which was measured by a LX-102 Light Meter (Lutron, Taipei,
Taiwan). After the illumination, the test tubes were incubated in the dark at 37 ◦C for 24 h and tested
visually for turbidity. MIC values were defined as the lowest concentration of Rose Bengal inhibiting
bacterial growth. In all cases, control experiments with bacteria in the presence of Rose Bengal in the
dark and in the absence of Rose Bengal under illumination were carried out.
4.7. MIC Determination of Sulfanilamide and Its New Derivatives for P. aeruginosa
MIC values of sulfanilamide and its derivatives IIa–n were determined by a standard broth
double dilutions procedure [41], when the concentration of sulfanilamide and of the derivatives IIa
–n
in the first test tube were correspondent to their solubility in water. The concentration of bacteria in
each sample was 5 × 104 CFU/mL. The samples were incubated for 24 h at 37 ◦C and visually tested
for turbidity. The MIC values were defined as the lowest concentration of antimicrobials that inhibited
visible growth of bacteria.
4.8. MIC Determination of Methicillin for S. aureus
MIC values of methicillin for S. aureus were determined in the medium 3 by a standard broth
double dilutions procedure [41] using concentration of 19 mg/L in the first test-tube. Concentration of
bacteria in all the test-tubes was 5
previous protocol.
×
104 CFU/mL. The rest of the procedure was as described in the
4.9. Combined PACT-Antibacterial Experiments
Experiments on the combined effect of PACT with methicillin for S. aureus and PACT with
sulfanilamide for P. aeruginosa were performed at sub-MIC concentrations of Rose Bengal which were
chosen as half-MIC concentrations for each strain. In each series concentration of Rose Bengal was
kept constant and methicillin (in the case of S. aureus) or sulfanilamide (in the case of P. aeruginosa)
were distributed by double dilutions using initial concentrations as described above. Concentration of
bacteria in all the test-tubes was 5
×
104 CFU/mL. The rest conditions were as described in the section
of PACT experiments. Control experiments on MIC determination in the absence of Rose Bengal, at the
sub-MIC concentrations of Rose Bengal without methicillin or sulfanilamide and in the absence of any
antimicrobial, were carried out for each series at the conditions of PACT.
4.10. SEM Imaging of Bacteria
Suspensions of bacterial cells were centrifugated at 10,000
×
g for 10 min and sediments were treated
with 4% paraformaldehyde under gentle mixing for 10 min. The suspensions were centrifugated again
and the sediments were washed 6 times by 0.05 M PBS, pH 7.4. The immobilized cells were coated with
gold [19]. Micrographs were obtained using a SEM microscope JSM-6510LV (JEOL, Tokyo, Japan).
4.11. Reproducibility of the Experiments
In each series, the results were obtained from at least 3 independent experiments with duplicates.
Standard errors were calculated for each value. In the case of testing correlation between various
factors correlation coefficient R was calculated. The factors were considered independent when R2 was
less than 0.5.

Molecules 2018, 23, 3152
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5. Conclusions
Photodynamic inactivation of S. aureus and P. aeruginosa by Rose Bengal may become an alternative
to antibiotic treatment of bacterial infections caused by both antibiotic-sensitive and antibiotic-resistant
strains. The antibiotic sensitivity of MRSA strains can be restored by combining methicillin with Rose
Bengal. Application of a combined antibiotic-PACT treatment of MRSA cells caused a decrease in
the MIC of methicillin. Multi-drug resistant P. aeruginosa strains can be eradicated by sulfanilamide.
The combination of Rose Bengal and sulfanilamide did not cause any increase in the cells’ sensitivity
to sulfanilamide.
Author Contributions: Conceptualization, M.N.; methodology, F.N. and I.M.; investigation, Y.I., A.F. and F.N.;
resources, M.N., I.M. and Y.P.; data curation, Y.I.; writing, M.N., I.M. and Y.P.; supervision, M.N.
Funding: This research was supported by the Research Authority of the Ariel University, Israel and by the Cherna
Moskowitz Foundation, California, USA.
Acknowledgments: We acknowledge Natalya Litvak and Miss Victoria Muzika (Ariel University, Israel) for their
assistance in the SEM imaging of the samples.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
The following abbreviations are used in this manuscript:
PACT
PS
Photodynamic Antibacterial Chemotherapy
photosensitizer
MSSA
MRSA
ROS
MIC
SEM
PBP
methicillin-sensitive Staphylococcus aureus
Methycillin-resistant Staphylococcus aureus
reactive oxygen species
minimum inhibitory concentration
scanning electron microscope
penicillin-binding protein
SOP
CLSI
CFU
standard operating procedure
The Clinical & Laboratory Standards Institute
colony forming unit
Appendix A
Characteristics of the Synthesized Sulfanilamide Derivatives
1
N-methyl-4-acetylamino benzenesulfonyl amide (Ia). Yield 60%; white powder, m.p. 183–185 ◦C. H NMR
(300 MHz, acetone-d6): δ = 1.85 (s, 3H, Me), 2.30 (s, 3H, Me), 5.40 (br. s, 1H, NH), 7.40–7.55 (m, 4H, Ar).
◦
N-(1-Methylethyl)-4-acetylamino benzenesulfonyl amide (Ib). Yield 85.5%, white powder, m.p. 165 C. 1H NMR
(300 MHz, CD3OD): = 1.10 (s, 9H, 3 Me), 2.10 (s, 3H, Me), 7.50 (d, J = 6.5 Hz, 2H, Ar), 7.60 (d, J = 6.5 Hz, 2H, Ar).
δ
◦
N-(1,1-Dimethylethyl)-4-acetylamino benzenesulfonyl amide (Ic), Yield 91%, white powder, m.p. 205 C. 1H NMR
(300 MHz, CD3OD): = 1.10 (s, 9H, 3 Me), 2.10 (s, 3H, Me), 7.50 (d, J = 6.5 Hz, 2H, Ar), 7.60 (d, J = 6.5 Hz, 2H, Ar).
δ
◦
N-Phenyl-4-acetylamino benzenesulfonyl amide (Id), Yield 67.2%, white powder, m.p. 207–209 C. 1H NMR
(300 MHz, acetone-d6): δ = 1.85 (s, 3H, Me), 6.80–7.30 (m, 9H, Ar), 7.43 (br. s, 1H, NH), 8.6 (br. s, 1H, NH).
N-(2-Methoxyphenyl)-4-acetylamino benzenesulfonyl amide (Ie), Yield 71.8%, white powder, m.p. 161 ◦C.
1H NMR (300 MHz, acetone-d6): δ = 2.10 (s, 3H, Me), 2.60 (s, 3H, Me), 3.80 (br. s, 1H, NH), 7.00–7.90 (m, 8H, Ar).
◦
N-Benzyl-4-acetylamino benzenesulfonyl amide (If), Yield 89.3%, white powder, m.p. 158 C. 1H NMR (300 MHz,
acetone-d6): δ = 2.10 (s, 3H, Me), 4.35–4.42 (m, 2H, CH2), 4.80 (br. s, 1H, NH), 7.00–7.90 (m, 9H, Ar).
◦
N-(3-Fluorobenzyl)-4-acetylamino benzenesulfonyl amide (Ig). Yield 86%, white powder, m.p. 139 C. 1H NMR
(300 MHz, DMSO-d6): = 2.09 (s, 3H, Me), 3.99 (s, 2H, CH2), 7.01–7.08 (m, 2H, Ar), 7.08 (d, J = 7.72 Hz, 1H, Ar),
7.25–7.33 (m, 1H, Ar), 7.70–7.75 (m, 4H, Ar), 8.07 (s, 1H, NH), 10.29 (s, 1H, NH).
δ
◦
N-(Carboxypropyl)-4-acetylamino benzenesulfonyl amide (Ih), Yield 75%, white powder, m.p. 145 C. 1H NMR
(300 MHz, DMSO-d6): = 1.57 (d, J = 7.23 Hz, 2H, CH2), 2.10 (s, 3H, Me), 2.19 (t, J = 7.36 Hz, 2H, CH2), 2.66
(q, J = 6.72 Hz, 2H, CH2), 5.50 (s, 1H, NH), 7.02–7.06 (m, 2H, Ar), 7.25–7.35 (m, 1H, Ar), 11.2 (s, 1H, NH).
δ

Molecules 2018, 23, 3152
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◦
1
N,N-Diphenyl-4-acetylamino benzenesulfonyl amide (Ii), Yield 91.6%, white powder, m.p. 233 C. H NMR
(300 MHz, acetone-d6): δ = 2.05–2.14 (m, 3H, Me), 2.90 (s, 1H, CH), 5.70 (br. s, 1H, NH), 7.20–07.6 (m, 14H, Ar).
1
N,N-Dimethyl-4-acetylamino benzenesulfonyl amide (Ij), Yield 80%, white powder, m.p. 149 ◦C. H NMR
(300 MHz, acetone-d6): δ = 2.20 (s, 3H, Me), 2.70 (s, 6H, 2 Me), 5.40 (br. s, 1H, NH), 6.60–7.50 (m, 4H, Ar).
◦
N-Benzyl-N-methyl-4-acetylamino benzenesulfonyl amide (Ik), Yield 78%, white powder, m.p. 154 C. 1H NMR
(300 MHz, acetone-d6):
(m, 4H, Ar).
δ = 2.20 (s, 3H, Me), 2.60 (s, 3H, MeCO), 4.20 (s, 2H, CH2), 7.25–7.36 (m, 5H, Ph), 7.70–7.90
◦
(4-Benzylpiperazino)-4-acetylamino benzenesulfonyl amide (Il), Yield 69%, white powder, m.p. 108 C. 1H NMR
(300 MHz, acetone-d6): = 2.10 (s, 3H, Me), 2.40–2.55 (m, 4H, 2 CH2), 2.90–3.00 (m, 4H, 2 CH2), 3.45–3.55 (m, 2H,
CH2-Ph), 7.10–7.25 (m, 5H, Ph), 7.60–7.80 (d, J = 6.5 Hz, 4H, Ar), 9.50 (br. s, 1H, NH).
δ
◦
(4-Methylpiperazino)-4-acetylamino benzenesulfonyl amide (Im), Yield 81%, white powder, m.p. 198 C. 1H NMR
(300 MHz, acetone-d6):
δ = 2.05 (s, 3H, Me), 2.13 (s, 3H, Me), 2.33 (s, 4H, 2 CH2), 2.78 (s, 4H, 2 CH2), 7.60
(d, J = 6.5 Hz, 2H, Ar), 7.70 (d, J = 6.5 Hz, 2H, Ar), 10.5 (br. s, 1H, NH).
N-(1-Methylbenzimidazole-2-yl)-4-acetylamino benzenesulfonyl amide (In). Yield 24%, white powder, m.p.
◦
168 C. 1H NMR (300 MHz, DMSO-d6):
δ = 2.05–2.15 (m, 3H, Me), 3.20 (s, 3H, Me), 4.70 (br. s, 1H, NH), 7.10–7.80
(m, 8H, Ar), 10.70 (br. s, 1H, NH).
◦
Sulfanilic acid N-methyl amide (IIa), Yield 48%; m.p. 110 C. 1H NMR (300 MHz, acetone-d6):
δ = 1.85 (s, 3H, Me),
5.00 (br. s, 2H, NH2), 6.60 (d, J = 6.5 Hz, 2H, Ar), 7.50 (d, J = 6.5 Hz, 2H, Ar).
1
Sulfanilic acid N-(1-methylethyl) amide (IIb). Yield 68%, white powder, m.p. 116 ◦C. H NMR (300 MHz,
acetone-d6):
δ = 1.04 (s, 3H, Me), 1.06 (s, 3H, Me), 3.25–3.35 (m, 1H, CH), 6.75 (d, J = 6.5 Hz, 2H, Ar), 7.55
(d, J = 6.5 Hz, 2H, Ar).
Sulfanilic acid N-(1,1-dimethylethyl) amide (IIc). Yield 87%, white powder, m.p. 126 ◦C. 1H NMR (300 MHz,
acetone-d6):
δ = 1.20 (s, 9H, 3 Me), 5.30 (br. s, 2H, NH2), 5.90 (br. s, 1H, NH), 6.70 (d, J = 6.5 Hz, 2H, Ar), 7.70
(d, J = 6.5 Hz, 2H, Ar).
Sulfanilic acid N-phenyl amide (IId). Yield 93%, white powder, m.p. 197 ◦C. 1H NMR (300 MHz, acetone-d6):
δ = 5.40 (br. s, 2H, NH2), 6.60–6.90 (m, 4H, Ar), 7.00–7.60 (m, 5H, Ar), 8.20 (br. s, 1H, NH).
Sulfanilic acid N-(2-methoxyphenyl) amide (IIe). Yield 97.2%, white powder, m.p. 205 ◦C. 1H NMR (300 MHz,
DMSO-d6):
δ = 3.70 (s, 3H, Me), 5.40 (br. s, 2H, NH2), 6.65 (d, J = 6.5 Hz, 2H, Ar), 6.90 (d, J = 6.5 Hz, 2H, Ar),
7.05–7.15 (m, 1H, Ar), 7.46–7.50 (m, 3H, Ar), 7.64 (br. s, 1H, NH).
◦
Sulfanilic acid N-benzyl amide (IIf). Yield 99%, white powder, m.p. 120 C. 1H NMR (300 MHz, DMSO-d6):
δ = 4.60 (s, 2H, CH2), 5.40 (br. s, 2H, NH2), 6.65–6.75 (m, 7H, Ar), 6.90 (d, J = 6.5 Hz, 2H, Ar).
1
Sulfanilic acid N-(3-fluorophenyl) amide (IIg). Yield 97%,white powder, m.p. 120 ◦C. H NMR (300 MHz,
DMSO-d6):
δ = 3.91 (d, J = 1.5 Hz, 2H, CH2), 5.90 (s, 1H, Ar), 6.60 (d, J = 8.63 Hz, 1H, Ar), 7.00–7.06 (m, 2H,
Ar), 7.08 (d, J = 7.69 Hz, 1H, Ar), 7.32 (dd, J = 7.35 and 14.33 Hz, 1H, Ar), 7.43 (d, J = 8.61 Hz, 2H, Ar), 7.69 (t,
J = 2.25 Hz, 1H, NH).
1
Sulfanilic acid N-(carboxypropyl) amide (IIh). Yield 86%, white powder, m.p. 129 ◦C. H NMR (300 MHz,
DMSO-d6):
δ = 1.57 (q, J = 7.23 Hz, 2H, CH2), 2.19 (t, J = 7.36 Hz, 2H, CH2), 2.66 (q, J = 7.23 Hz, 2H, CH2), 5.88
(br. s, 2H, NH2), 6.60 (d, J = 8.63 Hz, 2H, Ar), 7.01 (t, J = 5.92 Hz, 1H, NH), 7.39 (d, J = 8.62 Hz, 2H, Ar), 11.99 (br. s,
1H, OH).
Sulfanilic acid N-(bis-phenylmethyl) amide (IIi). Yield 97%, white powder, m.p. 179 ◦C. 1H NMR (300 MHz,
DMSO-d6):
δ = 2.90 (s, 1H, CH), 5.30 (br. s, 1H, NH), 5.60 (br. s, 2H, NH2), 6.60 (d, J = 6.5 Hz, 2H, Ar), 7.15–7.30
(m, 10H, Ar), 7.60 (d, J = 6.5 Hz, 2H, Ar).
◦
Sulfanilic acid N-(1,1-dimethyl) amide (IIj). Yield 94%, white powder, m.p. 175 C. 1H NMR (300 MHz, acetone
d6): δ = 2.50 (s, 6H, 2 Me), 5.40 (br. s, 2H, NH2), 6.60–7.50 (m, 4H, Ar).
◦
1
Sulfanilic acid N-methyl-N-benzyl amide (IIk). Yield 98%, white powder, m.p. 137 C. H NMR (300 MHz,
acetone-d6):
δ = 2.50 (s, 3H, Me), 4.20 (s, 2H, CH2), 5.50 (br. s, 2H, NH2), 6.70 (d, J = 6.5 Hz, 2H, Ar), 7.25–7.35
(m, 5H, Ph), 7.60 (d, J = 6.5 Hz, 2H, Ar).
Sulfanilic acid N-(4-benzylpiperazino) amide (IIl). Yield 89%, white powder, m.p. 244 ◦C. 1H NMR (300 MHz,
acetone-d66 + DMSO-d6 (1:1 v/v)):
δ = 2.35–2.45 (m, 4H, (CH2)2N), 2.40–2.50 (m, 4H, (CH2)2N-SO2), 3.30 (br. s, 2H,
NH2), 3.40 (s, 2H, CH2-Ph), 6.60–7.40 (m, 4H, Ar), 7.30–7.40 (m, 5H, Ph).
◦
Sulfanilic acid N-(4-methylpiperazino) amide (IIm). Yield 92%, white powder, m.p. 212 C. 1H NMR (300 MHz,
DMSO d6): = 2.13 (s, 3H, Me), 2.33 (s, 4H, (CH2)2N), 2.78 (s, 4H, (CH2)2N-SO2), 6.10 (br. s, 2H, NH2), 6.65 (d,
J = 8.45 Hz, 2H, Ar), 7.34 (d, J = 8.36 Hz, 2H, Ar).
δ

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Sulfanilic acid N-(1-methylbenzimidazole-2-yl) amide (IIn). Yield 45%, white powder, m.p. 265 ◦C. 1H NMR
(300 MHz, acetone-d6 + DMSO-d6 (1:1 v/v)):
δ = 3.40 (s, 3H, Me), 5.60 (br. s, 2H, NH2), 6.60 (d, J = 8.1 Hz, 2H, Ar),
7.50 (d, J = 8.2 Hz, 2H, Ar), 7.10–7.80 (m, 4H, Arim).
Appendix B
Yana A_B solub #97 [modified by LAB-HPLC]
mAU
1 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
400
1 - 4.620
350
IIa
300
250
200
150
100
50
0
min
15.0
-50
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
2.0ul
9.0
10.0
11.0
11.0
11.0
12.0
12.0
12.0
13.0
13.0
13.0
14.0
Yana A_B solub #98 [modified by LAB-HPLC]
mAU
2 2ul
-
UV_VIS_2
WVL:254 nm
1,400
1 - 7.077
1,200
1,000
800
600
400
200
0
IIb
min
15.0
UV_VIS_2
-200
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
3 2ul
8.0
9.0
10.0
14.0
Yana A_B solub #99 [modified by LAB-HPLC]
mAU
- 2.0ul
180
160
140
120
100
80
WVL:254 nm
1 - 9.343
IIc
60
40
20
0
min
15.0
-20
160
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
14.0
Yana A_B solub #100 [modified by LAB-HPLC]
mAU
4 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
1 - 11.283
140
120
100
80
IId
60
40
20
0
min
-20
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Figure A1. Cont.

Molecules 2018, 23, 3152
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Yana A_B solub #101 [modified by LAB-HPLC]
mAU
5 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
120
100
80
60
40
20
0
1 - 13.690
IIe
O
S
NH
H2N
OCH3
O
min
-20
140
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
19.9
Yana A_B solub #102 [modified by LAB-HPLC]
mAU
6 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
1 - 13.113
120
100
80
IIf
60
40
20
0
min
17.2
-20
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
Yana A_B solub #103 [modified by LAB-HPLC]
mAU
7 2ul
-
2.0ul
UV_VIS_2
WVL:254 nm
50.0
40.0
30.0
20.0
10.0
1 - 14.960
IIg
0.0
-5.0
250
min
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
8 2ul 2.0ul
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
Yana A_B solub #104 [modified by LAB-HPLC]
mAU
-
UV_VIS_2
WVL:254 nm
IIh
1 - 3.093
200
150
100
50
0
min
-50
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
11 2ul
8.0
- 2.0ul
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Yana A_B solub #156 [modified by LAB-HPLC]
mAU
UV_VIS_2
30.0
WVL:254 nm
1 - 31.253
IIi
25.0
20.0
15.0
10.0
5.0
0.0
min
45.0
-5.0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
17.5
20.0
22.5
25.0
27.5
30.0
32.5
35.0
37.5
40.0
42.5
Figure A1. Cont.

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Yana A_B solub #106 [modified by LAB-HPLC]
mAU
10 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
160
140
120
100
80
1 - 6.613
IIj
60
40
20
0
min
-20
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Yana A_B solub #118 [modified by LAB-HPLC]
mAU
21 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
1,200
1 - 15.100
1,000
800
600
400
200
0
IIk
min
-200
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
20.0
Yana A_B solub #121 [modified by LAB-HPLC]
mAU
24 2ul - 2.0ul
UV_VIS_2
WVL:254 nm
2,486
1 - 6.650
IIl
2,000
1,750
1,500
1,250
1,000
750
500
250
0
min
15.0
-260
2,500
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
27 2ul
8.0
2.0ul
9.0
10.0
11.0
12.0
13.0
14.0
Yana A_B solub #124 [modified by LAB-HPLC]
mAU
-
UV_VIS_2
WVL:254 nm
1 - 5.597
IIm
2,000
1,500
1,000
500
0
min
20.0
-500
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
Figure A1. Cont.

Molecules 2018, 23, 3152
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Yana A_B solub #119 [modified by LAB-HPLC]
mAU
22 2ul
-
2.0ul
UV_VIS_2
WVL:254 nm
3,000
2,500
2,000
1,500
1,000
500
1 - 3.133
IIn
0
min
20.0
-500
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
14.0
15.0
16.0
17.0
18.0
19.0
Figure A1. HPLC chromatograms of the synthesized sulfanilamide derivatives IIa–n.
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Sample Availability: Samples of the compounds IIa–n are available from the authors.
©
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/).