Silver(I) and gold(I) complexes with sulfasalazine: Spectroscopic characterization, theoretical studies and antiproliferative activities over Gram-positive and Gram-negative bacterial strains

Article abstract of DOI:10.1016/j.molstruc.2020.128158

The emergence of bacterial strains resistant to antibiotics, such as the sulfonamides (sulfa drugs), is currently a case of concern. The synthesis of metal complexes using well-known antibacterial agents and bioactive metals has proven to be an excellent strategy in the development of new and more active metallodrugs. Herein, we report the synthesis, structural characterization and antibacterial analysis of new gold(I) and silver(I) complexes with the sulfa drug sulfasalazine (ssz). Elemental, thermal and high-resolution mass spectrometric measurements indicated a 1:1:1 Au/ssz/Ph₃P molar composition for the gold(I) complex (Ph₃P - triphenylphosphine), while for the silver(I) complex the molar composition was 1:1 Ag/ssz. Solution state NMR and infrared spectroscopic data suggest that ssz coordinates to silver(I) and gold(I) by the oxygen atoms of the deprotonated carboxylic group. The coordination mode of the carboxylate was supported by density functional theory (DFT) calculations, which reinforces a monodentate coordination for the gold(I) complex and a bridged bidentate mode for the silver(I) one, with the molecular formulas [(Ph₃P)Au(ssz)] and [Ag(ssz)]₂, respectively. Antibacterial activity assays indicated the sensitivity of Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains to [Ag(ssz)]₂ and [(Ph₃P)Au(ssz)] complexes, while the free ligand was not able to inhibit the growth of any tested bacteria. The non-interaction of the complexes with deoxyribonucleic acid (DNA) was also demonstrated, which suggests that this biomolecule is not a preferential target for the compounds.

Full text of DOI:10.1016/j.molstruc.2020.128158

Journal Pre-proof
Silver(I) and gold(I) complexes with sulfasalazine: Spectroscopic characterization,
theoretical studies and antiproliferative activities over Gram-positive and Gram-
negative bacterial strains
Ana Thereza Fiori-Duarte, Raphael Enoque F. de Paiva, Carlos M. Manzano, Wilton
R. Lustri, Pedro P. Corbi
PII:
S0022-2860(20)30483-X
DOI:
https://doi.org/10.1016/j.molstruc.2020.128158
MOLSTR 128158
Reference:
To appear in: Journal of Molecular Structure
Received Date: 24 January 2020
Revised Date: 23 March 2020
Accepted Date: 29 March 2020
Please cite this article as: A.T. Fiori-Duarte, R.E.F. de Paiva, C.M. Manzano, W.R. Lustri, P.P. Corbi,
Silver(I) and gold(I) complexes with sulfasalazine: Spectroscopic characterization, theoretical studies
and antiproliferative activities over Gram-positive and Gram-negative bacterial strains, Journal of
Molecular Structure (2020), doi: https://doi.org/10.1016/j.molstruc.2020.128158.
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1
Silver(I) and gold(I) complexes with sulfasalazine: spectroscopic
characterization, theoretical studies and antiproliferative activities over
Gram-positive and Gram-negative bacterial strains
Ana Thereza Fiori-Duarte1,2, Raphael Enoque F. de Paiva3, Carlos M. Manzano1, Wilton
R. Lustri4, Pedro P. Corbi1*
1
Institute of Chemistry, University of Campinas – UNICAMP, P.O. Box 6154, 13083-
970 Campinas, SP, Brazil.
2 Faculty of Pharmaceutical Sciences, University of Campinas – UNICAMP, 13083-871
Campinas, SP, Brazil.
3
Institute of Chemistry, University of São Paulo – USP, 05508-000 São Paulo, SP,
Brazil.
4
Biological and Health Sciences Department – University of Araraquara-UNIARA,
14801-320 Araraquara, SP, Brazil.
* Corresponding author: ppcorbi@unicamp.br

2
ABSTRACT
The emergence of bacterial strains resistant to antibiotics, such as the
sulfonamides (sulfa drugs), is currently a case of concern. The synthesis of metal
complexes using well-known antibacterial agents and bioactive metals has proven to be
an excellent strategy in the development of new and more active metallodrugs. Herein,
we report the synthesis, structural characterization and antibacterial analysis of new
gold(I) and silver(I) complexes with the sulfa drug sulfasalazine (ssz). Elemental,
thermal and high-resolution mass spectrometric measurements indicated a 1:1:1
Au/ssz/Ph3P molar composition for the gold(I) complex (Ph3P - triphenylphosphine),
while for the silver(I) complex the molar composition was 1:1 Ag/ssz. Solution state
NMR and infrared spectroscopic data suggest that ssz coordinates to silver(I) and
gold(I) by the oxygen atoms of the deprotonated carboxylic group. The coordination
mode of the carboxylate was supported by density functional theory (DFT) calculations,
which reinforces a monodentate coordination for the gold(I) complex and a bridged
bidentate mode for the silver(I) one, with the molecular formulas [(Ph3P)Au(ssz)] and
[Ag(ssz)]2, respectively. Antibacterial activity assays indicated the sensitivity of Gram-
positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative (Escherichia
coli and Pseudomonas aeruginosa) bacterial strains to [Ag(ssz)]2 and [(Ph3P)Au(ssz)]
complexes, while the free ligand was not able to inhibit the growth of any tested
bacteria. The non-interaction of the complexes with deoxyribonucleic acid (DNA) was
also demonstrated, which suggests that this biomolecule is not a preferential target for
the compounds.
KEYWORDS
Sulfasalazine; Silver(I); Gold(I); Mass spectrometry; DFT; Antibacterial activity.

3
Abbreviation list (in alphabetical order)
[Ag(ssz)]2 – silver(I) complex with sulfasalazine;
ATCC - American Type Culture Collection;
BHI - Brain Heart Infusion;
CFU - Colony Forming Unit;
CisPt – Cisplatin;
DFT - Density Functional Theory;
DMSO - Dimethylsulfoxide;
DNA - Deoxyribonucleic acid;
ESI-QTOF-MS
Spectrometry;
-
Electrospray Ionization Quadrupole Time-of-Flight Mass
IR - Infrared vibrational spectroscopy;
MIC - Minimum Inhibitory Concentration;
NMR - Nuclear Magnetic Resonance;
(Ph3P) – Triphenylphosphine;
[(Ph3P)AuCl] - Chloro(triphenylphosphine)gold(I);
[(Ph3P)Au(ssz)] - Gold(I) complex with sulfasalazine;
ssz – Sulfasalazine;
SYBR Green - Nucleic acid gel electrophoresis stain;
TG/DTA - Thermogravimetric and Differential Thermal Analyses;

4
1. Introduction
The emergence of bacterial resistance to antibiotics is currently considered as a
case of concern by the World Health Organization (WHO). The lack of effective
antibiotics could have catastrophic effects on well-established modern medical
procedures, such as organ transplantation and general surgeries, cancer therapy and
chronic disease treatments [1,2]. The excessive use of the current antibiotics and the
lack of development of new drugs is among the main causes related to the crisis of
bacterial resistance [3].
In this context, one of the strategies for the development of new active
compounds for the treatment of infectious diseases is the combination of the already
known antibacterial drugs, such as the sulfonamides (sulfa drugs) with bioactive metals,
such as silver and gold, among others [4], seeking an improvement of activity by a
synergistic or additive effect [5–7]. In the viewpoint of coordination chemistry, the use
of sulfa drugs is an excellent strategy because they are endowed with coordinating
atoms and, in some cases, the metal complexes have shown to be more effective than
the free sulfas over bacterial strains [8–11].
The sulfonamides were the first chemotherapeutic agents used to treat bacterial
infections
[12,13].
Sulfasalazine
(ssz,
2-hydroxy-5-[[4-(pyridin-2-
ylsulfamoyl)phenyl]diazenyl]benzoic acid, C18H14N4O5S, Figure 1), is a sulfonamide
derived from mesalazine and it has shown to possess antibacterial and anti-
inflammatory activities, being classified as a nonsteroidal anti-inflammatory drug of the
salicylate class. It is used to treat chronic diseases such as rheumatoid arthritis and
inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease) [14–18].
According to the pH medium, sulfasalazine can exist in four forms in solution: neutral
(non-ionized) and three ionized forms associated with deprotonation of carboxyl group
(pKa1 = 2.35), sulfonamide and hydroxyl groups [19].
In addition to the antimicrobial activity, there are descriptions of sulfonamide-
derived drugs and its complexes that have shown to be effective in vitro and in vivo for
the treatment of various types of cancer [8,20–22]. Recently, the use of sulfasalazine to
improve the treatment of radio-resistant cancers was demonstrated in a study by Nagane
et al., where sulfasalazine decreased intratumoral glutathione content, increasing
radiosensitivity in an in vivo transplanted melanoma model [23].

5
16
15
13
9
10
17
O
N
OH 20
14
6
N
12
1
3
S
N
18
19
11
8
21
NH
7
O
O
2
5
9a
10a
HO
22
4
Fig. 1. Structure of sulfasalazine with carbon, hydrogen and nitrogen atoms
numbered.
Refat et al. synthesized and characterized sulfasalazine complexes and evaluated
their biological activities. The manganese(II), mercury(II), chromium(III) and
yttrium(III) complexes were prepared and evaluated for their antimicrobial and
antifungal activities by an antibiogram assay. In this study, the mercury(II) complex
was shown to be active against Tricoderma sp., with an inhibition zone of 20 mm of
diameter while sulfasalazine did not show inhibition in the same experimental
conditions [14]. There are also descriptions in the literature from the same group on the
synthesis of magnesium(II), calcium(II), strontium(II) and barium(II) complexes with
sulfasalazine. The strontium(II) complex showed higher activity than the free ligand and
the other complexes against Staphylococcus aureus (S. aureus), Escherichia coli (E.
coli), Bacillus anthracis (B. anthracis), Pseudomonas aeruginosa (P. aeruginosa) and
the fungi Candida albicans (C. albicans) [24]. A copper(II) complex with sulfasalazine
and
triphenylphosphine
as
ligands,
with
coordination
formula
[Cu(C18)H13N4O5S)(Ph3P)2], where Ph3P is triphenylphosphine, was also described in
the literature, but no biological studies were performed [25].
Gold(I) complexes with sulfonamides have also shown to be promising as
antimicrobial agents. Mizdal et al. presented the synthesis of five gold complexes with
sulfadiazine and sulfamethoxazole. Minimum inhibitory concentration (MIC) assays
against MRSA (methicillin-resistant S. aureus) and strains of the same species obtained
from clinical isolates showed the better activity of all five complexes in comparison to
the free ligands. The MIC of the gold(I) complexes with sulfamethoxazole against
MRSA were all between 2 and 8 µg/mL, while the MIC of sulfamethoxazole alone was
256 µg/mL [26].

6
Silver complexes with sulfonamides have also been extensively investigated as
antibacterial agents. In special, silver sulfadiazine (AgSSD) can be considered as the
most successful example of the use of sulfa in coordination chemistry. This complex is
used clinically worldwide in the treatment of bacterial skin infections in burn wounds
[27]. Since the discovery of the action of AgSSD, many other silver complexes have
been investigated for the treatment of bacterial infections. Some examples include the
silver(I)
complexes
with
sulfamoxole
(SMX),
[Ag2(SMX)2]·H2O
and
[Ag4(SCN)3(SMX)]·H2O, which exhibit antimicrobial activity against E. coli, S. aureus
and P. aeruginosa strains, and also antifungal action (against ten fungi strains) [28].
In the last years, our research group has dedicated efforts in the search of novel
and bioactive metal complexes with sulfonamides. Paiva et al. demonstrated the
antibacterial activity of a silver(I) complex with the anti-inflammatory nimesulide over
Gram-positive and Gram-negative bacteria [29]. Nunes et al. synthesized silver(I)
complexes with the sulfonamides sulfathiazole and sulfamethoxazole and evaluated
their antibacterial activity. The MIC values over S. aureus, P. aeruginosa and
Salmonella enterica ranged from 3.45 to 6.90 mmol L-1 for the sulfathiazole complex
and 1.74 to 13.9 mmol/L for the sulfamethoxazole one [30].
Similarly, silver(I) complexes with sulfisoxazole, sulfadimethoxine [31] and
sulfameter [32] were synthesized and exhibited antibacterial activities against S. aureus,
P. aeruginosa and E. coli with MIC values in the micromolar range. In all these
examples, the free sulfonamides were not able to inhibit any of the bacteria strains in the
considered experimental conditions. Yamamoto et al. reported copper(II) and silver(I)
complexes with sulfamethizole that were active over the same bacteria strains in the low
milimolar range, between 3.31 and 0.41 mmol L-1 [33].
More recently, Nakahata et al. studied the biological properties of three new
copper(II) complexes with the sulfonamides sulfameter and sulfadimethoxine, where the
nuclease activity of the complexes were correlated with their anti-M. tuberculosis
profiles and also with their antitumoral activities (total growth inhibition in the
micromolar range) over a variety of cancer cell lines, specially glioma (U251),
melanoma (UACC-62), ovarian (OVCAR-3) and colon (HT29) [34].
In this work, we present the synthesis of new gold(I) and silver(I) complexes
with sulfasalazine and its characterization combining theoretical and experimental
data. The antibacterial activities of the complexes and the biophysical studies about the
interaction of the compounds with DNA are also described here for the first time.

7
2. Materials and methods
2.1.Materials
Sulfasalazine (ssz ≥ 98%) and silver(I) nitrate (AgNO3 ≥ 99%) were purchased
from Sigma-Aldrich/Merck Laboratories. Tetrachloroauric(III) acid trihydrate (≥ 49%
Au) was obtained from Acros Organics. Sodium hydroxide (≥ 97%), triphenylphosphine
(≥ 98%) and triethylamine (N(CH CH ) ≥ 99%) were obtained from Vetec. SYBR
Green and cisplatin were also purchased from Sigma-Aldrich/Merck Laboratories.
Deoxyribonucleic acid (DNA) Ladder 1Kb Plus (1200 bp) was purchased from
Invitrogen. All chemical reagents were used as received.
2.2.Equipment
Elemental analyses for carbon, hydrogen and nitrogen were performed using a
Perkin Elmer 2400 CHNS/O Analyzer. Thermogravimetric and differential thermal
analyses (TG/DTA) were performed on a simultaneous TGA/DTA SEIKO EXSTAR
6000 thermal analyzer, using the following conditions: synthetic air, flow rate of 50 cm3
min-1 and heating rate of 10°C min-1, from 25°C to 900°C. X-Ray powder diffraction
analyses were performed on a Shimadzu XRD 7000 diffractometer. Infrared (IR)
spectroscopic analyses were performed in an Agilent Cary 630 FTIR spectrometer,
using the Attenuated Total Reflectance (ATR) method, in the range from 4000-400 cm-1
1
and with resolution of 4 cm-1. Solution state H and 13C nuclear magnetic resonance
(NMR) spectra were recorded in Bruker Avance III 400 MHz, 500 MHz and 600 MHz
spectrometers. The NMR spectra were acquired in DMSO-d6. The chemical shifts were
given relative to tetramethylsilane (TMS). Electrospray ionization quadrupole time-of-
flight mass spectrometric (ESI-QTOF-MS) measurements were performed on a XEVO-
QTOF-MS instrument operating in the positive mode. The samples were dissolved in 10
μL of DMSO and then diluted in a 1:1 H2O/CH3CN solution containing 0.1% of formic
acid (v/v). The instrumental parameters of the acquisition were: capillary voltage 3.5
kV, sampling cone voltage 30 V, source temperature 150°C, desolvation temperature
150 °C and collision energy 6 eV. Electrophoresis system BIO-RAD® (Sub-Cell® GT)
was used for the agarose gel electrophoresis studies.

8
2.3.Synthesis of the silver complex
The silver complex was synthesized by the reaction of 5.0 x 10-4 mol (0.0844 g)
of a freshly prepared 1:1 methanol/water solution of AgNO3 (10.0 mL) with a solution
containing 5.0 x 10-4 mol (0.1993 g) of sulfasalazine in 20.0 mL of a 1:1
methanol/water solution. The pH of the final solution was adjusted to 8-9 by the
addition of NaOH 0.10 mol/L solution. The synthesis was carried out under stirring and
at room temperature for 2 hours. A brownish solid was obtained and vacuum filtered,
washed with cold water and dried in a desiccator over P4O10. Elemental analysis led to a
1:1 Ag/ssz composition. Anal. Calcd. for AgC18H13N4O5S (%): C, 42.8; H, 2.59; N,
11.1. Found (%): C, 43.0; H, 2.59; N, 11.1. The silver complex is soluble in DMSO. No
single crystals were obtained considering crystal growth techniques such as mixture of
solvents or recrystallization for a detailed X-ray crystallographic study.
2.4.Synthesis of the gold(I) complex
First, the chloro(triphenylphosphine)gold(I) precursor ([(Ph3P)AuCl]) was
prepared by the reaction of 5.0 x 10-4 mol (0.1967 g) of tetrachloroauric(III) acid
trihydrate (HAuCl4·3H2O) dissolved in 1.0 mL of a 1:1 ethanol/acetone solution, with
1.0 x 10-3 mol (0.2626 g) of triphenylphosphine (Ph3P) dissolved in 1.0 mL of
chloroform. The synthesis was carried out under stirring at room temperature and after a
few minutes a white solid was obtained, filtered off and washed with ethanol.
Subsequently, a freshly prepared solution of [(Ph3P)AuCl] (1.25 x 10-4 mol,
0.0616 g) in 3.0 mL of chloroform was added to a solution of sulfasalazine (1.25 x 10-4
mol, 0.0498 g) and triethylamine (0.25 mL) in methanol (4 mL). The synthesis was
carried out under reflux for 24 hours. After slow evaporation an orange solid was
obtained. Elemental analysis led to a 1:1:1 Au/ssz/Ph3P composition, plus one
triethylamine molecule. Anal. Calcd. for AuPC36H28N4O5S∙N(CH CH ) (%): C,
52.7; H, 4.53; N, 7.31. Found (%): C, 51.8; H, 4.74; N, 7.45. The [(Ph3P)Au(ssz)]
complex is soluble in dimethylsulfoxide (DMSO) and chloroform and low soluble in
water, ethanol, methanol, acetonitrile and acetone. Again, no single crystals were
obtained for a detailed X-ray crystallographic study.

9
2.5.DFT studies
The structures of the silver(I) and gold(I) complexes were optimized using the
Density Functional Theory (DFT) as implemented in Orca 4.2 [35,36]. For the gold
compound, the hybrid functional PBE0, along with the triple-zeta def2-TZVP basis set
were used. Auxiliary Weigend basis were used [37]. Due to the size of the system, for
the silver compound the def2-SVP basis set was used. A grid size of 5 and a tight SCF
convergence criteria were used for both systems. The final figures were rendered with
JMol 14.29.53.
2.6.Antibacterial activity assays
The antibacterial activities of the precursors and the silver(I) and gold(I)
complexes were determined over four referenced bacterial strains, S. aureus ATCC
25923, Bacillus cereus (B. cereus) ATCC 14579, E. coli ATCC 25922 and P.
aeruginosa ATCC 27853 using the minimum inhibitory concentration (MIC) method.
The microorganisms were cultured in separate test tubes containing 10.0 mL of sterile
brain heart infusion (BHI) and incubated for 18h at 35-37 °C, following the
recommendations of the Clinical and Laboratory Standards Institute [38]. An inoculum
from each culture was added to new tubes with sterile BHI until the turbidity measured
was 1.0 McFarland (∼3.0·108 CFU·mL-1; CFU = Colony Forming Units). Stock
solutions of the sulfonamide and complexes were prepared in DMSO 10% (10.0
mg·mL-1). The tested compounds were sequentially diluted with BHI medium in 96
multi-well plate for a final volume of 100 µL/well. An inoculum of 100 µL of the
bacterial strains in BHI suspension at 1.0 McFarland scale was added to each serial
dilution reaching turbidimetric 0.5 McFarland (∼1.5·108 CFU·mL-1) in a final volume
of 200 µL/well at concentrations ranging from 2000 μg·mL-1 to 31.25 μg·mL-1. The
plate was incubated for 18h at 35-37 °C in a damp chamber under stirring at 100 rpm.
The first well of the microplate was used as a negative control, containing 60 μL of
sterile BHI, 40 μL of the DMSO 10% and 100 μL of the bacterial suspension on the 1.0
McFarland scale. As a positive control, a solution of silver nitrate was used. After the
incubation period, 15 μL of resazurin 0.02% in sterile aqueous solution was added to
each well. After 3h of re-incubation the MIC were estimated as reported in the literature
[38].

10
2.7.Agarose gel electrophoresis
The experimental procedure for the agarose gel electrophoresis assay was
similar to previous reports in the literature [39–41]. In this assay, samples of ssz,
silver(I) and gold(I) complexes were first solubilized in DMSO; AgNO3 and K2PdCl4
were solubilized in water and cisplatin (CisPt) was suspended in water. Then, all
2-
-
samples were diluted in phosphate buffer (HPO4 / H2PO4 , pH = 7.4) to 250 µmol·L-1.
Aliquots (6 or 3 µL) of each compound were mixed with pGEX-4T1 plasmid DNA
solution (19 µL) to final concentrations of 60 µmol·L-1 or 30 µmol·L-1 of the
compounds in phosphate buffer. After the incubation (37 °C, 18 hours), the loading
buffer (bromophenol blue 0.25 % m/v, xylene cyanol 0.25 % m/v, glycerol 30% m/v
and EDTA 30 mmol·L-1 in sterile distilled water; 5 µL per sample) was added to each
sample. The samples were applied to a 0.8% agarose gel matrix in Tris-Borate-EDTA
0.5x buffer at pH 8.3 and electrophoresed for 2 hours at 100 V. The pGEX-4T1 plasmid
was used as control and DNA Ladder 1Kb Plus (1200 bp) was used as a weight marker.
The gel was stained with 1 x SYBR green for 3 h and photodocumented using a UVDoc
400i Delpho equipment. The pGEX-4T1 plasmid DNA was provided by Professor
Wilton Rogério Lustri from the Biological and Health Sciences Department/ University
of Araraquara-UNIARA, Brazil.
3. Results and discussion
3.1.Thermal analysis
The gold(I) and silver(I) complexes and the free sulfasalazine were evaluated by
thermogravimetric (TG) and differential thermal analyses (DTA) in order to confirm the
proposed molar compositions of the complexes, and to evaluate the thermal stability of
the compounds.
The thermal decomposition of sulfasalazine occurs in two steps. The first starts
at 238 °C and ends at 322 °C, while the second one starts at 430 ºC and ends at 618 °C.
No residue was observed at 900 °C (Supplementary Material, Fig. S1). The thermal
decomposition of the silver(I) complex also occurs in two exothermic steps. The first
one starts at 230 °C and ends at 312 °C and the second starts at 403 °C and ends at 520
°C. The percentage of mass loss in the two steps is consistent with the release of one

11
ligand (C18H13N4O5S). Calcd. loss of C18H13N4O5S: 78.6%. Found: 76.4%. A residue of
23.6% is observed at 770 °C, which is consistent with the formation of metallic silver.
Calcd.: 21.3%; Found: 23.6% (Supplementary Material, Fig. S2). For the gold(I)
complex, the thermal decomposition also occurs in two exothermic steps. The first one
starts at 106 °C and ends at 380 °C and the second starts at 444 °C and ends at 676 °C.
The percentage of mass loss in the two steps is consistent with one ligand
(C18H13N4O5S), one triphenylphosphine (PC18H15) and one triethylamine (C6H15N).
Calcd. for organic mass loss of AuPC36H28O5N4S∙C6H15N: 79.4%. Found: 75.9%. A
residue of 24.1% is observed at 730 °C, which is consistent with the formation of
metallic gold. Calcd. for Au residue: 20.6%. Found: 24.1% (Supplementary
Material, Fig. S3). The identity of the thermal residue obtained from the decomposition
of the gold(I) complex was confirmed as metallic gold by powder X-ray diffraction
analysis of the residue (Supplementary Material, Fig. S4).
3.2.Infrared absorption spectroscopy
To confirm the coordination of the ligand to metal ions and to determine the
possible coordination sites, infrared (IR) spectroscopic measurements were performed.
The IR spectra (Supplementary Material, Fig. S5) obtained for the complexes were
analyzed in comparison to the spectrum of the ligand in its anionic form (sodium salt).
The coordination mode of the carboxylate (COO-) group to the metal can be
determined by the separation (Δν) between its asymmetric and symmetric stretching
modes. Absorption bands at 1627 and 1424 cm-1 (Δν 203 cm-1) in the IR spectrum of
the sodium salt of sulfasalazine were observed and attributed to the asymmetric (νas)
and symmetric stretching (νs) of the carboxylate group, respectively. For the silver(I)
and gold(I) complexes, bands at 1634 and 1430 cm-1 (Δν 204 cm-1) and at 1631 and
1391
cm-1 (Δν 240 cm-1), respectively, were observed and assigned to the
asymmetric (νas) and symmetric stretching (νs) of the carboxylate group.
It is well-established in the literature [42] that when Δν of the carboxylate group
in a metal complex is similar to the Δν of the ligand, a bridged bidentate mode of the
COO- group is expected, while when the Δν of the complex is greater than the ligand, a
monodentate coordination mode by the carboxylate group is suggested. Therefore,
based on the resolution of the equipment (4 cm-1), there are no changes in the Δν values
when the IR spectrum of the ligand is compared to the silver(I) complex, which is an

12
indication that the carboxylate group coordinates to the silver(I) by a bridged bidentate
mode. For the gold(I) complex, the higher Δν value for the carboxylate group when
compared to the ligand suggests a monodentate coordination mode. Moreover, no
significant changes in the asymmetric and symmetric stretching modes of the sulfone
group in the IR spectrum of the ligand when compared to the spectra of the complexes,
which indicates that the sulfonamide group does not participate in the coordination of
sulfasalazine to the metal atoms.
3.3.NMR spectroscopic measurements
1
13
Solution state H (Figure 2) and C NMR spectra (Supplementary Material,
Fig. S6 and S7) of the silver(I) and gold(I) complexes were obtained in order to identify
the coordination sites of sulfasalazine to silver(I) and gold(I). The spectra of the
complexes were analyzed by comparison with the NMR spectrum of the sulfasalazine.
1
13
By analyzing the H and C spectra of the silver(I) and gold(I) complexes and
comparing it to the spectra of the ligand, it was observed that the major chemical shifts
∆δ (δ complex – δ ligand) refer to the hydrogen and carbon atoms present in the
salicylic ring of sulfasalazine. The hydrogen atoms H15 and H16 and the carbon atoms
C14, C15, C17 and C18 have shown to be the most affected ones upon coordination
(Table 1). This result reinforces the suggested coordination modes of sulfasalazine to
silver(I) and gold(I) by the carboxylate group, which is present in the salicylic ring of
sulfasalazine, as suggested by the infrared spectroscopic data [43].
The 1H and 13C spectra also confirms the presence of a molecule of triethylamine
in the composition of the gold(I) complex as already stated by other techniques. There
1
were observed peaks at 1.15 and 3.03 ppm in the H NMR spectrum and at 9.36 and
46.1 ppm in the 13C spectrum of the gold(I) complex, which refer to triethylamine used
in the synthesis to deprotonate the ligand. Such spectra can be seen at the
Supplementary Material, Fig. S8 and S9.

13
Fig. 2. 1H NMR spectra in DMSO-d6 of (A) sulfasalazine, (B) silver(I) complex and (C)
gold(I). The insets (increased intensities) show the signals corresponding to H7 and
H20.

14
1
13
Table 1. H and C NMR assignments and chemical shifts for sulfasalazine, silver(I)
and gold(I) complexes in ppm. For hydrogen and carbon atoms numbering, see Figure
1.
Assignment
Sulfasalazine
Silver(I)
complex (δ)
8.05
Δδ (ppm)
Gold(I) complex
Δδ
(δ)
(δ)
H1
7.99
0.06
0.0
8.00
0.01
H2
6.87
6.87
6.86
-0.01
-0.02
-0.02
H3
7.77
7.75
-0.02
0.0
7.75
H4
7.24
7.24
7.22
H7
15.0 – 10.0
8.06
14.0 – 11.0
8.05
12.0 – 9.0
8.00
H9, H9a
H10, H10a
H15
H16
H19
H20
H22
C1
-0.01
-0.04
-0.12
-0.18
-0.02
-0.06
-0.08
-0.27
-0.41
-0.07
7.96
7.92
7.88
8.09
7.97
7.82
7.17
6.99
6.76
8.36
8.34
8.29
5.00 – 3.00
15.0 – 10.0
154.14
114.96
141.87
114.39
154.14
144.44
128.28
123.21
153.86
114.96
129.56
119.03
164.69
144.89
126.87
171.72
5.00 – 3.00
5.00 – 2.00
154.25
115.06
140.81
114.37
154.21
143.42
127.90
122.45
153.66
116.53
128.12
118.21
166.44
143.64
126.53
171.05
0.11
154.12
115.45
141.34
114.67
154.12
143.01
128.21
122.56
154.56
119.59
127.51
118.64
170.91
142.71
127.20
171.07
-0.02
0.49
C2
0.10
C3
-1.06
-0.02
0.11
-0.53
0.28
C4
C5
-0.02
-1.43
-0.07
-0.65
0.70
C8
-1.02
-0.38
-0.76
-0.20
1.57
C9, C9a
C10, C10a
C11
C14
C15
C16
C17
C18
C19
C21
4.63
-1.44
-0.82
1.75
-2.05
-0.39
6.22
-1.25
-0.34
-0.67
-2.18
0.33
-0.65

15
3.4.Mass spectrometric measurements
The silver(I) and gold(I) complexes were analyzed by ESI(+)-QTOF-MS and the
corresponding full spectra are presented in Figure 3.
For the silver complex, two monoprotonated ions were identified in the spectrum
(Figure 4). The first peak corresponds to the [Ag(C18H13N4O5S) + H]+ ion at m/z
506.98, while the second one is attributed to the [Ag2(C18H13N4O5S)2 + H]+ ion at m/z
1011.03. The peak at m/z 399.05 corresponds to the free ionized sulfasalazine
[(C18H14N4O5S) + H]+. This result confirms the proposed 1:1 metal/ligand composition
and that the complex might exist in a dimeric form, as it will be further discussed by
DFT studies in the 3.5 section.
The mass spectrum of the gold(I) complex shows a peak corresponding to the
[Au(C18H15P)(C18H13N4O5S) + H]+ ion at m/z 857.08. Ligand scrambling reactions of
the gold(I) complex can be observed with the formation of the
[Au2(C18H15P)2(C18H12N4O5S) + H]+ ion at m/z 1315.18, where two (phosphine)gold
units are present. This is a common feature found in the literature [44,45]. This species
probably does not exist in solid form, as confirmed by other techniques. The gold
isotopic pattern of the two species can be observed in Figure 5. The peak at m/z 399.05
corresponds to the free ionized sulfasalazine [(C18H14N4O5S) + H]+ and the peak at m/z
102.11 corresponds to the ionized triethylamine. This result confirms the proposed
composition for this complex as suggested by elemental and thermal analyses, and the
presence of one triethylamine molecule in the complex.

16
Fig. 3. ESI(+)-QTOF mass spectrum for (A) gold and (B) silver complex from m/z 90 to
1500.
Fig. 4. Theoretical isotopic distribution of (A) [Ag(C18H13N4O5S) + H]+ and (C)
[Ag2(C18H13N4O5S)2 + H]+ in comparison with their respective experimental data at (B)
m/z 506.98, (D) m/z 1011.03.

17
Fig. 5. Theoretical isotopic distribution of (A) [Au(C18H15P)(C18H13N4O5S) + H]+ and
(C) [Au2(C18H15P)2(C18H12N4O5S) + H]+
in comparison with their respective
experimental data at (B) m/z 857.13 and (D) m/z 1315.2.
3.5. DFT studies
The infrared, NMR and mass spectrometric data provided essential information
regarding the composition and coordination sphere for the silver(I) and gold(I)
complexes. This information was used to draw the starting geometries that were subject
to DFT optimization. The final optimized structures can be found in Figure 6. In the
case of the silver complex the combination of the theoretical (DFT) and experimental
data led us to propose a dimeric structure for the compound, where each sulfasalazine
bridges between two silver atoms by the carboxylate group. For the gold(I) complex a
monomeric structure has shown to be the most stable one. The bond lengths and angles
obtained for the optimized structures are shown in Table 2. Atomic coordinates for the
gold(I) complex are presented in the Supplementary Material File A, while for the
silver(I) complex the atomic coordinates can be found in the Supplementary Material
File B.

18
Fig. 6. DFT-optimized structures of (A) silver(I) and (B) gold(I) complexes. The atoms
that are part of the coordination spheres are labeled, as well as the metal ions. The
remaining atoms follow the typical color code: Nitrogen - blue; Carbon – dark grey;
sulfur - yellow.
Table 2. Selected bond lengths and angles for the DFT-optimized structures of silver(I)
and gold(I) complexes studied in this work.
silver complex
Atoms
Ag-O
Bond length (Å)
2.113
Atoms
Angle (°)
165.3
O-Ag-O
2.110
165.0
2.117
O-C-O
127.2
2.119
126.8
Ag-Ag
C-O
2.802
1.262
1.256
1.262
1.258

19
N=N
1.247
1.248
Gold(I) complex
Atoms
Au-P
Au-O
N=N
Bond length (Å)
2.226
Atoms
Angle (°)
176.4
P-Au-O
2.041
1.247
The cage-like arrangement observed for the silver(I) complex, which
corresponds to the [Ag(ssz)]2 coordination formula, is typical for silver complexes with
carboxylate-containing ligands and it is in good agreement with the experimental data
for the silver complex with sulfasalazine. One of the best examples of this kind of
arrangement is the silver-acetate complex, where the structure contains
[Ag2(carboxylate)2] dimer units [46]. This kind of carboxylate coordination can also be
seen in the crystal structure of a metal-organic framework containing silver and 4,4’-
bipyridine and salicylate as ligands. The salicylate acts as a bidentate bridging ligand
between two silver atoms in a similar way of that suggested in our case [47]. Another
example is the silver(I) complex with the anti-inflammatory drug ibuprofen, [Ag(ibu)]2,
where each molecule of the ligand bridges between two silver atoms by the carboxylate
group in a very similar fashion of the silver(I) complex described here [48].
The double carboxylate-supported dimeric silver complexes comprise one of the
classes of silver compounds that present argentophilic interactions [49]. The crystal
structure of a representative example of this class of compounds, the silver(I) 3,5-
dimethylbenzoate, has an Ag-Ag interaction of 2.7719 Å [50]. This short interaction
agrees with the Ag-Ag distance found by DFT for the [Ag(ssz)]2 complex, where the
argentophilic interaction is of 2.802 Å as observed in Table 2.
A monodentate coordination of carboxylate to the (phosphine)gold(I) motif is
also a common structural feature. It can be observed, for example, in the crystal
structure of [(Ph3P)Au(acetate)] [51], and in the structure of many (phosphine)gold
compounds with fluorocarboxylates [52]. In the review article of Grodzicki et al., a
series of (phosphine)gold(I) complexes are described and, in all cases, coordination by a
monodentate mode is observed [53]. Such evidences reinforce the suggested
coordination formula for the gold(I) complex as [(Ph3P)Au(ssz)].

20
3.6. Minimum inhibitory concentration (MIC)
Antibacterial activity assays were performed to evaluate the antibacterial activity
of the [Ag(ssz)]2 and [(Ph3P)Au(ssz)] complexes and to compare it with the activities of
the free ligand against Gram-positive (S. aureus and B. cereus) and Gram-negative (E.
coli and P. aeruginosa) strains. The experimental results are summarized in Table 3. As
a positive control, silver nitrate was used, which presented a MIC value < 31.25 μg·mL-
1 for all strains tested.
The free ligand did not exhibit antibacterial activity in vitro for any of the tested
bacterial strains in the considered experimental conditions. On the other hand, with
coordination of ssz to silver and gold metal ions, it was possible to observe inhibition of
the bacterial strains, as evidenced by the MIC values. The [Ag(ssz)]2 complex showed
better activity against S. aureus, with a MIC value of 500 μg·mL-1 (0.495 mmol L-1
For the [(Ph3P)Au(ssz)] complex, the lowest MIC found was of 62.5 μg·mL-1 (0.065
mmol L-1
against B. cereus, which was even more active than the gold(I) precursor
).
)
[(Ph3P)AuCl]. This particular result clearly demonstrates the contribution of
sulfasalazine and the gold(phosphine) moiety to the antibacterial activity of the
[(Ph3P)Au(ssz)] complex.
Comparing the results here presented with other silver(I) complexes with
sulfonamides described in the literature, it was possible to evaluate that the [Ag(ssz)]2
complex has similar MIC values and, in some cases, they have shown to be active in
lower concentrations than those already described. The sulfamethizole [33],
sulfamethoxazole and sulfathiazole [30] complexes with silver(I) presented MIC values
of 3.31, 13.9 and 6.90 mmol L-1, respectively, against S. aureus, and 0.41, 1.74 and 3.45
against P. aeruginosa. Silver sulfadiazine, which is clinically used, shows MIC values
from 0.045 to 0.360 mmol L-1 for S. aureus, a result that can be compared with that
obtained for the [Ag(ssz)]2 and [(Ph3P)Au(ssz)] complexes [54]. The solubility of the
complexes with sulfasalazine in DMSO may also be considered as an advantage over
AgSSD and warrants for further studies considering a possible application for the
treatment of skin infections.

21
Table 3. Minimum inhibitory concentration (MIC) values.
MIC µg mL-1 (mmol L-1)
Compounds
S. aureus
R
B. cereus
R
E. coli
R
P. aeruginosa
Ssz
R
[Ag(ssz)]2
[(Ph3P)Au(ssz)]
500 (0.495)
2000 (2.088)
500 (1.011)
1000 (0.990)
62.5 (0.065)
125 (0.253)
2000 (1.979)
1000 (1.044)
250 (0.505)
1000 (0.990)
500 (0.522)
125 (0.253)
[(Ph3P)AuCl]
AgNO3
< 31.25 (< 0.184) < 31.25 ( < 0.184) < 31.25 ( < 0.184) < 31.25 (< 0.184)
R = resistant.
Although silver nitrate exhibits MIC values lower than those observed for the
silver(I) and gold(I) complexes, it is already well-known in the literature that this salt is
toxic to health tissues in concentrations higher than 1%. Moreover, the light instability
and the fast and uncontrolled release of silver ions from silver nitrate have limited its
application in the clinics [31,55,56]. Nevertheless, further cytotoxic activity assays of
the complexes are necessary to compare with the cytotoxicity of silver nitrate and to
determine the safety of the complexes described here.
3.7. Interaction studies of the complexes with pGEX-4T1 DNA plasmid by agarose
gel electrophoresis
Considering the antibacterial activities exhibited by the new [Ag(ssz)]2 and
[(Ph3P)Au(ssz)] complexes, agarose gel electrophoresis assays were performed to
investigate if DNA is one of the possible targets for the compounds. In this case, pGEX-
4T1 plasmid DNA was used. For comparative purposes, K2PdCl4 and cisplatin
([PtCl2(NH3)2]), which are known for interacting with DNA, were used as positive
controls (Figure 7) [57–60].
The experimental results show modifications in the electrophoretic profile of the
plasmid with cisplatin at both concentrations, more specifically in the first band, which
is attributed to the open circular (OC) form of the plasmid, and in the middle bands that
are attributed to the linear form (LF) [61]. The interaction of K2PdCl4 with the pGEX-
4T1 plasmid is the most pronounced one, where we can see that all bands were affected
by the palladium(II) salt evidencing a strong interaction with DNA, as expected.
However, evidences of interaction of the ligand or the silver(I) and gold(I) complexes
with the plasmid were not detected. These results show that these compounds do not

22
interact with DNA as cisplatin or K2PdCl4. Silver nitrate and the gold precursor
[(Ph3P)AuCl] also did not show evidences of interaction with DNA. Further studies
with other biomolecules, such as proteins may also be envisaged to ascertain the
possible molecular target of the complexes with sulfasalazine.
Fig. 7. Results of the agarose gel electrophoresis of the pGEX-4T1 plasmid DNA in the
presence of K2PdCl4, ssz, [Ag(ssz)]2, AgNO3, [(Ph3P)Au(ssz)], [(Ph3P)AuCl] and
cisplatin (CisPt).
Conclusion
New complexes of silver(I) and gold(I) with sulfasalazine, with minimal
formulas AgC18H13N4O5S and AuPC36H28N4O5S∙N(CH CH ) , were obtained.
Infrared and 1H and 13C NMR studies permitted proposing that sulfasalazine coordinates
to silver(I) by the carboxylate group in a bidentate bridging mode, while for the gold(I)
complex coordination of the carboxylate occurs in a monodentate form. The presence of
the [Ag(C18H13N4O5S) + H]+ and [Ag2(C18H13N4O5S)2 + H]+ monoprotonated species in
the mass spectra suggests that the silver(I) complex exists in a dimeric form. For the
gold(I) complex, the [Au(C18H15P)(C18H13N4O5S) + H]+ ion was identified. DFT studies
combined with the experimental analyses led us to propose the coordination formulas of
the silver(I) and gold(I) species, where a dimeric structure for the silver(I) complex and
a monomeric one for the gold(I) complex have shown to be the most stable ones.

23
Antibacterial studies showed that both complexes are active against Gram-positive (S.
aureus and B. cereus) and Gram-negative (E. coli and P. aeruginosa) strains. The
gold(I) complex has shown to be more active over B. cereus, with MIC value of 62.5 µg
mL-1 (0.065 mmol L-1
), while the silver(I) complex has shown to be more active over S.
aureus bacterial strain, with MIC value of 500 µg mL-1 (0.495 mmol L-1). The
antibacterial activities observed for the [Ag(ssz)]2 and [(Ph3P)Au(ssz)] complexes in
comparison to the free ligand demonstrate that the coordination of metal ions to
bioactive ligands is an interesting strategy that can be further explored. The observed
results are closely related to those found for AgSSD, which is clinically used for
treatment of skin infections. Gel electrophoresis assays with pGEX-4T1 plasmid DNA
were performed, but no interactions of both complexes with DNA were observed. Such
results led us to conclude that DNA is not a preferential biomolecular target for the
complexes.
Disclosure statement
No potential conflict of interest was reported by the authors.
Acknowledgments
This study was supported by grants from the Brazilian Agencies FAPESP (São Paulo
State Research Council, Grants # 2015/09833-0, 2017/25995-6, 2018/12590-0
and
2018/12062-4) and CNPq (National Council of Scientific and Technological
Development, Grant # 407012/2018-4). Also, this study was financed in part by the
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) -
Finance Code 001. Dr. de Paiva also would like to thank the research developed with
high performance computation resources provided by the IT Superintendence of the
University of São Paulo, USP, Brazil.
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Fig. Captions
Fig. 1. Structure of sulfasalazine with carbon, hydrogen and nitrogen atoms numbered.
Fig. 2. 1H NMR spectra in DMSO-d6 of (A) sulfasalazine, (B) silver(I) complex and (C)
gold(I). The extrapolated spectra are given in detail to show the signals corresponding
to H7, H20 and H22.
Fig. 3. ESI(+)-QTOF mass spectrum for (A) gold and (B) silver complex from m/z 90 to
1500.
Fig. 4. Theoretical isotopic distribution of (A) [Ag(C18H13N4O5S) + H]+ and (C)
[Ag2(C18H13N4O5S)2 + H]+ in comparison with their respective experimental data at (B)
m/z 506.98, (D) m/z 1011.03.
Fig. 5. Theoretical isotopic distribution of (A) [Au(C18H15P)(C18H13N4O5S) + H]+ and
(C) [Au2(C18H15P)2(C18H12N4O5S) + H]+
in comparison with their respective
experimental data at (B) m/z 857.13 and (D) m/z 1315.2.
Fig. 6. DFT-optimized structures of (A) silver(I) and (B) gold(I) complexes. The atoms
that are part of the coordination spheres are labeled, as well as the metal ions. The