Crystal structure, antibacterial and cytotoxic activities of a new complex of Bismuth(III) with sulfapyridine

Article abstract of DOI:10.3390/molecules18021464

A new complex of Bi(III) and sulfapyridine was synthesized and characterized by elemental analysis, atomic absorption spectrometry, conductivity analysis, electrospray ionization mass spectrometry (ESI-MS), infrared spectroscopy, and single crystal X-ray diffraction methods. The antimicrobial and the cytotoxic activities of the compound were investigated. Elemental and conductivity analyses are in accordance to the formulation [BiCl₃(C₁₁H₁₁N₃O₂S) ₃]. The structure of the complex reveals a distorted octahedral geometry around the bismuth atom, which is bound to three sulfonamidic nitrogens from sulfapyridine, acting as a monodentate ligand, and to three chloride ions. The presence of the compound in solution was confirmed by ESI-MS studies. The complex is 3 times more potent than the ligand against Salmonella typhimurium, 4 times against Staphylococcus aureus, Shigella dysenteriae, and Shigella sonnei and 8 times more potent against Pseudomonas aeruginosa and Escherichia coli. The compound inhibits the growth of chronic myelogenous leukemia cells with an IC₅₀ value of 44 μM whereas the free ligand has no effect up to 100 μM.

Full text of DOI:10.3390/molecules18021464

Molecules 2013, 18, 1464-1476; doi:10.3390/molecules18021464
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Crystal Structure, Antibacterial and Cytotoxic Activities of a
New Complex of Bismuth(III) with Sulfapyridine
Ivana M. Marzano 1, Marina S. Franco 1, Priscila P. Silva 1, Rodinei Augusti 1,
Geandson C. Santos 1, Nelson G. Fernandes 1, Mônica Bucciarelli-Rodriguez 2,
Edmar Chartone-Souza 2 and Elene C. Pereira-Maia 1,*
1
Department of Chemistry, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627,
Belo Horizonte 31270-901, MG, Brazil
2
Department of General Biology, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627,
Belo Horizonte 31270-901, MG, Brazil
* Author to whom correspondence should be addressed; E-Mail: elene@qui.ufmg.br;
Tel.: +55-31-3409-5727; Fax: +55-31-3409-5700.
Received: 21 December 2012; in revised form: 17 January 2013 / Accepted: 17 January 2013 /
Published: 24 January 2013
Abstract: A new complex of Bi(III) and sulfapyridine was synthesized and characterized
by elemental analysis, atomic absorption spectrometry, conductivity analysis, electrospray
ionization mass spectrometry (ESI-MS), infrared spectroscopy, and single crystal X-ray
diffraction methods. The antimicrobial and the cytotoxic activities of the compound were
investigated. Elemental and conductivity analyses are in accordance to the formulation
[BiCl3(C11H11N3O2S)3]. The structure of the complex reveals a distorted octahedral
geometry around the bismuth atom, which is bound to three sulfonamidic nitrogens from
sulfapyridine, acting as a monodentate ligand, and to three chloride ions. The presence of
the compound in solution was confirmed by ESI-MS studies. The complex is 3 times more
potent than the ligand against Salmonella typhimurium, 4 times against Staphylococcus
aureus, Shigella dysenteriae, and Shigella sonnei and 8 times more potent against
Pseudomonas aeruginosa and Escherichia coli. The compound inhibits the growth of
chronic myelogenous leukemia cells with an IC50 value of 44 μM whereas the free ligand
has no effect up to 100 μM.
Keywords: Bi(III) complex; sulfapyridine; X-ray structure; antimicrobial activity;
cytotoxic activity

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1. Introduction
The excessive use of antibiotics caused the emergence of drug-resistant bacterial strains through a
variety of mechanisms. This problem has become quite alarming because almost all pathogenic
bacteria have acquired resistance against the presently known and widely used antibiotics [1–3].
Sulfonamides were the first synthetic antibiotics to be used in clinic and they exhibit interesting
pharmacological properties, such as selectivity to bacterial cells and low toxicity. Sulfapyridine was
the agent of choice for the treatment of pneumonia for a time. The mode of action of sulfonamides
involves the disruption of bacterial folic acid synthesis, which is essential for the biosynthesis of
N-methylated pyrimidines and purines as well as certain aminoacids, that are, in turn, necessary for
bacterial growth. Sulfapyridine mimics the paraaminobenzoic acid (PABA), which is a natural
substrate required for the biosynthesis of folic acid. More specifically, sulfapyridine inhibits the
activity of the enzyme dihydropteroate synthase (DHPS), which catalyzes the displacement of
pyrophosphate from a pteridine substrate resulting in a covalent bond to the amino group of PABA.
The competition between sulfapyridine and PABA for the enzyme results in sulfapyridine binding,
because it possesses a greater affinity for the enzyme. This inhibits the enzyme activity and
consequently the biosynthesis of folic acid [4]. Mammals obtain folic acid from food and are unable to
synthesize it. Because of this, sulfonamides are selective agents to bacteria and well tolerated by
humans. Unfortunately, resistance to sulfonamides, mainly due to the presence of drug-resistant
variants of the DHPS enzymes, limits its clinical use [5].
Bismuth compounds have been used for centuries in the treatment of a variety of microbial
infections due to their high efficacy and low toxicity. One can cite their use in the treatment of parasitic
infections, gastric disorders, and peptic ulcers. More recently, the discovery that bismuth inhibits the
growth of Helicobacter pylori and is effective in eradicating this bacterium when administered in
combination with antibiotics rehabilitated bismuth compounds in chemotherapy [6,7]. The bismuth
drugs most used in the treatment of gastrointestinal diseases related to the infection of Helicobacter
pylori are bismuth subsalicylate, coloidal bismuth subcitrate and ranitidine bismuth citrate [8–11].
However, the pharmaceutical formulations are not very well defined or stable, bismuth compounds are
not very soluble and the active species are not well characterized. The lability of the ligands, the Lewis
acidity and ability to expand the coordination sphere of the metal center make the control of the
reactions and characterization of the complexes a difficult task. A way of attenuating the hydrolysis of
bismuth complexes in aqueous solution with the formation of the [BiO+] cation is the incorporation of
N, O or S donor atoms into coordinating ligands. [12]. These aspects have stimulated the search for
new bismuth drugs presenting antibacterial activity, such as compounds containing non-steroidal anti-
inflammatory drugs [13], ciprofloxacin [14,15], thiosemicarbazones [16–21] or lipophilic thiols [22]. It
is worth notifying that the bismuth complex with 2-acetylpyrazine N(4)-pyridylthiosemicarbazone
described by Li-Zhi Zhang et al. exhibited effective antibacterial and anticancer activities [21]. More
recently, P.C. Andrews et al. prepared some tris-substituted bismuth(III) sulfonates, which exhibited
MIC (minimal inhibitory concentration) values in the nanomolar range against Helicobacter pylori [23].
The mechanism of the antibacterial action of bismuth drugs is not yet completely understood and
seems to involve enzyme inhibition. Bismuth inhibits fumarase (that catalyses the hydration of
fumarate to malic acid in the bacterial tricarboxylic acid cycle), cytosolic alcohol dehydrogenase

Molecules 2013, 18
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(ADH production of acetaldehyde), inhibits urease (that converts urea into ammonia and carbonic
acid), which is crucial for H. pylori survival in the low pH of stomach, induces oxidative stress and a
decrease in cellular protease activities [24,25].
The emergence of multidrug resistance to almost all known antibiotics makes the discovery of new
agents a crucial necessity. We have been working on the coordination of antibiotics to metal ions as a
promising strategy to obtain more active agents [26–28]. Our strategy is to ally the pharmacological
properties of bismuth compounds to those of sulfonamides in the search for better chemotherapeutic
compounds. A favorable characteristic of bismuth drugs is that there is no evidence of resistance as
observed with standard antibiotic compounds. We have synthesized, characterized and studied the
antimicrobial and cytotoxic activities of a new compound of bismuth(III) and sulfapyridine.
2. Results and Discussion
A new complex of Bi(III) with sulfapyridine was synthesized and characterized by elemental
analysis, atomic absorption spectrometry, conductivity analysis, electrospray ionization mass spectrometry
(ESI-MS), infrared spectroscopy, and single crystal X-ray diffraction methods.
Single-crystal X-ray analysis reveals that the complex crystallizes in the trigonal system, with space
group R 3. Crystallographic data and details of crystal data, data collection and refinement are summarized
in Table 1. Selected bond lengths, angles and geometrical parameters for hydrogen bonds are reported
in Table 2. As can be seen in Figure 1, the molecular structure of [BiCl3(C11H11N3O2S)3] shows
bismuth bound to three sulfapyridine molecules and three chloride ions in a distorted octahedral
environment. By symmetry imposition the three Cl2-Bi1-Cl2 angles are equal to each other, 87.74(3)°,
as well as the N6-Bi1-N6 angles, 108.79(5)°, however, they differ from the idealized octahedral
geometry, mainly the N6-Bi1-N6 angles. This distortion can be caused by steric and electronic effects
of the ligands combined with the presence of the bismuth(III) lone pair of electrons (6s2). The neutral
sulfapyridine ligands (deprotonated in the sulfonamidic nitrogen and protonated in the pyridinic
nitrogen) are coordinated to bismuth(III) in a unidentate way via its sulfonamidic nitrogen (N6). The
H8 atom has been found in the difference map, however, no significant differences can be found in the
C7-N6, C7-N8 and C9-N8 distances, which indicates that C7-N6 and C7-N8 bonds have the same
bond order. The 3+ charge of bismuth is neutralized by the chloride ions, being the Bi1-Cl2 bond
lengths 2.5442 Å, which falls within the range acceptable for other complexes [14,15,29]. The Bi1-N6
distance is 2.924 Å, which reveals an interaction of medium strength, the sum of covalent and van der
Waals radii for nitrogen and bismuth are Σrcov(Bi,N) 2.27 Å and ΣrvdW(Bi,N) 3.94 Å, respectively [29].
A Bi-N distance of 2.9(1) Å was also found in a similar compound, the trichlorotris-(3-sulphanilamido-
6-methoxypyridazine)-bismuth(III) [30]. Longer Bi-N distances were found for some distorted octahedral
organobismuth(III) compounds possessing a (C,N)3Bi core: in [2-{O(CH2CH2)2NCH2}C6H4]3Bi,
B-N = 3.170(7) Å, in [2-{MeN(CH2CH2)2NCH2}C6H4]3Bi, B-N = 3.211(5) Å [27] and in
[2-(Et2NCH2)C6H4]3Bi, Bi-N = 3.214(7) Å. [31].

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Table 1. Crystal data, data collection and structure refinement details for [BiCl3(C11H11N3O2S)3].
Empirical formula
Formula weight
Temperature
C33H33BiCl3N9O6S3
1063.22
293(2) K
Wavelength
0.71073 Å
Crystal system
Space group
Trigonal
R 3
a = 17.9557(3), b = 17.9557(3),
Unit cell dimensions
c = 10.19691 (12) Å, α = 90, β = 90, γ = 120°
2847.12(7) Å3
Volume
Z
3
Density (calculated)
Absorption coefficient
F(000)
1.860 Mg/m3
5.08 mm−1
1572
Crystal size
0.23 × 0.25 × 0.29 mm3
4.00 to 37.9°
Theta range for data collection
Index ranges
−27 ≤ h ≤ 30, −29 ≤ k ≤ 15,−9 ≤ l ≤ 17
6528
Reflections collected
Independent reflections
Absorption correction
Refinement method
Data/restraints/parameters
Goodness-of-fitonF2
4005(Rint = 0.024)
Empirical
Full-matrix least-squares on F2
4005/4/172
1.00
Final R indices (for3857reflectionswithI > 2σ(I)) R1 = 0.023, wR2 = 0.055
R indices (all data)
R1 = 0.024, wR2 = 0.057
1.37 and −0.55 eÅ−3
Largest diff. peak and hole
Absolute structure [5]
Flack parameter = 0.003(3)
Table 2. Selected bond lengths and hydrogen bonding geometries (Å, °).
Bi1—Cl2
C7—N6
2.5442 (7)
1.349 (4)
1.344 (4)
D—H
Bi1—N6
C7—N8
C16—N19
H···A
2.924 (2)
1.359 (4)
1.383 (4)
C9—N8
D—H···A
D···A
D—H···A
N8—H8···O4 i
N8—H8···Cl2 i
N19—H19A···O5 ii
0.86
2.13
2.858 (3)
3.340 (3)
142
122
0.86
2.81
0.85 (1)
2.14 (1)
2.83 (4)
3.12 (4)
2.995 (4) 177 (5)
3.568 (3) 146 (5)
3.702 (3) 127 (4)
N19—H19B···Cl2 iii 0.86 (1)
N19—H19B···Cl2 iv
0.85(1)
Symmetry codes: (i) −y, x − y, z; (ii) –x + y + 1/3, −x + 2/3, z + 2/3; (iii) x, y, z + 1; (iv) −y, x − y, z + 1.
Figure 2 shows the tridimensional network of hydrogen bonds which contribute to the crystal
structure stabilization of the title complex. There are three intermolecular hydrogen bonds, two of
them are bifurcated hydrogen bonds: N8-H8···O4(Cl2) and N19-H19B···Cl2(Cl2). On the other hand,
N19-H19A···O5 is almost linear, as shown in Figure 2 and Table 2.

Molecules 2013, 18
Figure 1. The molecular structure and the atom numbering scheme. Ellipsoids are drawn to
1468
include 50% probability, the hydrogen atoms have been omitted.
Figure 2. The tridimensional network of hydrogen bonds. Ellipsoids are drawn to include 50%
probability, the hydrogen atoms which are not involved in hydrogen bonds have been omitted.
Symmetry codes: (i) −y, x − y, z; (ii) –x + y + 1/3, −x + 2/3, z + 2/3; (iii) x, y, z + 1; (iv) −y, x−y, z + 1.
The molar conductivity value of 10−3 M solution in dimethylformamide at 25 °C was 10.35 Ohm−1
cm2 mol−1, which indicates that the complex is neutral. The literature values for a 1:1 electrolyte in
dimethylformamide are in the range 65–90 Ohm−1 cm2 mol−1 [32].
The modifications observed in the infrared spectrum of the complex in comparison to that of the
free ligand are in accordance with the coordination via the sulfonamidic nitrogen (Figure S1). The

Molecules 2013, 18
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spectrum of the complex is significantly modified in the region 3400–3000 due to the deprotonation of
the sulfonamidic nitrogen and protonation of the pyridinic nitrogen. In the sulfapyridine spectrum two
bands at 3418 and 3312 cm−1 are assigned to as(NH2) and s(NH2), respectively. Coordination to
Bi(III) shifts these absorptions to 3423 and 3333 cm−1. It is difficult to verify the deprotonation of the
sulfonamidic nitrogen because of the concomitant protonation of the pyridinic nitrogen in the complex,
which leads to the appearance of multiple absorptions in the region 3300–2960 cm−1 [33]. The
asymmetric and symmetric –SO2 group stretching vibrations in the sulfapyridine spectrum appear at
1369 and 1126 cm−1, respectively. The as (SO2) shifted from 1369 cm−1 in the free ligand to 1346 cm−1
in the complex, due to the deprotonation of the sulfonamidic nitrogen and bismuth coordination, while
the s(SO2) remained almost unchanged. The same effect was observed in the infrared spectra of other
sulfonamide complexes, in which coordination sites did not involve the SO2 group [34–36].
The presence of the complex in solution was confirmed by ESI-MS studies. In the mass
spectrum, positive mode, a peak at m/z 1063 refers to the molecular ion of the complex, i.e.,
[BiCl3(C11H11N3O2S)3]+● (M+●), which arises from the oxidation (removal of one electron) of the
neutral species [Figure S2]. The mass-selection and fragmentation (by collision induced dissociation)
of this ion yields an intense peak at m/z 815, whose structure is suggested to be formally
[BiCl3(C11H11N3O2S)2 + H]+. This product ion is proposed to be generated via a net loss of
[C11H10N3O2S]● (sulfapyridine-H●) from the molecular ion.
The main modification observed in the 1H-NMR spectrum of the complex in comparison to that of
the free ligand (Figure S3) is the absence of a resonance at  10.8 assigned to the proton of the
sulfonamidic nitrogen, indicating its deprotonation.
We have first checked that the solvent does not present an antibacterial effect in the concentrations
used. The antibacterial effect of the complex and the ligand was studied by determining the minimal
inhibitory concentration, MIC, which is the minimal concentration required to completely inhibit
bacterial growth. The complex inhibited the bacterial growth in a concentration-dependent way. The
MIC values obtained in six bacterial strains resistant to sulfapyridine are shown in Table 3. The MIC of
sulfapyridine against Escherichia coli was not attained up to the highest concentration tested (1,027 μM),
while for the complex the MIC value was 241 μM. The most important result is that the complex is
3 times more potent than the ligand against Salmonella typhimurium, 4 times against Staphylococcus
aureus, Shigella dysenteriae, and Shigella sonnei and 8 times against Pseudomonas aeruginosa.
Table 3. MIC of free sulfapyridine (sp) and the bismuth (III) complex.
MIC a/µM
Bacterial strains
sp
[Bi(sp)3Cl3]
E. coli
S. aureus
ATCC 25922 >1027
241
90
ATCC 25923
ATCC 24853
385
1027
96
P. aeruginosa
120
30
S. typhimur ium ATCC 13311
S. sonnei
ATCC 11060
ATCC 13313
1027
128
241
30
S. dysenteriae
a MIC is the minimal drug concentration required to inhibit bacterial growth. Number of assays = 4.

Molecules 2013, 18
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There are few works reporting on the study of metal compounds with sulfonamides. Kremer
et al. [34] studied the antibacterial activities of compounds with sulfonamides and Cu(II) ions. The
most important result was obtained with the compound [Cu(sulfametoxazol)2(H2O)4]·2H2O, which was
4 times more active than the ligand against S. aureus and E. coli. The authors suggested that the
improvement in the antimicrobial activity could be related to the higher lipophilicity of the complexes
in relation to free sulfonamides, which would increase their uptake in bacterial cells.
Mondelli et al. [35] synthesized and characterized a Ni(II) compound of sulfapyridine, in which
nickel is in a distorted octahedral environment, coordinated by two aryl amine N from two sulfonamides
acting as monodentate ligands and four N atoms (two sulfonamidic N and two heterocyclic N) from
two different sulfonamide molecules acting as bidentate ligands. Unfortunately, the complex was less
active against E. coli and S. aureus than the free ligand. The authors proposed that a reduced uptake
could be the reason for the lower activity.
The title complex is more active than the free ligand in six bacterial strains resistant to sulfapyridine.
This is an important result because bacterial resistance is the major obstacle for the treatment of
bacterial infections.
The fact that some bismuth compounds, such as tropolone or thiosemicarbazones complexes and
bismuth(III) dithiocarbamates present antitumor activities [19,37,38] motivated us to study the effect
of the synthesized complex in the growth of tumoral cells. The mechanism of the cytotoxic action of
some bismuth-containing drugs has been suggested to involve inhibition of enzymes, such as proteases,
lipases, glycosidases and phospholipases [39].
The sensitivity of chronic myelogenous leukemia to the compounds was evaluated by incubating
cells for three days in the presence of increasing complex concentration. Afterwards, cells were
counted and the concentration required to inhibit 50% of cell growth was calculated, the IC50.
The compound inhibited the growth of K562 cells with the IC50 value of 44 μM while sulfapyridine
was not active up to 100 μ (Figure 3). Mammalian cells do not have the enzyme dihydropteroate
synthase, which is the pharmacological target of sulfapyridine. Therefore, the cytotoxic action of the
compound should be related to bismuth(III) coordination.
Figure 3. Cell growth inhibition by [BiCl3(C11H11N3O2S)3] and sulfapyridine. Cells were
incubated with compounds for three days.
100
complex
sulfapyridine
80
60
40
20
0
20
40
60
80
100
Compound concentration (M)

Molecules 2013, 18
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3. Experimental
3.1. General and Instruments
Sulfapyridine (4-amino-N-pyridin-2-ylbenzenesulfonamide) and bismuth chloride were purchased
from Sigma Co. (St. Louis, MO, USA) All other chemicals were reagent-grade and were used without
further purification. Infrared spectra were recorded in KBr pellets over the region 400–4000 cm−1 with
a Perkin-Elmer 283 B spectrometer (Perkin Elmer Inc., Waltham, MA, USA). Full scan mass spectra
were obtained on a LCQ Fleet mass spectrometer from ThermoScientific (San Jose, CA, USA)
equipped with an electrospray source operating in the positive ion mode. Samples were dissolved in
MeOH containing 0.1% formic acid and were injected in the apparatus by direct infusion at a flow
rate of 10 μL min−1. The major ESI source conditions were: scan range 100–2,000 m/z, capillary
temperature 275 °C, spray voltage 25 kV, capillary voltage 25 V, and sheath gas (N2) flow rate
10 units. For ESI-MS2, the precursor ions of interest were first isolated by applying an appropriate
waveform across the end cap electrodes of the ion trap to resonantly eject all trapped ions except those
of m/z ratio of interest. The isolated ions were then resonantly excited with a supplementary AC signal
to cause collision-induced dissociation (CID). The relative collision energy was set to a value at which
the ions were produced in measurable abundance.
Single crystals of [BiCl3(C11H11N3O2S)3] suitable for an X-ray analysis were grown by slow evaporation
from an ethanolic solution and were obtained as yellow trigonal prismatic crystals. Single-crystal
X-ray diffraction data were collected at 293 K on an Oxford Gemini Atlas Ultra diffractometer with
graphite-monochromated, λ (MoKα) = 0.71073 Å using the CrysAlis-Pro data collection and data
processing software [40]. The structure was solved by the SIR97 program 1 [41] incorporated in the
WinGX program package [42]. All non hydrogen atoms were refined anisotropically on F2 using full
matrix least-square procedure [SHELXL-97] [43] with weight: w = 1/[σ2(Fo2) + (0.0347P)2], where
P = (Fo2 + 2Fc2)/3. The hydrogen atoms were found in the difference Fourier maps and were refined
using a riding model. A total of 6528 reflections have been measured and 4005 independent reflections
were obtained. Rint = 0.024, 172 parameters refined, R[F2 > 2σ(F2)] = 0.023, wR(F2) = 0.055, S = 1.00,
(Δ/σ)max = 0.001, Δρmin = −0.55, Δρmax = 1.37 e Å−3 at 0.74 Å from Bi1 site. The absolute structure was
determined giving a Flack parameter of 0.03(3) [44]. The structures were drawn by Mercury [45] and
ORTEP-3 for windows [46] programs.
X-ray crystallographic data: CCDC No. 828007 contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html
(or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax: +44 1223 336033;
E-Mail: deposit@ccdc.cam.ac.uk.
Carbon, nitrogen and hydrogen analysis were determined on a Perkin-Elmer 2400 CHN. Atomic
absorption analysis of the bismuth content was carried out on a model 8200 Hitachi Atomic
Absorption Spectrophotometer (Hitachi Ltd., Tokyo, Japan). Conductivity measurements were carried
out with a Digimed DM 31 (Digimed, São Paulo, Brazil) conductivity meter using a cell of constant
1.023 cm−1. The solvent used was spectroscopic grade dimethylformamide (Merck, Darmstadt,
Germany) (ΛM = 4.08 Ohm−1 cm2 mol−1).

Molecules 2013, 18
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The melting point/decomposition temperature was determined on a MQAPF-302 (Microquímica
Equipamentos Ltda, Palhoça, Brazil) instrument.
3.2. Synthesis
Sulfapyridine (p-amino-N-(2-pyridyl)benzenesulfonamide) (0,142 g or 5.7 × 10−4 mol) was dissolved
in water at pH 9 (the pH was adjusted with NaOH) and bismuth chloride (0.060 g or 1,9 × 10−4 mol)
was dissolved in water at pH 1 (the pH was lowered with HCl). The sulfapyridine solution was added
dropwise to that of BiCl3, under continuous stirring. A pale yellowish solid was formed and separated
by filtration, washed with water and dried under vacuum. The complex is soluble in dimethylformamide,
dimethyl sulfoxide and slightly soluble in ethanol or methanol. Yield: 0.107 g, 53%. Anal. calc. for
[Bi Cl3(C11H11N3O2S)3]: %C, 37.28; %H, 3.14; %N, 11.86; %Bi, 27.46. Found: C, 37.99; H, 3.18; N,
11.84; Bi, 27.98%. m.p. 505 K (decomposition). IR (cm−1): 3423s; 3333s; 3244m; 3211sh; 3148w;
3111w; 3087w; 3059w; 3026w; 2959w; 1628s; 1591s; 1529s; 1501s; 1464w; 1379s; 1346s; 1300s;
1279s; 1255s; 1241s; 1184w; 1167w; 1126s; 1082s; 1009m; 964s; 881w; 839m; 821w; 797s; 772m;
725w; 677s; 644w; 610w.
3.3. Microbial Strains and Growth Conditions
The bacterial strains selected from the bacteria collection of the Laboratory of Microbial Molecular
Genetics of the Department of General Biology, ICB-UFMG, to perform microbiological tests were:
E. coli ATCC 25922, S. aureus ATCC 25923, P. aeruginosa ATCC 24853, S. typhimurium ATCC
13311, S. sonnei ATCC 11060, S. dysenteriae ATCC 13313. Bacterial cultures were grown in the
medium specified at 37 °C. Culture stocks were performed on Lignières medium (0.8% w/v Difco
Nutrient Broth, 0.5% w/v Sigma gelatin, 0.7% w/v Difco agar).
3.4. Determination of Minimal Inhibitory Concentration
Sulfapyridine and the compound [BiCl3(C11H11N3O2S)3] were dissolved in N,N-dimethylformamide.
Stock solutions were diluted accordingly and added to Mueller Hinton Agar (Difco) previously melted
and cooled to 40 °C for the preparation of antibiotic plates. This agar was distributed onto Petri dishes so
as to obtain sulfapyridine (sp) concentrations of 8, 16, 32, 64, 96, 128, 193, 257, 385, 513 and 1,027 μM,
or [BiCl3(sp)3] concentrations of 2, 4, 8, 15, 23, 45, 60, 90, 120 and 241 μM. Plates for each concentration
were prepared in quadruplicate. Control plates containing only the solvent (dimethylformamide) or
Mueller Hinton Agar (Difco) without either drug or solvent were also prepared.
The bacterial strains selected were transferred to 2.0 mL of the Brain Heart Infusion (Difco) and
incubated at 37 °C for 24 h. The resulting cultures were diluted 100 times and inoculated onto the
plates previously prepared with antibiotics by means of a multi-inoculation apparatus (steer type) and
were incubated at 37 °C for 24 h. Bacterial growth was recorded after this period.
3.5. Cell Line and Culture
The K562 cell line was purchased from the Rio de Janeiro Cell Bank (number CR083 of the RJCB
collection). This cell line was established from pleural effusion of a 53 year-old female with chronic

Molecules 2013, 18
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myelogenous leukemia in terminal blast crisis. Cells were cultured in RPMI 1640 (Sigma Chemical Co.)
medium supplemented with 10% fetal calf serum (CULTILAB, São Paulo, Brazil) at 37 °C in a
humidified 5% CO2 atmosphere. Cultures grow exponentially from 105 cells mL−1 to about
8 × 105 cells mL−1 in three days. Cell viability was checked by Trypan Blue exclusion. The cell
number was determined by Coulter counter analysis.
3.6. Cytotoxicity Assays
For cytotoxicity assessment, 1 × 105 cells mL−1 were cultured for 72 h in the absence and the
presence of various concentrations of the tested compounds. The sensitivity to drug was evaluated by
the concentration that inhibits cell growth by 50%, IC50.
4. Conclusions
A new complex of Bi(III) with sulfapyridine was synthesized and its crystal structure was
determined. [BiCl3(C11H11N3O2S)3] was more active than the free ligand in six bacterial strains
resistant to sulfapyridine. In addition, the compound inhibited the growth of chronic myelogenous
leukemia cells while sulfapyridine was not active. Therefore, bismuth coordination enhances both
antibacterial and cytotoxic action.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/18/2/1464/s1.
Acknowledgments
We would like to thank FAPEMIG, CNPq and INCT-Catálise for financial support.
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Sample Availability: Samples of the compound [BiCl3(C11H11N3O2S)3] are available from the authors.
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