Synthesis, properties and biological activity of a novel phosphines ligand derived from ciprofloxacin

Article abstract of DOI:10.1016/j.poly.2013.04.059

Novel potential antibacterial agents derived from ciprofloxacin (HCp) were synthesized in the reaction of this antibiotic with hydroxymethyldiphenylphosphine. Detailed analysis by the NMR, IR and MS techniques attested to the identity and structure of the obtained compounds. In addition, the structure of phosphine PPh₂CH₂-Cp was unambiguously confirmed by X-ray crystal structural analysis. PPh ₂CH₂-Cp exhibited an antibacterial activity comparable to that of the original drug. Cytotoxicity studies revealed that this compound was characterized by lower toxicity against mammalian cells than HCp. Besides, it did not induce a morphological change in cells after its action and was unable to degrade plasmid DNA, as well as parent antibiotic. Our results open up new possibilities in designing novel, less toxic and comparably effective antibiotics drugs.

Full text of DOI:10.1016/j.poly.2013.04.059

Polyhedron 60 (2013) 23–29
Contents lists available at SciVerse ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, properties and biological activity of a novel phosphines ligand
derived from ciprofloxacin
a
a
a
b
_
Aleksandra Bykowska , Radosław Starosta , Anna Brzuszkiewicz , Barbara Bazanów ,
b
b
b
c
c
´
Magdalena Florek , Natalia Jackulak , Jarosław Król , Jakub Grzesiak , Krzysztof Kalinski ,
a,
⇑
_
Małgorzata Jezowska-Bojczuk
a Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14, 50-383 Wrocław, Poland
b Department of Veterinary Microbiology, Wrocław University of Environmental and Life Sciences, ul. Norwida 31, 50-375 Wrocław, Poland
c
_
Laboratory of Electron Microscopy, Wroclaw University of Environmental and Life Sciences, ul. Kozuchowska 5b, 50-631 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel potential antibacterial agents derived from ciprofloxacin (HCp) were synthesized in the reaction of
this antibiotic with hydroxymethyldiphenylphosphine. Detailed analysis by the NMR, IR and MS tech-
niques attested to the identity and structure of the obtained compounds. In addition, the structure of
phosphine PPh2CH2-Cp was unambiguously confirmed by X-ray crystal structural analysis. PPh2CH2-Cp
exhibited an antibacterial activity comparable to that of the original drug. Cytotoxicity studies revealed
that this compound was characterized by lower toxicity against mammalian cells than HCp. Besides, it
did not induce a morphological change in cells after its action and was unable to degrade plasmid
DNA, as well as parent antibiotic. Our results open up new possibilities in designing novel, less toxic
and comparably effective antibiotics drugs.
Received 6 March 2013
Accepted 28 April 2013
Available online 16 May 2013
Keywords:
Fluoroquinolone
Antibiotic
Ciprofloxacin
Diphenylphosphine
Microbial resistance
Cytotoxicity
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
against Gram-negative (Pseudomonas aeruginosa, Escherichia coli)
and some Gram-positive (Staphylococcus aureus, Mycobacterium
Increasing microbial resistance against antibiotics has been rec-
ognized by the World Health Organization as one of the significant
health dangers in the early twenty-first century [1,2]. Large genetic
variations of pathogenic organisms and excessive frequency of
antibiotics usage result in the development of increasingly effec-
tive defense mechanisms by microorganisms. Another problem is
the speed with which they inactivate drugs used in therapies [3–6].
For this reason, search for new antimicrobial agents is extre-
mely important and desirable. It can be performed in two ways.
One involves structural modifications of substances already used
in medicine, and the other – synthesis of completely new antibiot-
ics. The first approach is safer but it carries the risk of rapid resis-
tance acquisition. The other approach involves a much lower risk
but is more expensive and time consuming [7]. We have under-
taken a modification of ciprofloxacin (HCp), a second generation
fluoroquinolone, by enriching its molecule with a new functional
group.
tuberculosis) bacteria. The mechanism of its action is based on
blocking the DNA replication process by binding gyrase or topoiso-
merase IV [8–11].
In this paper we present the synthesis and the biological prop-
erties of HCp modified by substitution with diphenylphosphino-
methyl group. The presence of the –CH2PPh2 residue could
potentially generate additional interactions with bacteria. There-
fore it may alter the biological activity of the parent drug and im-
pact its bioavailability or toxicity level. Due to the presence of a
secondary nitrogen atom in the piperazine ring (Fig. 1), HCp can
be easily converted into phosphine ligand [12–14]. Possibility of
such modification seems especially attractive, because a number
of papers show that phosphines, as well as their derivatives and
the metal ion complexes (i.e. copper, platinum, gold) exhibit prom-
ising anticancer, antibacterial or antiarthritic activity [14–21]. On
the other hand, phosphines are very often characterized by a high
cytotoxicity [15].
HCp is a broad-spectrum antibiotic, commonly used in the
urinary tract, respiratory and digestive infections. It is active
HCp is an antibiotic that has already been subjected to struc-
tural modifications. They are associated with the introduction of
additional groups, including hydroxybisphosphonate [22], ben-
zenesulphoamide [23–29] or acyl [30]. Complexes in which HCp
is coordinated to the d-electron (Cu2+, Zn2+, Co2+, Cd2+) [31–34]
and f-electron (La3+, Eu3+, Tb3+) [35–37] metal ions have also been
⇑
Corresponding author. Tel.: +48 71 3757281.
E-mail address: malgorzata.jezowska-bojczuk@chem.uni.wroc.pl (
_
Małgorzata Jezowska-Bojczuk).
0277-5387/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.poly.2013.04.059

24
A. Bykowska et al. / Polyhedron 60 (2013) 23–29
2.1.2. Synthesis of O = PPh2CH2-Cp (OPCp)
OPcp was obtained in the reaction in dichloromethane-acetoni-
trile mixture (1:1) (10 ml) of PCp (0.1081 g; 0.204 mmol) placed in
ice bath with equimolar amount of H2O2 (30% solution in water)
(20.8 ll; 0.204 mmol). Solvent was evaporated under vacuum to
dryness and obtained yellowish solid was washed twice with
water. Yield 91%. Anal. Calc. for C30H29FN3O4P (545.5): C, 66.05;
H, 5.36; N, 7.70. Found: C, 65.88; H, 5.56; N, 7.67%.
NMR (CDCl3): 31P{1H}: 27.70 s; 1H: H3Pb: 14.49 s, H2P: 8.72 s, H5P
7.94 d J = 13.03, HPh(o),(m),(p): 7.80–7.49, H8P: 7.30 d J = 7.17, H1Pa
:
:
Fig. 1. Molecular scheme of a ciprofloxacin molecule (HCp).
0
0
0
3.50 m, H3P : 3.29, H1P : 3.34 d J = 6.31, H4P : 2.92, H1Pb: 1.37 m,
H1Pb: 1.17 m; 13C{1H}: C4P:177.18 s, C3Pa: 167.09 s, C6P: 154.74
and 152.73, C2P: 147.49 s, C7P: 145.87 d J = 10.35, C9P: 139.16 s,
CPh(i): 132.35 d J = 98.11, CPh(o): 132.13 d J = 2.69, CPh(p): 131.26 d
reported. Besides, there are known ternary systems containing this
drug, metal ion and diimine ligands [31,34,38]. It should be noted
that the modified antibiotic and HCp complexes with transition
metal ions often exhibit a higher activity against pathogens than
the parent drug [24,27,30–34].
J = 8.92, CPh(m): 128.75 d J = 11.54, C10P: 119.84 d J = 7.80, C5P
:
0
112.43 d J = 23.57, C3P: 108.19 s, C8P: 104.97 d J = 3.26, C1P : 58.05
0
0
d J = 87.34, C4P : 55.06 d J = 8.01, C3P : 49.90 d J = 4.93, C1Pa: 35.40
s, C1Pb: 8.33 s.
MS (CDCl3): 546.2 [MH]+; 344.1[CH2C17H17FN3O3]+;
m m
= 1725.2 cmꢀ1 (vs) –C@O; = 1186.2 cmꢀ1 (s) –P@O.
2. Experimental
IR (KBr):
2.1. General methods
2.1.3. X-ray structure of PPh2CH2-Cp (PCp)
The colourless plate shaped crystal of dimensions
0.21 ꢁ 0.14 ꢁ 0.10 mm was used for X-ray data collection. Data
were collected at low temperature (100 K) using an Oxford Cryo-
The reagents (ciprofloxacin, triethylamine, hydrogen peroxide,
diphenylphosphine, formaldehyde, hydrochloric acid) and solvents
were purchased from Sigma–Aldrich and used without further
purification. NMR spectra were recorded on a Bruker Avance
500 MHz spectrometer with traces of solvent as an internal refer-
ence for 1H and 13C spectra and 85% H3PO4 in H2O as an external
standard for 31P. Chemical shifts are reported in ppm (parts per
million) and coupling constants are reported in Hz. Mass spectra
were registered on a Bruker Daltonics micrOTOF–Q Mass spec-
trometer equipped with electrospray ionization (ESI) source and
operated in positive ion mode. IR spectra were recorded from
4000 to 400 cmꢀ1 on Bruker 113v FTIR spectrophotometer as a
KBr pellets. Elemental analysis was performed with a Vario EL3
CHN analyzer.
system device on a Kuma KM4CCD
j-axis diffractometer with
graphite-monochromated Mo K radiation (k = 0.71073 Å). The
a
crystal was positioned at 65 mm from the CCD camera. 612 frames
were measured at 0.75° intervals with a counting time of 10 s at
low theta angles and 764 frames were measured at 0.6° intervals
with a counting time of 30 s at high theta angles. ‘Multi-scan’
absorption correction was applied. Data reduction and analysis
were carried out with the Oxford Diffraction programs [40,41].
The structure was solved by direct methods (program SHELXS97
[42] and refined by the full-matrix least-squares method on all F2
data using the SHELXL97 [42] programs.
Crystal/refinement data: PCp ꢂ C30.02H29.02FN3O3P, Mr = 529.82,
Crystal size: 0.21 ꢁ 0.14 ꢁ 0.10 mm, Crystal system: Monoclinic,
Space group: P21/c, Unit cell: a = 7.595(3) Å, b = 28.100(12) Å,
c = 11.892(7) Å, b = 97.05(3)°, V = 2519(2) Å3, Dcalc(Z = 4) = 1.396 g/
Reactions were carried out under a nitrogen atmosphere using
standard Schlenk techniques. All solvents were deaerated prior to
use. [PPh2(CH2OH)2]+Clꢀ was synthesized according to the litera-
ture method [39].
cm3, h range for data collection: 3.07–36.99° deg, Mo K
(k = 0.71073 Å),
a
radiation
Tmin = 0.949, Tmax = 1.000,
Reflections collected/unique 45398/12212 [Rint = 0.0379], Final R
indices [I > 2 (I)] R1 = 0.0439, wR2 = 0.1060, R indices (all data)
l ,
Mo = 0.156 mmꢀ1
2.1.1. Synthesis of PPh2CH2-Cp (PCp)
r
Triethylamine (10 ml) was added drop wise to a solution of
1.2789 g of [PPh2(CH2OH)2]+Clꢀ (4.53 mmol) in 30 ml of methanol
on the ice bath. After 30 min of stirring water suspension (20 ml) of
ciprofloxacin HCp (1.5 g; 4.53 mmol) was added. The mixture was
stirred for 1 h at room temperature. The obtained white solid was
centrifuged, washed twice with water and dried under reduced
pressure. Yield 88%. Anal. Calc. for C30H29FN3O3P (529.5): C,
68.04; H, 5.52; N, 7.93. Found: C, 67.81; H, 5.75; N, 7.89%.
R1 = 0.0687, wR2 = 0.1125, Goodness-of-fit = 1.020, Largest differ-
ence in peak and hole: 0.580 and ꢀ0.314 eÅꢀ3, Data/restraints/
parameters: 12212/0/401; T = 100(2) K.
2.2. Antibacterial activity
The antimicrobial activity was evaluated by the serial dilutions
method using the Antibiotic Assay Medium (Assay Broth; pH
7.0 0.2; dextrose 1.0; K2HPO4 3.68; beef extract 1.5; peptone
5.0; KH2PO4 1.32; NaCl 3.5; yeast extract 1.5 g/L) according to
Grove and Randall [43]. The following strains were employed: S.
aureus PCM 2054 (=ATCC 25923), E. coli PCM 2057 (=ATCC
25922) from the Polish Collection of Microorganisms of the Insti-
tute of Immunology and Experimental Therapy in Wroclaw, as well
as P. aeruginosa isolated from clinical samples. The latter strain was
identified using conventional methods and miniaturized identifica-
tion systems (ID 32 C and API 20 NE [BioMérieux], respectively). An
overnight culture of strain tested was diluted 1:1000 in the AB. To
a series of tubes containing appropriate amounts of compounds (as
the films on the tube walls), 0.9 mL of AB and 0.1 mL of microbial
dilution were added. Following concentrations of each compound
NMR (CDCl3, 298 K): 31P{1H}: ꢀ27.42 s; 1H: H3Pb: 15.01 s, H2P
:
8.71 s, H5P: 7.95 d J = 13.80, HPh(o),(m),(p): 7.47–7.34, H8P: 7.47–
0
0
0
7.34, H1Pa: 3.53 m, H3P : 3.37, H1P : 3.29 d J = 2.87, H4P : 2.90,
H1Pb: 1.39 m, H1Pb: 1.18 m; 13C{1H}: C4P:177.15 s, C3Pa: 167.09 s,
C6P: 154.77 and 152.77, C2P: 147.44 s, C7P: 145.98 d J = 10.39, C9P
139.18 s, CPh(i): 138.12 d J = 12.40, CPh(o): 132.94 d J = 18.50, CPh(p)
:
:
:
128.85 s, CPh(m): 128.61 d J = 6.63, C10P: 119.76 d J = 7.93, C5P
0
112.39 d J = 23.50, C3P: 108.14 s, C8P: 104.91 d J = 3.35, C1P : 61.31
0
0
d J = 4.06, C4P : 54.20 d J = 9.29, C3P : 49.95 d J = 5.04, C1Pa: 35.41
s, C1Pb: 8.32 s.
MS (CHCl3): 344.1 [CH2C17H17FN3O3]+; 247.1 [C13H10FNO3]+;
199.1 [PPh2CH2]+; 530.2 [MH]+; 546.2 [MOH]+; 285.1 [PPh2CH2C4-
H10N2]+; 504.1 (503.1?) [PPh2CH2C15H16FN3O3]+;
IR (KBr):
m
= 1727.2 cmꢀ1 (vs) –C@O.
were obtained [lg/mL]: 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, 0.1.

A. Bykowska et al. / Polyhedron 60 (2013) 23–29
25
Drug-free purity and growth controls were included. The tubes
were incubated at 37 °C for 24 h. The minimal inhibitory concen-
tration (MIC) was defined as the lowest concentration of com-
pound that inhibited microbial growth. For each compound, tests
were performed in triplicate and results shown are the average
of independent experiments.
respect to not treated control wells. The results from all repeats
(n = 3) were averaged and compared with control cells [45–47].
2.4.2. Immunofluorescence
Cells were washed with PBS/1% FBS, fixed with 4% paraformal-
dehyde for 10 min at RT and permeabilized with 0,1% Triton X-100/
3%BSA for 10 min in room temperature. After that, cells were
washed again and incubated with primary antibody against active
caspase 3 c-terminal fragment (polyclonal IgG produced in rabbit,
Sigma) for 60 min in 37 °C/5%CO2. Cells were than washed again
three times and incubated with secondary antibody with fluores-
cent conjugate (AlexaFluor-594 nm, anti-rabbit IgG produced in
goat, Sigma) and with fluorescent-labeled phalloidine (Atto488,
Sigma) and were incubated for one hour in 37 °C/5%CO2. Finally,
cells were washed three times, counterstained with DAPI, treated
with mounting medium (Fluoromount, Sigma), observed and pho-
tographed with inverted, fluorescent microscope with digital cam-
era attached (Cannon PC1200). Picture of cells were taken also with
microscope contrast-phase light option enable [48,49–51]. Pictures
were merged and partially pseudocoloured for best visualization.
2.3. Cytotoxic assay on RK-13 according to EN 14675
2.3.1. Cells
The cell line (rabbit kidney), passages 119–156, incubated in
96-well polystyrene plate for 24 h were used in this project.
2.3.2. Cytotoxic assay
Substances were tested using EN 14675 [44] with own modifi-
cation. Product test solutions were prepared in Minimum Essential
Medium (MEM) supplemented with additional 2% Fetal Bovine Ser-
um (FBS) and L-glutamine. Solutions of the compounds in concen-
trations from 2 mM to 2 ꢁ 10ꢀ8 mM were prepared and transferred
(100 ll) into cell culture units (wells of microtitre plates) contain-
ing suspended cells in 100 ll. Final concentrations of the com-
plexes were: 1, 1 ꢁ 10ꢀ1, 1 ꢁ 10ꢀ2, 1 ꢁ 10ꢀ3, 1 ꢁ 10ꢀ4, 1 ꢁ 10ꢀ5
,
2.5. DNA strands break analysis
1 ꢁ 10ꢀ6, 1 ꢁ 10ꢀ7 and 1 ꢁ 10ꢀ8 mM. Eight units were inoculated
with each concentrations. Plates were incubated in 37 °C/5%CO2
and observed daily for up to 96 h for the development of cytopathic
effect (CPE), using an inverted microscope [44] (Olympus Corp.,
Hamburg Germany; Axio Observer, Carl Zeiss MicroImaging
GmbH).
The ability to induce strand breaks by the studied compounds
was tested with the application of pUC18 plasmid. The solution
of DNA in 5 mM sodium phosphate buffer (pH 7.4) was mixed with
compounds. After 1 h incubations at 37 °C, reaction mixtures
(20 lL) were mixed with 3 lL of loading buffer (bromophenol blue
in 30% glycerol) and loaded on 1% agarose gels, containing ethi-
dium bromide, in TBE buffer (90 mM Tris–borate, 20 mM EDTA,
pH 8.0). Gel electrophoresis was done at constant voltage of
100 V (4 V cmꢀ1) for 60 min. The gel was photographed and visual-
ized with a Digital Imaging System (Syngen Biotech).
2.4. Cytotoxic properties on mesenchymal stem cells (HMSC-ad)
Mesenchymal stem cells (HMSC-ad) isolated from horse adi-
pose tissue (passage 3) were seeded into 96-well plate (20 ꢁ 103
per well) and incubated in 37 °C/5%CO2 for 24 h for adhesion in
DMEM supplemented by 10% FBS and 1% penicillin/streptomycin/
amphotericin b [45–47]. HMSC-ad were treated with HCp and
PCp of various concentrations. Their influence on the cell metabo-
lism was determined by spectrophotometrical measurement of
resazurin bioreduction ratio at 600 nm, with distraction of absor-
bance at 690 nm wavelength as a background, according to manu-
facturer’s protocol. Additionally, control wells were included, with
addition of pure compounds solvent at concentrations reflecting
the research group. Viable cells reduce the amount of dye oxidized,
blue form and increase the amount of red, reduced. Non-toxic con-
centrations of the compounds were established in comparison to
normal, not treated control cells [46,47]. The research was sup-
ported by microscopic observations and fluorescent stainings. Ac-
tin chains, as active element of cell cytoskeleton were visualized
for evaluation of cell general morphology and condition, with fluo-
rophore-labeled phalloidine. Detection of apoptosis was done by
immunocytochemical assay, using antibodies against active cas-
pase 3 – protein directly committed in apoptosis. Additional DAPI
(40,6-diamidino-2-phenylindole dihydrochloride) staining showed
the form and condition of nuclei. Connection of these two ap-
proaches resulted in receiving detailed picture of pharmaceutics’
toxicity and their neutral concentrations for cells in vitro [48,49–
51].
3. Results and discussion
3.1. Chemistry
Phosphine derivative (PCp) of ciprofloxacin was obtained in the
reaction of HCp with hydroxymethyldiphenylphosphine. Then, this
new ligand was transformed into an oxide derivative (OPCp, Fig. 2).
The IR spectra confirm the presence of synthesized compounds.
On the HCp spectrum the C@O stretching vibrations of the group
from quinolone ring are observed at 1723.3 cmꢀ1. Since our modi-
fications do not include the ring, the band of C@O groups is also
present on the spectra of PCp and OPCp at 1727.2 cmꢀ1 and
1725.2 cmꢀ1 respectively. For OPCp derivative the band associated
with P@O bond stretching is observed at 1186.2 cmꢀ1. Further con-
firmation of the molecular structures of these derivatives was ob-
tained from the Mass spectra.
The reaction products were described in detail using NMR spec-
troscopy (see the Section 2 and Table S1 in Supplementary data).
We assigned all signals relying on the NMR data of HCp obtained
in other solvents [52,53]. Apparently, the formation of a C–N bond
in the course of the reaction of hydroxymethyldiphenylphosphine
with HCp leads to significant changes in the 31P{1H} NMR spectra
in CDCl3. A singlet at ꢀ11.46 ppm, characteristic for Ph2PCH2OH,
is moved to higher fields (ꢀ27.42 ppm) for PCp, which is associ-
ated with the change of the substituents at P atom. On the other
hand, oxidation of the phosphorus atom and – in consequence –
formation of the phosphine oxide (OPCp) causes a significant
downfield shift of the phosphorus atom signal. A similar trend
was observed for other diphenylphosphines [54,55].
2.4.1. Metabolic tests (alamar blue cytotoxic assay)
Examined substances were added to culture medium (end vol-
ume 200
l
l) at different concentrations (2 ꢁ 10ꢀ1
,
1 ꢁ 10ꢀ1
,
2 ꢁ 10ꢀ2, 1 ꢁ 10ꢀ2, 2 ꢁ 10ꢀ3, 1 ꢁ 10ꢀ3, 2 ꢁ 10ꢀ4 and 1 ꢁ 10ꢀ4 mM)
for 30 min. After that time, medium was replaced by fresh with an
addition of 10% resazurin (Sigma) and cultures were incubated for
4 h. After incubation, the changes in absorbance were measured
with plate reader (SpectroStar) in a wavelength of 600 nm, with
In contrast to the phosphorus spectra, 1H NMR spectra do not
undergo considerable changes. Signals of the Ph2P– group and of

26
A. Bykowska et al. / Polyhedron 60 (2013) 23–29
Fig. 2. Scheme of formation of PCp and OPCp.
–Cp fragment have similar values of chemical shifts for the
substrates and obtained derivatives. As expected, the largest
for the –CH2– group directly associated with the P atom, a signifi-
0
cant decrease of the 1J(C1P –P) coupling constant is observed. The
changes caused by the formation of the phosphine and its oxide
formation of the oxide triggers alterations in the chemical shifts
and coupling constants for carbon atoms of the phenyl rings and
0
are observed for a doublet originating from H1P protons. Forming
of a C–N bond results in a significant upfield shift of the signal with
the @PCH2– fragment. The signal of the latter atom shifts to higher
0
0
a simultaneous decrease of the 2J(H1P –P) spin–spin coupling con-
fields and 1J(C1P –P) dramatically increases from 4.06 to 87.34 Hz.
stant. After oxidation, this signal is shifted to lower fields with a
parallel increase of the above coupling constant.
The structure of PCp was unambiguously confirmed by the
crystallographic analysis. This compound crystallized as a strongly
disordered molecule in which the –N(CH2CH2)NCH2PPh2 fragment
occupies two completely different positions. The X-ray structure of
PCp in the dominating conformation is shown in Fig. 3. The
geometrical parameters of the shared parts (i.e. quinolone and
Due to the low solubility of HCp, 13C{1H} NMR spectra were
measured only for Ph2PCH2OH, PCp and OPCp. The available data
indicate that the formation of PCp ligand does not involve signifi-
cant changes of the phenyl rings carbon atoms signals. However,
Fig. 3. X-ray structure of a disordered PCp molecule.

A. Bykowska et al. / Polyhedron 60 (2013) 23–29
27
Table 1
Antibacterial activity (MIC [
cell line using the European standard EN 14675 [44]. A cytopathic
effect was observed after 24 h of incubation with different concen-
trations of the tested compounds and it remained unchanged
throughout the incubation time. HCp was cytotoxic in the concen-
trations range from 1 to 1ꢃ10ꢀ6 mM, whereas PCp only in the range
from 1 to 1ꢃ10ꢀ1 mM (Fig. 4). This indicates an unexpected low-
toxicity of the PCp derivative in comparison with the parent
antibiotic.
l
g/ml]) of HCp and its derivatives.
E. coli
S. aureus
P. aeruginosa
HCp
PCp
OPCp
<0.1
0.2
2
0.5
0.5
10
0.5
1
50
piperazine rings) of PCp and HCp hexahydrate [56] are similar (see
Tables S2 and S3 in the Supplementary data) however there are
some differences resulting from the variant character of these mol-
ecules. The molecule of HCp is zwitterionic while the molecule of
PCp is not. This is confirmed by the values of the angle between
the plane of the fused rings and the plane of the carboxylic group,
which is equal to 28.33° in HCp and to only 15.30° in PCp. The
lower value for PCp is most likely a result of a strong intramolec-
ular hydrogen bond: O2–H2ꢃ ꢃ ꢃO3 (dD–H = 0.82 Å; dH–A = 1.79 Å;
3.3.2. Metabolic test – alamar blue cytotoxic assay
Cell viability assays were also performed on the HMSC-ad cell
line. In contrast to the results obtained for the cell line RK-13,
the activities of HCp and PCp against HMSC-ad were not so diver-
gent (Fig. 5). However, we observed different concentration pro-
files of the cell response for both compounds. As can be seen
from the smooth course of the plot, the cell metabolic activities
at various concentrations of HCp do not differ so much at the full
range of concentrations. In the case of PCp, we observed a distinct
shape of the curve. This derivative does not affect the metabolic
activity up to 2 ꢁ 10ꢀ3 mM, while at 1 ꢁ 10ꢀ1 and 2 ꢁ 10ꢀ1 mM
it causes a significant decrease of the cell viability.
Cell imaging using three different techniques was performed to
visualize changes in the cells caused by the tested compounds
(Fig. 6). The phase contrast pictures (photos A 1–3) show that, after
treatment with HCp (A 2), the monolayer of the cell culture
thinned down with the necrosis effect and cell remnants are visi-
ble. In the case of PCp synthesized by us (A 3), normal cells mor-
phology with limited cluster formation was observed. There were
virtually no changes in comparison with the control cultures (A
1). These data are fully consistent with the images obtained using
immunofluorescent staining (B1–3). HMSC-ad cells treated with
HCp (B 2) are characterized by altered morphology, with cells de-
tached or undergoing necrosis. After treatment with PCp (B 3) the
cells did not undergo morphological changes and the only alterna-
tion was the formation of clusters. What is very important, their
image was more similar to that of the control group (B 1), than
to the image of the cells treated with HCp. Photos C 1–3 show
the nuclei of HMSC-ad cells stained with DAPI. The nuclei were
not damaged upon treatment with HCp (C 2) and PCp (C 3). For
both compounds their pictures look like the control (C 1). For
PCp, however, some clusters are formed with incidental occur-
rence of pyknotic nuclei [48].
dD–A = 2.551(2) Å;
a = 153.5°) in its molecule (Fig. 3).
3.2. Antibacterial activity
The antibacterial activity of HCp, PCp and OPCp was prelimi-
narily determined in vitro against Gram-negative (E. coli, P. aerugin-
osa) and Gram-positive (S. aureus) bacteria. MIC values are
presented in Table 1. The collected data demonstrate that PCp, a
phosphine derivative of ciprofloxacin, inhibits the growth of bacte-
rial strains at relatively low concentrations. Moreover, it exhibits
an activity against the tested strains comparable to the parent
drug. An analogous trend was observed for another derivative of
HCp: Cp–CH2–C2N3H–(CH2)3–C(PO(OH)3)2OH [22]. However, the
activity of OPCp is much lower and the inhibition of microbial
growth for all tested strains was observed at concentrations at
least one order of magnitude higher than for other compounds.
3.3. Cell viability studies
Due to the low antimicrobial activity of OPCp, cytotoxicity was
determined only for HCp and PCp. Both compounds were tested in
the rabbit kidney cell (RK-13) and mesenchymal stem cells isolated
from adipose tissue horse (HMSC-ad).
3.3.1. Cytotoxic assay according to EN 14675
A newly synthesized compound PCp and an unmodified antibi-
otic HCp were evaluated for their cytotoxic activities on the RK-13
Fig. 5. Metabolic tests performed on the mesenchymal stem cells (HMCS-ad)
isolated from horse adipose tissue for HCp (squares) and PCp (triangles). X-axis: the
concentration of the compounds [mM] in the logarithmic scale; Y-axis: the percent
of the metabolically active cells.
Fig. 4. Cytotoxic assay performed on RK-13 cell line for HCp (squares) and PCp
(triangles). X-axis: the concentration of the compound [mM] in the logarithmic
scale; Y-axis: the percent of the viable cells.

28
A. Bykowska et al. / Polyhedron 60 (2013) 23–29
Fig. 6. Imaging of activity of HMSC-ad cell line treated with PCp (3) (c = 2 ꢁ 10ꢀ2 mM) and HCp (2) (c = 2 ꢁ 10ꢀ2 mM) together with the untreated cells (1). (A) Phase-contrast
picture of monolayer HMSC-ad (Mag. 100ꢁ); (B) Immunofluorescent staining with phalloidine (Mag. 200ꢁ); (C) DAPI stained cells (Mag. 400ꢁ) (nuclei presented in white).
The results presented above suggest that the influence of a no-
vel phosphine derivative of ciprofloxacin on the morphology of the
investigated cells is considerably smaller than the impact of the
unmodified antibiotic, despite their comparable cytotoxicities at
2 ꢁ 10ꢀ2 mM.
focusing on interactions of PCp with biomolecules and verifying
its mechanism of action. We also work on syntheses and character-
istics of potentially active [17] copper(I) complexes with this inter-
esting molecule.
Acknowledgements
3.4. Interactions with pUC18 plasmid
The authors gratefully acknowledge financial support from the
Polish National Science Centre (NCN grant 2011/03/B/ST5/01557).
The influence of HCp, PCp and OPCp on the DNA plasmid was
determined for their different concentrations at the physiological
pH. None of the compounds were able to induce single and/or dou-
ble strand cleavage in a DNA molecule. The analysis of the electro-
pherogram shows that regardless of the concentration, the tested
compounds are unable to degrade the plasmid. This result clearly
indicates that PCp and OPCp are not mutagenic, which can be
important in the context of their potential medical applications.
Appendix A. Supplementary data
CCDC 922239 contains the supplementary crystallographic data
for PCp. These data can be obtained free of charge via http://
www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ,
UK; fax: +44 1223 336 033; or e-mail: deposit@ccdc.cam.ac.uk.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.poly.2013.04.059.
4. Conclusions
In this paper we presented the spectroscopic profile and biolog-
ical activity of the novel phosphine (PCp) and phosphine oxide
(OPCp) derived from ciprofloxacin (HCp). A detailed analysis using
the NMR, IR, MS and X-ray techniques attest to the identity and
structure of the obtained compounds.
References
[1] World Health Organization. The evolving threat of antimicrobial resistance –
Options for actions, 2012 (WHO/IER/PSP/2012.2).
[2] G. Alvan, C. Edlund, A. Heddini, Drug Resist. Update 14 (2011) 70.
[3] A.J. Alanis, Arch. Med. Res. 36 (2005) 697.
[4] A. Giedraitiene, A. Vitkauskiene, R. Naginiene, A. Pavilonis, Medicina 47 (2011)
137.
[5] L.K. Durham, M. Ge, A.J. Cuccia, J.P. Quinn, Eur. J. Clin. Microbiol. Infect. Dis. 29
(2010) 353.
[6] S. Senguputa, M.K. Chattopadhyay, Resonance 17 (2012) 177.
[7] P. Courvalin, J. Davies, Curr. Opin. Microbiol. 6 (2003) 425.
[8] P.M. Hawkey, J. Antimicrob. Chemother. 51 (2003) 29.
[9] D.C. Hopper, Drugs 58 (1999) 6.
Biological activity experiments showed that PCp was able to in-
hibit the growth of microorganisms at the same level as HCp. Addi-
tional studies revealed that phosphine in higher concentrations
was characterized by a lower in vitro toxicity against mammalian
cells than the parent drug. It did not induce any morphological
changes in cells after its action, which was definitely a positive re-
sult. In addition, PCp does not show any mutagenic activity similar
to HCp what was confirmed by testing its interactions with DNA.
This is relevant in terms of their potential medicinal usage.
The presented results confirm that the antimicrobial activity
and in vitro cytotoxicity of PCp are similar to those of the parent
drug. Therefore it makes this phosphine a smart choice for design-
ing new biological agents based on metal ions. We are currently
[10] K. Drlica, Curr. Opin. Microbiol. 2 (1999) 504.
[11] B.D. Bax, P.F. Chan, D.S. Eggleston, A. Fosberry, D.R. Gentry, F. Gorrec, I.
Giordano, M.M. Hann, A. Hennessy, M. Hibbs, J. Huang, E. Jones, J. Jones, K.
Koretke Brown, C.J. Lewis, E.W. May, M.R. Saunders, O. Singh, C.E. Spitzfaden, C.
Shen, A. Shillings, A.J. Theobald, A. Wohlkonig, N.D. Pearson, M.N. Gwynn,
Nature 466 (2010) 935.
[12] H. Coates, P.A.T. Hoye, Br. Pat. No. 842593.

A. Bykowska et al. / Polyhedron 60 (2013) 23–29
29
[13] A.A. Karasik, I.O. Georgiev, E.I. Musina, O.G. Sinyashin, J. Heinicke, Polyhedron
20 (2001) 3321.
[34] M.N. Patel, M.R. Chhasatia, P.A. Dosi, H.S. Bariya, V.R. Thakkar, Polyhedron 29
(2010) 1918.
[14] R. Starosta, M. Florek, J. Król, M. Puchalska, A. Kochel, New J. Chem. 34 (2010)
1441.
[35] A.V. Polishchuk, E.T. Karaseva, T. Korpela, V.E. Karasev, J. Lumin. 128 (2008)
1753.
[15] C. Marzano, M. Pellei, F. Tisato, C. Santini, Med. Chem. 9 (2009) 185.
[16] S.J. Berners-Price, P.J. Sadler, Bioinorg. Chem. 70 (1988) 27.
[17] R. Starosta, K. Stokowa, M. Florek, J. Król, A. Chwiłkowska, J. Kulbacka, J. Saczko,
[36] A.V. Polishchuk, E.T. Karaseva, M.A. Medkov, V.E. Karasev, Russ. J. Coord. Chem.
30 (2004) 828.
[37] L. Wei, G. Li, H. Li, Spectrochim. Acta, Part A: Mol. Biomol. Spectrosc. 75 (2010)
1486.
_
J. Skała, M. Jezowska-Bojczuk, J. Inorg. Biochem. 105 (2011) 1102.
[18] F.J. Ramos-Lima, A.G. Quiroga, J.M. Perez, M. Font-Bardia, X. Solans, C. Navarro-
Ranninger, Eur. J. Inorg. Chem. (2003) 1591.
[38] J. Hernandez-Gil, L. Perello, R. Ortiz, G. Alzuet, M. Gonzalez-Alvarez, M. Liu-
Gonzalez, Polyhedron 28 (2009) 138.
[19] K. Neplechova, J. Kasparova, O. Vrana, O. Novakova, A. Habtemarian, B.
Watchman, P.J. Sadler, V. Brabec, Mol. Pharm. 56 (1999) 20.
[20] W. Henderson, S.R. Alley, Inorg. Chim. Acta 322 (2001) 106.
[21] C.F. Shaw, Chem. Rev. 99 (1999) 2589.
[39] J. Fawcett, P.A.T. Hoye, R.D.W. Kemmitt, D.J. Law, D.R. Russell, Dalton Trans.
(1993) 2563.
[40] CrysAlis CCD, Oxford Diffraction, 2009.
[41] CrysAlis RED, Oxford Diffraction, 2009.
[22] J.C. McPherson III, R. Runner, T.B. Buxton, J.F. Hartmann, D. Farcasiu, I. Bereczki,
E. Roth, S. Tollas, E. Ostorházi, F. Rozgonyi, P. Herczegh, Eur. J. Med. Chem. 47
(2012) 615.
[42] G.M. Sheldrick, Acta Crystallogr., Sect. A 64 (2008) 112.
[43] D.C. Grove, W.A. Randall, Medical Encyclopedia, New York, 1955.
[44] EN 14675, 2006, Chemical disinfectants and antiseptics
– Quantitative
[23] R.H. Manzo, D.A. Allemandi, J.D. Perez, US Pat. No. 5395936.
[24] F. Alovero, M. Nieto, M.R. Mazzieri, R. Then, R.H. Manzo, Antimicrob. Agents
Chemother. 42 (1998) 1495.
suspension test for the evaluation of virucidal activity of chemical
disinfectants and antiseptics used in the veterinary Area-Test method and
requirements. European Committee for Standarization.
[25] M.J. Nieto, F.L. Alovero, R.H. Manzo, M.R. Mazzieri, Eur. J. Med. Chem. 34 (1999)
209.
[26] F.L. Alovero, X.S. Pan, J.E. Morris, R.H. Manzo, L.M. Fisher, Antimicrob. Agents
Chemother. 44 (2000) 220.
[45] S.M. Laster, J.M. Mackenzie Jr., Microsc. Res. Tech. 34 (1998) 272.
[46] U.J. Strotmann, B. Butz, W.R. Bias, Ecotoxicol. Environ. Saf. 25 (1993) 79.
[47] S. Perrot, H. Dutertre-Catella, Ch. Martin, P. Rat, J.M. Warnet, Toxicol. Sci. 72
(2002) 122.
[27] F. Alovero, A. Barnes, M. Nieto, M.R. Mazzieri, R.H. Manzo, J. Antimicrob.
Chemother. 48 (2001) 709.
[48] S. Namura, J. Zhu, K. Fink, M. Endres, A. Srinivasan, K.J. Tomaselli, J. Yuan, M.A.
Moskowitz, J. Neurosci. 18 (1998) 3659.
[28] M.J. Nieto, F.L. Alovero, R.H. Manzo, M.R. Mazzieri, Eur. J. Med. Chem. 40 (2005)
361.
[29] M.J. Nieto, A.B. Pierini, N. Singh, C.R. McCurdy, R.H. Manzo, A.R. Mazzieri, Med.
Chem. 8 (2012) 349.
[49] M. Noirot, P. Barre, J. Louarn, Ch. Duperray, S. Hamon, Ann. Bot. 89 (2002) 135.
[50] K.K. Kamo, R.J. Griesbach, Biotech. Histochem. 68 (1993) 350.
[51] R.U. Jänicke, M.L. Sprengart, M.R. Wati, A.G. Porter, J. Biol. Chem. 273 (1998)
9357.
[30] M.G. Rabbani, M.R. Islam, M. Ahmad, A.M.L. Hossion, Bangladesh J. Pharmacol.
6 (2011) 8.
[52] A.K. Chattah, Y. Garro Linck, G.A. Monti, P.R. Levstein, S.A. Breda, R.H. Manzo,
M.E. Olivera, Magn. Reson. Chem. 45 (2007) 850.
[31] Y. Wang, G.W. Lin, J. Hong, L. Li, Y.M. Yang, T. Lu, J. Coord. Chem. 63 (2010) 1.
[32] N. Jimenez-Garrido, L. Perello, R. Ortiz, G. Alzuet, M. Gonzalez-Alvarez, E.
Canton, M. Liu-Gonzalez, S. Garcia-Granda, M. Perez-Priede, J. Inorg. Biochem.
99 (2005) 677.
[53] A. Zieba, A. Maslankiewicz, J. Sitkowski, Magn. Reson. Chem. 42 (2004) 903.
[54] S.E. Durran, M.B. Smith, A.M.Z. Slawin, J.W. Steed, Dalton Trans. (2000) 2771.
[55] M.R.J. Elsegood, A.J. Lake, M.B. Smith, Dalton Trans. (2009) 30.
[56] I. Turel, P. Bukovec, M. Quirds, Int. J. Pharm. 152 (1997) 59.
[33] M.P. Lopez-Gresa, R. Ortiz, L. Perello, J. Latorre, M. Liu-Gonzalez, S. Garcia-
Granda, M. Perez-Priede, E. Canton, J. Inorg. Biochem. 92 (2002) 65.