Phosphine derivatives of ciprofloxacin and norfloxacin, a new class of potential therapeutic agents

Article abstract of DOI:10.1039/c3nj01243c

In this paper a new series of chalcogenides of diphenylmethylaminophosphine derived from ciprofloxacin (PPh₂CH₂Cp) and a new phosphine derived from norfloxacin (PPh₂CH₂Nr) are presented. The synthesized compounds were characterized by NMR, MS and X-ray techniques. Both phosphines exhibit antibacterial activity against: S. aureus, E. coli, K. pneumoniae and P. aeruginosa, similar to ciprofloxacin and norfloxacin. They inhibit the growth of microorganisms in relatively low concentrations. Chalcogenides are slightly less active than phosphines and unmodified antibiotics. All the derivatives were also tested in vitro as anticancer agents towards mouse colon carcinoma (CT26) and human lung adenocarcinoma (A549). Cytotoxicity studies revealed that phosphines and their chalcogenides are able to inhibit the proliferation of the cells at relatively low concentrations. Moreover, all the tested compounds are more active against tested cell lines than cisplatin-the main representative of antitumor drugs.

Full text of DOI:10.1039/c3nj01243c

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Phosphine derivatives of ciprofloxacin
and norfloxacin, a new class of potential
therapeutic agents†
Cite this: New J. Chem., 2014,
38, 1062
Aleksandra Bykowska,a Radosław Starosta,*a Urszula K. Komarnicka,a
Zbigniew Ciunik,a Agnieszka Kyzioł,b Katarzyna Guz-Regner,c
Gabriela Bugla-Płoskonskac and Małgorzata Jezowska-Bojczuk*a
´
˙
In this paper
a new series of chalcogenides of diphenylmethylaminophosphine derived from
ciprofloxacin (PPh2CH2Cp) and a new phosphine derived from norfloxacin (PPh2CH2Nr) are presented.
The synthesized compounds were characterized by NMR, MS and X-ray techniques. Both phosphines exhibit
antibacterial activity against: S. aureus, E. coli, K. pneumoniae and P. aeruginosa, similar to ciprofloxacin and
norfloxacin. They inhibit the growth of microorganisms in relatively low concentrations. Chalcogenides are
slightly less active than phosphines and unmodified antibiotics. All the derivatives were also tested in vitro as
anticancer agents towards mouse colon carcinoma (CT26) and human lung adenocarcinoma (A549).
Cytotoxicity studies revealed that phosphines and their chalcogenides are able to inhibit the proliferation
of the cells at relatively low concentrations. Moreover, all the tested compounds are more active against
tested cell lines than cisplatin – the main representative of antitumor drugs.
Received (in Montpellier, France)
9th October 2013,
Accepted 22nd November 2013
DOI: 10.1039/c3nj01243c
www.rsc.org/njc
diphenylmethylphosphine derivative (Ph2PCH2Cp; PCp) and its
oxide (Ph2P(O)CH2Cp; OPCp). The possibility of such a modifica-
Introduction
The large genetic variation of pathogenic organisms and the tion, benefiting from the presence of a secondary nitrogen atom in
excessive frequency of antibiotics usage has resulted in the the piperazine ring of HCp,4–6 seems to be especially attractive. This
development of increasingly effective defence mechanisms by is confirmed by a number of papers showing that phosphines,
microorganisms. It has been recognized by the WHO as one of as well as their derivatives and the metal complexes (i.e. copper,
the most significant health dangers.1,2 Although most bacteria platinum, gold) exhibit promising anticancer, antibacterial or
have remained susceptible to the fluoroquinolones with MICs antiarthritic activities.6–13
less than or equal to 1.0 mg mlꢀ1, low rates of resistance have
Our studies have shown that PCp is able to inhibit the growth
been observed in some strains of P. aeruginosa and Enterococci of microorganisms at the same level as HCp. Additional studies
and high rates in methicillin-resistant S. aureus. For this reason, have revealed that this phosphine at higher concentrations is
the search for new antimicrobial agents is extremely important characterized by a lower in vitro toxicity against mammalian cells
and desirable.
than the parent drug. Moreover, PCp, as previously shown for
In our previous paper3 we presented the spectroscopic profile HCp, did not show any mutagenic activity and this was confirmed
and biological activity of ciprofloxacin (HCp) modified to a novel by testing its interactions with DNA.3
The results described above confirmed that this phosphine
a Faculty of Chemistry, University of Wrocław, ul. F. Joliot-Curie 14,
was a good choice for designing new biological agents. Therefore
we decided to extend our studies. In this paper we present the
syntheses, structures, spectroscopic properties, antibacterial and
cytotoxic activities of a series of diphenylmethylphosphines and
their chalcogenides derived from HCp and norfloxacin (HNr). Both
drugs are 2nd generation fluorochinolones and are very similar to
each other. They are broad-spectrum antibiotics, commonly used
for the treatment of urinary tract, respiratory and digestive infec-
tions. HCp and HNr are active against gram-negative and some
gram-positive bacteria. Most gram-negative bacteria, including non-
fermentative bacteria such as P. aeruginosa, Acinetobacter spp.,
50-383 Wrocław, Poland. E-mail: malgorzata.jezowska-bojczuk@chem.uni.wroc.pl,
radoslaw.starosta@chem.uni.wroc.pl; Tel: +48-71-3757281, +48-71-3757273
b
´
Faculty of Chemistry, Jagiellonian University, R. Ingardena 3, 30-060 Krakow,
Poland
c Department of Microbiology, Institute Genetics and Microbiology,
University of Wroclaw, S. Przybyszewskiego str. 63/77, 51-148 Wroclaw, Poland
† Electronic supplementary information (ESI) available: Crystal packing pictures,
detailed NMR data, the selected photos of the cells after treatment with the tested
compounds and cytotoxic assay curves for all presented compounds. CCDC
948747 (OPCpꢁ2CH3COCH3), 948748 (SPCpꢁ0.5CH2Cl2) and 948749 (SePCpꢁ
0.79CH3OHꢁ0.11H2O). For ESI and crystallographic data in CIF or other electronic
format see DOI: 10.1039/c3nj01243c
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Burkholderia cepacia are highly susceptible in vitro and many changes are observed for H1P0 protons from the P–CH2–N group
gram-positive bacteria, including fastidious pathogens are sus- (Fig. 1) appearing as doublets (or broadened singlets for SePCp
ceptible or moderately susceptible.
and OPNr). Formation of a PQX bond (X = chalcogenide) shifts
The mechanism of their action is based on blocking the DNA this signal towards lower fields by 0.05–0.13 ppm in the case of
replication process by binding gyrase or topoisomerase IV.14–17 oxides, 0.25 ppm for sulfides and 0.45–0.43 for selenides.
Moreover, recent studies have proved that HCp showed cytotoxic
In the 13C{1H} NMR spectra, analogously to the 1H ones, the
properties against a few tumour cell lines and decreased their largest changes are observed for the C1P0 atom and CPh(i) – ipso
viability in vitro.18–20
carbon atom of the phenyl rings. A significant increase of the
1J(C1P0P) coupling constant is the most noticeable. The value of
this constant is equal to 4.1 Hz for PCp, whereas for the oxide
and the sulfide it is equal to 87.3 and 74.5 Hz, respectively. We
Results and discussion
1
did not observe a C1P0 signal for SePCp. The J(C1P0P) coupling
Ph2PCH2-Nr (PNr) – a diphenylphosphine derivative of norfloxacin,
similar to PCp,3 was obtained as a result of a two-step one-pot
synthesis from Ph2P(CH2OH)2Cl. Chalcogenide derivatives: oxides
(OPCp3 and OPNr), sulfides (SPCp and SPNr) and selenides (SePCp
and SePNr) were obtained from simple reactions of the corres-
ponding phosphines with hydrogen peroxide, resublimed sulfur
and metallic selenium, respectively (Fig. 1). The identities of all the
compounds were confirmed using elemental analyses, 1H, 31P{1H}
and 13C{1H} NMR spectroscopy and mass spectrometry.
constant for PNr is 3.4 Hz and for its chalcogenides: 85.6, 72.7
and 66.3 Hz for OPNr, SPNr and SePNr, respectively. The signal
for CPh(i) is slightly shifted towards higher fields after the
formation of a PQX bond with a simultaneous increase of the
1J(CPh(i)P) coupling constant, wherein the changes of the constant
value showed trends (12.4 and 11.7 Hz – PCp and PNr; 98.1 and
99.0 Hz – OPCp and OPNr; 79.0 and 79.0 Hz – SPCp and SPNr;
70.8 and 69.9 Hz – SePCp and SePNr;) like those observed for the
C1P0 carbon atom.
Summarizing, analysis of the NMR spectra suggests that
regardless of the modified antibiotic we observed similar changes
in the values of the chemical shifts and coupling constants for
the derivatives of both ciprofloxacin and norfloxacin. This is
fully justified because there is almost no difference between the
molecules of both compounds.
NMR spectroscopy of the phosphine chalcogenide derivatives
NMR spectra (see Experimental section and Table S1 in ESI†)
were measured at room temperature in deuterated chloroform
(CDCl3) as a solvent. In the 31P{H} NMR spectra both phosphines
gave signals at ꢀ27.42 ppm (PCp)3 and ꢀ27.50 ppm (PNr). The
formation of the chalcogenide derivatives of these phosphines
leads to significant changes in the spectra strongly shifting the
signal of the phosphorus atom to lower fields. The biggest shift is
observed for sulfide derivatives, and the chemical shift values are
equal to 35.74 ppm for SPCp and 35.84 ppm for SPNr. The oxides
give signals at 27.70 ppm for OPCp3 and 27.36 ppm for OPNr, while
the signals of phosphine selenides are placed at 26.11 ppm (SePCp)
and 26.20 ppm (SePNr). Phosphorus–selenium coupling constants
are equal to 726.5 and 731.4 Hz for SePCp and SePNr, respectively.
They are significantly larger than those determined for selenides
of tris(aminomethyl)phosphines (P(CH2NCH2CH2O)3: 709.8 Hz;
P(CH2NCH2CH2NMe)3 and P(CH2NCH2CH2NEt)3: 707.5 Hz)21 and
other aliphatic phosphines (PMe3:22 684.0 Hz, P(t-Bu)3:23 693.0 Hz)
and are comparable to the coupling constants determined for
selenides of aromatic phosphines: (PPh3:22 732.5 Hz).
X-ray structures
We obtained single crystals and resolved the X-ray structures of
all three derivatives of HCp: OPCp in OPCpꢁ2CH3COCH3, SPCp
in SPCpꢁ0.5CH2Cl2, and SePCp in SePCpꢁ0.79CH3OHꢁ0.11H2O.
Selected crystallographic and data collection parameters are
given in the Experimental section. Bond lengths and angles
are summarized in Table 1. Perspective views of the molecules
are depicted in Fig. 2.
As expected, all three molecules are similar to each other
and to PCp,3 they differ only by the chalcogenide atom bound to
phosphorus. Detailed analysis indicates however that binding of
the chalcogen causes significant changes in the geometry
around the P atom: P–C bonds are shortened and C–P–C bond
angles are enlarged. The largest changes are observed for OPCp,
probably due to the most electronegative character of oxygen.
Similar tendencies are observed also for other phosphines and
their derivatives (i.e.: P(CH2NCH2CH2Y)3, where Y = O, NMe
or NEt,6,21 PMe3,24–26 PPh3,27–30 P(t-Bu)3,23 P(CH2CH2CN)3,31,32
1,3,5-triaza-7-phosphaadamantane33,34).
The formation of chalcogenides does not cause significant
changes in the position of the proton signals. The only noticeable
A strong intramolecular hydrogen bond is observed in each
one of the OPCp, SPCp and SePCp molecules, as for PCp (Table 1;
O2–H2ꢁ ꢁ ꢁO3). This bond does not involve the hydroxyl and
carbonyl part of the same carboxyl group, but connects the OH
part of the carboxyl group and the neighbouring carbonyl
oxygen atom directly bound to the ring.
Despite their similar molecular structures, the presented
derivatives crystallize in different crystal systems (see Fig. S1–S3
in ESI†) and are characterized by different intermolecular
Fig. 1 Molecular structures of phosphines and their chalcogenides (X =
oxygen, sulfur or selenium atom) derived from ciprofloxacin (R = cyclo-
propyl) and norfloxacin (R = ethyl).
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Table 1 Selected bond lengths [Å] and angles [1] for OPCp, SPCp and SePCp together with PCp3
Geometry
PCp3
OPCp
SPCp
SePCp (A)
SePCp (B)
P(1)–Xa
P(1)–C(31)
P(1)–C(41)
P(1)–C(1)
F(12)–C(12)
O(1)–C(20)
O(2)–C(20)
O(3)–C(17)
1.487(3)
1.806(4)
1.805(4)
1.821(4)
1.371(4)
1.215(5)
1.324(5)
1.258(5)
1.811(4)
105.47(17)
1.959(1)
1.817(3)
1.826(3)
1.831(3)
1.363(3)
1.216(3)
1.320(3)
1.266(3)
1.825(3)
104.64(13)
2.1094(10)
1.825(3)
1.817(3)
1.842(4)
1.360(4)
1.219(5)
1.329(5)
1.251(4)
1.828(3)
105.18(15)
2.1088(12)
1.801(4)
1.802(4)
1.846(4)
1.355(4)
1.218(4)
1.327(4)
1.256(4)
1.816(4)
106.23(17)
1.831(1)
1.839(1)
1.855(1)
1.359(1)
1.212(1)
1.336(1)
1.264(1)
1.842(1)
100.49(5)
Av. P(1)–C(1,31,41)
Av. C(1,31,41)–P(1)–C(1,31,41)
Hydr.bond O2–H2ꢁ ꢁ ꢁO3
O2ꢁ ꢁ ꢁO3
2.551(2)
0.82, 1.79
153.5
2.531(4)
0.82, 1.77
154.2
2.533(3)
0.82, 1.77
153.0
2.501(4)
0.84, 1.72
154.2
2.510(4)
0.84, 1.73
154.4
O2–H2, H2ꢁ ꢁ ꢁO3
O2–H2ꢁ ꢁ ꢁO3
a
X = O(4), S(1) or Se(1).
Fig. 3 Dimer of OPCp with p-stacking interaction between the quinolone
rings (view perpendicular to the ring planes). The distance between the
planes is equal to 3.291 Å.
of highly disordered molecules of occluded dichloromethane
(see Fig. S2 in ESI†). On the other hand, the elemental cell of
SePCpꢁ0.79CH3OHꢁ0.11H2O contains two pairs of independent
molecules (A and B) differing not only in the selenide atom
disorder (Fig. S3 in ESI†), but also in the geometry around the
phosphorus atom. The crystal cell is also partially occupied by
molecules of methanol and water connected to one another and
to SePCp molecules by means of weak hydrogen bonds. Molecules
of SPCp and SePCp, in comparison to OPCp, are connected in the
crystal net only by weak interactions, mainly of C–Hꢁꢁꢁp, C–HꢁꢁꢁO
and C–Hꢁ ꢁ ꢁS(Se) character. No classic p-stacking interactions
are observed.
Fig. 2 The X-ray structures (50% ellipsoids) of OPCp (top), SPCp (middle)
and the ‘‘A’’ molecule of SePCp (bottom).
Antibacterial properties
interactions. OPCpꢁ2CH3COCH3 crystallizes in the triclinic
%
system (P1 space group). The elemental cell contains two The antibacterial activities of derivatives of fluorinated quino-
molecules of OPCp and four solvent molecules. The solvent lones against a reference gram-positive strain of S. aureus ATCC
molecules are unconnected to the phosphine oxide therefore a 6538 and three gram-negative strains of E. coli ATCC 25922,
high degree of observed structural disorder (see Fig. S1 in ESI†) K. pneumoniae ATCC 4352 and P. aeruginosa ATCC 27853 are
is observed. OPCp molecules form dimers bound by strong p- presented in Table 3. The results of MICs and MBCs tests show
stacking interactions which are typical for extended aromatic various degrees of the bacterial susceptibility to the derivatives
hydrocarbons35 and show carbon atoms both overlapping with compared to their native forms (Kurskal–Wallis test, P = 0.014).
others and placed in the centers of the stacked rings (Fig. 3). The most of the examined compounds were bactericidal
SPCpꢁ0.5CH2Cl2 and SePCpꢁ0.79CH3OHꢁ0.11H2O crystallize in against the tested microorganisms, especially enteric gram-
the monoclinic systems (both in the P21 space group). The negative rods (K. pneumoniae and E. coli) (Kurskal–Wallis test,
elemental cell of SPCpꢁ0.5CH2Cl2 contains four molecules of P = 0.0017). For HCp and all its derivatives the mean of the
phosphine sulfide (only one is independent) and two molecules MICs and MBCs was in the range from 0.004 to 0.125 mg mlꢀ1
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Table 2 Antibacterial activity of the investigated compounds against
Staphylococcus aureus 6538, Escherichia coli 25922, Klebsiella pneumo-
niae 4352 and Pseudomonas aeruginosa 27853 strains. MIC – minimal
inhibitory concentration, MBC – minimal bacteriocidal concentration
The mean of the MICs and MBCs of HNr and all its derivatives
ranged from 0.03 to 0.5 mg mlꢀ1 and 0.03 to 16.0 mg mlꢀ1
,
respectively. According to the recommendation of CLSI36 for
HNr, almost all of the tested bacterial strains were susceptible
to derivatives of HNr; their MICs were lower than 4.0 mg mlꢀ1
.
Cmpd
S. aureus
E. coli
K. pneumoniae
P. aeruginosa
MIC [mg mlꢀ1
0.125
0.125
1.0
2.0
0.5
]
The only exception were gram-negative rods of P. aeruginosa
resistant to OPNr and SPNr (Kurskal–Wallis test, P = 0.04) with
HCp
PCp
OPCp
SPCp
SePCp
0.008
0.008
1.0
0.25
0.0625
0.004
0.004
0.125
0.125
0.016
0.125
0.125
16
16
0.5
MICs accounted Z16.0 mg mlꢀ1
.
Among all the derivatives of HNr, PNr and SePNr were
characterized as the best antimicrobial agents against both gram-
positive and gram-negative bacteria, including the P. aeruginosa
strain. The mean of the MICs and MBCs of PNr were found to be in
HNr
PNr
OPNr
SPNr
SePNr
0.5
1.0
2.0
2.0
2.0
0.03
0.125
2.0
2.0
0.125
0.03
0.03
0.25
1.0
0.5
1.0
16
>16
2.0
the range from 0.03 to 1.0 mg mlꢀ1 and 0.0625 to 2.0 mg mlꢀ1
,
respectively. Whereas, the MICs and MBCs of SePNr were
0.125–2.0 mg mlꢀ1 and 0.25–4.0 mg mlꢀ1 range, respectively.
Remarkably, the antibacterial activity of OPNr and SPNr, observed
against gram-positive cocci and enteric gram-negative bacteria, were
similar to the activity of HNr (Kurskal–Wallis test, P = 0.09). The
values of the MICs and MBCs of SPNr were r2.0 and 4.0 mg mlꢀ1 for
S. aureus and E. coli and r1.0 and 1.0 mg mlꢀ1 for K. pneumoniae,
respectively. The mean of the MICs and MBCs of OPNr were almost
similar and were r2.0 mg mlꢀ1 and 2.0 mg mlꢀ1 for E. coli and
S. aureus and r0.25 mg mlꢀ1 and 0.5 mg mlꢀ1 for K. pneumoniae,
respectively. These compounds were ineffective against P. aeruginosa
strain (Kurskal–Wallis test, P = 0.045).
0.125
MBC [mg mlꢀ1
0.25
0.25
2.0
4.0
1.0
]
HCp
PCp
OPCp
SPCp
SePCp
0.008
0.016
2.0
0.25
0.125
0.008
0.008
0.25
0.25
0.03
0.25
0.25
>16
>16
1.0
HNr
PNr
OPNr
SPNr
SePNr
1.0
1.0
2.0
4.0
4.0
0.0625
0.125
2.0
4.0
0.25
0.03
0.0625
0.5
1.0
0.25
1.0
2.0
>16
>16
4.0
and 0.004 to 16.0 mg mlꢀ1, respectively. Therefore, according to
Cytotoxicity
the recommendation of CLSI36 for HCp, in most cases the tested To determine the cytotoxicity of the obtained derivatives we
bacterial strains were susceptible to derivatives of HCp (Kurskal– selected two cell lines, commonly used for preliminary in vitro
Wallis test, P = 0.04). The mean of the MICs of the HCp tests: mouse colon carcinoma (CT26) and human lung adeno-
derivatives was not greater than 1.0 mg mlꢀ1, except for that of carcinoma (A549). A study of biological activity was undertaken
OPCp for gram-positive cocci (with an intermediate susceptibility with concentrations of the newly synthesized compounds ranging
equal to 2.0 mg mlꢀ1) as well as that for OPCp and SPCp for non- from 0.1 to 0.001 mM. In parallel, the influence of the widely used
fermentative rods of P. aeruginosa strain (Kurskal–Wallis test, anticancer drug, cisplatin, was assessed. Treatment of CT26 and
P = 0.05). P. aeruginosa was resistant to these compounds with A549 cell lines with the tested compounds resulted in dose-
MICs of Z16.0 mg mlꢀ1 (Kurskal–Wallis test, P = 0.025).
dependent inhibition of cell proliferation. For both tested cell lines
It was found that the modification of native HCp by addition viability decreased with increasing concentration of the compounds.
of a Ph2PCH2-group did not alter its antimicrobial activity Fig. 4 and Fig. S8 and S9 in ESI† show the cytotoxic assays.
(Table 2). SePCp was the most effective compound against all
The obtained data indicates (Fig. 4) the statistically relevant
the tested bacteria, among the chalcogenated derivatives of PCp. differences in the cytotoxic action of the HCp and HNr derivatives.
The antibacterial activity of this compound was similar to the Generally, for both cell lines, all the tested compounds in our
activity of HCp (Kurskal–Wallis test, P > 0.05). The MICs and MBCs experimental system were found to have a higher cytotoxicity than
of SePCp for gram-positive and gram-negative bacteria were in the cisplatin. Moreover, phosphines and all chalcogenides showed
range from 0.016 to 0.5 mg mlꢀ1, and 0.03 to 1.0 mg mlꢀ1
respectively. Notably, a high antibacterial activity for OPCp or P = 0.00013) and HNr (ANOVA/MANOVA, P o 0.000001).
SPCp was observed against enteric gram-negative bacteria The best cytotoxic activity against the CT26 cell line was
,
a much better anticancer effect than HCp (ANOVA/MANOVA,
(Kurskal–Wallis test, P = 0.045). For SPCp the MICs and MBCs were observed for sulfides and selenides. SPCp and SePCp at c = 0.01 mM
found to be r0.125 mg mlꢀ1 and 0.25 mg mlꢀ1 for K. pneumoniae, induced a decrease of the cells viability by over 50% (Fig. 4A;
and r0.25 and 0.5 mg mlꢀ1 for E. coli, respectively. For OPCp the ANOVA/MANOVA, P o 0.00005). Analogously, SPNr and SePNr
mean of the MICs and MBCs were almost similar and were at c = 0.01 mM decreased the cells viability by approx. 65 and
found to be r0.125 and 0.25 mg mlꢀ1 for K. pneumoniae, and 55% (Fig. 4B; ANOVA/MANOVA, P = 0.00002).
r1.0 and 2.0 mg mlꢀ1 for E. coli, respectively. It should be noted
The A549 cell line was more resistant to the derivatives of
that these derivatives were not effective against the P. aeruginosa HCp. SPCp and SePCp at c = 0.01 mM caused a 25% decrease of
strain (Kurskal–Wallis test, P = 0.048).
the cells viability, but PCp and OPCp at the same concentration –
HNr the second investigated chemotherapeutic, also showed only 20% (Fig. 4C; ANOVA/MANOVA, P = 0.00002). Derivatives of
great antibacterial properties (Kurskal–Wallis test, P = 0.05). HNr were much more cytotoxic. The highest activity was observed
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Table 3 IC50 values [mM] for CT26 and A549 cells after treatment with
the studied compounds for 4 hours, determined by MTT assay
Cmpd
IC50 c [mM]
CT26
A549
HCp
PCp
OPCp
SPCp
SePCp
0.47 ꢂ 0.05
0.21 ꢂ 0.07
0.22 ꢂ 0.04
0.15 ꢂ 0.02
0.17 ꢂ 0.02
0.38 ꢂ 0.04
0.35 ꢂ 0.03
0.35 ꢂ 0.03
0.44 ꢂ 0.04
0.30 ꢂ 0.03
HNr
PNr
OPNr
SPNr
SePNr
0.47 ꢂ 0.03
0.16 ꢂ 0.09
0.17 ꢂ 0.02
0.18 ꢂ 0.02
0.18 ꢂ 0.02
0.33 ꢂ 0.05
0.16 ꢂ 0.04
0.15 ꢂ 0.02
0.15 ꢂ 0.02
0.32 ꢂ 0.04
Cisplatin
2.20 ꢂ 0.82
3.15 ꢂ 0.45
P o 0.00005). These two compounds lost their activity at c =
0.01 mM (ANOVA/MANOVA, P > 0.05) while PNr and OPNr
decreased viability by 25% ANOVA/MANOVA, P = 0.00002).
The IC50 parameter values (Table 3) were determined from
the fitting curve by calculating the concentration of a tested
compound that reduces the surviving fraction in the treated
cells by 50%, compared to control cells. For HCp and its
derivative compounds against CT26 and A549 cell lines the
IC50 values decreased in the following order: SPCp > SePCp >
PCp E OPCp c HCp and SePCp > OPCp = PCp > HCp c SPCp
for the tested cell lines respectively. Analogously, the values of
IC50 for HNr and its derivatives may be ordered as follows:
PNr E OPNr E SPNr = SePNr c HNr and OPNr = SPNr E PNr c
SePNr E HNr for CT26 and A549 cell lines, respectively.
Fig. 4 Cytotoxic assay performed on CT26 and A549 cell lines for HCp
and HNr and their derivatives. X-axis: the concentration of the compound
[mM] presented in the logarithmic scale; Y-axis: surviving fraction.
Fig. 5 Selected photos (magnification: 2000ꢃ; bar: 50 mm) of A549 cells
after treatment with the tested compounds (0.05 mM) for 4 hours. The
green cells with normal morphology are viable ones (AO+), while round red
cells are dead (PI+).
for OPNr and SPNr, which caused respectively a 60 and 55%
decrease of the viability at c = 0.05 mM (Fig. 4D; ANOVA/MANOVA,
1066 | New J. Chem., 2014, 38, 1062--1071
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Selected photographs of CT26 and A549 cells treated with
NJC
Now we are focusing on the synthesis and characteristics of
the tested compounds are displayed in Fig. 5 (and Fig. S4–S8 in copper(I) complexes obtained with phosphines. It is well known that
ESI†). The cell viability was examined by counting dead and alive phosphines are perfect binding ligands for this ion. Additionally,
cells stained with two fluorescent dyes: acridine orange (AO) and Cu(I) complexes are promising potential therapeutic agents
propidium iodide (PI). AO, in comparison ary to PI, penetrates with high anticancer activity.7,40,41
into viable cells and binds to DNA by intercalation. PI due to its
electric charge does not penetrate through the uninterrupted
cytoplasmic membrane of viable and early apoptotic cells. There-
fore, PI stains only cells with interrupted membranes – necrotic or
Experimental
Materials and methods
late apoptotic ones. Accordingly, green cells with normal nuclei
were treated as viable cells (AO+), while red ones as dead cells
Reactions were carried out under a nitrogen atmosphere using
(PI+). As can be noticed in the presented photographs, the tested
standard Schlenk techniques. The reagents and solvents were
purchased from Sigma-Aldrich and used without further
purification. All the solvents were deaerated prior to use.
derivatives of HCp and HNr caused a significant reduction of the
surviving fraction in both of the studied cell lines in comparison
[PPh2(CH2OH)2]+Clꢀ was synthesized according to the literature
with the original antibiotics.
method.42 PCp and OPCp were synthesized as described previously.3
NMR spectra were recorded on a Bruker Avance 500 MHz
spectrometer with traces of solvent as an internal reference for
1H and 13C{1H} spectra and 85% H3PO4 in H2O as an external
Conclusions
standard for 31P{1H}. Chemical shifts are reported in ppm and
coupling constants are reported in Hz. Mass spectra were registered
on a Bruker Daltonics micrOTOF-Q Mass spectrometer equipped
with an electrospray ionization (ESI) source and operated in the
positive ion mode. Elemental analysis was performed with a
Vario EL3 CHN analyzer.
We synthesized
a novel diphenylmethylaminophosphine
derived from HNr and a series chalcogenide derivatives of this
ligand as well as analogous derivatives of HCp. The identity and
structure of the obtained new compounds were confirmed by
elemental analysis, NMR, MS and X-ray techniques.
The antibacterial activity of the compounds was tested
against S. aureus ATCC 6538, E. coli ATCC 25922, K. pneumoniae
ATCC 4352 and P. aeruginosa ATCC 27853. The mean of the
Syntheses
Q
S
PPh2CH2Cp (SPCp). The sulfide was obtained from the
MICs of PCp and PNr were similar to those for HCp and HNr. reaction of PCp (0.287 g; 0.542 mmol) in chloroform (25 cm3)
Chalcogenide derivatives were found to be slightly less active, with equimolar amounts of resublimed sulfur (0.0174 g;
however selenides generally showed the highest antimicrobial 0.542 mmol). The mixture was placed in an ultrasonic bath
activity among all of the derivatives. Their MBCs values were for 0.5 h at room temperature. A yellowish solid was obtained
satisfactory, and were found to be 0.1 mg% for SePCp and after removing the solvent and purified by recrystallization
0.4 mg% for SePNr. Probably, the action mechanism of organo- from the chloroform–methanol mixture (1 : 2 V : V). The
selenium compounds is based on the release of the cytoplasmic obtained microcrystals were dried for 24 h under vacuum to
constituents from the cell and cell dehydration that could lead to remove any occluded solvent molecules.
disintegration. According to recent literature data such compounds,
Yield 87%. Anal. calcd for C30H29FN3O3PS (561.6): C, 64.15;
attached covalently to different biomaterials, inhibit the formation H, 5.16; N, 7.48%. Found: C, 64.12; H, 5.23; N, 7.45%. NMR
1
and growth of many bacterial biofilms.37–39
In vitro cytotoxicity studies showed that the investigated H5P: 7.90 d J = 13.00, HPh(o),(m),(p): 7.94–7.45, H8P: 7.28 d J = 7.2,
compounds exhibit potential anticancer properties. All of the H1Pa: 3.08 q J = 3.4, H3P0: 3.24 m, H1P0: 3.54 d J = 5.0, H4P0
(CDCl3, 298 K): 31P{1H}: 35.74 s; H: H3Pb: 14.98 s, H2P: 8.66 s,
:
tested derivatives of HCp and HNr were cytotoxic against human 2.91 m, H1Pb: 1.38 q J = 7.3, H1Pb: 1.15 td J = 6.7; 3.2; 13C{1H}:
lung adenocarcinoma (A549) and mouse colon carcinoma (CT26) C4P: 176.93 s, C3Pa: 166.85 s, C6P: 154.52 d J = 251.6; C2P: 147.29 s,
cell lines. For all of them we observed much better antitumor effect C7P: 145.60 d J = 10.0, C9P: 138.96 s, CPh(i): 131.83 d J = 79.0,
in comparison with the unmodified antibiotics. For example, the CPh(o): 131.73 d J = 2.7, CPh(p): 131.62 d J = 9.0, CPh(m): 128.53 d
IC50 value for HNr was determined to be 0.47 mM for CT26 cell J = 11.8, C10P: 119.63 d J = 7.3, C5P: 112.16 d J = 22.7, C3P: 107.93 s,
line and 0.33 mM for A549 cell line, while in the case of OPNr these C8P: 104.81 d J = 2.7, C1P0: 61.51 d J = 74.5, C4P0: 54.73 d J = 7.3,
values are 0.17 mM and 0.15 mM, respectively. In addition, the C3P0: 49.61 d J = 5.4, C1Pa: 35.26 s, C1Pb: 8.17 s. MS (CHCl3): 562.2
cytotoxicity of our compounds was much higher than that of [MH]+ 100%.
Q
Se PPh2CH2Cp (SePCp). An equimolar mixture of PCp
cisplatin, the main representative of anticancer drugs.
The presented data are relevant in the context of further (0.321 g; 0.607 mmol) and metallic selenium (0.048 g; 0.608 mmol)
studies and even potential applications in therapy because we in chloroform (25 cm3) was placed in an ultrasonic bath for 3 h.
have proven that modification of HCp or HNr by the addition of A yellowish solid was deposited at ꢀ18 1C. It was filtered off
the diphenylphosphinomethyl group as an interface to other and then purified by recrystallization from the chloroform–
functional moieties can selectively alter the biological activity methanol mixture (1 : 2 V : V). The obtained solid was dried for
of the parent drugs.
24 h under vacuum.
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Yield 86%. Anal. calcd for C30H29FN3O3PSe (608.5): C, 59.21; resublimed sulfur (0.0155 g; 0.483 mmol) was placed in an
H, 4.76; N, 6.91%. Found: C, 59.19; H, 4.79; N, 6.87%. NMR ultrasonic bath for 0.5 h at room temperature. The yellowish solid
1
1
(CDCl3, 298 K): 31P{1H}: 26.11 J(PSe) = 726.5; H: H3Pb: 14.97s, of sulfide derivative was filtered and purified by recrystallization
H2P: 8.74 s, H5P: 7.94 d J = 13.0, HPh(o),(m),(p): 7.98–7.48, H8P
7.30 d J = 7.1, H1Pa: 3.52 qn.t J = 4.0; 3.1, H3P0: 3.27, H1P0: 3.74* s, white microcrystals were dried for 24 h under vacuum.
:
from the chloroform–methanol solution (1 : 2 V : V). The obtained
H4P0: 2.90, H1Pb: 1.39 q J = 3.90, H1Pb: 1.19 td J = 6.7; 3.6; 13C{1H}:
C4P: 177.06 s, C3Pa: 166.92 s, C6P: 154.57 d J = 251.6, C2P
Yield 87%. Anal. calcd for C29H29FN3O3PS (549.3): C, 63.41;
:
H, 5.28; N, 7.65%. Found: C, 63.38; H, 5.31; N, 7.62%. NMR
147.40 s, C7P: 145.58 d J = 10.9, C9P: 139.00 s, CPh(i): 130.48 d (CDCl3, 298 K): 31P{1H}: 35.84 s; H: H3Pb: 15.10 s, H2P: 8.62 s,
1
J = 70.8, CPh(o): 131.88 d J = 2.7, CPh(p): 132.23 d J = 9.1, CPh(m)
:
H5P: 7.93 d J = 13.0, HPh(o),(m),(p): 7.95–7.44, H8P: 6.77 d J = 6.9,
128.60 d J = 11.8, C10P: 119.89 d J = 8.2, C5P: 112.39 d J = 23.6, H1Pa: 4.08 m, H3P0: 3.21 m, H1P0: 3.54 d J = 5.0, H4P0: 2.83 m,
C3P: 108.12 s, C8P: 104.84 d J = 2.70, C1P0: 62.04 d J = 65.95, C4P0
54.63 d J = 5.4, C3P0: 49.47 d J = 4.5, C1Pa: 35.26 s, C1Pb: 8.24 s. MS 154.32 d J = 252.5, C2P: 146.99 s, C7P: 145.79 d J = 10.9, C9P
(CHCl3): 610.1 [MH]+ 100%. 136.99 s, CPh(i): 131.76 d J = 79.0, CPh(o): 131.65 d J = 2.7, CPh(p)
PPh2CH2Nr (PNr):
:
H1Pb: 1.54 t d J = 7.2; 13C{1H}: C4P: 176.78 s, C3Pa: 167.03 s, C6P
:
:
:
A
solution of 0.8853
g
of 131.55 d J = 9.1, CPh(m): 128.46 d J = 11.8, C10P: 120.32 d J = 8.2,
[PPh2(CH2OH)2]+Clꢀ (3.13 mmol) in 25 cm3 of methanol was C5P: 112.43 d J = 23.6, C3P: 108.09 s, C8P: 103.85 d J = 3.6, C1P0
placed in an ice bath, NEt3 (4 ml) was added dropwise. After 61.52 d J = 72.7, C4P0: 54.68 d J = 7.3, C3P0: 49.68 d J = 4.5, C1Pa
:
:
30 min of stirring HNr (1.0 g; 3.13 mmol) was added. Then 49.66 s, C1Pb: 14.38 s. MS (CHCl3): 550.2 [MH]+ 100%; 332.1
the mixture was stirred for 1.5 h at room temperature. The [CH2Nr]+ 2.5%.
Q
obtained white solid was centrifuged, washed twice with water
and dried under reduced pressure.
Se PPh2CH2Nr (SePNr). The selenide derivative was pre-
pared by reacting PNr (0.281 g; 0.543 mmol) with an equimolar
Yield 89%. Anal. calcd for C29H29FN3O3P (517.2): C, 67.34; amount of metallic selenium (0.0429 g; 0.543 mmol) in chloro-
H, 5.61; N, 8.13%. Found: C, 67.31; H, 5.63; N, 8.11%. NMR form (25 cm3). The mixture was placed in an ultrasonic bath
(CDCl3, 298 K): 31P{1H}: ꢀ27.50 s; 1H: H3Pb: 15.13 s, H2P: 8.63 s, for 3 h. A yellowish solid was deposited at ꢀ18 1C. It was
H5P: 7.95 d J = 13.0, HPh(o),(m),(p): 7.46–7.33, H8P: 6.82 d J = 6.8, filtered and purified by recrystallization from the chloroform–
H1Pa: 4.31 m, H3P0: 3.36 m, H1P0: 3.29 d J = 2.8, H4P0: 2.89 m, methanol solution (1 : 2 V : V). The obtained microcrystals were
H1Pb: 1.56 m; 13C{1H}: C4P: 176.96 s, C3Pa: 167.26 s, C6P: 154.57 d dried for 24 h under vacuum.
J = 252.5; C2P: 147.13 s, C7P: 146.15 d J = 10.6, C9P: 137.21 s,
CPh(i): 138.09 d J = 11.7, CPh(o): 132.91 d J = 18.2, CPh(p): 128.82 s, H, 4.86; N, 7.05%. Found: C, 58.34; H, 4.90; N, 7.07%.
Yield 85%. Anal. calcd for C29H29FN3O3PSe (596.5): C, 58.39;
1
1
CPh(m): 128.58 d J = 6.6, C10P: 120.39 d J = 7.7, C5P: 112.61 d NMR (CDCl3, 298 K): 31P{1H}: 26.30 J(PSe) = 731.4; H: H3Pb
J = 23.2, C3P: 108.27 s, C8P: 103.95 d J = 3.2, C1P0: 61.27 d J = 3.4, 15.06 s, H2P: 8.64 s, H5P: 7.99 d J = 13.0, HPh(o),(m),(p): 8.00–7.47,
C4P0: 54.15 d J = 9.0, C3P0: 49.98 d J = 4.3, C1Pa: 49.82 s, C1Pb H8P: 6.78 d J = 6.94, H1Pa: 4.30 q J = 7.2, H3P0: 3.24 m, H1P0: 3.72 d
14.51 s. MS (CHCl3): 518.2 [MH]+ 100%; 443.2 2%; 332.1 J = 4.0, H4P0: 2.87 m, H1Pb: 1.57 t J = 7.3; 13C{1H}: C4P: 176.77 s,
:
:
[CH2Nr]+ 36%.
C3Pa: 167.02 s, C6P: 154.31 d J = 251.6, C2P: 146.99 s, C7P
:
:
OQPPh2CH2Nr (OPNr): The oxide derivative was prepared in 145.77 d J = 10.0, C9P: 136.98 s, CPh(i): 130.39 d J = 69.9, CPh(o)
the reaction of PNr (0.108 g; 0.209 mmol) with an equimolar 131.75 d J = 2.7, CPh(p): 132.14 d J = 9.1, CPh(m): 128.47 d J = 11.8,
amount of H2O2 (30% solution in water) in chloroform (25 cm3). C10P: 120.34 d J = 8.2, C5P: 112.44 d J = 23.6, C3P: 108.10 s, C8P
After 3 h of stirring at room temperature the solution was left 103.85 d J = 2.7, C1P0: 61.79 d J = 66.3, C4P0: 54.61 d J = 6.4, C3P0
:
:
at ꢀ18 1C overnight. The precipitated white solid was filtered 49.64 d J = 4.5, C1Pa: 49.73 s, C1Pb: 14.39 s. MS (CHCl3):
and purified by recrystallization from the chloroform–methanol 598.1 [MH]+ 100%.
mixture (1: 2 V : V). The obtained solid was dried for 24 h under
reduced pressure.
X-ray structures. The data were collected at 293 or 100 K
using an Oxford Cryosystem device on a Kuma KM4CCD k-axis
Yield 84%. Anal. calcd for C29H29FN3O4P (533.2): C, 65.32; H, diffractometer with graphite-monochromated Mo Ka radiation
5.44; N, 7.88%. Found: C, 65.27; H, 5.47; N, 7.87%. NMR generated from a diffraction X-ray tube operated at 50 kV and
1
(CDCl3, 298 K): 31P{1H}: 27.34 s; H: H3Pb: 15.07 s, H2P: 8.67 s, 20 mA. Data reduction and analysis were carried out with the
H5P: 8.03 d J = 13.1, HPh(o),(m),(p): 7.84–7.49, H8P: 6.81 d J = 6.9, Oxford Diffraction programs.43,44 The structures were analysed
H1Pa: 4.31 q J = 7.2, H3P0: 3.33 m, H1P0: 3.42 s*, H4P0: 3.01 m, by direct methods (program SHELXS9745) and refined by
H1Pb: 1.57 t J = 7.2; 13C{1H}: C4P: 176.98 s, C3Pa: 167.16 s, the full-matrix least-squares method on all F2 data using the
C6P: 154.45 d J = 251.6, C2P: 147.12 s, C7P: 145.79 d J = 10.0, SHELXL9745 program. Non H atoms were included in the
C9P: 137.05 s, CPh(i): 132.08 d J = 99.0, CPh(o): 132.16 d J = 2.7, refinement, with anisotropic displacement parameters and
CPh(p): 131.10 d J = 9.1, CPh(m): 128.71 d J = 10.9, C10P: 120.69 d the H atoms were included from the geometries of the mole-
J = 7.4, C5P: 112.78 d J = 22.7, C3P: 108.39 s, C8P: 103.92 d cules. The data for SePCpꢁ0.79CH3OHꢁ0.11H2O were corrected
J = 2.7, C1P0: 60.85 d J = 85.6, C4P0: 54.88 d J = 7.9, C3P0: 49.59 d for absorption,44 min/max absorption coefficients were 0.6634/
J = 4.5, C1Pa: 49.71 s, C1Pb: 14.44 s. MS (CHCl3): 534.2 [MH]+ 0.8436. In the solution of the OPCpꢁ2CH3COCH3 structure there
100%; 332.1 [CH2Nr]+ 5.5%.
is a very large maximum of electron density (2.21 e Åꢀ3) close to
SQPPh2CH2Nr (SPNr): A chloroform solution (25 cm3) con- one of the highly disordered molecules of occluded solvent
taining an equimolar amount of PNr (0.250 g; 0.483 mmol) and (d(Q1–C1S) = 1.431, d(Q1–O1S) = 1.803 Å).
1068 | New J. Chem., 2014, 38, 1062--1071
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NJC
Crystal/refinement data. OPCpꢁ2CH3COCH3RC36H41F- determined using the dilution of microarrays recommended by
N3O6P; Mr = 661.69, crystal size: 0.15 ꢃ 0.09 ꢃ 0.07 mm, crystal CLSI.46 96-well microtiter plates were filled with 100 ml of liquid
%
system: triclinic, space group: P1, unit cell: a = 8.650(2) Å, b = LB (1% yeast extract, 1% tryptone, 0.5% NaCl) medium containing
12.440(2) Å, c = 16.140(3) Å, a = 105.39(2)1, b = 90.16(2)1, g = serial dilutions of the tested compounds. All of the substances
94.95(2)1; V = 1667.6(6) Å3, Dcalcd(Z = 2) = 1.318 g cmꢀ3, y range for were diluted in DMSO to prepare 32.0 mg mlꢀ1 stocks solutions.
data collection: 2.91–25.001, Mo Ka radiation (l = 0.71073 Å), mMo
=
The bacterial suspensions (10 ml) were added into each well of the
0.139 mmꢀ1, reflections collected/unique 17 054/5882 [Rint = 0.0294], microtiter plates with the exception of the control and the plates
final R indices [I > 2s(I)] R1 = 0.0858, wR2 = 0.2339, R indices (all data) were incubated aerobically for 16–18 h at 34 1C. As a positive
R1 = 0.0986, wR2 = 0.2435, GOF = 1. 041, largest diff. peak and hole: control a suspension of bacteria in an LB broth medium was used,
2.208 (this large maximum of electron density is placed close to one of for the negative control a pure LB broth medium was used and the
the highly disordered molecules of occluded acetone solvent) and blank control was a suspension of bacteria in physiologic salt
ꢀ0.738 e Åꢀ3, data/restraints/parameters: 5882/0/444; T = 293(2) K. solution. Each control and dilution of tested compounds were
SPCpꢁ0.5CH2Cl2RC30.50H29ClFN3O3PS; Mr = 603.05, crystal tested in triplicate. The MIC values were determined visually and
size: 0.20 ꢃ 0.11 ꢃ 0.07 mm, crystal system: monoclinic, space spectrophotometrically. The OD (optical density) of each well was
group: P21, unit cell: a = 8.748(1) Å, b = 22.372(3) Å, c = 14.514(2) Å, measured at l = 590 nm using an automatic 96-well microplate
b = 94.60(2)1, V = 2831.4(6) Å3, Dcalcd(Z = 4) = 1.415 g cmꢀ3, y range reader Asys Hitachi 340, Driver version: 4.02 (Biogenet, Poland).
for data collection: 2.28–25.001, Mo Ka radiation (l = 0.71073 Å), MICs were taken as the lowest concentrations not showing
m
Mo = 0.310 mmꢀ1, reflections collected/unique 19 067/4988 [Rint
=
any visible growth or their values of absorbance in the
0.0470], final R indices [I > 2s(I)] R1 = 0.0539, wR2 = 0.1465, bacterial cultures after incubation were less than or equal to
R indices (all data) R1 = 0.0701, wR2 = 0.1528, GOF = 1.322, largest the blank control.
diff. peak and hole: 0.720 and ꢀ1.235 e Åꢀ3, data/restraints/
The minimum bactericidal concentration (MBC), i.e., the
lowest concentration of a substance that kills 99.9% of the
parameters: 4988/0/365; T = 293(2) K.
SePCpꢁ0.79CH3OHꢁ0.11H2ORC30.79H32.36FN3O3.89PSe; Mr = bacteria was also determined from the dilution of microarrays.
635.58, crystal size: 0.33 ꢃ 0.15 ꢃ 0.13 mm, crystal system: For growth inhibitory concentration (MIC) the presence of
monoclinic, space group: P21, unit cell: a = 8.102(3) Å, b = viable microorganisms was tested and the lowest concentration
25.993(6) Å, c = 14.216(4) Å, b = 92.42(3)1, V = 2991.2(16) Å3, causing a bactericidal effect was reported to be the MBC. The
Dcalcd(Z = 4) = 1.411 g cmꢀ3, y range for data collection: MBC was determined by removing a 10 ml volume of the medium
2.75–28.781, Mo Ka radiation (l = 0.71073 Å), mMo = 1.355 mmꢀ1
,
from each microtiter plate well used for MIC determinations and
Tmin = 0.6634, Tmax = 0.8436, reflections collected/unique 18 670/ spotting onto nutrient agar. Agar plates were incubated aerobically
11 096 [Rint = 0.0268], final R indices [I > 2s(I)] R1 = 0.0396, wR2 = for 18–24 h at 34 1C. The concentration causing a bactericidal effect
0.0810, R indices (all data) R1 = 0.0470, wR2 = 0.0844, GOF = was selected based on the absence of colonies on the agar plate. For
1.026, largest diff. peak and hole: 0.810 and ꢀ0.441 e Åꢀ3, data/ each test 3 replicates were undertaken.
restraints/parameters: 11 096/0/757; flack parameter: ꢀ0.001(5);
T = 100(2) K.
The mean values of MICs and MBCs were interpreted
as antimicrobial activity according to the recommendation of
the CLSI.36
The antimicrobial activity studies
Statistical analysis
Strains and bacterial suspensions. The antimicrobial activity
of HCp and HNr and their derivatives was tested at concentra- The antimicrobial activity of the studied derivatives was com-
tions from 0.002 mg mlꢀ1 to 16.0 mg mlꢀ1 against the reference pared to their native forms (HCp, HNr) by nonparametric
bacterial strains from the American Collections ATCC: Escherichia analysis of variance (Kruskal–Wallis ANOVA by ranks or Median
coli ATCC 25922, Staphylococus aureus ATCC 6538, Klebsiella test). The ANOVA (using STATISTICA for Windows; StatSoft,
pneumoniae ATCC 4352 and Pseudomonas aeruginosa ATCC Tulsa, OK, USA) was used to test for significant difference of the
27853. The bacterial strains, provided by the Institute of Genetics mean of the MICs and MBCs between bacterial strains. The
and Microbiology University of Wroclaw, were cultured aerobically P values less than or equal to 0.05 indicate that the values
at 34 1C in nutrient agar (0.5% enzyme-hydrolyzed proteins, being compared are statistically significantly different at a 95%
0.3% meat extract, 0.5% NaCl, 1.5% agar–agar) overnight confidence level.
and then three to four colonies of each of the strains were
suspended in 0.85% of NaCl solution to produce stock prepara-
Cytotoxicity studies
tions containing a log-phase cell density of approximately
Cell cultures. CT26 cell line (mouse colon carcinoma, morphol-
108 CFU per ml (colony forming units per milliliters). Each of ogy: fibroblast, ATCC: CRL-2638) and A549 cell line (human lung
the bacterial suspensions was diluted in physiologic salt adenocarcinoma, morphology: epithelial, ATCC: CCL-185) were
solution to 1 : 1000 (105 CFU per ml).46
obtained from Professor Luis G. Arnaut’s group (Chemistry
Determination of MIC and MBC. The minimum inhibitory Department, University of Coimbra, Portugal). Cells were
concentration (MIC), defined as the lowest concentration of cultured in Dulbecco’s Modified Eagle Medium (DMEM) with-
material that inhibits the growth of an organism, was determined out phenol red, supplemented with 10% fetal bovine serum
based on the broth dilution susceptibility tests. MIC values were (FBS) and with 1% streptomycin/penicillin. Cultures were
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incubated at 37 1C in a humidified atmosphere containing 5% CO2 European Regional Development Fund in the framework of
(standard conditions). Cells were passaged at preconfluent density, the Polish Innovation Economy Operational Program (contract
using a solution containing 0.05% trypsin and 0.5 mM EDTA. All no. POIG.02.01.00-12-023/08).
cell culture fluids were purchased from IMMUNIQ (Poland).
Cytotoxicity assay. Cells were seeded in the 96-well plate at a
density of 10 000 cells per ml and incubated for 24 h under
standard conditions. After that time, the culture medium was
Notes and references
changed for the tested compounds at a final concentration of
0.1–0.001 mM in PBS (IMMUNIQ, Poland). Cells with com-
pounds were incubated for 4 h, then washed with PBS and
finally a new medium was added. After 24 h of incubation the
cytotoxicity was assessed.
1 World Health Organization. The evolving threat of antimicrobial
resistance – Options for actions 2012 (WHO/IER/PSP/2012.2).
2 G. Alvan, C. Edlund and A. Heddini, Drug Resist. Updates,
2011, 14, 70–76.
´
3 A. Bykowska, R. Starosta, A. Brzuszkiewicz, B. Ba˙zanow,
´
´
The cytotoxicity of the tested compounds was determined by
the MTT assay. Briefly, 200 ml of MTT (5 mg mlꢀ1, Sigma-
Aldrich) was added to each of the wells and incubated for 4 h at
room temperature. Then the blue formazan reaction product
formed was dissolved in 100 ml of DMSO–CH3OH mixture
(1/1; v/v). The absorbance of the formazan solution was mea-
sured at 565 nm using an Infinite 200M PRO NanoQuant plate
reader (Tecan, Switzerland).
The results were determined as a cell survival fraction, S/S0
(%) and were calculated using the formula: S/S0 (%) = [abs565nm
of treated cells/abs565nm of untreated cells (control)] ꢃ 100.
Untreated cells, cultured only in medium, were treated as a
control. IC50 values (concentration of the tested agent causing
50% inhibition of cell growth) were calculated from the con-
M. Florek, N. Jackulak, J. Krol, J. Grzesiak, K. Kalinski and
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