Extending the family of quinolone antibacterials to new copper derivatives: self-assembly, structural and topological features, catalytic and biological activity

Article abstract of DOI:10.1039/c8nj05338c

A new series of copper(ii) compounds, [Cu(pef)₂(MeOH)] (1), [Cu(pef)(bipyam)Cl] (2), [Cu(pef)(phen)Cl] (3) and [Cu(pef)(bipy)Cl] (4), bearing the quinolone family member pefloxacin (Hpef) were self-assembled in the presence (optional) of N,N′-donor heterocyclic ligands such as 2,2′-bipyridylamine (bipyam), 1,10-phenanthroline (phen), or 2,2′-bipyridine (bipy). The products were fully characterized, including single-crystal X-ray diffraction analysis of 2-4. The structures are extended into 1D (2), 2D (3), or 3D (4) networks via multiple H-bonds between the monocopper(ii) units and guest water and/or methanol molecules; the latter are arranged into different types of water and hybrid water-methanol clusters. The resulting H-bonded networks were classified from a topological viewpoint, revealing diverse topologies that also include an undocumented type. Compounds 2-4 also act as homogeneous catalysts in a model oxidation reaction, namely the mild oxidation of C₆-C₈ cycloalkanes by H₂O₂ at 50 °C to give cyclic alcohols and ketones. The effects of various reaction parameters (substrate scope, temperature, and loadings of catalyst, cycloalkane, and oxidant) and selectivity features were investigated. Besides, products 1-4 also show remarkable antibacterial activity against four different microorganisms (Escherichia coli, Xanthomonas campestris, Staphylococcus aureus and Bacillus subtilis), which is superior to that of free Hpef. The interaction of the Cu(ii) compounds with calf-thymus DNA was studied suggesting intercalation as the most possible binding mode. Furthermore, the interaction of the obtained copper(ii) derivatives with human/bovine serum albumin was investigated by fluorescence emission spectroscopy and the corresponding albumin-binding constants were established. This study widens a limited family of transition metal pefloxacin derivatives.

Full text of DOI:10.1039/c8nj05338c

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Extending the family of quinolone antibacterials
to new copper derivatives: self-assembly,
structural and topological features, catalytic
and biological activity†
Cite this: New J. Chem., 2018,
42, 19644
a
b
b
c
Panagiotis Xerras, Anna-Maria Bacharidou, Stavros Kalogiannis, Franc Perdih,
d
de
c
Marina V. Kirillova, Alexander M. Kirillov,
*
Iztok Turel * and
a
George Psomas
*
2
A new series of copper(II) compounds, [Cu(pef) (MeOH)] (1), [Cu(pef)(bipyam)Cl] (2), [Cu(pef)(phen)Cl] (3)
and [Cu(pef)(bipy)Cl] (4), bearing the quinolone family member pefloxacin (Hpef) were self-assembled
0
0
in the presence (optional) of N,N -donor heterocyclic ligands such as 2,2 -bipyridylamine (bipyam),
0
1
,10-phenanthroline (phen), or 2,2 -bipyridine (bipy). The products were fully characterized, including
single-crystal X-ray diffraction analysis of 2–4. The structures are extended into 1D (2), 2D (3), or 3D (4)
networks via multiple H-bonds between the monocopper(II) units and guest water and/or methanol
molecules; the latter are arranged into different types of water and hybrid water–methanol clusters. The
resulting H-bonded networks were classified from a topological viewpoint, revealing diverse topologies
that also include an undocumented type. Compounds 2–4 also act as homogeneous catalysts in a
6 8 2 2
model oxidation reaction, namely the mild oxidation of C –C cycloalkanes by H O at 50 1C to give
cyclic alcohols and ketones. The effects of various reaction parameters (substrate scope, temperature,
and loadings of catalyst, cycloalkane, and oxidant) and selectivity features were investigated. Besides,
products 1–4 also show remarkable antibacterial activity against four different microorganisms (Escherichia
coli, Xanthomonas campestris, Staphylococcus aureus and Bacillus subtilis), which is superior to that of free
Hpef. The interaction of the Cu(II) compounds with calf-thymus DNA was studied suggesting inter-
calation as the most possible binding mode. Furthermore, the interaction of the obtained copper(II) derivatives
with human/bovine serum albumin was investigated by fluorescence emission spectroscopy and the corres-
ponding albumin-binding constants were established. This study widens a limited family of transition metal
pefloxacin derivatives.
Received 21st October 2018,
Accepted 2nd November 2018
DOI: 10.1039/c8nj05338c
rsc.li/njc
Introduction
a
Department of General and Inorganic Chemistry, Faculty of Chemistry,
Aristotle University of Thessaloniki, GR-54124 Thessaloniki, Greece.
E-mail: gepsomas@chem.auth.gr
Pefloxacin (Hpef, Fig. 1) is a synthetic antimicrobial agent
belonging to the group of quinolones which are well established
b
Department of Nutrition and Dietetics, Faculty of Agriculture, Food Technology
and Nutrition, Alexander Technological Educational Institution, Sindos,
Thessaloniki, Greece
1,2
and frequently used antibacterial agents studied in many fields.
In particular, two examples of such fields concern environmental
c
3
4
Faculty of Chemistry and Chemical Technology, University of Ljubljana,
Vecna pot 113, 1000 Ljubljana, Slovenia. E-mail: Iztok.Turel@fkkt.uni-lj.si
Centro de Qu ´ı mica Estrutural, Instituto Superior T ´e cnico, Universidade de Lisboa,
Av. Rovisco Pais, 1049-001, Lisbon, Portugal. E-mail: kirillov@tecnico.ulisboa.pt
Peoples’ Friendship University of Russia (RUDN University),
impact and toxicity that may cause side-effects which may both
be connected with metal ion interactions. Magnesium ions are
d
e
5
crucial for the antibacterial activity of quinolones, but, on the
other hand, complexes of other metals were reported to exert other
types of biological activity, for example cytotoxicity. Moreover,
6
6
Miklukho-Maklay st., Moscow, 117198, Russian Federation
†
Electronic supplementary information (ESI) available: Experimental protocols it was also reported that iron chelation by fluoroquinolones leads
for biological studies; tables with crystallographic data and selected bonding
parameters; the albumin constants; comparison of the biological activity; addi-
tional figures containing fluorescence emission UV-vis spectra and other data;
and crystallographic CIF files for 2–4. CCDC 1844910–1844912. For ESI and
crystallographic data in CIF or other electronic format see DOI: 10.1039/
c8nj05338c
to epigenetic effects through inhibition of alpha-ketoglutarate-
dependent dioxygenases that require iron as a co-factor. In the
7
mechanism of cellular death induced by quinolones, leaching of
iron from iron–sulfur clusters and the stimulation of the Fenton
8
reaction also take place. Although it is difficult to study the
19644 | New J. Chem., 2018, 42, 19644--19658 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018

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However, catalytic properties of metal–quinolone derivatives
remain largely unstudied. Hence, one of the aims of the
present work consisted of exploring the oxidation catalytic
potential of new copper pefloxacin derivatives. The conditions
used in our experiments are definitely far from physiological
conditions and it would be an oversimplification to suggest
that such reactions occur in the human body. However, we
believe it is worth obtaining more information on the catalytic
properties of metal–drug complexes and also about their
potential roles in living systems. It could not be excluded that
certain reactions in which metal complexes might be involved
(
which were not extensively studied) may also be related to
various effects, e.g. toxicity.
Fig. 1 The syntax formula of (A) Hpef, (B) bipyam, (C) bipy and (D) phen.
Herein, we present the synthesis and detailed characteriza-
tion of a new series of copper(II) compounds derived from
behavior of metal complexes under biological conditions, such the second-generation quinolone pefloxacin in the presence
0
information would be very important for understanding the (optional) of 2,2 -bipyridinylamine (bipyam), 1,10-phenanthroline
0
bioactivity of pefloxacin derivatives. As a second-generation quino- (phen), and 2,2 -bipyridine (bipy) as N-donor co-ligands
lone, Hpef is active against a broad spectrum of Gram-(ꢀ) and (Fig. 1(B)–(D)). Four compounds were isolated and formulated
9
Gram-(+) bacteria. With regard to coordination compounds, as [Cu(pef) (MeOH)] for 1 and [Cu(pef)(L)Cl] (L = bipyam, phen
2
10
11
12
limited examples of Ag, Mn, and Zn complexes with pefloxacin or bipy) for 2–4, respectively. The characterization of 1–4 was
were structurally characterized among those reported.
13–16
With achieved by standard physicochemical and spectroscopic tech-
particular regard to copper, some Cu pefloxacin derivatives were niques, as well as by single-crystal X-ray diffraction. Hence,
1
4,15
described;
crystal structures of the obtained compounds.
Copper is a d-block element that is essential for life but can 1–4 were screened as homogeneous catalysts for the mild
however, these studies do not report the X-ray single- compounds 2–4 represent unique examples of structurally
characterized Cu pefloxacin derivatives. In addition, products
1
7
also be toxic, depending upon the concentration. It has oxidation of C –C cycloalkanes as model substrates to give
6
8
unique properties and is specifically needed in a number of the corresponding cyclic alcohols and ketones. The in vitro
1
8,19
enzymes, especially those important for redox processes.
biological activity of 1–4 was also investigated in detail,
Copper is the strongest Lewis acid in Group 11 of the periodic including: (a) the evaluation of their antimicrobial activity
table and has the ability to mediate electron transport by against four Gram-positive or Gram-negative microorganisms
accepting and donating electrons. It took a long time to under- (i.e., Escherichia coli NCTC 29212 (E. coli), Xanthomonas cam-
stand the nature and role of copper–oxygen intermediates pestris ATCC 1395 (X. campestris), Staphylococcus aureus ATCC
2
0
within copper-containing enzymes and abiological catalysts.
6538 (S. aureus) and Bacillus subtilis ATCC 6633 (B. subtilis));
ꢁ
The formation of highly reactive hydroxyl radicals (OH ) in a (b) the investigation of their binding to bovine (BSA) and
Fenton-type reaction can also be promoted by copper. Sigman human (HSA) serum albumins; (c) the determination of their
2
1
+
and coworkers reported the first copper complex [Cu(phen)
2
]
binding mode and the binding strength to calf-thymus (CT)
(
phen = 1,10-phenantroline) that shows efficient DNA cleavage DNA; and (d) the investigation of their binding competition
2
2,23
activity.
Other copper(II) derivatives that can oxidatively with the well-known DNA-intercalator ethidium bromide (EB).
cleave DNA under physiological conditions were later prepared.
Frequently such complexes require a reducing agent and hydrogen
peroxide (or dioxygen) for their activity.
The copper-quinolone derivatives are the most popular
metal complexes of quinolones and have been thoroughly
Experimental
Materials, instrumentation and physical measurements
2
4,25
studied.
Examples reported so far include copper derivatives The reagents CuCl ꢂ2H O, pefloxacin, bipy, phen, bipyam,
2
2
2
6
with the first-generation quinolones, such as flumequine,
nalidixic acid and oxolinic acid, the second-generation, such purchased from Sigma-Aldrich Co, and all solvents were pur-
as cinoxacin, ciprofloxacin,
and ofloxacin,
and sparfloxacin. Most of these complexes have been structurally purification. DNA stock solution was prepared by dilution of CT
characterized showing in most cases a four- or a five-coordinate DNA in buffer (containing 15 mM trisodium citrate and 150 mM
KOH, NaCl, CT DNA, EB, BSA, HSA, and trisodium citrate were
2
7
28
2
9
30–32
33
34,35
enrofloxacin, norfloxacin
chased from ChemLab. All the chemicals and solvents were
reagent grade and were used as purchased without any further
2
9,36
37
and the third-generation such as gatifloxacin
38
environment around copper.
The development of abiological catalysts that can function kept at 4 1C for no longer than a week. The stock solution of CT
in biological systems and so-called bioorthogonal chemistry are DNA gave a ratio of UV absorbance at 260 and 280 nm (A260/A280
the hot research topics in recent years which clearly show the of 1.90, indicating that the DNA was sufficiently free of protein
NaCl at pH 7.0) followed by exhaustive stirring for three days, and
)
3
9–44
45
prospective catalytic applications of metal complexes.
contamination.
The DNA concentration was determined
This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2018 New J. Chem., 2018, 42, 19644--19658 | 19645

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by the UV absorbance at 260 nm after 1 : 20 dilution using r(C–H)bipyam: 769 (m). UV-vis: as nujol mull, l/nm: 735, 331,
ꢀ
1
ꢀ1 46
ꢀ1
ꢀ1
e = 6600 M cm
.
313; in DMSO, l/nm (e/M cm ): 730(125), 335(sh) (7500),
ꢀ
1
Infrared (IR) spectra (400–4000 cm ) were recorded on a 316(17 000). meff = 1.78 BM at room temperature. The complex is
Nicolet FT-IR 6700 spectrometer with samples prepared as KBr soluble in MeOH, EtOH, DMF and DMSO (LM = 7 S cm mol
2
ꢀ1
,
disks. UV-visible (UV-vis) spectra were recorded as nujol mulls and 1 mM DMSO solution) and partially soluble in H O.
2
ꢀ5
ꢀ3
in solution at concentrations in the range 1 ꢃ 10 –5 ꢃ 10 M on
[Cu(pef)(phen)Cl]ꢂMeOHꢂ2H
2
O, 3ꢂMeOHꢂ2H
2
O. Phen (0.25 mmol,
0
a Hitachi U-2001 dual beam spectrophotometer. Room tempera- 45 mg) was used as the N,N -donor. Dark green well-formed crystals
ture magnetic measurements were carried out by the Faraday of [Cu(pef)(phen)Cl], 3 (130 mg, yield: 85%), suitable for X-ray
method. C, H and N elemental analyses were performed on a structure determination, were collected after ten days. Anal.
Perkin-Elmer 240B elemental analyzer. Molar conductivity calcd. for [Cu(pef)(phen)Cl]ꢂMeOHꢂ2H
2 5 6
O, C30H35ClCuFN O
measurements were carried out with a Crison Basic 30 conducto- (M = 679.62): C 53.02, H 5.19, N 10.31%; found: C 52.84, H
W
ꢀ
1
meter. Fluorescence spectra were recorded in solution on a Hitachi 4.97, N 10.57%. IR (KBr disk): vmax/cm : v(CQO)pyridone, 1631
F-7000 fluorescence spectrophotometer. Viscosity experiments (vs); v (CO ), 1613 (vs); n (CO ), 1376 (s); Dn(CO ) =
asym
ꢀ
2
sym
2
2
1
were carried out using an ALPHA L Fungilab rotational viscometer 237 cm ; r(C–H)phen: 725 (m). UV-vis: as nujol mull, l/nm:
ꢀ
1
ꢀ1
equipped with an 18 mL LCP spindle and the measurements were 670, 331, 313; in DMSO, l/nm (e/M cm ): 685 (60), 327 (sh)
performed at 100 rpm. For the analysis of the reaction mixtures in (10 900), 316(12 500). meff = 1.79 BM at room temperature.
catalytic tests, gas chromatography (GC) analyses were run on The complex is soluble in MeOH, EtOH, DMF and DMSO
2
ꢀ1
M
an Agilent Technologies 7820A series gas chromatograph (He as (L = 10 S cm mol , 1 mM DMSO solution).
the carrier gas) equipped with an FID detector and BP20/SGE
[Cu(pef)(bipy)Cl]ꢂ0.5MeOHꢂ4H O, 4ꢂ0.5MeOHꢂ4H O. Bipy
2
2
0
(
30 m ꢃ 0.22 mm ꢃ 0.25 mm) capillary column.
Synthesis of the compounds
Synthesis of [Cu(pef)
(0.25 mmol, 39 mg) was used as the N,N -donor. Dark
green well-formed crystals of [Cu(pef)(bipy)Cl]ꢂ0.5MeOHꢂ4H O,
2
4
2
ꢂ0.5MeOHꢂ4H O (110 mg, yield: 75%), suitable for X-ray structure
2
(MeOH)], 1. Pefloxacin (0.5 mmol, 166 mg) determination, were collected after two weeks. Anal. calcd for
5 W
37ClCuFN O7.5 (M =
was dissolved in methanol (10 mL) and deprotonated by KOH [Cu(pef)(bipy)Cl]ꢂ0.5MeOHꢂ4H
2
O, C27.5
H
(0.5 mmol, 0.5 mL 1 M). After 30 min stirring, the initial solution 675.21): C 48.89, H 5.52, N 10.37%; found: C 48.75, H 5.38,
ꢀ
1
was added to a methanolic solution (5 mL) of CuCl
2
ꢂ2H
2
O
N 10.69%. IR (KBr disk): vmax/cm : v(CQO)pyridone, 1632 (vs);
ꢀ1
(0.25 mmol, 43 mg). The reaction mixture was stirred for vasym(CO ), 1609 (vs); n (CO ), 1388 (s); Dn(CO ) = 221 cm
;
2
sym
2
2
3
0 min and left for slow evaporation. A green-blue precipitate r(C–H)bipy, 775 (medium (m)). UV-vis: as nujol mull, l/nm: 675,
ꢀ1
ꢀ1
of [Cu(pef) (MeOH)], 1 (150 mg, 75%), was collected after 332, 311; in DMSO, l/nm (e/M cm ): 685 (30), 330 (shoulder (sh))
2
two weeks. Anal. calcd for [Cu(pef)
2
(MeOH)], C35
H
42CuF
2
N
6
O
7
(8900), 313(13 200). meff = 1.98 BM at room temperature. The complex
2
ꢀ1
(M
W
= 760.30): C 55.29, H 5.57, N 11.05%; found: C 55.12, is soluble in MeOH, EtOH, DMF and DMSO (L
M
= 6 S cm mol , in
1 mM DMSO solution) and partially soluble in H O.
ꢀ
1
H 5.45, N 10.75%. IR (KBr disk): vmax/cm : v(CQO)pyridone
633 (very strong (vs)); vasym(CO ), 1598 (vs); nsym(CO ), 1372
strong (s)); Dn(CO ) = nasym(CO ) = 226 cm . UV-vis: as
) ꢀ nsym(CO
nujol mull, l/nm: 740, 335, 310; in DMSO, l/nm (e/M cm ): Single-crystal X-ray diffraction data were collected on an Agilent
35(75), 332(11 500), 315(14 500). meff = 1.84 BM at room temperature. Technologies SuperNova Dual diffractometer using Mo-Ka
,
2
1
2
2
ꢀ
1
X-ray crystal structure determination
(
2
2
2
ꢀ
1
ꢀ1
7
2
ꢀ1
The complex is soluble in DMF and DMSO (LM = 8 S cm mol
mM DMSO solution).
Synthesis of [Cu(pef)(L)Cl], 2–4 (L = bipyam for 2, phen for 3, processed using CrysAlis Pro. The structures were solved by
and bipy for 4). Complexes 2–4 were prepared in a similar way direct methods and refined by a full-matrix least-squares procedure
,
radiation (l = 0.71073 Å) at room temperature (2ꢂ2H O and
2
1
4ꢂ0.5MeOHꢂ4H
2
O) and at 150 K (3ꢂMeOHꢂ2H
2
O). The data were
47
0
2
48
with the use of the corresponding N,N -donor ligand. More based on F with SHELX-97. All the non-hydrogen atoms were
specifically, a methanolic solution (20 mL) of Hpef (0.25 mmol, refined anisotropically. Hydrogen atoms were readily located
83 mg) and KOH (0.25 mmol, 14 mg) was stirred for 30 min and in difference Fourier maps and were subsequently treated as
then added, simultaneously with a methanolic solution (5 mL) riding atoms in geometrically idealized positions with Uiso(H) =
0
of the corresponding N,N -donor (0.25 mmol), to a methanolic kU (C or O), where k = 1.5 for hydroxyl and methyl groups, which
eq
solution (5 mL) of CuCl
obtained reaction mixture was stirred for 20 min and left for unless otherwise noted. In the crystal structure 2ꢂ2H
slow evaporation. gen atoms bonded to water solvate molecules O4 and O5 and to
Cu(pef)(bipyam)Cl]ꢂ2H O. Bipyam (0.25 mmol, bipyam amino atom N3 were refined restraining the bonding
2
ꢂ2H
2
O (0.25 mmol, 43 mg). The were permitted to rotate but not to tilt, and 1.2 for all other H atoms
2
O, hydro-
[
2
O, 2ꢂ2H
2
0
4
3 mg) was used as the N,N -donor. Dark green crystals of distances with Uiso(H) = 1.5Ueq(O) and Uiso(H) = 1.2Ueq(N). In
[
Cu(pef)(bipyam)Cl]ꢂ2H O, 2ꢂ2H O (105 mg, yield: 70%), suita- the crystal structure 3ꢂMeOHꢂ2H O, hydrogen atoms bonded to
2
2
2
ble for X-ray structure determination, were collected after three water solvate molecules O4 and O5 were refined freely with
weeks. Anal. calcd. for [Cu(pef)(bipyam)Cl]ꢂ2H O, C H ClCuFN O Uiso(H) = 1.5U (O). In the crystal structure 4ꢂ0.5MeOHꢂ4H O,
2
27 32
6
5
eq
2
W
(M = 638.58): C 50.78, H 5.05, N 13.16%; found: C 50.92, H 4.89, hydrogen atoms bonded to water solvate molecules O7–O14 were
ꢀ
1
N 12.82%. IR (KBr disk): vmax/cm : v(CQO)pyridone, 1639 (vs); refined restraining the bonding distances with Uiso(H) = 1.5Ueq(O).
v
ꢀ1
asym(CO
2
), 1586 (vs); nsym(CO
2
), 1380 (s); Dn(CO
2
) = 206 cm
;
Crystallographic data are listed in Table S1 (ESI†).
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Topological analysis
by fluorescence emission spectroscopy. Detailed procedures
regarding the study of the biological activity of the compounds
are given in the ESI† (Sections S1–S5).
Topological analysis of H-bonded networks was carried out
using Topos software and following the concept of the simplified
49–52
underlying net.
Such underlying nets were generated by con-
tracting Cu molecular units and water or water–methanol aggre-
gates to the corresponding centroids, maintaining their connectivity
via hydrogen bonds. Only strong D–Hꢂ ꢂ ꢂA hydrogen bonds
Results and discussion
Synthesis and physicochemical characterization
were considered, wherein Hꢂ ꢂ ꢂA o 2.50 Å, Dꢂ ꢂ ꢂA o 3.50 Å, Compounds 1–4 were synthesized in good yield via the self-
and +(D–Hꢂ ꢂ ꢂA) 4 1201; D and A stand for donor and acceptor assembly reaction of pefloxacin, deprotonated by KOH, with
4
9,50
atoms.
The obtained nets were then classified from the CuCl ꢂ2H O in the absence (reaction (1) for 1) or presence of the
2
2
0
topological viewpoint.
corresponding N,N -donor co-ligand (L = bipy, phen, and
bipyam for 2–4, respectively; reaction (2)):
Mild catalytic oxidation of alkanes
CuCl
2
ꢂ2H
2
O + 2Hpef + 2KOH + MeOH - [Cu(pef)
2
(MeOH)]
Alkane oxidation reactions were typically performed in air
atmosphere in thermostated glass reactors equipped with a
condenser under vigorous stirring at 50 1C and using MeCN as
the solvent (up to 2.5 mL total volume). In a typical experiment,
copper(II) catalyst (5.0 mmol) and gas chromatography (GC)
+
2KCl + 4H O
(1)
2
CuCl ꢂ2H O + Hpef + KOH + L - [Cu(pef)(L)Cl] + KCl + 3H O
2
2
2
(2)
2
internal standard (MeNO , 25 mL) were introduced into MeCN
solution, followed by addition of an alkane substrate (1 mmol).
The reaction started upon introduction of hydrogen peroxide
Products 1–4 were characterized by elemental analysis, IR
and UV-vis spectroscopies and magnetic measurements at
room temperature. The structures of 2–4 were determined by
X-ray crystallography. The complexes are mainly soluble in
DMSO and DMF and partially in organic solvents, while they
(
50% in H
were monitored by withdrawing small aliquots after different
periods of time, which were treated with PPh for the reduction
of remaining H O and alkyl hydroperoxides that are typically
2
O, 5 mmol) in one portion. The oxidation reactions
3
remain insoluble in H O. For the biological experiments, the
2
2
2
compounds were initially dissolved in DMSO (1 mM); there-
after a solution of the complexes in a mixture of DMSO : water
5
3,54
formed as primary products in alkane oxidations.
The
samples were then analyzed by GC using nitromethane as an
internal standard. The formation of alkyl hydroperoxides as
primary intermediate products was also confirmed by GC
analyses of the reaction mixtures before and after the treatment
(
5% in DMSO) was used. The values of the molar conductivity
of 1 mM DMSO solutions of the complexes (L ) are in the
range of 6–10 S cm mol . Thus, we may consider that 1–4 do
not dissociate in DMSO solution, since for a 1 : 1 electrolyte,
the LM value should be B70 S cm mol
M
2
ꢀ1
5
3,54
2
ꢀ1
with PPh
3
(Shul’pin’s method).
Attribution of peaks was
.
made by comparison with chromatograms of authentic samples.
Blank tests confirmed that alkane oxidations do not proceed in the
absence of copper catalyst. Compound 1 was also tested and found
to be inactive in the present type of catalytic reactions, resulting
in negligible yields of products (o1%). The inactivity of 1 can
be explained by its quick degradation in the course of catalytic
experiments.
Compounds 1–4 are paramagnetic and show meff values
= 1.78–1.98 BM) that are higher than the theoretically
(
expected spin-only value (= 1.73 BM) at room temperature
9
for a mononuclear Cu(II) complex with d configuration
5
5
(
S = 1/2). Similar meff values were found for reported Cu(II)–
2
6,37
quinolone derivatives.
Stability of the complexes in solution
Biological activity studies
The UV-vis spectra of complexes 1–4 were recorded in DMSO
In order to study in vitro the biological activity of Hpef and solutions and as nujol mulls. In the solution spectra, there is a low-
ꢀ1
ꢀ1
its complexes 1–4 (i.e., antimicrobial activity and interaction intensity band in the 685–735 nm region (e = 30–125 M cm
)
with DNA or albumins), they were initially dissolved in DMSO which can be attributed to the d–d transitions; this band is
5
5
(
1 mM). Mixing of such solutions with the aqueous buffer DNA typical for distorted square pyramidal geometry. Further
solutions used never exceeded 5% DMSO (v/v) in the final bands appearing in the UV region of the spectra are attributed
solution, which was needed due to low aqueous solubility of to intraligand transitions, confirming the presence and the
most compounds.
coordination of ligands. The UV-vis spectra of 1–4 in nujol are
The antimicrobial activity of the compounds was evaluated similar to those in DMSO solution, suggesting the preservation
by determining the MIC values toward two Gram-(ꢀ) (E. coli of the structure upon dissolution. The UV-vis spectra of 1–4
and X. campestris) and two Gram-(+) (S. aureus and B. subtilis) were also recorded in the presence of a series of buffer solu-
bacterial species. The albumin binding studies were performed tions with pH in the 6–8 range (150 mM NaCl and 15 mM
by tryptophan fluorescence quenching experiments. The inter- trisodium citrate at pH values regulated by HCl(aq)). These
action of the compounds with CT DNA was investigated by spectra show no significant change (i.e., shift of lmax of the
UV-vis spectroscopy and viscosity measurements and via the existing bands or appearance of new bands), thus also confirm-
evaluation of the EB-displacing ability of the complexes studied ing the integrity of the complexes in the buffer solutions used
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5
for the biological experiments. The observed behavior of 1–4 is values of tetragonality T and trigonality index t (Table S2, ESI†).
5
in good agreement with recent reports on related metal– The determination of T is based on the changes in bond
2
6,37
56
quinolone derivatives.
lengths, while t is calculated by the equation t = (j ꢀ j )/601,
1
2
where j and j are the largest angles in the coordination sphere.
1
2
The t values can vary from 0 to 1, with t = 0 corresponding to a
5
7
perfect square pyramid and t = 1 to a perfect trigonal bipyramid.
Structural features
X-ray crystal structures of 2–4
5
The T and t values are 0.772 and 0.068 for 2, 0.786 and 0.036 for 3,
and 0.776 and 0.041 for 4, respectively, suggesting a slight
distortion from the regular square-based pyramidal geometry
as observed in related compounds. For such an arrangement
around the copper ion, the quinolone oxygen atoms O(1) and
The crystal structures of complexes 2–4 are depicted in Fig. 2
and selected bond distances and angles are cited in Table S2
2
5
(ESI†). The structures are discussed together along with their
similarities and differences. There are water solvate molecules
in all three complexes and methanol solvate molecules in
complexes 3 and 4.
0
O(3) and the N,N -donor nitrogen atoms N(1) and N(2) occupy
the vertices of the basal plane, with the copper atom being
displaced from the basal plane toward the Cl(1) atom which lies
at the apical position of the pyramid.
Compounds 2–4 are mononuclear and bear a deprotonated
pefloxacin ligand that is bound to the Cu(1) atom in a bidentate
chelating mode through the carboxylate O(1) and pyridone O(3)
oxygen atoms, leading to the formation of a six-membered
Proposed structure of 1
Our continuous attempts to grow crystals of complex 1 suitable
for X-ray crystallography were not successful. Therefore, we may
propose a structure for complex 1 on the basis of the experi-
mental data (IR and UV-vis spectroscopy, elemental analysis,
molar conductivity and magnetic measurements) and after
a comparison with the reported structurally characterized
Cu(II)–quinolone complexes. According to the magnetic data,
the complex is mononuclear and the d–d band observed in the
UV-vis spectra suggests a distorted square pyramidal geometry
around the Cu(II) ion. On the basis of IR spectra, the deproto-
nated pefloxacin ligands are in a chelate bidentate binding
manner through the pyridone and a carboxylato oxygen. There-
fore, complex 1 is expected to have a structure similar to those
0
chelate ring. The N,N -donor ligands bipyam, phen and bipy
are coordinated bidentately to Cu(1) through the two aromatic
nitrogen atoms N(1) and N(2) forming a six-membered (in 2)
and a five-membered (in 3 and 4) chelate ring. In all three
structures 2–4, the Cu(1)–O distances [= 1.912(2)–1.9548(16) Å]
are shorter than the Cu(1)–N distances [= 2.005(2)–2.023(2) Å],
2
6,37
being comparable to those of related compounds.
The five-coordinate Cu(1) atom presents a slightly distorted
square pyramidal {CuN O Cl} geometry as indicated by the
2
2
3
6
58
of [Cu(oflo)
2 2 2 2
(H O)] (Hoflo = ofloxacin), [Zn(oflo) (H O)], and
1
2
[
Zn(pef) (H O)].
2
2
Water and hybrid water–methanol clusters in structures of 2–4
An interesting feature of the obtained crystal structures con-
sists of the H-bonding interactions of crystallization water and
5
9
methanol molecules. These are arranged into different types
of solvent aggregates (Fig. S1, ESI†) which include a water
dimer (H
2 2 2
O) in 2 and an infinite zigzag (H O)5n chain in 4;
this chain can be classified within the C4 type according to a
5
9a
classification of water clusters.
are also hybrid water–methanol associates (H
H O) (MeOH), respectively; these can be classified within the
In complexes 3 and 4, there
2
O) (MeOH) and
2
(
2
3
5
9a
D3 type.
monocopper(II) molecular units, thus resulting in generation of
D (2), 2D (3) or 3D (4) networks.
All these clusters are multiply H-bonded to the
1
Topological description of H-bonded networks
To get further insight into the complex H-bonded nets, we
simplified them by reducing the copper(II) complex units and
solvent clusters into the corresponding centroids. Then, the
obtained underlying nets were analyzed and classified from a
topological perspective. An underlying 1D H-bonded chain in
complex 2 (Fig. 3(A)) can be described as a uninodal 2-connected
net with the decorated 2C1 topology. An underlying 2D H-bonded
layer in compound 3 (Fig. 3(B)) is composed of the 3-connected
Fig. 2 Molecular structures of complexes (A) 2, (B) 3 and (C) 4 with only
the heteroatom labeling. Hydrogen atoms and solvate (methanol and
water) molecules are omitted for clarity.
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Mild homogeneous catalytic oxidation of cycloalkanes
The copper(II) compounds 2–4 were tested as homogeneous
catalysts for the oxidation of C –C cycloalkanes to give a
6
8
mixture of the corresponding cyclic alcohols and ketones.
Cycloalkanes were selected as model substrates given the
presence of only one type of aliphatic carbon atom in their
structure as well as the practical importance of alkane oxidation
6
0–62
reactions,
including the biological hydroxylation of alkanes by a
63
copper containing enzyme, particulate methane monooxygenase.
The catalytic reactions were typically performed at 50 1C in air and
in a MeCN/H O medium, using aqueous 50% H O as an oxidant.
2
2 2
As a solvent system, aqueous acetonitrile is widely considered as
the best option in the mild homogeneous oxidation of alkanes by
2 2
H O , especially given the stability of MeCN toward oxidation as
well as the solubility of both alkane substrates and metal-complex
53,54,60–62,64–68
catalysts.
The obtained results are presented in Fig. 4
and summarized in Table 1; all the product yields are based on the
cycloalkane substrate.
The compounds 2–4 catalyze the oxidation of cycloalkanes
and show a similar trend of substrate reactivity. Hence, for all
the tested catalysts, the highest total yields of products were
observed in the oxidation of cycloheptane (26–32%), followed
by cyclooctane (20–29%) and cyclohexane (16–21%) (Table 1
and Fig. 4). In the oxidation of all cycloalkane substrates, the
best activity is exhibited by 4 (Fig. 4(C)), while compounds 2
and 3 are slightly less efficient catalysts and show similar
results. In all cases, cyclic alcohols are formed in slightly higher
amounts than cyclic ketones (Table 1), and the typical alcohol/
ketone molar ratios lie between 1.2 : 1 and 2.3 : 1.
The analysis of the kinetic curves in catalytic systems (Fig. 4)
reveals that the oxidation reactions are rather quick; they are
essentially complete within 40–60 min of the reaction time.
The product yields do not increase appreciably on prolonging the
Fig. 3 Topological representation of the underlying H-bonded networks.
(
A) 1D chains in 2 showing a uninodal 2-connected net with the decorated 2C1 reaction time up to 120 min. Besides, there is also no decline in
topology; view along the a axis; color codes: centroids of [Cu(pef)(bipyam)Cl] the total product yields at a prolonged reaction time, thus suggest-
2 2
molecular nodes (green balls), centroids of (H O) clusters (red). (B) 2D
ing that overoxidation of the products does not occur.
An important feature of the present catalytic systems con-
cerns their ability to catalyze the oxidation of cycloalkanes
layer in 3 showing a uninodal 3-connected net with the fes [Shubnikov
2
plane net (4ꢂ8 )] topology; view along the a axis; color codes: centroids
of 3-connected [Cu(pef)(phen)Cl] molecular nodes (green balls), centroids
of 3-connected (H
in complex showing
unique topology; view along the c axis; color codes: centroids of 4- and
-connected [Cu(pef)(bipy)Cl] molecular nodes (green balls), centroids
of 4-connected (H O) (MeOH) cluster nodes (yellow), centroids of
-connected (H O)5n nodes (red).
2 2
O)
(MeOH) cluster nodes (yellow). (C) 3D framework without requiring any type of additive or co-catalyst. In fact,
4
a
tetranodal 4,4,5,7-connected net with
a
addition of trifluoroacetic acid as a recognized additive into the
catalytic systems containing 2–4 does not lead to better product
yields in comparison to those achieved in the absence of any
additive (Table 1). This behavior significantly contrasts with the
majority of Cu-based catalysts previously applied for the mild
5
2
3
7
2
5
3,54,60–62,64–66
oxidation of cycloalkanes,
Cu(pef)(phen)Cl] molecular nodes and the 3-connected acid promoter (strong mineral or carboxylic acid) to show an
O) (MeOH) cluster nodes. These are topologically equivalent appreciable level of activity.
and give rise to a uninodal 3-connected net with the fes
To evaluate the effects of various reaction parameters (amounts
which usually need an
[
(H
2
2
2
[
(
Shubnikov plane net (4ꢂ8 )] topology and the point symbol of catalyst, oxidant, substrate, and reaction temperature) on the
2
4ꢂ8 ). The 3D H-bonded framework of 4 is significantly catalytic activity of the present systems, we selected catalyst 4 and
more complex (Fig. 3(C)) due to the presence of the 4- and cycloheptane as a model system given the highest product yield
5
-connected [Cu(pef)(bipy)Cl] molecular nodes, 4-connected achieved among the tested catalysts and substrates (Table 1).
O) (MeOH) cluster nodes, and the 7-connected (H
The effect of the catalyst amount on the total yield of
nodes. Topological analysis reveals a tetranodal 4,4,5,7-connected the products and the maximum initial reaction rate (W ) in
(H
2
3
2
O)5n
0
4
2 3 5
net with a unique topology and the point symbol (3ꢂ4 ꢂ5)(3 ꢂ4 ꢂ5 ꢂ the cycloheptane oxidation catalyzed by 4 is shown in Fig. 5.
5
6
2
2
2
4
5
6
ꢂ7 )(4 ꢂ5 ꢂ7 )(4 ꢂ5ꢂ7 ).
Both the total yield of cycloheptanol and cycloheptanone and
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Fig. 4 Oxidation of cyclohexane, cycloheptane, and cyclooctane (total yield of the products vs time) with H
2
O
2
catalyzed by complex (A) 2, (B) 3 and
(
2 2 3
C) 4. Reaction conditions: catalyst (5 mmol), cycloalkane (1.0 mmol), H O (5.0 mmol), CH CN (up to total volume of 2.5 mL), 50 1C, 2 h.
the initial reaction rate gradually increase on raising the concentration (Fig. 7(C)) is close to linear in the low range of
catalyst amount from 0.5 to 2.0 mM. Such a trend indicates concentrations (up to 0.4 M C 14); after that, W reaches a
first order dependence of W on the concentration of 4, thus plateau and does not depend on the concentration of C
indicating involvement of one type of the Cu-containing Such a type of W
species in the rate-limiting catalytic step of the cycloheptane is typical for the catalytic systems that operate with HO
7
H
0
0
7 14
H .
0
dependence on the substrate concentration
ꢁ
5
3,54,61
oxidation.
radicals.
The total product yield, in % based on the
The oxidant amount also has an important effect on the substrate, is higher at a low substrate loading (35% yield at
product yield. An increase of the H concentration from 0.2 M C 14) due to increased concentrations of other reagents
.4 to 2.0 M leads to growth of the maximum total yield from relative to the substrate (Fig. 7(B)). Besides, the reaction system
to 32% (Fig. 6). At the same time, at lower peroxide concen- is restricted by a limited solubility of C 14 in the CH CN/H
2
O
2
7
H
0
8
7
H
3
2
O
trations the oxidation reactions end very quickly (in B10 min), reaction medium.
thus preventing further yield growth. In contrast, at a higher Reaction temperature is also an important parameter in the
concentration (2 M) the maximum total yield of 32% is mild oxidation of cycloalkanes. Prior studies showed that
attained in B60 min. gentle heating of the reaction mixture (from ambient tempera-
2 2
H O
The total product yield (in mmol, Fig. 7(A)) increases on ture up to 50 1C) accelerates the alkane oxidation reactions,
changing the cycloheptane concentration from 0.2 to 0.4 M, without providing a notable contribution to overoxidation of
6
1,64–67
whereas further increase of the C
practically does not affect the reaction kinetics. The depen- is in agreement with the present 4/C
dence of the initial reaction rate on the cycloheptane oxidation of cycloheptane proceeds much faster at 50 1C in
7
H
14 loading (up to 0.6 M) products, and decomposition of catalyst or oxidant.
This
7
H
14 system, wherein the
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Table 1 Mild oxidation of C
6
–C
8
cycloalkanes catalyzed by compounds
a
2
–4
b
Product yield , %
Alcohol
c
Substrate
Ketone
Total
Catalyst 2
Cyclohexane
Cycloheptane
Cyclooctane
9.5
14.9
11.0
6.6
11.5
8.5
16.1
26.4
19.5
Catalyst 3
Cyclohexane
Cycloheptane
Cyclooctane
11.9
17.0
11.0
5.2
10.5
9.2
17.1
27.5
20.2
Catalyst 4
Cyclohexane
Cycloheptane
Cyclooctane
14.8
18.5
16.9
5.7
13.2
12.3
20.5
31.7
29.2
a
Fig. 6 Effect of the oxidant amount on the total yield of the products
2 2
Reaction conditions: cycloalkane (1 mmol), catalyst (5 mmol), H O
(
50% aq., 5 mmol), MeCN (up to 2.5 mL total volume), 50 1C, 2 h. (cycloheptanol and cycloheptanone) in the cycloheptane oxidation cata-
b
Yields are based on the cycloalkane substrate: (moles of product per lyzed by 4. Reaction conditions: catalyst 4 (5.0 mmol), C
7
H
14 (1.0 mmol),
c
mol of cycloalkane) ꢃ 100%. Sum of the yields of alcohol and ketone H O (0.4–2 M), CH CN (up to 2.5 mL total volume), 50 1C.
2
2
3
products.
comparison with the reaction at 30 1C (Fig. 8). Besides, the or 4, respectively. In the oxidation of methylcyclohexane, the
maximum total yield of 32% can be achieved in 60 min at 50 1C, bond selectivity parameters 11 : 21 : 31 of 1 : 5 : 10 (2), 1 : 5 : 7 (3),
whereas the reaction at 30 1C requires a much longer reaction or 1 : 4 : 8 (4) are also rather similar and indicate that the tertiary
time to attain a comparable yield (25% in 300 min).
C atom is oxidized with some preference over the secondary
To get some information on the nature of the oxidizing carbon atoms. Such regioselectivity and bond selectivity para-
species and on the selectivity parameters in the present meters are comparable to those reported for other copper-
reactions, we investigated the mild oxidation of n-heptane complex catalysts, suggesting free radical mechanisms and an
and methylcyclohexane as model substrates (Table 2). For all involvement of hydroxyl radicals as a powerful and rather
6
4–67
ꢁ
the catalysts, the oxidation of n-C
7
H16 proceeds without a indiscriminate oxidizing species.
Thus, the HO radicals
specific preference for any secondary C atom of the n-heptane abstract the H atoms from an alkane (RH) to generate alkyl
ꢁ
chain and leads to indiscriminative regioselectivity parameters radicals (R ). These then react with O
2
(e.g., from air or formed
ꢁ
C(1) : C(2) : C(3) : C(4) of 1 : 6 : 6 : 6, 1 : 5 : 5 : 5, or 1 : 4 : 4 : 6 for 2, 3, from H
2
O
2
) to form alkyl peroxy radicals (ROO ). The latter then
0
Fig. 5 Effect of the catalyst amount on (A) the total yield of the products (cycloheptanol and cycloheptanone) and (B) initial reaction rate (W )
7 2 2 3
in the cycloheptane oxidation catalyzed by 4. Reaction conditions: catalyst 4 (5 mmol), C H14 (1.0 mmol), H O (5.0 mmol), CH CN (up to 2.5 mL
total volume), 50 1C.
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Fig. 7 Effect of the substrate (C
7
H
14) amount on (A and B) the total yield (in mmol and %) of the products and (C) the initial reaction rate (W
0
) in the
cycloheptane oxidation catalyzed by 4. Reaction conditions: catalyst 4 (5 mmol), C
.5 mL total volume), 50 1C.
7
H
14 (0.5–1.5 mmol; 0.2–0.6 M), H (5.0 mmol), CH CN (up to
2
O
2
3
2
give alkyl hydroperoxides (ROOH) as primary intermediate
products; their formation was confirmed by GC analysis follow-
Antimicrobial activity
5
3,54
ing a method developed by Shul’pin.
Alkyl hydroperoxides The antimicrobial potential of pefloxacin and its copper(II)
quickly decompose to the corresponding alcohols and ketones derivatives 1–4 was evaluated against two Gram-positive
6
4–67
as final oxidation products.
(B. subtilis, S. aureus) and two Gram-negative (X. campestris,
E. coli) bacteria (Table 3). Hpef and complexes 1–4 are highly
active against all four bacteria tested and exhibit MIC values in
Biological activity studies
ꢀ1
We also performed standard biological experiments with the range of 0.25–2.0 mg mL (0.33–3.41 mM). Compounds 1–4
isolated copper(II) pefloxacin complexes. Such studies include are thus among the most active antimicrobials if compared
evaluation of the antimicrobial activity of the compounds and with other examples of Cu(II)-quinolone derivatives [Cu(Q)(N,N-
2
5,28,33,37,38,69–71
their interaction with serum proteins (HSA, BSA) and CTA DNA. donor)Cl].
In particular, complex 1 (the only
0
This enables us to compare the results with our previous data compound without a N,N -donor) is as active as Hpef against
on other copper(II) quinolone complexes. The bioactivity tests the microorganisms tested and more active than complexes 2–4
were performed under standard conditions, without addition of against the Gram-(ꢀ) bacteria. In addition, when the MIC
reducing agents or hydrogen peroxide. Herein only the most values expressed in mM are considered, complexes 2–4 are
important results are briefly reported and the corresponding equally active to Hpef, while 1 is an even more potent anti-
figures are given in the ESI.†
microbial than pefloxacin. There are five commonly accepted
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we may suggest that the coordination of pefloxacin to Cu(II) which
results in the formation of the chelate ring is the most important
one for the enhanced antimicrobial activity of 1–4 if compared to
Hpef. Further, the antimicrobial activity of the complexes seems to
be dependent on the number of pef ligands in the complex.
Interaction of the compounds with serum albumins
The study of the interaction of bioactive compounds with serum
albumins (SAs), proteins that participate in the transportation of
74
ions and drugs toward cells and tissues, is a first approach to
explain the potential biological activity, since their binding to SAs
may influence the biological properties or may reveal novel alter-
75
native transportation pathways or mechanisms. The quenching
of the SA-fluorescence emission band in the presence of copper(II)
Fig. 8 Effect of the reaction temperature on the total yield of the products compounds is much more pronounced for BSA, with the max-
cycloheptanol and cycloheptanone) in the cycloheptane oxidation catalyzed
by 4. Reaction conditions: catalyst 4 (5.0 mmol), C 14 (1.0 mmol), H
CN (up to 2.5 mL total volume), 30 or 50 1C.
(
imum quenching observed in the presence of 2 (up to 51.0% and
8.0% for HSA and BSA, respectively, Fig. S2 and S3, ESI†). This
quenching may be attributed to possible changes in the trypto-
phan environment of SA, probably due to changes in the albumin
7
H
2 2
O
7
(5.0 mmol), CH
3
7
5
Table
methylcyclohexane
2
Selectivity parameters in the oxidation of n-heptane and secondary structure.
a
The SA-quenching constants (k
q
) of the compounds were
calculated from the corresponding Stern–Volmer plots (Fig. S4
and S5, ESI†) and the Stern–Volmer quenching equation
(eqn (S2) and (S3)). These constants are much higher
Selectivity parameter
Regioselectivity
C(1) : C(2) : C(3) : C(4) (n-C
Bond selectivity
2
3
4
b
7
H
16
)
1 : 6 : 6 : 6
1 : 5 : 10
1 : 5 : 5 : 5
1 : 5 : 7
1 : 4 : 4 : 6
1 : 4 : 8
1
0
ꢀ1 ꢀ1
(
Table 4) than the value of 10
M
s
and suggest the
c
76
11 : 21 : 31 (methylcyclohexane)
existence of a static quenching mechanism with the binding
7
7
a
q
of the compounds to SA. The k constants of the complexes
2 2
Reaction conditions: catalyst (5 mmol), alkane (1.0 mmol), H O
(
5.0 mmol), MeCN up to 2.5 mL total volume, 2 h, 50 1C. All parameters are higher than the corresponding values for free Hpef. Com-
were calculated based on the ratios of isomeric alcohols. The calculated
plexes 1 and 2 feature the highest k
q
constants for HSA and
constants are within the
s ) found for a series of metal-complexes
parameters were normalized, i.e. recalculated taking into account the number
of H atoms at each carbon atom. Parameters C(1) : C(2) : C(3) : C(4) are the
b
BSA, respectively. On average, the k
q
12
13
ꢀ1 ꢀ1
relative reactivities of hydrogen atoms at carbons 1, 2, 3, and 4 of the range (10 –10
M
c
n-heptane chain. Parameters 11 : 21 : 31 are the relative normalized
25,26,37,69
bearing quinolones as ligands.
reactivities of the hydrogen atoms at primary, secondary, and tertiary
carbon atoms of methylcyclohexane.
The SA-binding constants (K), calculated from the Scatchard
plots (Fig. S6 and S7, ESI†) and the Scatchard equation
(
eqn (S4)), are moderate-to-high (Table 4) and are of the same
Table 3 Antimicrobial activity of Hpef and complexes 1–4 expressed in
MIC in mg mL or mM (the values in parentheses)
magnitude as those reported for other metal–quinolone
complexes.
ꢀ1
25,26,37,69
Complexes 1–4 bind tighter to BSA than
to HSA. Compound 2 has the highest K constant for BSA,
whereas complexes 2 and 3 display the highest K constants
for HSA among the present compounds. The K constants are in
the 1.56 ꢃ 10 –8.65 ꢃ 10 M range, being indicative of the
SA-binding and migration of the compounds to the sites of
potential biological targets. On the other hand, a comparison of
these constants with the K value of the strongest non-covalent
Compound
E. coli
X. campestris S. aureus B. subtilis
Pefloxacin (Hpef)
0.5 (1.50) 0.5 (1.50)
0.5 (0.66) 0.5 (0.66)
0.25 (0.75) 0.5 (1.50)
0.25 (0.33) 1 (1.32)
0.5 (0.83) 1 (1.66)
0.5 (0.82) 1 (1.64)
0.5 (0.85) 2 (3.41)
[
[
[
[
2
Cu(pef) (MeOH)], 1
Cu(pef)(bipyam)Cl], 2 1 (1.66)
Cu(pef)(phen)Cl], 3
Cu(pef)(bipy)Cl], 4
4
5
ꢀ1
1 (1.66)
1 (1.64)
1 (1.70)
1 (1.64)
1 (1.70)
1
5
ꢀ1
factors that influence the antimicrobial activity of the interaction, i.e. K B 10
M
regarding the interaction of
72,73
78
complexes,
namely: (i) the chelate effect of ligands, (ii) the avidin with diverse compounds, may reveal a reversibility of
nature of ligands, (iii) the nuclearity, (iv) the total charge, and (v) the binding of the compounds to the SAs which is essential for
the presence and the nature of counterions. Among these factors, their potential release upon arrival at biotargets.
Table 4 The albumin constants for Hpef and complexes 1–4
ꢀ
1
ꢀ1
ꢀ1 ꢀ1
q(HSA) (M
ꢀ1
(BSA) (M
ꢀ1
Compound
Hpef
k
q(BSA) (M
s
)
k
s
)
K
)
K
(HSA) (M
)
11
13
13
13
12
12
12
12
12
12
5
5
5
4
5
5
4
5
5
4
8.46(ꢄ0.10) ꢃ 10
1.69(ꢄ0.05) ꢃ 10
1.26(ꢄ0.07) ꢃ 10
1.04(ꢄ0.05) ꢃ 10
5.52(ꢄ0.26) ꢃ 10
1.15(ꢄ0.12) ꢃ 10
2.64(ꢄ0.15) ꢃ 10
6.05(ꢄ0.40) ꢃ 10
5.46(ꢄ0.25) ꢃ 10
1.53(ꢄ0.11) ꢃ 10
2.51(ꢄ0.12) ꢃ 10
1.77(ꢄ0.06) ꢃ 10
8.65(ꢄ0.37) ꢃ 10
9.50(ꢄ0.10) ꢃ 10
1.14(ꢄ0.08) ꢃ 10
5.13(ꢄ0.35) ꢃ 10
1.56(ꢄ0.43) ꢃ 10
1.23(ꢄ0.10) ꢃ 10
1.24(ꢄ0.06) ꢃ 10
4.78(ꢄ0.40) ꢃ 10
[
[
[
[
2
Cu(pef) (MeOH)], 1
Cu(pef)(bipyam)Cl], 2
Cu(pef)(phen)Cl], 3
Cu(pef)(bipy)Cl], 4
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Interaction of the compounds with CT DNA
The interaction of metal complexes with CT DNA, and especially
the ones bearing quinolones as ligands, is of increasing research
interest due to the biological role of quinolones in targeting the
enzymes DNA gyrase and topoisomerase IV, which are responsible
1,2,9
for DNA-replication.
In general, the interaction of metal com-
plexes may take place covalently or noncovalently (including inter-
calation, groove-binding or electrostatic interactions) and depends
79,80
mainly on the stability and the nature of the complexes.
The changes observed in the UV spectra of CT DNA solution
in the presence of Hpef and 1–4 (Fig. S8, ESI†), as well as in the
spectra of similar solutions having increasing amounts of CT
DNA (Fig. S9, ESI†), indicate the interaction of the studied
8
1
compounds with CT DNA. The percentage of hypochromism
in most cases is not sufficiently intense (Table 5) to conclude
unambiguously the DNA-interaction mode of the complexes.
1
/3
Fig. 9 Relative viscosity (Z/Z
0
)
of CT DNA ([DNA] = 0.1 mM) in buffer
solution (150 mM NaCl and 15 mM trisodium citrate at pH 7.0) in the presence
of Hpef and complexes 1–4 in increasing amounts (r = [complex]/[DNA]).
The DNA-binding constants (K ) of complexes 1–4, as calcu-
b
82
lated by the Wolfe–Shimer equation (eqn (S5)) and the respective
plots (Fig. S10, ESI†), are higher than that of Hpef suggesting their
tighter binding to CT DNA upon coordination to Cu. The highest
5
92 nm (up to 79.5% of the initial EB fluorescence for complex 1,
Fig. S11(B) and Table 6, ESI†).
This quenching is in good agreement (R B 0.99, Fig. S12,
ESI†) with the linear Stern–Volmer equation (eqn (S2)) and may
be assigned to the displacement of EB from the EB–DNA
conjugate by the compounds, as a possible result of their
competition with EB for the intercalation DNA-sites. The KSV
constants (Table 6) are in the range reported for other metal–
quinolones,
constant among the pefloxacin derivatives. The quenching
constants (k ) for the compounds (Table 6) are significantly
higher than 10 s , suggesting a static mechanism for
K
b
constant among the compounds is observed for complex 2
(
Table 5), which is among the tightest DNA-binders reported for
25,26,37,69
metal–quinolone complexes.
The DNA-viscosity changes (Z/Z ) depend on the relative
0
DNA-length changes (L/L ) in the presence of a DNA-binder
0
1
/3 83
and are related by the equation L/L
0
= (Z/Z
0
)
.
Therefore, the
2
6,37,69
with complex 1 having the highest KSV
DNA-binding mode of the compounds may be clarified
by monitoring the DNA-viscosity changes in the presence of
the compounds. The viscosity changes of a DNA solution
q
1
0
ꢀ1 ꢀ1
M
(0.1 mM) were monitored in the presence of increasing
the quenching of the EBꢀDNA fluorescence; they also prove
the EB-displacement and the simultaneous binding of the
compounds under study. Therefore, the intercalation of the
amounts of the compounds (up to the value of r = 0.35). Upon
addition of the compounds, a significant increase of the
relative DNA-viscosity is observed (Fig. 9), indicating that the
interaction of the compounds with DNA takes place probably
7
6
compounds to DNA may be indirectly verified.
2
6,37,69,83
via intercalation.
Interaction of copper(II)–quinolone complexes with
biomolecules
EB is a typical intercalation marker since its intercalation
in-between adjacent base pairs on the double DNA-helix via its
planar EB phenanthridine ring results in the formation of the Considering the reported copper(II)–quinolone complexes, we may
EB–DNA conjugate which presents an intense fluorescence compare the biological behavior (antimicrobial activity and bind-
7
4,84
emission band at 592 nm, when excited at 540 nm.
ing with DNA and albumins) of the present pefloxacin compounds
The fluorescence emission spectra of pre-treated EB–DNA conju- 1–4 with their copper(II) analogues bearing the quinolones enro-
3
3
26,69
27
gate ([EB] = 20 mM, [DNA] = 26 mM) were recorded for increasing floxacin (Herx), flumequine (Hflmq),
gatifloxacin (Hgati),
34
34
28
amounts of the compounds and are representatively shown for norfloxacin (Hnorf), ofloxacin (Hoflo), oxolinic acid (Hoxo),
complex 1 in Fig. S11(A) (ESI†). The addition of each compound pipemidic acid (HPPA), N-propyl-norfloxacin (Hpr-norf) and
resulted in significant quenching of the EB–DNA emission band at sparfloxacin (Hsf) as ligands (Tables S4 and S5, ESI†). It should
85
85
70
Table 5 Spectral features of the interaction of Hpef and its copper(II) complexes 1–4 upon addition of DNA. UV-band (lmax, in nm) (percentage of the
observed hyper-/hypo-chromism (DA/A , %), blue-/red-shift of lmax (Dl, nm)) and the corresponding DNA-binding constants (K )
0 b
a
b
ꢀ1
Compound
pefloxacin
Band (DA/A
0
, Dl )
b
K (M )
4
5
6
4
5
285 (+3.5, ꢀ4); 318 (ꢀ2, +1)
286 (ꢀ6, ꢀ2); 320 (ꢀ4.5, +3)
319 (ꢀ5, ꢀ3)
6.05(ꢄ0.12) ꢃ 10
4.99(ꢄ0.14) ꢃ 10
8.46(ꢄ0.05) ꢃ 10
7.83(ꢄ0.35) ꢃ 10
6.86(ꢄ0.20) ꢃ 10
[
[
[
[
2
Cu(pef) (MeOH)], 1
Cu(pef)(bipyam)Cl], 2
Cu(pef)(phen)Cl], 3
Cu(pef)(bipy)Cl], 4
291 (ꢀ4.3, 0); 326 (ꢀ16, +3)
c
291 (ꢀ4.5, ꢀ2); 313(sh) (ꢀ30, elm ); 333 (ꢀ21, +5)
a
b
c
‘
‘+’’ denotes hyperchromism, ‘‘ꢀ’’ denotes hypochromism. ‘‘+’’ denotes red-shift, ‘‘ꢀ’’ denotes blue-shift. ‘‘elm’’ = eliminates.
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Table 6 Percentage of EB–DNA fluorescence quenching (DI/I
o
, %) and the intracellular H
O
concentration is relatively uniform in
2
2
ꢀ1
ꢀ1 ꢀ1
the Stern–Volmer (KSV in M ) and EB–DNA quenching constants (k
for pefloxacin and its complexes 1–4
q
, M
s
)
aerobic forms of life, and appears to vary from B0.001 mM to
.7 mM. Low endogenous levels of H O are maintained by the
0
2
2
ꢀ
1
ꢀ1 ꢀ1
Compound
Hpef
DI/I
o
(%) Ksv (M
)
k
q
(M
s
)
catalase enzyme that catalyzes its decomposition to water and
8
7
4
12
13
13
13
12
oxygen. Although the conditions used in our catalytic experi-
ments are clearly very different from those used in the above
described biological experiments (higher concentration of
70.8
7.00(ꢄ0.36) ꢃ 10 3.04(ꢄ0.16) ꢃ 10
5
[
[
[
[
Cu(pef)
Cu(pef)(bipyam)Cl], 2 74.3
Cu(pef)(phen)Cl], 3
Cu(pef)(bipy)Cl], 4
2
(MeOH)], 1 79.5
9.91(ꢄ0.26) ꢃ 10 4.31(ꢄ0.12) ꢃ 10
5
2.37(ꢄ0.07) ꢃ 10 1.03(ꢄ0.03) ꢃ 10
5
74.6
74.7
2.53(ꢄ0.04) ꢃ 10 1.10(ꢄ0.02) ꢃ 10
5
2 2
H O , higher temperature, different solvent), there are some
1.67(ꢄ0.05) ꢃ 10 7.26(ꢄ0.20) ꢃ 10
observations which are valid for both types of results. It seems
that our complexes containing Cu(II) ions and quinolone but no
0
be noted that Hflmq, Hoxo and HPPA are first-generation N,N -donor ligand are not good catalysts and can also not
quinolones, Herx, Hnorf, Hoflo and Hpr-norf are second- substantially cleave DNA without addition of hydrogen peroxide.
generation quinolones (similar to Hpef), and Hgati and Hsf It also seems probable that formation of reactive oxygen species is
are third-generation quinolones.
important for both types of activities. Without any doubt, further
As seen in Table S4 (ESI†), the antimicrobial activities of the studies would be needed to claim that such copper complexes can
copper–quinolone complexes are similar or slightly better than catalyze important reactions in living systems. However, it could
those of the corresponding free quinolones. The main factor also not be totally excluded that various types of copper (or other
affecting the range of the antimicrobial activity of these com- metal) complexes are formed in the human body and that some
plexes is the generation of the quinolone ligand; the higher the might be involved in processes that are important for side effects
generation of the quinolone, the more active the compound. A of quinolone drugs.
0
general conclusion of whether the presence of an N,N -donor
ligand enhances the biological activity cannot be drawn, since
its presence may ameliorate the activity (as for Cu(II)–gatifloxacin
complexes) and may not induce significant variances.
Conclusions
25
In regard to the DNA-binding constants (Table S5, ESI†), In the present work, we described the synthesis and character-
the generation of the quinolone does not seem to affect the ization of mononuclear copper compounds with a second-
magnitude of K . On the other hand, the presence of the generation quinolone antibacterial drug (pefloxacin) in the
b
0
N,N -donor ligand seems, on average, to enhance the affinity absence or presence of nitrogen-donor heterocyclic ligands
of the compounds to DNA. The conclusions regarding the such as bipyam, phen or bipy. In the resultant copper(II) com-
binding affinity for the albumins are quite similar in general plexes, the quinolone ligands are deprotonated and coordinated to
(
Table S5, ESI†), i.e. copper(II)–quinolone complexes with the copper in a bidentate chelating mode through the pyridone and a
CuN Cl coordination sphere have higher SA-binding con- carboxylato oxygen. The crystal structures of [Cu(pef)(bipyam)Cl],
stants than the complexes with the CuO environment. [Cu(pef)(phen)Cl] and [Cu(pef)(bipy)Cl] were determined by X-ray
2 2
O
5
We would also like to mention some previous biological crystallography, revealing a distorted squareꢀpyramidal geometry
studies which might be important for better understanding of for Cu(II) and presenting a similar arrangement of the atoms
catalytic results in the present work. For example, DNA cleavage around copper.
activity of various copper ciprofloxacin (cfH) complexes was
also tested. It was observed that after 4 h incubation in the are extended into diverse 1D, 2D, or 3D H-bonded networks via
We also showed that the discrete monocopper(II) complexes
3
2
presence of a reducing agent ascorbate, a ternary complex multiple hydrogen bonding interactions with crystallization
[
CuCl(cfH)(phen)]Clꢂ2H
2
O completely fragmented DNA. Apart solvent molecules. In turn, water and/or methanol molecules
from this complex, the measurable nuclease activity was of crystallization are arranged into different types of H-bonded
only observed with the mixed-valence Cu(II)/Cu(I) compound clusters, including hybrid water–methanol associates and
II
I
[Cu (cfH) (Cu Cl ) ] but not with the copper(II) complex con- infinite 1D water tapes. Topological analysis and classification of
2 2 2
taining only quinolone ciprofloxacin ([Cu(cfH) Cl ]ꢂ6H O). the simplified H-bonded nets was performed. Hence, the present
2
2
2
2 2
In an additional experiment, H O was added and after that study also contributes to the detection of different water assemblies
all the studied compounds exert measurable nuclease activity. trapped by various metal–organic materials as well as to topological
Interesting results were recently obtained also by Kyziol et al. classification of complex H-bonded nets.
who studied the biological properties of Cu(II) and Cu(I) com-
Besides, we have also disclosed an application of the obtained
8
6
plexes of various quinolone derivatives. They have found that copper(II) compounds as efficient homogeneous catalysts for the
Cu(I) complexes were much more active but Cu(II) complexes mild oxidation of cycloalkanes to give a mixture of cyclic alcohols
were more efficient in producing reactive oxygen species. and ketones. These reactions proceed rather quickly and under
Obviously, the oxidation state of the metal and conditions used mild conditions (50 1C, atmospheric pressure), in the absence of
in experiments (e.g., use of reducing agent, hydrogen peroxide) any additional promoter or co-catalyst, and lead to maximum
are very important in such systems. Various reactive oxygen product yields up to 35% based on a cycloalkane substrate. Such a
species are formed in all aerobic organisms but the levels are level of product yields is rather high in the field of mild alkane
low and are regulated. For example, the physiological range for oxidation, especially if the high inertness of these hydrocarbons
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and mild reaction conditions are considered. In fact, an industrial HSA
Human serum albumin
SA-binding constant
DNA-binding constant
SA-quenching constant
Stern–Volmer constant
Medium
process (DuPont) for the oxidation of cyclohexane uses a homo-
K
geneous cobalt naphthenate catalyst and proceeds with only B5% Kb
88
cyclohexane conversion under harsher reaction conditions. Inter- kq
estingly, complex 1 appeared to be inactive in the present type of KSV
cycloalkane oxidations, which is most likely associated with its fast
m
decomposition in the reaction medium. In comparison with 2–4, MIC
such a behavior of 1 can be related to the absence of chloride and pef
N,N -donor aromatic ligands in its structure. Furthermore, the phen
Minimum inhibitory concentration
Anion of pefloxacin
1,10-Phenanthroline
[compound]/[DNA] ratio
[DNA]/[compound] ratio
Strong
0
effects of different reaction parameters on the catalytic activity of
catalyst 4 in the oxidation of cycloheptane (a model system) were
investigated. We believe that the present work might open up a
r
r
s
0
novel application of the Cu–quinolone coordination compounds S. aureus
in catalysis. Further research on the application of such copper(II) SA
catalysts in the oxidative functionalization of alkanes and on sh
Staphylococcus aureus ATCC 6538;
Serum albumin
Shoulder
widening the substrate scope of such reactions will be pursued.
All the obtained compounds also showed significant X. campestris
antimicrobial activity, with MIC values ranging from 0.25 to Dn(CO
vs
very strong
Xanthomonas campestris ATCC 1395
2
)
nasym(CO
2
) ꢀ nsym(CO
2
).
ꢀ1
2.0 mg mL (0.33–3.64 mM). Such a level of activity in most
cases is enhanced in comparison to free pefloxacin. The inter-
action of the complexes with albumins showed that they exhibit Conflicts of interest
high affinity for albumins and especially for BSA. All the
There are no conflicts to declare.
SA-binding constants of the compounds are of the order
4
5
ꢀ1
1
0 –10 M , proving their ability to bind reversibly to the
albumins in order to get transferred toward their potential
biological targets. The copper(II)ꢀpefloxacin complexes can
bind to CT DNA in an intercalative mode as revealed by the
techniques employed in the present study. The complexes
exhibit higher binding constants to CT DNA than free peflox-
Acknowledgements
This work has been supported by the FCT and Portugal 2020
LISBOA-01-0145-FEDER-029697, IF/01395/2013/CP1163/CT005,
(
CEECIND/03708/2017, UID/QUI/00100/2013). We would like to
acknowledge networking support by the COST action CA15106
acin, with complex 2 having the highest calculated K
among the present compounds, which also lies among the
highest K values reported for metal–quinolone complexes.
b
constant
(CHAOS). The publication has also been prepared with the
b
support of the RUDN University Program 5-100. Financial
support from the Slovenian Research Agency (ARRS) through
project P1-0175 and infrastructure (use of SuperNova diffracto-
meter) of the EN–FIST, Center of Excellence, Trg OF 13,
Ljubljana, Slovenia is gratefully acknowledged.
In conclusion, the present work extended a still very limited
family of structurally characterized pefloxacin coordination
compounds to their copper(II) examples. Apart from the struc-
tural significance of 1–4, the Cu(II)ꢀpefloxacin derivatives 2–4
exhibit notable catalytic activity. Besides, all the obtained
compounds feature a very promising biological profile in
regard to their interaction with biomacromolecules and their
antibacterial activity, which deserve further attention and
investigation. These studies will be pursued in our laboratories.
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