Antimicrobial activity and urease inhibition of Schiff bases derived from isoniazid and fluorinated benzaldehydes and of their copper(II) complexes

Article abstract of DOI:10.3390/molecules21121742

In order to evaluate the influence of substitution on biological properties of Schiff bases and their metal complexes, a series of differently substituted fluorine-containing Schiff bases starting from the drug isoniazid (isonicotinylhydrazide) were prepared and their structures were established by single-crystal X-ray diffraction. Also, four copper(II) complexes of these Schiff bases were synthesized. The prepared compounds were evaluated for their antimicrobial activity and urease inhibition. Two of the Schiff bases exerted activity against C. albicans. All copper(II) complexes showed excellent inhibitory properties against jack bean urease, considerably better than that of the standard inhibitor acetohydroxamic acid.

Full text of DOI:10.3390/molecules21121742

molecules
Article
Antimicrobial Activity and Urease Inhibition of
Schiff Bases Derived from Isoniazid and Fluorinated
Benzaldehydes and of Their Copper(II) Complexes
Ladislav Habala 1,*, Samuel Varényi 1, Andrea Bilková 2, Peter Herich 1,3, Jindra Valentová 1,
Jozef Kožíšek 3 and Ferdinand Devínsky 1
1
Department of Chemical Theory of Drugs, Faculty of Pharmacy, Comenius University, Kalincˇiakova 8,
SK-832 32 Bratislava, Slovakia; varenyi@fpharm.uniba.sk (S.V.); herich@fpharm.uniba.sk (P.H.);
valentova@fpharm.uniba.sk (J.V.); devinsky@fpharm.uniba.sk (F.D.)
Department of Cellular and Molecular Biology of Drugs, Faculty of Pharmacy, Comenius University,
2
Kalincˇiakova 8, SK-832 32 Bratislava, Slovakia; bilkova@fpharm.uniba.sk
Department of Physical Chemistry, Faculty of Chemical and Food Technology,
3
Slovak University of Technology, Radlinského 9, SK-812 37 Bratislava, Slovakia; jozef.kozisek@stuba.sk
*
Correspondence: habala@fpharm.uniba.sk; Tel.: +421-2-50-117-325
Academic Editor: Patrick Gamez
Received: 13 November 2016; Accepted: 13 December 2016; Published: 17 December 2016
Abstract: In order to evaluate the influence of substitution on biological properties of Schiff bases and
their metal complexes, a series of differently substituted fluorine-containing Schiff bases starting from
the drug isoniazid (isonicotinylhydrazide) were prepared and their structures were established by
single-crystal X-ray diffraction. Also, four copper(II) complexes of these Schiff bases were synthesized.
The prepared compounds were evaluated for their antimicrobial activity and urease inhibition.
Two of the Schiff bases exerted activity against C. albicans. All copper(II) complexes showed excellent
inhibitory properties against jack bean urease, considerably better than that of the standard inhibitor
acetohydroxamic acid.
Keywords: copper(II) complexes; urease inhibitors; antimicrobial activity; fluorine; isoniazid
1. Introduction
Over the last few decades, the research field of medicinal inorganic chemistry has experienced
continuous growth. The application areas encompass antibacterial, anti-inflammatory, and anticancer
therapies [1], among others. The early metallodrugs often exerted their biological activity via
direct covalent binding to the target biomolecule (e.g., cisplatin). Recently, the research increasingly
concentrates on metallodrug candidates acting by enzyme inhibition, in other words, by reversible
binding to the active centre of an enzyme, thus influencing its biological function [2–4].
One of the potential targets for new metallodrugs is urease, an enzyme catalysing hydrolysis
of urea to ammonia and carbon dioxide [
the pathogenesis of several diseases [ ]. The most prominent example is the urease found in
Helicobacter pylori, a bacterium presumably involved in several pathological conditions (gastric ulcers,
gastritis, gastric cancer) [ 11]. This bacterium is able to colonize the acidic milieu of the stomach
5]. Activity of this enzyme has been implicated in
6–8
9
–
by increasing its pH in the course of ammonia production. Another important pathogen expressing
urease is Mycobacterium tuberculosis [12]. Thus, the inhibition of urease represents an effective way to
combat these pathogens.
A number of urease inhibitors have been reported [13]. Besides solely organic substances, a very
efficient group of urease inhibitors is formed by metal complexes. The “free” metal ions themselves
(i.e., their aqua complexes) show marked urease inhibition [14]. The aquated transition metal ions were
Molecules 2016, 21, 1742; doi:10.3390/molecules21121742
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1742
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2+
found to inhibit urease in the order Cu2+ > Ni2+ > Co2+ Zn
[
15]. The complexes of these metals
∼
=
follow roughly the same order of efficiencies, although the inhibitory activity depends to a large degree
on the nature of the ligands and the overall structure of the complexes. Copper(II) complexes are
among the most efficient urease inhibitors [16]. Besides this, they exhibit a number of other interesting
biological activities, for example, anticancer activity [17].
As part of our research, we are interested in the inhibitory properties of metal complexes towards
enzymes, such as urease [16] or protein tyrosine phosphatase [18,19]. Fluorine-containing compounds
constitute a class of compounds with unique pharmacological properties [20]. Schiff bases are
another interesting type of substance, both as organic substances and as ligands for the synthesis of
complexes [21,22]. There have been several reports [23–27] on transition metal complexes of Schiff bases
derived from isoniazid, a highly efficient tuberculostatic [28]. The aim of our study was to investigate
hydrazine derivates of isoniazid with fluoro and trifluoromethyl substituted aromatic aldehydes, as
well as their copper(II) complexes. The ligands were synthesized according to established synthetic
procedures. The prepared substances were screened for their urease inhibition against jack bean urease
and also for their antimicrobial properties, with the intent to evaluate the effect of the substituents
and their location on the bioactivity of the prepared substances. Also, we successfully recorded X-ray
structural data for the Schiff bases as an important tool for evaluating various pharmacological
parameters. This publication utilizes research results of the CEBV project (Centrum Excelencie
Bezpecˇnostného Výskumu/Centre of Excellence of Security Research), ITMS: 26240120034.
2. Results and Discussion
2.1. Synthesis
The Schiff base ligands were prepared by condensation reactions between isoniazid and
appropriately substituted benzaldehydes (Scheme 1). The reaction is straightforward and provides
the products in good to excellent yields. Use of methanol-chloroform mixture as a reaction medium
often leads directly to X-ray quality crystals. Alternatively, the monocrystals can be obtained upon
recrystallization from ethanol and they generally crystallize in the form of monohydrates as white
needles stable in air and are soluble in polar organic solvents.
R1
O
R1
NH
O
O
N
H2N
NH
R2
R4
R2
R4
+
N
R3
R3
N
Scheme 1. Synthesis of the Schiff bases L1–L6 (
L1: R2 = CF3, R1 = R3 = R4 = H; L2: R4 = CF3,
R1 = R2 = R3 = H; L3: R2 = F, R1 = R3 = R4 = H; L4: R1 = F, R4 = OH, R2 = R3 = H; L5: R3 = F, R4 = OH,
R1 = R2 = H; L6: R1 = R2 = R3 = R4 = H).
The synthesis of the copper(II) complexes was carried out by combining ethanolic solutions of the
particular Schiff base ligand with copper(II) acetate in a metal:ligand molar ratio of 2:1. The complexes
are green-coloured solids and are poorly soluble in common organic solvents with the exception of
dimethyl sulfoxide (DMSO) and dimethylformamide (DMF). The attempts to grow crystals suitable
for X-ray structure analysis were unsuccessful. The complexes were non-electrolytes as proven by the
measurement of their conductivities in 1 mM DMSO solutions (molar conductivities ranging from
7.2 to 26.9 S
·
mol−1 cm2). The composition of the complexes corresponds to [Cu(Ln-H)2], as seen from
·
the results of elemental analysis and infrared spectroscopy.

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2.2. Analytical Characterization of the Compounds
The products were characterized by appropriate analytical methods—elemental analysis, NMR,
and infrared spectroscopy. The characteristic infrared (IR) vibrational bands are listed in the
Experimental part and are in accordance with the expected values [29]. Infrared spectra of the
Schiff bases contain a strong and sharp peak near 1670 cm−1, characteristic of the valence vibration
of the carbonyl group (amide I). Signals from the valence vibrations of the azomethine group can be
seen around 1660 cm−1. The medium peak near 1415 cm−1 accounts for the deformation vibration
of the C–N bond of amide (amide II). The strong and sharp peak near 1200 cm−1 originates from the
valence vibration of the C–F bond. The very strong peak near 750 cm−1 corresponds to the deformation
vibration of the aromatic C–H bond. The N–N bond causes infrared absorption in the ligand spectra
near 850 cm−1. Hydroxyl containing Schiff base ligands also show a very strong peak near 1280 cm−1
for phenol C–O valence vibration.
In the infrared spectrum of the ligands, a signal of valence vibration of the N–H bond can be
detected near 3200 cm−1. The disappearance of this signal in the infrared spectra of the complexes
clearly indicates deprotonation at the nitrogen due to enolization of the adjoining carbonyl group
in the course of complex formation. The strong signal of the carbonyl group found in the range of
1667–1691 cm−1 disappears upon coordination and the new medium-strong signals corresponding
to enolic C–O stretching vibration appear at 1056–1066 cm−1. Vibrational bands of the O–H group
originating from crystal water are found in some ligands. These bands also disappear in the course of
the coordination to the central atom. Absence of signals of the water molecules in the infrared spectra
disproves the possibility of any additional aqua ligands present in the copper complexes. Thus, the
structure of the complexes seems to correspond to the general formula [ML2] with a square-planar
coordination at the central atom, unlike the complexes reported in [23,25], supposed to possess two
extra aqua ligands and an octahedral geometry. This assumption is further supported by the results of
elemental analysis of the Cu(II) complexes, confirming the expected composition with metal-to-ligand
ratio 1:2 and the absence of aqua ligands in the structure of the complexes. A definite assignment of
particular coordination geometry to the complexes would require the preparation of a single crystal
and its X-ray structure analysis, which has not yet met with success, mainly due to the problems
with solubility.
2.3. X-ray Structures of the Schiff Bases
The three ligands L1, L2, and L4 crystallized in the monoclinic P21/n (for L1) and P21/c
(
for L2 and L4) space groups (No. 14). The ligand L3 crystallized in the orthorhombic P212121 space
group (No. 19). All four ligands crystallized as one independent molecule in the asymmetric unit.
The molecular structures for (L1–L4) are shown in Figure 1.
The molecule for all ligands comprises three chemical moieties: pyridine, benzene (substituted
with –OH, –F and –CF3), and hydrazone. The ligand molecules are not planar, with the angles between
the benzene and pyridine rings being 4.05◦ for L1, 51.62◦ for L2, 11.55◦ for L3, and 10.19◦ for L4. C–N
and N–N bond distances (Table 1) are shorter than a typical C–N, N–N single bond 1.405(6) Å [30
indicating significant delocalization along the NNC(O)C moiety. The shorter C–N and N–N bond
distances (Table 1) were observed in other compounds; for C–N, the bond distance is in the range of
1.343(2) Å–1.362(6) Å, and for N–N, the bond distance is in the range of 1.359(5) Å–1.384(3) Å [33 42].
The N=C bond distances (Table 1), are typical for a double bond. For all ligands, the central torsion
angles C1–N2–N3–C7 are listed in Table 1. The crystal structures of ligands L1 L4 are stabilized
–32],
π
–
–
by a system of intermolecular and intramolecular (for L2 and L4) hydrogen interactions (Table 2,
Figures 2–5).

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Figure 1. The molecular structure of ligands (L1–L4), showing the atom-labelling scheme. Displacement
ellipsoids are drawn at the 50% probability level.
◦
Table 1. Selected bond distances (Å) and angles ( ) for hydrazide group of ligands (L1–L4).
Ligand
L1
L2
L3
L4
Bond distances
C1–N2
N2–N3
N3–C7
1.3509(17)
1.3800(15)
1.2690(18)
1.353(2)
1.3679(18)
1.270(2)
1.347(2)
1.389(2)
1.273(3)
1.3500(17)
1.3786(14)
1.2764(17)
Torsion angles
C1–N2–N3–C7
169.57(13)
−
173.01(16)
−171.94(19) −176.96(11)

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Table 2. Hydrogen-bond (interaction) geometry of ligands (L1–L4).
Ligand
L1
D–H...A
C7–H7A...O1 i
N2–H2A... O1 i
d(D–H)
d(H...A)
d(D...A)
<(DHA)
0.93
0.86
2.45
2.15
3.2591(16)
3.9797(14)
145.4
161.0
C7–H7...F2
C7–H7...F5
0.93
0.93
0.89(2)
2.46
2.10
2.17(2)
2.980(2)
2.757(13)
3.0482(18)
115.4
126.6
172.5(19)
L2
N2–H2N...N1 i
O2–H2A...O1
O2–H2A...N3
C7–H7...O2 i
N2–H2...O2 i
O2–H2B...N1 ii
C4–H4...F1 iii
0.8396(19)
0.8396(19)
0.93
0.86
0.835(18)
0.93
2.274(16)
2.565(14)
2.64
2.02
2.06(2)
2.55
2.954(2)
3.271(2)
3.338(2)
2.834(2)
2.861(2)
3.349(3)
138(2)
142(2)
132.3
157.8
161(2)
144.6
L3
O2–H2O...N3
C4–H4...F1 i
0.82
0.93
0.93
0.86
1.92
2.64
2.51
2.19
2.6316(15)
3.281(2)
3.2566(18)
2.8861(15)
145.3
126.9
138.1
138.1
L4
C12–H12...O1 ii
N2–H2N...N1 iii
For L1: (i) x, y
−
1, z; for L2: (i) x + 1, y, z; for L3: (i) x
−
1, y, z; (ii)
−
x + 1, y + 1/2,
−
z + 1/2; (iii) –x + 3/2,
−y + 1, z − 1/2; for L4: (i) x + 1,y + 1, z; (ii) –x + 1, y − 1/2, −z + 1/2; (iii) –x + 2, y − 1/2, −z + 3/2.
Figure 2. The crystal packing of ligand L1, viewed along the b axis. Red dashed lines indicate
hydrogen interactions.
Figure 3. The crystal packing of ligand L2, viewed along the b axis. Red dashed lines indicate
hydrogen interaction.

Molecules 2016, 21, 1742
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Figure 4. The crystal packing of ligand L3, viewed along the b axis. Red dashed lines indicate
hydrogen interactions.
Figure 5. The crystal packing of ligand L4, viewed along the b axis. Red dashed lines indicate
hydrogen interactions.
2.4. Bioactivity of the Ligands and Complexes
Antimicrobial activity of the Schiff bases and the Cu(II) complexes was evaluated in vitro
against Gram-negative (E. coli) and Gram-positive (S. aureus) bacterial strains, and against C. albicans.
Also, the standard drugs ciprofloxacin and isoniazid were co-assayed for comparison. The results
are summarized in Table 3 as minimum inhibitory concentration (MIC) values. Two Schiff bases
(
L1 and L4) showed marked activity against C. albicans (MIC 0.037 mM and 0.048 mM, respectively).
The Schiff base L4 also exhibited weak activity against E. coli (MIC 1.55 mM). Among the copper(II)
complexes, only Cu L6 showed any noteworthy activity (against S. aureus, MIC 0.76 mM). The
-
hydrophobic fluoro and trifluoromethyl groups confer marked lipophilic properties to the molecules of
ligands. Upon complexation, the lipophilicity becomes even more pronounced, as the polar hydrophilic
groups are used in coordination to the central atom and hidden inside the complex molecule, while the
hydrophobic moieties are exposed to the environment. Although this could be expected to increase the
penetration through biomembranes, a marked increase in activity upon complexation was not observed.
It is possible that high lipophilicity might lead to decreased mobility of the substances in aqueous
cell media and impair the interaction with potential targets in the cell. In the two ligands containing
hydroxyl (L4 and L5), the Schiff base with fluoro substituent in para position to the hydroxyl group had
significantly higher activity than with the same group in the ortho position. Also, in trifluoromethyl
containing ligands (L1 and L4), para substitution shows a positive effect on antimicrobial activity
compared to the ortho substitution.

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Table 3. Antimicrobial bioassay (minimum inhibitory concentration (MIC) values) for ligands and
Cu(II) complexes.
Compound
S. aureus Mau 82/78
E. coli 327/73
C. Albicans 59/91
L1
L2
L3
L4
L5
37.5
37.5
n.i.
9.37
18.76
25.0
1.55
25.0
12.5
6.25
12.5
12.5
6.25
n.i.
0.037
9.37
25.0
0.048
12.5
12.5
6.25
6.25
12.5
6.25
50.0
n.a.
n.i.
50.0
50.0
6.25
6.25
12.5
0.76
n.i.
L6
MIC [mM]
Cu-L3
Cu-L1
Cu-L2
Cu-L6
Isoniazid
Ciprofloxacin
−4
−4
6.8 × 10
<3 × 10
n.a. = not applicable, n.i. = no inhibition observed at the highest used concentration (100 mM).
Jack bean urease is a urease isolated from Canavalia ensiformis (Jack bean). Because of their
highly conserved structure, ureases from different sources share similar structural features, and
conclusions may be drawn concerning the properties of the other ureases (such as the Helicobacter pylori
urease). The inhibition of Jack bean urease by Schiff bases, copper(II) complexes and the standard
substance acetohydroxamic acid was studied, and the results are expressed in the form of their IC50
values (Table 4). All Cu(II) complexes showed excellent inhibitory properties, whereas the ligands
exerted only weak activity. The best activity (0.49
of N-[[(4-(trifluoromethyl)phenyl]methylidene]pyridine-4-carbohydrazide (Cu
±
0.01
µ
M) was observed in the Cu(II) complex
-
L1). The inhibition
is clearly influenced by the structure of the ligand, particularly on the substitution of the phenyl
ring, as the overall structure of the complex is analogous and the low electric conductivity of the
DMSO solutions of the complexes renders improbable the dissociation of the complexes and the
formation of charged Cu2+ species to any significant degree. Nevertheless, at this stage, it is difficult to
draw clear conclusions regarding the structure–activity relationships for the inhibition of urease by
these copper(II) complexes. More extensive studies on the mechanism of urease inhibition by metal
complexes are needed to accomplish this, including data on lipophilicity and stability constants of the
complexes, amongst others.
Table 4. Inhibition of jack bean urease by ligands and Cu(II) complexes.
Compounds
IC50 ± SD (µM)
ligands
Cu-L3
Cu-L1
Cu-L2
Cu-L6
>500
1.17 ± 0.02
0.49 ± 0.01
1.25 ± 0.03
1.01 ± 0.02
185 ± 6.2
Acetohydroxamic acid
3. Materials and Methods
3.1. General
All chemicals purchased from commercial sources were of analytical grade and were used
without further purification. The chemicals 3-fluoro-2-hydroxybenzaldehyde and 5-fluoro-2-
hydroxybenzaldehyde were prepared by a modified procedure according to Ferguson et al. [43]. Cell
cultures were purchased from the Czech National Collection of Type Cultures. Infrared spectra were
recorded using the ATR (attenuated total reflectance) technique on a Nicolet 6700 FT-IR spectrometer
from Thermo Scientific (Waltham, MA, USA) in the 600–4000 cm−1 range. Elemental analysis was
1
performed with the help of a Flash2000 instrument from Thermo Scientific. H-, 13C- and 19F-NMR
spectra were measured on a Varian Mercury 300 MHz instrument (Palo Alto, CA, USA) using DMSO-d6
as solvent and TMS (tetramethylsilane) as internal standard. Melting points were determined on

Molecules 2016, 21, 1742
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a Büchi B-540 apparatus (Büchi Labortechnik AG, Flawil, Switzerland). The conductivities of the
◦
copper(II) complexes were evaluated in DMSO solutions (10−3 mol/L, 25 C) on a WTW LF 530
conductometer (Weilheim, Germany).
3.2. Synthesis of the Schiff Bases
General procedure for the synthesis of the Schiff bases.
N-[[(4-(trifluoromethyl)phenyl]methylidene]pyridine-4-carbohydrazide L1),
The ligand,
was prepared
(
in the following way. A 1.37 g portion (10.0 mmol) of isoniazid and 1.74 g (10.0 mmol) of
4-trifluoromethylbenzaldehyde were dissolved in 40 mL of methanol-chloroform mixture (1:1 v/v).
The solution was then refluxed for 80 min and then left to freely crystallise at room temperature. After
three days, colourless crystals suitable for X-ray structure analysis were obtained. They were filtered
off, washed with ether, and sucked dry in open air. If the crystals were of unsatisfactory quality for
crystallography, they were recrystallized from methanol-chloroform or methanol-dichloromethane
mixtures. The other ligands were prepared in an analogous way.
N-[[(4-(trifluoromethyl)phenyl]methylidene]pyridine-4-carbohydrazide (L1). Yield 91% (white needle-like
crystals); m.p. 198–200 ◦C. IR (neat, cm−1): 1667 (vs, C=O, amide I), 1172 (vs, C–F), 1119 (vs, C–F),
1
766 (s, C–H, arom.), 689 (s, C–H, arom.). H-NMR (DMSO-d6,
δ, ppm): 7.83–7.86 (m; 4H; aromatic);
8.00 (d; 2H; J = 8.1 Hz; aromatic); 8.54 (s; 1H; azomethine); 8.82 (d; 2H; J = 6 Hz; aromatic); 12.27
(s; 1H; N–H). 13C-NMR (DMSO-d6,
, ppm): 121.54 (CF3); 125.74; 127.84; 137.97; 140.23 (C=N); 147.21;
150.36; 161.86 (C=O). 19F-NMR (282.3 MHz, DMSO-d6,
, ppm): 61.20 (s). Elemental anal. calcd. for
δ
δ
−
C14H10N3F3O (293.25): C 57.34, H 3.44, N 14.33; found: C 57.01, H 3.57, N 14.40.
N-[[(2-trifluoromethyl)phenyl]methylidene]pyridine-4-carbohydrazide (L2). Yield 61% (white needle-like
◦
crystals); m.p. 210–212 C. IR (neat, cm−1): 3078 (w, C–H, arom.), 1672 (vs, C=O, amide I), 1410 (w, C–N,
1
amide II), 1115 (vs, C–F), 770 (vs, C–H, arom.). H-NMR (DMSO-d6,
δ, ppm): 7.68 (t; 1H; J = 7.2 Hz;
J = 7.5 Hz; aromatic); 7.68–7.78 (m; 4H; aromatic); 8.27 (d; 1H; J = 7.8 Hz; aromatic); 8.82 (d; 2H;
J = 6.0 Hz; aromatic); 8.86 (d; 1H; J = 2.1 Hz; azomethine); 12.38 (s; 1H; N–H). 13C-NMR (DMSO-d6,
ppm): 122.02 (CF3); 126.45; 127.47; 130.91; 132.34; 133.43; 140.59 (C=N); 144.42; 150.86; 162.34 (C=O).
19F-NMR (282.3 MHz, DMSO-d6,
, ppm): 56.71 (s). Elemental anal. calcd. for C14H10N3F3O (293.25):
δ,
δ
−
C 57.34 H 3.44 N 14.33; found: C 57.38 H 3.41 N 14.62.
N-[(4-fluorophenyl)methylidene]pyridine-4-carbohydrazide monohydrate (L3). Yield 75% (white needle-like
◦
crystals); m.p. 192–193 C. IR (neat, cm−1): 1685 (m, C=O, amide I), 1655 (vs, C=N, azomethine),
1
1232 (s, C–F), 836 (s, C–H, arom.), 689 (vs, C–H, arom.). H-NMR (DMSO-d6,
δ, ppm): 7.32 (td; 3H;
J = 2.1 Hz; J = 6.9 Hz; aromatic); 7.85–7.80 (m; 3H; aromatic); 8.47 (s; 1H; azomethine); 8.80 (dd; 2H;
J = 1.8 Hz; J = 2.7 Hz; J = 4.5 Hz; aromatic); 12.08 (s; 1H; O–H). 13C-NMR (DMSO-d6,
, ppm): 116.32;
116.61; 121.99; 129.90; 130.02; 131.10; 140.91 (C=N); 148.36; 150.82; 162.09 (C=O); 165.42 (C–F). 19F-NMR
(282.3 MHz, DMSO-d6, , ppm): 110.04 to 110.54 (m). Elemental anal. calcd. for C13H10N3FO H2O
(311.25): C 59.77 H 4.63 N 16.08; found C 59.42 H 5.22 N 16.62.
δ
δ
−
−
·
N-[(5-fluoro-2-hydroxyp◦henyl)methylidene]pyridine-4-carbohydrazide (L4). Yield 43% (white needle-like
crystals); m.p. 250–252 C. IR (neat, cm−1): 2933 (w, C–H, arom.), 1683 (vs, C=O, amide), 1556 (s, N–H,
1
amide II), 1285 (vs, C–F), 1261 (s, C–O, phenolic), 781 (s, C–H, arom.), 682 (s, C–H, arom.). H-NMR
(DMSO-d6, δ, ppm): 6.97 (q; 1H; J = 4.5 Hz; aromatic); 7.20 (td; 1H; J = 3.3 Hz; J = 9 Hz; J = 11.7 Hz;
aromatic); 7.48 (dd; 1H; J = 3 Hz; J = 3.3 Hz; J = 9.3 Hz; J = 9.6 Hz; aromatic); 7.85 (dd; 2H; J = 1.5 Hz;
J = 4.5 Hz; aromatic); 8.67 (s; 1H; azomethine); 8.81 (dd; 2H; J = 1.5 Hz; J = 4.5 Hz; aromatic); 10.86
(s; 1H; O–H); 12.32 (s; 1H; O–H). 13C-NMR (DMSO-d6,
δ
, ppm): 113.78; 114.11; 118.10; 118.20; 118.71;
119.03; 120.24; 120.34; 121.99; 123.29; 140.43 (C=N); 147.36; 147.39; 150.11; 150.87; 154.11; 154.09; 154.30
(C–O); 157.41 (C–F); 161.95 (C=O). 19F-NMR (282.3 MHz, DMSO-d6,
, ppm): 124.73 (s); 124.93 to
δ
−
−
−125.01 (m). Elemental anal. calcd. for C13H10N3O2F (259.24): C 60.23 H 3.89 N 16.21; found C 59.96
H 3.73 N 16.46.

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N-[(3-fluoro-2-hydroxyphenyl)methylidene]pyridine-4-carbohydrazide (L5). Yield 38% (white needle-like
◦
crystals); m.p. 276–278 C. IR (neat, cm−1): 2995 (w, C–H, arom.), 1680 (vs, C=O, amide I), 1418 (m, C–N,
1
amide II), 1278 (vs, C–O), 1244 (vs, C–F, arom.), 733 (vs, C–H, arom.), 691 (vs, C–H, arom.). H-NMR
(DMSO-d6,
7.46 (d; J = 8.1 Hz; 1H; aromatic); 7.86 (q; J = 1.5 Hz; J = 3 Hz; 2H; aromatic); 8.7 (s; 1H; azomethine);
8.82 (d; J = 5.1 Hz; 2H; aromatic); 11.38 (s; 1H; O–H); 12.42 (s; 1H; O–H). 13C-NMR (DMSO-d6,
, ppm):
118.14; 118.37; 119.77; 119.86; 121.74; 121.99; 125.12; 125.16; 140.26 (C=N); 145.66; 145.84; 148.73; 148.78;
149.86; 150.90 (C–F); 153.06 (C–O); 161.96 (C=O). 19F-NMR (282.3 MHz, DMSO-d6,
, ppm): 135.96
(m); 136.65 (dd; J = 4.8 Hz; J = 5.65 Hz). Elemental anal. calcd. for C13H10N3O2F (259.24): C 60.23 H
3.89 N 16.21; found C 59.33 H 3.84 N 15.93.
δ, ppm): 6.90–6.97 (m; 1H; aromatic); 7.31 (td; J = 1.5 Hz; J = 6.6 Hz; J = 6.9 Hz; 1H; aromatic);
δ
δ
−
−
N-(benzylidene)pyridine-4-carbohydrazide (L6). Yield 79% (white needle-like crystals); m.p. 198–201 ◦C.
IR (neat, cm−1): 3199 (w, N–H), 3026 (w, C–H arom.), 1691 (vs, C=O, amide), 1565 (s, N–H, amid II),
1
686 (vs, C–H, arom.). H-NMR (DMSO-d6,
δ
, ppm): 7.49–7.46 (m; 3H; aromatic); 7.84–7.74 (m; 4H;
aromatic); 8.48 (s; 1H; azomethine); 8.80 (d, 2H, J = 6 Hz, aromatic); 12.06 (s, 1H, N–H). 13C-NMR
(DMSO-d6, , ppm): 116.44; 118.69; 119.44; 121.46; 121.60; 129.15; 129.26; 131.75; 139.98 (C=N); 148.89;
δ
149.02; 150.33; 150.44; 157.47; 161.33 (C=O). Elemental anal. calcd. for C13H11N3O (225.25): C 69.32 H
4.92 N 18.66; found C 68.75 H 4.78 N 18.54.
3.3. Synthesis of the Complexes
General procedure for the synthesis of the copper(II) complexes. The Schiff base ligand
N-[(4-fluorophenyl)methylidene]pyridine-4-car◦bohydrazide (L3, 0.243 g; 1.0 mmol) was dissolved in
50 mL ethanol and the solution was heated to 50 C. Then, a solution of copper(II) acetate monohydrate
(0.5 mmol; 0.100 g) in 20 mL ethanol was added dropwise under constant stirring. In the course of
its addition, a green precipitate started to appear. The reaction mixture was stirred at the same
temperature for 2 h. After this, the mixture was allowed to cool to room temperature and the product
was isolated by filtration on a glass filter crucible, washed several times with water and ethanol, and
dried in vacuum to yield a green solid complex substance (Cu-L3). The remaining complexes were
prepared in the same way.
Cu(L3-H)2] (Cu-L3) Yield 93% (green powder). IR (neat, cm−1): 1520 (s, C=N), 1061 (m, enolic C–O),
823 (s, N–N). Elemental anal. calcd. for C26H18N6CuF2O2 (259.24): C 56.98 H 3.31 N 15.33; found
C 56.45 H 3.36 N 14.97.
[Cu(L1-H)2] (Cu-L1). Yield 78% (green powder). IR (neat, cm−1): 1519 (s, C=N), 1066 (m, enolic C–O),
828 (s, N–N). Elemental anal. calcd. for C28H18N6F6O2Cu (648.02): C 51.90 H 2.80 N 12.97; found
C 50.91 H 2.89 N 12.40.
[Cu(L2-H)2] (Cu-L2). Yield 95% (green powder). IR (neat, cm−1): 1511 (s, C=N), 1059 (m, enolic C–O),
854 (s, N–N). Elemental anal. calcd. for C28H18N6F6O2Cu (648.02): C 51.90 H 2.80 N 12.97; found
C 51.45 H 2.66 N 12.73.
[Cu(L6-H)2] (Cu-L6). Yield 92% (green powder). IR (neat, cm−1): 1513 (s, C=N), 1056 (m, enolic C–O),
843 (s, N–N). Elemental anal. calcd. for C26H20N6O2Cu (512.02): C 60.99 H 3.94 N 16.41; found 60.29 H
3.84 N 16.25.
3.4. X-ray Single-Crystal Structure Determination
All X-ray measurements were performed on an Oxford Diffraction Gemini R four-circle
diffractometer (Oxford Diffraction Ltd.: Abingdon, UK) with CrysAlis [44], using Mo-K
α radiation
for L1 L3 L4, and Cu-K radiation for L2 at 293(1) K. Data reduction was done by CrysAlis RED [44].
,
,
α
The structure was solved by the charge-flipping algorithm superflip [45] using OLEX2 [46]. Refinement
was carried out on F2 and scattering factors incorporated in SHELXL-2013 [47] were used. All

Molecules 2016, 21, 1742
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non-hydrogen atoms were refined with anisotropic thermal parameters. Crystal data for L1, L2, L3, L4,
data collection procedures, structure determination, and refinement are summarized in Table 5 and
Figure 2. The positions of all other H atoms were geometrically optimized and constrained to ride on
their parent atoms, with bond length C–H = 0.93 Å (aromatic), N–H = 0.86 Å (hydrazide for L1, L3, L4),
N–H = 0.89 Å (hydrazide for L2), O–H = 0.82 Å (hydroxyl for L4), and O–H = 0.84 Å (hydrogens in
water for L3) with hydrogen‘s temperature factors Uiso(H) = 1.2 Ueq(C,N,O). The DIAMOND program
package was used for molecular structure drawing [48]. Cambridge Crystallographic Data Centre
(CCDC) 1498952–1498955 contains the supplementary crystallographic data for this article. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/conts/retrieving.html, or from the Cambridge
Crystallographic Data Centre, 12 Union Road, CambridgeCB2 1EZ, UK; fax: +44-1223-336-033; or
e-mail: deposit@ccdc.cam.ac.uk.
Table 5. Crystal data and structure refinement for Schiff base ligands (L1–L4).
L1
C14H10F3N3O
293(2) K
L2
C14H10F3N3O
293(2) K
L3
C13H12FN3O2
293(2) K
L4
C13H10FN3O2
293(2) K
Empirical formula
Temperature (K)
Wavelength (Å)
Crystal system
Space group
0.71073
1.54184
0.71073
0.71073
Monoclinic
P21/n
Monoclinic
P21/c
Orthorhombic
P212121
Monoclinic
P21/c
a = 11.7577(7) Å
b = 5.1630(2) Å
c = 21.6108(13) Å
a = 8.1335(2) Å
b = 7.2718(2) Å
c = 22.2575(6) Å
a = 6.4785(4) Å
b = 7.0244(10) Å
c = 27.373(4) Å
a = 8.9048(3) Å
b = 10.1067(3) Å
c = 13.6087(5) Å
Unit cell dimensions
◦
◦
◦
◦
a = 90
b = 98.143(5)
a = 90
b = 98.686(2)
a = 90
b = 90
a = 90
b = 103.420(4)
◦
◦
◦
◦
◦
◦
◦
◦
g = 90
g = 90
g = 90
g = 90
Formula weight
Volume (Å3)
293.25
293.25
261.26
259.24
1298.66(12)
1301.33(6)
1245.7(3)
1191.32(7)
Z/Calculated density
(mg/m3)
4/1.500
4/1.497
4/1.393
4/1.445
−1
0.127
1.101
0.107
0.111
Absorption coefficient (mm
F (000)
)
600
600
544
536
Crystal size (mm)
0.97 × 0.78 × 0.21
0.30 × 0.15 × 0.05
4.018 to 75.559
0.37 × 0.11 × 0.08
2.977 to 30.497
0.62 × 0.23 × 0.12
3.097 to 26.368
◦
◦
◦
◦
◦
◦
◦
◦
Theta range for data collection 3.132 to 26.361
−14 ≤ h ≤ 14
−10 ≤ h ≤ 10
−9 ≤ k ≤ 9
−9 ≤ h ≤ 9
−11 ≤ h ≤ 11
−12 ≤ k ≤ 12
−16 ≤ l ≤ 16
Index ranges
−6 ≤ k ≤ 6
−10 ≤ k ≤ 10
−27 ≤ l ≤ 27
−27 ≤ l ≤ 27
−39 ≤ l ≤ 39
Reflections collected
Independent reflections
Completeness to 2Θ = 25.000
Refinement method
19468
19144
27502
3797
[R(int) = 0.0352]
18517
2654
[R(int) = 0.0218]
99.7%
2675
[R(int) = 0.0418]
100.0%
2432
[R(int) = 0.0292]
99.8%
◦
99.9%
Full-matrix
Full-matrix
Full-matrix
Full-matrix
least-squares on F2
least-squares on F2
least-squares on F2
least-squares on F2
Data/restraints/parameters
Goodness-of-fit on F2
2654/268/218
1.089
2675/268/221
1.034
3797/3/178
1.015
2432/0/174
1.066
R1 = 0.0405
wR2 = 0.1063
R1 = 0.0410
wR2 = 0.1133
R1 = 0.0428
wR2 = 0.0946
R1 = 0.0382
wR2 = 0.1035
Final R indices (I > 2σ(I))
R1 = 0.0474
wR2 = 0.1120
R1 = 0.0530
wR2 = 0.1227
R1 = 0.0633
wR2 = 0.1040
R1 = 0.0461
wR2 = 0.1097
R indices (all data)
Extinction coefficient
0.023(4)
n/a
n/a
0.012(2)
Largest difference peak and
0.177 and −0.189
0.181 and −0.254
0.202 and −0.145
0.245 and −0.239
−3
hole (e·Å
)

Molecules 2016, 21, 1742
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3.5. Urease Inhibition Assay
Both ligands and their copper(II) complexes were tested for their urease inhibition. The
measurement of urease inhibitory activities was carried out using the modified method reported
by Tanaka et al. [49]. Generally, the assay mixture containing 75
tested compounds (dissolved in DMSO) with various concentrations was pre-incubated for 15 min on
a 96-well assay plate. Afterwards, 75 L of phosphate buffer at pH 6.8 containing phenol red (0.18 mM)
µL of jack bean urease and 75 µL of
µ
and urea (400 mM) were added and the mixture was incubated at room temperature. The reaction
time required for enough ammonium carbonate to form to raise the pH phosphate buffer from 6.8
to 7.7 was determined by Biotek Synergy HT micro-plate reader (560 nm) with end-point indicated
by the colour change of the phenol-red indicator. Acetohydroxamic acid was used as the standard
reference substance.
3.6. Determination of Antimicrobial Activity
For testing of antimicrobial activity, bacteria E. coli (CNCTC 327/73), S. aureus (CNCTC Mau
82/78), and yeast C. albicans (CNCTC 59/91) were used. Bacteria were grown aerobically in nutrient
broth (NB, Imuna, Šarišské Michal’any, Slovakia) and yeast in Sabouraud dextrose broth (SDB, Difco,
Bordeaux, France) for 18 h at 37 ◦C or 48 h at 24 ◦C, respectively. Cultures were then maintained at
4 ◦C on appropriate solid medium: Endo agar (EA, Oxoid, Basingstoke, UK) for E. coli, blood agar
(BA, Biomark, Pune, India) for S. aureus, and Sabouraud dextrose agar (SDA, Difco) for C. albicans.
Working cultures were prepared by incubation of a single colony of each microorganism in NB
◦
◦
(bacteria) or SDB (yeast) for 18 h at 37 C or 48 h at 24 C, respectively. A microbial suspension
was prepared in saline solution (0.85% NaCl) according to the McFarland standard No. 0.5 using
Lambda 35 UV/VIS Spectrophotometer (PerkinElmer, Waltham, MA, USA), to obtain a concentration
of ca. 1.5 × 108 cfu/cm3. After dilution in an appropriate liquid medium (NB for bacteria and SDB
for yeast), a working concentration 1.5 × 107 cfu/cm3 was prepared.
The antimicrobial activity assay was performed according to Bilková et al. [50] with some
modifications. Stock solutions (200 mM) of test compounds and the standards (isoniazid and
ciprofloxacin) were prepared in DMSO immediately before use. Working test compounds and the
standards were prepared by serial dilution of stock solutions in sterile doubly concentrated NB or SDB
to a final volume 100 mL within the 96-well microplates. Freshly prepared inoculum (5 mL) of the tested
microorganism was added to each appropriate well (bacteria into the plates with NB, C. albicans into the
plates with SDB). The final concentration of each organism in each well was ca. 7.5 × 105 cfu/cm3. The
concentration of tested compounds ranged from 100 mM to 0.2
µM, except for compounds L1 and L2,
which were tested in a range from 75 mM to 0.2 M due to their lower available amount. Each
µ
concentration was tested in triplicate. For each assayed sample and microorganism, the following
controls were applied: blank, uninoculated media without test compound to account for changes
in the media during the experiment; negative control, uninoculated media containing only the test
compound; positive control 1, inoculated media without compound, the positive control 2, inoculated
media with serial dilution of DMSO without test compound, thereby assessing any activity of the
◦
◦
solvent. The 96-well plates were incubated aerobically for 24 h at 37 C or 24 C, depending on
whether bacteria or yeast were grown, respectively. Afterwards, 5 mL of each well was inoculated on
the corresponding agar plate (BA for S. aureus, EA for E. coli, and SDA for C. albicans). Bacteria and
yeast were grown aerobically for 24 h at 37 ◦C or 24 ◦C, respectively. The MIC was expressed as the
lowest concentration of the compound inhibiting growth of the microorganism on the agar plates for
all parallel samples in comparison with that of the positive control after 24 h.
4. Conclusions
Six Schiff base ligands and four copper(II) complexes from these ligands were prepared and
characterized. The X-ray crystallographic analysis of four Schiff bases was performed and their spatial

Molecules 2016, 21, 1742
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structure determined in solid state. Interestingly, the ligands are not planar, showing varying degrees
of distortion of the conjugated system. The Schiff bases L1 and L4 exhibited interesting in vitro activity
against C. albicans. All four copper(II) complexes showed excellent inhibition of Jack bean urease.
Acknowledgments: This work was supported by the Slovak Research and Development Agency under the
contract no. APVV-0516-12. This study was financially supported by the Research and Development Agency of
the Slovak Republic under the contract No. APVV-15-0079 and the Scientific Grant Agency of the Slovak Republic
(VEGA Project 1/0871/16).
Author Contributions: L.H. designed and coordinated the research, synthesized some of the compounds, and
prepared several monocrystals for X-ray crystal structure analysis, wrote the manuscript (except crystallographic
and antimicrobial activity parts); S.V. performed part of the synthesis and obtained several monocrystals for
X-ray crystal structure analysis; A.B. carried out the microbiological experiments and wrote the corresponding
paragraph; P.H. performed single-crystal X-ray structure analysis and wrote the paragraph concerning the
crystallographic data; J.V. provided data on urease inhibition; J.K. supported the data acquisition for single-crystal
X-ray structure analysis; F.D. provided support with the discussion of data and manuscript preparation.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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