Pyrazoline derivatives of acryloyl substituted ferrocenyl ketones: Synthesis, antimicrobial activity and structural properties

Article abstract of DOI:10.1016/j.ica.2017.11.061

A series of ferrocenyl ketones were synthesized in reaction with ferrocene and corresponding substituted acryloyl chlorides, following previously described procedure. Synthesized products have conjugated enone system, which is suitable for further transformations. In a reaction with hydrazine in acidic medium (acetic acid) new pyrazoline derivatives were obtained. Their antimicrobial properties have been tested. Synthesized pyrazoline derivatives demonstrated expressed in vitro antimicrobial activity towards 12 strains of microorganisms inhibiting all tested bacteria and fungi. The most potent compound in all cases was sorbyl derivative; for bacteria activity was very close to streptomycin, and for fungi in one case the same as ketoconazole. It is established that this compound can be a new, potential antimicrobial agent with minimum inhibitory concentrations from 0.039 to 0.312 mg/mL. One of the starting compounds and two products were crystal substances, suitable for the single crystal X-ray diffraction analysis, which confirmed undoubtedly their structures.

Full text of DOI:10.1016/j.ica.2017.11.061

Inorganica Chimica Acta 471 (2018) 570–576
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Research paper
Pyrazoline derivatives of acryloyl substituted ferrocenyl ketones:
Synthesis, antimicrobial activity and structural properties
a
a
a,
⇑
b
b
ˇ
´
´
´
Adrijana Burmudzija , Jovana Muškinja , Zoran Ratkovic , Marijana Kosanic , Branislav Rankovic ,
c
c
´
´
Sladjana B. Novakovic , Goran A. Bogdanovic
a Department of Chemistry, Faculty of Science, University of Kragujevac, Radoja Domanovi´ca 12, 34000 Kragujevac, Serbia
b Department of Biology and Ecology, Faculty of Science, University of Kragujevac, Radoja Domanovi´ca 12, 34000 Kragujevac, Serbia
c
ˇ
Vinca Institute of Nuclear Sciences, Laboratory of Theoretical Physics and Condensed Matter Physics, University of Belgrade, P.O. Box 522, 11001 Belgrade, Serbia
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of ferrocenyl ketones were synthesized in reaction with ferrocene and corresponding substituted
acryloyl chlorides, following previously described procedure. Synthesized products have conjugated
enone system, which is suitable for further transformations. In a reaction with hydrazine in acidic med-
ium (acetic acid) new pyrazoline derivatives were obtained. Their antimicrobial properties have been
tested. Synthesized pyrazoline derivatives demonstrated expressed in vitro antimicrobial activity towards
12 strains of microorganisms inhibiting all tested bacteria and fungi. The most potent compound in all
cases was sorbyl derivative; for bacteria activity was very close to streptomycin, and for fungi in one case
the same as ketoconazole. It is established that this compound can be a new, potential antimicrobial
agent with minimum inhibitory concentrations from 0.039 to 0.312 mg/mL. One of the starting com-
pounds and two products were crystal substances, suitable for the single crystal X-ray diffraction analy-
sis, which confirmed undoubtedly their structures.
Received 15 September 2017
Received in revised form 27 November 2017
Accepted 28 November 2017
Available online 2 December 2017
Keywords:
Ferrocenyl ketones
Enone system
Pyrazoline derivatives
Crystal structure
Antimicrobial activity
Ó 2017 Elsevier B.V. All rights reserved.
1. Introduction
and the ferrocene scaffold into the same molecule might have an
attracting structural result for development of novel antimicrobial
Chalcones (1,3-diaryl-2-propen-1-ones), and chalcone-like
compounds (with similar enone system) are an important class
of organic compounds, since they often represent core structure
of many natural products and exhibit various pharmacological
and biological activities. Antimicrobial [1–6], antioxidant [7–9],
antifungal [5,6,10], antimalarial [11–13], anti-inflammatory
[14,15] and anticancer activity [16–21] are well expressed and
explored.Enone system presented in chalcones is the often a key
part of substrates; it is almost planar and have trans-double bond.
This structure enables various transformations of enone system,
which could be easily converted into different heterocyclic deriva-
tives, in reactions with urea, thiourea, hydroxylamine, hydrazine,
guanidine [22,23], forming heterocyclic unit between aromates.
Ferrocenyl derivatives are among the most promising
organometallic compounds which can be used in microbiological
research. In continuation of our interest in synthesis of ferrocene
containing heterocycles exhibiting some biological activities
[24–27], we expected that incorporation of pyrazoline fragment
agents.Herein we wish to report on synthesis, spectral characteri-
zation and evaluation of antimicrobial activity on some strains of
microorganisms a series of novel Fc-pyrazoline derivatives, pre-
pared from chalcone-like ketones (2a–e) and heterocyclic chalcone
2f.All new products were characterized by their spectral data (IR,
MS, 1H NMR and 13C NMR). Compounds 2c, 3c and 3f gave crystals
suitable for the X-ray analysis
2. Experimental
2.1. Chemistry
2.1.1. Materials and measurements
All starting chemicals were commercially available and used as
received, except for the solvents being purified by distillation. Col-
umn chromatography were carried out using silica gel 60 (Merck,
230–400 mesh ASTM); for TLC was used Silica gel 60 F254-pre-
coated plates (Merck); layer thickness 0.2 mm. IR spectra: Per-
kin-Elmer Spectrum One FT-IR spectrometer with a KBr disc,
m in
cmÀ1. NMR spectra: Varian Gemini 200 MHz spectrometer (200
MHz for 1H and 50 MHz for 13C), using CDCl3 as the solvent and
⇑
Corresponding author.
E-mail address: wor@kg.ac.rs (Z. Ratkovic´).
https://doi.org/10.1016/j.ica.2017.11.061
0020-1693/Ó 2017 Elsevier B.V. All rights reserved.

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A. Burmudzija et al. / Inorganica Chimica Acta 471 (2018) 570–576
571
TMS as the internal standard. 1H and 13C NMR chemical shifts were
reported in parts per million (ppm) and were referenced to the sol-
vent peak; CDCl3 (7.26 ppm for 1H and 76.90 ppm for 13C). Multi-
plicities are represented by s (singlet), d (doublet), t (triplet), q
(quartet) and m (multiplet). Coupling constants (J) are in Hertz
(Hz). Mass spectrometry was performed by Waters Micromass
ZQ mass spectrometer and MassLynx software for control and data
processing. Electro spray ionization in the positive mode was used.
The electro spray capillary was set at 4.3 kV and the cone at 40 V.
The ion source temperature was set at 125 °C and the nitrogen flow
rates were 400 L/h and 50 L/h, for desolvation and cone gas flow
respectively. The collision energy was 40 eV. The melting point of
products was determined by using MelTemp1000 apparatus.
(s, 2H); 13C NMR: d 20.3, 21.9, 41.6, 51.8, 67.2, 67.5, 69.4, 70.2,
75.7, 156.0, 168.3 (CO). ESI-MS (40 eV): m/z (%) = 310 (100%)
[M]+, 268 (31%), 185 (10%), 121 (27%), 43 (8%).
2.1.3.3. 1-(5,5-Dimethyl-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)
ethanone (3c). Cinnabar red crystals; mp 142 °C; Yield 61.2%; IR
(cmÀ1): 3086, 2929, 1651 (CO), 1498, 1405, 1314, 1105, 1012;
NMR: d 1H NMR: d 1.65 (s, 6H), 2.29 (s, 3H), 2.99 (s, 2H), 4.18 (s,
5H), 4.37 (t, J = 1.8 Hz, 2H), 4.57 (t, J = 2.0 Hz, 2H); 13C NMR: d
23.3, 26.3, 50.6, 62.8, 67.1, 69.3, 70.1, 72.3, 76.0, 153.6, 169.0
(CO). ESI-MS (40 eV): m/z (%) = 324 (100%) [M]+, 283 (27%), 267
(63%), 185 (9%), 121 (27%), 43 (9%).
2.1.3.4. 1-(4-Methyl-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)etha-
none (3d). Cinnabar red crystals; mp 78–79 °C; Yield 81.2%; IR
(cmÀ1): 3099, 2974, 1655 (CO), 1496, 1449, 1308, 1160, 1106,
1028, 997; 1H NMR: d 1.14–1.85 (m, 2H), 1.71 (s, 3H), 2.33 (s,
3H), 3.29–3.45 (m, 1H), 4.18 (s, 5H), 4.41 (m, 2H), 4.59 (m, 1H),
4.69 (m, 1H); 13C NMR: d 19.8, 21.3, 40.5, 51.9, 67.4, 67.5, 69.6,
69.9, 70.2, 75.7, 161.4, 168.9 (CO). ESI-MS (40 eV): m/z (%) = 310
(100%) [M]+, 268 (32%), 185 (19%), 121 (33%), 44 (30%).
2.1.2. Procedure for the synthesis of 1-ferrocenyl-2,4-hexadien-1-one
(2e)
The ferrocenyl ketone, 1-ferrocenyl-2,4-hexadien-1-one (sorbyl
ferrocene), 2e, was prepared by following procedure: sorbic acid,
1.12 g (10 mmol) was dissolved in a 150 mL of dried CH2Cl2, and
1 mL of PCl3 was added. Closed vessel with solution was stirred
overnight at room temperature. To this solution 1.98 g of ferrocene
(10 mmol) was added following with 1.44 g (10 mmol) of anhy-
drous AlCl3. Solution became deep blue from formed complex,
and stirring was continued for next 2–3 h. Reaction mixture was
poured out in 100 mL of 2 M HCl solution and shaken well. Organic
phase was separated, and water layer was extracted with 50 mL of
CH2Cl2. Combined organic layers were washed with 2 Â 100 mL of
water and dried over anhydrous Na2SO2. The main part of solvent
was removed by distillation and concentrated crude mixture was
filtered through SiO2 pad. Separation of product was performed
on SiO2 column using CH2Cl2 as eluent. Deep red band belongs to
ferrocenyl ketone 2e. Solvent was evaporated by distillation and
products crystalizes on standing.
2.1.3.5. (E)-1-(3-Ferrocenyl-5-propenyl-4,5-dihydro-1H-pyrazol-1-yl)
ethanone (3e). Red-orange oil; Yield 78.4%; IR (cmÀ1): 3086, 1654
(CO), 1497, 1412, 1378, 1106, 1007; 1H NMR: d 1.71 (dt, J = 6.4,
1.3 Hz, 3H), 2.82 (dd, J = 17.0, 3.8 Hz, 1H), 3.32 (dd, J = 17.2, 11.2
Hz, 1H), 4.12 (s, 5H), 4.39 (m, 2H), 4.54 (dt, J = 3.8, 1.6 Hz, 1H),
4.66 (dt, J = 3.8, 1.8 Hz, 1H), 5.01 (ddd, J = 10.3, 5.6, 4.0 Hz, 1H),
5.48 (ddq, J = 15.2, 6.0, 1.0 Hz, 1H), 5.69 (ddd, J = 15.2, 6.1, 1.0 Hz,
1H); 13C NMR: d 21.8, 43.7, 55.9, 58.9, 67.1, 67.6, 69.3, 70.2, 70.4,
75.4, 108.9, 111.6, 117.2, 134.7, 148.4, 149.3, 155.8, 168.2 (CO).
ESI-MS (40 eV): m/z (%) = 336 (100%) [M]+, 294 (17%), 185 (6%),
121 (21%), 43 (7%).
Cinnabar red crystals; mp 139–140 °C; Yield 75%; IR (cmÀ1):
3118, 3017, 1652, 1627, 1583, 1457, 1376, 1267, 1104, 1072,
1001; 1H NMR: d 1.89 (d, J = 5.4 Hz, 3H), 4.18 (s, 5H), 4.54 (t, J =
2.2 Hz, 2H), 4.83 (t, J = 1.8 Hz, 2H), 6.22–3.38 (m, 2H), 6.48 (d, J =
15.4 Hz, 1H), 7.27–7.44 (m, 1H); 13C NMR: d 18.8, 69.6, 70.0,
72.4, 80.7, 124.3, 130.5, 139.7, 141.2, 193.3.
2.1.3.6. 1-(5-(Furan-2-yl)-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)
ethanone (3f). Cinnabar red crystals; mp 153 °C; Yield 59.5%; IR
(cmÀ1): 3101, 1651 (CO), 1498, 1416, 1376, 1309, 1149, 1156,
1018, 1006; 1H NMR: d 2.32 (s, 3H), 3.39 (m, J = 17.2, 11 Hz, 2H),
4.21 (s, 5H), 4.41 (m, 2H), 4.51 (m, 1H), 4.75 (m, 1H), 5.63 (m, J
= 11, 0.23 Hz, 1H), 6.35 (s, 2H), 7.34 (s, 1H); 13C NMR: d 21.8,
39.3, 52.6, 66.8, 67.9, 69.5, 70.2, 70.5, 75.2, 107.5, 110.6, 141.7,
156.1, 168.2 (CO). ESI-MS (40 eV): m/z (%) = 362 (100%) [MÀ16]+,
320 (29%), 185 (4%), 121 (21%), 43 (10%).
2.1.3. Synthesis of pyrazoline derivatives (3a–f)
To a stirred solution of 2a–f (10 mmol), in acetic acid (10 mL)
hydrazine monohydrate (1.25 mL, 25 mmol) was added and reac-
tion mixture was heated to reflux for 3 h. The solvent was evapo-
rated under reduced pressure and water (50 mL) was added to
the colored residue. Products were extracted from the reaction
mixture with toluene or toluene/EtOAc (95:5) mixture. After
removal of the main part of solvent the residue was filtered over
SiO2 pad. After evaporation of solvent oily residue was dissolved
in ether, from which some of products 3a–f crystallize on standing
in deepfreeze.
2.2. Antimicrobial activity
Antimicrobial activities of tested compounds were evaluated
against five strains of bacteria: Staphylococcus aureus (ATCC
25923), Bacillus subtilis (ATCC 6633), B. cereus (ATCC 10987),
Escherichia coli (ATCC 25922) and Proteus mirabilis (ATCC 29906)
and seven species of fungi: Aspergillus flavus (ATCC 9170), A. fumi-
gatus (ATCC 1022), Candida albicans (ATCC 10259), Penicillium itali-
cum (ATCC 10454) and Trichophyton mentagrophytes (ATCC 9533),
Geotrichum candidum (ATCC 34614) and Mucor mucedo (ATCC
20094) obtained from the American Type Culture Collection
(ATCC).
The bacteria isolates were picked from overnight cultures in
Mueller-Hinton agar and suspensions were prepared in sterile dis-
tilled water. The turbidity of suspensions was adjusted by compar-
ing with 0.5 McFarland’s standard to approximately 108 CFU/mL.
Fungal suspensions were prepared from 3- to 7-day-old cul-
tures that grew on a potato dextrose agar except for C. albicans that
was maintained on Sabourad dextrose (SD) agar. The spores were
rinsed with sterile distilled water, used to determine turbidity
spectrophotometrically at 530 nm NCCLS [28]. The resulting sus-
pensions were approximately 106 CFU/mL.
2.1.3.1. 1-(3-Ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)ethanone (3a).
Light orange crystals; mp 184–185 °C; Yield 85%; IR (cmÀ1): 3085,
1650 (CO), 1502 (C@Carom.), 1418, 1311, 1104, 1029, 1011; 1H
NMR: d 2.33 (s, 3H), 3.06–3.15 (m, 2H), 3.91–4.01 (m, 2H), 4.18
(s, 5H), 4.39 (t, J = 1.8 Hz, 2H), 4.61 (t, J = 2.0 Hz, 2H); 13C NMR: d
21.4, 33.0, 43.3, 67.3, 69.4, 70.2, 75.4, 157.3, 168.5 (CO). ESI-MS
(40 eV): m/z (%) = 296 (100%) [M]+, 254 (35%), 185 (12%), 121
(39%), 43 (7%).
2.1.3.2. 1-(5-Methyl-3-ferrocenyl-4,5-dihydro-1H-pyrazol-1-yl)etha-
none (3b). Red-orange oil; Yield 81.2%; IR (cmÀ1): 3086, 1648
(CO), 1498, 1413, 1314, 1106, 1027, 1006; 1H NMR: d 1.38 (d, J =
6.6 Hz, 3H), 2.31 (s, 3H), 2.65 (dd, J = 17.2, 3.4 Hz, 1H), 3.32 (dd, J
= 17.2, 10.8 Hz, 1H), 4.19 (s, 5H), 4.39 (s, 2H), 4.57 (m, 1H), 4.63

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Table 1
The 96-well microtiter assay using resazurin as the indicator of
Crystallographic data for the crystal structures of 2c, 3c and 3f.
cell growth [29,30], was employed for the determination of the
minimum inhibitory concentration (MIC) of the active compo-
nents. Starting solutions of tested compounds were obtained by
dissolving it in 5% DMSO. Next, serial twofold dilutions of tested
compounds were made in a concentration range from 10 to
0.004 mg/mL in sterile 96-well plates containing Mueller-Hinton
broth for bacterial cultures and a SD broth for fungal cultures. After
that, diluted bacterial and fungal suspensions were added to
appropriate wells and finally, resazurin solution was added as an
indicator to each well. The inoculated plates were incubated at
37 °C for 24 h for bacteria and 28 °C for 72 h for fungi. The MIC
was determined visually and defined as the lowest concentration
of tested compounds that prevented resazurin color change from
blue to pink. Streptomycin and ketoconazole were used as a posi-
tive control. Solvent control test was performed to study an effect
of 5% DMSO on the growth of microorganism.
Empirical formula
Formula weight
C15H16FeO
268.13
C17H20FeN2O
324.20
C19H18FeN2O2
362.20
Colour, crystal shape
Orange, prism Orange, prism Orange, prism
Crystal size (mm3)
0.19 Â 0.20 Â
0.45 Â 0.54 Â
0.20 Â 0.28 Â
0.41
0.61
0.45
Temperature (K)
Wavelength (Å)
Crystal system
Space group
Unit cell dimensions
a (Å)
293(2)
293(2)
293(2)
0.71073
Monoclinic
P21/c
0.71073
Monoclinic
P21/n
0.71073
Triclinic
P1À
7.4494(3)
16.2301(7)
10.6694(5)
90
10.1531(5)
15.5466(7)
10.8705(5)
90
10.6668(4)
10.9147(4)
15.2710(6)
109.838(4)
100.165(3)
94.941(3)
1625.6(1)
4
b (Å)
c (Å)
a
(°)
b (°)
96.858(3)
90
1280.7(1)
4
1.391
1.157
112.631(6)
90
1583.8(2)
4
1.360
0.952
c
(°)
V (Å3)
Z
Dcalc (Mg/m3)
1.480
0.941
l
(mmÀ1
)
2.3. X-ray crystallography
h range for data collection 2.51–29.06
(°)
2.62–29.06
2.58–29.07
Reflections collected
Independent reflections,
Rint
7073
2954, 0.0215
14,032
3773, 0.0214
28,480
7740, 0.0281
Single-crystal X-ray diffraction data for compounds 2c, 3c and
3f were collected on an Oxford Gemini S diffractometer equipped
with a CCD detector, using monochromatized Mo K
a radiation
Data/
2954/0/160
3773/36/221
7740/36/463
restraints/parameters
Goodness-of-fit
Final R1/wR2 indices
[I > 2r(I)]
Final R1/wR2 indices (all
(k = 0.71073 Å). Data reduction and empirical absorption correction
were performed with CrysAlisPRO [31]. The structures were solved
by direct methods using SHELXS and refined on F2 by full-matrix
least-squares using SHELXL [32]. All non-H atoms were refined
anisotropically. H atoms were placed at geometrically calculated
positions with the CAH distances fixed to 0.93 from Csp2 and
0.98, 0.97 and 0.96 Å from methine, methylene and methyl Csp3,
respectively. The corresponding isotropic displacement parameters
of the H atoms were equal to 1.2 Ueq and 1.5 Ueq of the parent Csp2
and Csp3, respectively. In compound 3c the substituted cyclopenta-
dienyl ring was found disordered over two sites with occupancies of
0.59(2) and 0.41(2), also in compound 3f the unsubstituted
cyclopentadienyl ring in the crystallographically independent
molecule B was found disordered over two sites with occupancies
of 0.56(2) and 0.44(2). In both compounds the disordered rings
were modeled by AFIX 56, while the thermal parameters of the cor-
responding carbon atoms were restrained by SIMU instruction from
the SHELXL [32]. Crystallographic details for structure analysis of
compounds 2c, 3c and 3f are summarized in Table 1. The crystal
structures of compounds including the atom labeling schemes are
presented in Fig. 1, while selected geometrical parameters are listed
in Tables 3 and 4. Compound 3f crystalizes with two crystallograph-
ically independent molecules in the asymmetric unit. There is no
symmetry relation between two independent molecules (Fig. 1c).
Figures were produced using ORTEP-3 [33] and MERCURY [34].
The software used for the preparation of the materials for publica-
tion: WINGX [35], PLATON [36], PARST [37].
1.115
1.014
0.0365/
0.0865
0.0505/
0.0940
0.269/À0.215
1.047
0.0390/
0.0948
0.0519/
0.1020
0.440/À0.320
0.0382/
0.0861
0.0530/
0.0938
data)
Largest diff. peak and hole 0.223/À0.453
(e ÅÀ3
)
heterocyclic ring is more reactive and polarized (heteroatom close
to the conjugated double bonds) than common aromatic ring, and
we suppose that will have different activity from chalcone like
compounds.
Synthesized enone compounds 2a–f (acyl-ferrocenes) reacts
with hydrazine in boiling acetic acid [38], forming new, crystalline
pyrazoline derivatives 3a–f, Scheme 2.
3.2. Spectral characterization
All synthesized compounds were characterized by IR, 1H and
13C NMR spectral data. In IR spectra of the synthesized compounds
the most recognizable band appears in the carbonyl group region
1661–1648 cmÀ1 (CH3COA).
In 1H NMR spectra of compounds 3a–f could be recognized
characteristic signals corresponding to the 4H- and 5H-protons of
pyrazoline ring. Those protons appear at 2.65–4.57 region, as sin-
glet (2.99, 2H, compound 3c), multiplet (3.06–3.15 or 3.91–4.01,
2H, compound 3a) or doublet of doublet (2.65, 1H, compound 3c,
2.82, 1H compound 3e). Position and structure of signals are
dependent on presence of substituents at 4- and 5-position of
pyrazoline ring.
3. Results and discussion
3.1. Synthesis
As mentioned in the Introduction section, we prepared, starting
from conjugated ferrocenyl ketones 2a–f, a series of ferrocenyl
pyrazoline derivatives 3a–f following the literature procedure
[38], and tested their microbial activity.
Ferrocene 1 was acylated in situ with prepared acyl chlorides
under Friedel-Crafts conditions yielding corresponding acyl-fer-
rocenes 2a–e on which enone system is presented. Compounds
2a–d were earlier described [39–43] whereas 2e is a new com-
pound, Scheme 1, and their spectral data are given. The main rea-
son why chalcone 2f was included in the experiments is fact that
3.3. Antimicrobial activity
Antimicrobial activity of compounds 3a–f were tested against
12 strains of microorganisms, including the agents of human, ani-
mal and plant diseases, mycotoxin producers, and food spoilage
agents. The results of in vitro testing of antibacterial and antifungal
activities of tested compounds in relation to selected species of
microorganisms are shown in Table 2. The MIC values are the same
in several cases because a series of double dilutions of the tested

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A. Burmudzija et al. / Inorganica Chimica Acta 471 (2018) 570–576
573
Fig. 1. The molecular structure of: (a) 2c, (b) 3c and (c) 3f with the atom-labeling scheme. Only the major component of molecules 3c and 3f is presented (see Figs. S2 and S4,
for disordered 3c and 3f, respectively). Displacement ellipsoids are drawn at the 30% probability level.
Table 2
Antibacterial and antifungal activity of compounds 3a–f, minimum inhibitory concentration (MIC).
Compound Microorganisms
Staphylococcus
aureus
Bacillus
subtilis
Bacillus
cereus
Escherichia
coli
Proteus
mirabilis flavus
Aspergillus
Aspergillus
fumigatus
Candida
albicans
Penicillium
italicum
Trichophyton
mentagrophytes candidum
Geotrichum
Mucor
mucedo
3a
3b
3c
3d
3e
3f
0.312
0.156
0.312
0.312
0.078
0.625
0.156
0.039
0.078
0.078
0.039
0.156
0.016
0.078
0.039
0.078
0.078
0.039
0.156
0.016
0.625
0.156
0.312
0.312
0.156
1.25
0.312
0.156
0.312
0.312
0.156
1.25
2.5
0.312
2.5
1.25
0.312
2.5
2.5
1.25
2.5
0.312
1.25
0.625
0.312
1.25
1.25
1.25
2.5
0.312
0.625
0.625
0.312
1.25
0.312
0.625
0.625
0.156
0.625
0.039
0.312
0.625
0.312
0.156
1.25
0.156
0.625
0.625
0.312
0.625
0.078
0.312
0.625
1.25
0.156
1.25
Antibiotics 0.031
0.062
0.062
0.156
0.156
0.156
0.078
0.156
Values given as mg/mL.
Antibiotics: Streptomycin (for bacteria) and Ketoconazole (for fungi).
compounds was used in the experiment against every tested
microorganism.
The other tested fungi have shown sensitivity at the higher concen-
trations (MIC ranged from 0.312 to 5 mg/mL). Solvent control
showed that 5% DMSO had no inhibitory effect on the tested
microorganisms.
Among tested compounds, 3e showed marked inhibitory activ-
ity of the growth of selected bacteria and fungi in low concentra-
tions. This compound can be a new, potential antimicrobial agent
since these selected tested microorganisms have been developing
resistance to common antibiotics.
Regarding the tested bacteria, the MIC for tested compounds
ranged from 0.039 to 2.5 mg/mL. The most sensitive bacteria were
B. subtilis and B. cereus with MIC values between 0.039 and 0.312
mg/mL. Moderate activity was shown against S. aureus with MIC at
0.078–1.25 mg/mL, while the highest resistance was shown in P.
mirabilis and E. coli.
Antifungal activity was a slightly weaker than antibacterial. The
MIC for tested compounds relative to the fungi ranged from 0.156
to 5 mg/mL. C. albicans and T. mentagrophytes appeared to be the
most sensitive fungi (MIC values were from 0.156 to 1.25 mg/mL).
In this study, the noticeable results were that the tested com-
pounds inhibited the growth of Gram-negative bacteria and fungi
at the highest tested concentrations than Gram-positive bacteria.

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A. Burmudzija et al. / Inorganica Chimica Acta 471 (2018) 570–576
574
Table 3
non-H atoms of 2c molecule is C12AC13 bond of the aliphatic frag-
ment [1.331(3) Å]. The bonds involving carbonyl C11 atom have
similar lengths [C1AC11 1.468(3) and C11AC12 1.476(3) Å], both
shorter than a single CAC bond length (1.54 Å [44]). The approxi-
mately planar aliphatic fragment noticeably deviates from the level
of Cp ring forming the dihedral angle with the corresponding Cp
mean plane of 23.0(2)°. While the carbonyl O1 alone deviates from
the Cp plane for 0.093(4) Å [torsion angle C2AC1AC11AO1 = À19.8
(3)°] the deviation gradually increases toward the terminal atoms
resulting in maximal deviation for the methyl C15 [1.458(8) Å].
The carbonyl O1 as well as the terminal methyl groups of the ali-
phatic fragment plays a important role in intermolecular interac-
tions, stabilizing the crystal structure of 2c.
Selected bond lengths (Å) and bond angles (°) in crystal structures of 3c and 3f.
3c
3f
A
B
N1AN2
N1AC13
N1AC14
N2AC11
O1AC14
C11AC12
C12AC13
C14AC15
N1AN2AC11
N1AC13AC12
N1AC14AO1
N2AN1AC13
N2AC11AC12
C11AC12AC13
1.386(2)
1.492(3)
1.346(3)
1.286(3)
1.232(3)
1.485(3)
1.532(3)
1.494(3)
108.1(2)
100.8(2)
120.7(2)
112.8(2)
114.4(2)
103.5(2)
1.400(3)
1.484(3)
1.352(3)
1.286(3)
1.222(3)
1.505(3)
1.529(3)
1.498(4)
107.4(2)
100.5(2)
120.0(2)
112.3(2)
114.0(2)
102.2(2)
1.392(3)
1.481(3)
1.347(3)
1.285(3)
1.222(3)
1.505(3)
1.543(3)
1.499(4)
107.8(2)
100.6(2)
120.8(2)
112.9(2)
114.1(2)
102.3(2)
Due to the absence of polar hydrogen bonding donors, the crys-
tal packing of 2c is mainly stabilized by weak CAH. . .O and
CAH. . .p interactions (Table 4). The bifurcated hydrogen bond
C5AH5. . .O1 and C12AH12. . .O1 involving Cp and aliphatic CAH
donors is the shortest intermolecular interaction which connects
the molecules into a chain extending along the c crystallographic
axis (Table 4, see also Supplementary material, Fig. S1a). Within
the chain the Fc units of linked molecules have nearly orthogonal
mutual orientation, so that the dihedral angle between the substi-
tuted Cp rings of the neighboring units equals to 87.2°. In this
These results are not unexpected, considering that is known that
Gram-negative bacteria, in contrast to Gram-positive bacteria, con-
tain the outer membrane which serves as a permeability barrier
and prevent the entry of noxious compounds including antibacte-
rial compounds. The cell walls of fungi are the least permeable and
consist of polysaccharides such as chitin and glucan, and it is the
reason for their greater resistance [29,30,28].
The experimental results of antimicrobial activity of tested
compounds may open the possibility of their potentially use in
medicine, food production and pharmaceutical industry.
arrangement, each Cp ring of Fc units serves as
methyl donors in two weak interactions, C14AH14b. . .
C15AH15b. . . (Table 4, Fig. S1b). The 2D layers developed by
these CAH. . . interactions in ab plane interlink by CAH. . .O chains
p-acceptor of
p
and
p
p
to form 3D crystal structure of 2c.
The ferrocene-based compound 3c (Fig. 1b) comprises the pyra-
zoline ring bonded to one Cp ring of the Fc unit. Selected bond dis-
tances and angles are summarized in Table 3.
3.4. Description of crystal structures 2c, 3c and 3f
The pyrazoline ring attached to the disordered Cp ring is fully
ordered. The geometry of this ring is normal and closely compara-
ble with those of previously reported Fc derivatives comprising the
N-acetyl-pyrazoline substituent [45–47] The single C12AC13 bond
[1.532(3) Å] is the longest bond of the pyrazoline ring, while the
remaining bonds display intermediate lengths reflecting the exis-
In the precursor compound 2c (Fig. 1a) the cyclopentadienyl
(Cp) rings of the ferrocene unit (Fc) adopt nearly eclipsed geome-
try, with the C1–Cg1–Cg2–C6 torsion angle of 3.2° (Cg1 and Cg2
are centroids of the substituted and unsubstituted Cp rings, respec-
tively). The Cp rings show a slight mutual tilting as evidenced by a
dihedral angle between their mean planes of 2.5(2)°. The Fe. . .Cg
distances display typical features of the monosubstituted ferroce-
nes where the distance of the Fe atom towards centroid of the sub-
stituted Cp ring is somewhat shorter than a distance to the
centroid of unsubstituted ring (1.639 and 1.652 Å, respectively).
The aliphatic fragment attached to the Fc unit is essentially planar
with the r.m.s. deviation of non-H atoms from the mean plane of
0.027 Å and the largest deviation of atom C11 [0.044(2) Å]. Besides
the carbonyl C11AO1 [1.227(2) Å] the shortest bond involving
tence of the
p-electron delocalization over the ring system
(Table 3). The r.m.s. deviation of the non-H atoms constituting
the pyrazoline ring is 0.026 Å with the largest deviation from the
mean plane observed for atom C13 [0.035(2) Å] leading to an
envelope-like conformation. As expected, the acetyl group
attached to N1 atom lies in the pyrazoline plane [dihedral angles
between the planes is 1.5(3)°], while the oxygen atom takes trans
position relative to pyrazoline N2 [torsion angle N2AN1AC14AO1
Table 4
Geometrical parameters for intermolecular CAH. . .O and CAH. . .
C16aAO2a and C16bAO2b ring, respectively).
p interactions in crystal structures of 2c, 3c and 3f (Cg1, Cg2, Cg3 and Cg4 are centroids of the C1AC5, C6AC10,
DAH. . .A
DAH (Å)
H. . .A (Å)
DAH. . .A (°)
Symmetry codes:
2c
C5AH5. . .O1
C12AH12. . .O1
C15AH15b. . .Cg1
C14AH14b. . .Cg2
0.93
0.93
0.96
0.96
2.45
2.49
3.20
3.25
158
174
151
131
x, Ày + 0.5, z À 0.5
x, Ày + 0.5, z À 0.5
x + 1, y, z
x, y + 0.5, Àz + 1.5
3c
C3AH3. . .O1
C16AH16c. . .Cg1
0.93
0.96
2.51
3.15
128
121
x, y, z + 1
Àx, Ày, Àz + 1
3f
C18aAH18a. . .O1a
C7bAH7b. . .O2b
C13aAH13a. . .O1b
C7aAH7a. . .Cg3
C10bAH10b. . .Cg3
C19aAH19a. . .Cg4
C12aAH12a. . .Cg4
C15aAH15b. . .Cg1a
0.93
0.93
0.98
0.93
0.93
0.93
0.98
0.96
2.50
2.51
2.55
2.73
2.72
2.83
3.21
3.10
166
163
133
158
157
144
134
144
Àx, Ày, Àz + 1
Àx + 1, y, z
Àx + 1, Ày + 1, Àz + 1
Àx + 1, Ày + 2, Àz + 1
x, y À 1, z
x, y + 1, z
Àx + 1, Ày + 1, Àz + 1
Àx + 1, Ày, Àz + 1

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A. Burmudzija et al. / Inorganica Chimica Acta 471 (2018) 570–576
575
Scheme 1. Synthesis of ketone 2e.
Fig. 2. Overly of crystallographically independent molecules of 3f based on a least-
squares fit of atoms N1, N2 and C11 from the corresponding pyrazoline rings. The
molecule shown in red is molecule A and that shown in blue is molecule B (major
component). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)
and B, respectively. The conformation of 3f molecules is, as
expected, markedly different from that of previously reported 3-
(2-furyl)-5-ferrocenyl-2-pyrazoline isomer [47], where the Fc unit
and furyl ring attach to sp3 and sp2 C atoms of pyrazoline ring,
respectively. In contrast to 3f, such arrangement of molecular com-
ponents leads to coplanarity between the pyrazoline and furyl
rings (reported dihedral angle of 5.7(1), while 80.2° in average
for 3f) and orthogonal arrangement of pyrazoline and Cp ring [re-
ported dihedral angle 81.4(1), while 17.0° in average for 3f].
Crystal packing of 3f is stabilized by a complex network of
Scheme 2. Reaction of ferrocenyl ketones 2 with hydrazine in AcOH.
176.4(2)°]. Finally, the plane C13/C16/C17 containing the non-H
atoms of methyl substituents is orthogonally positioned with
respect to the pyrazoline ring (89.5(1)°]. The torsion angle
N2AC11AC1AC2 relating the substituent and adjacent Cp ring
has the values of À20.1(7) and À6.7(10)° for major and minor com-
ponent of the ring.
In the crystal packing of 3c the acetyl O1 atom represents the
most important H-bonding acceptor. The shortest C3AH3. . .O1
interaction actually involves the CAH donor from the disordered
Cp ring, thus the geometry of this interaction differs for the major
and minor component of Cp ring: H3. . .O1 2.51/2.33 Å and
C3AH3. . .O1 126/155°. Though displaying more favorable parame-
ters the minor component also implies the increased sterical inter-
action between the H atoms. The occurrence of such an interaction
conflict might explain why this Cp ring is disordered between two
positions. Nevertheless, the C3AH3. . .O1 is the leading intermolec-
ular interaction which connects the molecules of 3c into a chain
extending along the c crystallographic axis (Fig. S3). The molecules
from the neighboring chains are interconnected by pairs of cen-
CAH. . .O and CAH. . .
p interactions (Table 4). By means of
C18aAH18a. . .O1a interaction formed between the furyl donor
and carbonyl O1 acceptor the A molecules arrange into cyclic, cen-
trosymmetric dimers (Fig. S5a). The molecules B do not display this
type of dimer, instead there is a tendency of B molecules to form
centrosymmetric dimer by C7bAH7b. . .O2b interaction involving
the donor from disordered Cp ring and furyl oxygen as an acceptor.
Unlike the carbonyl acceptor O1a, the acceptor O1b is employed in
C13AH13a. . .O1b interaction (Table 4) that links the A and B
dimers into a corresponding chain of dimers (Fig. S5a). The furyl
rings from both independent molecules serve as
p acceptors in four
dissimilar CAH. . . interactions (Table 4) which interconnect the
p
chains of dimers toward 3D crystal structure (Fig. S5b).
4. Conclusion
trosymmetric C16AH16c. . .p interactions where ordered Cp ring
has the role of acceptor for the methyl donor (Table 4).
p
We described herein synthesis, spectral, analytical, biological
activity of series of compounds 3a–f, and single crystal X-ray char-
acterization of 2c (starting ketone) and pyrazoline products 3c and
3f with ferrocene core. Synthesized compounds show significant
antimicrobial activity, very close to streptomycin and ketoconazole
(compound 3e have equal activity as ketoconazole on Mucor
mucedo fungi). From the experimental results, we suppose that
substituent at position 5- in pyrazoline ring is responsible for ele-
vated activity.
X-ray structural analysis has shown that the Fc-pyrazoline frag-
ment of the novel 3c and 3f compounds exhibits similar conforma-
tional properties. In both compounds the incorporated pyrazoline
ring is characterized by an envelope-like conformation with the
somewhat increased puckering in 3f. The crystal packing of 3c
and 3f molecules are both stabilized by weak CAH. . .O and
The CAC and CAN distances in the pyrazoline rings of indepen-
dent molecules (Table 3) closely agree with the values observed for
3c. In comparison to 3c, the pyrazoline rings in A and B show
somewhat higher deviation of the constituting atoms from the cor-
responding mean planes (r.m.s deviation of 0.083 and 0.067 in A
and B, respectively), with the largest deviation observed for C13
atom attaching the furyl ring [À0.112(2) and À0.090(2) for C13a
and C13b, respectively]. The C2AC1AC11AN2 torsion angle of
À12.3(3) and À18.3(3)° in A and B, respectively, shows that in both
molecules the pyrazoline ring remains close to the level of substi-
tuted Cp ring. The overlay of independent molecules presented in
Fig. 2 reveals difference in molecular conformation, which mainly
concerns the different orientation of their furyl rings. This is
reflected in N1AC13AC16AO2 torsion angle, which has the values
of 74.6(2) and 54.2(3)° in A and B molecules, respectively. The
angle between the least-squares planes of the furyl and the substi-
tuted Cp ring is 85.1(1) and 75.2(1)°, while the angle between
planes of furyl and pyrazoline ring is 85.1(1) and 87.2(1)° in A
CAH. . .
p interactions, nevertheless the different additional sub-
stituents at the pyrazoline ring (dimethyl and furyl in 3c and 3f,
respectively) significantly influence the manner of molecular inter-
action with the crystalline surrounding.

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A. Burmudzija et al. / Inorganica Chimica Acta 471 (2018) 570–576
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Appendix A. Supplementary data
}
The supplementary Crystallographic data for the structural
analysis have been deposited with Cambridge Crystallographic
Data Centre: Deposition number CCDC 1567081, 1567082 and
1567083 for compounds 2c, 3c and 3f respectively. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/data_
request/cif or from the Cambridge Crystallographic Data Centre,
12, Union Road, Cambridge, CB2 1EZ, UK; Fax: +44 1223 336033;
e-mail: deposit@ccdc.cam.ac.uk.
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