Synthesis, spectral characterization, electrochemical properties and antimicrobial screening of sulfur containing acylferrocenes

Article abstract of DOI:10.1016/j.poly.2010.03.002

Several known and eight new sulfur containing acylferrocenes of the general formula FcCO(CH₂)_nSR (where Fc = ferrocenyl, n = 1 or 2 and R = alkyl, 4-bromobenzyl or 2,6-dichlorobenzyl group) were synthesized in order to test their in vitro antimicrobial activity against 11 bacterial and three fungal/yeast strains. It has been shown that only four of the 14 ketones are completely inactive at the tested dose, while the activities of the other ones were noteworthy. All new compounds were well characterized by IR and NMR spectral data, and their electrochemical properties were investigated by cyclic voltammetry. The X-ray crystal structures of two representative ketones are also presented.

Full text of DOI:10.1016/j.poly.2010.03.002

Polyhedron 29 (2010) 1863–1869
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, spectral characterization, electrochemical properties
and antimicrobial screening of sulfur containing acylferrocenes
a
b
b
c
d,
*
´
´
´
´
´
´
Danijela Ilic , Ivan Damljanovic , Dragana Stevanovic , Mirjana Vukicevic , Niko Radulovic
,
e
f
b,
**
´
´
Volker Kahlenberg , Gerhard Laus , Rastko D. Vukicevic
a Technical Faculty Kosovska Mitrovica, University of Priština, Kneza Miloša 7, 38220 Kosovska Mitrovica, Serbia
b Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovi´ca 12, 34000 Kragujevac, Serbia
c Department of Pharmacy, Faculty of Medicine, University of Kragujevac, S. Markovi´ca 65, 34000 Kragujevac, Serbia
d Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbia
e Institute of Mineralogy and Petrography, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austria
f Institute of Inorganic Chemistry, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Several known and eight new sulfur containing acylferrocenes of the general formula FcCO(CH2)nSR
(where Fc = ferrocenyl, n = 1 or 2 and R = alkyl, 4-bromobenzyl or 2,6-dichlorobenzyl group) were synthe-
sized in order to test their in vitro antimicrobial activity against 11 bacterial and three fungal/yeast
strains. It has been shown that only four of the 14 ketones are completely inactive at the tested dose,
while the activities of the other ones were noteworthy. All new compounds were well characterized
by IR and NMR spectral data, and their electrochemical properties were investigated by cyclic voltamme-
try. The X-ray crystal structures of two representative ketones are also presented.
Received 9 February 2010
Accepted 2 March 2010
Available online 6 March 2010
Keywords:
Ferrocene
Friedel-Crafts acylation
S-Alkylthioglycolic acids
2-Alkylthiopropanoic acids
Cyclic voltammetry
Antimicrobial activity
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
site etc. [3]. Despite the fact that early attempts to apply ferrocene
derivatives in medicine were discouraging [4,5], chemists did not
Antimicrobials have played an important role in health care
since the middle of the 20th century. However, in the recent time
the success in application of antibiotics in treating of infections has
been eroded by increased resistance of bacteria. Therefore, there is
a permanent interest in finding more and more novel substances
exhibiting antimicrobial activity [1,2]. It is well known that substi-
tution of an aromatic nucleus of certain organic compounds with a
ferrocenyl group can lead to products possessing unexpected prop-
erties which are less manifest or even absent in the parent mole-
cule. Many new molecules containing a ferrocene unit were
synthesized following this idea, i.e. they have been designed to
be derivatives of known compounds (that already possess desired
properties) in which a certain group was replaced by a ferrocene
unit (expecting an improved property). Thus, bioconjugates con-
taining this metallocene represent a new class of biomaterials, in
which organometallic unit serves as a molecule scaffold, sensitive
probe, chromophore, biological marker, redox-active site, catalytic
abandon this idea, and it is known now that many ferrocenes exhi-
bit different biological activities, promising thus significant appli-
cation in medicinal practice [6–14]. In continuation of our
permanent interest in ferrocene chemistry, particularly in the
investigation of the biological activity of ferrocene derivatives
[15–18], we at present synthesized several known [19], and several
novel acylferrocenes containing sulfur in the side chain (1 and 2,
Fig. 1) in order to test their antimicrobial activity. For these inves-
tigations we have been prompted by the literature findings con-
cerning the antimicrobial activities of sulfur containing ketones:
2-(methylthio)-1-(pyridin-3-yl)ethanone (3), 2-(methylthio)-1-
(pyridin-4-yl)ethanone (4) and 2-(methylthio)-1-(pyrazin-2-
yl)ethanone (5) derivatives of pyridine and pyrazine (Fig. 1) [20].
Namely, in the light of the above text, we find that compound 1
(R = CH3) exactly fits the mentioned synthetic logic regarding fer-
rocene derivatives – the corresponding aromatic units (3- and 4-
pyridyl and 2-pyrazinyl groups) from the three mentioned com-
pounds (3–5), have been substituted by the ferrocene one. In fact,
such a type of compounds has been synthesized by us several years
ago [19], but at that time their antimicrobial activity has not been
assessed. Moreover, we set our goal to vary the structural design of
the ketones by inserting an additional methylene group between
* Corresponding author. Fax: +381 18 533 014.
** Corresponding author. Fax: +381 34 33 50 40.
E-mail addresses: vangelis0703@yahoo.com (N. Radulovic´), vuk@kg.ac.rs (R.D.
Vukic´evic´).
0277-5387/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2010.03.002

´
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
1864
Calc. for C14H16FeOS (288.03): C, 58.35; H, 5.60; Fe, 19.38; O, 5.55;
S, 11.13. Found: C, 58.30; H, 5.57%.
2.2.2. 1-Ferrocenyl-4-methyl-3-thiapentan-1-one (1c)
Yield 57%; m.p. 68–69 °C; 1H NMR (CDCl3, ppm) d: 1.29 (d,
J = 6.8 Hz, 6H,(CH3)2CH–), 3.05 (td, J = 13.4, 6.7 Hz, 1H, –CH–S),
3.59 (s, 2H, –COCH2–S), 4.23 (s, 5H, Cp), 4.54 (t, J = 2.0 Hz, 2H,
Cp), 4.81 (t, J = 2.0 Hz, 2H, Cp). 13C NMR (CDCl3, ppm) d: 22.90
(C-2000 and 3000), 35.40 (C-1000), 37.33 (C-2), 69.84 (C-30 and 40),
69.95 (C-100-500), 72.50 (C-20 and 50), 77.56 (C-10), 199.49 (C-1). IR
(KBr, cmꢀ1): 3120, 2961, 1645, 1455, 1413, 1377, 1293, 1070,
820, 682, 635. Anal. Calc. for C15H18FeOS (302.21): C, 59.61; H,
6.00; Fe, 18.48; O, 5.29; S, 10.61. Found: C, 59.67; H, 5.92.
2.2.3. 1-Ferrocenyl-3-thiahexan-1-one (1d)
Yield 41%; m.p. 65–67 °C; 1H NMR (CDCl3, ppm) d: 0.99 (t,
J = 7.3 Hz, 3H, CH3), 1.62 (td, J = 10.82, 7.29 Hz, 2H, CH2–CH3),
2.59 (t, J = 7.4 Hz, 2H, CH2–CH2), 3.55 (s, 2H, S-CH2), 4.23 (s, 5H,
Cp), 4.54 (t, J = 2.0 Hz, 2H, Cp), 4.81 (t, J = 2.0 Hz, 2H, Cp). 13C
NMR (CDCl3, ppm) d: 13.21 (C-3000), 22.22 (C-2000), 34.40 (C-1000),
38.13 (C-2), 69.72 (C-30 and 40), 69.84 (C-100-500), 72.42 (C-20 and
50), 77.36 (C-10), 199.03 (C-1). IR (KBr, cmꢀ1): 3120, 2959, 2921,
1645, 1458, 1412, 1380, 1295, 1072, 838, 821, 682, 635. Anal. Calc.
for C15H18FeOS (302.21): C, 59.61; H, 6.00; Fe, 18.48; O, 5.29; S,
10.61. Found: C, 59.67; H, 6.06.
Fig. 1. Structures of target acylferrocenes 1a–g and 2a–g.
the carbonyl carbon and the sulfur atoms, and replacing the S-
methyl group with other alkyl groups, as it has been shown in
the Fig. 1.
2.2.4. 1-Ferrocenyl-4-methyl-3-thiahexan-1-one (1e)
Yield 36%; m.p. 59–60 °C; 1H NMR (CDCl3, ppm) d: 0.91 (t,
J = 7.2 Hz, 3H, CH3), 1.38 (td, J = 13.9 and 6.9 Hz, 2H, CH2–CH2)
1.49–1.67 (m, 2H CH2–CH3), 2.61 (t, J = 7.3 Hz, 2H, CH2–S), 3.55
(s, 2H, CO–CH2–S), 4.23 (s, 5H, Cp), 4.54 (t, J = 2.0 Hz, 2H, Cp),
13
4.81 (t, J = 1.9 Hz, 2H, Cp). C NMR (CDCl3, ppm) d: 13.55 (C-4000),
2. Experimental
21.85 (C-3000), 31.07 (C-2000), 32.21 (C-1000), 38.28 (C-2), 69.83 (C-30
and 40), 69.94 (C-100-500), 72.51 (C-20 and 50), 77.47 (C-10), 199.12
(C-1). IR (KBr, cmꢀ1): 3112, 3083, 2959, 2927, 1645, 1453, 1410,
1375, 1290, 1074, 826, 681, 636. Anal. Calc. for C16H20FeOS
(316.06): C, 60.77; H, 6.37; Fe, 17.66; O, 5.06; S, 10.14. Found: C,
60.70; H, 6.39%.
2.1. Materials and instruments
All chemicals were commercially available and used as re-
ceived, except that the solvents were purified by distillation. Chro-
matographic separations were carried out using Silica Gel 60
(Merck, 230–400 mesh ASTM), whereas Silica Gel 60 on Al plates,
layer thickness 0.2 mm (Merck) was used for TLC. Melting points
were determined on a Mel-Temp capillary melting points appara-
tus, model 1001 and are uncorrected. Microanalysis of carbon
and hydrogen were carried out with a Carlo Erba 1106 microanaly-
ser; their results agreed favorably with the calculated values. IR
measurements were carried out with a Perkin–Elmer FTIR 31725-
X spectrophotometer. NMR spectra were recorded on a Varian
Gemini (200 MHz) spectrometer, using CDCl3 as the solvent and
TMS as the internal standard. Chemical shifts are expressed in d
(ppm).
2.2.5. 1-Ferrocenyl-4-thiahexan-1-one (2b)
Yield 86%; 1H NMR (CDCl3, ppm) d: 1.30 (t, J = 7.4 Hz, 3H, CH3),
2.62 (q, J = 7.4 Hz, 2H, CH2–CH3), 2.96 (m(AA0BB0), 4H, CO–CH2CH2–
S), 4.23 (s, 5H, Cp), 4.52 (t, J = 1.9 Hz, 2H, Cp), 4.80 (t, J = 2.0 Hz, 2H,
Cp). 13C NMR (CDCl3, ppm) d: 14.68 (C-2000), 25.88 (C-1000 or 3), 26.38
(C-1000 or 3), 39.79 (C-2), 69.21 (C-30 and 40), 69.78 (C-100-500), 72.28
(C-20 and 50), 78.65 (C-10), 202.38 (C-1). IR (KBr, cmꢀ1): 3097, 2966,
2926, 1667, 1455, 1411, 1379, 1253, 1106, 1080, 824, 697, 638.
Anal. Calc. for C15H18FeOS (302.21): C, 59.61; H, 6.00; Fe, 18.48;
O, 5.29; S, 10.61. Found: C, 59.69; H, 6.04%.
2.2.6. 1-Ferrocenyl-5-methyl-4-thiahexan-1-one (2c)
2.2. Preparation of ketones 1a–g and 2a–g
Yield 36%; 1H NMR (CDCl3, ppm) d: 1.31 (d, J = 6.6 Hz, 6H,
(CH3)2CH–), 2.8–3.05 (m, 4H from CO–CH2CH2–S and 1H from
CH–(CH3)2), 4.23 (s, 5H, Cp), 4.52 (t, J = 2.0 Hz, 2H, Cp), 4.80 (t,
Ketones 1a–g and 2a–g were synthesized by the known proce-
dure [19]. Spectral data of compounds 1a,f,g and 2 a,f,g were pre-
viously described [19], whereas the data for the newly synthesized
compounds follow.
13
J = 1.9 Hz, 2H, Cp). C NMR (CDCl3, ppm) d: 23.32 (C-2000 and 3000),
24.71 (C-3), 35.36 (C-1000), 39.90 (C-2), 69.23 (C-30 and 40), 69.80
(C-100-500), 72.28 (C-20 and 50), 78.67 (C-10), 202.43 (C-1). IR (KBr,
cmꢀ1): 3098, 2960, 2926, 1668, 1455, 1411, 1380, 1255, 1107,
1078, 823, 694, 642. Anal. Calc. for C16H20FeOS (316.06): C, 60.77;
H, 6.37; Fe, 17.66; O, 5.06; S, 10.14. Found: C, 60.81; H, 6.40%.
2.2.1. 1-Ferrocenyl-3-thiapentan-1-one (1b)
Yield 61%; m.p. 49–51 °C; 1H NMR (CDCl3, ppm) d: 1.28 (t,
J = 7.4 Hz, 3H, CH3), 2.64 (q, J = 7.4 Hz, 2H, CH2–CH3), 3.57 (s, 2H,
CH2–S), 4.23 (s, 5H, Cp), 4.55 (t, J = 1.9 Hz, 2H, Cp), 4.81 (t,
2.2.7. 1-Ferrocenyl-4-thiaheptan-1-one (2d)
13
J = 2.0 Hz, 2H, Cp). C NMR (CDCl3, ppm) d: 14.17 (C-2000), 26.50
Yield 45%; 1H NMR (CDCl3, ppm) d: 1.02 (t, J = 7.3 Hz, 3H, CH3),
1.65 (td, J = 14.4, 7.3 Hz, 2H, CH2–CH3), 2.58 (t, J = 7.6 Hz, 2H, CH2),
2.95 (m(AA0BB0), 4H, CO–CH2CH2–S), 4.23 (s, 5H, Cp), 4.52 (t,
J = 1.9 Hz, 2H, Cp), 4.80 (t, J = 1.9 Hz, 2H, Cp). 13C NMR (CDCl3,
(C-1000), 37.97 (C-2), 69.82 (C-30 and 40), 69.93 (C-100-500), 72.51 (C-
20 and 50), 77.64 (C-10), 198.93 (C-1). IR (KBr, cmꢀ1): 3120, 2959,
2912, 1645, 1458, 1411, 1380, 1294, 1071, 838, 821, 688, 630. Anal.

´
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D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
ppm) d: 13.24 (C-3000), 22.69 (C-2000), 26.11 (C-3), 34.47 (C-1000),
2.4. Electrochemical measurements
39.68 (C-2), 69.04 (C-30 and 40), 69.61 (C-100-500), 72.11 (C-20 and
50), 78.50 (C-10), 202.14 (C-1). IR (KBr, cmꢀ1): 3098, 2961, 2929,
1667, 1455, 1411, 1379, 1254, 1078, 823, 691, 636. Anal. Calc. for
C16H20FeOS (316.06): C, 60.77; H, 6.37; Fe, 17.66; O, 5.06; S,
10.14. Found: C, 60.78; H, 6.42%.
Electrochemical measurements were performed at ambient
temperature (ca. 25 °C) by using a CH Instruments (Austin, TX)
potentiostat CHI760b Electrochemical Workstation. A standard
three-electrode cell (5 mL) equipped with a platinum wire and a
silver wire immersed in 0.1 M LiClO4 solution in CH3CN as the
counter and reference electrode, respectively. A platinum disk
(d = 2 mm) was used as the working electrode.
2.2.8. 1-Ferrocenyl-4-thiaoctan-1-one (2e)
Yield 73%; 1H NMR (CDCl3, ppm) d: 0.93 (t, J = 7.2 Hz, 3H, CH3),
1.41 (td, J = 13.7, 7.0 Hz, 2H, CH2–CH2) 1.52–1.69 (m, 2H CH2–CH3),
2.60 (t, J = 7.3 Hz, 2H, CH2), 2.95 (m(AA0BB0), 4H, CO–CH2CH2–S),
4.23 (s, 5H, Cp), 4.52 (t, J = 1.9 Hz, 2H, Cp), 4.80 (t, J = 1.9 Hz, 2H,
2.5. Biological assay
The in vitro antimicrobial activities of 1a–g and 2a–g were
tested against a panel of laboratory control strains belonging to
the American Type Culture Collection Maryland, USA. Antibacterial
activity was evaluated against six Gram positive and five Gram
negative bacteria. The Gram positive bacteria used were: Bacillus
subtilis (ATCC 6633), Clostridium pyogenes (ATCC 19404), Enterococ-
cus sp. (ATCC 25212), Micrococcus flavus (ATCC 10240), Sarcina lu-
tea (ATCC 9341) and Staphylococcus aureus (ATCC 6538). The
Gram negative bacteria utilized in the assays were: Escherichia coli
(ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Proteus vulgaris
(ATCC 8427), Pseudomonas aeruginosa (ATCC 27857) and Salmonella
enteritidis (ATCC 13076). The antifungal activity was tested against
three organisms Aspergillus niger (ATCC 16404), Candida albicans
(ATCC 10231) and Saccharomyces cerevisiae (ATCC 9763).
A disk diffusion method, according to NCCLS [23], was em-
ployed for the determination of the antimicrobial activity of 1a–g
and 2a–g. The following nutritive media were used: Antibiotic
Medium 1 (Difco Laboratories, Detroit Michigan USA) for growing
Gram positive and Gram negative bacteria and Tryptone soy agar
(TSA – Institute of Immunology and Virology ‘‘Torlak”, Belgrade,
Serbia) for C. albicans and A. niger. Nutritive media were prepared
according to the instructions of the manufacturer. All agar plates
were prepared in 90 mm Petri dishes with 22 ml of agar, giving a
final depth of 4 mm. One-hundred microliters of a suspension of
the tested microorganisms (108 cells per ml) were spread on the
solid media plates. Sterile filter paper disks (‘‘Antibiotica Test Blatt-
chen”, Schleicher and Schuell, Dassel, Germany, 12.7 mm in diam-
13
Cp). C NMR (CDCl3, ppm) d: 13.57 (C-4000), 21.91 (C-3000), 26.38
(C-3), 31.68 (C-2000), 32.31 (C-1000), 39.86 (C-2), 69.22 (C-30 and 40),
69.79 (C-100-500), 72.27 (C-20 and 50), 78.69 (C-10), 202.40 (C-1). IR
(KBr, cmꢀ1): 3098, 2958, 2929, 1668, 1455, 1411, 1379, 1254,
1080, 823, 692, 638. Anal. Calc. for C17H22FeOS (330.07): C, 61.82;
H, 6.71; Fe, 16.91; O, 4.84; S, 9.71. Found: C, 61.80; H, 6.67%.
2.3. Crystal structure determination
X-Ray diffraction data were collected with an Oxford Diffraction
Gemini-R Ultra diffractometer using graphite-monochromated Mo
K
a radiation (k = 0.71073 Å). The structure was solved by direct
methods and refined using full-matrix least squares on F2. The
crystallographic data and refinement details are listed in Table 1.
Table 1
Crystal data and structure refinement details.
Compound
1a
2a
CCDC No.
Formula
Mr
759144
C13H14FeOS
274.15
759145
C14H16FeOS
288.18
Crystal shape, color
irregular fragment, red-
brown
0.16 ꢁ 0.12 ꢁ 0.08
monoclinic
P21/n
5.7288(4)
19.7336(11)
10.3223(6)
98.037(6)
1155.47(12)
4
prismatic fragment, red-
brown
0.34 ꢁ 0.08 ꢁ 0.08
monoclinic
P21/c
5.7723(2)
12.9582(4)
16.5834(5)
94.450(3)
1236.68(7)
4
Crystal size, (mm3)
Crystal system
Space group
a (Å)
b (Å)
c (Å)
eter) were impregnated with 40
(65 mg/ml) in DMSO (all solutions were filter-sterilized using a
0.45 m membrane filter), i.e. 500 g per disk, and placed on inoc-
ll of the samples solutions
l
l
ulated plates. These plates, after standing at 4 °C for 2 h, were incu-
bated at 37 °C for 24 h for bacteria and at 30 °C for 48 h for the
fungi. Standard disks of Tetracycline and Nystatine (origin – Insti-
b (°)
V (Å3)
Z
Dcalc (g cmꢀ3)
1.58
1.46
1.55
1.37
tute of Immunology and Virology ‘‘Torlak”, 30
ponent, diameter 6 mm) were used individually as positive
controls, while the disks imbued with 50 l of pure DMSO were
lg of the active com-
l
(mmꢀ1
)
F(0 0 0), e
568
600
l
Diffractometer
Oxford Diffraction
Gemini-R Ultra
Oxford Diffraction
Gemini-R Ultra
used as a negative control. The diameters of the inhibition zones
were measured in millimeters (to the nearest mm) using a ‘‘Fish-
er-Lilly Antibiotic Zone Reader” (Fisher Scientific Co., USA). Each
test was performed in quintuplicate. In order to evaluate statisti-
cally any significant differences among mean values, a one-way
ANOVA test was used. In all tests the significance level at which
we evaluated critical values differences was 5%.
Broth microdilution susceptibility assay was used, as recom-
mended by NCCLS, for the determination of MIC values (NCCLS,
[24]). All tests were performed in Mueller Hinton broth (MHB;
BBL) supplemented with Tween 80 detergent (final concentration
of 0.5% (v/v)), with the exception of the fungal organisms (Sabou-
raud dextrose broth-SDB + Tween 80), and with 2 ꢁ 105 colony
forming units (CFU) mLꢀ1 of the bacteria/fungi in the exponential
phase. Test compounds (2a, 2b, 1c, 2c, 1d, 2d, 1e and 2e) were dis-
solved in dimethyl sulfoxide and this stock solution used to pre-
pare the exact agar dilutions. Final concentrations of the
compounds in the broth ranged from 0.012 to 256 mg/l and these
Radiation type
Data collection
method
Mo K
a
Mo K
x scans
a
x
scans
T (K)
173
173
hmax (°)
25.4
25.3
h, k, l range
Absorption correction multi-scan
Measured reflections
Independent
ꢀ6 ? 5, 23, ꢀ12 ? 9
6, ꢀ15 ? 14, ꢀ19 ? 16
multi-scan
7022
7441
2107 (Rint = 0.040)
2244 (Rint = 0.032)
reflections
Observed reflections
1691
1885
[I > 2r(I)]
Data/restraints/
2107/0/146
2244/0/155
parameters
R1/wR2 [I > 2
R1/wR2 (all data)
r
(I)]
0.026/0.048
0.040/0.050
0.026/0.056
0.036/0.058
0.95
Goodness-of-fit (GOF) 0.94
on F2
D
q
max/
D
)
q
min
0.29/ꢀ0.22
0.26/ꢀ0.25
(e Åꢀ3

´
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
1866
were prepared in a 96-well microtiter plate. The dilutions were
based on a geometrical order (factor 2) and were related to concen-
trations 1 mg/l and 1.5 mg/l (0.012, 0.016, 0.012, 0.031, 0.047,
0.063, 0.094, 0.125, 0.19, 0.25, 0.38, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8,
12, 16, 24, 32, 48, 64, 96, 128, 192, 256 mg/l). In order to get more
accurate values of MIC further dilutions were additionally pre-
pared in the concentration range between the first well without
visible growth and the one next to it with lower test compound
concentration. Plates were incubated at 37 °C for 24 h for bacteria
and at 30 °C for 48 h for the yeasts/mold. Each test was performed
in duplicate and repeated twice. Amikacin and Bifonazole were
used as positive controls, while DMSO + Tween 80 (in the form of
a blank) were the negative control.
H-bonded b-thioalkyl counterparts. The NMR spectra utterly con-
firm the structures of compounds 1b–d and 2b–d. Thus, in the
1H NMR spectra of all eight compounds signals of the cyclopenta-
dienyl rings protons appear at almost the same positions in the
spectra (d = 4.23 ppm for the unsubstituted, and 4.52–4.54 and
4.80–4.81 ppm for the substituted rings, respectively). Protons of
the –CH2–S– unit in 1b–d also have almost the same chemical
shifts (3.55–3.59 ppm). On the other hand, a specific AA0BB0 second
order multiplet signal in the 1H NMR spectra of the compounds
2b–d, originating from the –CO–CH2–CH2–S- moiety, appears (its
center) always at 2.95 ppm.
3.3. X-ray analysis of compounds 1a and 2a
Single crystal X-ray diffraction structure analysis of the com-
pounds 1a and 2a revealed, not unexpectedly, that these homolo-
gous molecules exhibit very similar conformations (Fig. 2). Thus,
the ferrocene units in 1a and 2a adopt almost eclipsed conforma-
tions, with average torsion angles of 4.14 and 2.06°, respectively.
The respective cyclopentadienide (Cp) rings are tilted by 2.44
and 2.29°. The carbonyl groups deviate from the planes of the adja-
cent Cp rings by only 0.16 Å (C) and 0.03 Å (O) in 1a, and 0.12 Å (C)
and 0.06 Å (O) in 2a, respectively. However, the packing arrange-
ments in the crystals are quite different. Compound 1a forms di-
mers by weak contacts between the carbonyl oxygen atom O1
and H12a of the methylene group at the symmetry position (ꢀx,
ꢀy, 1 ꢀ z), with a HꢂꢂꢂO distance of 2.58 Å, a CꢂꢂꢂO distance of
3.524 Å, and a C–HꢂꢂꢂO angle of 160° (Fig. 3a). In contrast, the mol-
ecules of compound 2a are linked by weak interactions between
the carbonyl oxygen atom O1 and H14c of the methyl group at
(ꢀ1 + x, y, z) into infinite chains in the direction of the crystallo-
graphic a axis, with a HꢂꢂꢂO distance of 2.64 Å, a CꢂꢂꢂO distance of
3.597 Å, and a C–HꢂꢂꢂO angle of 165° (Fig. 3b).
3. Results and discussion
3.1. Synthesis of 1-ferrocenyl-1-(hydroxyimino)thialkanes 2a,b and
3a,b
The synthesis of acylferrocenes 1a–g and 2a–g has been
achieved by modified Friedel–Crafts acylation of ferrocene with
chlorides of the corresponding carboxylic acids (Scheme 1)
[19,21,22]. In this improved method the acylating agents – acyl
chlorides – were generated in situ from the corresponding carbox-
ylic acids and phosphorus trichloride, and have not been isolated
or purified for subsequent use. Namely, the phosphorus species lib-
erated in the course of this reaction, whatever they are (most prob-
ably derivatives of phosphorous acid, H3PO3), do not prevent the
acylation. We are not sure if this is caused by the known high reac-
tivity of ferrocene towards Friedel–Crafts acylation (which is
thought to be similar to the reactivity of phenols) so that the inac-
tivation of the catalyst (aluminium chloride) is not of such an ex-
tent to prevent the onset of the reaction, or if it is due to the
complete lack of the catalyst inactivation. The yields of the corre-
sponding ketones were in the range of 36–86% (see Scheme 1).
3.4. Electrochemistry
Cyclic voltammetry in acetonitrile containing 0.1 mol/L lithium
perchlorate as a supporting electrolyte has been used for the eval-
uation of electrochemical properties of all the synthesized com-
3.2. Spectral characterization
The structures of compounds 1a–g and 2a–g were spectroscop-
ically characterized by IR, 1H, and 13C NMR. These data were in
complete agreement with the reported ones for the known com-
pounds (1a,f,g 2 a,f,g). The main common feature of the IR spectra
of all of the new ketones is a strong band at 1640–1645 for com-
pounds 1b–d, and at 1667–1668 cmꢀ1 for compounds 2b–d, which
we attributed to the carbonyl group stretching vibrations. Appar-
ently, this difference is the consequence of the vicinity of S-alkyl
pounds
– the known and new ones. All ketones exhibit a
reversible one-electron redox couple at the similar potential (oxi-
dation waves appear at 647–680 mV and the reduction ones at
671–601 mV, Table 2). These potentials are considerably more po-
sitive than that of the unsubstituted ferrocene, as a consequence of
the presence of an electron-withdrawing acyl group. As the repre-
sentative examples we give the voltammograms of compounds 1a
and 2a (Fig. 4). The difference between anodic and cathodic peak
potentials (Table 2) is close to the theoretical value and indepen-
dent of the scan rate v. Both, anodic and cathodic peak currents
are proportional to the square root of the scan rate, and their ratio
is independent of the scan rate, indicating a diffusion-controlled
process.
group in the case of compounds 1b–d (a position of S-alkyl group).
This could also likely be an influence of the stronger hydrogen
bonding holding together the dimeric species in the crystal lattice
of
a
-thioalkyl ketones (see 2.3. for C–HꢂꢂꢂO@C lengths supporting
this assumption) compared to the infinite array of intermolecularly
3.5. Biology
Antibacterial activity against six Gram positive (B. subtilis, C.
pyogenes, Enterococcus sp., Micrococcus flavus, Sarcina lutea and S.
aureus) and five Gram negative bacteria (E. coli, K. pneumoniae, P.
vulgaris, P. aeruginosa and S. enteritidis), as well as the antifungal
activity against three fungal organisms (A. niger, C. albicans and
S. cerevisiae as the representative species of fungi) were assessed
for compounds 1a–g and 2a–g. The antimicrobial activity screen-
ing was performed by means of a disk diffusion assay (NCCLS,
[23]), and the obtained results are listed in Table 3. As it can be
seen from the diameters of the growth inhibition zones presented
Scheme 1. Synthesis of sulfur containing acylferrocenes.

´
1867
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
Fig. 2. Ortep plot of compound 1a (a) and 2a (b) (ellipsoid probability 50%).
Fig. 3. (a) Dimers formed by weak C–HꢂꢂꢂO contacts in the crystal structure of 1a. (b) Infinite chains formed by weak C–HꢂꢂꢂO contacts in the crystal structure of 2a.
Table 2
The electrochemical data for compounds 1a–g and 2a–g.
D
Ec (mV)
a
a
b
Compound
Epa (mV)
Epa (mV)
E1/2 (mV)
1a
1b
1c
1d
1e
1f
1g
2a
2b
2c
2d
2e
2f
656
668
656
681
659
659
668
656
647
650
662
647
649
656
577
595
577
601
577
583
589
568
574
571
583
574
571
574
616
631
616
641
618
621
628
612
610
610
622
610
610
615
79
73
79
80
82
76
79
88
73
79
79
73
77
82
2g
a
b
c
Epa and Epc anodic and cathodic peak potentials, respectively, at 100 mV sꢀ1
E1/2 = (Epa + Epc)/2.
.
D
E = Epa ꢀ Epc
.
in Table 3 the prepared compounds showed a wide range of activ-
ity (from completely inactive ones to those of medium activity).
Fig. 4. Cyclovoltammograms of compounds 1a, 2a and ferrocene at Pt disc in 0.1 M
LiClO4 in acetonitrile at v = 0.1 V/s.
The compounds that showed (at a dose of 500 lg per disk) some
degree of activity in this test were further subjected to a dilution
test to get more precise results on their antimicrobial nature. The
minimal inhibitory concentration (MIC, considered to be the low-
used as positive controls. The values of MIC for the currently syn-
thesized active sulfur containing acylferrocene derivatives (1c, 1d,
est concentration of the tested compound in
lg/mL which inhib-
1e, 2a, 2b, 2c, 2d and 2e) ranged from 102 to 237
lg/mL (Table 4).
ited growth of bacteria or fungi) was determined using
a
Concentrations above 256 g/mL were not tested.
l
microdilution method based on the recommendations of NCCLS
[24]. Under the same conditions solutions of differing concentra-
tion of Amikacin (antibacterial) and Bifonazole (antifungal) were
The determined MIC concentrations are similar in efficacy range
compared to other published [15] ferrocene derivative MIC values.
The results of the dilution method confirmed the findings of the

´
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
1868
Table 3
The antimicrobial activity (diameters of growth inhibition zones of compounds 1a–g and 2a–g in a disc diffusion assay at a 500 lg per disc dose).
Microorganism
Compound
1a
2a
1b
2b
1c
2c
1d
2d
1e
2e
1f
2f
1g
2g
Tetracycline
Nystatine
A. niger
–
–
–
–
–
–
–
–
–
–
–
–
–
–
28a
32
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
34
32
–
–
–
–
36
34
35
34
32
34
–
31
34
32
35
35
36
33
33
35
32
30
33
38
35
31
32
–
30
33
36
–
–
40
–
–
–
–
30
–
–
–
–
–
32
33
–
36
37
35
35
35
–
36
–
34
37
38
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
nt
nt
nt
17
17
16
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
nt
C. albicans
S. cerevisiae
S. lutea
M. flavus
B. subtilis
35
35
33
35
32
34
34
29
32
35
38
33
35
34
36
36
38
35
28
37
30
40
34
34
30
34
35
35
36
–
26
30
25
27
23
24
24
26
26
26
29
–
32
34
36
31
36
–
–
35
–
S. aureus
E. coli
P. aeruginosa
C. pyogenes
K. pneumoniae
S. enteritidis
P. vulgaris
Enterococcus sp.
–
–
35
34
35
35
33
–
–
–, Not active, nt – Not tested.
a
Mean values (in mm) of five experiments, including the disc diameter (12.7 nm).
Table 4
Minimal inhibitory concentration (MIC,
lg/ml) of the selected compounds (that have shown activity in the disk diffusion assay, 2a, 2b, 1c, 2c, 1d, 2d, 1e, 2e).
Microorganism
Compound
2a
2b
1c
2c
1d
2d
1e
2e
Amikacin
Bifonazole
A. niger
229
166
>256
161
164
173
157
155
155
172
207
172
139
147
178
170
>256
126
167
155
130
132
139
130
237
138
129
123
>256
>256
>256
141
165
169
177
172
164
>256
>256
>256
161
180
154
211
146
164
144
183
163
152
172
212
163
138
156
192
206
>256
155
168
142
160
152
156
141
>256
152
143
188
198
168
159
>256
>256
>256
>256
>256
194
161
155
177
155
181
155
>256
153
138
152
148
135
>256
124
>256
160
120
129
n.t.
n.t.
n.t.
3
12
32
7
C. albicans
S. cerevisiae
S. lutea
M. flavus
B. subtilis
>256
>256
102
>256
>256
>256
>256
204
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
4
31
15
4
44
17
8
9
8
58
S. aureus
E. coli
P. aeruginosa
C. pyogenes
K. pneumoniae
S. enteritidis
P. vulgaris
Enterococcus sp.
>256
>256
161
>256
>256
>256
164
>256
n.t. – Not tested.
diffusion test. It is quite obvious (Tables 3 and 4) that the com-
pounds have a more profound effect upon the growth of bacteria
than fungal organisms. None of the tested microorganisms were
susceptible to compounds 1a, 1b, 1f, 1g, 2f and 2g. These are the
pairs of compounds having a 4-bromobenzyl (f) and dichloroben-
compounds to the enzyme(s), and though it seems that the length
of the side chain is important, the position of the sulfur atom also
counts (1b and 2a have a chain of 4 atoms, but 1b had no activity
what so ever).
We performed agglomerative hierarchical clustering (AHC) on
the mentioned samples (Table 3, the method was applied utilizing
the values of diameters of growth inhibition zones as original vari-
ables without any recalculation), using the Excel program plug-in
XLSTAT version 2008.6.07. However, the mentioned analysis
showed no logical grouping of the compounds except for making
a clear distinction of 1e and 1c (completely inactive towards the
three tested fungal strains) from the bulk of the other compounds
that showed a bigger impact on the growth of fugal organisms.
zyl group (g), as well as the compounds containing an
(1a) and -Et (1b) side chains. The best results (MIC = 102
a
-MeS
a
lg/mL,
also a growth inhibition zone of 40 mm) have been obtained in
the case of compound 2d against B. subtilis, while a similar effect
was noted for 2b against a strain of Enterococcus sp. with a
123
lg/mL inhibitory concentration and a diameter of the inhibi-
tion zone of 40 mm at 500
lg per disk. Generally, compound 2b,
FcCO(CH2)2SEt, was the most active, but showed no inhibition of
the growth of the yeast S. cerevisiae. However, the active com-
pounds demonstrated non-selectiveness towards the bacteria, ex-
cept that compound 2d that appeared to have
a
more
4. Conclusion
pronounced affect against the fungi but still maintained a distinc-
tive activity against B. subtilis. Additionally, Gram negative and
Gram positive strains have not been differentiated by the synthe-
sized compounds. From the analysis of Tables 3 and 4 it is perhaps
possible to establish some SARs. Comparing compounds with n = 1
and n = 2, the latter show to be more active or similar in activity
(1a and 1b were completely inactive, while the 2a and 2b counter-
parts had shown significant activity). Perhaps the bulkiness of the
side chains in f and g compounds prevented the docking of the
In summary, we synthesized fourteen sulfur containing acylfer-
rocenes, eight of them being new ones. The new compounds have
been characterized by spectral means, whereas the cyclic voltam-
metry technique showed that all the compounds exhibit one well
defined redox couple (attributed to the ferrocene unit) at a very
mutually similar potential. Most of them have also shown a certain
degree of activity against the microorganisms among which some
are notorious human pathogens, however compared to the tested

´
1869
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
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Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033;
or e-mail: deposit@ccdc.cam.ac.uk.
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This work was supported by the Ministry of Science and Tech-
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