Sulfur-containing ferrocenyl alcohols and oximes: New promising antistaphylococcal agents

Article abstract of DOI:10.1002/cbdv.201200029

A small library containing four different series of new ferrocene derivatives, 2-(alkylsulfanyl)-1-ferrocenylethan-1-ols, 3-(alkylsulfanyl)-1- ferrocenylpropan-1-ols, (E)- and (Z)-2-(alkylsulfanyl)-1-ferrocenylethan-1-one oximes, and (E)- and (Z)-3-(alkylsulfanyl)-1-ferrocenylpropan-1-one oximes (36 different compounds in total) was synthesized starting from ferrocene and the corresponding sulfanyl acids. All compounds were spectrally (IR and NMR) and electrochemically characterized. In general, the obtained compounds were found to exhibit very strong antimicrobial activities (broth microdilution assay) against the tested microorganisms (six common human pathogens). For the majority of the tested compounds, the determined MIC values were either under the 10μg/ml MIC limit recognized to delimit efficient antimicrobials or were comparable to/lower than those of the used positive controls (tetracycline/nystatin). The most susceptible organism was found to be Staphylococcus aureus with MIC values even reaching 0.001μg/ml. The presence of iCH(OH)(CH₂)_nSi and iCH(iNOH)(CH₂)_nSi (n=1 or 2) structural fragments seems to be essential for the observed strong activity (introduction of hydroxyimino and alcohol functionalities, instead of the keto function, resulted in a more than 10⁵-fold increase in antistaphylococcal activity in some instances). Nevertheless, a possible influence of the ferrocenyl-core redox chemistry (Fe²⁺/Fe ³⁺) should not be disregarded. The studied alcohols exhibited a reversible one-electron redox couple at almost the same position as ferrocene, while the hydroxyimino group conjugated with cyclopentadienyl ring considerably shifted the redox potential of the ferrocene unit in oximes.

Full text of DOI:10.1002/cbdv.201200029

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Sulfur-Containing Ferrocenyl Alcohols and Oximes: New Promising
Antistaphylococcal Agents
by Danijela Ilic´ a), Ivan Damljanovic´ b), Dragana Stevanovic´ b), Mirjana Vukic´evic´ c),
Polina Blagojevic´ d), Niko Radulovic´*d) and Rastko D. Vukic´evic´*b)
a
ˇ
ˇ
) Technical Faculty Kosovska Mitrovica, University of Pristina, Kneza Milosa 7,
RS-38220 Kosovska Mitrovica
) Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovica 12,
b
´
RS-34000 Kragujevac
(phone: þ38134300286; fax: þ38134335040; e-mail: vuk@kg.ac.rs)
c
´
) Department of Pharmacy, Faculty of Medicine, University of Kragujevac, S. Markovica 69,
RS-34000 Kragujevac
d
ˇ
ˇ
) Department of Chemistry, Faculty of Science and Mathematics, University of Nis, Visegradska 33,
ˇ
RS-18000 Nis
(phone: þ381628049210; fax: þ38118533014; e-mail: nikoradulovic@yahoo.com)
A small library containing four different series of new ferrocene derivatives, 2-(alkylsulfanyl)-1-
ferrocenylethan-1-ols, 3-(alkylsulfanyl)-1-ferrocenylpropan-1-ols, (E)- and (Z)-2-(alkylsulfanyl)-1-fer-
rocenylethan-1-one oximes, and (E)- and (Z)-3-(alkylsulfanyl)-1-ferrocenylpropan-1-one oximes (36
different compounds in total) was synthesized starting from ferrocene and the corresponding sulfanyl
acids. All compounds were spectrally (IR and NMR) and electrochemically characterized. In general, the
obtained compounds were found to exhibit very strong antimicrobial activities (broth microdilution
assay) against the tested microorganisms (six common human pathogens). For the majority of the tested
compounds, the determined MIC values were either under the 10 mg/ml MIC limit recognized to delimit
efficient antimicrobials or were comparable to/lower than those of the used positive controls
(tetracycline/nystatin). The most susceptible organism was found to be Staphylococcus aureus with
MIC values even reaching 0.001 mg/ml. The presence ofꢀCH(OH)(CH2)nSꢀandꢀCH(¼NOH)(CH2)nSꢀ
(n¼1 or 2) structural fragments seems to be essential for the observed strong activity (introduction of
hydroxyimino and alcohol functionalities, instead of the keto function, resulted in a more than 105-fold
increase in antistaphylococcal activity in some instances). Nevertheless, a possible influence of the
ferrocenyl-core redox chemistry (Fe2þ/Fe3þ ) should not be disregarded. The studied alcohols exhibited a
reversible one-electron redox couple at almost the same position as ferrocene, while the hydroxyimino
group conjugated with cyclopentadienyl ring considerably shifted the redox potential of the ferrocene
unit in oximes.
Introduction. – Over the past few decades, the number and extent of infections
caused by multi-drug resistant (MDR) microorganisms has reached an alarming level.
The rapidly developing drug resistance and drug side-effects limit the use of a large
number of antimicrobial agents. All of this represents a serious challenge to the medical
community [1]. Hence, the discovery and development of new antimicrobial agents
became an important goal. Certain organometallic compounds offer a particular
promise in this respect, because they are kinetically inert, often uncharged, and fairly
lipophilic [2]. The metal centers exist in low oxidation states, which limit the danger of
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2237
oxidative damage inside cells [2]. Ferrocene (1; Scheme 1) and some of its derivatives
(e.g., ferrocifen) were found to have strong and useful pharmacological properties, and
are considered as promising lead structures. Even ferrocene itself was found to have
some antiproliferative activity. Its mechanism of action remains elusive despite several
efforts; however, it is assumed by some researchers that the redox chemistry (Fe2þ/Fe3þ
)
is at the heart of its activity [2]. Synthesis of new ferrocene-containing antimicrobial/
cytotoxic agents is one of the main objectives of our research program, and, up to now,
we managed to obtain a considerable number of new ferrocene derivatives, some of
which were shown to possess interesting biological/pharmacological properties [3–12].
In continuation of our work, we wanted to synthesize some additional (new) ferrocene
derivatives that would contain moieties known to be potent pharmacophores.
Scheme 1
Many S- and N-containing organic compounds exhibit strong antimicrobial and
antioxidant properties [10]. For example, the 2- and 3-(alkylsulfanyl)-1-hydroxy moiety
is a common structural fragment in a vast group of natural products that were proven to
have useful biological and pharmaceutical activities [13–18]. Leukotrienes LTC4 and
LTD4 could serve as an example of the previously mentioned. On the other hand, we
assumed, for several reasons, that the introduction of the hydroxyimino group could
further enhance antimicrobial properties of the designed ferrocenyl derivatives. First of

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
all, it is a quite common and stable functional group frequently employed in current
drugs [19]. Second, the oxime group has an electron lone pair of at the N-atom that can
participate in H-bonding with the drug target. And finally, this functional group can be
readily obtained from the corresponding ketone in a single synthetic step [20].
Thus, encouraged by all of the previously mentioned findings and with the aim of
finding new antimicrobial agents, we have synthesized, and spectrally and electro-
chemically characterized four different, analogous series of ferrocene derivatives, 2-
(alkylsulfanyl)-1-ferrocenylethan-1-ols,
3-(alkylsulfanyl)-1-ferrocenylpropan-1-ols,
(E)- and (Z)-2-(alkylsulfanyl)-1-ferrocenylethan-1-one oximes, and (E)- and (Z)-3-
(alkylsulfanyl)-1-ferrocenylpropan-1-one oximes (36 different compounds in total;
Scheme 1). All of the synthesized compounds were assayed for antimicrobial activities
(broth microdilution susceptibility test). The set of compounds prepared and tested
varied in structure in such a manner as to additionally allow the examination of
structureꢀactivity relationships. By changing the the alkyl group at the S-atom, we
anticipated that we could finetune the lipophilicity of the novel compounds in order to
attain the highest potency and most favorable physical properties. Moreover, the
electrochemical properties of synthesized compounds (that possibly could also be
important with respect to the aims of this study) were investigated by cyclic
voltammetry.
Results and Discussion. – Library Design. A series of S-containing acyl ferrocenes
was previously studied and characterized in terms of their antimicrobial properties
(broth microdilution assay) [3]. Despite the presence of keto, ferrocenyl, and sulfanyl
moieties, at least some of which are considered to induce potentially strong biological
activities in a number of different, previously studied molecules, these compounds have
shown moderate-to-low antimicrobial activity [3]. One of the reasons for the obtained
unexpectedly poor results of the performed activity assays could be possibly found in
the fact that the electrophilicity of the keto group is significantly reduced by
conjugation with the strongly electron-donating ferrocene core, directly attached to the
CO C-atom. Regardless of the mentioned, somewhat discouraging (average) results, we
decided to use this series of acylferrocenes as the starting point for the design of new
compounds that would potentially have enhanced target properties. We assumed that
simple chemical modifications of the mentioned ketones would result in significant
improvement of the desired biological activity. Based on the fact that 2- and 3-
(alkylsulfanyl)-1-hydroxy unit is a common structural motif in a number of biologically
and pharmacologically active natural products [14–18], we decided to convert the CO
function in the S-containing acylferrocenes to the alcohol group. On the other hand, for
the already mentioned reason (a stable functional group frequently employed in
current drugs, the presence of a N lone pair of electrons that can participate in H-
bonding with the drug target), we supposed that the introduction of (hydr)oxyimino
group could further enhance antimicrobial properties of the designed ferrocenyl
derivatives [19]. Replacement of the CO (H-acceptor) by alcohol and hydroxyimino
groups (H-acceptor/donor) could have a significant impact on the binding of these new
analogs with the target biomolecule(s). A further assumption was that the sulfanyl and
alcohol/oxime groups would not ꢂactꢃ separately, as the distance between these two
moieties (one or two CH2 groups apart) could allow potential anchimeric assistance of

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2239
S in the reactions that would involve nucleophilic attack at the C-atom directly attached
or part of the alcohol/oxime functionalities.
Alongside with the derivatives with alkyl groups (normal and branched chains;
different number of C-atoms) on the S-atom, we decided to also include analogs
bearing a (2,6-dichlorophenyl)methyl moiety. This was conducted to possibly test the
influence of an additional aromatic core on the activity (corresponding additional
binding interactions with the target(s) might be important for optimal docking).
As previously shown, the length and bulkiness of the side chain, as well as the
position of the S-atom, were important issues that influenced the antimicrobial
activities of the studied S-containing ferrocenyl ketones [3]. Our aim was to test if new
derivatives would follow the same trends, or if the newly introduced functional groups,
oxime and alcohol, would have a predominant impact on the expected activity.
Chemistry. The synthesis of S-containing ferrocenyl alcohols, 4a–4l, and oximes,
5a–5l, is outlined in Scheme 1. Ferrocenyl ketones 3a–3l were obtained via the
modified FriedelꢀCrafts acylation of ferrocene (1) with in situ prepared chlorides of the
corresponding sulfanyl acids 2a–2l, respectively, according to the previously published
procedure [3][8][9]. Then, they were either reduced with NaBH4 [6] or were refluxed
in MeOH with NH2OH·HCl [7]. In this way, a library of 24 different S-containing
ferrocenyl derivatives (alcohols: series I, compounds 4a–4f; series II, compounds 4g–
4l; and oximes: series III, compounds 5a–5f; series IV, compounds 4g–4l) was
prepared. Nevertheless, the hydroxyimination of ketones gave mixtures of the
corresponding (E)- and (Z)-isomers in quantitative yields. The ratio of these
1
diastereoisomers was inferred from H-NMR data of the mixtures and was found to
be in the range from (E)/(Z) 2.08 to (E)/(Z) 6.85. Single-crystal X-ray-analysis of the
pure isomers confirmed that the (E)-isomer is the one obtained in higher yield [7],
while calculations performed on the semi-empirical level of theory (PM3 method;
PolakꢀRibiere (conjugate gradient) minimization method; energy convergence crite-
rion of 0.01 kJ/mol) showed that, generally speaking, in the gas phase, the (E)-isomer is
the one thermodynamically more favorable (e.g., for compound 5b, calculated
equilibrium (E)/(Z) ratio was 40.33). In addition, it has been previously shown that
(E)- and (Z)-isomers undergo interconversion on prolonged standing in solution [7].
Thus, since the dissolution of any of the pure isomers would eventually give an
equilibrium mixture of these two isomers, they were not further separated from one
another, and their mixtures (obtained after the appropriate workup of the correspond-
ing reaction mixtures) were used for the antimicrobial assays (this equilibration is
expected to go to completion in the nutrient broth during the antimicrobial tests).
Spectral Characterization of the Obtained Compounds. All synthesized compounds
were characterized by means of IR, and 1H- and 13C-NMR spectroscopy. IR Spectra of
the compounds of series I and II, i.e., 4a–4l, displayed broad bands between 3550 and
3200 cmꢀ1 (OH), and strong adsorptions in the range of 970–1150 cmꢀ1 (CꢀO),
characteristic for alcohols. This, together with the absence of the C¼O absorption,
confirmed the transformation of the ketones to the corresponding alcohols. As
expected, IR spectra of the compounds of the series III and IV, i.e., 5a–5l, were
characterized by the presence of intensive bands in the ranges of 3600–2800 (OH) and
1640–1615 cmꢀ1 (C¼N), which corroborated the presence of the hydroxyimino moiety
in the structures of these compounds. Once again, a band characteristic for ketones

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(1760–1670 cmꢀ1) was not observed. Spectra of some of the studied compounds
(especially oximes 5a–5l) were further characterized by a strong band around 490 cmꢀ1
that most probably corresponds to the ferrocene ring tilt.
1
In the H-NMR spectra of all obtained alcohols, the signals of H-atoms of the
ferrocene ring were observed at 4.17–4.28 ppm. Five H-atoms from Cp5 H ring
(cyclopentadienyl ring with 5 H-atoms) gave a broad singlet at ca. 4.20 ppm, while the
signals of Cp4 H (cyclopentadienyl with 4 H-atoms) H-atoms appeared as two broad
pseudo-triplets (in some cases, even more complex multiplets) at ca. 4.18 and 4.25, with
coupling constant of ca. 1.8 Hz. For compounds 4a–4f (series I), signals of the two
diastereotopic H-atoms (b-H atoms), observable at ca. 2.73 and 2.88, characterized by a
strong geminal coupling, were well-defined doublets of doublets (Ja ꢁ13 and Jb ꢁ4 Hz,
i.e., Ja ꢁ13 and Jb ꢁ8 Hz), and they could be straightforwardly assigned. With the other
alcohol series (series II, 4g–4l), these H-atoms were a part of a much more complex
spin system (XCHCH2CH2Y). Each of them was coupled with four other different H-
atoms. It seems, from the shape and complexity of these and of the signals of other H-
atoms from the same spin system, that some of the coupling constants had close, but still
not completely equal values. For the mentioned reasons, detailed analyses of the
mentioned multiplets at ca. 1.85–2.00 (b-H atoms) were not possible. In some cases, the
signal(s) corresponding to the g-H-atoms (at ca. 2.66) had an appearance of a broad
triplet, but in fact it was a multiplet that was not further analyzed. It could be interesting
to note that the spectra of some of the alcohols (e.g., 4f, 4h, 4j, etc.) showed couplings of
the a- and the H-atoms from the alcohol groups, with the value of ca. 3 Hz. After the
addition of a few drops of D2O to the mentioned samples, the signal assigned to the OH
H-atom disappeared, and the structure of that corresponding to the a-H atom was
simplified (i.e., in some cases, the hard-to-interpret multiplet, at ca. 4.3 ppm, became a
sharp doublet of doublets). Worth mentioning is the finding that a low exchange rate on
the NMR time scale of analogous exchangeable H-atoms (attached to heteroatoms)
has previously been already noticed for some other ferrocene derivatives, while their
phenyl analogs displayed a swift exchange [10][11].
As mentioned before, the hydroxyimination of ketones gave mixtures of (E)- and
(Z)-isomers. Previously, it was shown that only the (E)-isomer is the one obtained in
higher yield, and that the two isomers undergo interconversion easily on the prolong
standing in solution [7]. Therefore, the isomers were not separated from one another
prior to further analysis/assays. In general, NMR signals corresponding to (E)- and
(Z)-isomers were well-resolved, and their intensities differed significantly. This
enabled their (almost complete) assignments, even through the analysis of the NMR
spectra of mixtures of isomers 5a–5l. The ratio of the isomers was also estimated on the
basis of 1H-NMR. This was accomplished via the ratios of the integrals corresponding
to the matching appropriate signals. In the case of the compounds belonging to the
series III, singlets corresponding to the H-atoms of the CH2S moieties (at ca. 3.55 and
3.72) were used, while for those from the series IV, signals of the two H-atoms from the
Cp4 H ring (appearing at higher shifts, ca. 4.56 and 4.91) were chosen.
As in the case of alcohols, the shifts corresponding to the H-atoms from the
repeating structural units of all of the compounds belonging to series III and IV were
mutually comparable. Shifts of the analogous H-atoms from the ferrocene core differed
only slightly. The same is true for b- (and g-H-atoms) or those from the alkylꢀS

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2241
substituents. Multiplets corresponding to b- and g-H-atoms of the compounds 5g–5l
(series IV; ca. 2.78–3.00) overlapped completely, and their complete assignment was
not possible. As expected, it was much simpler to interpret the spectra of the
compounds belonging to the series III.
The structures of the synthesized compounds were additionally corroborated by the
analysis of their 13C-NMR spectra. The 13C-NMR spectra of the studied compounds
contained groups of signals corresponding to the ferrocene ring (one signal that
originated from five C-atoms of the Cp5 H ring and five signals of the C-atoms of
Cp4 H; signals of the C-atoms of Cp4 H were not equivalent, due to the presence of a-
stereogenic center, alkyl chains, and C-atoms of the oxime/carbinol functionalities,
which could be easily distinguished and almost completely unambiguously assigned. In
the case of compound 5l, with the higher (E)/(Z)-ratio, some signals corresponding to
the C-atoms of the (Z)-isomer were not observed, but the shifts of those detected in the
spectrum had expected values, based on the structure and the spectra of other studied
compounds.
Electrochemistry. As previously mentioned, it is assumed by some researchers that
the redox chemistry (Fe2þ/Fe3þ ) of the ferrocene core is the source of its biological/
pharmacological activity [2]. Bearing this in mind, electrochemical investigations
undertaken in the present study were aimed to evaluate redox features of the
synthesized compounds appearing due to the presence of the ferrocene nucleus. For
this purpose, cyclic voltammetry of the synthesized compounds in MeCN, containing
0.1m LiClO4 as the supporting electrolyte, has been utilized. Cyclic voltammetry was the
method of choice, as it is a very versatile electrochemical technique, which allows
probing the mechanisms of redox reactions and transporting properties of a system in
solution [21]. Since ferrocene itself exhibits, under given conditions (a Pt working
electrode, scanning rate n¼0.1 V·sꢀ1), a reversible redox couple at E1/2(n)¼0.358 V,
we chose the potential window 0.000–1.000 V for our measurements. As the
representative examples, we give here the voltammograms of alcohol 4h and oxime
5h (Fig. 1), whereas the data for all other alcohols and oximes are compiled in Table 1.
It is observable from this data that all alcohols exhibit a reversible one-electron redox
couple at almost the same position as ferrocene. However, as it can be expected from an
electron-withdrawing group, the hydroxyimino functionality conjugated with the
cyclopentadienyl ring considerably shifts the redox potential of the ferrocene unit in
oximes (by ca. 0.100 V). This shift, however, is considerably less than that of the CO
group in the same position (by ca. 0.300 V) [3]. The differences between anodic and
cathodic peak potentials (cf. Table 1) are close to theoretical values, and independent of
the scan rate n. 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.
Biological Activity. Antibacterial activities against two Gram-positive (S. aureus
and B. cereus) and three Gram-negative bacteria (E. coli, K. pneumoniae, and P.
vulgaris), as well as the antifungal activity against one fungal organism (C. albicans)
were assessed for compounds 4a–4l and 5a–5l. The minimal inhibitory concentration
(MIC; the lowest concentration of the tested compound in mg/ml which inhibited the
growth of bacteria or fungi) values were determined using a microdilution method
based on the recommendations of NCCLS [22]. Under the same conditions, solutions

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Fig. 1. Cyclic Voltammograms of a) 5 mm ferrocene, b) 5 mm 3b, c) 5mm 4h, and d) electrolyte at the Pt
electrode (2 mm diameter) with a 0.1 Vsꢀ1 scan rate in a 0.1m LiClO4 solution in MeCN
of differing concentration of tetracycline (antibacterial) and nystatin (antifungal) were
used as positive controls. The results of the broth microdilution susceptibility assay are
compiled in Table 2. As presumed during the library design, compounds 4a–4l and 5a–
5l showed significantly higher antimicrobial activities than the corresponding starting
ketones 3a–3l [3]. The MIC values of the synthesized compounds ranged from 0.001 to
5000 mg/ml. Concentrations above 5000 mg/ml were not tested. Every compound tested
showed a very high inhibitory activity against at least one microorganism. For some of
the tested compounds, MIC values against all of the microbial strains (apart from for E.
coli) were close to or under the 10-mg/ml MIC limit set for an efficient antimicrobial
[3]. It must be stressed that the activity of some of them was of the same order of
magnitude, or even better, compared to the positive controls that were used. Moreover,
some of the compounds could be considered as being quite selective in their action
(their activity varied significantly from one microorganism to another). For example,
compound 5e was much more active against B. cereus and C. albicans (MICꢂ0.400 mg/
ml) than against other tested strains (MICꢃ39.000 mg/ml). Just the opposite could be
said for compound 4b. It was characterized with lower MIC values than tetracycline in
the case of S. aureus and K. pneumoniae. It showed notable activities against all other
considered microorganisms as well (MICꢂ1.000 mg/ml, except against E. coli; see
Table 2).
To better interpret the results of the biological assays, we performed agglomerative
hierarchical clustering (AHC) and principal component analysis (PCA) on the
mentioned samples (Table 2; the methods were applied utilizing the MIC values as

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2243
Table 1. Electrochemical Data for Compounds 4a–4l and 5a–5l
Compound
Epaa) [V]
Epca) [V]
E1/2b) [V]
DEc) [mV]
Alcohols
4a
4b
4c
4d
4e
4f
4g
4h
4i
0.385
0.406
0.394
0.388
0.388
0.394
0.381
0.378
0.381
0.400
0.385
0.400
0.314
0.327
0.320
0.327
0.320
0.327
0.311
0.308
0.311
0.320
0.314
0.320
0.350
0.366
0.357
0.357
0.354
0.360
0.346
0.343
0.346
0.360
0.350
0.360
71
79
74
61
68
67
70
70
70
80
71
80
4j
4k
4l
Oximes
5a
5b
5c
5d
5e
5f
5g
5h
5i
0.479
0.473
0.479
0.476
0.476
0.485
0.479
0.488
0.485
0.482
0.485
0.485
0.406
0.403
0.403
0.403
0.403
0.418
0.409
0.409
0.409
0.406
0.406
0.409
0.442
0.438
0.441
0.440
0.440
0.452
0.444
0.448
0.447
0.444
0.446
0.447
73
70
76
73
73
67
70
79
76
76
79
76
5j
5k
5l
a) Epa and Epc are anodic and cathodic peak potentials, respectively, at 0.1 Vsꢀ1
.
b) E1/2 ¼(Epa þEpc)/2.
c) DE¼Epa ꢀEpc.
original variables without any recalculation). The results of the statistical analyses
showed that the level of activity of a great number of the tested compounds was not
statistically different from that of the controls. In addition, compound 5f was clearly
separated from all others as the least active one. A possible explanation for this could
be sought in the bulkiness of the particular S-substituent, which could have a negative
impact on the 3f/5f docking to the target biomolecule. In contrast to 5f, compound 5l
was much more active against the tested strains. Once again, for this pair of compounds,
this is in agreement with the previous results, which revealed that there are significant
differences in the activity of 2- and 3-(alkylsulfanyl)acylferrocenes [3]. It could be
interesting to note, however, that alcohols 4f and 4l were both active in rather low
concentrations against the tested strains.
Multivariate statistical analyses performed using an inversed observation/variable
table as the input data set clearly separated E. coli, the least susceptible strain, from all
other microorganisms. This type of analysis also indicated that the studied active
compounds demonstrate some level of selectivity towards bacteria or fungi. However,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Table 2. MIC Values [mg/ml] of the Studied Compounds, 4a–4l and 5a–5l, Obtained from a Broth
Microdilution Susceptibility Assay
Compound Bacteria
Gram-positive
Fungus
Gram-negative
S. aureus
B. cereus
E. coli
K. pneumoniae
P. vulgaris C. albicans
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
5a
5b
5c
5d
5e
5f
310
39
156
156
0.30
156
1250
2500
156
1.000
620
70
70
0.10
0.20
0.40
0.20
0.90
0.030
0.15
0.20
0.90
0.20
0.003
0.20
0.20
0.003
0.15
156
0.20
0.20
0.40
0.070
0.40
0.070
39
0.070
9.000
0.001
0.070
0.050
0.001
0.001
9.0
0.050
0.050
0.40
19
0.10
19
9.0
1250
0.001
0.003
9.0
0.10
19
>5000
>5000
>2500
>2500
1250
>2500
>5000
>5000
>2500
>2500
>5000
>5000
>5000
>5000
>5000
750
0.003
0.003
0.003
0.780
0.150
156
0.20
0.003
0.30
156
0.90
0.020
0.050
1.0
35
70
0.400
0.020
0.40
560
0.10
310
70
560
70
35
70
0.90
0.070
0.070
0.030
0.030
0.070
0.15
0.070
0.15
0.070
0.070
156
0.40
0.90
0.030
0.070
0.40
39
156
78
0.110a)
0.10
0.10
0.070
0.20
0.40
0.10
78
>2500
5g
5h
5i
5j
5k
5l
>5000
>2500
>5000
>5000
>5000
325
0.070
0.80
0.030
0.030
0.070
0.030
0.039b)
0.40
310
1.0
0.110a)
4.0
0.003a)
Control
0.230a)
0.050a)
a) Tetracycline. b) Nystatin.
it seems that Gram-negative and Gram-positive strains were not differentiated by their
susceptibility towards the synthesized compounds.
For all of the prepared compounds, log Po/w values were calculated (this was
performed in the following way: log Po/w of the corresponding phenyl analogs was
assessed using Chem3D ultra 10.0 Software (CambridgeSoft), and the obtained values
were then corrected according to [22]). But, it seems that the hydrophilicity of the
compounds does not play an important role, when antimicrobial activity of this set of
ferrocenyl derivatives is in question. In addition, to check if there is any correlation
between the redox properties of these ferrocene derivatives and the observed activity,
we plotted the redox potentials (Epa, Epc, E1/2, and DE) against log(1/MIC) and
performed linear regression analyses (least-squares method). In all cases, the
correlation coefficients (r2) had values considerably less than 0.8 (maximal r2 value
found was 0.6). These results indicate that the redox properties were not essential for
the observed differences in the activities (in any potential QSAR equation, the
corresponding coefficients would probably have relatively low values).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2245
Based on all of the previously mentioned findings, it seems that the significant
increase of activity for the alcohols and oximes, compared to the starting ketones, is not
(predominantly) due to the changes in the hydro/lipophilicity and/or electrochemical
properties, but that the newly introduced functional groups are directly involved in
some novel interaction with the target biomolecules. Possible mechanisms of
antimicrobial action of the herein studied series of compounds are depicted in
Schemes 2 and 3. After the initial protonation of the OH groups, and a concomitant
intramolecular attack of the S-atom (anchimeric aid), H2O could be eliminated from
the molecule to give the highly electrophilic substituted 1-alkylthiiranium or 1-
alkylthietanium cations. The two reactive species could then react with a (bio)nucleo-
phile and give two possible products (Scheme 2) that would represent covalently
modified (inactive) enzymes for example. An analogous mechanism could be pictured
for the oximes as well (Scheme 3): after the protonation, S (intramolecular
nucleophile) could attack the oxime electrophilic C-atom and form a potent electro-
philic species, i.e., intramolecular tetrahedral intermediate. The obtained substituted 1-
alkylthiiranium/1-alkylthietanium cations could then react with some available
nucleophile (bacterial metabolite).
Scheme 2. Possible Mechanism of Antimicrobial Action of the Studied Alcohols through Anchimeric
Assistance (Nu: nucleophile)
Although further studies to test the correctness of this potential mechanism of
action were not carried out, we have performed a simple test which showed that
anchimeric assistance of the S-atoms in these molecules is quite probable. Compound
4b (m/z 290 (Mþ )), dissolved in Et2O, was allowed to stand in the presence of BuNH2
for 1 h. Then, the reaction mixture was directly injected into a GC/MS, and the
concomitant analysis showed that the dominantly produced compound was the
corresponding elimination product (ethyl(2-ferrocenylvinyl)sulfane (m/z 272 (Mþ ));
identification of this compound was based on the MS fragmentation pattern (Fig. 2),
and the correspondence of the experimentally determined and expected values of the
retention indices. It seems that there are no other reasonable mechanisms, except for

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
Scheme 3. Possible Anchimeric Mechanism of Antimicrobial Action of the Studied Oximes (Nu:
nucleophile)
Fig. 2. Mass spectra of compounds 4b (top) and ethyl(2-ferrocenylethenyl)sulfane (bottom)
that involving the anchimeric assistance of the S-atom that could explain the
elimination of H2O from the alcohol (4b) under basic conditions.
It is interesting to note that, except 4l/4f and 5l/5f (log Po/w >5), all other herein
studied ferrocene derivatives are in agreement with the Lipinskiꢃs rule of five, related
to orally active drugs. This rule gives only rough guidelines regarding molecules
considered for oral pharmaceuticals. Nevertheless, the finding that the studied series of
biologically active molecules fulfills at least these basic general requirements could be
considered as a justification for some further, more in-depth studies.
Conclusions. – To summarize, taking into account the properties of hydroxyimino
and hydroxy functional groups and the results of a previous study focused on the

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2247
biological activity of a series of S-containing acyl ferrocenes [3], we have designed a
small library comprising four different, but analogous groups of compounds. All of the
obtained compounds were spectrally and electrochemically fully characterized, and
their antimicrobial activities against some of the common human pathogens were
determined (broth microdilution assay). The results of the antimicrobial assays showed
that the studied compounds are strong and promising antimicrobial agents (except in
the case of E. coli). Also, the results of the activity studies, as well as the calculated log
Po/w values, indicated that the introduction of alcohol/oxime groups was essential for the
onset of activity of the studied ferrocenyl derivatives, leading to a more than 105-fold
increase in antistaphylococcal activity in some instances, and that correct assumptions
were used during the library design. Furthermore, as can be seen from Table 2, although
some correlation between the activity of compounds and the position of the S-atom
could be found, it seems that this is not as important for the observed antimicrobial
properties as in the case of the corresponding ketones. However, similarly as for
starting acyl ferrocenes, the effect of bulkiness of the side chains could be easily
deduced (compare the activities of compounds with Pr and iPr groups attached to the S-
atom (Table 2)). The finding that the studied compounds were generally active in very
low concentrations (e.g., MIC values against S. aureus were only 0.001 mg/ml for
compounds 4e, 4h, 4j, and 5g), which were either under the 10 mg/ml MIC limit or were
comparable/lower than those determined for the used positive controls (tetracycline
and nystatin), renders them as candidates for further studies, even as potential oral
antimicrobial drugs especially against S. aureus.
This work was funded by the Ministry of Education and Science of Serbia (Project 172034).
Experimental Part
General. All chemicals were commercially available and used as received, except that the solvents
were purified by distillation. The protected sulfanyl acids 2a–2l (Scheme 1) were synthesized by the
known procedure from the corresponding sulfanyl acids (thioglycolic and 3-sulfanylpropanoic acid) and
org. halides [9]. Column chromatography (CC): silica gel 60 (Merck, 230–400 mesh ASTM). TLC: silica
gel 60 on Al plates, layer thickness 0.2 mm (Merck). M.p. (uncorrected): Mel-Temp cap. melting-points
apparatus, model 1001. IR Spectra: Perkin-Elmer FTIR 31725-X spectrophotometer. 1H- and 13C-NMR
spectra: in CDCl3, Varian Gemini (200 MHz) spectrometer; chemical shifts (d) [ppm] rel. to the residual
1
solvent H-atoms CHCl3 (7.26 ppm) for H, and 13CDCl3 (77.0 ppm) for 13C as the internal standards.
Cyclic voltammetry experiments: at r.t., under Ar in a three-electrode cell using an Autolab potentiostat
(PGSTAT 302N). The working electrode was a Pt disk (2 mm diameter). The counter electrode was a Pt
wire, and a Ag/AgCl electrode was used as the reference electrode.
Synthesis of Ketones 3a–3l [3][8][9]. A mixture of the corresponding carboxylic acid (3 mmol),
PCl3 (0.3 ml; ca. 1 mmol), and ferrocene (558 mg; 3 mmol) in 50 ml of CH2Cl2 was stirred 3 h at r.t. under
Ar, then anh. AlCl3 (600 mg) was added. The obtained dark-violet mixture was stirred for 3 h, and the
reaction was quenched with 50 ml of cold 2m KOH soln. The org. layer was separated, and the H2O phase
was extracted with CH2Cl2 (two 30-ml portions). The collected org. layers were washed with H2O and
brine, and dried (Na2SO4) overnight. After the evaporation of the solvent, the residue was purified by CC
(SiO2; petroleum ether (PE)/AcOEt 9 :1 (v/v)). A small amount of the unchanged ferrocene eluted as
the first component, followed by the corresponding ketones 3a–3k (36–86%) [3][8][9].
Synthesis of Alcohols 4a–4l [6]. To a soln. of the corresponding ketone 3a–3k (1 mmol) in MeOH
(30 ml), an excess of NaBH4 was added in several portions, and the resulting mixture was stirred at r.t.,
monitoring the reaction progress by TLC. After the reaction was completed (ca. 2 h), the solvent was

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
evaporated, and H2O (30 ml) was added to the residue. The mixture was extracted with CH2Cl2 (two
30 ml portions), the collected org. layers washed with H2O and brine, and dried (anh. Na2SO4) overnight.
After the evaporation of the solvent, the crude product was subjected to CC (SiO2; toluene/AcOEt 9 :1
(v/v)). The starting ketone was eluted first, then the corresponding alcohols 4a–4k, followed by small
amounts of 1-ferrocenylethanol (in the case of ketones 3a–3f) or 1-ferrocenylpropan-1-ol (in the case of
ketones 3g–3l).
1-Ferrocenyl-2-(methylsulfanyl)ethanol (4a). Yield: 72%. IR (KBr): 3442, 3092, 2915, 1410, 1224,
1
1105, 1049, 1000, 817. H-NMR (200 MHz): 2.11 (s, Me); 2.70 (br., OH); 2.71 (dd, J¼13.7, 8.1, 1 H of
CH2S); 2.82 (dd, J¼13.7, 4.5, 1 H of CH2S); 4.17 (br., 2 H of Cp4 H); 4.19 (s, Cp5 H); 4.27 (br., 2 H of
Cp4 H); 4.49 (br. dd, J¼8.1, 4.5, CHOH). 13C-NMR: 16.0 (C(1’)); 42.5 (C(2)); 65.6 (1 C of Cp4 H); 66.7
(1 C of Cp4 H); 67.8 (1 C of Cp4 H); 67.9 (1 C of Cp4 H); 68.0 (1 C of Cp4 H); 68.4 (Cp5 H); 91.3 (C(1)).
Anal. calc. for C13H16FeOS (276.18): C 56.54, H 5.84; found: C 56.58, H 5.86.
2-(Ethylsulfanyl)-1-ferrocenylethanol (4b). Yield: 94%. IR (KBr): 3438, 3093, 2963, 2924, 2870,
1451, 1410, 1265, 1224, 1105, 1045, 1022, 1000, 816. 1H-NMR (200 MHz): 1.26 (br. t, J¼7.3, Me); 2.56 (br.
q, J¼7.3, MeCH2); 2.68 (br., OH); 2.73 (dd, J¼13.6, 8.2, 1 H of CH2S); 2.88 (dd, J¼13.6, 4.2, 1 H of
CH2S); 4.18 (br., 2 H of Cp4 H); 4.20 (s, Cp5 H); 4.28 (br., 2 H of Cp4 H); 4.44–4.50 (m, CHOH).
13C-NMR: 14.7 (C(2’)); 26.4 (C(1’)); 40.0 (C(2)); 65.7 (1 C of Cp4 H); 66.8 (1 C of Cp4 H); 67.8 (1 C of
Cp4 H); 67.9 (1 C of Cp4 H); 68.3 (1 C of Cp4 H); 68.4 (Cp5 H); 91.5 (C(1)). Anal. calc. for C14H18FeOS
(290.20): C 57.94, H 6.25; found: C 57.97, H 6.23.
1-Ferrocenyl-2-(propylsulfanyl)ethanol (4c). Yield: 79.5%. IR (KBr): 3445, 3094, 2960, 2920, 2871,
1456, 1410, 1382, 1223, 1105, 1046, 1022, 1000, 816. 1H-NMR (200 MHz): 0.98 (t, J¼7.3, Me); 1.60 (br. qt,
J¼7.3, MeCH2); 2.50 (br. t, J¼7.3, CH2CH2S); 2.71 (dd, J¼13.5, 8.3, 1 H of CH2S); 2.76 (br., OH); 2.85
(dd, J¼13.5, 4.3, 1 H of CH2S); 4.15 (br. t, J¼1.6, 2 H of Cp4 H); 4.18 (s, Cp5 H); 4.27 (br. t, J¼1.6, 2 H
of Cp4 H); 4.46 (br. dd, J¼8.3, 4.3, CHOH). 13C-NMR: 13.3 (C(3’)); 22.8 (C(2’)); 34.5 (C(1’)); 40.4
(C(2)); 65.6 (1 C of Cp4 H); 66.6 (1 C of Cp4 H); 67.7 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 68.4 (Cp4 H);
68.4 (5 C of Cp5 H); 91.3 (C(1)). Anal. calc. for C15H20FeOS (304.23): C 59.22, H 6.63; found: C 59.25, H
6.66.
1-Ferrocenyl-2-[(1-methylethyl)sulfanyl]ethanol (4d). Yield: 78.4%. IR (KBr): 3445, 3094, 2959,
2923, 2865, 1453, 1410, 1383, 1365, 1223, 1105, 1046, 1000, 816. 1H-NMR (200 MHz): 1.27 (br. d, J¼6.8,
2 Me); 2.69 (br., OH); 2.74 (dd, J¼13.2, 8.1, 1 H of CH2S); 2.89 (dd, J¼13.2, 4.2, 1 H of CH2S); 2.94
(sept., J¼6.8, Me2CH); 4.17 (br., 2 H of Cp4 H); 4.20 (s, Cp5 H); 4.29 (br., 2 H of Cp4 H); 4.45 (dd, J¼
8.1, 4.2, CHOH). 13C-NMR: 23.4 (C(2’)); 23.4 (C(2’)); 35.2 (C(1’)); 38.9 (C(2)); 65.7 (1 C of Cp4 H); 66.8
(1 C of Cp4 H); 67.9 (1 C of Cp4 H); 68.4 (Cp5 H); 68.5 (1 C of Cp4 H); 68.6 (1 C of Cp4 H); 91.6 (C(1)).
Anal. calc. for C15H20FeOS (304.23): C 59.22, H 6.63; found: C 59.24, H 6.67.
2-(Butylsulfanyl)-1-ferrocenylethanol (4e). Yield: 99%. IR (KBr): 3447, 3094, 2956, 2927, 2871, 1464,
1
1411, 1273, 1221, 1106, 1047, 1022, 1000, 816. H-NMR (200 MHz): 0.91 (t, J¼7.2, Me); 1.24–1.47 (m,
MeCH2); 1.48–1.66 (m, CH2CH2Me); 2.53 (t, J¼7.2, CH2S); 2.66 (br., OH); 2.72 (dd, J¼13.4, 8.2, 1 H of
CH2S); 2.86 (dd, J¼13.2, 4.2, 1 H of CH2S); 4.17 (br., 2 H of Cp4 H); 4.20 (s, Cp5 H); 4.28 (br., 2 H of
Cp4 H); 4.46 (br. dd, J¼8.1, 4.2, CHOH). 13C-NMR: 13.6 (C(4’)); 21.9 (C(3’)); 31.7 (C(2’)); 32.3 (C(1’));
40.5 (C(2)); 65.7 (1 C of Cp4 H); 66.9 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 67.9 (1 C of Cp4 H); 68.5
(Cp5 H); 68.5 (1 C of Cp4 H); 91.5 (C(1)). Anal. calc. for C16H22FeOS (318.26): C 60.38, H 6.97; found: C
60.40, H 6.96.
2-{[(2,6-Dichlorophenyl)methyl]sulfanyl}-1-ferrocenylethanol (4f). Yield: 79.6%. IR (KBr): 3447,
3092, 2920, 1580, 1560, 1436, 1219, 1105, 1087, 1047, 1000, 819, 778, 759. 1H-NMR (200 MHz): 2.60 (d, J¼
3.0, OH), 2.83 (dd, J¼13.6, 8.4, 1 H of CH2S); 2.98 (dd, J¼13.6, 4.2, 1 H of CH2S); 4.06 (s, ArCH2S); 4.17
(br., 2 H of Cp4 H); 4.18 (s, Cp5 H); 4.21 (br., 2 H of Cp4 H); 4.45–4.55 (m, CHOH); 7.11 (dd, J¼7.2,
8.8, 1 arom. H); 7.30 (br. d, Jꢁ7.6, 2 arom. H). 13C-NMR: 32.0 (C(1’)); 40.8 (C(2)); 65.7 (1 C of Cp4 H);
66.9 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 67.9 (1 C of Cp4 H); 68.5 (Cp5 H); 68.6 (1 C of Cp4 H); 91.5
(C(1)); 128.5; 128.6; 135.2; 135.4. Anal. calc. for C19H18Cl2FeOS (421.16): C 54.18, H 4.31; found: C 54.22,
H 4.34.
1-Ferrocenyl-3-(methylsulfanyl)propan-1-ol (4g). Yield: 96.3%. IR (KBr): 3420, 3092, 2915, 1426,
1411, 1276, 1262, 1236, 1105, 1046, 1023, 1000, 960, 882, 815. 1H-NMR (200 MHz): 1.87–2.00 (m,
CH2CH2S); 2.09 (br., OH); 2.11 (s, Me); 2.57–2.67 (m, CH2S); 4.15–4.19 (m, 2 H of Cp4 H); 4.20 (br. s,

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2249
Cp5 H); 4.22–4.26 (m, 2 H of Cp4 H); 4.43–4.54 (m, CHOH). 13C-NMR: 15.5 (C(1’)); 30.7 (C(3)); 37.1
(C(2)); 65.3 (1 C of Cp4 H); 66.9 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 68.0 (1 C of Cp4 H); 68.3 (Cp5 H);
68.4 (1 C of Cp4 H); 93.4 (C(1)). Anal. calc. for C15H20FeOS (290.20): C 57.94, H 6.25; found: C 57.98, H
6.22.
3-(Ethylsulfanyl)-1-ferrocenylpropan-1-ol (4h). Yield: 79.4%. IR (KBr): 3420, 3093, 2961, 2922,
1449, 1411, 1375, 1262, 1105, 1044, 1019, 1000, 889, 815. 1H-NMR (200 MHz): 1.26 (t, J¼7.3, Me); 1.87–
1.99 (m, CH2CH2S); 2.09 (d, J¼3.2, OH), 2.54 (br. t, J¼7.3, MeCH2S); 2.60–2.70 (m, CH2CH2S); 4.17
(br. t, J¼1.6, Cp4 H); 4.20 (s, Cp5 H); 4.24 (br. t, J¼1.6, Cp4 H); 4.43–4.53 (m, CHOH). 13C-NMR: 14.7
(C(2’)); 25.9 (C(1’)); 28.1 (C(3)); 37.5 (C(2)); 65.3 (1 C of Cp4 H); 66.9 (1 C of Cp4 H); 67.8 (1 C of
Cp4 H); 68.0 (1 C of Cp4 H); 68.3 (Cp5 H); 68.4 (1 C of Cp4 H); 93.4 (C(1)). Anal. calc. for C15H20FeOS
(304.23): C 59.22, H 6.63; found: C 59.23, H 6.67.
1-Ferrocenyl-3-(propylsulfanyl)propan-1-ol (4i). Yield: 83.6%. IR (KBr): 3420, 3093, 2959, 2928,
2871, 1412, 1377, 1289, 1238, 1105, 1044, 1021, 1000, 888, 815. 1H-NMR (200 MHz): 0.99 (t, J¼7.3, Me);
1.64 (br. qt, J¼7.3, MeCH2); 1.87–2.00 (m, CH2CH2S); 2.09 (d, J¼3.2, OH); 2.51 (br. t, J¼7.3,
MeCH2CH2S); 2.59–2.68 (m, CH(OH)CH2CH2S); 4.16 (br. t, J¼1.8, 2 H of Cp4 H); 4.20 (s, Cp5 H);
4.24 (br. t, J¼1.6, 2 H of Cp4 H); 4.43–4.54 (m, CHOH). 13C-NMR: 13.4 (C(3’)); 22.84 (C(2’)); 28.5
(C(3)); 34.1 (C(1’)); 37.5 (C(2)); 65.3 (1 C of Cp4 H); 66.9 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 67.9 (1 C
of Cp4 H); 68.3 (Cp5 H); 68.4 (1 C of Cp4 H); 93.4 (C(1)). Anal. calc. for C16H22FeOS (318.26): C 60.38,
H 6.97; found: C 60.41, H 6.94.
1-Ferrocenyl-3-[(1-methylethyl)sulfanyl]propan-1-ol (4j). Yield: 70.6%. IR (KBr): 3447, 3094, 2956,
1
2927.32, 2871, 1464, 1411, 1273, 1221, 1106, 1047, 1022, 1000, 816. H-NMR (200 MHz): 1.26 (d, J¼6.8,
Me); 1.27 (d, J¼6.8, Me); 1.85–1.99 (m, CH2CH2S); 2.08 (d, J¼3.2, OH), 2.61–2.71 (m, CH2S); 2.94
(sept., J¼6.8, Me2CH) 4.16 (br. t, J¼1.6, 2 H Cp4 H); 4.20 (s, Cp5 H); 4.24 (br. t, J¼1.6, 2 H of Cp4 H);
4.43–4.54 (m, CHOH). 13C-NMR: 23.3 (C(2’)); 27.0 (C(3)); 34.8 (C(1’)); 37.7 (C(2)); 65.4 (1 C of
Cp4 H); 66.9 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 67.9 (1 C of Cp4 H); 68.3 (Cp5 H); 68.5 (1 C of Cp4 H);
93.4 (C(1)). Anal. calc. for C16H22FeOS (318.26): C 60.38, H 6.97; found: C 60.40, H 6.98.
3-(Butylsulfanyl)-1-ferrocenylpropan-1-ol (4k). Yield: 80.5%. IR (KBr): 3433, 3094, 2956, 2927,
2871, 1465, 1412, 1382, 1271, 1227, 1105, 1044, 1022, 1000, 816. 1H-NMR (200 MHz): 0.92 (t, J¼7.2, Me);
1.30–1.46 (m, MeCH2); 1.47–1.65 (m, CH2CH2Me); 1.85–2.00 (m, CH(OH)CH2CH2S); 2.09 (d, J¼3.4,
OH); 2.53 (t, J¼7.6, MeCH2CH2CH2S); 2.60–2.69 (m, CH2S); 4.14–4.19 (m, 2 H of Cp4 H); 4.22–4.25
(s, Cp5 H); 4.24 (m, 2 H of Cp4 H); 4.43–4.54 (m, CHOH). 13C-NMR: 13.6 (C(4’)); 21.6 (C(3’)); 28.6
(C(3)); 31.7 (C(2’)); 31.8 (C(1’)); 37.6 (C(2)); 65.3 (1 C of Cp4 H); 66.9 (1 C of Cp4 H); 67.8 (1 C of
Cp4 H); 67.9 (1 C of Cp4 H); 68.3 (Cp5 H); 68.4 (1 C of Cp4 H); 93.4 (C(1)). Anal. calc. for C17H24FeOS
(332.28): C 61.45, H 7.28; found: C 61.44, H 7.26.
3-[(2,6-Dichlorophenyl)methylsulfanyl]-1-ferrocenylpropan-1-ol (4l). Yield: 83.6%. IR (KBr): 3427,
1
3091, 2923, 1580, 1560, 1435, 1105, 1087, 1051, 1022, 1000, 891, 817, 777, 760, 692. H-NMR (200 MHz):
1.89–2.02 (m, CH2CH2S, OH); 2.70–2.80 (m, CH(OH)CH2CH2S); 4.04 (br. s, ArCH2S); 4.15–4.17 (m,
2 H of Cp4 H); 4.19 (s, Cp5 H); 4.20–4.23 (m, 2 H of Cp4 H); 4.42–4.53 (m, CHOH); 7.11 (dd, J¼7.2,
8.8, 1 arom. H); 7.30 (br. d, J ca. 7.6, 2 arom. H). 13C-NMR: 29.1 (C(3)); 31.6 (C(1’)); 37.7 (C(2)); 65.3
(1 C of Cp4 H); 67.0 (1 C of Cp4 H); 67.8 (1 C of Cp4 H); 67.9 (1 C of Cp4 H); 68.3 (Cp5 H); 68.4 (1 C of
Cp4 H); 93.4 (C(1)); 128.4; 128.5; 135.3; 135.4. Anal. calc. for C20H20Cl2FeOS (435.19): C 55.20, H 4.63;
found: C 55.23, H 4.62.
Synthesis of Oximes 5a–5l [7]. The soln. of 1 mmol of the corresponding ketone (2a–2l) in 20 ml of
MeOH was added to a soln. of 820 mg (10 mmol) of AcONa and 695 mg (10 mmol) of NH2OH·HCl in
10 ml of H2O. The mixture was refluxed for 2 h, cooled to r.t., and then MeOH was evaporated in vacuo.
The residue was extracted with CH2Cl2 (two 30-ml portions), org. layers were washed with H2O and
brine, and dried (Na2SO4) overnight. After the evaporation of the solvent, the solid was filtered through a
short pad (5 g of SiO2; toluene/AcOEt 9 :1 (v/v)) to obtain mixture of (Z)- and (E)-oximes 5a–5k. The
ratios of the obtained stereoisomers were determined by using the integrals of appropriate corresponding
non-overlapping 1H-NMR signals.
(E)- and (Z)-1-Ferrocenyl-2-(methylsulfanyl)ethan-1-one Oxime (5a). Yield: quant. Spectral data in
accordance with those previously published for the same compound(s) [7]. (E)/(Z) 67.7:32.3. Anal. calc.
for C13H16FeNOS (289.02): C 53.99, H 5.23; found: C 53.98, H 5.25.

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(E)- and (Z)-2-(Ethylsulfanyl)-1-ferrocenylethanone Oxime (5b). Yield: quant. (E)/(Z) 67.5 :32.5.
IR (KBr): 3205, 2965, 2929, 1634, 1442, 1404, 1298, 1230, 1106, 1030, 1021, 1002, 976, 940, 877, 824, 768,
707, 576, 486.
(E)-Isomer. 1H-NMR (200 MHz): 1.31 (t, J¼7.4, Me); 2.69 (q, J¼7.4, MeCH2); 3.74 (s, CH2S); 4.19
(s, Cp5 H); 4.34 (t, J¼1.9, 2 H of Cp4 H); 4.64 (t, J¼1.9, 2 H of Cp4 H); 9.78 (s, OH). 13C-NMR: 14.7
(C(2’)); 25.2 (C(1’)); 26.9 (C(2)); 66.9 (2 C of Cp4 H); 69.6 (2 C of Cp4 H); 69.4 (5 C of Cp5 H); 79.3 (1 C
of Cp4 H); 156.5 (C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 1.24 (t, J¼7.3, Me); 2.58 (q, J¼7.3, MeCH2); 3.54 (s, CH2S); 4.18
(s, Cp5 H); 4.39 (t, J¼1.9, 2 H of Cp4 H); 5.0 (t, J¼1.9, 2 H of Cp4 H); 9.78 (s, OH). 13C-NMR: 14.3
(C(2’)); 25.9 (C(1’)); 34.4 (C(2)); 69.5 (5 C of Cp5 H); 70.1 (2 C of Cp4 H); 71.0 (2 C of Cp4 H); 72.8 (1 C
of Cp4 H); 153.4 (C(1)). Anal. calc. for C14H17FeNOS (303.20): C 55.46, H 5.65; found: C 55.48, H 5.66.
(E)- and (Z)-1-Ferrocenyl-2-(propylsulfanyl)ethanone Oxime (5c). Yield: quant. (E)/(Z) 67.9 :32.1.
IR (KBr): 3420, 3165, 2961, 2925, 1619, 1454, 1403, 1382, 1298, 1106, 1029, 1000, 956, 883, 821, 514, 504,
486.
1
(E)-Isomer. H-NMR (200 MHz): 0.98 (t, J¼7.3, Me); 1.67 (br. qt, J¼7.3, MeCH2); 2.64 (t, J¼7.3,
MeCH2CH2S); 3.72 (s, C(NOH)CH2S); 4.18 (s, Cp5 H); 4.32 (t, J¼1.8, 2 H of Cp4 H); 4.64 (t, J¼1.8, 2 H
of Cp4 H); 10.07 (s, OH). 13C-NMR: 13.4 C(3’)); 22.8 (C(2’)); 25.4 (C(2)); 34.9 (C(1’)); 66.8 (2 C of
Cp4 H); 69.5 (2 C of Cp4 H); 69.3 (Cp5 H); 79.2 (1 C of Cp4 H); 156.4 (C(1)).
1
(Z)-Isomer. H-NMR (200 MHz): 0.97 (t, J¼7.3, Me); 1.64 (br. qt, J¼7.3, MeCH2); 2.56 (t, J¼7.3,
MeCH2CH2S); 3.55 (s, C(NOH)CH2S); 4.17 (s, Cp5 H); 4.37 (t, J¼1.8, 2 H of Cp4 H); 5.0 (t, J¼1.8, 2 H
of Cp4 H); 10.07 (s, OH). 13C-NMR: 15.1 (C(3’)); 22.4 (C(2’)); 34.0 (C(1’)); 34.6 (C(2)); 69.4 (Cp5 H);
70.0 (2 C of Cp4 H); 71.0 (2 C of Cp4 H); 72.7 (1 C of Cp4 H); 153.4 (C(1)). Anal. calc. for C15H19FeNOS
(317.23): C 56.79, H 6.04; found: C 56.82, H 6.04.
(E)- and (Z)-1-Ferrocenyl-2-[(1-methylethyl)sulfanyl]ethan-1-one Oxime (5d). Yield: quant. (E)/
(Z) 70.1:29.9. IR (KBr): 3202, 2959, 2924, 2865, 1623, 1452, 1411, 1382, 1296, 1106, 1030, 1001, 952, 881,
820, 502, 487.
(E)-Isomer. 1H-NMR (200 MHz): 1.31 (t, J¼6.8, 2 Me); 3.1 (sept., J¼6.8, Me2CH); 3.73 (s, CH2S);
4.19 (s, Cp5 H); 4.32 (t, J¼1.9, 2 H of Cp4 H); 4.61 (t, J¼1.9, 2 H of Cp4 H); 10.08 (s, OH). 13C-NMR:
23.0 (C(2’)); 36.1 (C(1’)); 24.6 (C(2)); 66.8 (2 C of Cp4 H); 69.5 (2 C of Cp4 H); 69.3 (Cp5 H); 79.3 (1 C of
Cp4 H); 156.6 (C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 1.30 (t, J¼6.8, 2 Me); 3.02 (sept., J¼6.8, Me2CH); 3.60 (s, CH2S);
4.17 (s, Cp5 H); 4.37 (t, J¼1.9, 2 H of Cp4 H); 5.0 (t, J¼1.9, 2 H of Cp4 H); 10.08 (s, OH). 13C-NMR: 23.3
(C(2’)); 33.6 (C(1’)); 34.9 (C(2)); 69.4 (Cp5 H); 70.0 (2 C of Cp4 H); 71.0 (2 C of Cp4 H); 72.6 (1 C of
Cp4 H); 153.6 (C(1)). Anal. calc. for C15H19FeNOS (317.05): C 56.79, H 6.04; found: C 56.81, H 6.02.
(E)- and (Z)-2-(Butylsulfanyl)-1-ferrocenylethanone Oxime (5e). Yield: quant. (E)/(Z) 68.4 :31.6.
IR (KBr): 3206, 2957, 2929, 1627, 1456, 1412, 1381, 1297, 1225, 1107, 1030, 1001, 951, 881, 820, 503. 488.
(E)-Isomer. 1H-NMR (200 MHz): 0.90 (t, J¼7.4, Me); 1.40 (br. qt, J¼7.4, MeCH2); 1.63 (tt, J¼7.4,
CH2CH2Me); 2.66 (t, J¼7.4, MeCH2CH2CH2S); 3.72 (s, C(NOH)CH2S); 4.18 (s, Cp5 H); 4.32 (br., 2 H
of Cp4 H); 4.64 (br., 2 H of Cp4 H); 10.03 (s, OH). 13C-NMR: 13.6 (C(4’)); 21.9 C(3’)); 25.4 (C(2)); 31.5
(C(2’)); 32.6 (C(1’)); 66.8 (2 C of Cp4 H); 69.3 (Cp5 H); 69.5 (2 C of Cp4 H); 79.2 (1 C of Cp4 H); 156.4
(C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 0.90 (t, J¼7.4, Me); 1.40 (br. qt, J¼7.4, MeCH2); 1.63 (tt, J¼7.4,
CH2CH2CH2Me); 2.58 (t, J¼7.4, MeCH2CH2CH2S); 3.55 (s, C(NOH)CH2S); 4.17 (s, Cp5 H); 4.37 (br.,
2 H of Cp4 H); 5.0 (br., 2 H of Cp4 H); 10.03 (s, OH). 13C-NMR: 13.6 (C(4’)); 21.9 C(3’)); 31.2 (C(2’));
31.6 (C(1’)); 34.6 (C(2)); 69.4 (Cp5 H); 69.9 (2 C of Cp4 H); 71.0 (2 C of Cp4 H); 73.7 (1 C of Cp4 H);
153.3 (C(1)). Anal. calc. for C16H21FeNOS (331.24): C 58.01, H 6.39; found: C 58.00, H 6.40.
(E)- and (Z)-2-{[(2,6-Dichlorophenyl)methyl]sulfanyl}-1-ferrocenylethan-1-one Oxime (5f). Yield:
quant. (E)/(Z) 68.9 :31.1. IR (KBr): 3202, 2937, 1638, 1579, 1559, 1436, 1299, 1106, 1088, 1026, 1003, 983,
953, 892, 876, 814, 776, 760, 512, 499, 488.
(E)-Isomer. 1H-NMR (200 MHz): 3.88 (s, C(NOH)CH2S); 4.25 (s, ArCH2S); 4.17 (s, Cp5 H); 4.30 (t,
J¼1.8, 2 H of Cp4 H); 4.53 (t, J¼1.8, 2 H of Cp4 H); 9.40 (s, OH); 7.11 (dd, J¼7.2, 8.8, 1 arom. H); 7.30
(br. d, J ca. 7.6, 2 arom. H). 13C-NMR: 26.8 (C(2)); 33.6 (C(1’)); 66.8 (2 C of Cp4 H); 69.3 (Cp5 H); 69.6
(2 C of Cp4 H); 79.3 (1 C of Cp4 H); 155.8 (C(1)).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2251
(Z)-Isomer. 1H-NMR (200 MHz): 3.74 (s, C(NOH)CH2S); 4.13 (s, ArCH2S); 4.16 (s, Cp5 H); 4.36 (t,
J¼1.8, 2 H of Cp4 H); 4.92 (t, J¼1.8, 2 H of Cp4 H); 9.40 (s, OH); 7.11 (dd, J¼7.2, 8.8, 1 arom. H); 7.30
(br. d, Jꢁ7.6, 2 arom. H). 13C-NMR: 32.2 (C(1’)); 35.8 (C(2)); 69.5 (Cp5 H); 70.0 (2 C of Cp4 H); 70.8
(2 C of Cp4 H); 72.9 (1 C of Cp4 H); 153.1 (C(1)). Anal. calc. for C19H17Cl2FeNOS (434.16): C 52.56, H
3.95; found: C 52.57, H 3.94.
(E)- and (Z)-1-Ferrocenyl-3-(methylsulfanyl)propan-1-one Oxime (5g). Yield: quant. (E)/(Z)
77.5 :22.5. IR (KBr): 3220, 3111, 2916, 1632, 1446, 1383, 1318, 1296, 1106, 1035, 1022, 999, 959, 934, 888,
809, 518, 503, 486.
(E)-Isomer. 1H-NMR (200 MHz): 2.23 (s, Me); 2.76–3.01 (m, CH2CH2); 4.19 (s, Cp5 H); 4.34 (t, J¼
1.8, 2 H of Cp4 H); 4.56 (t, J¼1.8, 2 H of Cp4 H); 9.26 (s, OH). 13C-NMR: 15.6 (C(1’)); 28.2 (C(3)); 30.9
(C(2)); 66.6 (2 C of Cp4 H); 69.3 (Cp5 H); 69.8 (2 C of Cp4 H); 80.2 (1 C of Cp4 H); 157.9 (C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 2.19 (s, Me); 2.76–3.01 (m, CH2CH2); 4.18 (s, Cp5 H); 4.38 (t, J¼
1.8, 2 H of Cp4 H); 4.91 (t, J¼1.8, 2 H of Cp4 H); 9.26 (s, OH). 13C-NMR: 15.7 (C(1’)); 32.3 (C(3)); 34.2
(C(2)); 69.5 (5 C of Cp5 H); 70.1 (2 C of Cp4 H); 70.7 (2 C of Cp4 H); 73.5 (1 C of Cp4 H); 150.3 (C(1)).
Anal. calc. for C14H17FeNOS (303.20): C 55.46, H 5.65; found: C 55.45, H 5.69.
(E)- and (Z)-3-(Ethylsulfanyl)-1-ferrocenylpropan-1-one Oxime (5h). Yield: quant. (E)/(Z)
77.9 :22.1. IR (KBr): 3415, 3197, 3140, 2923, 1641, 1407, 1105, 1026, 1000, 971, 937, 886, 820, 774, 520,
506, 490.
(E)-Isomer. 1H-NMR (200 MHz): 1.33 (t, J¼7.4, Me); 2.67 (q, J¼7.4, MeCH2); 2.78–3.00 (m,
CH2CH2); 4.19 (s, Cp5 H); 4.34 (t, J¼1.8, 2 H of Cp4 H); 4.56 (t, J¼1.8, 2 H of Cp4 H); 9.26 (s, OH).
13C-NMR: 14.7 (C(2’)); 26.0 (C(1’)); 28.2 (C(3)); 28.7 (C(2)); 66.6 (2 C of Cp4 H); 69.3 (Cp5 H); 69.5
(2 C of Cp4 H); 80.2 (1 C of Cp4 H); 157.9 (C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 1.30 (t, J¼7.4, Me); 2.63 (q, J¼7.4, MeCH2); 2.78–3.00 (m,
CH2CH2); 4.18 (s, Cp5 H); 4.34 (t, J¼1.8, 2 H of Cp4 H); 4.91 (t, J¼1.8, 2 H of Cp4 H); 9.26 (s, OH).
13C-NMR: 14.7 (C(2’)); 26.2 (C(1’)); 29.7 (C(3)); 30.9 (C(2)); 69.8 (Cp5 H); 70.1 (2 C of Cp4 H); 70.7
(2 C of Cp4 H); 73.6 (1 C of Cp4 H); 150.3 (C(1)). Anal. calc. for C15H19FeNOS (317.23): C 56.79, H 6.04;
found: C 56.82, H 6.00.
(E)- and (Z)-1-Ferrocenyl-3-(propylsulfanyl)propan-1-one Oxime (5i). Yield: quant. (E)/(Z)
77.2 :22.8. IR (KBr): 3197, 2925, 2871, 1636, 1452, 1409, 1382, 1106, 1025, 1000, 936, 883, 819, 518, 506, 490.
1
(E)-Isomer. H-NMR (200 MHz): 1.01 (t, J¼7.2, Me); 1.65 (br. qt, J¼7.2, MeCH2); 2.61 (t, J¼7.2,
CH2S); 2.75–3.00 (m, CH2CH2S); 4.17 (s, Cp5 H); 4.32 (br., 2 H of Cp4 H); 4.56 (br., 2 H of Cp4 H); 9.90
(s, OH). 13C-NMR: 13.4 (C(3’)); 22.7 (C(2’)); 28.5 (C(3)); 28.7 (C(2)); 34.0 (C(1’)); 66.5 (2 C of Cp4 H);
69.1 (Cp5 H); 69.6 (2 C of Cp4 H); 80.0 (1 C of Cp4 H); 157.9 (C(1)).
1
(Z)-Isomer. H-NMR (200 MHz): 1.00 (t, J¼7.2, Me); 1.64 (br. qt, J¼7.2, MeCH2); 2.57 (t, J¼7.2,
CH2S); 2.75–3.00 (m, CH2CH2S); 4.17 (s, Cp5 H); 4.36 (br., 2 H of Cp4 H); 4.91 (br., 2 H of Cp4 H); 9.90
(s, OH). 13C-NMR: 13.4 (C(3’)); 22.8 (C(2’)); 30.1 (C(3)); 34.2 (C(2)); 34.5 (C(1’)); 69.3 (Cp5 H); 69.9
(2 C of Cp4 H); 70.6 (2 C of Cp4 H); 73.5 (1 C of Cp4 H); 154.7 (C(1)). Anal. calc. for C16H21FeNOS
(331.25): C 58.01, H 6.39; found: C 58.00, H 6.43.
(E)- and (Z)-1-Ferrocenyl-3-[(1-methylethyl)sulfanyl]propan-1-one Oxime (5j). Yield: quant. (E)/
(Z) 77.4 :22.6. IR (KBr): 3416, 3205, 2956, 2923, 1639, 1442, 1409, 1382, 1154, 1105, 1025, 937, 883, 820,
746, 520, 508, 494.
1
(E)-Isomer. H-NMR (200 MHz): 1.33 (d, J¼6.6, 2 Me); 2.78–2.96 (m, CH2CH2); 3.11 (sept., J¼
6.6, Me2CH); 4.19 (s, Cp5 H); 4.34 (t, J¼1.8, 2 H of Cp4 H); 4.55 (t, J¼1.8, 2 H of Cp4 H); 9.12 (s, OH).
13C-NMR: 23.4 (C(2’)); 27.2 (C(3)); 28.9 (C(2)); 34.8 (C(1’)); 66.6 (2 C of Cp4 H); 69.3 (Cp5 H); 69.5 (2 C
of Cp4 H); 80.2 (1 C of Cp4 H); 158.0 (C(1)).
1
(Z)-Isomer. H-NMR (200 MHz): 1.31 (d, J¼6.6, 2 Me); 2.78–2.96 (m, CH2CH2); 3.11 (sept., J¼
6.6, Me2CH); 4.18 (s, Cp5 H); 4.38 (t, J¼1.8, 2 H of Cp4 H); 4.91 (t, J¼1.8, 2 H of Cp4 H); 9.12 (s, OH).
13C-NMR: 23.5 (C(2’)); 30.2 (C(3)); 34.7 (C(2)); 35.1 (C(1’)); 69.8 (2 C of Cp4 H); 70.1 (Cp5 H); 70.7
(2 C of Cp4 H); 73.6 (1 C of Cp4 H); 150.3 (C(1)). Anal. calc. for C16H21FeNOS (331.25): C 58.01, H 6.39;
found: C 58.03, H 6.42.
(E)- and (Z)-3-(Butylsulfanyl)-1-ferrocenylpropan-1-one Oxime (5k). Yield: quant. (E)/(Z)
79.0 :21.0. IR (KBr): 3223, 2952, 1637, 1437, 1411, 1381, 1320, 1105, 1033, 1006, 983, 937, 889, 836, 817,
585, 520, 504.

2252
CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
(E)-Isomer. 1H-NMR (200 MHz): 0.94 (t, J¼7.2, Me); 1.44 (br. qt, J¼7.2, MeCH2); 1.64 (tt, J¼7.2,
CH2CH2Me); 2.64 (t, J¼7.2, CH2S); 2.78–2.99 (m, CH2CH2S); 4.19 (s, Cp5 H); 4.34 (t, J¼1.8, 2 H of
Cp4 H); 4.55 (t, J¼1.8, 2 H of Cp4 H); 9.40 (br., OH). 13C-NMR: 13.7 (C(4’)); 22.0 (C(3’)); 28.7 (C(3));
28.7 (C(2)); 31.7 (C(2’)); 31.8 C(1’)); 66.6 (2 C of Cp4 H); 69.3 (Cp5 H); 69.8 (2 C of Cp4 H); 80.2 (1 C of
Cp4 H); 157.92 (C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 0.94 (t, J¼7.2, Me); 1.44 (br. qt, J¼7.2, MeCH2); 1.63 (tt, J¼7.2,
CH2CH2Me); 2.61 (t, J¼7.2, CH2S); 2.78–2.99 (m, CH2CH2S); 4.18 (s, Cp5 H); 4.37 (t, J¼1.8, 2 H of
Cp4 H); 4.91 (t, J¼1.8, 2 H of Cp4 H); 9.40 (br., OH). 13C-NMR: 13.7 (C(4’)); 22.0 (C(3’)); 30.2 (C(3));
31.8 (C(2’)); 32.1 C(1’)); 34.6 (C(1)); 69.5 (Cp5 H); 70.1 (2 C of Cp4 H); 70.7 (2 C of Cp4 H); 73.6 (1 C of
Cp4 H); 150.3 (C(1)). Anal. calc. for C17H23FeNOS (345.28): C 59.14, H 6.71; found: C 59.17, H 6.74.
(E)- and (Z)-3-{[(2,6-Dichlorophenyl)methyl]sulfanyl}-1-ferrocenylpropan-1-one Oxime (5l).
Yield: quant. (E)/(Z) 87.2 :12.8. IR (KBr): 3436, 3204, 2931, 1629, 1435, 1420, 1410, 1284, 1106, 1028,
999, 939, 867, 833, 816, 777, 760, 580, 512, 502, 486.
(E)-Isomer. 1H-NMR (200 MHz): 2.93–3.03 (m, CH2CH2); 4.21 (s, CH2S); 4.17 (s, Cp5 H); 4.34 (t,
J¼1.8, 2 H of Cp4 H); 4.54 (t, J¼1.8, 2 H of Cp4 H); 7.11 (dd, J¼7.2, 8.8, 1 arom. H); 7.30 (br. d, J ca. 7.6,
2 arom. H); 9.11 (s, OH). 13C-NMR: 28.6 (C(3)); 29.8 (C(2)); 32.2 C(1’)); 66.6 (2 C of Cp4 H); 69.3
(Cp5 H); 69.8 (2 C of Cp4 H); 80.2 (Cp4 H); 128.4; 128.5; 135.3; 135.5; 157.9 (C(1)).
(Z)-Isomer. 1H-NMR (200 MHz): 2.93–3.03 (m, CH2CH2); 4.11 (s, CH2S); 4.16 (s, Cp5 H); 4.36 (t,
J¼1.8, 2 H of Cp4 H); 4.88 (t, J¼1.8, 2 H of Cp4 H); 7.11 (dd, J¼7.2, 8.8, 1 arom. H); 7.30 (br. d, Jꢁ7.6, 2
arom. H); 9.11 (s, OH). 13C-NMR: 69.5 (Cp5 H); 70.0 (2 C of Cp4 H); 70.7 (2 C of Cp4 H); 73.6 (1 C of
Cp4 H); 128.4; 128.5; 135.3; 135.5; 150.3 (C(1)). Anal. calc. for C20H19Cl2FeNOS (448.19): C 53.60, H
4.27; found: C 53.59, H 4.28.
Electrochemical Measurements. Electrochemical measurements were performed at r.t. (ca. 258) with
an Autolab potentiostat (PGSTAT 302N). A standard three-electrode cell (5 ml) equipped with a Pt wire
and a Ag/AgCl electrode immersed in 0.1m LiClO4 soln. in MeCN as the counter and reference electrode,
resp. A Pt disk (d¼2 mm) was used as the working electrode.
Antimicrobial Assay. The in vitro antimicrobial activities of 4a–4l and 5a–5l were tested against a
panel of laboratory control strains (food isolates or those belonging to the American Type Culture
Collection, Maryland, USA). Antibacterial activities were evaluated against two Gram-positive and
three Gram-negative bacteria. The following Gram-positive bacteria used were Bacillus cereus (food
isolate) and Staphylococcus aureus (ATCC 6538). The Gram-negative bacteria utilized in the assays were
Escherichia coli (ATCC 25922), Klebsiella pneumoniae (ATCC 10031), and Proteus vulgaris (ATCC
8427). The antifungal activity was tested against Candida albicans (ATCC 10231). Bacterial isolates were
ˇ
obtained from the Institute of Public Health, Nis, Serbia, and are stored in the microbiological collection
at the Microbiology Laboratory (Department of Biology, Faculty of Science and Mathematics, University
ˇ
ˇ
of Nis, Nis, Serbia). Broth microdilution susceptibility assay was used, as recommended by NCCLS, for
the determination of MIC values (NCCLS [23]). All tests were performed in MuellerꢀHinton broth
(MHB; BBL) supplemented with Tween 80 detergent (final concentration of 0.5% (v/v)), with the
exception of the fungal organism (Sabouraud dextrose broth-SDBþTween 80), and with 2ꢄ105 colony-
forming units (CFU) mlꢀ1 of the bacteria/fungi in the exponential phase. Test compounds, 4a–4l and 5a–
5l, were dissolved in DMSO, and this stock soln. was used to prepare the exact agar dilutions. Final
concentrations of the compounds in the broth ranged from 0.001 to 5000 mg/ml, and these were prepared
in a 96-well microtiter plate. The dilutions were based on a geometrical order (factor 2). To obtain more
accurate MIC values, further dilutions were additionally prepared 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 378 for 24 h for bacteria, and at 308 for 48 h for the yeasts. Each test was performed in
duplicate and repeated twice. Tetracycline and nystatin were used as positive controls, while DMSOþ
Tween 80 (in the form of a blank) were the negative control.
Statistical Analysis. Principal component analysis (PCA) and agglomerative hierarchical clustering
(AHC) were performed using the Excel program plug-in XLSTAT version 2011.3.02 [24]. Both methods
were applied utilizing the MIC values as original variables without any recalculation. AHC was
determined using Pearson dissimilarity, where the aggregation criterion were simple linkage, unweighted
pair-group average and complete linkage, and Euclidean distance where the aggregation criterion were

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
2253
weighted pair-group average, unweighted pair-group average, and Wardꢃs method. PCA of the Pearson
(n) type was performed.
REFERENCES
[1] D. P. Fidler, Emerg. Infect. Dis. 1998, 4, 169.
[2] N. Metzler-Nolte, U. Schatzschneider, ꢂBioinorganic chemistry: a practical courseꢃ, Walter de
Gruyter GmbH & Co KG, Berlin, 1990, pp. 120–123.
´
´
´
´
´
´
[3] D. Ilic, I. Damljanovic, D. Stevanovic, M. Vukicevic, N. Radulovic, V. Kahlenberg, G. Laus, R. D.
´
´
Vukicevic, Polyhedron 2010, 29, 1863.
´
´
´
´
´
´
´
[4] Z. Ratkovic, Z. D. Juranic, T. Stanojkovic, D. Manojlovic, R. D. Vukicevic, N. Radulovic, M. D.
´
Joksovic, Bioorg. Chem. 2010, 38, 26.
´
´
´
´
´
´
[5] M. D. Joksovic, V. R. Markovic, Z. D. Juranic, T. P. Stanojkovic, L. S. Jovanovic, I. S. Damljanovic,
´ ´
´
´
´
´
´
´
K. Mesaros Szecsenyi, N. Todorovic, S. Trifunovic, R. D. Vukicevic, J. Organomet. Chem. 2009, 694,
3935.
´
´
´
´
´
´
´
[6] I. Damljanovic, M. Vukicevic, N. Radulovic, R. Palic, E. Ellmerer, Z. Ratkovic, M. D. Joksovic,
´
´
R. D. Vukicevic, Bioorg. Med. Chem. Lett. 2009, 19, 1093.
´
´
´
´
[7] M. D. Vukicevic, K. Wurst, A. G. Mꢁller, G. Laus, R. D. Vukicevic, Polyhedron 2005, 24, 533.
´
´
´
´
´
´
´
[8] M. D. Vukicevic, Z. R. Ratkovic, A. V. Teodorovic, G. S. Stojanovic, R. D. Vukicevic, Tetrahedron
2002, 58, 9001.
´
´
´
´
´
´
[9] R. D. Vukicevic, D. Ilic, Z. Ratkovic, M. Vukicevic, Monatsh. Chem. 2001, 132, 625.
´
´
´
´
´
´
´
[10] I. Damljanovic, D. Stevanovic, A. Pejovic, M. Vukicevic, S. B. Novakovic, G. A. Bogdanovic, T.
´
´
´
Mihajlov-Krstev, N. Radulovic, R. D. Vukicevic, J. Organomet. Chem. 2011, 696, 3703.
´
´
´
´
´
´
´
[11] A. Pejovic, I. Damljanovic, D. Stevanovic, M. Vukicevic, S. B. Novakovic, G. A. Bogdanovic, N.
´
´
´
Radulovic, R. D. Vukicevic, Polyhedron 2012, 31, 789.
´
´
´
´
´
´
´
[12] A. Pejovic, D. Stevanovic, I. Damljanovic, M. Vukicevic, S. B. Novakovic, G. A. Bogdanovic, T.
´
´
´
Mihajilov-Krstev, N. Radulovic, R. D. Vukicevic, Helv. Chim. Acta 2012, 95, 1425.
[13] I. A. Dzhafarov, E. G. Mamedbeili, T. G. Kyazimova, K. I. Gasanov, E. I. Suleimanova, Russ. J.
Appl. Chem. 2010, 83, 854.
[14] P. Deetae, H.-E. Spinnler, P. Bonnarme, S. Helinck, Appl. Microbiol. Biotechnol. 2009, 82, 169.
[15] G. Vernin, C. Parkanyi, H. Casabianca, in ꢂFood flavor and Chemistry; Exploration into the 21st
Centuryꢃ (Special Publication), Eds. A. M. Spanier, F. Shahidi, T. H. Parliament, C. Mussinan, C.-T.
Ho, Royal Society of Chemistry, 2005, p. 115.
[16] S. Schulz, J. S. Dickschat, B. Kunze, I. Wagner-Dobler, R. Diestel, F. Sasse, Marine Drugs 2010, 8,
2976.
[17] J. R. Luly, G. Bolis, N. BaMaung, J. Soderquist, J. F. Dellaria, H. Stein, J. Cohen, T. J. Perun, J. Greer,
J. J. Plattner, J. Med. Chem. 1988, 31, 532.
[18] J. R. Luly, N. Yi, J. Soderquist, H. Stein, J. Cohen, T. J. Perun, J. J. Plattner, J. Med. Chem. 1987, 30,
1609.
[19] C. Y. Hong, Y. K. Kim, J. H. Chang, S. H. Kim, H. Choi, D. H. Nam, Y. Z. Kim, J. H. Kwak, J. Med.
Chem. 1997, 40, 3584.
´
´
´
´
´
[20] I. Damljanovic, M. Vukicevic, R. D. Vukicevic, Monatsh. Chem. 2006, 137, 301.
[21] J. Bard, L. R. Faulkner, ꢂElectrochemical methods: fundamentals and applicationsꢃ, John Wiley &
Sons, New York, 2001.
[22] R. Ahmedi, T. Lanez, Int. J. Pharm. Pharm. Sci. 2009, 1, 183.
´
´
´
´
´
[23] N. S. Radulovic, M. S. Dekic, Z. Z. Stojanovic-Radic, S. K. Zoranic, Chem. Biodiversity 2010, 7,
2783.
´
´
[24] N. S. Radulovic, P. D. Blagojevic, Chem. Biodiversity 2010, 7, 2856.
Received January 27, 2012