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

2236

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

) 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

) 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 (Fe^2þ/Fe^3þ) 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 (Fe^2þ/Fe^3þ

)

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

2238

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 CH₂groups 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 cm^ꢀ1(OH), and strong adsorptions in the range of 970–1150 cm^ꢀ1(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 cm^ꢀ1(C¼N), which corroborated the presence of the hydroxyimino moiety

in the structures of these compounds. Once again, a band characteristic for ketones

2240

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)

(1760–1670 cm^ꢀ1) was not observed. Spectra of some of the studied compounds

(especially oximes 5a–5l) were further characterized by a strong band around 490 cm^ꢀ1

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 (J_aꢁ13 and J_bꢁ4 Hz,

i.e., J_aꢁ13 and J_bꢁ8 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 (Fe^2þ/Fe^3þ) 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 LiClO₄as 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·s^ꢀ1), a reversible redox couple at E_1/2(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

2242

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 Vs^ꢀ1scan rate in a 0.1m LiClO₄solution 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

E_pa^a) [V]

E_pc^a) [V]

E_1/2^b) [V]

DE^c) [mV]

Alcohols

4a

4b

4c

4d

4e

4f

4g

4h

4i

0.385

0.406

0.394

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

0.354

0.360

0.346

0.343

0.346

0.360

0.350

0.360

71

79

74

61

68

67

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

0.485

0.479

0.488

0.485

0.482

0.485

0.406

0.403

0.418

0.409

0.406

0.409

0.442

0.438

0.441

0.440

0.452

0.444

0.448

0.447

0.444

0.446

0.447

73

70

76

73

67

70

79

76

79

76

5j

5k

5l

^a) E_paand E_pcare anodic and cathodic peak potentials, respectively, at 0.1 Vs^ꢀ1

.

^b) E_1/2¼(E_paþE_pc)/2.

^c) DE¼E_paꢀE_pc.

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,

2244

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

0.30

156

1250

2500

156

1.000

620

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

0.003

0.15

156

0.20

0.40

0.070

0.40

0.070

39

0.070

9.000

0.001

0.070

0.050

0.001

9.0

0.050

0.40

19

0.10

19

9.0

1250

0.001

0.003

9.0

0.10

19

>5000

>2500

1250

>2500

>5000

>2500

>5000

750

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

0.030

0.070

0.15

0.070

0.15

0.070

156

0.40

0.90

0.030

0.070

0.40

39

156

78

0.110^a)

0.10

0.070

0.20

0.40

0.10

78

>2500

5g

5h

5i

5j

5k

5l

>5000

>2500

>5000

325

0.070

0.80

0.030

0.070

0.030

0.039^b)

0.40

310

1.0

0.110^a)

4.0

0.003^a)

Control

0.230^a)

0.050^a)

^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 P_o/wvalues were calculated (this was

performed in the following way: log P_o/wof 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 (E_pa, E_pc, E_1/2, 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 r²value

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 (M^þ)), 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 (M^þ));

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

2246

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 P_o/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

P_o/wvalues, 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 CH₂Cl₂was 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 NaBH₄was 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

2248

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.

2250

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 LiClO₄soln. 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

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 2ꢄ10⁵colony-

forming units (CFU) ml^ꢀ1of 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

Article Doi

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

DOI: 10.1002/cbdv.201200029

Source and publish data:

Authors:

Article abstract of DOI:10.1002/cbdv.201200029

Full text of DOI:10.1002/cbdv.201200029

Products guided by the article

R&D Labs maybe for 1415678-94-2

Relevant to this article

Hot Product