Polyhedron 29 (2010) 1863–1869
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier .com/locate /poly
Synthesis, spectral characterization, electrochemical propertiesand antimicrobial screening of sulfur containing acylferrocenes
Danijela Ilic a, Ivan Damljanovic b, Dragana Stevanovic b, Mirjana Vukicevic c, Niko Radulovic d,*,Volker Kahlenberg e, Gerhard Laus f, Rastko D. Vukicevic b,**
a Technical Faculty Kosovska Mitrovica, University of Priština, Kneza Miloša 7, 38220 Kosovska Mitrovica, Serbiab Department of Chemistry, Faculty of Science, University of Kragujevac, R. Domanovica 12, 34000 Kragujevac, Serbiac Department of Pharmacy, Faculty of Medicine, University of Kragujevac, S. Markovica 65, 34000 Kragujevac, Serbiad Department of Chemistry, Faculty of Science and Mathematics, University of Niš, Višegradska 33, 18000 Niš, Serbiae Institute of Mineralogy and Petrography, University of Innsbruck, Innrain 52, 6020 Innsbruck, Austriaf Institute of Inorganic Chemistry, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
a r t i c l e i n f o a b s t r a c t
Article history:Received 9 February 2010Accepted 2 March 2010Available online 6 March 2010
Keywords:FerroceneFriedel-Crafts acylationS-Alkylthioglycolic acids2-Alkylthiopropanoic acidsCyclic voltammetryAntimicrobial activity
0277-5387/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.poly.2010.03.002
* Corresponding author. Fax: +381 18 533 014.** Corresponding author. Fax: +381 34 33 50 40.
E-mail addresses: [email protected] (N. RVukicevic).
Several known and eight new sulfur containing acylferrocenes of the general formula FcCO(CH2)nSR(where Fc = ferrocenyl, n = 1 or 2 and R = alkyl, 4-bromobenzyl or 2,6-dichlorobenzyl group) were synthe-sized in order to test their in vitro antimicrobial activity against 11 bacterial and three fungal/yeaststrains. It has been shown that only four of the 14 ketones are completely inactive at the tested dose,while the activities of the other ones were noteworthy. All new compounds were well characterizedby IR and NMR spectral data, and their electrochemical properties were investigated by cyclic voltamme-try. The X-ray crystal structures of two representative ketones are also presented.
� 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Antimicrobials have played an important role in health caresince the middle of the 20th century. However, in the recent timethe success in application of antibiotics in treating of infections hasbeen eroded by increased resistance of bacteria. Therefore, there isa permanent interest in finding more and more novel substancesexhibiting antimicrobial activity [1,2]. It is well known that substi-tution of an aromatic nucleus of certain organic compounds with aferrocenyl group can lead to products possessing unexpected prop-erties which are less manifest or even absent in the parent mole-cule. Many new molecules containing a ferrocene unit weresynthesized following this idea, i.e. they have been designed tobe derivatives of known compounds (that already possess desiredproperties) in which a certain group was replaced by a ferroceneunit (expecting an improved property). Thus, bioconjugates con-taining this metallocene represent a new class of biomaterials, inwhich organometallic unit serves as a molecule scaffold, sensitiveprobe, chromophore, biological marker, redox-active site, catalytic
ll rights reserved.
adulovic), [email protected] (R.D.
site etc. [3]. Despite the fact that early attempts to apply ferrocenederivatives in medicine were discouraging [4,5], chemists did notabandon this idea, and it is known now that many ferrocenes exhi-bit different biological activities, promising thus significant appli-cation in medicinal practice [6–14]. In continuation of ourpermanent interest in ferrocene chemistry, particularly in theinvestigation of the biological activity of ferrocene derivatives[15–18], we at present synthesized several known [19], and severalnovel acylferrocenes containing sulfur in the side chain (1 and 2,Fig. 1) in order to test their antimicrobial activity. For these inves-tigations we have been prompted by the literature findings con-cerning the antimicrobial activities of sulfur containing ketones:2-(methylthio)-1-(pyridin-3-yl)ethanone (3), 2-(methylthio)-1-(pyridin-4-yl)ethanone (4) and 2-(methylthio)-1-(pyrazin-2-yl)ethanone (5) derivatives of pyridine and pyrazine (Fig. 1) [20].Namely, in the light of the above text, we find that compound 1(R = CH3) exactly fits the mentioned synthetic logic regarding fer-rocene derivatives – the corresponding aromatic units (3- and 4-pyridyl and 2-pyrazinyl groups) from the three mentioned com-pounds (3–5), have been substituted by the ferrocene one. In fact,such a type of compounds has been synthesized by us several yearsago [19], but at that time their antimicrobial activity has not beenassessed. Moreover, we set our goal to vary the structural design ofthe ketones by inserting an additional methylene group between
Fig. 1. Structures of target acylferrocenes 1a–g and 2a–g.
1864 D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
the carbonyl carbon and the sulfur atoms, and replacing the S-methyl group with other alkyl groups, as it has been shown inthe Fig. 1.
2. Experimental
2.1. Materials and instruments
All chemicals were commercially available and used as re-ceived, except that the solvents were purified by distillation. Chro-matographic separations were carried out using Silica Gel 60(Merck, 230–400 mesh ASTM), whereas Silica Gel 60 on Al plates,layer thickness 0.2 mm (Merck) was used for TLC. Melting pointswere determined on a Mel-Temp capillary melting points appara-tus, model 1001 and are uncorrected. Microanalysis of carbonand hydrogen were carried out with a Carlo Erba 1106 microanaly-ser; their results agreed favorably with the calculated values. IRmeasurements were carried out with a Perkin–Elmer FTIR 31725-X spectrophotometer. NMR spectra were recorded on a VarianGemini (200 MHz) spectrometer, using CDCl3 as the solvent andTMS as the internal standard. Chemical shifts are expressed in d(ppm).
2.2. Preparation of ketones 1a–g and 2a–g
Ketones 1a–g and 2a–g were synthesized by the known proce-dure [19]. Spectral data of compounds 1a,f,g and 2 a,f,g were pre-viously described [19], whereas the data for the newly synthesizedcompounds follow.
2.2.1. 1-Ferrocenyl-3-thiapentan-1-one (1b)Yield 61%; m.p. 49–51 �C; 1H NMR (CDCl3, ppm) d: 1.28 (t,
J = 7.4 Hz, 3H, CH3), 2.64 (q, J = 7.4 Hz, 2H, CH2–CH3), 3.57 (s, 2H,CH2–S), 4.23 (s, 5H, Cp), 4.55 (t, J = 1.9 Hz, 2H, Cp), 4.81 (t,J = 2.0 Hz, 2H, Cp). 13C NMR (CDCl3, ppm) d: 14.17 (C-20 0 0), 26.50(C-10 0 0), 37.97 (C-2), 69.82 (C-30 and 40), 69.93 (C-10 0-50 0), 72.51 (C-20 and 50), 77.64 (C-10), 198.93 (C-1). IR (KBr, cm�1): 3120, 2959,2912, 1645, 1458, 1411, 1380, 1294, 1071, 838, 821, 688, 630. Anal.
Calc. for C14H16FeOS (288.03): C, 58.35; H, 5.60; Fe, 19.38; O, 5.55;S, 11.13. Found: C, 58.30; H, 5.57%.
2.2.2. 1-Ferrocenyl-4-methyl-3-thiapentan-1-one (1c)Yield 57%; m.p. 68–69 �C; 1H NMR (CDCl3, ppm) d: 1.29 (d,
J = 6.8 Hz, 6H,(CH3)2CH–), 3.05 (td, J = 13.4, 6.7 Hz, 1H, –CH–S),3.59 (s, 2H, –COCH2–S), 4.23 (s, 5H, Cp), 4.54 (t, J = 2.0 Hz, 2H,Cp), 4.81 (t, J = 2.0 Hz, 2H, Cp). 13C NMR (CDCl3, ppm) d: 22.90(C-20 0 0 and 30 0 0), 35.40 (C-10 0 0), 37.33 (C-2), 69.84 (C-30 and 40),69.95 (C-10 0-50 0), 72.50 (C-20 and 50), 77.56 (C-10), 199.49 (C-1). IR(KBr, cm�1): 3120, 2961, 1645, 1455, 1413, 1377, 1293, 1070,820, 682, 635. Anal. Calc. for C15H18FeOS (302.21): C, 59.61; H,6.00; Fe, 18.48; O, 5.29; S, 10.61. Found: C, 59.67; H, 5.92.
2.2.3. 1-Ferrocenyl-3-thiahexan-1-one (1d)Yield 41%; m.p. 65–67 �C; 1H NMR (CDCl3, ppm) d: 0.99 (t,
J = 7.3 Hz, 3H, CH3), 1.62 (td, J = 10.82, 7.29 Hz, 2H, CH2–CH3),2.59 (t, J = 7.4 Hz, 2H, CH2–CH2), 3.55 (s, 2H, S-CH2), 4.23 (s, 5H,Cp), 4.54 (t, J = 2.0 Hz, 2H, Cp), 4.81 (t, J = 2.0 Hz, 2H, Cp). 13CNMR (CDCl3, ppm) d: 13.21 (C-30 0 0), 22.22 (C-20 0 0), 34.40 (C-10 0 0),38.13 (C-2), 69.72 (C-30 and 40), 69.84 (C-10 0-50 0), 72.42 (C-20 and50), 77.36 (C-10), 199.03 (C-1). IR (KBr, cm�1): 3120, 2959, 2921,1645, 1458, 1412, 1380, 1295, 1072, 838, 821, 682, 635. Anal. Calc.for C15H18FeOS (302.21): C, 59.61; H, 6.00; Fe, 18.48; O, 5.29; S,10.61. Found: C, 59.67; H, 6.06.
2.2.4. 1-Ferrocenyl-4-methyl-3-thiahexan-1-one (1e)Yield 36%; m.p. 59–60 �C; 1H NMR (CDCl3, ppm) d: 0.91 (t,
J = 7.2 Hz, 3H, CH3), 1.38 (td, J = 13.9 and 6.9 Hz, 2H, CH2–CH2)1.49–1.67 (m, 2H CH2–CH3), 2.61 (t, J = 7.3 Hz, 2H, CH2–S), 3.55(s, 2H, CO–CH2–S), 4.23 (s, 5H, Cp), 4.54 (t, J = 2.0 Hz, 2H, Cp),4.81 (t, J = 1.9 Hz, 2H, Cp). 13C NMR (CDCl3, ppm) d: 13.55 (C-40 0 0),21.85 (C-30 0 0), 31.07 (C-20 0 0), 32.21 (C-10 0 0), 38.28 (C-2), 69.83 (C-30
and 40), 69.94 (C-10 0-50 0), 72.51 (C-20 and 50), 77.47 (C-10), 199.12(C-1). IR (KBr, cm�1): 3112, 3083, 2959, 2927, 1645, 1453, 1410,1375, 1290, 1074, 826, 681, 636. Anal. Calc. for C16H20FeOS(316.06): C, 60.77; H, 6.37; Fe, 17.66; O, 5.06; S, 10.14. Found: C,60.70; H, 6.39%.
2.2.5. 1-Ferrocenyl-4-thiahexan-1-one (2b)Yield 86%; 1H NMR (CDCl3, ppm) d: 1.30 (t, J = 7.4 Hz, 3H, CH3),
2.62 (q, J = 7.4 Hz, 2H, CH2–CH3), 2.96 (m(AA0BB0), 4H, CO–CH2CH2–S), 4.23 (s, 5H, Cp), 4.52 (t, J = 1.9 Hz, 2H, Cp), 4.80 (t, J = 2.0 Hz, 2H,Cp). 13C NMR (CDCl3, ppm) d: 14.68 (C-20 0 0), 25.88 (C-10 0 0 or 3), 26.38(C-10 0 0 or 3), 39.79 (C-2), 69.21 (C-30 and 40), 69.78 (C-10 0-50 0), 72.28(C-20 and 50), 78.65 (C-10), 202.38 (C-1). IR (KBr, cm�1): 3097, 2966,2926, 1667, 1455, 1411, 1379, 1253, 1106, 1080, 824, 697, 638.Anal. Calc. for C15H18FeOS (302.21): C, 59.61; H, 6.00; Fe, 18.48;O, 5.29; S, 10.61. Found: C, 59.69; H, 6.04%.
2.2.6. 1-Ferrocenyl-5-methyl-4-thiahexan-1-one (2c)Yield 36%; 1H NMR (CDCl3, ppm) d: 1.31 (d, J = 6.6 Hz, 6H,
(CH3)2CH–), 2.8–3.05 (m, 4H from CO–CH2CH2–S and 1H fromCH–(CH3)2), 4.23 (s, 5H, Cp), 4.52 (t, J = 2.0 Hz, 2H, Cp), 4.80 (t,J = 1.9 Hz, 2H, Cp). 13C NMR (CDCl3, ppm) d: 23.32 (C-20 0 0 and 30 0 0),24.71 (C-3), 35.36 (C-10 0 0), 39.90 (C-2), 69.23 (C-30 and 40), 69.80(C-10 0-50 0), 72.28 (C-20 and 50), 78.67 (C-10), 202.43 (C-1). IR (KBr,cm�1): 3098, 2960, 2926, 1668, 1455, 1411, 1380, 1255, 1107,1078, 823, 694, 642. Anal. Calc. for C16H20FeOS (316.06): C, 60.77;H, 6.37; Fe, 17.66; O, 5.06; S, 10.14. Found: C, 60.81; H, 6.40%.
2.2.7. 1-Ferrocenyl-4-thiaheptan-1-one (2d)Yield 45%; 1H NMR (CDCl3, ppm) d: 1.02 (t, J = 7.3 Hz, 3H, CH3),
1.65 (td, J = 14.4, 7.3 Hz, 2H, CH2–CH3), 2.58 (t, J = 7.6 Hz, 2H, CH2),2.95 (m(AA0BB0), 4H, CO–CH2CH2–S), 4.23 (s, 5H, Cp), 4.52 (t,J = 1.9 Hz, 2H, Cp), 4.80 (t, J = 1.9 Hz, 2H, Cp). 13C NMR (CDCl3,
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869 1865
ppm) d: 13.24 (C-30 0 0), 22.69 (C-20 0 0), 26.11 (C-3), 34.47 (C-10 0 0),39.68 (C-2), 69.04 (C-30 and 40), 69.61 (C-10 0-50 0), 72.11 (C-20 and50), 78.50 (C-10), 202.14 (C-1). IR (KBr, cm�1): 3098, 2961, 2929,1667, 1455, 1411, 1379, 1254, 1078, 823, 691, 636. Anal. Calc. forC16H20FeOS (316.06): C, 60.77; H, 6.37; Fe, 17.66; O, 5.06; S,10.14. Found: C, 60.78; H, 6.42%.
2.2.8. 1-Ferrocenyl-4-thiaoctan-1-one (2e)Yield 73%; 1H NMR (CDCl3, ppm) d: 0.93 (t, J = 7.2 Hz, 3H, CH3),
1.41 (td, J = 13.7, 7.0 Hz, 2H, CH2–CH2) 1.52–1.69 (m, 2H CH2–CH3),2.60 (t, J = 7.3 Hz, 2H, CH2), 2.95 (m(AA0BB0), 4H, CO–CH2CH2–S),4.23 (s, 5H, Cp), 4.52 (t, J = 1.9 Hz, 2H, Cp), 4.80 (t, J = 1.9 Hz, 2H,Cp). 13C NMR (CDCl3, ppm) d: 13.57 (C-40 0 0), 21.91 (C-30 0 0), 26.38(C-3), 31.68 (C-20 0 0), 32.31 (C-10 0 0), 39.86 (C-2), 69.22 (C-30 and 40),69.79 (C-10 0-50 0), 72.27 (C-20 and 50), 78.69 (C-10), 202.40 (C-1). IR(KBr, cm�1): 3098, 2958, 2929, 1668, 1455, 1411, 1379, 1254,1080, 823, 692, 638. Anal. Calc. for C17H22FeOS (330.07): C, 61.82;H, 6.71; Fe, 16.91; O, 4.84; S, 9.71. Found: C, 61.80; H, 6.67%.
2.3. Crystal structure determination
X-Ray diffraction data were collected with an Oxford DiffractionGemini-R Ultra diffractometer using graphite-monochromated MoKa radiation (k = 0.71073 Å). The structure was solved by directmethods and refined using full-matrix least squares on F2. Thecrystallographic data and refinement details are listed in Table 1.
Table 1Crystal data and structure refinement details.
Compound 1a 2a
CCDC No. 759144 759145Formula C13H14FeOS C14H16FeOSMr 274.15 288.18Crystal shape, color irregular fragment, red-
brownprismatic fragment, red-brown
Crystal size, (mm3) 0.16 � 0.12 � 0.08 0.34 � 0.08 � 0.08Crystal system monoclinic monoclinicSpace group P21/n P21/ca (Å) 5.7288(4) 5.7723(2)b (Å) 19.7336(11) 12.9582(4)c (Å) 10.3223(6) 16.5834(5)b (�) 98.037(6) 94.450(3)V (Å3) 1155.47(12) 1236.68(7)Z 4 4Dcalc (g cm�3) 1.58 1.55l (mm�1) 1.46 1.37F(0 0 0), e 568 600Diffractometer Oxford Diffraction
Gemini-R UltraOxford DiffractionGemini-R Ultra
Radiation type Mo Ka Mo KaData collection
methodx scans x scans
T (K) 173 173hmax (�) 25.4 25.3h, k, l range �6 ? 5, ±23, �12 ? 9 ±6, �15 ? 14, �19 ? 16Absorption correction multi-scan multi-scanMeasured reflections 7441 7022Independent
reflections2107 (Rint = 0.040) 2244 (Rint = 0.032)
Observed reflections[I > 2r(I)]
1691 1885
Data/restraints/parameters
2107/0/146 2244/0/155
R1/wR2 [I > 2r(I)] 0.026/0.048 0.026/0.056R1/wR2 (all data) 0.040/0.050 0.036/0.058Goodness-of-fit (GOF)
on F20.94 0.95
Dq max/Dq min(e �3)
0.29/�0.22 0.26/�0.25
2.4. Electrochemical measurements
Electrochemical measurements were performed at ambienttemperature (ca. 25 �C) by using a CH Instruments (Austin, TX)potentiostat CHI760b Electrochemical Workstation. A standardthree-electrode cell (5 mL) equipped with a platinum wire and asilver wire immersed in 0.1 M LiClO4 solution in CH3CN as thecounter and reference electrode, respectively. A platinum disk(d = 2 mm) was used as the working electrode.
2.5. Biological assay
The in vitro antimicrobial activities of 1a–g and 2a–g weretested against a panel of laboratory control strains belonging tothe American Type Culture Collection Maryland, USA. Antibacterialactivity was evaluated against six Gram positive and five Gramnegative bacteria. The Gram positive bacteria used were: Bacillussubtilis (ATCC 6633), Clostridium pyogenes (ATCC 19404), Enterococ-cus sp. (ATCC 25212), Micrococcus flavus (ATCC 10240), Sarcina lu-tea (ATCC 9341) and Staphylococcus aureus (ATCC 6538). TheGram negative bacteria utilized in the assays were: Escherichia coli(ATCC 25922), Klebsiella pneumoniae (ATCC 10031), Proteus vulgaris(ATCC 8427), Pseudomonas aeruginosa (ATCC 27857) and Salmonellaenteritidis (ATCC 13076). The antifungal activity was tested againstthree organisms Aspergillus niger (ATCC 16404), Candida albicans(ATCC 10231) and Saccharomyces cerevisiae (ATCC 9763).
A disk diffusion method, according to NCCLS [23], was em-ployed for the determination of the antimicrobial activity of 1a–gand 2a–g. The following nutritive media were used: AntibioticMedium 1 (Difco Laboratories, Detroit Michigan USA) for growingGram positive and Gram negative bacteria and Tryptone soy agar(TSA – Institute of Immunology and Virology ‘‘Torlak”, Belgrade,Serbia) for C. albicans and A. niger. Nutritive media were preparedaccording to the instructions of the manufacturer. All agar plateswere prepared in 90 mm Petri dishes with 22 ml of agar, giving afinal depth of 4 mm. One-hundred microliters of a suspension ofthe tested microorganisms (108 cells per ml) were spread on thesolid media plates. Sterile filter paper disks (‘‘Antibiotica Test Blatt-chen”, Schleicher and Schuell, Dassel, Germany, 12.7 mm in diam-eter) were impregnated with 40 ll of the samples solutions(65 mg/ml) in DMSO (all solutions were filter-sterilized using a0.45 lm membrane filter), i.e. 500 lg per disk, and placed on inoc-ulated plates. These plates, after standing at 4 �C for 2 h, were incu-bated at 37 �C for 24 h for bacteria and at 30 �C for 48 h for thefungi. Standard disks of Tetracycline and Nystatine (origin – Insti-tute of Immunology and Virology ‘‘Torlak”, 30 lg of the active com-ponent, diameter 6 mm) were used individually as positivecontrols, while the disks imbued with 50 ll of pure DMSO wereused as a negative control. The diameters of the inhibition zoneswere measured in millimeters (to the nearest mm) using a ‘‘Fish-er-Lilly Antibiotic Zone Reader” (Fisher Scientific Co., USA). Eachtest was performed in quintuplicate. In order to evaluate statisti-cally any significant differences among mean values, a one-wayANOVA test was used. In all tests the significance level at whichwe evaluated critical values differences was 5%.
Broth microdilution susceptibility assay was used, as recom-mended by NCCLS, for the determination of MIC values (NCCLS,[24]). All tests were performed in Mueller Hinton broth (MHB;BBL) supplemented with Tween 80 detergent (final concentrationof 0.5% (v/v)), with the exception of the fungal organisms (Sabou-raud dextrose broth-SDB + Tween 80), and with 2 � 105 colonyforming units (CFU) mL�1 of the bacteria/fungi in the exponentialphase. Test compounds (2a, 2b, 1c, 2c, 1d, 2d, 1e and 2e) were dis-solved in dimethyl sulfoxide and this stock solution used to pre-pare the exact agar dilutions. Final concentrations of thecompounds in the broth ranged from 0.012 to 256 mg/l and these
1866 D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
were prepared in a 96-well microtiter plate. The dilutions werebased on a geometrical order (factor 2) and were related to concen-trations 1 mg/l and 1.5 mg/l (0.012, 0.016, 0.012, 0.031, 0.047,0.063, 0.094, 0.125, 0.19, 0.25, 0.38, 0.5, 0.75, 1.0, 1.5, 2, 3, 4, 6, 8,12, 16, 24, 32, 48, 64, 96, 128, 192, 256 mg/l). In order to get moreaccurate values of MIC further dilutions were additionally pre-pared in the concentration range between the first well withoutvisible growth and the one next to it with lower test compoundconcentration. Plates were incubated at 37 �C for 24 h for bacteriaand at 30 �C for 48 h for the yeasts/mold. Each test was performedin duplicate and repeated twice. Amikacin and Bifonazole wereused as positive controls, while DMSO + Tween 80 (in the form ofa blank) were the negative control.
3. Results and discussion
3.1. Synthesis of 1-ferrocenyl-1-(hydroxyimino)thialkanes 2a,b and3a,b
The synthesis of acylferrocenes 1a–g and 2a–g has beenachieved by modified Friedel–Crafts acylation of ferrocene withchlorides of the corresponding carboxylic acids (Scheme 1)[19,21,22]. In this improved method the acylating agents – acylchlorides – were generated in situ from the corresponding carbox-ylic acids and phosphorus trichloride, and have not been isolatedor purified for subsequent use. Namely, the phosphorus species lib-erated in the course of this reaction, whatever they are (most prob-ably derivatives of phosphorous acid, H3PO3), do not prevent theacylation. We are not sure if this is caused by the known high reac-tivity of ferrocene towards Friedel–Crafts acylation (which isthought to be similar to the reactivity of phenols) so that the inac-tivation of the catalyst (aluminium chloride) is not of such an ex-tent to prevent the onset of the reaction, or if it is due to thecomplete lack of the catalyst inactivation. The yields of the corre-sponding ketones were in the range of 36–86% (see Scheme 1).
3.2. Spectral characterization
The structures of compounds 1a–g and 2a–g were spectroscop-ically characterized by IR, 1H, and 13C NMR. These data were incomplete agreement with the reported ones for the known com-pounds (1a,f,g 2 a,f,g). The main common feature of the IR spectraof all of the new ketones is a strong band at 1640–1645 for com-pounds 1b–d, and at 1667–1668 cm�1 for compounds 2b–d, whichwe attributed to the carbonyl group stretching vibrations. Appar-ently, this difference is the consequence of the vicinity of S-alkylgroup in the case of compounds 1b–d (a position of S-alkyl group).This could also likely be an influence of the stronger hydrogenbonding holding together the dimeric species in the crystal latticeof a-thioalkyl ketones (see 2.3. for C–H���O@C lengths supportingthis assumption) compared to the infinite array of intermolecularly
Scheme 1. Synthesis of sulfur containing acylferrocenes.
H-bonded b-thioalkyl counterparts. The NMR spectra utterly con-firm the structures of compounds 1b–d and 2b–d. Thus, in the1H NMR spectra of all eight compounds signals of the cyclopenta-dienyl rings protons appear at almost the same positions in thespectra (d = 4.23 ppm for the unsubstituted, and 4.52–4.54 and4.80–4.81 ppm for the substituted rings, respectively). Protons ofthe –CH2–S– unit in 1b–d also have almost the same chemicalshifts (3.55–3.59 ppm). On the other hand, a specific AA0BB0 secondorder multiplet signal in the 1H NMR spectra of the compounds2b–d, originating from the –CO–CH2–CH2–S- moiety, appears (itscenter) always at 2.95 ppm.
3.3. X-ray analysis of compounds 1a and 2a
Single crystal X-ray diffraction structure analysis of the com-pounds 1a and 2a revealed, not unexpectedly, that these homolo-gous molecules exhibit very similar conformations (Fig. 2). Thus,the ferrocene units in 1a and 2a adopt almost eclipsed conforma-tions, with average torsion angles of 4.14 and 2.06�, respectively.The respective cyclopentadienide (Cp) rings are tilted by 2.44and 2.29�. The carbonyl groups deviate from the planes of the adja-cent Cp rings by only 0.16 Å (C) and 0.03 Å (O) in 1a, and 0.12 Å (C)and 0.06 Å (O) in 2a, respectively. However, the packing arrange-ments in the crystals are quite different. Compound 1a forms di-mers by weak contacts between the carbonyl oxygen atom O1and H12a of the methylene group at the symmetry position (�x,�y, 1 � z), with a H���O distance of 2.58 Å, a C���O distance of3.524 Å, and a C–H���O angle of 160� (Fig. 3a). In contrast, the mol-ecules of compound 2a are linked by weak interactions betweenthe carbonyl oxygen atom O1 and H14c of the methyl group at(�1 + x, y, z) into infinite chains in the direction of the crystallo-graphic a axis, with a H���O distance of 2.64 Å, a C���O distance of3.597 Å, and a C–H���O angle of 165� (Fig. 3b).
3.4. Electrochemistry
Cyclic voltammetry in acetonitrile containing 0.1 mol/L lithiumperchlorate as a supporting electrolyte has been used for the eval-uation of electrochemical properties of all the synthesized com-pounds – the known and new ones. All ketones exhibit areversible one-electron redox couple at the similar potential (oxi-dation waves appear at 647–680 mV and the reduction ones at671–601 mV, Table 2). These potentials are considerably more po-sitive than that of the unsubstituted ferrocene, as a consequence ofthe presence of an electron-withdrawing acyl group. As the repre-sentative examples we give the voltammograms of compounds 1aand 2a (Fig. 4). The difference between anodic and cathodic peakpotentials (Table 2) is close to the theoretical value and indepen-dent of the scan rate v. Both, anodic and cathodic peak currentsare proportional to the square root of the scan rate, and their ratiois independent of the scan rate, indicating a diffusion-controlledprocess.
3.5. Biology
Antibacterial activity against six Gram positive (B. subtilis, C.pyogenes, Enterococcus sp., Micrococcus flavus, Sarcina lutea and S.aureus) and five Gram negative bacteria (E. coli, K. pneumoniae, P.vulgaris, P. aeruginosa and S. enteritidis), as well as the antifungalactivity against three fungal organisms (A. niger, C. albicans andS. cerevisiae as the representative species of fungi) were assessedfor compounds 1a–g and 2a–g. The antimicrobial activity screen-ing was performed by means of a disk diffusion assay (NCCLS,[23]), and the obtained results are listed in Table 3. As it can beseen from the diameters of the growth inhibition zones presented
Fig. 2. Ortep plot of compound 1a (a) and 2a (b) (ellipsoid probability 50%).
Fig. 3. (a) Dimers formed by weak C–H���O contacts in the crystal structure of 1a. (b) Infinite chains formed by weak C–H���O contacts in the crystal structure of 2a.
Table 2The electrochemical data for compounds 1a–g and 2a–g.
Compound Epaa (mV) Epa
a (mV) E1/2b (mV) DEc (mV)
1a 656 577 616 791b 668 595 631 731c 656 577 616 791d 681 601 641 801e 659 577 618 821f 659 583 621 761g 668 589 628 792a 656 568 612 882b 647 574 610 732c 650 571 610 792d 662 583 622 792e 647 574 610 732f 649 571 610 772g 656 574 615 82
a Epa and Epc anodic and cathodic peak potentials, respectively, at 100 mV s�1.b E1/2 = (Epa + Epc)/2.c DE = Epa � Epc.
Fig. 4. Cyclovoltammograms of compounds 1a, 2a and ferrocene at Pt disc in 0.1 MLiClO4 in acetonitrile at v = 0.1 V/s.
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869 1867
in Table 3 the prepared compounds showed a wide range of activ-ity (from completely inactive ones to those of medium activity).
The compounds that showed (at a dose of 500 lg per disk) somedegree of activity in this test were further subjected to a dilutiontest to get more precise results on their antimicrobial nature. Theminimal inhibitory concentration (MIC, considered to be the low-est concentration of the tested compound in lg/mL which inhib-ited growth of bacteria or fungi) was determined using amicrodilution method based on the recommendations of NCCLS[24]. Under the same conditions solutions of differing concentra-tion of Amikacin (antibacterial) and Bifonazole (antifungal) were
used as positive controls. The values of MIC for the currently syn-thesized active sulfur containing acylferrocene derivatives (1c, 1d,1e, 2a, 2b, 2c, 2d and 2e) ranged from 102 to 237 lg/mL (Table 4).Concentrations above 256 lg/mL were not tested.
The determined MIC concentrations are similar in efficacy rangecompared to other published [15] ferrocene derivative MIC values.The results of the dilution method confirmed the findings of the
Table 3The antimicrobial activity (diameters of growth inhibition zones of compounds 1a–g and 2a–g in a disc diffusion assay at a 500 lg per disc dose).
Microorganism Compound
1a 2a 1b 2b 1c 2c 1d 2d 1e 2e 1f 2f 1g 2g Tetracycline Nystatine
A. niger – 28a – 34 – 31 31 30 – 32 – – – – nt 17C. albicans – 32 – 32 – 34 32 33 – 33 – – – – nt 17S. cerevisiae – – – – – 32 – 36 – – – – – – nt 16S. lutea – 35 – 33 36 35 34 – – 36 – – – – 26 ntM. flavus – 35 – 35 34 35 34 – – 37 – – – – 30 ntB. subtilis – 33 – 34 35 36 30 40 32 35 – – – – 25 ntS. aureus – 35 – 36 34 33 34 – 34 35 – – – – 27 ntE. coli – 32 – 36 32 33 35 – 36 35 – – – – 23 ntP. aeruginosa – 34 – 38 34 35 35 – 31 – – – – – 24 ntC. pyogenes – 34 – 35 – 32 36 – 36 36 – – – – 24 ntK. pneumoniae – 29 – 28 – 30 – 30 – – – – – – 26 ntS. enteritidis – 32 – 37 – 33 35 – – 34 – – – – 26 ntP. vulgaris – 35 – 30 35 38 35 – 35 37 – – – – 26 ntEnterococcus sp. – 38 – 40 34 35 33 – – 38 – – – – 29 nt
–, Not active, nt – Not tested.a Mean values (in mm) of five experiments, including the disc diameter (12.7 nm).
Table 4Minimal inhibitory concentration (MIC, lg/ml) of the selected compounds (that have shown activity in the disk diffusion assay, 2a, 2b, 1c, 2c, 1d, 2d, 1e, 2e).
Microorganism Compound
2a 2b 1c 2c 1d 2d 1e 2e Amikacin Bifonazole
A. niger 229 178 >256 180 192 198 >256 181 n.t. 12C. albicans 166 170 >256 154 206 168 >256 155 n.t. 32S. cerevisiae >256 >256 >256 211 >256 159 >256 >256 n.t. 7S. lutea 161 126 141 146 155 >256 >256 153 3 n.t.M. flavus 164 167 165 164 168 >256 >256 138 4 n.t.B. subtilis 173 155 169 144 142 102 194 152 31 n.t.S. aureus 157 130 177 183 160 >256 161 148 15 n.t.E. coli 155 132 172 163 152 >256 155 135 4 n.t.P. aeruginosa 155 139 164 152 156 >256 177 >256 44 n.t.C. pyogenes 172 130 >256 172 141 >256 155 124 17 n.t.K. pneumoniae 207 237 >256 212 >256 204 >256 >256 8 n.t.S. enteritidis 172 138 >256 163 152 >256 >256 160 9 n.t.P. vulgaris 139 129 161 138 143 >256 161 120 8 n.t.Enterococcus sp. 147 123 164 156 188 >256 >256 129 58 n.t.
n.t. – Not tested.
1868 D. Ilic et al. / Polyhedron 29 (2010) 1863–1869
diffusion test. It is quite obvious (Tables 3 and 4) that the com-pounds have a more profound effect upon the growth of bacteriathan fungal organisms. None of the tested microorganisms weresusceptible to compounds 1a, 1b, 1f, 1g, 2f and 2g. These are thepairs of compounds having a 4-bromobenzyl (f) and dichloroben-zyl group (g), as well as the compounds containing an a-MeS(1a) and a-Et (1b) side chains. The best results (MIC = 102 lg/mL,also a growth inhibition zone of 40 mm) have been obtained inthe case of compound 2d against B. subtilis, while a similar effectwas noted for 2b against a strain of Enterococcus sp. with a123 lg/mL inhibitory concentration and a diameter of the inhibi-tion zone of 40 mm at 500 lg per disk. Generally, compound 2b,FcCO(CH2)2SEt, was the most active, but showed no inhibition ofthe growth of the yeast S. cerevisiae. However, the active com-pounds demonstrated non-selectiveness towards the bacteria, ex-cept that compound 2d that appeared to have a morepronounced affect against the fungi but still maintained a distinc-tive activity against B. subtilis. Additionally, Gram negative andGram positive strains have not been differentiated by the synthe-sized compounds. From the analysis of Tables 3 and 4 it is perhapspossible to establish some SARs. Comparing compounds with n = 1and n = 2, the latter show to be more active or similar in activity(1a and 1b were completely inactive, while the 2a and 2b counter-parts had shown significant activity). Perhaps the bulkiness of theside chains in f and g compounds prevented the docking of the
compounds to the enzyme(s), and though it seems that the lengthof the side chain is important, the position of the sulfur atom alsocounts (1b and 2a have a chain of 4 atoms, but 1b had no activitywhat so ever).
We performed agglomerative hierarchical clustering (AHC) onthe mentioned samples (Table 3, the method was applied utilizingthe values of diameters of growth inhibition zones as original vari-ables without any recalculation), using the Excel program plug-inXLSTAT version 2008.6.07. However, the mentioned analysisshowed no logical grouping of the compounds except for makinga clear distinction of 1e and 1c (completely inactive towards thethree tested fungal strains) from the bulk of the other compoundsthat showed a bigger impact on the growth of fugal organisms.
4. Conclusion
In summary, we synthesized fourteen sulfur containing acylfer-rocenes, eight of them being new ones. The new compounds havebeen characterized by spectral means, whereas the cyclic voltam-metry technique showed that all the compounds exhibit one welldefined redox couple (attributed to the ferrocene unit) at a verymutually similar potential. Most of them have also shown a certaindegree of activity against the microorganisms among which someare notorious human pathogens, however compared to the tested
D. Ilic et al. / Polyhedron 29 (2010) 1863–1869 1869
standards their MIC values in vitro lie in a range one to two ordersof magnitude higher than the ones exhibited by the antibiotics.Likewise one can conclude that the ferrocene unit does not pre-clude the activity of the previously synthesized pyridine and pyra-zine analogs [20]. These results should provide a good startingpoint and should be regarded as a good learning set for the devel-opment of new more potent ferrocene containing antimicrobials.
5. Supplementary data
CCDC 759144 and 759145 contain the supplementary crystallo-graphic data for compounds 1a and 2a. These data can be obtainedfree of charge via http://www.ccdc.cam.ac.uk/conts/retriev-ing.html, or from the Cambridge Crystallographic Data Centre, 12Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223-336-033;or e-mail: [email protected].
Acknowledgements
This work was supported by the Ministry of Science and Tech-nological Development of the Republic of Serbia (Grant No142042).
References
[1] P.M. Hawkey, J. Antimicrob. Chemother. 62 (S1) (2008) 1.[2] R. Cantón, Clin. Microbiol. Infect. 15 (2009) 20.[3] T. Moriuchi, T. Hirao, Top. Organomet. Chem. 17 (2006) 143.[4] B. Loev, M. Flores, J. Org. Chem. 26 (1961) 3595.[5] F.D. Popp, S. Roth, J. Kirby, J. Med. Chem. 6 (1963) 83.
[6] C. Biot, G. Glorian, L.A. Maciejewski, J.S. Brocard, J. Med. Chem. 40 (1997) 3715.[7] D. Osella, M. Ferrali, P. Zanello, F. Laschi, M. Fontani, C. Nervi, G. Cavigiolio,
Inorg. Chim. Acta 306 (2000) 42.[8] G.JaouenS. Top, A. Vessières, R. Alberto, J. Organomet. Chem. 600 (2000) 23.[9] D.R. van Staveren, N. Metzler-Nolte, Chem. Rev. 104 (2004) 5931.
[10] E.W. Neuse, J. Inorg. Organomet. P15 (2005) 3.[11] C.S. Allardyce, A. Dorcier, C. Scolaro, P.J. Dyson, Appl. Organomet. Chem. 19
(2005) 1.[12] G. Jaouen, W. Beck, M.J. McGlinchey, in: G. Jaouen (Ed.), Bioorganometallics:
Biomolecules, Labeling, Medicine, Wiley-VCH, Weinheim, 2006.[13] M.F.R. Fouda, M.M. Abd-Elzaher, R.A. Abdelsamaia, A.A. Labib, Appl.
Organomet. Chem. 21 (2007).[14] O. Payen, S. Top, A. Vessières, E. Brulé, M.-A. Plamont, M.J. McGlinchey, H.
Müller-Bunz, G. Jaouen, J. Med. Chem. 51 (2008) 1791.[15] I. Damljanovic, M. Vukicevic, N. Radulovic, R. Palic, E. Ellmerer, Z. Ratkovic,
M.D. Joksovic, R.D. Vukicevic, Bioorg. Med. Chem. Lett. 19 (2009) 1093.[16] I. Damljanovic, M Colovic, M. Vukicevic, D. Manojlovic, N. Radulovic, K. Wurst,
G. Laus, Z. Ratkovic, M.D. Joksovic, R.D. Vukicevic, J. Organomet. Chem. 694(2009) 1575.
[17] M.D. Joksovic, V. Markovic, Z. Juranic, M. Vukicevic, T. Stanojkovic, Lj.S.Jovanovic, I.S. Damljanovic, K.M. Mésáros Szécényi, N. Todorovic, S. Trifunovic,R.D. Vukicevic, J. Organomet. Chem. 694 (2009) 3935.
[18] Z. Ratkovic, T. Stanojkovic, D. Manojlovic, R.D. Vukicevic, N. Radulovic, M.D.Joksovic, Bioorg. Chem. 38 (2010) 26.
[19] R.D. Vukicevic, D. Ilic, Z. Ratkovic, M. Vukicevic, Chem. Month. 132 (2001) 625.[20] G.A. Wächter, M.C. Davis, A.R. Martin, S.G. Franzblau, J. Med. Chem. 41 (1998)
2436.[21] R.D. Vukicevic, M. Vukicevic, Z. Ratkovic, S. Konstantinovic, Synlett (1998)
1329.[22] M.D. Vukicevic, Z. Ratkovic, A. Teodorovic, G. Stojanovic, R.D. Vukicevic,
Tetrahedron 58 (2002) 9001.[23] NCCLS (National Committee for Clinical Laboratory Standards). Performance
standards for antimicrobial disk susceptibility testing; 6th InternationalSupplement, 1997, Wayne Pa. M2-A6.
[24] NCCLS (National Committee for Clinical Laboratory Standards). Performancestandards for antimicrobial susceptibility test, 9th International Supplement,1999, Wayne PA. M100-S9.
Recommended
