(CH3C5H4)2Vcl2 with Α-, Β-Andγ- Cyclodextrines: Synthesis, EPR Study and Microbiological Behavior Toward Escherichia Coli

CEJC 3(1) 2005 72–81

Inclusion compounds of cystostatic active (C5H5)2VCl2 and (CH3C5H4)2VCl2 with α-, β-andγ- cyclodextrines: Synthesis, EPR study and microbiological behavior toward Escherichia coli.

Jarom´ır Vinklárek∗, Jan Honz´ıˇcek, Jana Holubová Department of General and Inorganic Chemistry, University of Pardubice, nám. Cs.ˇ legi´ı 565, 532 10 Pardubice, Czech Republic

Received 21 July 2004; accepted 7 October 2004

Abstract: The inclusion of vanadocene dichloride (VDC) and 1,1’-dimethyl vanadocene dichloride (MeVDC) into cyclodextrines (α-CD, β-CD and γ-CD) was studied by EPR spectroscopy. It was found that VDC and MeVDC with β-CD and γ-CD form true inclusion compounds, but with α-CD, VDC and MeVDC gave only ﬁne dispersion mixtures. The inclusion was validated by anisotropic EPR spectra of solid samples. In addition, the antimicrobial behavior (against E. coli) of each of the complexes was determined. It was established that not only did VDC and MeVDC cause elongation of E. coli, but also the new vanadocene inclusion complexes were eﬀective in this regard. c Central European Science Journals. All rights reserved.

Keywords: EPR spectroscopy, vanadocene complexes, cyclodextrine, inclusion compounds, Escherichia coli, bent metallocene

1 Introduction

5 Metallocene dihalide complexes, (η -Cp)2MX2 , of early transition metals are currently undergoing intensive studies due to their biological activity [1-4]. Titanocene dichloride (TDC) and vanadocene dichloride (VDC, 1) have been found to be the most active metallocene complexes in many biological experiments [5]. Inclusion compounds of cyclodextrines are known to form stable inclusion compounds with a variety of organometallics, including ferrocene and its derivatives [6-9], half-sandwich complexes of molybdenum[10,11]

∗ E-mail: [email protected] J. Vinkl´arek et al. / Central European Journal of Chemistry 3(1) 2005 72–81 73

and titanocene or molybdenocene dihalide complexes [12-14]. These inclusion compounds are being investigated for pharmaceutical uses owing to their ability to increase the aqueous solubility of drugs, improve oral absorption, and enhance chemical stability with respect to oxidation by air, sensitivity to light etc [15]. For the validation of inclusion compounds it is necessary to choose an appropriate experimental method. The preparation of a single-crystal, which is indispensable for X-ray structure analysis, is very difficult for these type of compounds and therefore another experimental method has to be cho- sen. In the case of inclusion compounds of titanocene dichloride with cyclodextrines, 1H-NMR spectroscopy has successfully been used for verification of inclusion compounds [12]. Unfortunately, use of this method for paramagnetic substances is very limited. The aim of this work is to find a method that could be used to verify paramagnetic compounds, such as vanadocene dichloride (VDC; 1) and its ring substituted derivative, 1,1’-dimethyl vanadocene dichloride (MeVDC; 2) as inclusion cyclodextrine compounds. The combination of vanadocene molecules with various dimensions and cyclodextrines with different cavity size’s gives either true inclusive complexes or only fine dispersion mixtures of starting compounds. In our previous work, we have studied paramagnetic bent metallocenes by means of electron paramagnetic resonance (EPR) and we have found that magnetic parameters are very sensitive to small changes in the surroundings of paramagnetic central metal d1 atom V (IV) [16,17]. In this paper, we intend to show how to distinguish true inclusion compounds from those that are just a fine dispersion of paramagnetic compound and cyclodextrine by means of EPR spectroscopy. The finding of elongation of Escherichia coli by Rosenberg at al. [18], was the first hint to the biological activity of the inorganic platinum complexes (cisplatin) and led to the detection of pronounced antitumor effectivity against animal and human tumors. Elongation is generally interpreted as evidence that the agents are effecting cell division and DNA replication [19].

2 Results and discussion

Solid samples of 3-8 were prepared by evaporation of aqueous solutions of vanadocenes 1 and 2 with appropriate cyclodextrines (α-CD, β-CD or γ-CD). These compounds were green, air stable paramagnetic powders that were soluble only in water and insoluble in the other common solvents. Isolated compounds 3-8 were characterized by the elemental analysis, IR and Raman spectroscopy and especially by the EPR spectroscopy. The results from infrared and Raman spectra measurements evidenced the keeping of vanadocene (methylvanadocene) fragment in compounds 3-8. Particularly, the pres- 5 ence of η -bonded C5H5 ring (in compounds 3-5) is validated by vibrations C5H5:C-H −1 −1 stretch (Raman: ∼ 3110 cm ), C5H5:C-C breath (Raman: ∼ 1135 cm )andC5H5:C-H −1 5 bend (IR: ∼ 833 cm ). The keeping of η -bonded CH3C5H4 ring (in compounds 6-8) −1 is validated by vibrations C5H4:C-H stretch (∼ 3110 cm ), CH3: C-H stretch (Raman: −1 −1 ∼ 2930 cm ), C5H4: C-H bend (IR: ∼ 833 cm ). The tilting of cyclopentadiene rings (Raman: ∼ 300 cm−1) validates the presence of both rings in the molecule [20].The vibra- 74 J. Vinkl´arek et al. / Central European Journal of Chemistry 3(1) 2005 72–81

tion spectra, however, does not give any information about inclusion. This information was obtained from anisotropic EPR spectra. The anisotropic spectra of vanadocene dichloride (1) is rhombic symmetric with a signiﬁcant g-tensor and A-tensor anisotropy, that are characteristic for d1-metallocene dihalides (Tab. 1). The orientation of principal directions for magnetic tensors with respect to the molecular geometry according to related complexes was used [21]. The EPR parameters of compounds 1 and 2 are very similar (Tab. 1). Thus, the substitution by one methyl group on each of the Cp rings does not change the magnetic properties around the paramagnetic center of vanadium. The knowledge of the magnetic behavior of compounds 1 and 2 is important for discussion about the inclusion of vanadocene moiety into the cavity of cyclodextrine.

Ax Ay Az gx gy gz

α−CD-VDC (3) Single asymmetric peak β−CD-VDC (4) 73.6 115.9 18.0 1.982 1.971 2.011 γ−CD-VDC (5) 74.2 115.9 21.0 1.986 1.971 2.007 α−CD-MeVDC (6) Single asymmetric peak β−CD-MeVDC (7) 73.3 115.9 18.3 1.982 1.971 2.008 γ−CD-MeVDC (8) 76.1 115.6 21.0 1.988 1.971 2.011

Table 1 Hyperﬁne coupling tensors (104cm−1)andg-tensors for compounds 3-8.

Overall, two different patterns of EPR spectra were obtained for compounds 3-8 (Fig. 1, 2). The compounds 3 (α-CD-VDC, Fig. 1 spectrum A) and 6 (α-CD-MeVDC, Fig. 2 spectrum A) gave similar anisotropic EPR spectra, in which the hyperfine interaction with vanadium nucleus is not perceptible. The compounds 4 (β-CD-VDC, Fig. 1 spectrum B), 5(γ-CD-VDC, Fig. 1 spectrum C), 7(β-CD-MeVDC, Fig. 2 spectrum B) and 8 (γ-CD-MeVDC, Fig. 2 spectrum C) gave spectra with distinct hyperfine interactions. The observed g-tensors and A-tensors are summarized in Table 1. The observed difference’s in spectrum patterns can only be explained in one way. Samples of compounds 3 and 6 have not been magnetically diluted, because they are not true inclusion compounds. The α-CD forms with vanadocene dichlorides 1 and 2 are only fine dispersion mixtures. In contrast, cyclodextrines with larger cavities (β-CD and γ- CD) produced with the vanadocene dichlorides 1 and 2, are true inclusion compounds (see scheme). The inclusion of a paramagnetic compound into the cavity of cyclodextrine causes a dilution of the paramagnetic centers. The obtained spectra have similar patterns to the EPR spectra of the frozen solutions and to the solid EPR spectra in isostructural diamagnetic matrix. Table 2 summarizes the results of microbiological studies and gives the minimum inhibitory concentrations (MIC), effective concentration as well as the relative amounts of elongated cells (in %) and maximal elongation at the most effective concentration. All studied complexes are effective in the cell division and embodies a strong inhibition of bacterial growth. From the obtained data, it can be estimated that complexes 1 and J. Vinklárek et al. / Central European Journal of Chemistry 3(1) 2005 72–81 75

Fig. 1 EPR spectra of compounds: (A) α-CD-VDC (3), (B) β-CD-VDC (4),γ-CD-VDC (5); ν0= 9.544 GHz.

2 are more effective than the antitumor agent TDC [22], which has undergone clinical testing before [4,23]. The true inclusion compounds 4, 5, 7 and 8 show similar biological activity as free vanadocene complexes 1 and 2. The minimum inhibitory concentrations (MIC) were found to be 4.5 mmol/l and the effective concentration was in the range 0.25 - 1.8 mmol/l for all studied compounds. It was shown that inclusion into the cavity of cyclodextrine does not adversely influence the germicidal activity and nor probably the cytostatic activity.

MICa Eﬀective conc. Rel. % Max. elong. (mmol/l) (mmol/l) elong. cells

1 4.5 1.34-0.28 25-50 80x 2 4.5 1.03-0.33 20-40 60x 4 4.5 1.13-0.24 25-40 70x 5 4.5 1.79-0.24 25-50 70x 7 4.5 1.13-0.28 25-50 60x 8 4.5 1.82-0.28 25-45 70x

a minimum inhibitory concentration.

Table 2 Microbiological activity of compounds 1, 2, 4, 5, 7 and 8. 76 J. Vinkl´arek et al. / Central European Journal of Chemistry 3(1) 2005 72–81

Fig. 2 EPR spectra of compounds: (A) α-CD-MeVDC (6), (B) β-CD-MeVDC (7),γ-CD- MeVDC (8); (ν0= 9.544 GHz).

CH3 Cl V Cl V Cl Cl

CH3

β 4 β-CD 7 -CD γ 5 γ-CD 8 -CD

Scheme 1 The proposed structures of true inclusion compounds.

3 Conclusion

From EPR spectra measurements, the inclusion of bent metallocenes is essentially a steric question connected with the size of the cyclodextrine cavity and the particle included. The inclusion does not activate changes on the paramagnetic center that would manifest in diﬀerences in magnetic parameters between inclusive molecules and mechanical mixtures. A true inclusion can be unambiguously determined by means of the pattern of the anisotropic EPR spectra. Only in the case of a true inclusion does the isolation of indi- vidual paramagnetic centers occur, which leads to an EPR spectrum with characteristic J. Vinkl´arek et al. / Central European Journal of Chemistry 3(1) 2005 72–81 77

hyperﬁne splitting of paramagnetic molecules. The results of microbiological tests with E.coli B established that the inclusion into the cyclodextrine cavity does not decrease the biological activity of the including compound. By contrast the inclusion compound may actually have its application features (e.g. solubility, stability...) enhanced by the cyclodextrine cavity. A suitable cyclodextrine molecule may also be possible to use as an inert carrier for application in the organism.

4 Experimental section

4.1 Methods and materials

All operations were performed under argon by using conventional Schlenk-line techniques. The solvents were purified and deoxygenated by standard methods. Water was deionised double-distilled and saturated by argon. Cyclodextrines (α-CD, β-CD, γ-CD) were obtained from Fluka and used without further purification. Vanadocene dichloride (1) and 1,1’-dimethyl vanadocene dichloride (2)werepre- pared by the published methods [21,24] and purified by anaerobic Soxhlet extraction with dichloromethane. Their purity was checked by elemental analysis, IR, Raman, EPR and UV/VIS spectroscopy. Complex 1 EPR: not diluted powder-single asymmetric peak; dilute powder (5 % in (C5H5)2TiCl2) Ax = 78.6 G, Ay = 125.9 G, gx = 1.983, gy = 1.971; frozen CHCl3 solution Ax = 78.5 G, Ay = 125.9 G, gx = 1.985, gy = 1.971. Complex 2 EPR: not diluted powder - single asymmetric peak; dilute powder (5 % in (CH3C5H4)2TiCl2) Ax = 78.7 G,Ay = 126.0 G, gx = 1.980, gy = 1.969; frozen CHCl3 solution Ax = 79.0 G, Ay = 125.7 G, gx = 1.981, gy = 1.972.

4.2 Antimicrobial studies

Escherichia coli strain B was used for all microbiological experiments. These bacteria were grown aerobically on a blood agar at 37˚C/24 hour. From this culture a suspension was prepared according to McFarland (2nd degree) in a M9 broth (15.40 g Na2HPO4.12 H2O, 3.00 g KH2PO4,5.00gNaCl,1.00gNH4Cl, 0.25 g MgSO4.7H2O, 0.11 g CaCl2, 8.00 g glucose and 10.00 g casamino acids as carbon and amino source per litre of distilled water; pH=7.4) [19] and it was incubated for 18 h at 37˚C. The appropriate amounts of 1, 2, 4, 5, 7, 8 (c = 9 mmol/l) were dissolved in 2 ml M9 and these working solutions were then diluted with sterile media M9. Then a 100 µl bacterial suspension was added to each tube of the concentration line. After incubation (37˚C/24 h), the native preparations from each tube and cells were counted at a magnification of 1000x with a phase-contrast microscope. The filamentous growth was described subjectively and categorized by esti- mating the relative percentage of filamentous cells. The relative length of the filaments is also estimated by comparing them to a control of normal size cells. 78 J. Vinklárek et al. / Central European Journal of Chemistry 3(1) 2005 72–81

4.3 Spectral measurements

EPR spectra were measured on ERS 221 (ZWG Berlin) apparatus in microwave X-band (∼ 9.5 GHz). The apparatus was gauged on DPPH value (g = 2.0036 ± 2) [25]. Solid samples were measured in quartz capillaries (width 0.5 mm) at room temperature. The EPR spectra obtained were computer simulated using EPR simulation software SimFonia v.1.2 (Bruker). Second-order perturbation theory for interaction of unpaired electronic spin with vanadium nuclear spin anisotropic linewidths and mixed Lorentzian/Gaussian lineshapes were used. IR spectra were recorded at 4000 - 350 cm−1 region on a Perkin-Elmer 684 using Nujol mulls between KBr windows. Raman spectra were recorded on a Bruker IFS 55 s with extension FRA 106 at 50 - 3500 cm−1.

4.4 Synthesis of compounds 3-8

All compounds (3-8) were prepared as follows. 1.98 mmol of 1 (or of 2) was dissolved in 10 ml water. Then added gradually an equimolar amount of the appropriate cyclodextrine (α-CD, β-CD or γ-CD). The solution was stirred for ten minutes, ﬁltered and the solvent was evaporated to dryness.

α-CD-(C 5H5)2VCl 2 · 9H2O(3 ) Yield: 79.5 %; Calc. for C46H90Cl2O39V (MW 1386.86): C 39.80, H 6.49, H2O 11.68; Anal. Found: C 40.00, H 6.50, H2O 11.70; Raman: 3376m, 2921w, 1136m, 295m; IR: 3424s, 2721w, 2668w, 2588w, 1754w, 1649s, 1375vs, 1372s, 1328m, 1292w, 1239w, 1195w, 1152vs, 1076s, 1025vs, 999s, 949m, 938w, 834m, 752w, 720m, 601w, 573m, 538w. EPR: powder, single asymmetric peak.

β-CD-(C 5H5)2VCl 2 · 10 H 2O(4 ) Yield: 83.7 %; Calc. for C52H102Cl2O45V (MW 1567): C 39.82, H 6.51, H2O 11.49; Anal. Found: C 40.00, H 6.50. H2O 11,50; EPR: powder Ax = 79.5 G, Ay = 125.9 G; gx = 1.982, gy = 1.971; Raman: 3369w, 2921m, 1136m, 307m; IR: 3339s, 2352w, 1634m, 1388vs, 1152s, 1078vs, 1054vs, 1038vs, 940m, 833w, 728m, 605w, 531w, 408w.

γ-CD-(C 5H5)2VCl 2 · 11 H 2O(5 ) Yield: 90.0 %; Calc. for C58H114Cl2O51V (MW 1747.14): C 39.84, H 6.52, H2O 11.33; Anal. Found: C 39.90, H 6.50, H2O 11.30; EPR: powder Ax = 80.0 G, Ay = 125.9 G; gx = 1.986, gy = 1.971; Raman: 3319m, 2922w, 1132w, 298m; IR: 3543w, 1634m, 1389vs, 1258m, 1152s, 1078vs, 1030vs, 931w, 833w, 760w, 719m, 614w, 596w, 531w, 441w.

α-CD-(CH 3C5H4)2VCl 2 · 8H2O(6 ) Yield: 89.2 %; Calc. for C48H90Cl2O38V (MW 1396.86): C 41.24, H 6.44, H2O 10.31; Anal.Found: C 41.20, H 6.50, H2O 10.30; Raman: 3166m, 2921w, 301m; IR: 3545w, 2729w, 2668w, 2348w, 1636s, 1496m, 1375vs, 1337s, 1310m, 1267m, 1213w, 1152vs, 1025vs, 949m, 938w, 854m, 831w, 796m, 760w, 739m, 701m, 605m, 580s, 523m, 484w, 453w, 402w. EPR: powder, single asymmetric peak.

β-CD-(CH 3C5H4)2VCl 2 · 10 H 2O(7 ) Yield: 91.7 %; Calc. for C54H104Cl2O45V (MW 1595): C 40.63, H 6.52, H2O 11.29; Anal.Found: C 40.50, H 6.50, H2O 11.30; EPR: J. Vinkl´arek et al. / Central European Journal of Chemistry 3(1) 2005 72–81 79

powder Ax = 79.2 G, Ay = 125.9 G; gx = 1.982, gy = 1.971; Raman: 3107w, 2924w, 297m; IR: 3436s, 2730w, 2676w, 1825w, 1649m, 1629w, 1501w, 1375vs, 1343m, 1301w, 1256w, 1248w, 1203w, 1159s, 1097m, 1076vs, 1053vs, 1031vs, 999s, 936m, 854w, 757m, 726m, 707m, 605m, 580m, 535w, 453w.

γ-CD-(CH 3C5H4)2VCl 2 · 9H2O(8 ) Yield: 85.0 %; Calc. for C60H112Cl2O49V (MW 1739.14): C 41.40, H 6.44, H2O 9.31; EPR: powder Ax = 82.0 G, Ay = 125.6 G; gx = 1.988, gy = 1.971; Raman: 3109w, 2915m, 294m; IR: 3443s, 2730w, 2676w, 1649m, 1638m, 1488m, 1375vs, 1343s, 1301m, 1248w, 1152s, 1106s, 1076vs, 1025vs, 999vs, 936m, 848w, 758w, 706w, 701m, 600m, 580m, 529w, 440w.

Acknowledgement

This work was supported by grant from the Ministry of Education of the Czech Republic (No. 340003).

References

[1] V.J. Moebus, R. Stein, D.G. Kieback, I.B. Runnebaum, G. Sass and R. Kreienberg: ”Antitumor activity of new organometallic compounds in human ovarian cancer cell lines and comparison to platin derivatives”, Anticancer Research, Vol. 17, (1997), pp. 815–821. [2] G. Lummen,¨ H. Sperling, H. Luboldt, T. Otto and H. Rubben:¨ ”Phase II trial of titanocene dichloride in advanced renal-cell carcinoma”, Cancer Chemother. Pharmacol., Vol. 42, (1998), pp. 415–417. [3] P. Köpf-Maier: ”Antitumor activity of titanocene dichloride in xenografted human renal-cell carcinoma”, Anticancer Research, Vol. 19, (1999), pp. 493–504. [4] N. Kroger, U.R. Kleeberg, K. Mross, L. Edler, G. Sass and D.K. Hossfeld: ”Phase II clinical trial of titanocene dichloride in patients with metastatic breast cancer”, Onkologie, Vol. 23, (2000), pp. 60–62. [5] P. Köpf-Maier and H. Köpf: ”Non-Platinum-Group Metal Antitumor Agents: History, Current Status, and Perspectives”, Chem. Rev., Vol. 87, (1987), pp. 1137–1152. [6] A. Harada and S. Takahashi: ”Preparation and Properties of Cyclodextrin - Ferrocene Inclusion Complexes”, J. Chem. Soc.-Chem. Commun., (1984), pp. 645–646. [7] A. Harada, Y. Hu, S. Yamamoto and S. Takahashi: ”Preparation and Properties of Inclusion-Compounds of Ferrocene and Its Derivatives with Cyclodextrins”, J. Chem. Soc.-Dalton Trans., (1988), pp. 729–732. [8] Y. Odagaki, K. Hirotsu, T. Higuchi, A. Harada and S. Takahashi: ”X-Ray Structure of the α-Cyclodextrin Ferrocene (2-1) Inclusion Compound”, J. Chem. Soc.-Perkin Trans. 1, (1990), pp. 1230–1231. [9] P. Ferreira, I.S. Goncalves, M. Pillinger, J. Rocha, P. Santos and J.J. C. Teixeira- Dias: ”Modification of β-cyclodextrin with ferrocenyl groups by ring opening of an encapsulated 1 ferrocenophane”, Organometallics, Vol. 19, (2000), pp. 1455–1457. 80 J. Vinklárek et al. / Central European Journal of Chemistry 3(1) 2005 72–81

[10] S.S. Braga, I.S. Goncalves, A.D. Lopes, M. Pillinger, J. Rocha, C.C. Romao and J.J.C. Teixeira-Dias: ”Encapsulation of half-sandwich complexes of molybdenum with beta-cyclodextrin”, J. Chem. Soc.-Dalton Trans., (2000), pp. 2964–2968. [11] S. Lima, I.S. Goncalves, P. Ribeiro-Claro, M. Pillinger, A.D. Lopes, P. Ferreira, J.J.C. Teixeira-Dias, J. Rocha and C.C. Romao: ”Interactions of cationic and neutral molybdenum complexes with β-cyclodextrin host molecules”, Organometallics,Vol. 20, (2001), pp. 2191–2197. [12] I. Turel, A. Demsar and J. Kosmrlj: ”The interactions of titanocene dihalides with alpha-, beta- and gamma-cyclodextrin host molecules”, J. Incl. Phenom. Macrocycl. Chem., Vol. 35, (1999), pp. 595–604. [13] C.S. Lu, Y. Zou, J.L. Xie, Z.P. Ni, H.Z. Zhu, Q.J. Meng and Y.G. Yao: ”An antitumor agent of β-cyclodextrin-modified titanocene complex: Synthesis and characterization”, Chem. Pharm. Bull., Vol. 51, (2003), pp. 864–866. [14] S.S. Braga, I.S. Gon¸calves, M. Pillinger, P. Ribeiro-Claro and J.J.C. Teixeira-Dias: ”Experimental and theoretical study of the interaction of molybdenocene dichloride (Cp2MoCl2)withβ-cyclodextrin”, J. Organomet. Chem., Vol. 632, (2001), pp. 11–16. [15] E. Fenyvesi, L. Szente, N.R. Russell and M. McNamara: Comprehensive Supramolecular Chemistry, J.L. Atwood, J.E.D. Davies, D.D. MacNicol, F. Vögtle, J.M. Lehn, J. Szejtli and T. Osa (Eds.), Pergamon, Oxford, 1996, Vol. 3, chap. 10. [16] I. Pavl´ık and J. Vinklárek: ”Hydrolysis Behavior of the Antitumor Agent Vanadocene Dichloride in Strongly Acid, Neutral and Alkaline Aqueous-Media - an EPR Study”, Eur. J. Solid State Inorg. Chem., Vol. 28, (1991), pp. 815–827. [17] J. Honz´ıˇcek, P. Nachtigall, I. C´ısaˇrova and J. Vinklárek: ”Synthesis, characterization and structural investigation of the first vanadocene(IV) carboxylic acid complexes prepared from the vanadocene dichloride”, J. Organomet. Chem., Vol. 689, (2004), pp. 1180–1187. [18] B. Rosenberg and L. van Camp: ”The inhibition of growth and cell division in Escherichia coli)”, J. Biol. Chem., Vol. 242, (1967), pp. 1347–1352. [19] I.C. Tornieporth-Oetting and P.S. White: ”Ionic Titanocene(IV) α-Amino- Acid Complexes of Dl- Phenylalanine and Dl-4-Fluorophenylalanine - Synthesis, Characterization, and Investigation of the Antimicrobial Behavior toward Escherichia-Coli”, Organometallics, Vol. 14, (1995), pp. 1632–1636. [20] E. Diana, R. Rossetti, P.L. Stanghellini and S.F.A. Kettle: ”Vibrational study of (η5-cyclopentadienyl) metal complexes”, Inorg. Chem., Vol. 36, (1997), pp. 382–391. [21] J. Petersen and F. Dahl: ”Synthesis and Structural Characterization by X-Ray Diffraction and Electron Paramagnetic Resonance Single-Crystal Techniques of V(η5- 5 C5H4CH3)2Cl2 and Ti(η -C5H4CH3)2Cl2. A Study of the Distribution of the Unpaired 5 Electron in a V(η -C5H5)2L2-Type Complex”, J. Am. Chem. Soc., Vol. 97, (1975), pp. 6422–6233. [22] P. Gowik and T. Klapötke: ”Why Do Organometallic Hexafluoropnicogenate Complexes Show Biocidal Activity - a Study to the Structure Activity Correlation”, Monatshefte Fur¨ Chemie, Vol. 120, (1989), pp. 711–714. [23] K. Mross, P. Robben-Bathe, L. Edler, J. Baumgart, W.E. Berdel, H. Fiebig and C. Unger: ”Phase I clinical trial of a day-1,-3,-5 every 3 weeks schedule with titanocene dichloride (MKT 5) in patients with advanced cancer - A study of the phase I study group of the Association for Medical Oncology (AIO) of the German Cancer Society”, Onkologie, Vol. 23, (2000), pp. 576–579. J. Vinklárek et al. / Central European Journal of Chemistry 3(1) 2005 72–81 81

[24] G. Wilkinson and J.M. Birmingham: ”Bis-Cyclopentadienyl Compounds of Ti, Zr, V, Nb and Ta”, J. Am. Chem. Soc., Vol. 76, (1954), p. 4281. [25] J. Krzystek, A. Sienkiewicz, L. Pardi and L.C. Brunel: ”DPPH as a Standard for High-Field EPR”, J. Magn. Reson., Vol. 125, (1997), pp. 207–211.