Synthesis , Electrochemistry and Antitumor Activity of 1 ’ H , 3 ’ H ( Me )-spiro-[ ( aza ) benzimidazoline-2 ’ , 3-( 1 , 2-diferrocenylcyclopropenes ) ] , 2-( 1 , 2-Diferrocenylvinyl ) benz-and Azabenzimidazoles

A new method of synthesis of 2-(1,2-diferrocenylvinyl)benzand azabenzimidazoles (3a-f), (4a-f) and 1’H,3’H(Me)-spiro-[(aza)benzimidazoline-2’,3-(1,2-diferrocenylcyclopropenes)] (5a-f) via reactions of diferrocenyl(methylsulfanyl)cyclopropenylium iodide (1) with aromatic o-diamines (2a-f) in the presence of Et3N (80 ̊C 82 ̊C) is described. The structures of the resultant compounds are established using IR, 1H and 13C NMR spectroscopy, mass spectrometry and elemental analysis. The structure of one compound, cis-2-(1,2-diferrocenylvinyl)-1-methylbenzimidazole (3b), is confirmed by X-ray diffraction analysis. The electrochemical properties of compounds 3a, 3b, 3d and 5f are investigated using cyclic square wave voltammetry. Two electrochemical processes (I-II), attributed to oxidation of the ferrocene moieties, and the values of E0’(I), E0’(II), ∆E0’(II-I) and comporportionation constant Kcom are reported. The bioactivities of seven compounds 3a, 3c-f, 5d, 5f are evaluated. Compound 5f is the most active compound with a modest cytotoxic activity against six human cancer cell lines: U-251 (glioma), PC-3 (prostate cancer), K-562 (leukemia), HCT-15 (colon cancer), MCF-7 (breast cancer) and SKLU-1 (lung cancer). Corresponding author. J. J. Sánchez García et al.


Introduction
Diaryl-and diferrocenylcyclopropenilium cations with dialkylamino and methylsulfanyl groups in the small cycle are successfully used in organic synthesis as diferrocenyl-substituted three-carbon building blocks [1]- [7].Reactions of such cations with carbon and nitrogen nucleophiles, which proceed via the opening of the threemembered cycle and the formation of diferrocenylvinylcarbenes, have been described [1]- [13].On the basis of intramolecular transformations of such carbenes, researchers have developed new methods of synthesizing fiveand six-membered carbo-and heterocycles, polyene compounds with two ferrocene substituents and functional groups in the molecules [4]- [13].The interaction of diferrocenylcyclopropenylium salts with 1,2-diamines has scarcely been studied.The first study concerning the use of 2,3-diferrocenyl-1-methylsulfaylcyclopropeny-lium iodide in reactions with aliphatic 1,2-diamines for the synthesis of 2-(1,2-diferrocenylvinyl)imidazoline and imidazolidine derivatives has recently been published [13].Relatively stable spiro-(imidazolidine)-2',3-(1,2-diferrocenylcyclopropenes) were first isolated at the same time [13].It is known that researchers pay much attention to the synthesis of new derivatives of mono-and polycyclic imidazoles, since the imidazole ring is an important structural element in many natural compounds, alkaloids, proteins, herbicides, vitamins, medications, etc. [14]- [21].
Ferrocene-substituted nitrogen heterocycles are of special interest in search for bioactive substances [22]- [28].Investigations of interactions between diferrocenylcyclopropenylium cations and aromatic 1,2-diamines must apparently lead to the synthesis of new diferrocenyl-substituted polycyclic imidazole derivatives, which is of interest with regard to the search for practical applications of the novel compounds in pharmacology, electrochemistry, etc.

Instruments
All the solvents were dried according to the standard procedures and freshly distilled before use [29].Column chromatography was carried out on alumina (Brockmann activity III); TLC, on silica gel.The 1 H and 13 C NMR spectra were recorded on a Unity Inova Varian spectrometer (300 and 75 MHz) for solutions in CDCl 3 , with Me 4 Si as the internal standard.The IR spectra were measured on an FT-IR spectrophotometer (Spectrum RXI Perkin Elmer instruments) using KBr pellets.The mass spectra were obtained on a Varian MAT CH-6 instrument (EI MS, 70 eV).Elementar Analysensysteme LECO CHNS-900 was used for elemental analyses.All the electrochemical measurements were performed at sample concentrations of about 5 × 10 −4 M in the acetonitrile solution in the presence of 0.1 M tetra-N-butylammonium hexafluorophosphate (TBAPF 6 ) using a Biologic SP-50 potentiostat/galvanostat.A typical three-electrode array was employed: a platinum disk as the working electrode, a platinum wire as the counter electrode, and a pseudo-reference electrode of silver.All the solutions were bubbled with nitrogen prior to each measurement.The cyclic wave voltammetry experiments were initiated from the open circuit potential (E ocp ) in the positive direction, using the scan rates from 0.1 to 2 Vs −1 .The current interrupt method was used for iR compensation.All the potentials were reported versus the Fc/Fc + couple according to the IUPAC convention [30].The unit cell parameters and the X-ray diffraction intensities for 3b were recorded on a Gemini diffractometer (detector Atlas CCD, Cryojet N 2 ).The crystallographic data, the parame-ters of the X-ray diffraction experiments, and the refinements are listed in Table 1.The structure of compound 3b was solved by the direct method (SHELXS-97 [31]) and refined using the full-matrix least-squares on F 2 .

Determination of the Crystal Structure
The unit cell parameters and the X-ray diffraction intensities were recorded on a on a Gemini diffractometer (detector Atlas CCD, Cryojet N 2 ).The structure of compound 3b was solved by the direct method (SHELXS-97 [31]) and refined using full-matrix least-squares on F 2 .Crystal data for C 30 H 26 CCDC 981930 contains the supplementary crystallographic data for this paper (compound 3b).These data can be obtained free of charge at www.ccdc.cam.ac.uk/const/retrieving.html [or from the Cambridge Crystallographic Data Centre, 12, Union Road, Cambridge DB2 1EZ, UK; fax: (internet.)+44 1223/336 033; E-mail: deposit@ccdc.cam.ac.uk].

Cytotoxicity Assay
The compounds were screened in vitro against human cancer cell lines HCT-15 (human colorectal adenocarcinoma), MCF-7 (human mammary adenocarcinoma), K562 (human chronic myelogenous leukemia), U251 (human glioblastoma), PC-3 (human prostatic adenocarcinoma), SKLU-1 (human lung adenocarcinoma).The cell lines were supplied by the National Cancer Institute (USA).The human tumor cytotoxicity was determined using the protein-binding dye sulforhodamine B (SRB) in the microculture assay to measure the cell growth, as is described in the protocols established by the NCI [32] [33].The cell lines were cultured in the RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10,000 units/ml penicillin G sodium, 10 µg/ml streptomycin sulfate, 25 µg/ml amphotericin B (Gibco) and 1% non-essential amino acids (Gibco).The cultures were maintained at 37˚C in a humidified 5% CO 2 atmosphere.As was determined using trypan blue, the viability of the cells used in the experiments exceeded 95%.The cells were removed from the tissue culture flasks by treatment with trypsin and diluted with fresh media.100-ml cell suspension aliquots, containing 5000 -10,000 cells per well, were transferred to 96-well microtiter plates (Costar) and incubated at 37˚C for 24 h in a 5% CO 2 atmosphere.
Stock solutions of the test compounds initially dissolved in DMSO (20 mM) were prepared and further diluted in the medium to produce the desired concentrations.100-ml aliquots of the diluted solutions of the test compounds were added to each well.The cultures were exposed to the compound at concentrations 50 µM for 48 h.After the incubation period, the cells were fixed to a plastic substratum by the addition of 50 µl of cold 50% aqueous trichloroacetic acid.The plates were incubated at 4˚C for 1 h, washed with tap H 2 O, and air-dried.The cells fixed with trichloroaceticacid were stained by the addition of 0.4% SRB.Free SRB solution was removed by washing with 1% aqueous acid acetic.The plates were air-dried, and the bound dye was solubilized by the addition of 100 µL of 10 mM unbuffered tris base.The plates were placed on a shaker for 5 min prior to analysis.The optical densities were determined using an Ultra Microplated Reader (El x 808: Bio-Tek Instruments, Inc., Winooski, VT, USA) at a test wavelength of 515 nm.
The products of each reaction were separated using Al 2 O 3 (activity grade III) column chromatography.The structures of the compounds were established using IR, 1 H and 13 C NMR spectroscopy, mass spectrometry and elemental analysis.For example, the 1 H NMR spectra of compounds 3a-c and 4a-c contain the characteristic signals from protons of the ferrocenyl, aryl, and methyl groups, as well as the singlets of protons from the -NH and =CH fragments.An important feature of the 1 H NMR spectra of compounds 3a-c is the fact that they contain signals from the hydrogen atom of the CH = fragment in a stronger field (δ = 7.93, 6.56, 8.12) than the corresponding signals in compounds 4a-c (δ = 8. 16, 6.71, 8.26).
The benzimidazole structure is additionally confirmed by the fact that the 13 C NMR spectra contain the corresponding numbers of signals from the quaternary carbons, as well as from the CH=, CH 3 , C 6 H 4 , C 6 H 3 , C 6 H 5 , and 2 Fc groups.
To establish the geometrical configuration of compounds 3a-c, we performed X-ray diffraction analysis of single crystals of 3b, which were isolated via crystallization from CH 2 Cl 2 .The general form of molecule 3b is shown in Figure 1, and the main geometrical parameters of compound 3b are listed in Table 1.
The X-ray findings show that the structure of 3b is that of cis-2-(1,2-diferrocenylvinyl)-1-methylbenzimidazole.By analogy with it, compounds 3a and 3c were also regarded as having the cis-configuration.Hence, compounds 4a-c contain the 1,2-diferrocenylvinyl fragment with the trans-orientation of the ferrocenyl groups.A characteristic feature of the crystal structure of 3b is the presente of two molecules in the unit cell differing in the orientation of the four ferrocenyl substituents.Scheme 1. Reaction of 2,3-diferrocenyl-1-methylsulfanil-cyclopropenylium iodide 1 with aromatic carbo-and heterocyclic diamines 2a-f.
The structures of compounds 5a-c were established using IR, 1 H and 13 C NMR spectroscopy, mass spectrometry, and elemental analysis.For example, the 1 H NMR spectrum of compound 5a characterizes it as a molecule with a symmetrical structure, whose spectrum contains one signal from the protons of two C 5 H 5 rings and two signals from the protons of two C 5 H 4 fragments of two ferrocene sandwiches, as well as two signals from the protons of the o-phenylene ring and one singlet from the protons of two NH groups.The 13 C NMR spectrum of compound 5a contains one signal from the two C ipso Fc carbons (δ = 66.74), one from the quaternary C spiro carbon atom (δ = 65.15), and two signals from four C ipso carbon atoms (δ = 130.03, 137.05).The data of the 1 H and 13 C NMR spectra of compounds 5b and 5c are provided in the Experimental section.The IR spectra of compounds 5a-c contain bands at 1828 -1835 cm −1 , which are characteristic of the cyclopropenyl group.Taken as a whole, these spectral data confirm the structures of compounds 5a-c to be those of 1'H,3'H(or Me)-spiro[benzimidazoline-2',3-(1,2-diferrocenylcyclopropenes)].
Spiranes 5a-c are fine crystals of red color, stable upon storage at room temperature (20˚C -25˚C) in the inert atmosphere.At an elevated temperature or upon exposure to air, they decompose via the opening of the threecarbon cyclopropene ring and form a mixture of benzimidazoles 3a-c and 4a-c, with the amount of the transisomer 4a-c in the mixture increasing over time.
On the basis of these preliminary data, we studied the behavior of compounds 3a-c, 4a-c, and 5a-c upon heating.It turned out that boiling of spiroimidazolines 5a-c in benzene (~3 -4 h) leads to the formation of a mixture of cis-3a-c and trans-4a-c benzimidazoles (yield ~85%, ~2:1); subsequent boiling of the resultant mixture in toluene yields the pure trans isomer 4a-c (~71%).Trans-4a-c are also formed as a result of direct boiling of spiranes 5a-c in toluene (~4 -5 h, ~77%) (Scheme 2).
One of the nitrogen atoms of 1,2-diamines 2a-f first attacks atom C-1 of 2,3-diferrocenyl-1 methylsulfanylcyclopropenylium iodide 1 and substitutes the MeS group with the formation of 1-aminocyclopropenylium cations 6a-f.The repeated attack of another nitrogen atom on the C-1 carbon atom in cations 6a-f yields the spirane benz-and azabenzimidazoles 5a-f.The opening of the small cycle in the cyclopropene fragments of spi- ranes 5a-f with the formation of vinylcarbene intermediates 7a-f and subsequent intramolecular transformation of the carbenes lead to the formation of bicyclic imidazoles 3a-f and 4a-f.

Electrochemistry
Figure 2 shows the typical cyclic voltammogram of compound 3a.One can observe two oxidation signals (I a and II a ) with the corresponding reduction signals (I c and II c ).For process I, the resultant anodic and cathodic peak potentials E pa (I) and E pc (I) were −0.033V/Fc-Fc + and 0.0259 V/Fc-Fc + , respectively.On the other hand, the corresponding potentials E pa (II) and E pc (II) for process II were 0.130 and 0.189 V/Fc-Fc + .For both processes, a ΔE p value of 0.059 V was estimated, without any dependence on the scan rate.The peak current values I p adhered to a linear relationship with v 1/2 .Therefore, two-step reversible oxidation of the ferrocene moieties for processes I and II was suggested.The formal electrode potential was evaluated as the half-sum of the anodic and cathodic peak potentials, E 0' = (E pa + E pc )/2 [30].Its values for processes I and II were E 0' (I) = 0.003 V/Fc-Fc + and E 0' (II) = 0.159 V/Fc-Fc + .These values enabled us to estimate the comproportionation constant K com = 4.32 × 10 2 [34]- [36].The electrochemical response of compounds 3b and 3d is very similar to that observed for compound 3a, although there are slight changes in the values of the formal electrode potentials for processes I and II.In the case of compound 5f, a different electrochemical response was observed.
The typical cyclic voltammogram of compound 5f presents two oxidation signals (I a and I * ) and one reduction process (I c ), see Figure 3.When the scan rate was increased, an increase in the current values was detected for all the signals.A linear relationship between I p and v 1/2 , characteristic for reversible electrochemical processes, was observed for I a and I c .These results, as well as the absence of the corresponding reduction signal for I * , indicate that I * is associated with an adsorption-desorption process; meanwhile, I a and I c are attributed to the electron transfer for ferrocene moieties.The difference between the potential peak values ΔE p = 0.10 V and the for-  mal electrode potential E 0' (I) = 0.426 V/Fc-Fc + were calculated.According to literature, the difference between the formal electrode potentials ∆E(II-I) = 0.073 V was evaluated for the E r E r mechanism using the working curve ΔE p vs. ∆E [33] [34].On the basis of this value, it was possible to calculate the formal electrode potential for process II: E 0' (II) = 0.353 V/Fc-Fc + .Table 2 summarizes the electrochemical behaviour of all the compounds studied in this work.
The estimated values of K com for all the compounds suggest that the electronic charge is slightly delocalized in the electrochemically generated mixed valence, according to the Robin-Day classification [35] [36].For compound 5f, the lowest electronic communication in the electrochemically generated mixed-valence species was observed.It can be noticed that the presence of different substituents around each ferrocene moiety in compounds 3a, 3b and 3d makes a considerable contribution to each formal electrode potential, and therefore the K com values are increased because of this effect.

Pharmacology
In order to examine the applicability of two types of compounds (3a, 3c-f and 5d, 5f) as antitumor agents, they were tested in vitro against six human tumor cell lines: U-251 (glioma), PC-3 (prostate cancer), K-562 (leukemia), HCT-15 (colon cancer), MCF-7 (breast cancer) and SKLU-1 (lung cancer).Primary screening at a fixed concentration showed cytotoxicity against the six tested human tumor cell lines and besides against human lymphocytes (MT2).Cisplatin at the same concentration was used as the positive control.The compounds were used as 50 µM solutions in DMSO (Table 3).Compound 5f showed 100% inhibition of cell growth at 50 µM for five human tumor cell lines, 86.8% inhibition for SKLU-1, and also 90.8% inhibition for human lymphocytes (MT2).Compound 3f showed higher activity than cisplatin for U-251 and MCF-7 (Table 3).

Figure 2 .
Figure 2. Cyclic voltammogram obtained for 3a in the presence of 0.1 M TBAPF6 in CH3CN.Scan rate 0.1 V/s, platinum working electrode.

Figure 3 .
Figure 3. Cyclic voltammogram obtained for 5f in the presence of 0.1 M TBAPF6 in CH3CN.Scan rate 0.1 V/s, platinum working electrode.

Table 3 .
Inhibition of the growth (%) of human tumor cell lines and human lymphocytes (MT2) cell for compounds 3a, b, c-f, 5d, f at 50 µM in DMSO.
NA: not active.