Symmetric 1,1'-Dimethylferrocene-Derived Amino Acids: Their Synthesis, Characterization, Ligational and Biological Properties With Cu(II), Co(II) and Ni(II) Ions

Some novel symmetric 1,1′-dimethylferrocene derived amino acids have been prepared by the reaction of 1,1′-ferrocenedimethyldichloride with amino acids (glycine, alanine, phenylalanine and tyrosine). Their Cu(II), Co(II) and Ni(II) complexes, of the type [M(L)] where [M = Cu(II) and L = L1-L5] and [M(L)Cl2] where [M-Co(II)and Ni(II), L = L1-L5] have been prepared. The dicarboxylic acids and their metal complexes were characterized by their physical, analytical and spectral data. The [M(L)] complexes showed a square planar geometry whereas an octahedral geometry was observed for [M(L)Cl2] complexes. The title dicarboxylic acids and their metal complexes have also been screened for their antibacterial activity.


INTRODUCTION
There are significant evidences 1-4 that amino acid complexes are potentially used in the treatment of tumors. Various tumors tend to have poor blood supplies and therefore, amino acids have been effectively used to direct nitrogen mustards into the cancer cells. For example, phenylalanine mustard is used in controlling malignant myeloma and Burkitts' lymphoma and, similarly sarcolysine is used to treat wide range of tumors. Indeed, certain tumors and cancer cells are unable to produce all the amino acids synthesized by the normal cells. Therefore, these cells require an external supply of such essential amino acids to pass on to the cancer cells by the blood stream. In the recent past a number of studies 82 have highlighted the utility of ferrocene and its derivatives in various applications 137. Very few ferrocene-derived compounds have been used as ligands for the complex formation reactions. Keeping in view the significance of amino acids and their complexes as chemotheraptic agent and the chemistry of ferrocene or ferrocene-containing compounds as stable intermediates, a successful effort to join the chemistry of amino acids and ferrocene is made. For this purpose, some novel symmetric 1,1'-dimethylferrocene derived amino acids (Figure 1) have been synthesized and studied for their physicochemical, ligational behavior with Cu(II), Co(II) and Ni(II) metal ions and also for their antibacterial properties against bacterial strains, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Material and Methods All solvents were used as Analar grade. 1, l'-Ferrocenedimethanol was obtained from Merck. l,l'-Ferrocenedimethyldichloride derivative was prepared by a reported method 8 using thionyl chloride in triethylamine. All metals were used as chlorides. IR, 1H NMR and 13C NMR spectra were recorded on Philips Analytical PU 9800 FTIR and Brucker 250 MHz instruments. UV-Visible spectra were obtained on a Hitachi U-2)00 double-beam spectrophotometer. Conductance of the metal complexes was determined in DMF at 10"dilution on a YSI-32 model conductometer. Magnetic measurements were done on solid complexes using the Gouy method. The synthesized dicarboxylic acids and their metal complexes were analyzed for C, H and N by Butterworth Laboratories Ltd. Melting points were recorded on a Gallenkamp apparatus and are uncorrected. Synthesis of Dicarboxylic acids A mixture of l,l'-ferrocenedimethanol (0.65 g, 3.0 mmol), triethylamine (0.60 g, 6.0 mmol) and dichloromethane (20 mL) was cooled in an ice bath. Thionyl chloride (0.71 g, 6.0 mmol) in dichloromethane Symmetric 1,1-Dimethylferrocene-Derived Amino Acids: Their Synthesis, Characterization, Ligational and Biological Properties with Cu(II), Co(II) and Ni(II) Ions (20 mL) was added into this mixture under N2 at such a rate to keep the temperature between 15-20 C. After complete addition, the reaction mixture was kept at 20 C for 30 minutes and then stirred at 40 C for another 30 minutes. Ice was added and mixture stirred for another 5 minutes. A small amount of NaHCO3 was then added to obtain pH 6.0. The organic layer was separated and dried over CaClz. Filtration and evaporation of the solvent gave a dark brown solid which was dissolved in dichloromethane (20 mL) and each amino acid (glycine, alanine, phenylalanine or tyrosine) (1.25 mmol) in dichloromethane (20 mL) was individually added to it. The reaction mixture was refluxed for 5 h under a slow stream of N2. After allowing to cool to room temperature solvent was evaporated to give a yellow-orange solid which was recrystallized from chloroform.

Synthesis of Metal Complexes
Dicarboxylic acid (1.0 mmol) was dissolved in ethanol (30 mL) and warmed for several minutes. A solution of metal (II) chloride (1.0 mmol) in ethanol (20 mL) was added to the above solution. Then 2-3 drops of conc H2SO4 were added and mixture was refluxed for 3 h. During this time, precipitate was formed that was filtered, washed several times with warm ethanol and diethyl ether and, then dried over anhydrous CaCI2.

Antibacterial Studies
The synthesized metal chelates in comparison to the ligands were screened for their antibacterial activity against pathogenic bacterial strains, Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa and Klebsiella pneumonae. The paper disc diffusion method was adopted for the determination of antibacterial activity19'2.  IR Spectra

RESULTS AND DISCUSSION
The important infrared frequencies of the uncomplexed dicarboxylic acids and its complexes along with their assignments are given in Tables and 3, respectively. The IR spectra of the dicarboxylic acids show characteristic absorption bands at~3372,~1822 and~1550 cm due to the v(NH), v(COOH) and v(C=C) stretching vibrations 22 respectively. The bonding of the dicarboxylic acids to the metal atoms was investigated by comparing IR spectra of the free dicarboxylic acids with those of their metal complexes. The spectra of the complexes show significant changes as compared to that of the dicarboxylic acids. It can be seen that the bands due to the v(NH) move towards lower frequency by 5-10 cm indicating their coordination to the metal atoms through the v(NH) group. Deprotonation of the v(COOH) was also indicated in the spectra of the complexes as the band due to the v(COO) was observed at~1575 cm which in turn, showed complexation through a deprotonated (COOH) group. Moreover, in the far IR region, three new bands around 365, 415, 450 cm 1 assigned to the v(M-CI), v(M-N) and v(M-O) modes 23 respectively were found in the spectra of the Co(lI) and Ni(II) complexes and not in the spectra of the dicarboxylic acids. The stretches due to the v(M-N) and v(M-O) were only found in the spectra of the Cu(II) complexes, however, bands due to the v(M-CI) in the far IR region were not found in the spectra of the Cu(II) complexes which indicated that the Co(II) and Ni(II) complexes possess an octahedral and the Cu(II) complexes a square planar geometry.

NMR Spectra
The H NMR and 3C NMR spectra of the free dicarboxylic acids as well as some of their metal complexes, taken in DMSO-d6 are listed in Table 4. The free dicarboxylic acids exhibited signals due to all the expected protons and carbons in their expected region and have been identified from the integration curve found to be equivalent to the total number of protons deduced from their proposed structures. These were identical to those reported 24"28 signals of the known compounds and therefore, gave further support for the compositions of these new dicarboxylic acids and their complexes as suggested by their IR and elemental analyses data. When these shifts were compared to those of the corresponding complexes, they exhibited a shift of some resonances. In each case, a broad singlet occurring downfield at 5 8.7-8.9 ppm assigned to (NH) undergoes a shift towards higher field by 0.15-0.2 ppm in the complexes. Also, the protons due to (COOH) found in the spectra of the dicarboxylic acids at 5 11.7-11.9 ppm suggested 29 Table 4. The spectra of other metal complexes showed similar characteristic features except the shift (0.5-1.5 ppm) of signals and therefore, are not included in Table 4.

Electronic Spectra and Magnetic Moments
The electronic spectra of the Cu(II) complexes showed two weak low-energy bands at 15150-16355 cm and 18770-19585 cm and a strong high-energy band at 30345-31770 cm. The low-energy bands are in positions characteristic for a square planar configuration and may be assigned to 2Big 2Ag and 2Blg -4 2Eg transitions, respectively3'3. The strong high-energy band is assigned to metal -4 ligand charge transfer.
Also, the magnetic moment values (1.5-1.9 B.M) for the Cu(II) complexes were found to be consistent with the proposed square planar structure (Fig 2A).
Characterization, Ligational and Biological Properties with Cu(II), Co(II) and Ni(II) Ions Table 3 36 also an octahedral geometry for the Ni(ll) complexes ( Fig 2B).
Furthermore, a broad band centered at 22150-22500 cm " observed for every complex was assigned 3 to the transition IAig ---) IElg in the iron atom of the ferrocenyl group indicate that there is no magnetic interaction between the Cu(II), Co(ll) and Ni(II) ions and the diamagnetic Fe(lI) ion.
Based on the above observations, it is proposed that the Cu(II) complexes have a square planar geometry (Fig   2A) whereas the Co(II) and Ni(II) complexes are octahedral (Fig 2B). The synthesized dicarboxylic acids and their metal complexes were evaluated for their antibacterial activity against Escherichia coli (a), Pseudomonas aeruginosa (b), Staphylococcus aureus (c) and Klebsiella pneumonae (d). The compounds were tested at a concentration of 30 lag/0.01 mL in DMF solution using the paper disc diffusion method. The susceptibility zones measured in mm are reported in Table 5. The susceptibility zones were the clear zones around the discs. All the dicarboxylic acids were found to be biologically active and their metal complexes showed more significant antibacterial activity against one or more bacterial species in comparison to the uncomplexed ligands. In most of the cases chelation tends to make the dicarboxylic acids act as more powerful and potent bactericidal, thus killing more of the bacteria than the parent dicarboxylic acids. A possible explanation for the increased activity of the complexes is proposed. It may be suggested that in the chelated complex, the positive charge of the metal is partially shared with donor atoms and there is x-electron delocalization over the whole chelate ring. This increases the lipophilic character of the metal chelate and favors its permeation through lipoid layers of the bacterial membranes.